2 2-? t: .«Jw . ; F—"* a . as. a i! A . .4 inks. .m 2.. xx. ,. 2:... $15 I. .5 t .3. 5.2.. r. hm wanna? aifianfim.fi. 5 .. . Ed .84.. 3.? Em ' V lv‘ «:1 1i tdhw'fi‘ 2 v a“ .. . v.- h9...»fi.ufi...~ .a." .1 a... #4,...run‘flh L12... 5.. .‘r an. n n . .f x I 44‘ 3, IL“. I . P “4”,» i .21 LIBRARY ”U .. I Michiga State 1%” Universuy This is to certify that the thesis entitled ANALYSIS OF LlGAND-BINDING DOMAINS OF THE MOSQUITO VITELLOGENIN RECEPTOR presented by LIQUN MAO has been accepted towards fulfillment of the requirements for the M. 8. degree in Genetics Graduate Program >7M /% ’71 M... / Major Professor’s S/ignature QC\;41/57 QPK‘B Date MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6’01 cJClRC/DatoDue.p65-p.15 ANALYSIS OF LlGAND-BINDING DOMAINS OF THE MOSQUITO VITELLOGENIN RECEPTOR BY Liqun Mao A THESIS Submitted to Michigan State University In partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Genetics Graduate Program 2003 ABSTRACT ANALYSIS OF LIGAND—BINDING DOMAINS OF THE MOSQUITO VITELLOGENIN RECEPTOR By Liqun Mao To determine which of the two clusters (CLI and CLII) of complement-type repeats (CR5) in A. aegipti vitellogenin receptor (AanR) is responsible for the binding of Aan, mini-receptors encoding either CLI or CLII were constructed and expressed in Drosophila cells. Saturation-binding assays indicated one binding site on each cluster, with dissociation constants (Kd) of 25.9 nM and 53 nM, respectively. AanR showed at least two binding sites with the apparent Kd of 3.2 nM. Thus, both clusters contribute to the high affinity to Vg, probably in a synergistic way. Protein modeling shows that both clusters have strong negative surfaces, and the surface of CLI is more negative than that of CLII. This indicates that the CLI has higher affinity and that the force mediating AanR-Aan interaction is predominantly electrostatic complementation. The modeled CLIIs of both A. gambiae VgR and AanR have similar surface charge distribution. Modeled AanR EGF-like repeats lack strong negative surfaces. Modeled three AanR YWTD B-propellers have several surface histidine residues that may bring significant change to the surface charge, which implicates YWTD propellers as false ligands for the ligand-binding domain in regulating Aan release and AanR recycling. Lamprey Iipovitellin has an omni-positive surface. The modeled Aan small subunit has a moderately positive surface, which supports the complementation hypothesis. The modeled Aan large subunit has the helical domain, C-sheet, and A-sheet. Copyright by LIQUN MAO 2003 ACKNOWLEDGMENTS I want to thank heartedly my mentor, Dr. Alexander S. Raikhel, for his patient and insightful guidance throughout my graduate research. I thank my present advisor, Dr. Suzanne M. Thiem. I appreciate support from my Guidance Committee, Dr. Leslie Kuhn, and Dr. Rebecca Grumet. I wish to thank director of the Genetics Program, Dr. Helmut Bertrand, for his support during these years. I want to thank Mr. Alan R. Hays for his technical support and Dr. J ianxin Sun for his nice suggestions on experiments. I also appreciate suggestions and help from Dr Guoqiang Sun, Dr. Shenfu Wang, Dr. Jinsong Zhu, Dr. Chao Li, and many other members of Dr. Raikhel’s laboratory. Finally, I thank Geoffrey Attardo for editing the abstract. iv TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES KEY TO ABBREVIATIONS Chapter 1 Literature Review LDLR FAMILY AND INSECT YOLK PROTEIN RECEPTORS Introduction LDLR Family Insect Yolk Protein Receptors Oligomerization of Some LDLR Family Members MOSQUITO YOLK PROTEINS AND INTERNALIZATION OF VITELLOGENIN BY ITS RECEPTOR Mosquito Vitellogenesis and Yolk Proteins Aa VCP A a VCR AaLp and AaLpR Aan Synthesis in Fat Bodies and Internalization by Ovaries Forces Mediating Receptor-Ligand Interaction INTRODUCTION TO THIS THESIS WORK Questions Raised on AanR and My Hypotheses Methodology and Predicted Outcomes Chapter 2 Both Clusters of Complement-type Repeats in AanR Bind Aan INTRODUCTION MATERIALS AND METHODS RESULTS The CLII of CR5 in Insect YPR and the Single Cluster of CR5 in VgRs/YPRs from Other Egg-Laying Animals, VLDLR, LDLR, and ApoER2 Share Homologous Modules in Different Combinations ----------- Construction, Expression, and Purification of AanR Minireceptors ------- Construction of the Aa VgR mini-receptors Expression of mini-receptors in vitrc Expression of mini-receptors in the insect cell line and purification- Binding of The AanR Mini-receptors to Aan Both clusters of CR5 contribute to the high aflinity of the Au VgR to Ad Vg DISCUSSION CONCLUSIONS vii viii ”\INNN— 13 14 14 15 16 21 25 25 28 33 34 34 48 48 53 54 6O 60 67 68 72 75 Chapter 3 Protein Modeling of AanR and Aan: Structural Basis of The Aan-AanR Interaction INTRODUCTION MATERIALS AND METHODS RESULTS AanR Protein Modeling The CLI of CRs in Aa VgR has stronger negative surface electrostatic potential than the CLII The surface charge distribution of the A. gambiae VgR CLII is similar to that of the Aa VgR CLII The E GF -like repeats of Aa VgR very possibly do not contribute to the binding of Ad Vg The Aa VgR YWT D fl propellers and their role in Vg dissociation and receptor recycling The Insect VgR/YPR has one or two kind(s) of endocytosis signal(s)- Aan Modeling-Evidence of A Positive Surface The “receptor-binding motif” on blue tilapia Vg is not a receptor- binding site The lamprey Iipovitellin has an omni—positive surface EP -------------- The Aa Vg small subunit has a positively charged surface The Aa Vg large subunit has the helical domain, C-sheet, and A- sheet DISCUSSION The CLI of CRs in AanR Has A More Strongly Negative Surface Than The CLII The Role of the AanR EGF Homology Domain in Aan Release --------- Sorting Signals in The Cytoplasmic Tail of LDLR Family Members ------- Potential Receptor-Binding Sites on Aan CONCLUSIONS Chapter 4 Summary and Future Research Perspectives SUMMARY FUTURE RESEARCH PERSPECTIVES Bibliography REFERENCES vi 76 77 77 79 79 79 79 79 103 111 116 117 117 122 124 124 135 135 136 138 140 142 144 145 146 152 LIST OF TABLES Chapter 1 Table 1. Tissue expression of the LDLR family members 4 Table 2. Functions and mutational phenotypes of LDLR family members ------------ 5 Table 3. Ligand spectrums of the mammalian LDLR family members 6 Table 4. Number of CR3 of LDLR family members 10 Table 5. Sequences in various ligands critical for receptor binding 24 Chapter 2 Table 6. Kd values for saturation binding assays 72 Chapter 3 Table 7. Comparison of surface charge distribution of the CLII of CR5 in AanR with that in AngR 103 vii LIST OF FIGURES Chapter 1 Fig. 1. Structural organization of members of the low-density lipoprotein receptor family Fig. 2. Conservation of vitellogenin sequences among egg laying animals ----------- Fig. 3. Synthesis and processing of Aan in the fat body Fig. 4. Intemalization of Vg and recycling of VgR in mosquito oocytes .............. Chapter 2 Fig. 5. The CLII of CR5 in insect VgR/YPR and the single cluster of CR5 in egg- laying vertebrate and nematode VgR/YPR share homologous modules in different combinations Fig. 6. The structural organization of the VgR/YPR, VLDLR, LDLR, and ApoER2 Fig. 7. The schematic representation of Aan minireceptors Fig. 8. Construction of AanR minireceptors Fig. 9. In vitro expression of the VgR minireceptors in a coupled transcription and translation system Fig. 10. Transcription and translation of minireceptor genes in the Drosophila SZ cell line Fig. 11. Nonreducing Western blot of VgR minireceptors Fig. 12. The solid phase saturation binding assays of the minreceptors Chapter 3 Fig. 13. Surface electrostatic potential of thirteen modeled modules from two clusters of CR5 in AanR Fig. 14. The surface EPs of seven modules in the CLII of CR5 of the Anopheles gambiae VgR Fig. 15. Comparison of surface charge distribution of the CLII of CR3 in AanR viii 17 20 22 49 51 54 55 61 63 66 68 80 94 Fig. 16. Fig. 17. Fig. 18. Fig. 19. Fig. 20. Fig. 21. Fig. 22. Fig. 23. Fig. 24. Fig. 25. with that in AngR The Surface EPs of seven EGF repeats in AanR Ribbon diagram and topology of the AanR YWTD B propellers ----------- The surface EPs of the AanR YWTD B propellers at pH7 Potential sorting signals in the cytoplasmic tails of invertebrate VgRs/YPRs, human LPRl, and human LPR2 The claimed “receptor-binding motif” of blue tilapia Vg is not a receptor- binding site Lamprey Iipovitellin has a strongly positive surface surrounding the lipid- binding cavity 2D structural alignment of the Aan small subunit with the N-sheet domain of lamprey Iipovitellin The Aan small subunit has a positively charged surface Sequence alignment of the N terminal region of the Aan large subunit with other vitellogenins The Aan large subunit has the helical domain, C-sheet, and A-sheet ------ Chapter 4 Fig. 26. Proposed future design on mosquito minireceptors 104 106 112 114 117 118 123 125 126 127 134 147 APP ApoE ApoER2 APS At BSA Brno bp CAPS Cc cDNA Ce CHAPS CCP KEY TO ABBREVIATIONS 20-hydroxyacdysone 0L2 macroglobulin activated form of a2 macroglobulin Aedes aegypti Acanthogobiusflavimanus Anopheles gambiae amyloid precursor protein apolipoprotein E apolipoprotein E receptor-2 Aedes physical saline Acipenser transmontanus bovine serum albumin Bombyx mori base pair 3-cyclohexylamino- l -propane sulfonic acid Cyprinus carpio complementary DNA Caenorhabditis elegans 3-[3-Cholamidopropyl)-dimethylammonio]- 1 -propanesulfonate clathrin-coated pits CPM CR Dm DMSO DNA Dr EDTA EGF EP ER FACE Fh FH FN3 HDL HDLp HRP Hs IDL IgG IMAC count per minite complement-type repeat Drosophila melanogaster dimethyl sulfoxide deoxyribonucleic acid Danio rerio ethylenediaminetetraacetic acid epidermal growth factor electrostatic potential endoplasmic reticulum formaldehyde agarose gel electrophoresis F undulus heteroclitus familial hypercholesterolemia fibronectin type 3 Gallus gallus high density lipoprotein High-density lipophorin horseradish peroxidase Homo sapiens incubation buffer intermediate-density lipoproteins immunoglobulin G immobilized metal affinity chromatography xi Iu kb kDa La LDLp LDLR LL/LI Lp LpR LRl l LRP LV mRNA ngR N CBI N MR NPxY 0! 0m Pa Ichthyomyzon unicuspis kilobase pair dissociation constant kilo-dalton Larus argentatus low-density lipophorin low-density lipoprotein receptor leucine-leucine or leucine-isoleucine lipophorin lipophorin receptor LDLR relative with l 1 ligand-binding repeats low density-lipoprotein receptor-related protein Iipovitellin Melanogrammus aeglefinus mitogen activated protein messenger RNA VgR minireceptor national center for biotechnology information nuclear magnetic resonance asparigine-proline-x-tyrosine (x can be any amino acid residue) Oryzias latipes Oncorhynchus mykiss Periplaneta Americana xii PAGE PAI-l PBS PEG PTU PVDF PCR RAP Rc RNA RT. 82 cell SDS-PAGE 5] SorLA SRP TC TNT uPA VCB VCP Vg polyacrylamide gel electrophoresis plasminogen activator inhibitor 1 phosphate buffered saline polyethylene glycol Pimephales promelas phenylthiourea polyvinylidene difluoride polymerase chain reaction receptor associated protein Riptortus clavatus ribonucleic acid room temperature Drosophila melanogaster Schneider 2 cell sodium dodecylsulfate-polyacrylamide gel electrophoresis Sillagojaponica sorting protein-related receptor containing LDLR class A repeats surface plasmon resonance tubular compartments transcription and translation urokinase-type plasminogen activator vitellogenic cathepsin-B vitellogenic carboxypeptidase vitellogenin xiii VgR VHDLp VLDLR Vn vitellogenin receptor very high-density lipophorin very low-density lipoprotein receptor vitellin Xenopus laevis yolk protein precursor yolk protein receptor vacuolar protein sorting tyrosine-x-x-d) (d) is a residue with a bulky hydrophobic side chain) tyrosine-tryptophan-threonine-aspartic acid xiv Chapter 1 Literature Review LDLR FAMILY AND INSECT YOLK PROTEIN RECEPTORS Introduction In mosquitoes, the egg maturation is activated by a blood meal that initiates the accumulation of yolk proteins— mainly vitellogenin (Vg)——in the developing oocyte, which increases in size over 300-fold within 36 hours post blood meal (PBM) during Vitellogenesis. Coupled with Vitellogenesis, mosquitoes transmit numerous pathogens to humans and animals. Every year over one million people die worldwide from mosquito- bome malaria. Mosquito-vectored diseases include parasitic diseases, such as malaria and filarial diseases (such as dog heart worm), and virus diseases, such as dengue, encephalitis, and yellow fever. Therefore, elucidating the fundamental mechanisms of mosquito Vitellogenesis is critical in controlling mosquito-bome diseases. During Vitellogenesis of a female mosquito, Aedes aegypti, Vg is synthesized in trophocytes of fat bodies (insect metabolic tissue analogous to the vertebrate liver), circulated in the hemolymph, and accumulated via receptor-mediated endocytosis into the developing oocyte. This internalization process is mediated by the Vg receptor (VgR), a member of the low-density lipoprotein receptor (LDLR) family. LDLR Family The LDLR family is a group of constitutively recycling cell-surface receptors that recognize and internalize extracellular ligands for degradation by lysosomes or storage as yolk granules that provide essential nutrients for cellular functions. The prototype of this family, LDLR, plays a major role in cholesterol homeostasis. Low-density lipoproteins (LDL) contain apolipoprotein B-100 (apoB-100) and are cholesteryl ester-rich, triglyceride-poor macromolecules, arising from the intravascular lipolysis of triacylglycerol-rich very-low-density lipoproteins (VLDL) produced in small intestine and liver by lipoprotein lipase. The LDLR is responsible for the uptake of cholesterol- containing lipoprotein into cells. Mutations in the LDLR gene result in the accumulation of LDL cholesterol in the circulation, which leads to familial hypercholesterolemia (FH), a genetics autosomal dominant disorder. Apart from the basic cargo fimction, quite a few family members bind cytosolic signaling proteins and scaffold proteins with cytoplasmic tails, and thus have signaling function (Willnow et. al. 1999, Howell and Herz 2001). The LDLR-related protein 2 (LRP2, previously named megalin) is also involved in the regulation of biological function of retinoids and steroids by endocytosis (Howell and Herz 2001). Tables 1 through 3 list tissue specificities, functions, mutational phenotypes, and ligand spectrums of selected members of the LDLR family. Each member of the LDLR family has a long extracellular portion, one transmembrane helix, and a short cytoplasmic tail. The extracellular portion contains at least three kinds of modules: the cysteine-rich complement-type repeats (CR, or called LDLR class A repeat) that are candidates for the ligand-binding site, the cysteine-rich epidermal growth factor (EGF)-like repeats, and the tyrosine-tryptophan-threonine- aspartic acid (YWTD) B-propeller fold. The YWTD B-propeller and juxtaposed EGF -1ike repeats compose the EGF precursor-homology domain. Some family members also have a serine/threonine-rich, O-linked sugar domain in the juxta—transmembrane region, but this sugar domain is not important for the ligand binding. 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Such redistribution of yolk protein receptors upon the onset of vitellogenin uptake also occurs in vertebrates. In previtellogenic oocytes of chicken, Gallus gallus (Gg), GngR/VLDLR is detected in vesicular structures of oocytes. During fast yolk protein uptake, GngR/VLDLR relocalizes mainly to cortex of the oocyte (Shen et. al. 1993). Mosquito AanR migrates as a 205 kDa band under a non-reducing condition or a 214 kDa under a reducing condition, a size similar to that of DmYPR. AanR is likely a dimer in vivo. In the mosquito, AanR presents in the pre-vitellogenic ovary as early as the day of eclosion, and its level rises and decreases paralleling the rate of Vg uptake after initiation of Vitellogenesis by a blood meal (Sappington et. al. 1995). Oligomerization of Some LDLR Family Members Oligomeric forms have been observed in several LDLR family members, including LDLR, AaLpR, AanR. On a nonreducing western blot, apart from the monomer-formed AanR band, a secondary band was observed with native AanR preparation (Sappington et. al. 1995, Figure 7), and a 390 kDa rather than a 205 kDa band was observed to be the native-formed AanR on a native western blot (Sappington et. al. 1995, Figure 6A). These secondary bands could represent aggregates or polymerized forms of receptors. The ligand blot (or called far-western blot) result showed strong binding of Aan to the monomer form of AanR, indicating sufficient affinity of monomer to Aan (Sappington et. al. 1995). In Aedes aegypti, another LDLR family member, the ovary-formed lipophorin receptor (AaLpR, insect equivalent of the mammalian VLDLR) also forms a dimer. The AaLpR protein expressed in a TNT system showed as a 145 kDa band on a reducing SDS-PAGE. When AaLpR purified from the ovary membrane was subjected to a non- reducing SDS-PAGE, a 250 kDa receptor band was detected by its ligand, lipophorin (personal communication from Jianxin Sun), which suggests the dimmer-formed AaLpR under the nonreducing condition. In addition to AanR and AaLpR, the LDLR was also found to have Oligomeric forms. In van Driel’s experiment, after the bovine LDLR was solubilized from bovine adrenal cortex membranes and immuno-precipitated, most receptors were found to be presenting as dimers via disulfide bonds (van Driel et. al. 1987). These bonds were formed only after homogenization, because covalent bonds could not be formed if tissue was homogenized in the presence of sulflrydryl alkylating agents (alkylated cysteines do not form S-S). The authors demonstrated that the cytoplasmic domain of LDLR is responsible for self-association, and stated that native formed bovine and human LDLRs have capacity to self-associate noncovalently and form dimers and higher order structures (van Driel et. al. 1987). Because monomeric forms of LDLR from SDS-PAGE gels bind LDL and B-VLDL on ligand blots, it appears that Oligomerization is not crucial for ligand binding (van Driel et. al. 1987). In another experiment, when an LDLR mini-receptor covering the ligand-binding domain and one following EGF-like repeat was subjected to a non-reducing SDS-PAGE, at least three bands were observed, with the smallest band corresponding to the size of a monomer (Dirlam et. a1. 