THESlS lllllilllllllllllillllllillHilllllllllllllllllllllllll 31293 01570 6355 This is to certify that the thesis entitled MOLECULAR CLONING OF MAIZE ENDOSPERM SOLUBLE STARCH SYNTHASE I presented by Bing Li has been accepted towards fulfillment of the requirements for M. S . degree in Biochemistry ”.112. @W Major professor Date é/Iajg?’ 0-7 639 MS U is an A ffirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE II I i j MSU Is An Affirmative Action/Equal Opportunity Institution o:\e|rc\ddedae.nm3-o.1 MOLECULAR CLONING OF MAIZE ENDOSPERM SOLUBLE STARCH SYNTHASE I By Bing Li A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE Department of Biochemistry 1997 ABSTRACT MOLECULAR CLONING OF MAIZE ENDOSPERM SOLUBLE STARCH SYNTHASE I By Bing Li Soluble starch synthase I of maize endosperm is involved in the chain elongation of starch synthesis. In order to correlate the structure of soluble starch synthase I to its functions, efforts have been made to establish the primary structure of soluble starch synthase I from maize endosperm. The partial cDNA clone coding for maize endosperm soluble starch synthase I has been isolated from a maize endosperm cDNA library using plaque hybridization as well as PCR technique. The deduced amino acid sequence shares high sequence identity with the sequence of rice seed soluble starch synthase 1. It shares a significant but low sequence identity with maize granule-bound starch synthase and E.coli glycogen synthase, respectively. However, several regions, including the substrate-binding site with a Lys-X- Gly-Gly consensus sequence for E.coli glycogen synthase, are highly conserved among these four enzymes. Therefore, it can be concluded that this protein corresponds to one form of soluble starch synthase in maize endosperm. To my family iii ACKNOWLEDGMENTS First of all, I would like to thank Dr. Preiss, for accepting me to work in this lab and for his guidance and support throught the length of this project. Thanks are also due to Dr. Sivak for her discussion on starch synthase, and my guidance committee members, Dr. Kaguni, Dr. Kuhn, Dr. McIntosh, and Dr.Nadler, for their insightfiJl advice on my experiments. I wish to express my appreciation to Dr. H.P. Guan and Mr. Brian Smith-White for their valuable suggestion on molecular cloning. I would also like to express my gratitude to other members in this lab for their always kind help. Special thanks are due to Dr. Tadashi Baba in Japan for providing us with rice soluble starch synthase I cDNA, and to Dr. Gerard Barry in Monsanto Co. for the maize endosperm cDNA library and maize seeds. Finally, I wish to thank all my Chinese friends for the precious friendship. TABLE OF CONTENTS LIST OF TABLES ................................... vii LIST OF FIGURES ........................... ....... viii LIST OF ABBREVIATION ................................ ix 1. LITERATURE REVIEW ................ . ................. 1 1.1 The structure and synthesis of starch .................................................. 2 1.1.1 The component and structure of starch ........................................ 2 1.1.2 The enzymatic mechanism of starch synthesis ............................. 6 1.2 Starch synthase ......................................... 7 1.2.1 Classification and properties of starch synthase ......................... 7 1.2.2 Genetic and kinetic studies on enzymes involved in starch synthesis ......................................................................... 9 1.3 A tentative model of starch synthesis ....... 12 1.4 Objective of the thesis ................ .. .13 2. MOLECULAR CLONING OF MAIZE ENDOSPERM SOLUBLE STARCH S Y N lHASE I ................................................. 15 2.1 Introduction .................................................. 15 2.2 Experimental procedures .................................................................. 20 2.3 Results and Discussion ..................................................................... 33 2.3.1 Cloning ofMSSS I ..................... 33 2.3.2 Sequence analysis of MSSS I ................................................... 33 2.3.3 Expression of the partial MSSSI cDNA in E. coli ..................... 42 3. SUMMARY AND PERSPECTIVE ........................................................ 