1996). This suggested that LDLR exists as both monomers and multimers, formed possibly throught intermolecular 12 disulfide cross-linking. The fact that on ligand blots all three bands bound human LDL in a calcium-dependent manner supported the notion that Oligomerization is not very important for ligand binding. MOSQUITO YOLK PROTEINS AND INTERNALIZATION OF VITELLOGENIN BY ITS RECEPTOR Mosquito Vitellogenesis and Yolk Proteins In the mosquito A. aegypti, Vitellogenesis process can be divided into the pre-, post-, and vitellogenic phases. The previtellogenic phase can be further split into a 3-day long preparatory developmental stage when the fat body and ovary gain competence to a blood meal, and a developmental arrest stage when the mosquito seeks a blood meal. The vitellogenisis phase is triggered by a blood meal ingestion, when yolk protein genes are activated and massive yolk proteins are produced in fat bodies and deposited into ovaries. In the postvitellogenic phase, production of yolk proteins is shut off, and the mosquito is ready for another blood meal and a subsequent new cycle of Vitellogenesis (Raikhel 1987). In the female mosquito, A. aegypti, most yolk protein precursors (Y PPS) are synthesized in fat bodies during vitellogenisis. These YPPs include the most abundant Vg, the second most abundant proenzyme, vitellogenic carboxypeptidase (VCP) (Cho et. al. 1991), the proenzyme, vitellogenic cathepsin B (VCB) (Cho et. al. 1999), and the lipophorin (Lp) (Sun et. al. 2000). During pre-and post-vitellogenisis, these four YPPs are expressed at low levels in female mosquitoes. Following a blood meal that initiates commencement of Vitellogenesis, the expressions of YPPs increase rapidly and reach their peak levels around 24 hours PBM, then decline gradually. This expression pattern is also similar to those of the AanR (Sappington et. al. 1995) and AaLpR (Cheon et. al. 2001) An VCP: A. aegipti VCP is a serine carboxypeptidase that is synthesized as an inactive latent prenzyrne 53 kDa in size by fat bodies of female mosquitoes during Vitellogenesis, and secreted to the hemolymph and internalized into the develop oocytes. At the onset of embryonic development, AaVCP is activated and its size is reduced to 48 kDa (Cho et. al. 1991). This active form of VCP is maximally present in the middle of embryonic development and disappears by the end (Cho et. al. 1991). Noticing that some of the serine carboxypeptidases are implicated in proteolytic activation of a number of enzymes or biologically active molecules, it was suspected that AaVCP activates hydrolytic enzymes involved in the degradation of yolk proteins in developing embryos, or directly degrades yolk proteins (Cho et. al. 1991). On a preliminary ligand blot from Cho’s laboratory (personal communication from Wenlong Cho), radioactively labeled AaVCP bound an ovarian membrane protein band larger than the 214 kDa AanR monomer, which suggested a putative new receptor on the ovary membrane that recognizes AaVCP. AaVCB: A. aegypti VCB was given its name for the sake of its high similarity to mammalian cathepsin B (Cho et. a1. 1999). The secreted AaVCB in the hemolymph is a large proenzyme, likely a hexamer that is consisted of 44 kDa subunits. Afier internalization into oocytes, the size of AaVCB decreases to 42 kDa. In the mature yolk body, AaVCB is located in the matrix surrounding AaVn. At the onset of embryogenesis, AaVCB is further processed to a 33 kDa active form that is possibly involved in the embryonic degradation of Vg (Cho et. al. 1999). AaLp and AaLpR: The oocyte development in insects involves the accumulation of large amounts of lipids mostly originated extra-ovarianly and delivered by Lp in the insect hemolymph. Lp is basically composed of a 230~250 kDa apoLipophorin-l (apoLp- 1) and a 70~85 kDa apoLp-II. Based on the lipid amount it carries, Lp could be classified as high-density lipophorin (HDLp) or low-density lipophorin (LDLp). In the insect body, Lp mainly functions as a reusable lipid shuttle transporting lipids among lipid loading sites in the midgut, storage sites, and metabolism sites. The Lp—mediated lipid delivery into the developing oocytes could take place in two ways. The major vehicle is LDLp. In response to a stimulatory signal, HDLp associated with two apoLp-IIIs loads its major lipid component, diacylglycerol, from fat bodies and converts to LDLp. LDLp is associated with apoLp-III in proportional to the amount of diacylglycerol incorporated (Soulages and Wells 1994). Upon reaching delivery sites, LDLp unloads its lipid, releases apoLp-III subunits, and converts back to HDLp. Intemalization by cells is not needed for this lipid loading and unloading event. There are also some lipids that are delivered by HDLp, and internalized by the developing oocytes via receptor-mediated endocytosis. The internalized HDLp unloads lipid and apoLp-III, and is converted to very-high-density Lipophorin (VHDLp). The Lps from mosquitoes, A. aegypti, Anopheles albimanus (Wiedemann), and Culex quinquefasciatus (Say) are all HDLps. These mosquito Lps have triacylglycerol as their major neutral lipid component, in contrast to diacylglycerol in other insect species. AaLp has a hydrated density of 1.113 g/ml and contains 49% of lipid and 3.2% of 15 carbohydrate (Ford and Van Heusden 1994). The native formed AaLp is 480 kDa in size, and contains one 238 kDa apoLp-l and one 73 kDa apoLp-II subunits that are both glycosylated (Ford and Van Heusden 1994). The ovary-formed AaLpR is a 129 kDa long membrane protein that is present only in female germ line cells and expressed early during oocyte development (Cheon et. al. 2001). AaLpR shares highest similarity with the locust LpR and high homology with numerous VLDLRs. The ovarian AaLpR intemalizes AaLp into the developing oocyte (Cheon et. al. 2001). Data from Alexander Raikhel’s laboratory suggested that both AaVCP and AaVCB bind AaLp, which binds the AaLpR. In another word, the AanR binds only Aan but not other three minor yolk proteins on the oocyte cell surface. Aan Synthesis in Fat Bodies and Internalization by Ovaries In egg-laying animals, Vg is a very-high-density lipoprotein secreted by the liver of vertebrates, the intestine of nematode, and the fat body of insect. Vg plays a fundamental role during Vitellogenesis (Wallace 1985; Byrne et. al. 1989). Insect Vg is a high molecular weight oligometric phosphoglycolipoprotein with 7-15% lipids, mainly phospholipids and diacylglycerol (Raikhel and Dhadialla 1992). In most Insects, Vg precursor is cleaved in fat bodies into the large and small subunits that oligomerize to form a monomer Vg, while in vertebrate, Vg is synthesized in the liver and cleaved into several subunits only after internalization by the oocyte. Figure 2 shows the conservation of Vg sequences among invertebrate and vertebrate animals. Fig. 2. Conservation of vitellogenin sequences among egg laying animals. The vitellogenin (Vg) sequences from insects, nematode, and vertebrates share five homologous regions (shaded regions) and first four were numbered. The region I in the Iqu forms the N sheet, which is corresponding to the small subunit of Aan. Numeral, amino acid position; S, polyserine region; RXRR, cleavage signal in insect Vgs; Aa, Aedes aegypti (yellow fever mosquito); Ag, Anopheles gambiae (African malaria mosquito); At, Acipenser transmontanus (white sturgeon); Bmo, Bombyx mori (domestic silkworm); Ce, Caenorhabditis elegans (nematode); F h, F undulus heteroclitus (mummichog), Gg, Gallus gallus (chicken); Iu, Ichthyomyzon unicuspis (lamprey); XI, Xenopus laevis (African clawed frog). Insect Ang Bmng Aan Fish Iqu Ath Fth Bird 0ng Amphibian XIVg Nematode CeVgS CeVg6 25 335 360 13 834 870 1058 1466 16741790 M’ ij 1 In!“ 11 111 IV 36 334L363 27 845 874 1056 1449 1637 1782 1 1 LL 15 . - i: 1 RSRR 11 111 IV 88 400 456 582 1012 1087 1285 1740 1948 2139 1 \1 i 1 1 1 1 IS FT?“ if"! 1 i I 1 RYRR 11 111 IV ”8“..-” ._., 20 295 69 733 934 1131 1343 1564 1731 1823 l l 8 1a.»:- 1 s 1 I II 111 IV w ‘ {n1 '2” ."_,--A“'." .3 ‘ ‘_ I" _~- . .;’ m N sheet on domain C sheet \A sheet/7 19 293 687 714 911 1097 1288 1490 1664 l l I s 1 i I II III IV 24 69 719 916 1076 1232 1442 16161704 1 l l I—F'r'mliifllflml l S l ‘j/ I II III IV 26 300 696 723 920 1125 1319 1580 1756 1851 isl #5 1 11 111 26 300 696 723 920 1126 1321 1536 1713 1807 l l 1 EM 1 s 1 jj/ 1 11 111 IV 26 309 325 718 761 945 1306 1474 1603 1 11 111 IV 36 327 342 749 793 982 1340 1514 1651 I_l 1.181. - warm. I I II III IV 18 In A. aegypti, a blood meal activates the translation of the 224 kDa long pre-pro- Vg in the rough endoplasmic reticulum (ER). This pre-pro-Vg is cotranslationally glycosylated and posttranslationally phosphorylated in the rough ER to produce a 250 kDa long pro-Vg (Dhadialla and Raikhel 1990), which is rapidly cleaved into the 190 and 62 kDa long subunits by a Vg convertase (Chen and Raikhel 1996). This Vg convertase is a member of a subtilisin-like proprotein endoprotease family that recognizes the motif, (R/K)x(R/K)R or RxxR, with a juxtaposed B turn for optimal recognition (Brakch et. a1. 1993), and cleaves immediately after this motif (Barr 1991; Rouille et. al. 1995). Both Vg fragments then enter the Golgi complex, are sulfated and further glycosylated to form a 200 kDa long large subunit and a 66 kDa long small subunit. In the Golgi complex, two subunits oligomerize to form the 380 kDa long mature Aan, which is packaged into condensing vacuoles that develop further into large secretory granules, and is finally released into the hemolymph (Dhadialla and Raikhel 1990). Figure 3 depicts the synthesis and processing of Aan in the fat body. In vertebrates, Vg is synthesized in the liver, secreted into the blood stream and internalized by oocytes. In oocytes, Vg is proteolytically cleaved into several polypeptide chains. The lipid-binding product is renamed Iipovitellin (LV), which is comprised of a heavy chain (LVl) of approximately 120 kDa and a light chain (LV2) of about 30 kDa (Byme et. al. 1989). LV can load varying amounts (up to 16% w/w) of noncovalently bound lipid, 2/3 of which are phospholipids (Ohlendorf et. al. 1977; Norberg and Haux 1985). The polyserine region of vertebrate Vg is also released. This released small peptide is named phosvitin (28-35 kDa) or phosvette (13-19 kDa), because it is serine- l9 w mRNA (6.5 kb) pre-prng (224 kDa) cleavage site ’1 1% small (62 kDa) Vg dimer (380 kDa) vitellin (Vn) crystal Fig. 3. Synthesis and processing of Aan in the fat body. (modified after Dhadialla and Raikhel 1990) 20 Translation Cotranslationa/ glycosylation Phosphorylation Cleavage Sulfation Dimen'zation Secretion VgR mediated endocytosis Crystal/iation 83 ufinoa X8|dLUOO 15109 udLu/llowaH 81/1300 enriched (up to 50%), and most of serine residues are phosphorylated (Byme et. al. 1989). Afier Aan is internalized into the oocyte by the AanR, it is discharged from the receptor, stripped of its lipids, and crystallized in the storage form called vitellin (Vn). The AanR is then recycled to the oocyte cell surface (Snigirevskaya et. al. 1997). The details of this process are shown in Figure 4. Forces Mediating Receptor-Ligand Interaction All of the CRs of LDLR family members have a highly conserved acidic CaZ+- binding motif that contributes to the negatively charged surface of the C terminal moiety. Negative charges have long been proposed to be primarily responsible for the recognition of ligands by receptors. Comparisons among 3-D structures of CR5 and CR6 of LDLR and CRII-6 (the sixth module in the CLII of CR3) of LRP showed a region of negatively charged surface electrostatic potential (EP) surrounding the coordinated Ca2+ ion (North and Blacklow 2000, Figure 5). In contrast to CRs, The two EGF-like repeats of LDLR lack the concentration of negatively charged residues found in CRs. Among numerous ligands of the LDLR family members, only ApoE and receptor- associated protein (RAP) bind to all mammalian LDLR family members. The several identified potential receptor-binding sites on various ligands share no significant sequence homology except that they are all rich in basic residues (lysine (K) and arginine (R)) (Table 5). More than one decade ago, Roehrkasten and Ferenz reported K/R residues on locust Vg to be important for binding of VgR on the oocyte membrane (Roehrkasten and Ferenz 1992). Suramin is a negatively charged compound called polysulfated 21 Fig. 4. Internalization of Vg and recycling of VgR in mosquito oocytes. After mosquito Vg is delivered to the oocyte though the hemolymph, Vg enters between follicle cells and through pores of the vitelline envelope, and binds to VgR on the oocyte plasma membrane. Vg/VgR complexes cluster in clathrin-coated pits (CCP), which pinch-off to form coated vesicles. Upon losing the clathrin coat, the coated vesicles are transformed into early endosomes, which fuses with one another to form late endosomes, or transitional yolk bodies. At endosomal pH, Vg is released from VgR and delivered to the mature yolk bodies where it is crystallized and stored until the onset of embryonic development. Released VgRs are recycled to the oocyte surface via tubular compartments (TC). The figure was drawn after Snigirevskaya et. al. (Snigirevskaya et. al. 1997). 22 '; follicle cell j _' - o o microvillus 23 Am .9 888288 0828 .8882 888% 283 AM .vc In 888828 888 8882888 6889 .8882 6885 283 . 888m .888 a 88 88838588 $8-688 8885882828., N38 .3 .3 888888802 HngnHfimfin Sign < E88898 actofiowzmmm M308 .3 .86 mammafiofi zxmozquwquguo glam 2.286 8 825.2888 8988388 88808888888888 Emammmufliuoamogmnu 38788. So— .3 .86 888—22 9853 vwmuéwm 88: 58888an 33 .3 .3 8885802 «9.6322830 Sulwm 888858988 8888 .3 .3 882088 8886888888588 $2.82 882? 88 a .3 .53 8888 .3 a 888883 88388888888888.88588858 8818.: M828... 8888 8.3.8082 Emmfiammgmmmgmfifim 82.82 8.82.83 2-83 .6 88:08 389,883. 88886.83— ...oocozcom 8828804 8ng .9553 838.898 .88 .3ch mafia: 8:83.888.» 888 8.835889% .m 03:. 24 polycyclic hydrocarbon. Suramin blocks the binding of many ligands (including vertebrate and insect Vgs) to their receptors, and dissociates binding by competition. This implies that ionic bonds play a crucial role in Vg/VgR interaction. Recently, Li et. al. claimed a K/R-rich VgR-binding site on tilapia Vg (Li et. al. 2003). INTRODUCTION TO THIS THESIS WORK Questions Raised on AanR and My Hypotheses It is already accepted that the single cluster of CR5 in one-cluster LDLR family members mediates binding of ligands, while for two- and four-cluster family members things are much more complicated. Taking LRP as an example, LRP has 4 clusters of CR5 with 2, 8, 10, 11 modules in each cluster. Studies with LRP mini-receptors showed that both the CLII and CLIV bind the same 12 kinds (including az-M“) of known ligands (Neels et. al. 1999; Willnow et. al. 1994). In contrast, the CLIII binds only ApoE and binds RAP weakly (Neels et. al. 1999), and the CLI has only low affinity to az-M“ (Mikhaihenko et. al. 2001). In comparison to either the CLI or CLII, a LRP mini-receptor covering both clusters has much higher affinity to az-M“ (Mikhaihenko et. al. 2001), which indicates that the CLI and CLII cooperate to generate a high-affinity binding site for az-M“. From the LRP binding results, we learned that for four-cluster family members such as LPR, different clusters of CR5 could have anywhere from nearly the same to very different ligand spectrum(s). But how about the two clusters in insect VgRs/YPRs? The VgRs in egg-laying vertebrate animals (for example, bird, fish, and amphibian) have only 25 one cluster of eight CRs, and the nematode YPR also has one cluster of merely five CRs. Is it possible that the first cluster of five CRs in the insect VgR/YPR and the single cluster of five CRs in nematode YPR are evolved from a common ancestor, while the second cluster of CR5 in the insect VgR/YPR and the single cluster of CR3 in some one- cluster LDLR family members are evolved from another common ancestor? Another question on the ligand-binding domain of AanR concerns the function of one additional cluster of CR5 in insect VgRs/YPRS. One possible answer is that there are different ligand spectrums for CLI and CLII. While a surprising fact is, unlike chicken VgR/VLDLR that also imports VLDL, riboflavin-binding protein, and az-M in addition to Vg into oocytes (Schneider 1996), Vg is the only ligand for known insect (mosquito, fruitfly, and cockroach) VgRs. Another possible explanation is the strengthened binding of Vg through forming either a large interdomain ligand-binding site with higher affinity to Vg or two separate ligand-binding sites, with each site binding one copy of Vg independently. Until now, no comparison on the ligand-binding properties of CLI and CLII of CRs in insect VgR/YPR has been reported. An interesting question on AanR is which one of the two clusters of CR5 is predominantly or exclusively responsible for high affinity to its ligand, Aan? The negative surface charges of some LDLR family members have been proposed to be primarily responsible for recognition of ligands. While F ass et. al. solved the structure of the CR5 of LDLR, and showed that acidic residues in the conserved acidic motif of CR5 are buried to participate in Ca2+ coordination, rather than being exposed to the surface (F ass et. al. 1997). Based on nearly neutral surface EP of the LDLR CR5 they 26 showed (Fass et. al. 1997, Figure 4), they suggested a hydrophobic concave face of the LDLR CR5 rather than a negative surface to be responsible for interaction with ligands. To elucidate the force mediating the AanR—Aan interaction, a check on the molecular surface EPs of individual modules from both clusters of CR3 in AanR can provide a good answer. Comparison of surface charges of modules from both clusters can also tell us which cluster has a stronger negatively charged surface and thus higher affinity to Aan, given that most modules have predominantly negative surfaces. Modeling the small and large subunits of Aan can also allow me to evaluate, from the ligand side, the hypothesis that the AanR-Aan interaction is mediated mainly by the negative-positive electrostatic attraction. The YWTD B propeller of LDLR has a negatively charged top face on the cell surface. In the crystallized form, this top face contacts the Ca2+-binding loops of CR4 and CR5 of LDLR at endosomal pH (pH5.3) (Rudenko et. al. 2002). Because both CR4 and CR5 are critical for lipoprotein binding, their association with YWTD B propellers (rather than ligands) at endosomal pH proposed a mechanism for lipoprotein release in the endosome by replacing a real ligand of LDLR with a false one, the YWTD B propeller of LDLR (Rudenko et. al. 2002). An investigation of the CR4 and CR5 docking face of the LDLR YWTD B propeller found on the docking face two histidine residues that potentially change the surface charge of this face from negative to positive at endosomal pH (Jeon and Blacklow 2003, Figure 4). To evaluate whether the proposed mechanism of LDLR ligand release is applicable to other LDLR members—for example, AanR—an investigation must be done of the surface properties of three assumed YWTD B propellers of AanR and evidence of histidine residues must be sought of the 27 surface of assumed YWTD B propellers of AanR that can potentially switch the surface charge to positive. Although CRs of the LDLR family members are primarily responsible for ligand binding, the EGF precursor homology domain of LDLR (including the YWTD B propeller and its flanking EGF-like repeats) were shown to be also important for binding of LDLR to some ligands. Davis et. al. reported that an LDLR mini-receptor lacking the EGF precursor homology domain showed markedly reduced affinity to LDL on the cell surface, and complete degradation in the transfected cells afler incubation with B-VLDL on the cell surface (Davis et. al. 1987). The EGF precursor homology domain was also found to be important for in vivo binding of LDLR to LDL on the cell surface, but not for in vitro binding (Davis et. a1. 1987). In another experiment, binding analyses of LDLR mini-receptors showed that the first EGF-like repeat was required for binding of LDL, but not B-VLDL, while the second EGF-like repeat was not required for ligand binding (Esser et. al. 1988). To investigate whether any of seven EGF-like repeats of AanR can contribute to the assumed electrostatic complementarities between AanR and Aan, it is necessary to check the surface charge distribution of each EGF-like module in AanR. Recently, a “VgR-binding motif" on the small subunit of blue tilapia Vg was reported by Li et. al. (Li et. al. 2003). It would be usefiil to investigate the corresponding regions on lamprey Vg and .4an and evaluate whether these corresponding regions could be the VgR-binding motifs or not. Methodology and Predicted Outcomes 28 To find evidence to support or oppose my assumption that the CLI in AanR and the cluster of five CRs in nematode YPR are evolved from one common ancestor, while the CLII in AanR and the single cluster of CR5 in some of the one-cluster LDLR family members are evolved from another common ancestor, a multiple—sequence alignment of CRs from investigated species can provide a sound prediction. If the alignment shows that the five CRs of nematode YPR are more homologous to the CLI than the CLII of CR3 in insect VgR/YPR, and the single cluster of CR5 in some one-cluster members is more homologous to the CLII in insect VgR/YPR, then my assumption is correct. If the alignment shows that the five CRs of nematode VgR are more homologous to the CLII than the CLI in insect VgR/YPR, then the CLI of five CRs in insect VgR/YPR is evolved from a different ancestor. To test whether one cluster of CR3 in AanR is predominantly or exclusively responsible for the binding of Aan, saturation binding assays need to be done to directly measure the dissociation constant for each cluster. For this purpose, mini-receptors with either one or both clusters of CR5, together with the flanking EGF-precursor homology domain, need to be constructed. To make sure mini-receptors have correct post- translational modification and thus biological activities, mini-receptor genes need to be expressed in an insect cell line. Because the Drosophila melanogaster VgR (DngR) is structurally very similar to the AanR, and because the Drosophila S2 cell line is commercially available, it is reasonable to express mini-receptor genes in Drosophila cells. Even though the reproduction system of Drosophila is somewhat similar to that of the mosquito, it is still possible that the truncated VgR may not be fully processed and modified in the Drosophila system. If saturation ligand-binding assays show no affinity 29 of mini-receptors to Aan and there are no mutations in the coding regions of mini- receptors, other insect expression systems (preferably mosquito), with the exception of fruit fly, should to be tried later. If ligand-binding results show high affinity for one cluster and much lower affinity for another and the higher affinity approaches that of the full-length AanR, then one cluster is predominantly responsible for Aan binding. If the affinities of both clusters are much lower than that of the full-length AanR, then both clusters contribute to the high affinity, probably in a synergistic way. To test whether the negative-positive charge attraction is the main force mediating the AanR-Aan interaction, evidence from both sides needs to be obtained. On the receptor side, it will be necessary to check to see if most of the CRs of AanR have negatively charged surfaces and if some of them have strongly negatively charged surfaces. This can be done by modeling thirteen CRs of AanR and calculating surface EP of each module. If the results show that some modules do have negatively charged surfaces, and some modules have strong negative surfaces, then I can compare the surface EPs of the two clusters. Theoretically, the cluster with stronger negative surface has higher affinity to Aan. If the results show that most modules have nearly neutral or even positive surfaces, then the force mediating the AanR—Aan interaction is primarily not negative-positive charge attraction. On the ligand side, a partial or whole Aan needs to be modeled, and surface EP of the modeled region needs to be calculated to find expected positively charged surface patches. Because the 3-D structure of the mature form of lamprey Vg has been solved by X-ray crystallography (Anderson et. al. 1998; Raag et. al. 1988; Thompson and Banaszak 2002), modeling Aan becomes possible. If 30 the surface of mature lamprey Vg has no large positively charged patches, then electrostatic attraction is not the dominant force mediating the VgR-Vg interaction. To investigate whether EGF-like repeats of AanR contribute to the assumed electrostatic complementarities between AanR and Aan, models for each module of seven EGF-like repeats will be made, and then the surface EP of each modeled module will be calculated. One possible result would be that some of the EGF-like repeats, especially the EGF-like repeats juxtaposed the boundary CRs in each cluster, may have concentrated negatively charged residues. If that is true, then these EGF-like repeats are possibly involved in binding to Aan, together with CRs. A second possibility is that there are no concentrated negatively charged surface patches on any EGF-like repeats of AanR. It could then be concluded that no EGF-like repeats contribute to negative- positive charge complementarities between AanR and Aan. To evaluate whether any of the assumed three YWTD B propellers of AanR potentially act as a false ligand of the ligand-binding domain of AanR, three assumed YWTD B propellers of AanR will be modeled, if possible. If they really have B propeller structures, then calculation of the surface EP of each YWTD domain will be done and a search for surface histidine residues will be performed. If the three YWTD domains do not have six-bladed B propeller structures, then it is impossible for YWTD domains of mosquito VgR to play a critical role in Vg release and VgR recycling. If at least some YWTD domains have six-bladed B propeller structures but there is only one histidine residue on the surface, or if there are several histidine residues on the surface but they are scattered and located not in positive or nearly neutral surface areas, then it would still be somewhat possible that the YWTD B propellers of AanR contact a 31 ligand-binding domain in endosomes, even though the conversion to the false ligand in eondosomes is not switched by histidine residues. If some YWTD domains have six- bladed B propeller structure, there are at least two histidine residues on the surface, and some surface histidine residues are gathered and located in a negative or nearly neutral surface area, then these gathered surface histidine residues potentially could switch the YWTD B propeller into false ligand of the ligand-binding domain of AanR at endosomal pH. To test whether in AanR there is a counterpart of the “VgR-binding motif’ found in tilapia Vg, a multiple alignment of Vgs will need to be done. An inspection of the corresponding region of lamprey Vg would be very persuasive in evaluating popularity of this “VgR-binding motif” in fishes and insects. If in some insect Vg the corresponding regions lack the feature of a receptor-binding motif, and/or if the corresponding region in lamprey Vg does not make the surface EP of this region more positive than most other surface regions, then this clamed “VgR-binding motif” is at least not applicable to the species investigated. If the opposite is the case, then it is quite possible that the counterpart in Aan is a VgR-binding motif. 32 Chapter 2 Both Clusters of Complement-type Repeats in AanR Bind Aan 33 INTRODUCTION This chapter focuses on two questions. First, a multiple alignment of the CLI and CLII of CR5 in insect VgRs/YPRs with the single cluster of CR5 in one-cluster LDLR family members will be performed to evaluate whether it is possible that the single cluster of five CRs in nematode YPR and the CLI of five CRs in insect YPRsNgRs are evolved from a common ancestor. Another question is whether one cluster of CR5 in AanR is predominantly or exclusively responsible for the binding of Aan. If both the CLI and CLII in AanR bind Aan, then we should try to find out which cluster has higher affinity to Aan. To answer this question, mini-receptors with either one or both cluster(s) of CRs will be constructed and transfected into a Drosophila line, and the expressed mini-receptor proteins will be used in saturation binding assays to measure the dissociation constant for each cluster. MATERIALS AND METHODS Animals Mosquitoes, Aedes aegpti, were reared as described (Hays and Raikhel 1990). Vitellogenesis was initiated 3~5 days afier eclosion with a blood meal on rats. Construction of Mini-receptors Two Aedes aegpti vitellogenin mini-receptor (AangR) genes, which encode the signal peptide, one of the two clusters of CR3, and one following B propeller fold with its three juxtaposed EGF-like repeats, were obtained by Polymerase Chain Reaction (PCR) with PCR SuperMix High Fidelity (Invitrogen). The third m VgR gene encoding the 34 extracellular portion of AanR except for the 0-linked sugar domain was constructed as a positive control. The plasmid pBlue-VgR2.7 was constructed earlier by inserting a 2.7 kb long AanR cDNA fragment from an A. aegpti ovary cDNA library to a pBluescript vector (Stratagene) (Sappington et al. 1996). The plasmid pG-VgR3.2 was built earlier by inserting a 3.2 kb long AanR cDNA fragment from a 3' RACE of an A. aegpti ovary total RNA into the vector pGEM-S (Promega) (Sappington et al. 1996). Identities of all constructs were checked by both restriction analysis and sequencing. Mini-receptor m VgR] : A 1920 bp-long AanR cDNA fragment encoding the CLI of CR5 and the first YWTD B propeller (YWTDl) juxtaposed by three EGF-like repeats (EGF 1, EGF2, and EGF3) was amplified from the template pBlue-VgR2.7. The primer set was composed of the forward primer P1, 5'- AATGAGCTCTCGAGTTTGATGGGCGCGATC-3' with restriction sites Sac I and Xho I introduced, and the reverse primer P3, 5'-GAAGGGCCCGCGGCAAGAATGCTTG-3’ with Apa I site introduced. The 1897 bp—long Sac I-Apa I fragment of the amplified DNA was inserted to the same sites on the cloning vector, pGEMSZf(+) (3003 bp, Promega) to construct the 4831 bp-long plasmid, meVgRl. The 1898 bp—long Xho I-Apa I fragment of meVgRl was inserted to the same sites on the expression vector, pAc5.1N5-HisA (Invitrogen), in the 5’ end of a 83 bp-long sequence encoding a V5 epitope and a six- residue polyhistidine (6xHis) tag. The constructed 7266 bp-long plasmid was named pAngRl. Mini-receptor ngRZ: A 115 bp-long cDNA encoding the signal peptide of AanR was amplified from the template, pBlue-VgR2.7, with the forward primer P1, and the reverse primer P2, 5'-TGCCTGCAGGATCCACGCTTCCGAAG-3’, with BamH I 35 and Pst I sites introduced. The 97 bp-long Sac I-Pst I fragment of the amplified DNA was inserted to the same sites on pGEMSZf(+) to construct the 3088 bp-long plasmid, pG- signal. A BamH I site was conveniently introduced in the 3' end of the signal peptide coding sequence. The 2223 bp-long AanR cDNA fragment encoding the CLII of CR5 and YWTD3 juxtaposed by 3 EGF-like repeats (EGFS, EGF 6, and EGF7) was amplified from the template, pG—VgR3.2. The primer set was composed of the forward primer P6, 5’- GCGGATCCCTGCGAGTTCAAGTGTACC -3’ with BamH I site introduced, and the reverse primer P7, S'- GTTGGGCCCTTGCGGACAGATGTCC-3' with Apa I site introduced. The 2206 bp-long BamH I-Apa I fragment of the amplified DNA was inserted to the same sites on pG-signal to construct the 5233 bp-long plasmid, meVgR2(a). The 2305 bp-long Xho I-Apa I fragment of meVgR2 was inserted to the same sites on pAc5.1/V5-HisA to construct the 7668 bp-long plasmid, pAngRZ. Mini-receptor m VgRI-Z: A 2254 bp—long AanRZ. 7 fragment was obtained by PCR with the template, pBlue-VgR2.7, and the primer set, Pl/P5. The sequence of the reverse primer, P5, is 5'-CCCGGTGGCGAGTCTGGAG-3'. The 2866 bp-long AanR3.2 fragment was obtained by PCR with the template, pG-VgR3.2, and the primer set, P4/P7. The sequence of the forward primer, P4, is 5'- CCCGGTGGCGAGTCTGGAG-3'. The purified Aa VgRZ. 7 and Aa VgR3.2 PCR fragments were taken as two overlapping templates in the second tier of PCR with the primer set, P1/P7. The obtained 5059 bp-long Sac I-Apa I fragment encoding the extracellular portion of AanR except for the O-linked sugar domain was inserted to the same sites on pGEMSZf(+) to construct the 8008 bp-long plasmid, meVgRl-2(a). A 36 5080 bp-long Xho I—Apa I fragment of meVgRl-Z was inserted to same sites on pAcS. l/VS-HisA to construct the 10443 bp-long plasmid, pAngRl-Z. Reversion of Point Mutations on Mini-receptors Sequencing was performed in the Genomics Technology Support Facility at Michigan State University. Three mini-receptor genes were fully sequenced, and four clones of VgR3.2 cDNA—pG-VgR3.2, pG-VgR3.2-la, pG-VgR3.2-2a, and pG-VgR3.2- 2b—were partially sequenced. A nonsense mutation (C3046G) was found near the 5' portion of a sequence coding for the CLII of CR5 in plasmids meVgRZ, meVgRl-Z, pG-VgR3.2, pG-VgR3.2-2a, and pG-VgR3.2-2b. pG-VgR3.2-la does not have this mutation. In the CLII of CR5 in the m VgR1-2 gene, one more point mutation (A333 16) was detected that replaced the asparagines (N) residue with the aspartic acid (D) residue. Reversion of pG ngRZ mutants: The ngR2 coding sequence was reamplified from an alternative template, pG-VgR3.2-1a, to avoid the nonsense mutation from the original template, pG-VgR3.2. The amplified fragment was cloned on pG-signal to construct the plasmid, meVgR1-2(b). Sequencing of three clones of meVgR2(b) and the template, pG-VgR3.2-1a, detected in the C terminal region eight common point mutations from pG-VgR3.2-1a that are absent in pG-VgR3.2. To reverse these eight point mutations in the C terminal region of the m VgR2(b) gene, a 887 bp—long Xho I-Pst I fragment of meVgR2(a), which encodes the signal peptide and % of the CLII of CR5, was replaced with its equivalent on pG ngR2(b) to construct the reversed plasmid, meVgRZ. 37 Reversion of pG m VgRI-Z mutants: There are respectively 1, 2, and 3 EcoR I site(s) on p6 ngRZ, pG ngRl-Z and pG-VgR3.2-1a. A 1122 bp—long fragment from EcoR I digestion of pG-VgR3.2-1a was ligated to a 6886 bp-long fragment from EcoR I digestion of pG ngRl-Z to reverse the nonsense mutation. Preferred orientation of the insert was indicated by a 468 bp (rather than 653 bp) fiagment from EcoR V/Pst I double digestion. The reversed construct was named pG ngRl-Z. In Vitro Expression of Mini-receptor Genes in a Coupled Transcription and Translation System The mini-receptor gene driven by a phage SP6 RNA polymerase promoter on the pGEMS vector was expressed in the TNT® SP6 quick-coupled reticulocyte lysate transcription and translation system (Promega), and labeled with [35S] methionine (PerkinElmer) as described in technical manual. The Luciferase SP6 Control DNA (Promega) was used as a positive control. Reactions were subjected to the reducing sodium dodecylsulfate—polyacrylamide gel (SDS-PAGE) and autoradiography. Maintenance of Insect Cell Line Drosophila melanogaster Schneider 2 (82) cell (Invitrogen) culture was initiated from frozen stock and maintained generally as described in the manual. The GIBCOTM Drosophila Serium-Free Medium (Drosophila-SFM, Invitrogen) with L-glutamine, penicillin, and streptomycin at the final concentrations of 16.4 mM, 10 U/ul, 10 ug/ul, respectively, was used for cell maintenance. Before long storage of cells, three-day-old cells were loosen by taping shock and the cell concentration was determined on a 38 hemacytometer. Cells were then pelleted and colleted by spinning at 840g for 4 min at 4°C. Afier washing in the Phosphate Buffered Saline (PBS) and respinning, cells were resuspended at a density of 1.1x107 cells/ml in the freezing medium (11 ml of SFM, 7 ml of conditioned medium, and 2ml of dimethyl sulfoxide (DMSO, Sigma». Aliquot of lml was dispensed to each 2 ml cryogenic vial, and chilled gradually to -80°C. Frozen vials were then submerged in liquid nitrogen for the long-term storage. Stable Transfection of Insect Cell Line The Drosophila Expression System (DES, Invitrogen) was applied to the stable transfection of S2 cells. The plasmids used for transfection were, the constitutive expression vector, pAc5. l/VS-HisA, the selection vector, pCoHYGRO, which carries a hygromycin resistance gene, and the positive control vector, pAc5.1N5-His/lacZ, which expresses B-galactosidase protein. Stable transfection of S2 cells with E ffeetene reagent: 106 S2 cells were suspended in 5 ml of Schneider’s Drosophila complete medium (Invitrogen) and seeded in a 25 ml flask or a 60 mm Petri dish. After cultured at 28°C for one day, the flask is expected to be 40-80% confluent on the day of transfection. 1 pg of pAcS. 1/V5-HisA (mock transfection), pAngRl , pAngRZ, pAngRl-Z, or pAc5. 1N S-His/lacZ (positive control), 50 ng of pCoHygro, and the DNA condensation buffer (Qiagen) were mixed to make a total volume of 150 pl. 8 ul of enhancer (Qiagen) was then added and mixed by vortexing for 1 sec. After the mixture was incubated at room temperature (RT) for 5 min, 10 ul of Effectene transfection reagent (Qiagen) was added, and the mixture was further incubated for 10-15 min to form a complex. While complex formation was 39 taking place, cells were washed once in the flask with PBS, and 4 ml of the fresh medium was added to the flask. When incubation was done, the transfection complex was mixed with lml of the Drosophila complete medium by pipetting up and down twice, and immediately applied to the cell layer in the flask. 