45 Bibliography ............................................................................................... 48 LIST OF TABLES Table 1.1-Various properties of soluble starch synthase of maize endosperm .................... 9 vii LIST OF FIGURES Figure 1.1-Amylose and amylopectin, the polysaccharides of starch .................. Figure 1.2-The cluster model for amylopectin Figure 1.3-A tentative model of starch synthesis starch sy “ hybridization Figure 2.5-Fragmentation and subcloning for sequencing Figure 2.6-Construction of the expression plasmid ............... 3 5 14 Figure 2.1-Conserved regions of amino acid sequences of E.coli glycogen synthase, soluble starch synthase I and various granule-bound 18 Figure 2.2-Structure of ADP-glucose, ADP-pyridoxal and PLP, and chemical modification of the substrate-binding site -K-X-G—G- of E. coli glycogen synthase by ADP-pyridnxal 19 Figure 2.3-Restriction maps of R888 I cDNA (A) and the partial MSSSI cDNA (B), and restriction fragments with the conserved regions used in screening maize endosperm cDNA libraries via plaque 22,23 Figure 2.4-Strategy of screening a cDNA library for 5’-ends via PCR ............................ 25 26 30 Figure 2.7-Nucleotide sequence and deduced amino acid sequence of the partial MSSSI cDNA 35,36,37 Figure 2.8-Sequence alignment of the partial maize endosperm soluble starch synthase I (MSSSI) with rice soluble starch synthase 1 (RSSSI), maize granule-bound starch synthase OVIGBSS), and E.coli 39 glycogen synthase (glgA) viii LIST OF ABBREVLATION ADP-glucose: adenosine diphosphate glucose BE: branching enzyme GBSS: granule-bound starch synthase GS: glycogen synthase PCR: polymerase chain reaction SS: starch synthase ( GBSS and SSS) SSS: soluble starch synthase Chapter 1 Literature Review Starch not only is a critical primary source of dietary carbohydrates but also is used extensively for various industrial purposes. Despite its wide availability in nature and its many industrial applications, the mechanisms by which starch is formed in plant endosperm tissue are not well understood. Therefore, the biochemical mechanisms of starch biosynthesis are of great interest for understanding fundamental aspects of plant physiology and also for their potential utility in manipulating plant growth for practical purposes. Amylose and amylopectin, the two major components of starch, have distinct physical and chemical properties (36). Biosynthesis of specific starch forms in maize endosperm occurs by the coordinated activity of three types of enzymes, namely, starch synthases, branching enzymes, and debranching enzymes (26). Starch synthase catalyzes the elongation of or-l,4-linked glucose chains by transfer of the glucosyl unit of ADP-glucose to the nonreducing end of or-l,4-glucan primer. Starch synthase is present in two forms: soluble and granule-bound enzymes. The detailed roles 2 of the multiple forms of starch synthase and the potential interaction between individual forms of soluble starch synthase and branching enzyme in the synthesis of amylopectin is unclear because of the lack of the information concerning these enzymes. 1.1 The structure and synthesis of starch 1.1.1 The component and structure of starch Starch is the principal storage form of glucose in plants and occurs as large water- insoluble clusters or granules that are confined to plastids. It has been well known that starch contains two types of glucose polymer, amylose and amylopectin (Figure 1.1). Amylose consists of basically linear chains of between 600 and 3,000 D-glucose units connected by oc(1->4) linkages with very few branch chains. Amylopectin is generally much larger and more highly branched, containing 6,000-60,000 glucosyl residues. The glucosidic linkages joining successive glucose residues in amylopectin are or(1->4), and or- 1,6-glucosyl branch points occur about every 20 to 26 glucose units (30,36,40,41,45). The or(1->6) linkages comprise only about 4-5% of the total linkages in amylopectin, but they impart a branched structure to the molecule. The biological effect of branching is to make the amylopectin in molecule more soluble and to increase the number of nonreducing ends (one without a free anomeric carbon), thus making the starch more reactive to both starch synthase and degradative enzymes. Because of the most stable conformation of adjacent rigid chairs, the or(1->4) linkages of amylose and amylopectin cause these polymers to assume a tightly coiled helical structure. This compact structure produces the dense, sernicrystalline granules of stored starch seen in many cells. SM d .2-: as»; .828 EN B5285 Mo 836:5 $8: “So .8202 .3353 ”SE “Sou €55 64:38 ca «0 88.08% 3 . Santana. 