24-48 hours after transfection, cells were passed into selective media with penicillin/streptomycin and hygromycin at a concentration of 100-200 pig/ml depending on the cell density. Stable transfection of S2 cells with Cellfectin reagent: S2 cells were cultured in the Drosophila-SFM without antibiotics. 106 cells were seeded in each 25 cm2 flask and cultured for one day. On the day of transfection, 30 ug of plasmid construct, 1.5 pg of pCoHygro, and 2.5 ml of the Drosophila-SFM were mixed in a tube, while 120 pl of the CellFectin reagent (Invitrogen) was mixed with 2.5 ml of the SFM in another tube. Solutions from two tubes were mixed gently, and the mixture was incubated for 15-45 min at R.T.. S2 cells were washed once in the flask with the PBS or SFM, and overlaid with the lipid-DNA complex. After 5-24 hours, the transfection mixture was replaced with the SFM, and cells were cultivated for two days before passed into the selective medium. B-Galactosidase Enzyme Assay To monitor the expression of B-galactosidase in the 82 cells transfected with positive control vector, pAc5.1/V5-His/lacZ, adherent cell lysate was prepared using the reporter lysis buffer (Promega) as described in the technical bulletin. The B-galactosidase assay was done using the B-Gal Assay Kit (Invitrogen) as described in the manual. 40 Extraction of poly A+ mRNA from Transfected 82 Cells and Northern Blot of Mini-receptors Extraction of poly A” mRNA: S2 cells stably transfected with mini-receptor genes were harvested 7, 25, 49, and 72 hours after activation by passage of cells, and were used for extraction of poly A+ mRN As with the Oligotex Direct mRNA Kit (QIAGEN) as described in the protocol. Extracted mRN A was then subjected to formaldehyde agarose gel electrophoresis (PAGE). Northern blot of mini-receptors: The 3-morpholinopropanesulfonic acid (MOPS, Sigma) buffer (Pata and Truve 1998) was used to be the buffer system for the formaldehyde agarose gel (2.5 M formaldehyde and 2mM Ethylenediaminetetraacetic acid (EDTA) in the MOPS buffer), the RNA gel loading buffer (final 3 M formaldehyde, 34% forrnamide, 4.5% glycerol, and bromophenol blue in the MOPS buffer), and the gel running buffer (0.61 M formaldehyde and 2 mM EDTA in the MOPS buffer). The poly A+ mRNA extracted from the same mass of cells was mixed with sheared salmon sperm DNA (Invitrogen), denatured at 68°C for 10 min, and subjected to FAGE. After run at 25 volts overnight, mRNA bands were transferred to a positively charged nylon filter (Hybond-N+, Amersham Biosciences) overnight as described (Pata and Truve 1998). The nylon blot was rinsed briefly in 2x SSC, and crosslinked in a Stratalinker® UV crosslinker (Stratagene) as described in the manual. The RNA marker slot was cut off the blot, and stained with methylene blue as described (Sambrook and Russell 2001). The Cross-linked blot was rinsed briefly in H20 to remove salts, and prehybridized in the ULTRAhbe hybridization buffer (Ambion) overnight at 42°C. The linearized m VgRI-Z DNA was used as the template to make the DNA probe with the random primed DNA 41 labeling kit (Roche) as described in the manual. The labeled probe was purified by passage through a spin column packed with Sephadex G-50 (Amersham Biosciences). The purified probe was denatured at 95°C for 10 min and added to the hybridization bottle. The hybridization was carried out at 42°C overnight. After intensive washing as described (Pata and Truve 1998), the blot was subjected to autoradiography. Purification of Mini-receptors by Immobilized Metal Affinity Chromatography His-tagged mini-receptor proteins expressed in stably transfected S2 cells and secreted to media were purified with the cobalt-based TALON® resin (CLONTECH) packed in a gravity-flow column. The conditioned cell culture media were harvested by spinning for 5 min at 800g, and 20 min at 1200g at 4°C. Proteins were precipitated with 18% of polyethylene glycol (PEG) 6000-8000 with rotation at 4°C overnight followed by centrifugation at the highest speed for 40-50 min. Pellet was resuspended in the PBS with 5-10 mM imidazole, the 100x diluted protease inhibitor cocktail for mammalian tissues (PI-tissue, Sigma), and 12.5 mM EDTA, and was dialyzed against the PBS with 5 mM imidazole in a lS-ml Slide-A-Lyzer® dialysis cassette (Pierce). The 10 ml chromatography columns (Bio-Rad) were packed with the PBS equilibrated TALON® metal affinity resin (CLONTECH) and loaded with dialyzed samples. After washing with 5 mM imidazole in the PBS, the bound proteins were eluted with 150 mM imidazole in the PBS. Fractions with protein eluate were detected by a Bradford protein microassay (1.25-25 ug/ml) as described in the technical note (Bio-Rad), pooled, and diafiltrated against the Incubation Buffer (IB, Sappington et al. 1995) in an Amicon® Ultra-4 42 centrifiigal filter device (Millipore) as described in the user guide. The protein concentration was determined by 3 Bradford protein assay with bovine serum albumin (BSA) as a standard. Isolation of Aedes Ovary Membranes and Enrichment of AanR The preparation of mosquito ovary membrane and enrichment of AanR was essentially according to previous descriptions (Dhadialla et al. 1992; Sappington et al. 1995). Purification was done either on ice or at 4°C. Isolation of mosquito ovary membranes: Ovaries were dissected from female mosquitoes fed with a blood meal 20-24 hours before dissection as described previously (Sappington et al. 1997) and stored at -85°C until use. 2000 pairs of frozen ovaries were suspended in 5 ml of the Aedes Physiological Saline (APS, Hagedom et al. 1977) with 50x diluted PI-tissue and 12.5 mM EDTA and homogenized in a glass-Teflon homogenizer for 5 bouts. After centrifugation at 500g for 10 min, the surface lipid was wiped off, if possible, and the supernatant was saved. The pellet was extracted one more time with 5 ml of the APS (PI+), and two times with 5 ml of the APS/suramin (APS with P1, EDTA, and 5 mM suramin (EMD Biosciences)) as above to release bound VgR from Vn. The supernatants from above steps were combined, and spun at 100,000g for 45 min. The supernatant and lipid layer were carefully removed, and the pellet was extracted with 5 ml of the B2 buffer/suramin (B2 buffer with 100x diluted PI-tissue and 5 mM suramin at pH 8.4) to release crystallized and bound Vn. After spinning at 100,000g for 45 min, the pellet was extracted one more time with the B2 buffer/suramin, two times with the B2 buffer (PI+) to wash off suramin, one time with the APS (PI+, pH7) to change buffer and 43 neutralize, and one time with the Tris-Maleate buffer (with 100x diluted PI at pH6) to change buffer and fiirther reduce the pH. The pellet was resuspended in a minimum volume of the Tris/Maleate buffer (PI+) by pipetting and sonicating (Vibra CellTM VC40, Sonics & Materials) at the lowest setting for 5 sec. The protein concentration of the suspension was determined by a Bradford protein microassay, and adjusted to 5-6 ug/ul by dilution. Solubilization of the ovary membrane and enrichment of Ad VgR: The above membrane preparation was mixed with an equal volume of Tris-Maleate/CHAPS (Tris- Maleate buffer with P1 and 80 mM CHAPS (Sigma)) and incubated for 1 hour to solubilize membrane proteins. After spinning at 100,000g for 1 hour, the supernatant was recovered and diluted with the Tris-Maleate buffer to reduce the concentration of CHAPS to 15 mM. After spinning at 100,000g for 45 min to precipitate partially soluble proteins, the supernatant was recovered and treated with 9% of PEG4600 for 1—2 hours with rotation to selectively precipitate soluble proteins. After another spinning at 100,000g for 40 min, the VgR-enriched pellet was washed in 100 pl of the IB by pipetting and sonicating as above, and spun at 100,000g for 30 min. The pellet was finally resuspended in the IB and stored at -80°C. Metabolic Radiolabelling of Vitelloginin in the Fat Body Tissue Culture Fat body tissues were dissected from female mosquitoes 17-22 hour PBM and cultured in vitro generally as described previously (Raikhel et al. 1997; Dhadialla et al. 1991). In the complete fat body culture medium, 200x diluted PI cocktail for tissue culture media (PI-culture, Sigma), 10'5 M B-ecdysone, and 0.16 mM phenylthiourea (PTU, Sigma) resolved in ethanol were included to diminish protein degradation, reinforce the induction of total protein synthesis, and prevent the oxidation of amino acids, respectively. Vg was metabolically labeled with 0.4 11M [35S]-methionine (PerkinElmer) in the complete culture medium with 8 uM unlabled methionine for 2-3 hours. The conditioned medium was collected and diluted in 1/5 volume of the B2 buffer (Dhadialla et al. 1992) with the 6x diluted PI-tissue and 75 mM EDTA. After clarification by spinning at 19,000g for 8 min at 4°C, the supernatant was stored at -85°C until use. Purification of Vitelloginin and Vitellin by Anion Exchange Chromatography The Vg samples were from the mosquito fat body tissue culture, and the Vn sample was made by resolving crystallized Vn from the homogenized mosquito ovaries with the B2 buffer as described (Koller et al. 1989). The raw protein preparation was diafiltrated against the 150 mM anion exchange buffer (with 150 mM salt, Dhadialla et al. 1991) in an Amicon® Ultra-15 centrifugal filter device (Millipore) as described in the user guide. The 100x PI-tissue was added back to the sample and the sample was loaded three times to the DEAE-Sepharose Fast Flow (Amersham Biosciences) resin equilibrated with the 150 mM AE buffer and packed in an Econo—Pac® chromatography column (Bio-Rad). After washing the column with 150 mM and 200 mM AE buffers (with 150 mM and 200 mM salt), pure Vg was eluted with the 400 mM AE buffer (with 400 mM salt). The elution peak was determined by transferring 2 ul from each fraction to a 20 ml scintillation vial with 3 ml of the BioCountTM complete counting scintillation 45 cocktail (Research Products International) and measuring the counts per minute (CPM) with the Beckman Liquid Scintillation System (LS 1801, Beckman). The peak fractions were pooled and diafiltrated against the 1B. The final protein concentration was determined and the CPM measured to calculate the labeling strength of Vg. Silver Staining of Polyacrylamide Gel After electrophoresis, the polyacrylamide gel was fixed in the fixing solution (50% ethanol, 12% acetic acid, and 0.05% formaldehyde) for at least 90 min. The gel was washed with 50% ethanol several times, and rinsed with pure H20 for 20 see only. The gel was transferred to another tray, submerged in 0.02% sodium thiosulfate for exactly 1 min, and washed with pure H20 three times each for 20 sec. The gel was submerged in the chilled silver solution (0.1% silver nitrate and 0.075% formaldehyde) for 15 min at 4°C and rinsed with pure H20 two times each for 20 sec at R.T.. The stained gel was transferred to a new tray and incubated in the developing solution (3% NazCO3, 0.05% formaldehyde, and 0.0005% Na2S203) until the development was done. The reaction was stopped with 5% acetic acid. Western Blot of Mini-receptors The purified mini-receptor proteins were subjected to a nonreducing CriterionTM 4-15% Tris-HCl SDS-PAGE gel (Bio-rad) at 4°C according to the instruction manual and blotted at 30 volts overnight to a polyvinylidene difluoride (PVDF) membrane in the transfer buffer (10 mM 3-cyclohexylamino-l-propane sulfonic acid (CAPS, EMD Biosciences) and 10% methanol) at 4°C. After rinsing for several times to renature 46 proteins, the blot was treated with the blocking solution (0.5% casein-Hammersten (US Biochemical) and 1% BSA in the IB) overnight at 4°C. After intensive washing in the IB with 0.05% Tween20, the blot was incubated with rabbit anti-native-formed AanR IgGs (in the blocking solution) for at least 3 hours at 4°C. After intensive washing, the blot was incubated with the horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (Cappel Research Reagents) for 1-2 hours at 4°C. After intensive washing, the blot was incubated with the SuperSignal® West Pico chemiluminescent substrate (Pierce) for 5 min, and promptly exposed to a Biomax light film (Kodak). After exposion, the blot was stained with the Colloidal Gold Total Protein Stain (Bio-Rad) solution as a reference. Solid Phase Saturation-Binding Assay of Mini-receptors The test wells on 96-well high-affinity Reacti-BindTM EIA plates (Pierce) were coated with the same amount (no more than 400 ng) of protein for the same receptor sample in 100 pl overnight at 4°C. After the sample solution was aspirated, the wells were washed briefly with the IB for two times and blocked with 330 pl of the blocking solution (0.05% Tween-20 and 5% BSA in the IB) first for 1 hour at RT. and then for several hours at 4°C. The blocking solution was aspirated, and the wells were washed briefly with the blocking solution for 2 times to remove residue receptor proteins. Aliquots of the 100 pl blocking solution with geometrically increasing amounts of [35S]- labeled Vg were loaded successively, and the plates were incubated for 2-5 hours at 4°C. One hundred times of unlabeled Vn was added to the geometrically diluted “hot” Vg solutions as a parallel, nonspecific binding control for each receptor. To calculate the concentration of unbound Vg, aliquots of 60 ul were transferred from wells, mixed with 5 47 ml of the BioCountTM complete counting scintillation cocktail, and counted with the Liquid Scintillation System (Beckman). After washing promptly with the blocking solution for three times in a well-to-well way, the bound Vg was lifted with 0.1 M NaOH in the diluted RADIACWASH radiodecontamination solution (Biodex Medical Systems) at R.T.. To calculate the concentration of bound Vg, aliquots were removed from wells for liquid scintillation accounting. The radioactive saturation binding data were fitted into 1- or 2-site binding equations to get regression curves in the nonlinear regression analysis with the GraphPad Prism® software. Either 6 or 9 data points were used in the regression analysis, and each data point represents the mean of either 3 or 4 determinations. Nonlinearly regressed nonspecific binding was deduced from the total binding to obtain the specific binding, and was not shown. RESULTS The CLII of CR5 in insect YPR and the single cluster of CRs in VgRs/YPRs from other egg-laying animals, VLDLR, LDLR, and ApoER2 share homologous modules in different combinations The potential ligand-binding domains of insect VgR/YPR are composed of a varying number of CR5 in different combinations (Figure 5 and 6). Although all modules in the second cluster (CLII) of CR5 of Anopheles gambiae (malaria mosquito) VgR (AngR) are more homologous to their counterparts in AanR than in DngR, the CLII of AngR lacks CRII-S (fifth CRs in the CLII). There is no possibility of alternative splicing, because on the A. gambiae genome the region from the C terminal 2/3 of CRII-4 to the C terminus of the protein is coded by one large exon (strain PEST, genome ID: 48 Fig. 5. The CLII of CRs in insect VgR/Y PR and the single cluster of CRs in egg- laying vertebrate and nematode VgR/YPR share homologous modules in different combinations. CRs from the CLII of insect VgR/YPR and from egg-laying vertebrate and nematode VgR/YPR were aligned and grouped by homology. The five CRs in CeYPR were found less homologous to those from the CLI of insect VgR/Y PR (alignment not shown). Each CR was assigned a group number. In the consensus sequences: a, aromatic (F, H, W, Y); (1, (D,N); h, hydrophobic (A, F, G, H, I, K, L, M, P, R, T, V, W, Y); 1 , aliphatic (I,L,V); p, polar (D, E, H, K, R, S, T); t, tumlike (D, E, G, H, K, Q). Disulfide bonds are diagramed as lines on top. --, missing residues; V , acidic Ca2+-coordinating residues with side chain carboxylate oxygen in human LDLR (Fass et. al. 1997); A, position of residues coordinate Ca2+ through backbone carbonyl oxygen in human LDLR (E, R, W); GngR, Gallus gallus (chicken) VgR/VLDLR (Bujo et. al. 1994); XIV gR, Xenopus laevis (African clawed frog) VgR (Okabayashi et. al. 1996); CeYPR, Caenorhabditis elegans (nematode) YPR (Brant and Hirsh 1999). DmYPR, Drosophila melanogaster (fruit fly) YPR (Schonbaum et. a1. 1995); PanR, Periplaneta americana (American cockroach) VgR (Tufail and Takeda 2002, protein ID: BAC02725); AngR, Anopheles gambiae (malaria mosquito) VgR (Holt et. al. 2002). The AngR sequence is from conceptual translation (ID: EAA06264) except that the last exon starts at 3682837 instead of 3682852 on the genome sequence AAAB01008846. 49 r—fi-f—f 1——-——. l + 1 1 l A 1' '1 W 1 TD AKAKCEES cs R C Pu. WK CD DEDCSD SDES AC CR 1 GngR TTT CEES C R C TSl WK CD DEDCSJ‘SDFS SC CR 1 XlVgR APA: STCTT',AK EFL‘I C RR CiPAE 2‘4, C’) ifAl‘ICLZ‘ RDES C CR 1 CeYPR UEATDC EPR C S E CiTV R C RRDC D SDEV C CRII-l DmYPR .DA.UC EEK CT S E C TLS KR C KDCAD SDEK C CRII-l AanR TDAAAC A&R CA S E CQAR QR C R UCVD,SDE; C CRII-l AngR VKKTCAES DE? C S CCP R W, CD DPDCED SDESAE C CR 2 GngR TKKTCAFS 1F CR C PSR WE CD DPDCED SDETPEIC CR 2 XIVgR S-A CSTS CK CTA E EK CD EE CRD SDE, C CR 2 CeYPR DEE R KPKU CSPS ;EA C 51E, C DKE RR CD RKDC D SDE; C CRII-Z DmYPR ------------------------------------------ CR3 SEE” YC CRII-2 PanR DEA .PK_T C_ID EV? CA DKS’ C D.T RR CDE TDC D SDEK KC CRII-2 AanR ESKTP A TT CRH EER CA D SR CiAAT SR ESRPDCAJRSDEA C CRII-2 AngR TRTCRY E,S C P-ST, C P”S WK CD EKDCDS EDEE C CR 3 GngR YHRTCRAT EES C VRST. C PAS W CD ERDCA AEDEE 'C CR 3 XlVgR lKSRED S PSAPTTF? PEC PP RlR CR 8 . CT.PD UV CD TDCS DDEU C CR 3 CeYPR EKFDKSKKC Y _ CD K C DSS TU CD T DC D SDEL 1C CRII-3 DmYPR FEEEC EDR _FK CR T D C EKS WY CD SKDCED SDEE C CRII-3 PanR E TLR T C E _ i CP D F CTDT T? CD EPDCTD SDEU C CRII-3 AanR E Y RRT CTRY , S CA D F C HAT AR CD, PDCPD SEE, EC CRII-3 AngR VTCSAA EFT CS S _ CiSKS EV C _DDCSD SDEL EC CR 4 GngR TTCSPS ExT CS S R C SST F? C’ . DCSD SDEU C CR 4 XIVgR SSTDE DDT L DPTFFA ED KCRS YTJ C S D“ C.PUS FL CD DLDCDDASDEK C CR 4 CeYPR EAT RCEP *9, C S S C‘A S WE CD R.DCSD SDE D KC CRII-4 DmYPR EETTCEPS AFK CA L _ C;PEE WT CD _SDCFEDTDE, C CRII-4 PanR TD.T EKS ATTC Pl WFR C H ; CfPKN WE CD PDCTD SDE D KC CRII-4 AanR A Y K ”KATTCAA MFR C S P C SSA 1? CD DDC D TDEE C CRII-4 AngR APPTC T E?. CK SST CTP S W? CDDDADCSD SDESLE.C CR 5 GngR TPPTC A ER, CK 'ES C PJS WT CDDEPDCAD SDESlE,C CR 5 XlVgR ' RSCPPD H R CL S C C1DRS ;V CD- DC DKSDEL 'C CRII-S DmYPR APPTC P APS C R CL3,T L; C YDDC DRSDED PC CRII-S PanR uTKTDC A FTK CA ;- C.ETR 1? CD ”DC D SDEL C CRII-5 AanR RPPAPP KCSTS ET, C S E C7 KK HR CD DPBCKD SUE; C CR 6 GngR R,P AP_RCSA EVP C S E Cl KK NR CV-DADCKDKSDE? C CR 6 XlVgR ,T APSEEFVHS ;AD fl SCSAA HTS C TK SE‘ IC.PL AT C IKECPT DDESK C CR 6 CeYPR TDSSTV iSCAED ,T_ CT S .KlC PSA VR C TTECPR‘EDEA DC CRII-6 DmYPR RKPA EEEERiSTlLCKE YET C P K ?T1CIPSS R C TAECPV DDER C CRII-6 PanR KVEiEPCT IEED PTKYL CPR S K C DTA R C TAECPD EDEA C CRII-6 AanR “ R2 ATAA,CSE;A A TAYR CAR S C PAA AR C TAECP EDET C CRII-6 AngR PSRTCRPD _§‘R CE 1.2 c- s R c "IRDCLD TDEA '0 CR 7 GngR PSRTC,PD _rK CE D CE 8 R. CD TRDC D TEE: RC CR 7 XIVgR DTCSTY EEK CR S RE C RRE FR CD ,KDC D SDE1 SC CRII—7 DmYPR C 7" .ET CT K C PSE WU CD 1 DC 3 SDE ARC CRII-7 PanR 8 C'T_ EV, CK S K C RKE ER CDKETDCDD SDE“ DC CRII-7 AanR 8 C IR E?_ CS D . C.R,E ER CD D_ZCDU SUE C CRII-7 AngR Y1;CS P KFK CRS E C T K” C DCKDWSDEPNKEC CR 8 GngR K V.,CS P K K CK S F C ES KG C K KDCKDWSDEPTKEC CR 8 XlVgR ESEK .S;i;PHSTSSRSCRP 'FD C. D E C L S R“ C EPDCT DE P KC CRII-8 DmYPR .TPSST TP PCT DYA C E _ C'SUS 1A C KR CED SSE .C CRII-8 PanR . TAAE IE? VAC E TEE CK P ” C E’S _' C KKLCDD KDE K C CRII-B AanR TA AD STA T ATDC RD TEE C P E C PTA KC CD RRDCT DEE AC CRII-8 AngR 100% consensus: C C Cl Cdt C pm; C 65% consensus: C a C Cl h L: :th I SEE C 50 Fig. 6. The Structural organization of the VgR/YPR, VLDLR, LDLR, and ApoER2. CRs from the CLII of insect VgR/YPR and CRs from other VgR/YPR, VLDLR, LDLR, and ApoER2 were assigned into one of eight groups by homology. Abbreviations: ApoER2, apolipoprotein E receptor 2; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Gg, Gallus gallus; HS, Homo sapiens; 0m, Oncorhynchus mykiss; Pa, Periplaneta Americana; XI, Xenopus laevis; LDLR, low-density lipoprotein receptor; VgR, vitellogenin receptor; VLDLR, very low-density lipoprotein receptor; YPR, yolk protein receptor; Y W'I'D, tyrosine-tryptophan-threonine-aspartic acid. PanR 0 CR in cluster I 0 CR in cluster 11 . EGF-like repeat fl s 113.; YWTD B-propeller HH- O-linked sugar domain NV TM domain A! Cytoplasmic tail “one” 55“” XIVgR, OngR, 8. Gg VLDLRNgR GgApoER2 6050906 HsApoERZ HsLDLR 52 AAAB01008846.1, join 3682837..3684961). Periplaneta americana (American cockroach) VgR (PanR, Tufail and Takeda 2002) has at least six intact CRs (CRII-3 to CRII-8) and the C terminal 'A remnant of CRII-2 in the CLII (Figure 5 and 6). The juxtaposed EGF repeat N terminal to the CL 11 has only an N terminal remnant. A surprising observation is that the five CRs of Caenorhabditis elegans (nematode) YPR (CeYPR, Grant and Hirsh 1999) apparently have higher homology to modules from the CLII than to those from the CLI in insect VgR/YPR (Figure 5 and 6). CeYPR has homologues of CRII-l to CRII-4 and CRII-6 of the insect VgR/YPR, and no homologues of CRII-S, CRII-7, and CRII-8. A similar observation comes from human LDLR and ApoER2, which lack homologues of the first and eighth CRs of VLDLR (Figure 5 and 6). Construction, Expression, and Purification of AanR Mini-receptors Construction of the AanR mini-receptors: To determine which cluster of CR5 in AanR is responsible for exclusive or predominant Aan binding, two mini-receptor genes (m VgR] and m VgRZ) encoding either the CLI or CLII of CR5 and its C terminal adjacent EGF precursor homology domain were constructed by PCR and cloned into cloning vectors and expression vectors (Figure 7 and 8). A third mini-receptor gene (m VgRI-Z) encoding the extracellular portion (except for the O-linked polysaccharide domain) was also constructed as a positive control (Figure 7 and 8). Point mutations found in the constructs were reversed either by a copy-and-paste work, or by amplification with different cDNA clones. The details of construction and reversion of point mutations are described in the Materials and Methods in this chapter. 53 09906669“ /_ minireceptor ngR1-2 igll" :1 minireceptor ngR1 minireceptor ngR2 4 - signal peptide 1“) YWTD B-propeller CR in cluster I % O-linked sugar domain CR in cluster II 9"; TM domain 0002 EGF-like repeat all Cytoplasmic tail Fig. 7. The schematic representation of Aan mini-receptors. 54 Fig. 8. Construction of AanR mini-receptors. Mini-receptors, ngRl , ngR2, and ngR1 -2, were constructed as described in the Materials and Methods. For the legend, refer Figure 7. A. Amplification of the signal PCR fragment, ngRl PCR fragment, ngRZ PCR fragment, and ngRl-Z PCR fragment. B. Construction of the mini- receptor mVgRl. pGEMSZf(+) and pAc5.l-V5-HisA were cloning vector and expression vector respectively. C. Construction of the mini-receptor ngRZ. D. Construction of the mini-receptor ngRl-2. 55 Ememé «on. 395 .858: mom E9... «JV 7mw:...°% .l. V % EoEmmt «Ed .956 RE 32. E mafia mm: E — — mEE ESQ V \...U. m; 5.3339060; E / \ H l SW 7%.: / V 28.8: mom «$95 \.. Any. \FV 7:.” Farm»; 7.93%; i/. Av A \. 56 an 8% Em>e Qcflm .. saw? . cm... . a o w E L < _m_ U qmw _m \/ Vqud Po ".3 3 H1 WWo En Ema -~.moEOQ ms mER> :Emfi 0mm .. c a. i a l . .....m \iyfi . .. INN.“ i... ... a O can u m. E a < m: u mvxaa m < W E > d um m w um. wwwvdu 0 p I Al I an? ~omm as am: new «8 Em>a E0 . a :o w E —/ E 95 a < . c W E > xs m. "H2 new 57 as most NMm>E :Ewi EH. i 9< 3:00 4 93¢: _ \.\_/ 8a o< . mm mm awn“... ... r m mmWo I Am as :8 -_.mo 393m 0mm llldwmffll E 5. . _ 5 98 av 3 E as. v SEE am a? E c mmMm m. M mu mwm I 0 H mm WWI 8 S E? ~IEwm an $09 Bewaém an 8% .93 MB SHE/e ..|ll|lAllu n .. .. to hnm< who C V1 NMTUT/C. find—r0 d 0 S B 0 1 au 1 m1 m H an 83 rwNmzmon. as 5 new m8 acme . a E Which; a < to c damus Mm“ WNW .— m < H a as $3: EMS/SE m8 £fin> Ecwzm - - _ _ I L _ — _ < 530 av 91sz d d3 535:. J” _ 8a mo< w o m Wm “mm Am an 28 55.9. _ .32 WW 3 2:?» 3:00 8” ”fl _ 05 no.4. V as HMWWm an 823 N-Em>80m I Am Ecwmm 0mm P J_ _ _ _ 1 V 29¢: misa wan Em>e .m. o H Do XS m m mm E? 3% E 8on rawmzmom an ace .wfi «2 N-Em>a :23 F _ to 1 1 u _ . P 95 .3 to a .w 27.4: mm ”arm.” Ew>e _/ W. o I Do “8 VdS m m WW .0 w. w WI1 59 Expression ofmini-receptors in vitro: The constructed mini-receptor genes were expressed in vitro in a coupled reticulocyte lysate Transcription and Translation (TNT®) system. The ngRl gene product gave a major band at anticipated size of around 80 kDa and a minor band with a slightly lower molecular weight (Figure 9, lane 3). The ngRZ gene product was a broad band with a molecular weight between 115 and 140 kDa (Figure 9, lane 4), which is higher than the anticipated 92 kDa before modification of the protein. This is probably because of the limited co-translational and post-translational modification (phosphorylation, glycosylation, and sulfation). A major band of around 210 to 230 kDa was shown to be the ngRI-Z gene product (Figure 9, lane 5), although the size virtually exceeds the size limit of 176 kDa for the TNT system. Expression of mini-receptors in the insect cell line and purification: To get mini-receptor proteins properly modified after translation, three mini-receptor genes were stably transfected into the D. melanogaster 82 cell line with either Effectene reagent (Qiagen) and Cellfectin reagent (Invitrogen). The Vector plasmid, pAc5. 1/V5-HisA, and the expression control plasmid, pAc5. 1N5-His/lacZ, were also transfected into 82 cells as the negative and positive controls, respectively. The efficiencies of two transfection methods were roughly equivalent as indicated by a B-galactosidase enzyme assay (data not shown). Stably transfected 82 cells were harvested at different time intervals and the poly A+ mRNAs were extracted from cell lysates. The poly A+ mRNAs extracted from the same mass of cells were subjected to formaldehyde agarose gel electrophoresis and northern blot with m VgR1-2 DNA as a probe. Auto-radiography of the blot showed a 60 lucnferase meVgR1 -250- -150- Fig. 9. In vitro expression of the VgR mini-receptors in a coupled transcription and translation system. Each mini-receptor gene driven by a SP6 promoter on the pGEMS vector was expressed in the TNT SP6 coupled reticulocyte lysate transcription and translation system (Promega) in the presence of [35S] methionine. Reactions were subjected to a reducing SDS—polyacrylamide (PAGE) gel and autoradiography. The anticipated sizes of nascent unmodified products are 78-, 92-, and l94-kDa for the ngRI (lane 3), the ngR2 (lane 4), and the ngRl-Z (lane 5) respectively. The size of the product of the luciferase control gene should be 61-kDa (lane 1). 61 clear single band for each mini-receptor gene transcript in the correct size (Figure 10A: lanes 1-4, 2337 bases for ngR1 mRNA; lanes 6-9, 2739 bases for ngR2 mRNA; lanes 10-13, 5541 bases for ngRI—Z mRN A), which suggested the normal initiation and termination of transcription. Overall, the transcripts of three mini-receptor genes from the same cell mass increased in quantity over a period of three days, which implied the stability of mini-receptor transcripts and/or increased transcription rates afier activation of cells by passage. No band was observed fiom the mock transfection control (Figure 10A, lane 5). Total proteins in the transfected 82 cell media were precipitated with 18% PEG, and the His-tagged mini-receptor proteins were purified by the cobalt-based immobilized metal affinity chromatography (IMAC). Figure 103 shows the purified ngRI and ngR2 proteins on an SDS-PAGE gel stained with Coomassie blue. The abundances of both ngRI and ngR2 were very low in the transfected 82 media (Figure 108, compare lane 1 with lane 2, and lane 5 with lane 6), as indicated by virtually the same band spectra of 82 media before and after loaded to the affinity column. The ngRl eluate gave a major ngRl band of around 80 kDa, which accounted for nearly 40% of total proteins (Figure 108, lane 3). The ngRZ eluate also gave a major band of around 100 kDa (Figure 10B, lane 7), although the ngR2 preparation was less pure than the ngRI preparation. The purified His—tagged ngRl-Z protein, which was predicted to be around 200 kDa, was invisible on the gel (Figure 103, lane 10), indicating either the very low efficiency of expression of the around 200 kDa protein in 82 cells or lack of an intact His-tag at the C terminus of ngRl-Z, possibly caused by the premature 62 Fig. 10. Transcription and translation of mini-receptor genes in the Drosophila 82 cell line. Drosophila 82 cells were transfected with mini-receptor genes. Mock transfection was made with vector plasmid only. A, Stably tranfected 82 cells were harvested 7, 25, 49, and 72 hours after passage, and poly A+ mRN As were extracted. Mini—receptor mRNAs from the same mass of cells were subjected to a Northern blot and detected with the m VgRI-Z probe. The anticipated sizes of transcripts (excluding poly A tail added during polyadenylation) are 2337, 2739, and 5514 bases for ngR1 (lane 1-4), m VgRZ (lane 6-9), and ngRI-Z (lane 10-13) respectively. Lane 5 is mock transfection with vector plasmid only. B, His-tagged VgR mini-receptor proteins were purified from 32 cell culture media by TALON immobilized metal affinity chromatography (IMAC). The eluates were subjected to SDS-PAGE, and the gel was stained with Coomassie brilliant blue. Arrows, ngRl (lane 3) and ngR2 (lane 7) protein bands. 63 ngRl mRNA mock ngRZ mRNA ngRl-Z mRNA F I I II I Time 4 éé 6948bases— 4742bases— ‘1’ 2661bases— “'1... ‘33.. 1812bases— ‘ lSl7bases— 1 2 3 4 5 6 7 8 910111213 0 a a a a a E“ 8 ° 8 0 U E v E .5 v E g s E "E s g a a :3 a a v a g '«3 a s a a s .5 % ‘3. go a go ‘3 a. 9:, a E. E. E E is E E E ‘5 ”t5 E m m m E m m m E 2 w termination of translation. The 5,154 bp-long coding region of the ngRl-Z gene was not fully sequenced afier reversion of a nonsense mutation found in the CLII of CR5. All of the visible bands from ngRl-Z sample eluate had equivalent bands from both ngRI and ngR2 eluates (Figure 103, compare lane 10 with lane 3 and lane 7). Multi- bands demonstrated insufficiency of one-step purification of mini-receptors. Another reason is the inclusion of 5 mM instead of 10 mM imidazole in both the sample loading buffer and the washing buffer, which increased the nonspecific binding to columns. To make the mini-receptor preparation purer, a gel filtration step could be added. To substitute ngRl-Z with a new positive control, the full length AanR was enriched from A. aegypti vitellogenic ovaries 20-24 hours PBM as described in the experimental procedures. The purified ngR1 and ngR2 were subjected to a reducing Tris-HCl SDS-PAGE and blotted to a PVDF membrane. After the blot was incubated with the HRP conjugated anti-V5 antibody (Invitrogen), a chemiluminescence reaction detected anticipated-sized single band for both the ngRI and ngR2 mini-receptors (data not shown). This indicated that the full length ngRI and ngR2 have been purified from 82 cells. Two purified mini-receptors and AanR were further subjected to a nonreducing Tris-HCI SDS-PAGE and immunofluorescent blot under a nondenaturing condition as described in the Materials and Methods. After the blot was incubated with the rabbit anti-native-formed AanR IgG, and the HRP conjugated goat anti-rabbit IgG, protein bands were detected with a chemiluminescent substrate and exposed to a light film (Figure 11). The anti-native-formed VgR antibody bound to both the ngR1 (Figure 11, lanes 1-2) and the ngR2 bands (Figure 11, lanes 5-6) at anticipated sizes. A much weaker band at anticipated size was also detected to be potentially the ngRl-Z protein 65 d) d) ‘5 ‘5 :3 :3 B 3 u o < < E E B ‘25.) B E > E —- 'O as. a %. > .0 > E E E N N CD LL] (I) I 11 II I 5" ~25 - 150 ' ~ -100 ~75 -50 Fig. 11. Nonreducing Western blot of VgR mini-receptors. Enrichment of native VgR from vitellogenic mosquito ovaries and western blot are described in the Materials and Methods. Protein samples were subjected to a nonreducing 4~15 % Tris-HCl SDS- PAGE, and blotted onto a PVDF membrane. The blot was incubated with the primary antibody, rabbit anti-native AanR IgG, and the HRP conjugated secondary antibody. Vg receptor bands were detected with a chemiluminescent substrate and exposed to a Biomax light film. 66 on the blot by anti-VgR IgG, and was visualized on the blot afier colloidal gold staining (data not show), which suggested that a trace amount of the His-tagged ngRl-Z protein has been obtained. For each of the three mini-receptors and AanR, there was always one additional, very faint band with a much higher molecular weight in proportion to the size of the major band (Figure 11, lane 3 for clear secondary band, too week to be seen here for the ngRI and ngR2, and not shown for the ngRl-Z). Binding of The AanR Mini-receptors to Aan Both clusters of CRs contribute to the high affinity of the AanR to Aan: To prepare for VgR/V g binding assays, Vg was metabolically radiolabeled with [35S]- methionine in the Aedes aegypti fat body tissue culture. The crystallized Vn was resolved from homogenized Aedes aegypti ovaries. Both Vg and Vn were purified by anion exchange chromatography, and detected by SDS-PAGE (data not shown). The 96-well microtiter plate-based, solid phase saturation-binding assays were performed to measure the binding affinities of ngRI , ngR2, and AanR to Aan (Figure 12). One hundred times of unlabeled (cold) Vn was added to geometrically diluted [35S]-Vg solutions as a parallel, nonspecific binding control for each receptor. The radioactive saturation binding data for ngR1 (Figure 12A) and ngR2 (Figure 128) fit one-site nonlinear regression, and the dissociation constants (Kd) of 25.9 i 3.3 nM and 53 i 8.5 nM were calculated for ngR1 and ngR2, respectively (Table 6). In contrast to two mini-receptors, the binding data for AanR fit two-site nonlinear regression better than one-site (Figure 12C), which indicated the presense of at least two binding sites on the full-length AanR. The apparent Kd of VgR was calculated to be 3.2 67 Fig. 12. The Solid phase saturation binding assays of the mini-receptors. 96-well microtiter plates were used in the solid phase saturation binding assay. Test wells were coated with same amount of protein for the same kind of receptor. After blocking test wells, geometrically increasing amount of [35S]-Vg were loaded in order. After incubation, aliquots of incubation solution were accounted for free [35S]-Vg signal. After washing, the bound [3SS]-Vg was lified and aliquots were counted. Nonspecific binding controls were set by involving 100 times of cold Vn in the binding solutions for each receptor. 1- and 2-site nonlinear regression analyses were performed with software Prism (GraphPad), and a primary model was chosen based on the significance of 2-site model over the l-site one on data fitting. Either six or nine data points were included in the regression analysis, and each data point represents the mean of three or four determinations. Graphs show the specific binding and nonspecific binding data. A. Nonlinear regression of ngR1 specific and nonspecific binding. B. Nonlinear regression of ngR2 specific and nonspecific binding. C. Nonlinear regression of VgR specific and nonspecific binding. 