3V owe: $0-; 3 5 3E: emcee—mAH mo 5:53 58:: a .8385. A8 203?. mo mowEAoommbom 2t “Egoomoiaw was 08354. H; onE E Edge see. .0 0 0 0 C. :8on O. .0 I e e e O O O O O O O. 0 mode to O momoovoucoz .0 O O O O O O O 3 30 m 0 u a m mo v m m m 0 4 The branches themselves in amylopectin form an organized structure. There have been many models about how the branches are arranged in amylopectin. The cluster model for amylopectin proposed in 1986 by Hizukuri (24, Figure 1.2) represents the best overall picture of branch structure, and is generally accepted today. In the model, some chains have no branch points (A chains) and are linked via an ot(l->6) bond at its reducing end to inner branches (B chains). B chains are linked in the same manner but carry A- or othe B- chains and may be branched at one or several points. A single chain per amylopectin molecule functions like B chains and has the only free reducing end group, 4), and is called the C chain. The branches are not randomly arranged but are clustered at 7-10-nm intervals. The B3 chains are longer than the B2 chains which are longer than the B1 chains. The BZ, B3 and B4 chains extend into 2, 3, and 4 cluster regions, respectively. The average chain lengths are 19 for B1, 41 for B2, 69 for B3, and 104 for B4 (24,32,36,4l). Starch isolated from different plants are composed of distinct amylose and amylopectin fractions (30). Usually, amylose makes up from 10% to 25%, the rest being the branched amylopectin. The amylose/amylopectin ratio and the distribution of low and high molecular weight D-glucose chains can affect starch granule properties, such as gelatinization temperature, retrogradation, and viscosity, which in turn, determine its economic value (4,45). Therefore, understanding the mechanism of starch biosynthesis is of theoretical and commercial importance. amaze: G A--- c - a “A ..... Ease :wQEw-Q - o - Av A-- C H ------ anoqoiaw com 3on 5330 BE- N; oar-H mmrsw u ._.o T218 0 —_ u--. —— ---- —— o--- —— 6 1.1.2 The enzymatic mechanism of starch synthesis Starch is synthesized in chloroplasts in photosynthetic tissues and in amyloplasts in non- photosynthetic tissues (32). The enzymatic mechanism of starch synthesis is believed to be the ADP-glucose pathway. It involves three processes: initiation, chain elongation, and branching, which include three important enzymes: ADP-glucose pyrophosphorylase (EC 2.7.7.23), starch synthase (EC 2.4.1.21), and starch branching enzyme (EC 2.4.1.28) (40,41,45,46). ADP-glucose pyrophosphorylase catalyzes the formation of ADP-glucose and inorganic pyrophosphate from glucose—l-phosphate and ATP, and is thought to be the regulatory enzyme and the rate-limiting step. Starch synthase, a glucosyl-transferase, is involved in the elongation process and catalyzes the stepwise addition of glucosyl unit from ADP-glucose to the non-reducing end of an or-l,4-glucan primer. Starch branching enzyme cuts an or (1->4)-linked glucan chain and form an or (1->6) linkage between the reducing end of the cut chain and the C6 of another glucose residue in an or(1->4)-linked chain, thus creating a branch (30,32,36,40,41). The presence of multiple starch synthases and branching enzymes distinguishes plant starch synthesis from those of bacterial, fiingal, and mammalian glycogen. This complexity has complicated genetic assignment of functions to the various starch synthases and branching enzymes in the building of amylopectin (29), and substantiates the need for further research to solve the enigma. How the initial primers for the synthesis of glucan chains are produced in plants is not 7 known. In mammal, which synthesize glycogen via a pathway that has many similarities with starch synthesis in plants, a protein called glycogenin, a Mn2+/Mg2+-dependent UDP- glucose-requiring glucosyltransferase, has been identified as the primer which initiates the glycogen synthesis in mammal (47). The first step is catalyzed by a protein tyrosine glucosyltransferase and involves the formation of the covalent attachment of glucose to Tyrl94 on glycogenin. The next step is the extension of the glucan chain from Tyrl94 to seven further glucosyl residues autocatalyzed by glycogenin. The further elongation of the glucan primer is carried out by glycogen synthase(47). 