68 20000“ ngR1 specific binding A 15000- E n. 9. a: > 10000- 1': C 3 o m 5000- . . . — 1-51te nonlinear regressron Vg specifically bound to ngRI 0 l 1 I I l l o 50 100 150 200 250 300 35 [er61 S]V9] (HM) 300‘ ngR1 nonspecific binding 700- A 600~ E Q 500- a: > 400- u S o 300- In 200- 100 - Vg nonspecifically bound to ngR1 I o I l l l l l l l o 25 50 75 100 125 150 175 200 [free [353] v9] (CPM) 69 30001 ngRZ specific binding 5 2000- 9, U) > '0 C 8 an 1000‘ — l-site nonlinear regression Vg specifically bound to ngR2 0 l l l l l l o 50 100 150 200 250 300 Ifreet3581vm (nM) ngR2 nonspecific binding control 400‘ E 300- 9 O > E 2004 3 O m 1001 - Vg nonspecifically bound to ngR2 l f F l l l 50 75 100 125 150 175 200 [free [358] V9] nM 70 25000“ 20000‘4 15000- 10000- Bound Vg (CPM) 5000-, VgR specific binding 1 site nonlinear regression '_ 2 sites nonlinear regression ' Vg specifically bound to VgR 2000- 1500- 1000- Bound [V9] CPM 500d 50 160 160 260 260 [free [353] V9] (nM) VgR nonspecific binding control - Vg nonspecifically bound to VgR 50 160 150 260 250 360 [fret-21358] V9] (nM) 71 Table 6. K, values for saturation binding assays. Receptor No of determinations No of points (Kd)app orKd Kd (high) sample control nM nM VgR 3 2 9 3.17 i: 0.27 2.50 :t 0.26 ngRI 3 2 6 25.9 :L' 3.3 n/a ngR2 4 3 9 53.0 i 8.5 n/a i 0.3 nM in one experiment and 3.49 nM on average over three independent experiments (data not shown). The apparent Kd of AanR to Aan is much higher than those of ngR1 and ngR2 and is also much higher than those reported earlier (Sappington et. al. 1995; Dhadialla and Raikhel 1991). Based on the two-site binding model, there are at least one very high-affinity (Kd (high) of 093-25 nM) Vg-binding site and one very low- affinity site on AanR based on the results from three independent binding assays. The high-affinity site was responsible for 70%-90% of specific binding. The calculated Kd of the very-low-affinity Vg-binding site on AanR varied significantly in three independent binding assays. DISCUSSION In the LDLR family, insect VgRs/YPRS represent a subfamily with two clusters of CR3 (Figure 5). Noticing that the cDNA clone of PanR encodes a protein missing most of the C terminal 5/6 of the EGF-like module right before the CLII of CR5 and the N terminal % of CLII-2 (or even CLII-l, if it has), there is a possibility that PanR 72 transcript was subjected to an alternative splicing event (personal communication from M. Tufail). Quite a few alternative splicing examples have been reported in the LDLR family, which include VLDLR in human (Christie et. al. 1996) and ApoER2 in mouse (Brandes et. al. 2001), chicken (Brandes et. al. 1997), and human (Brandes et. al. 1997). Another possibility is that PanR lacks CRII-l and most of CRII-2. This is also possible because compared with AanR, which has eight contact CRs in the CLII, the CLII in AngR has only seven contact modules. To summarize, the CLII of CR5 in insect VgR/Y PR can have a varying amount of CR5, and the maximum number of modules in the CLII is eight, because in both the single cluster of one-cluster members and the CLII of four-cluster members the maximum number of CR3 is eight. A striking observation is that the five CR5 of nematode CeYPR have apparently higher homology to modules from the CLII than those from the CLI in insect VgR/YPR (Figure 5 and 6, the multiple alignment of CR3 from the CLI is not shown). This implies that the single cluster of CR8 in the one-cluster VgR/YPR (including five-CR-bearing CeYPR) and the CLII in the two-cluster insect VgR/YPR evolve from a common ancestor and that the more conserved CLIs of insect VgRs/YPRs evolve from a different common ancestor. One conspicuous fact is that some VgRs/YPRs have fewer CRs than others. For example, CeYPR has homologues of CRII-1,2,3,4 and CRII-6 of insect VgR/YPR, but no homologue of CRII-S, CRII-7, or CRII-8. AngR has homologues of most modules of AanR except CRII-S. These findings suggest that not all CRs are important for yolk protein binding. In other words, some CRs are unimportant to binding of Vg. The CRII-S and CRII-8 of VgRs might be such unimportant modules. 73 The microtiter plate-based solid-phase saturation binding results showed that, both the CLI and CLII in AanR have one binding site for Aan, while the full length AanR has at least two binding sites (Figure 12). The affinity of the full-length AanR is much higher than that of either cluster, which suggests a synergistic effect of two clusters in achieving the high affinity to Aan. Because the affinities of both clusters are not low, it is apparent that one additional cluster of CR5 in AanR supplies strengthened binding of Aan. For the LDLR family members with multiple clusters, LRP is one that has been studied extensively to elucidate the roles of modules from different clusters of CR5 in the binding of different ligands. LRP has four clusters of CR5 with 2, 8, 10, and 11 modules per cluster, respectively. Studies with LRP mini-receptors have shown that both the CLII and the CLIV bind twelve kinds (including az-M“) of known ligands (Neels et. al. 1999; Willnow et. al. 1994) and both clusters have only minor differences in ligand-binding kinetics (Neels et. al. 1999, Table I and II). This suggests a functional redundancy within LRP. In the CLII of LRP, Neels et. al. found that CRII-l to -5 are essential for the binding of ligands (Neels et. a1. 1999, Table III). In contrast to the CLII and CLIV, the CLIII only bound ApoE and weakly bound RAP, both of which also bound the CLII and CLIV. Although Neels et. al. did not find any ligand for the CLI of LRP, Mikhaihenko et. al. reported that not only the CLII (and CLIV) of LRP, but also the CLI have affinity to az-M“, albeit lower affinity (Mikhaihenko et. al. 2001). In comparison with the CLI and CLII, a LRP mini-receptor covering both the CLI and CLII-1,2,3 has much higher affinity to az-M“ (Mikhaihenko et. al. 2001). This indicates that the CLI and CLII cooperate to generate a high-affinity binding site for az-M*. The results from Neels et. al. 74 also confirmed the discovery that the CRII-1,2,3 is important to binding of az-M’“, because CRII-l,2,3,4,5 bound seven ligands (including O.2-M*), while CRII-1,2 and CRII-3,4,5 did not (Neels et. al. 1999). CONCLUSIONS First, modules in the CLII of CR3 in insect VgRs/YPRs are more homologous than those in the CLI to modules in the single cluster of CR5 in VgRs/YPRs from other egg laying animals, VLDLR, LDLR, and ApoER2. The CLII of CR5 in insect VgRs/YPRs has from six to eight modules. The five CRs of nematode CeYPR have apparently higher homology to modules from the CLII than those from the CLI in insect VgR/YPR. This implies that the one-cluster VgRs/YPRs and the CLII of insect receptors might evolve from a common ancestor. Second, both the CLI and CLII in AanR have one binding site for Aan, while the full-length AanR has at least two binding sites. The CLI has higher affinity to Aan than the CLII, and both clusters have moderate affinity. The affinity of the full-length AanR is much higher than either cluster, which suggests a synergistic effect of two clusters in achieving the high affinity to Aan. One additional cluster of CR5 in AanR supplies strengthened binding of Aan. 75 Chapter 3 Protein Modeling of AanR and Aan: Structural Basis of The Aan- AanR Interaction 76 INTRODUCTION In this chapter, sequence analyses and protein modeling efforts will be performed in order to answer several questions. First, each module in both the CLI and CLII of CR5 will be modeled, and the surface charges of modules from both clusters will be compared to see if some CRs in CLI and/or CLII of CR5 have negatively charged surfaces and to determine which cluster of CR5 has stronger negatively charged surface. Second, both the small and large subunits of Aan will be modeled to see if there is a positive surface on Aan and to learn the 3-D structure feature of Aan. Third, supposed three YWTD B propeller folds in AanR will be modeled, and the surface charges of each model will be investigated. The distribution of histidine residues will also be checked to see if there are enough histidine residues on the propeller surface to potentially switch a surface patch from weak negative, nearly neutral, or weak positive to strong positive. This will help to determine the mechanism of Aan release and AanR recycling. Fourth, all of the EGF- like repeats in AanR will be modeled, and their surface charge distribution will be checked to see if any of them can potentially contribute to the assumed electrostatic complementarities between AanR and Aan. Finally, the claimed VgR-binding motif on the small subunit of blue tilapia Vg will be investigated by multiple-sequence alignment, by checking its counterpart on lamprey Vg, and by modeling tilapia Vg and Aan. MATERIALS AND METHODS Protein Sequence/Structure Analysis and Protein Modeling 77 Protein sequence and structure analyses were performed with the National Center for Biotechnology Information (NCBI) services (hgp://www.ncbi.nlm.nih.gov[ ), the Squeb® package (Version 2.02, Accelrys), and the PredictProtein Service at Columbia University (http://cubic.bioc.columbia.edu/pp/). 3-dimensional (3-D) protein modelings of AanR were done on the bioinformatics server at Ben Gurion University (http://wwwcsbguac.il/~bioinbgu/) for protein structure fold recognition (threading) modeling, with the LOOPP program at Cornell University (hgpwser- loopptc.comell.edu/loopp_old.html) for sequence/structure alignments, and on the SWISS-MODEL protein modeling server (Schwede et. al. 2003) for protein structure homology modeling. The 3-D figures were generated and molecular surface electrostatic potential calculated with the Swiss-deViewer Wersion 3.7). A target protein sequence was firstly aligned with protein sequences from other species by hand. Based on the multiple-alignment, the target protein sequence was ligned with all potential 3-D templates with known 3-D structures by hand and alignments were submitted to the SWISS-MODEL server. The returned models were evaluated based on three disulfide bond connections, the conservation on secondary structures, the degree of homology to the template sequences, the total energy, the additional secondary structures, and the number and sizes of uncertain regions. Improvement in hand alignment was made until no better model could be obtained with each potential template. For each target sequence, acceptable models predicted with different templates were compared and a best model was chosen for that target sequence. 78 RESULTS AanR Protein Modeling The CLI of CRs in Aa VgR has stronger negative surface electrostatic potential than the CLII: All of the thirteen modules from both clusters of CR8 in AanR were modeled (Figure 13). With the exception of CRII-l, which missed the C1-C3 disulfide bond in the model, the modules were all modeled with three S-S bonds (C1-C3, C2-C5, and C4-C6) correctly linked. All modeled modules have, overall, similar 3-D structures, with two anti-parallel B strands in the [3 loop region near the N terminus (Figure 13), and most models have negative total molecular energy. For eleven modules that have alternative models, the alternative models gave similar molecular surface electrical potentials (EPs). Comparing surface EPs of modules from two clusters showed that all thirteen modeled modules from both clusters have, overall, negative surface EP. In the CLI, CRI-l and CRI—S are the two modules with the highest negatively charged surfaces, and CRI-3 has a very strongly negative surface. In the CLII, CRII-3 has the most strongly negatively charged surface, and CRII-2, CRII-4, and CRII-7 also have strongly negative surfaces. In general, the CLI has more strongly negative EP than the CLII, and the surface charges of both clusters fall within the same scale. The surface charge distribution of the A. gambiae VgR CLII is similar to that of the AanR CLII: All seven modules from the CLII of CR5 in AngR were modeled (Figure 14). Alternative models were generated for all modules in the AngR CLII, and each chosen model had negative total energy. For all seven modules, alternative models for each module gave similar molecular surface EPs. In the CLII of AngR, CRII-7 had the most strongly negatively charged surface. Both CRII-3 and CRII-4 had very strongly 79 Fig. 13. Surface electrostatic potentials (EPs) of thirteen modeled modules from two clusters of CR3 in AanR. The top row shows ribbon representation of AanR CRs colored by model B-factors (model confidence factors, shown as color temperature). The green and red colors represent regions with certainty and uncertainty respectively. The second and fourth rows show ribbon diagrams of modeled AanR CRs. B strands are colored in golden and or helices in aqua. Only side chains of cysteine residues are displayed to show three disulfide bonds in yellow. All the modules are oriented with N terminals on the left and C terminals on the right. The third and fifth lines show corresponding molecular surfaces colored with EP (Red: -12, strongly negative; white: neutral; blue: +6 positive). Acceptable models for each module were chosen by alternative hand alignments with all potential templates and by comparison of models made on Swiss-Model modeling server. Graphs were drawn with the Swiss-deViewer. Alternative models for CRI-l, CRI-2, CRI-3, CRI-S, CRII-2, CRII-3, CRII-4, CRII-S, CRII-6, CRII-7, and CRII-8 were compared to choose the best model for each module. Surface charges of best models for all AanR CRs were compared. 80 comtmano 600E 7E0 mm>m< .89: E“: 585 d9 .89: mm: .89: $6? 68.: RE .82: 2.5 $09.38 $5308 $wm§§m $mo§wm canteen? $953 3. ”SEE“: 5.53. NS- .252 NS- .253. m5- .952 8? .253. our 6.53. Bo- ”scam mvé 032$. mYm 3me 91¢ 9..ng me 0223. 31m 2.23. m: 6323.. “.2265. «9‘ Eofifié E 38.8 .263 E0: Ea 81 «was front view, colored by B-factors Modeled residue: 3-43 residue: 2-44 residue 1-41 Etot: -136 KJ/mol -203 KJ/mol -152 KJ/mol ldn/Sim: 32%l59% 30%l63% 31 %l55% 1F8Z model 1CR8 model 1F5Y model AanR CRl-2 model comparison 82 S P ‘ front view, colored by B-factors front back Modeled residue: 4-46 residue 1—46 residue 6-45 Etot: 78 KJ/mol 942 KJ/mol 806 KJ/mol ldn/Simi: 39%]70% 40%/60% 40%/58% 1J8E model 1F5Y model 1F82 model AanR CRl-3 model comparison 83 WWW/i205 front view. colored by B-factors W WWW- front i Modeled residue 343 residue 145 residue 1-45 residue 1-46 Etot3 -20 KJ/mol 500 KJ/mol 630 KJ/mol 485 KJ/mol ldn/Sim: 34%/59% 39%l67% 38%l62% 36%l62% 1AJJ model 102L model 1CR8 model 1F5Y model AanR CRl-5 model comparison 84 cowtmano .25.: N-__mo mm>m< EUOE ”—0.: EUOE 2.5. .28.: mmow .muoE an: $9553. $mw§m¢ axommaxbv aortas: .852 «9.. 6:51 to? .953. Rm. .253. man. 3.» 032mm. 519 2.292 mYm 0:23.. wa 3ng a .u gag. .39: Nan: sets? Ewen. .055. 8c. ”Em wfiim 9.630. 02060—2 288;. .3 3.0.8 .263 “co: m? 85 fir/WWW front view, colored by B-factors Modeled residue: 7-43 Etot: -77 KJ/mol ldn/Sim: 43%l65% 1AJJ model residue 5-45 residue 3—45 residue 1-42 393 KJ/mol 207 KJ/mol 26 KJ/mol 44%l73% 37%/67% 36%/60% 1CR8 model 1DZL model 1F5Y model AanR CRIl-3 model comparison 86 82.89.00 .89: EEO mm>m< EUOE 4mm: 600E >mn:. EUOE .5..— EDGE qu: 600E mm: .mUoE 2.4: {ammuxvmm $3539. o\oo\.\$ S. okmmuwomv $mt$m¢ okmtfiifi 359:2 _oE=.v_ 9b. 6.53. Em- _oE=.v._ mum- 38:! mm T _oE=.v_ cum- 6:5,! m5- LEM 91m 2652 t; 0323. tum 32mm: 910 2.292 91v 2650.. 31. 6:28.. “.2822 290$.m B no.0.oo .so_> E0: amxfisgfisee .82: an: 5.890..” 553. a: NY... 26.8. comrmano .000E 9:10 102?. .009: >9: .009: N0“... .000E wmow .009: ......0 00.0.00 .303 “:0: 88 \ f \f/ front view, colored by B—factors % 1‘8— Modeled residue: 5-50 residue 5-50 Etot: 167 KJ/mol 52 KJ/mol ldn/Sim: 33%[50% 31%l52% 1F5Y model 1LDL model AanR CRll-6 model comparison 89 50:09:00 .000E EEO 1023. .009: 0:9 .009: 40.: .009: >0“: .009: 2...: .009: an: .000E No“: 9.. E0000 $3.09.? $099.? $055.. 5 $099.00 $059.00 “£90. .953. mom- .053. $0. .953. 000. .953. 00:- .953. 00 ..- .953. max. new 91 030.00. El 030.00: :1 030.00: wm-~ “000.00. 004 000.00. 004 “030.00. 00.0005. 0.200;. >0 00.5.00 .30; E9. 1 so 90 50000800 .000E m-..m.o Ma>0< .009: wmov .009: mm: .009: an: .009: N0”: .009: 2.4: $05.8 9.9st $00.95 5.00.5.8 $89.8 ”5.9%. .253. 80 .253. 8 .025. 0% .953. .00 .953. mi ”.20 00.0 030.00. 91.0 000.00.. 00.0 000.00. 00.0 030.00.. 31: ”030.00. 00.0005. .000 ES. ‘- .. .s an .. : ._.. n‘. 9%? 0.200;. .3 00.0.8 .303 E05 55... 91 0023.0 #6 .o _ .2020 m. «.0 .1 mo m. mu m. m0 .00oE 2;: .009: Nan... .00oE Mm: .009: Nan: $mm§§m $065.09” nxbtokmm $95003 .953. om- .953. oov. .953. an .953. mm T ‘ . 0.0 ..-. mo .89: 2.5 9.5.5:. E000. _oeexmmm.usm IN, .omn E0... {50% 0.2005». .3 00.0.8 .30... E0... 9 92 «.90... .o 000 .o ._ .2020 w-.. m0 5... «.0 o-.. mu m-.. «.0 5... mu m-.. m0 N-.. «.0 T: KO .009: 2:0... .00oE Mm”: .009: >9: .009: 4mm: .009: ..._.<—. .00OE 03: .00OE qu: .00oE an: $®o\o\omm QOmwQOVm 5oom§hm o\oot$wv obmtoanv 0569.59. .x.mto\omv $m9$mm .953. m: .953. Nwm- .oE\...v. 5.. _oE\3.mmN- .oE\3_ m5- .oE53. R- .953. com. .oE\3. mmm 2900.0 .3 00.0.8 .503 Eo... $55053... 93 Fig. 14. The surface EPs of seven modules in the CLII of CRs of the Anopheles gambiae VgR. The alternative models for each module in the CLII of CR5 of Anopheles gambiae VgR (AngR) were made and compared in the same way as AanR models. Alternative models for AngR modules CRII-l, CRII-Z, CRII-3, CRII-4, CRII-6, CRII-7, and CRII-8 were compared, and the surface charges of the best models for all the AanR CRs were compared. Graphs are arranged in the same way as in Figure 13. The color scale for molecular surfaces is, red: -12, strongly negative; white: 0, neutral; blue: +6, positive. 94 A front view, colored by B-factors Modeled residue: 2-37 residue 2-40 residue 1.40 Etot‘ -815 KJ/mol -1266 KJ/mol -597 KJ/mol ldn/Simi: 39%/71% 34%/64% 31 %/64% 1F5Y model 1J8E model 1DZL model A. gambiae VgR CRll-1 model comparison 95 EUOE >mu= $03.8 .85. £2- 91. 828. comtmano EuoE N-__mo mm> mmBEmo. .< :69: an: 609.: Ne": 68E wmor EvoE 2.<_. $8.2»; $3ng $83.? sags: Ewan. 655. 9:- .953. 82- .952 v3: .253. «ma- ”~on mYm 32mm: 910 2.38.. ml 267.9 31 32mm: UQovoE xomn .3... ' ._1 g Bofifii B “.228 .263 E0: gm E0: 96 front view, colored by B-factors back Modeled residue: 7-43 residue 4-42 Etot: 4150 KJ/mol -550 KJ/mol Identity/similarity: 46%l73% 39%/61% 1AJJ model 1LDR model A. gambiae VgR CRII—3 model comparison 97 .muoE >mu: $o9$mm .253. 3m- 91. 32mm. cowtmano .308 1.10 mm> 858% .< _oUoE qu: _0UOE wmow 600E moi: £30}wa fievmuwoom ob 2.3%.. 3 .953. 97 _oE\_..¥ man. .953. cm? mg 2.wa 91m 2.28.. 91v 923.. leg .39: 2:3 eEeE. Ewen. .953. New- Loam $1» “2652 uoficoE xomn 220m“; B 2:28 .263 E0: 98 front view, colored by B-factors back residue 4-49 residue 5-51 residue 7-50 residue 5-51 residue 6-51 Em: -431 KJ/mol -772 KJ/mol -489 KJ/mol -297 KJ/mol -678 KJ/mol Idn/sim: 28%]55% 26%]60% 27%/55% 26%l55% 26%]54% 1LDR model 1CR8 model 1AJJ model 1D2J model 1F8Z model A. gambiae VgR CRll-6 model comparison 99 c0wthEoo .mooE EEO mm> mmBEmu .< .0UOE .._Nn_—. _0UoE >mu:. .009: mmor EDGE an: $59: Nmn: EDGE .231. $mw§ov nevtfiowv «aegis? $nt$mv .xktgav o\o Eax. Fm UE_.wE_u_ _oE=.v_ vow? .0522 own? _oE=.v_ mum? 6.53. NVNT _oE=.v_ m5? .953. mwNN- ”Bum 9.4 032m? 3; maniac El 6328. mm; 0.669 mm; 9.669 mm-m 2.2mm. uflouoE 2209; >9 v9.0.8 .26; “co: §§§%,§ . 100 :owtmnEoo _ouoE m-__mo mm> 039.23 .< 780E Mm: 68E >9: 68E wmor _mnoE Na": .0008 291.. .x. 5E3” $Nw§¢m $wm§wm .xbwuxsmm .x. F933 ”SEER 66:! 2o. 6.53. mm- 6E2! RT BEG. ET .953. :8. mew 91v 033mm: 9...? $28.. $6 6328.. 36 632mm. $4. 3ng BEBE anomum E 8.28 .263 E9: Ea 101 w-.. «.0 .ovoE 2...: £0 53%... .953. Foo- mm> 355% .< 5 mac .6 __ .286 2.10 $4.10 v... mo m-__ «.0 .wuoE 2;: $59: ”5.: .08.: 2.5. .362. 2;: £0 531m QamQQOwN o\o wheat» o\om...\$ov .953.. mwNN- .oE\_..v. 5v. .953. Nmm- .953. omS- N-.. mo .98.: 2...: {30305. .oE:.v. mm FN- F-.. mo .89: .6“: $E§mm .952 m5- 102 negative surfaces. Comparison of the surface charge distribution of the AanR CLII with that of the AngR CLII (Figure 15) showed three common modules with strongly negative surface charges: CRII-3, CRII-4, and CRII-7. One big difference is that in the AngR CLII, CLII-7 rather than CLII-3 has the most strongly negative surface EP. Table 7 summarized modules with strongly negative surface charges in AanR and AngR. Table 7. Comparison of surface charge distribution of the CLII of CRs in AanR with that in AngR. negative surface charge module AanR AngR CLII-2 strong nearly neutral CLII-3 strongest very strong CLII-4 strong very strong CLII—7 strong strongest The E GF -like repeats of Au VgR very possibly do not contribute to the binding of Au Vg: The EGFl-Z, 3, 4, 5-6, and 7 of AanR were modeled with Swiss-Model server (Figure 16). All the EGF-like modules modeled have three correctly linked S-S bonds (C1-C3, C2-C4, and C5-C6) and overall similar 3-D structures, with two [3 sheets each made of two anti-parallel [3 strands. Only EGF 4 and EGF 7 have alternative models, and these alternative models are only approximately 30% identical to their templates. Despite the limited homology, the alternative models showed similar surface EPs for both EGF4 and EGF7. The total energy of each selected model for the seven modules is negative. EGF 1 has a very strongly positive surface, and EGF4, EGFS, and EGF 6 have 103 Fig. 15. Comparison of surface charge distribution of the CLII of CRs in AanR with that in AngR. The lefi two columns are the front and back views of surface EPs of best models for modules from the CLII of CR5 in AanR, and the right two columns show their counterparts in AngR. The color scale for molecular surfaces is, red: -12, strongly negative; white: 0, neutral; blue: +6, positive. 104 . 4“”.h » "I" CLII-5 5’" 4'” “g. 71 fw) % I" r I! ##3" .-.:.s CLII-6 11:? 74m ' s s A. aegypti VgR 105 a, ‘ A. gambiae VgR Fig. 16. The surface EPs of seven EGF repeats in AanR. The surface EPs of models for EGF 1,2, EGF 3, EGF 4, EGF 5,6, and EGF 7 are shown. Alternative models for EGF4 and EGF 7 are compared. EGF1,2, EGF3, and EGF5,6 each had only one acceptable model. In each graph, the top row shows the models of AanR EGF-like repeats colored by B-factors (model confidence factors, shown as color temperature). The green and red colors represent regions with certainty and uncertainty respectively. The second and fourth rows are ribbon diagrams of the modeled AanR EGF-like repeats. The four [3 strands in each module are colored either in golden or in pink, and a helices are colored in aqua. Only side chains of cysteine residues are displayed to show three disulfide bonds in yellow. Calcium ions are represented by small spheres. All the EGF repeats are oriented with N terminals on left and C terminals on right. The third and fifth rows show corresponding molecular surfaces colored with EP (Red: -12, negative; blue: +6 positive). 106 We: %% side 1. colored by B-factors side 1 Etot: ~11 KJ/mol 152 KJ/mol ldn/Sim: 31%l58% Idn/Sim: 31%l55% 1HJ7 model 1|0U model AanR EGF4 model comparison 107 a-%ex/}? side 1, colored by B-factors Modeled residue: 6-47 residue: 6-47 residue 4.45 Etoti -354 KJ/mol -68 KJ/mol -209 KJ/mol ldn/Sim: 29%l52% 29%l52% 29%l57% 1HJ7 N' model 1|0U N' model 1HJ7 C' model AanR EGF7 model comparison 108 (%W side 1. colored by B-factors Etot5 -1427 KJ/mol -101 KJ/mol Idn/Sim: 34%/63% 35%l60% EGF1,2 1|0U model EGF3 1J8E model 109 .mUoE fife NLOM {ammuxemm _oE=.v_ 3m- .cuoE Do... Qmmwm $8§5 .953. 8:. EDGE 211—. quw $3...» 5 ”SEE“: 625. :. Eu «2% Eofiflm >2 no.0.oo 32m 110 weakly positive surfaces. In comparison, EGF 3 has a nearly neutral surface, and both EGFZ and EGF7 have weakly negative surfaces. The positive or weakly negative surface properties of EGF-like modules in AanR exclude them from being involved in the binding of Aan through primarily electrostatic complementarities. The Ad VgR YW T D ,6 propellers and their role in Vg dissociation and receptor recycling: The three YWTD B propellers of AanR were modeled with Swiss-Model server (Figure 17 and 18). The topology of the three propellers is the same as that of the six-bladed YWTD B propeller in human LDLR. There are 24 highly compact B strands organized in six anti-parallel B sheets (called blades), with four strands in each sheet (Figure 17). The YWTD] has, overall, highly negative surface EP, although the side surface, composed of B1 (green) and B6 (blue), is weakly positive. There are five histidine residues on both the top and bottom faces of YWTD]. Because histidine residues convert from neutral on the cell surface at around pH7 to bearing positive charges in the endosome at pHS, there is a possibility that these histidine residues can create a patch of positive surface area on the top and/or bottom of YWTD] in the endosome. In comparison with the YWTDl, the YWTDZ has, overall, a weakly to moderately positive surface, although there is a moderately negative side face composed of B4 (pink), which breaks the continuity of the positive surface. Significantly, there are eight histidine residues on the surface of the YWTD2, and five of them aggregate around the B4 sheet (four on the B4 sheet and one on the last B strand of B3) in a region with moderately negative EP. When receptors transfer from the cell surface to endosomes, the low pH may cause a significant change to the EP of this negatively charged surface patch, lll Fig. 17. Ribbon diagram and topology of the AanR YWTD B propellers. The top threes rows are the top, bottom, and side views of models of the AanR YWTD B- propeller 1,2, and 3. The B strands are shown as arrows. The six B sheets (propeller ‘blades’) are marked in different colors and numbered and the loop regions are colored in grey. The top view looks over the pseudosymmetry axis, with the N terminus coming out of the page and the C terminus entering the page. The bottom row is the topology diagram of three models. The six B sheets are colored and numbered as above, with centripetal and centrifugal arrows representing ascending and descending B strands, respectively. 112 i g I ‘N side N- C' YWTD1 :35 B3 B sheet 1 C' N' AanR YWTD topology 113 Fig. 18. The surface EPs of the AanR YWTD B propellers at pH7. The top row shows the ribbon diagrams of three AanR YWTD B-propeller models colored by B- factors. The green and red colors represent regions with certainty and uncertainty, respectively. The second and fourth rows show models with B sheets colored as in Figure 17. The third and fifth rows show the corresponding molecular surfaces colored with EP (Red: -6 kEV, negative; white: 0, neutral; blue: +6 kEV, positive) at pH7. Histidine residues are drawn in red. 114 top 8: front. colored by B-factors Etot: -7307 KJ/mol 552 KJ/mol 78 KJ/mol ldn/high sim: 31 .1%/57.2% 18.2%]39.1% 16.8%l50.2% Similarity: 64.8% 51.6% 63.7% YWTD-1 YWTD-2 YWTD-3 115 and may possibly switch most of the surface of YWTD2 to positive, making YWTD2 a strong competitor to Aan for the ligand-binding domain of AanR. The YWTD3 has a very strongly negative bottom surface. On the side of B5 (yellow), there is a strongly negative surface patch, and the rest of the surface is nearly neutral. On the surface of YWTD3, there are seven histidine residues: two on B2 (indigo) and five on the top. These five histidine residues may act as pH-sensitive switches that convert the surface charge of the top face from nearly neutral at the cell surface to negative in the endosome. The Insect VgR/YPR has one or two kind(s) of endocytosis signal(s): Searches for potential sorting signals in the cytoplasmic tails of insect and nematode VgRs/YPRs found at least one or two LL/Ll internalization motif(s) (Figure 19). Yxxd) motifs and NPxY-like (or NxxY) motifs exist in vertebrate VgRs, while, in contrast, they are not found in invertebrate VgRs/YPRs, possibly because the task of VgR/YPR is simply to transfer yolk proteins to oocyte yolk storage vesicles. Both AanR and DmYPR have an FxNPxY-homologous sequence, yet a non-aromatic residue occupies the sixth position in both receptors, and thus it cannot form a tight turn essential to recognition by adaptor proteins. Unlike AanR and DmYPR, which both have a nonfunctional NPxY motif; both PanR and CeYPR have one functional NPxY motif. The NPxY motif in CeYPR (YGNPMY) theoretically has the highest efficacy of internalization, and the one in PanR (FKNPTF) should have a less-than-typical efficiency of endocytosis with no tyrosine residue at either position 1 or position 6. 116 Aan modeling—Evidence of A Positive Surface The “receptor-binding motif” on blue tilapia Vg is not a receptor-binding site: Recently a “receptor-binding motif ’, HLTKTKDL (position 182-189), in the small subunit of Orechromis aureus (blue tilapia) Vg was reported (Li et. al. 2003). Mutation of either H182 or K187 had no effect on VgR binding, and K185A mutant attenuated the binding to approximately 50% (Li et. al. 2003, Figure 8), which suggested that the basic AanR ......... 14 F ‘Pel 98 -59}. AngR ......... 14 F Ntel ...85 939%. PanR ......... 17 FkNPtF ...89 a9}; DmYPR ......... 14 FiNPla ...76 359% ........................... 92 3§P§ CeYPR ......... 79 Y NPmY ..111 gent. HsLRPl ......... 24 I NPtY ...41 ..... I_. _I_. ............. 58 Ftnpvm ..84 Egg. HsLRPZ 6 _S_LL_ ..75 FeNPTrY ......... 101 mm Y ..147 FeNPim Fig. 19. Potential sorting signals in the cytoplasmic tails of invertebrate VgRs/YPRs, human LPRl, and human LPR2. A number on the lefi of each underlined sequence indicates the distance from the leftmost residue in the motif to the transmembrane helix of receptor. AanR, Aedes aegypti VgR; AngR, Anopheles gambiae VgR; CeYPR, Caenorhabditis elegans YPR; DmYPR, Drosophila melanogaster YPR; HsLRPl, Homo sapiens LRP]; HsLRPZ, Homo sapiens LRP2; PanR, Periplaneta americana VgR; single and double underlines, NPxY motif and NPxY-like motif respectively; framed residues, Yxx¢ motif; bald upper case and lower case, conserved and not conserved residues in a motif; dotted line, omitted sequence. 117 SD 5383 :08 hfincxm. maficrcfizw ...mV Xmas :88 8.96.: “firefigm 6% ”$39 08.8 :88 :cw:.3-:>»o:£ .82. 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>>q;vemmma¢mamzomemmqe>g>mmm>zgx-mxmgmrmmqw>m.Hm>mmoHemm--xgzne>fl>mm>mmmmqmg-u-mqruemmmmmmmmmmp-md¢M>mogemyamm>>z;x-omwxqrmm>>uxrhh>mmoHewm--oqm_mH>>mm>mmx44mo-a-zms-m:ao>m>ommadmwmmwmo>meHm.492.>odw>z¢x-omx>ammm>>>Mn>Hm>moHwa>--3;aaqwzoe>mmxesz-0-20m-ermxm-Qmmmm w>qo>m>xqomdmvzm>mawmw>o.492u>o¢w>4¢m-mxu>amm4H¢>xxHzmmmoHBmxw--34”eqwzwm>qum¢>n-w-20g-awmmm-Qmwwe :uamx>:m9¢mmxozgevmwmmmeamonwmy>mu>mmeemeng-w----ohc>qH-mm>psrmHmoxmeuwrde-ez-q¢wmm mqumx>rmhdmmxozgeqmxmmHmmnmqg>mmg>mo>em99m¢szmu----omgqu-mm>mmmmqmoommmMm¢Bw-93-mmwgmz;>mm»>m«x-oxrwarmg>n>emeem¢xwflmu----mxw94H-mmwhdm»quomoaxmdem-ez-m4w>m wqarxquammmxmzmm0530m>mmgmHz>mmm>m>>9meem¢wamu----oxw«QH-mm>¢mmgmw:>;mg>max-oxwwqum:>o>amhhm¢x»Haw----OXuqu-mm>9mq mm>mo Hm>mo max m>flm ~m>w< Hm>m¢ m>eo m>mm m>u¢ m>ou mm>zm Hm>nm mm>Ho Hm>Ho mm>mz ¢m>mz mm>uo Hm>uo m>su m>mo m>2q m>mm mm>mm m>mm m>co m>um¢ m>om mm>mm mm>mm Hm>mm m>cm m>u¢ m>mm4 m>a¢ m>nq m>uom m>OEm m>>< m>m¢ 121 residues in this motif are not very conserved. A multiple alignment among 40 Vgs from sixteen insects, thirteen fishes, one amphibian, and two birds showed that the two basic residues in this motif are not highly conserved. In some species, only the first basic residue is conserved, and in some others, there is not even a basic residue in the corresponding region. The surface area of Ichthyomyzon unicuspis Iipovitellin (IuLV) corresponding to the “VgR-binding motif” of Oan is on the left side of the N sheet domain, which corresponds to the small subunit of Aan (Aan-S). On IuLV, this region is not more positive than most other areas on the N sheet, and it is much less positive than the A sheet domain, which corresponds to a portion of the big subunit of insect Vg (Figure 20A). Because tilapia Vg and lamprey LV are highly homologous, this motif in the modeled tilapia Vg is also not more positive than most other regions in the N-sheet (data not shown). The most important thing is that in IuLV this motif is located at the monomer interface and is very near a disulfide bond. Just beside highly conserved first basic residue (K175) in this motif, threonine 176 in one monomer interacts with alanine 562 in another (Anderson et. al. 1998, in table 2). These are two reasons why this motif is conserved. Because the motif does not have to be positive and is buried in the docking face of two monomers, it is virtually impossible for this motif to be a part of the receptor- binding site. The lamprey Iipovitellin has an omni—positive surface EP: IuLV has an omni- positive surface, and the surface of the N-sheet domain is much less positive than that of the lipid-binding cavity structure, which corresponds to the large subunit of Aan (Figure 21). The lamprey vitellogenin has a very strongly positive surface around the lipid- 122 .Aozzma Ema—ohm .o_+ 6:3 3830: 6 “BE? 633mg bszm J .o- 635 mm 55 coho—8 8035.0. 83838 wEvcommth 05 950% 38 Eaton 2:. .boZHoonB >8» Ea .035 $8 .33 .52» E .505 8a “85%, can @356 .5582. 30:0: 485-2 2:. 58288 Em: A: 03983 50on 5&5 5:833: 989:8: $3.53: .~ mo mfiflwflv =25: $6.7. 38 no“ 2:. $326 «52.5-13: o5 mica—Eta 38...; 95:8.— bugbm a ma: £3325 huh—.55 AN .uE 123 binding cavity (bottom row in Figure 21). The strongest positive surface is located on the A-sheet, which corresponds to a part of the large subunit of Aan (Aan-L). The Aa Vg small subunit has a positively charged surface: The Aan-S was aligned with the N-sheet domain of Ichthyomyzon unicuspis Iipovitellin (IuLV) by hand (Figure 22 and Figure 208). Aan-S residue 85 to 399 was modeled based on the alignment (Figure 23). The structure of Aan—S is very similar to that of the N-sheet domain of IuLV. Because the [311 strand of the C sheet joins the A-sheet and the [312 strand of the C sheet joins the C-sheet in the Aan—S regional model, the B12 and [313 strands do not show up. Two significant differences between Aan-S and IuLV are two insertion sequences in Aan-S. The first gap in the alignment was predicted to be a pair of additional anti-parallel B strands (referred as B-A and B-B). Because these two additional [3 strands are beside B7 in the model, it is possible and quite reasonable that they join the N-sheet by contacting [37 at the left rim of the N-sheet. The second gap was predicted to be a long loop with an internal helix (or-A) by the Swiss-Model server. The 2-D structure prediction program, PROF, also predicted this region to have a helix with low reliability, while the program PHD made no prediction for this region. A check on the surface charge of the modeled Aan-S showed that Aan-S has a moderately positive surface and that the front and right sides are more positive (Figure 23). The Aa Vg large subunit has the helical domain, C-sheet, and A-sheet: Regions in 40 Vg sequences corresponding to the helical domain, the C-sheet, and a part of the N terminal region of the A-sheet of IuLV were aligned by hand (Figure 24). Residue 601 to 1301 of Aan-L was modeled based on the multi-alignment (Figure 25). The structure of 124 IuLV Aan IuLV Aan IuLV Aan IuLV Aan IuLV Aan IuLV Aan 131 132 B3 W "-295551151531353 135-195? BBQSZEVW EL ._T ART—5515 515L311? swmp rarvr VTSerrALAELDD,WT rrTRAvlviRPRSRDerArvKQPEYAUE ERLP Bl 62 B3 B4 a1 B5 B6 a2 50.3;ng -’:’D r*'_._~iE;z'-pi:g:;isa 13;;“rDégAPPSITDTAX’ T‘v."R :1 727.333; YATKFY DHEKF,PJPMSSKPF ER! K ATE WIVEKTIP War TLKAW.S;LQUDT B4 a1 B5 B6 a2 [37 BB ---------------- KK {QIEELgEIgVE_iQBIIEVIgE Y-------—‘-‘-------‘ R A TV SSKP: PSK EN YKuflEPLVT ECET YD? L:PAYNI;A K;WVPQ 91R E B-A B-B B7 B8 a-A J r l [39 B10 [311 [312 r~fim3 B13 RT:LATKTKQ; __5CD K1315???“-TA-‘x’AFRCPTc.Kf--t K_;R§T_A;’§:-_'I’B_"F‘ EPS DDLF.::TKTL FDRCULRI i F FT YSDFRP T ,I YASKS;FSYHYJT WY B9 310 a3 513 914 B15 B16 ;;;LKSA SE§;;;%§YFD7KE ”Vfgs5;x§:;§_itsgPAAs;AAsrgmg--"gerEP FT1,5331: K AiAPsav KEPA,VYA;U FTL DH PEDKYPI PAED K FTDLVYSY $14 B15 B16 a4 SSAJTKY EPSDKK Fig. 22. 2—D structural alignment of the Aan small subunit with the N-sheet domain of lamprey Iipovitellin. The Aan small subunit sequence was aligned with the N-sheet domain of Ichthyomyzon unicuspis Iipovitellin (IuLV) by hand. Two disulfide bonds in the N-sheet of IuLV are represented as gray loops. 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Hfirm:E_m\b_..m=c.__m 53:33:02 ) fi a, g \ ._-m>m< . . ,/. 34 1 the modeled Aan-L is very similar to that of IuLV. In the modeled helical domain, all 18 helices in the IuLV are conserved in Aan-L. Between two Aan counterparts corresponding to the 09 and 0110 on IuLV, an Aan—L insertion sequence was predicted to contain an extra a helix by the Swiss-Model server (Figure 25, golden-colored helix). The exact orientation of this extra helix is unsure because IuLV lack this helix. It is possible that this extra helix joins one layer of helices by interacting with the Aan-L counterparts to helices 09 and 0:10, but this is beyond the modeling capability of the Swiss-Model server. In the modeled N terminal part of the A-sheet, there is an insertion that was predicted to have two short anti-parallel B strands (Figure 25, golden-colored helix). The structure and location of this extra tiny B sheet look nice. Because most of the A-sheet was not predicted, it is still too early to check the surface EP of the Aan-L. DISCUSSION The CLI of CR8 in AanR Has A More Strongly Negative Surface Than The CLII All the thirteen modules from both clusters of CR3 were modeled. Comparisons of surface EPs of modules from both clusters led to a prediction that the CLI has a more strongly negative EP than the CLII (Figure 13). The calcium atom was excluded during modeling. Because the five to six calcium-coordinating residues are highly conserved in each module, although the calcium atom will attenuate the negative surface potential somewhat, the trend of the surface EP of each module will not change much. In addition, the influence of calcium on the surface potential is roughly equal for each module, so calcium can be overlooked during module comparison. 135 The prediction from protein modeling of two clusters of CR5 in AanR is a good fit to the experimental results from ligand-binding assays. With regard to the AanR, the protein modelings predicted that CRI-l, CRI-S, and CRII-3 would have the most strongly negatively charged surfaces; CRI-3, CRII-Z, and CRII-4 would have strongly negative surfaces; and CRII-7 would have a somewhat strongly negative surface. To sum up, the CLI has a more strongly negative EP than the CLII of AanR, and the surface charges of both clusters fall in the same scale. The results of saturation ligand-binding assays also showed that, in AanR, the affinity of the CLI is approximately 100% higher than that of the CLII, while the affinity of the full-length AanR is much higher than either cluster. In AanR, the surface EP of CRII-S is one of the four weakest CRs (the other three are those of CRII-l, CRII-6, and CRII-8) in the CLII. In A. gambiae VgR, counterparts of almost all AanR CRs are present, except that of CRII-S. Nematode YPR (CeYPR) lacks not only CRII-S but also CRII-7 and CRII-8. The Role of the AanR EGF Homology Domain in Aan Release The decision to include one B propeller fold together with its three flanking EGF- like repeats C terminal to the CLI and CLII in currently constructed mini-receptors was based on two earlier reports. Davis et. al. found that a truncated LDLR lacking the EGF precursor homology domain showed markedly reduced affinity to LDL but not B-VLDL compared to native LDLR on the surface of transfected Chinese hamster ovary cells, and complete degradation in the transfected cells 4 hours after incubation with B-VLDL on the cell surface (Davis et. a1. 