1.2 Starch synthase 1.2.1 Classification and properties of starch synthase Starch synthase is present in two forms: soluble or granule-bound forms. Multiple forms of soluble starch synthase have been found and partially purified in many species: maize endosperm and leaf (7,37,39), pea embryos (10,33,34), potato tubers (1,22), rice seeds (2), sorghum seeds (5), spinach leaf (23), castor bean endosperm (20), wheat endosperm (9), and Chlamydomonas (8,14,28,29). These soluble forms are classified into two types; one (type I, SSSI) is able to catalyze D-glucan formation without added primer in the presence of a high concentration of salts, such as sodium citrate, while the other requires exogenous primers for the activity and is called type II (88811) (40,41,45). In maize endosperm, the two types of soluble starch synthase, 72-76 KDa and 92-95 KDa, respectively, have different apparent affinities to primers, kinetic properties, and 8 immunological properties (summarized in Table 1.1) (40). It is possible that they are the products of two different genes (40,41,45). The properties for maize endosperm soluble starch synthase I and II in Table 1.1 are essentially representative. The apparent affinity for the substrate, ADP-glucose, for both forms (Km) is about the same. However, the two forms have different apparent affinities with respect to primers. The Km for Type I enzyme for amylopectin is 9-times lower that that for Type II enzyme for amylopectin. Citrate, at 0.5 M, can decrease the Km of amylopectin for both SSSI and SSSH: l60-fold for SSSI, and about 16-fold for SSSII (40). There are also differences in the relative activities (Vmax) between SSSI and SSSII. The Vmax of SSSI is greater with rabbit liver glycogen than with amylopectin. In contrast, SSSII is less active with glycogen than with amylopectin. Citrate stimulation of the primed reaction (with amylopectin) is greater with SSSI than with SSSII. And, SSSI is active without added primer in the presence of 0.5 M citrate while SSSII is inactive. Both SSSI and SSSII can use the oligosaccharides, maltose and maltotriose, as primers at high concentrations. SSSI seemed to be more active with the oligosaccharides than did SSSII. Maize endosperm SSSI and SSSII are also immunologically distinct. Antibody prepared with SSSI showed very little reaction with SSSII in neutralization tests. Becauseof their different kinetic properties and their different specificities with respect to primer activities, they may have different functions in the formation of starch granule. 9 Table 1.1 Various properties of soluble starch synthase of maize endosperm soluble starch synthase I soluble starch synthase [I molecular weight ( KDa ) 72---76 92---95 substrate Km values ADP-glucose 0.1 mM 0.1 mM Amylopectin 0.16 mg/ml 1.5 mg/ml Amylopectin+0.5M Citrate <0.001 mg/ml 0.09 rug/ml primer Relative activities to primer Amylopectin 1.0 1 .0 rabbit liver glycogen 2.1 0.6 Mylopectin+05M Citrate 4.4 1.8 0.5 M Citrate _ 5.8 < 0.02 1.0 M Maltose 1.6 1.2 0.1 M Maltotriose 0.9 0.5 1.2.2 Genetic and kinetic studies on enzymes involved in starch synthesis Granule-bound starch synthase (GBSS) has been demonstrated to be responsible for amylose synthesis based on the studies of the waxy mutation of cereals and the amf mutation of potato (32,36,40,45). For example, in waxy maize endosperm, which has low amount of starch, the almost complete absence of amylose is correlated with a massive reduction in the activity of granule-bound starch synthase. By contrast, amylase extender is deficient in branching enzyme 11 (BE II). This line is 10 characterized by an increase in the proportion of amylose in the granule from less than 30% in normal maize to about 60%. Furthermore, the amount of starch is low and the amylopectin from this mutant is far less branched (with longer branches) (3 2,36,40,45). Besides starch synthase and branching enzyme, it has been shown that a debranching enzyme (or(l->6)-glucosidase)is also very impbrtant in starch synthesis. Pan and Nelson (1984) (38) postulated that synthesis of amylopectin could result from an equilibrium between the actions of starch branching enzymes and starch debranching enzymes. The sugary mutant of maize endosperm has reduced amylopectin content as well as low starch amount but accumulates a highly branched water-soluble polysaccharide called phytoglycogen. The sul mutant is deficient in a debranching enzyme activity (26,38). Previous work in this lab has shown that soluble starch synthase and branching enzyme has different specificities in terms of chain length of primer. The BB isoforrns fiom maize endosperm distinguish from each other in the rates of branching amylose and amylopectin, and in the structure of the products formed, (21, 48). Maize BEI preferentially transfers longer chains than BEII (48). These results suggest that the BE isoforrns could play distinct roles in amylopectin synthesis. The properties of the two isoforms of maize endosperm soluble starch synthase are summarized in Table 1.1 (40). It could be concluded that SSSI may have a higher preference for the short exterior A—chains that are more prevalent in glycogen than in ll amylopectin. And SSSII may have a preference for the longer chains (B chains) seen in amylopectin (40, 46). Previous work showed that SSSI and SSSH were involved in the synthesis of amylopectin of different chain length (Libal-Weskler, unpublished data). Steven Ball’s group has studied the mutations in starch synthesis in monocellular algal Chlamydomonas and provided some interesting evidence of the potential roles of starch synthases. Growth-arrested Chlamydomonas cells accumulate a storage polysaccharide that has strong structural and functional similarity to storage starch in higher plants(3). It is synthesized by similar enzymes and responds in an identical way to the mutations affecting these enzyme activities. Therefore, it is an ideal model to study plant starch synthesis. GBSS-deficient mutant (8), SSSII—deficient mutant (14) and double mutants deficient both in GBSS and in SSSII (29) by UV or X-ray radiation have been isolated. The results can be summarized as follows: (1) In the GBSS-deficient mutant, Chlamydomonas is defective in amylose biosynthesis and accumulates a structurally modified amylopectin. Therefore, GBSS could be also involved in the synthesis of amylopectin (8,29). (2) SSSII may be necessary for the synthesis or maintenance of intermediate size chains (B chain?) that are the main components of the branched clusters of amylopectin. So, SSSH may prefer the longer chains (B chains) in amylopectin (14). 12 (3) In the GBSS-SSSII-deficient double mutant (i.e., in the sole presence of SSSI), Chlamydomonas directs the synthesis of a major water-soluble polysaccharide fraction and minute amounts of a new type of highly branched granular material, a structure that is intermediate between those of glycogen and amylopectin (29). 1.3 A tentative model of starch synthesis Fromlthe genetic and kinetic studies on enzymes involved in starch synthesis, it is conceivable that different forms of SS coordinated with BEs may help to determine both the fine structure and relative proportions of amylose and amylopectin within starch. A tentative model of the synthesis of amylose and amylopectin has been proposed by Dr. Preiss and Dr. Sivak (41, Figure 1.3), based on the studies of the Chlamydomonas mutants, and the work on the maize mutants deficient in starch synthase, branching enzyme and debranching enzyme, respectively, as well as the kinetic data on maize endosperm SSS and BE isoforrns. In the model, initiation of ot-l,4-glucan synthesis may involve the synthesis of a maltodextrin attached covalently to an acceptor protein. This putative protein-a-glucan then can act as a primer and accept glucose from ADP-glucose via GBSS catalysis to form an amylose structure. At the surface of the developing granule, SSSII and BEI could interact with the maltodextrin/amylose product synthesized via the GBSS reaction, to form a branched long chain polysaccharide with intermediate-size chains and complete the cluster structure of branched glucan. GBSS could be involved in