1987). The EGF precursor homology domain was also shown to be important for in vivo binding of the LDLR to LDL on the cell surface, but 136 not for in vitro binding, because a truncated LDLR did bind LDL on a ligand blot (Davis et. al. 1987). Deletion analysis in the EGF precursor homology domain of LDLR by Esser et. al. also suggested its role in efficient binding of LDL (Esser et. al. 1988). Binding analyses of their mutant receptors showed that the first EGF-like repeat was required for binding of LDL, but not B-VLDL, while the second EGF-like repeat was not required for the ligand binding (Esser et. al. 1988). Each modeled EGF-like repeat of AanR has a surface EP varying from strongly positive to weakly negative (Figure 16). Although the bound calcium was not included during protein modeling, calcium will not make the actual surface EP of each EGF-like module more negative, and thus the conclusion that EGF-like repeats have no overall strongly negative surfaces is correct. Because until now the negative-positive electrostatic attraction is still thought to dominate the receptor-ligand interaction in the LDLR family, and because AanR EGF-like repeats do not have strongly negative surfaces, EGF -1ike repeats in AanR surely could not bind Aan via electrostatic attraction, and quite possibly they do not bind Aan at all. The acid-dependent conformational change in the EGF-like and /or YWTD repeats of the LDLR has long been reported to alter the ligand binding properties of the LDLR, allowing dissociation from bound ligand after acidification of endocytic vesicle and recycling of LDLR (Davis et. al. 1987). Rudenko et. al. have resolved the extracellular domain of the LDLR at endosomal pH (pH5.3), and showed that both the CR4 and CR5 interact with the top face of the YWTD B propeller via hydrophobic contacts and salt bridges (Rudenko et. al. 2002). 137 The three YWTD B propellers of AanR were modeled and surface EPs at pH7 calculated (Figure 17 and 18). The YWTD1 has five histidine residues on the top face and another five on the bottom face; the YWTD2 has five histidine residues on the B4 sheet with moderately negatively charged surface; and the YWTD3 also has five histidine residues on the nearly neutral top face. These gathered histidine residues on three YWTD propellers potentially could significantly change the surface charge at endosomal pH (pHS), even though the calculation of the surface EPs of the AanR YWTD propellers at pHS (rather than pH7) has not been performed yet. These histidine residues might possibly act as pH-sensitive switches that turn B propellers into false ligands of AanR at acidic pH. The change in the surface charges of AanR YWTD B propellers should be far more significant than the predicted limited change on the weakly negative top face of the human LDLR YWTD fold via merely two histidine residues (Jeon and Blacklow 2003, Figure 4). Sorting Signals in The Cytoplasmic Tail of LDLR Family Members The sorting signals in the cytoplasmic tails of membrane proteins mediate targeting of membrane proteins to the endosomes, the lysosomes, the apical plasma membrane, the basolateral plasma membrane, and the trans-Golgi network (Ohno et. al. 1995; Takeda et. al. 2003). Four sorting signals have been reported so far, which include the NPxY motif, the NPxY-like motif, the Yxxd) motif, and the LL/LI motif. The internalization efficiency of the FxNPxY signal increases when the xNPx core sequence is flanked by two aromatic residues (F, H, and Y), including at least one Y (Paccaud et. al. 1993). Y at the first and/or the last position of this motif has a stronger propensity than 138 F in inducing a tight turn conformation and hence stronger internalization efficiency (Paccaud et. al. 1993). The Yxx¢ motif ((1) is an residue with a bulky hydrophobic side chain, e. g. I, L, F) mediates not only internalization but also targeting to the trans-Golgi network (Bos et. al. 1993; Wong and Hong 1993). The LL/LI motif is an endocytosis motif present within many transmembrane cell surface proteins and functions as an internalization and lysosomal-targeting signal. The LL motif and a serine phosphorylation within the LRP tail were found to contribute to the receptor-mediated endocytosis (Li et. a1. 2000 and 2001). The NPxY-like motif (where P is replaced with other residues) in the megalin cytoplasmic tail was recently found to be an apical sorting signal of LRP2, which is expressed on the apical plasma membrane of polarized epithelia cells (Takeda et. al. 2003). The sorting signals of membrane proteins can be recognized by clathrin adaptor protein complexes, which in turn associate with clathrin and other accessory molecules to generate clathrin coats and coated transport vesicles during the clathrin-coated and pit-mediated receptor internalization (Hirst and Robinson 1998). A set of cytoplasmic adaptor and scaffold proteins bind to the cytoplasmic tails of LDLR family members, which suggests the participation of quite a few family members in several signal transduction pathways. For example, The VLDLR and ApoER2 are involved in the reelin signaling pathway (N impf and Schneider 2002; Howell and Herz 2001; Herz et. a1. 2000; Herz and Beffert 2000). The VLDLR is also involved in the urokinase-type plasminogen activator (uPA)-PAI-1 complex initiated cell signaling (Strickland et. al. 2002). LRP is involved in the signaling pathway initiated with the signaling protein, amyloid precursor protein (APP) (Herz et. al. 2000A and 20008). The LRP is also involved in the midkine (a growth factor with migration - and survival- 139 promoting activites) pathway, in the regulation of neuronal calcium influx mediated by azM, and perhaps in the Ras-extracellular signal regulated kinase-mitogen activated protein (MAP) kinase pathway (Strickland et. al. 2002). The LRP5/6 function in the Wnt/Wingless signaling pathway initiated with Wnt proteins (a family of secreted cysteine-rich proteins) (Howell and Herz 2001). Potential Receptor-Binding Sites on Aan Alignment among Vg sequences showed a varying number of basic residues around the corresponding region of the “receptor-binding motif” in several insect Vgs (Figure 20). The vicinity of this motif on Aan has a roughly equal number of basic and acidic residues (lEgEDqu igytfl, tgnfDEchfi). Although the claimed “receptor- binding motif” on 0an could be aligned with receptor-binding sites on ApoB and ApoE (Li et. al. 2003), a common pattern of these three motifs is not conserved among Vgs in investigated species (data not shown). Moreover, in 0an, this motif is on a B strand in the N-sheet, while the receptor—binding motif on both ApoB and ApoE is on an a helix. Although the receptor-binding motif on ApoB and ApoE share high sequence homology, the protein sequence of ApoE shares no homology with that of Vg. Although the N terminal portion of ApoB-lOO has a Iipovitellin-like domain, which is homologous to Vg and LV, the receptor-binding site on ApoB is near the C terminus, which has no homology to Vg or LV (Segrest et. al. 2001, Figure 2). Searches for an amphipathic receptor-binding helix-homologous sequence on Aan were also unsuccessful (data not shown). 140 The corresponding region of the claimed “receptor-binding site” on IuLV has the positive surface patch comparable to most other areas on the N-sheet, and it is much weaker than the outer surface of the lipid-binding cavity (Figure 21). This cavity is composed primarily of the A-sheet, the C-sheet, and a small portion of the helical domain. Because the interface of a homodimer is formed mainly by the N-sheets and helical domains from two monomers, and most of the A-sheet is not supported by the monomer contact (Anderson et. al. 1998, Figure 6 and Table 2), the strongly positive surface on the A-sheet is exposed to solvent even in a dimer. Earlier binding studies showed that AaVn-S has lower affinity to AanR than to AaVn-L and that both subunits have lower affinity than the intact Aan and AaVn (Dhadialla et. al. 1992; Sappington et. al. 1995). The weakened affinities of both AaVn-S and AaVn-L indicate that both subunits participate in binding to the AanR through either more than one binding site or a large binding face spanning both subunits. The Aan-S has a moderately positive surface, which supports the hypothesis that the force mediating AanR-Aan interaction is negative-positive charge interaction. The surface EP of Aan-L was not checked because most of the A-sheet domain was not in the model. An interesting observation is that there are many tyrosine-enriched sequences in Aan. In IuLV, tyrosine residues in the N-sheet mostly face inward, and tyrosine residues in the C-sheet and A-sheet domains mostly face toward the lipid-binding cavity (figure not shown). Thompson et. al. (Thompson and Banaszak 2002) reported 12 tyrosine residues on IuLV that interacted with loaded lipids (Y90, Y187, Y194, Y644, Y669, Y800, Y807, Y742, Y888, Y1048, Y1382, Y1518). Dhadialla (Dhadialla et. al. 141 1992) reported that dephosphorylation of Aan decreased its binding to AanR to less than 20% (Dhadialla et. al. 1992, Figure 9). These evidences suggested that most tyrosine residues in Aan might also interact with loaded lipids in the lipid-binding cavity and that some tyrosine residues may be involved in maintaining the receptor—binding site in the correct conformation and keeping it approachable. CONCLUSIONS Sequence analysis and protein modeling work in this thesis gave several predictions. First, both the CLI and CLII of CR5 in the AanR have predominantly negatively charged surfaces. CRI-l, CRI-S, and CRII-3 have most strongly negatively charged surfaces, CRI-3, CRII-2, and CRII-4 have strongly negative surfaces, and CRII-7 has a somewhat strongly negative surface. CRII-3, CRII-4, and CRII-7 are three modules with strongly negatively charged surfaces in CLIIs of both AanR and A. gambiae VgR. The CLI of AanR has more strongly negative EP than the CLII, and the negative surface charge of both clusters falls into the same scale. Second, AanR has three YWTD B propellers with quite a few histidine residues on each propeller surface. These many surface histidine residues potentially could significantly change the surface charge of propellers at endosomal pH. Third, all modeled EGF-like repeats in AanR lack strongly negatively charged surfaces, and thus they do not contribute to the negative- positive charge interaction between the AanR and Aan. Fourth, the modeled small subunit of Aan has a moderately positively charged surface. The modeled portion of the large subunit of Aan shows that it has one helical domain, one C-sheet domain, and one A-sheet domain. Fifth, the claimed “receptor-binding motif” on the N-sheet of tilapia Vg 142 is not basic residue-enriched in some Vg species and does not give a more positively charged surface patch than most other surface regions on the N-sheet of the modeled N- sheet of tilapia Vg; its counterparts in lamprey Vg and the Aan small subunit are also the case. This motif in lamprey Vg is on the docking face of two monomers. Sixth, Insect VgR/YPR has either one or two kinds(s) of endocytosis signal(s). 143 Chapter 4 Summary and Future Research Perspectives 144 SUMMARY Probably one of the most amazing biological phenomena known so far is the egg yolk accumulation in the mosquito oocytes during Vitellogenesis, when the developing oocyte increases in size over 300-fold within 36 hours, mostly through the VgR-mediated endocytosis of mosquito Vg. Coupled with Vitellogenesis, mosquitoes pass to human and animals numerous diseases, including dengue fever and malaria, which are threatening around half the world population. Therefore, investigation on the mosquito Vg-VgR interaction can elucidate the molecular mechanism of Vg internalization that is crucial to the mosquito-borne disease control. AanR is an ideal representative for the two-cluster LDLR subfamily. Unlike A. gambiae and P. Americana VgR, AanR has eight contact modules in the CLII of CR5, and even better than Drosophila VgR, AanR has an O-linked sugar domain. In addition, A. aegypti is the first model animal in elucidating the mechanism of receptor-mediated endocytosis. In this work, three mini-receptor genes were constructed and transfected into Drosophila cells. The saturation binding assay results showed that both the CLI and CLII of AanR have one binding site each with moderate binding affinity and that they are responsible for the high affinity of the full-length AanR to Aan. The AanR has at least two binding sites, and both clusters supply strengthened binding of Aan, probably in a synergistic way. The CLI of AanR is predicted to have, overall, a stronger negative surface than the CLII, which is a good fit to the results from the saturation binding assays. Both clusters have overall strongly negative surfaces, which also supports the conclusion from experimental results that two clusters supply strengthened binding of Aan. Negatively 145 charged surfaces of both clusters highly support the hypothesis that the AanR—Aan interaction is mediated mainly by the negative-positive electrostatic attraction. In comparision with the CRs in both clusters, no EGF-like modules in AanR have strongly negative surfaces. All three YWTD B propellers in AanR have numerous histidine residues on their surfaces, implicating their critical role in regulating Aan release and AanR recycling through acid-induced change of surface charges in mosquitoes. The reported “receptor-binding motif” on tilapia Vg is not a real receptor-binding site. The modeled Aan small subunit has a moderately positively charged surface. The Aan large subunit model shows that it has a helical domain, a C-sheet domain, and an A-sheet domain. Evidence of the positively charged surface of Aan supports the hypothesis of electrostatic complementarities from the ligand side. Finally, hypotheses were cast as a guidance to further studies. FUTURE RESEARCH PERSPECTIVES Investigation of individual CRs of Au VgR on binding Aan: We now know that both the CLI and CLII in the AanR bind Aan. The next step would be to determine which module or combination of modules in each cluster is/are critical for Vg binding. Figure 26 shows the proposed future construction of mosquito mini-receptors. This design is based on two hypotheses. The first hypothesis is that the interaction between Aan and AanR is predominantly positive-negative electrostatic complementation. Based on this assumption, CRs with most negatively charged surfaces compose the core region of newly designed mini-receptors. The second hypothesis supposes that there are no intro-domain interactions between modules within the cluster and that each CR binds 146 .cwaon £5 :8: 32:88 2a @882 oxzdcm wcflcwc m: was mEom 95;. @6235 386% 32080:-m0m 502833: mw>uw mo 50 no 30 $56 Eot $2:on £3» 83883 gawk: Ea _mw>Ev mcoEoootfiE mo 3:3 95 30% $603 Emu new to. 2:. .2 Same 5 mm .33 08mm 05 E 5.86 803 can .38 EoCOQEOo mo 98 :03 CO 832 $32 was 5&3 moon.“ x23 95 60¢ 05 mo mam 8&5; 5322: mm 230% Sn 363.528 coaoootfiuz £338.:EE 3258:. :e 53.36 255 632.95 .3 .wE 147 66% a my... 0 6.3.2; >5 6% . . :9 ‘56" ‘56 . .. E . 3 ‘\§ i “r; s? a: ".3 “é > 94% 9612,66; . n: q 9 66a 66 646 $6.36 M 6, 6 6 6:»; 66‘ * ‘ it." "2 i 8 ., “E . .2- g 66?»; 66 66, 66. 6%: .1 My: \ \\ A” >'5 1 £56: 66 M 66:» I a’ a a’ E 148 ligand independently. Based on this assumption, mini-receptor ngR1135 would be designed, with CRI-2 and CRI—4 kicked out except for their linker regions (Figure 26). This assumption is based on several observations. First, the crystal structure of LDLR shows that the ligand-binding domain is arranged in an extended way in the crystal form, with CR2 to CR7 separated by linkers, and modules do not interact with each other directly (Rudenko et. al. 2002). Second, Beglova et. al. observed in an NMR study that the linker connecting CR5 and CR6 is substantially flexible, and the covalent connection between CR5 and CR6 did not essentially change the intrinsic dynamic behavior of each repeat (Beglova et. a1. 2001). Third, an NMR study of the CRl-CRZ pair of LDLR also demonstrated flexibility in this concatermer (Kurniawan et. al. 2000). Seeking more structural evidences in support of hypotheses: The direct evidence of intensified and/or expended positively charged surface areas on YWTD [3 propellers would be sought by calculating the surface EP at pHS. The docking interface between each YWTD [3 propeller and its contacting CR(s) would be made, with reference to Rudenko’s X-ray result (Rudenko et. al. 2002). Modeling of the whole-length A-sheet domain of the Aan large subunit needs to be finished, after which checking the surface EP of the Aan-L will become possible. Finding residues on CRs critical for Vg binding: Afier we know which CR or combination of CR5 is/are crucial for the Vg binding, a further study on residues critical to the Vg binding would be done by both biochemical and genetics methods. Mutations would be made on candidate residues and in vitro and in viva binding assays would be performed. As a more powerful technology, surface plasmon resonance (SPR), would be used for several advantages over traditional binding assays. SPR can measure not only 149 affinity constants, but also reaction kinetic rate constants; SPR can monitor biomolecular interactions in real time without labeling requirements; SPR can also measure the amount of binding complex at equilibrium even in the presence of unbound ligand (Rich and Myszka, 2001). Finally, results from binding assays and theoretical modeling should be supported by 3-D structures of critical modules solved by solution nuclear magnetic resonance (N MR) or X-ray crystallography. 150 Bibliography 151 REFERENCES Anderson, T. A., Levitt, D. G., and Banaszak, L. J. (1998). The structural basis of lipid interactions in Iipovitellin, a soluble lipoprotein. Structure. 6, 895-909. Barr, P. J. (1991). Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66, 1-3. Beglova, N., North, C. L., and Blacklow, S. C. (2001). Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor. Biochemistry 40, 2808- 2815. 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