the formation of the basic 13 (internal) structure of the ultimate amylopectin. SSSI along with BEII could be involved in the synthesis of the A chains and exterior B chains to form the exterior of the amylopectin structure and produce the final amylopectin product. The enlargement of the amylopectin could continue by repeating reaction 11 and III, to produce pre-amylopectin which is more highly branched than amylopectin and water-soluble. Then, isoamylase participates in a debranching process to catalyze the formation of the final amylopectin from pre- amylopectin (26,38) and the resultant chains (pre—amylose) are used as primers by GBSS to form amylose. It is possible that most of the amylose is produced in the internal part of the granule where BEs may be absent. However, the temptation to assign specific roles to individual forms of SS and BE should be resisted until more detailed information on how such enzymes interact in the synthesis of starch granules is available. 1.4 Objective of the thesis In order to understand the structure—function relationships of SSS in the cause of their potential roles in elongation process and their cooperation with the multiple isoforms of branching enzymes, it is essential to establish the primary structures of SSS from maize endosperm. l4 mam-2th? seawee- mo 3on 968:8 < m; char-H Aoomoobmoumnmv chaos-am 58033:“?er SoEEoo 05 E8 8 .md meowoebm Mo Begum omflmegm v m 8325a .6320 Eocene—mecca + Segue-0E cuoomcvnfiaa ouoEEoo 8 ”"5an m eotoam 8“ <8 seesaw ammo mew m = mm ewe—ages Bonn-m €295: 888328 ammo 92:20 .5 £26 cum awmm =35?me Escape: .«0 03608..an use 32 £14 £855? Mo cowaEE ounces-am Mo mmwoficmw 20%? N _ €283. Chapter 2 Molecular Cloning of Maize Endosperm Soluble Starch Synthase I 2.1 Introduction Starch is the most significant form of carbon reserve, and plays a key role in energy metabolism during the growth and development in higher plants. Amylose and amylopectin are the major components of starch, and have distinct structural, physical, and chemical properties which affect the quality of starch (36). Starch synthase catalyzes the elongation of a-1,4-glucosidic chains on amylose and amylopectin by transfer of the glucosyl moiety of ADP-glucose, and is present in two major forms, soluble starch synthase (SSS) and granule-bound starch synthase (GBSS). It is generally accepted that GBSS is responsible for the synthesis of amylose in starch. However, at present, little information is known concerning the detailed roles of SSS in starch synthesis, even though this enzyme has been implicated to play an important role in the synthesis of amylopectin in presumably regulated coordination with branching enzyme, and/or to participate in the amylose synthesis together with GBSS (49). 15 16 In order to understand the biochemical mechanisms of starch synthesis, starch synthase in many species has been identified, characterized, and cloned (41). Most of them have been granule-bound starch synthases from various plants. Recently, cDNA clones of soluble starch synthase have been isolated from immature rice seeds (2), pea embryos (12), and maize endosperm (unpublished data). The rice seed soluble starch synthase I cDNA is the first one to be cloned, but has been proven to be incomplete at the 5’ ends (49). Pea embryo granule-bound starch synthase H (GBSSII) has been localized both in soluble phase and on the granule phase. The N-temiinal “flexible arm” which is rich in serine could affect the partitioning of pea embryo GBSSII between the soluble and granule-bound phases,(12, 32). Therefore, it is believed to be soluble starch synthase II (SSSH) that might. lose the “flexible arm” and be bound to starch granules, perhaps as a result of entrapment as the granule grows (12, 32). The sequence comparison between E. coli glycogen synthase, plant granule-bound starch synthases, and rice soluble starch synthase I (RSSSI) indicates three well-conserved homologous boxes (2,50, Figure 2.1). The homologous boxes include the consensus sequence (Lys-Ser/Thr-Gly-Gly). The. LyslS in the Lys-Thr-Gly-Gly sequence in E. coli glycogen synthase has been reported to function as the substrate ADP-glucose binding site based on the chemical modification of the site by the substrate analog ADP-pyridoxal (17, Figure 2.2). It was found, via site-directed mutagenesis and kinetic studies, that the two glycyl residues in the conserved sequence Lys-Thr-Gly-Gly, especially the one closer to 17 the ADP-glucose binding lysyl residue, participate in catalysis by assisting conformational change(s) of the active site or stabilizing the transition state (18). The Ly5277 of the active site in E. coli glycogen synthase could participate in the catalytic reaction rather than binding of the substrate(l9). To facilitate further studies of starch synthesis, it is necessary to establish the primary structure of maize endosperm soluble starch synthase I (MSSSI). It is believed that the three homologous boxes could be also conserved in maize endosperm soluble starch synthases (MSS S), so, to isolate cDNA coding for M888, the restriction fragments of the RSSSI cDNA containing one or two of the homologous boxes were used as a probe to screen maize endosperm cDNA libraries (Figure 2.3). Reported below is the isolation of the partial cDNA clone encoding maize endosperm soluble starch synthase 1. In this study, a partial cDNA clone has been characterized. The nucleotide sequence shares high identity with that of rice soluble starch synthase I cDNA clone, and the deduced amino acid sequence contains the three highly conserved regions, including a Lys-X-Gly-Gly sequence in the amino-terminal region, which could act as the substrate-binding site. Therefore, it can be concluded that this partial cDNA clone encodes one isoform of soluble starch synthase in maize endosperm. 18 880m 89a 835% as, Bee 8838 05 8 magma—“8.2 2583 05 80¢ 598:: 2668 2: 88:3: 8539. 05 95829 $385: 2:. ommfiam mega “ca—05-2283” 32.5, Ea H 0358mm nobfim 0328 .0358? nowoobm zoom we $3253 28 8.58 mo £8th 320300 fim 8:me >aqmquawmvv "__ ___. 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Enoch—n :8 d gwfinWaomMmHS um .50 -30 -an. -aM .. 945E amoeba :3 .H M «MD mo M“malolmio. /o o 382w .. ma Afiaafiaoa acacia 20 2.2 Experimental procedures Materials Maize plants, Zea may (B73), were grown in a field in 1993 at the Experiment Station at Michigan State University in East Lansing, MI. The kernels were obtained on days 18-20 after pollination, immediately fi'ozen in liquid nitrogen, and stored at -80°C. A cDNA library (Zea mays, cultivar B73, endosperm hand-dissected, 30 days after pollination) was prepared in UNI-ZAP1M XR (Stratagene, LA Jolla, CA) and kindly provided by Dr. Gerard Barry at Monsanto Co, St. Louis. The rice soluble starch synthase I cDNA (RSSSI, 2,533-bp, subcloned at EcoRI site on pUC119) was a kind gift from Dr. Tadashi Baba at the Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan. a-32P-dCTP (3000 Ci/mmol) was purchased from Du Pont or Amersham. Oligonucleotides were synthesized using a DNA synthesizer (MSU Macromolecule Structure Facility). 14C-ADP-glucose (9.8 mM, 390 cpm/nrnol) and a glgA-deficient E.coli B strain was kindly provided by Dr. Sivak. 21 Screening of maize endosperm cDNA library 0 Plague hybridization (43) The maize endosperm cDNA library from Monsanto Co. was screened with RSSSI cDNA as a heterogeneous probe. The restriction fragments of RSSSI cDNA containing the highly conserved regions (Figure 2.3) were labeled with Klenow fragment of E. coli DNA polymerasel and a-32P-dCTP using random primers. Plaque lifts were prehybridized at 60°C in 6 X SSC (1 X SSC = 15 mM sodium citrate, pH 7.0, and 0.15 M sodium chloride ), 5 X Denhardt’s reagent (1 X Denhardt’s reagent= 0.05% Ficoll-type 400, 0.05% polyvinylpyrrolidone, and 0.05% BSA), and 0.5% SDS. Hybridization was carried out at 60°C overnight in the prehybridization bufl‘er containing denatured or-32P-probe (~106 cpm/ml). The filters were washed in 2 X SSC at room temperature for 10 minutes twice, in 2 X SSC plus 0.1% SDS at 60°C for 30 minutes twice, and in 1 X SSC plus 0.1% SDS at 60°C for 30 minutes twice, before autoradiography at -80°C. Positive clones were plaque purified, and pBluescriptSK(-) vector containing the insert was excised invivo from UNI-ZAPTM XR following Stratagene’s protocol using helper phage R408. The recombinant phagemid was identified by restriction mapping and sequencing (Figure 2.5). 22 80.58%an 883m «3 $55: 320 bps long / fill-in reaction of digested PCR fragments by T4 DNA polymerase subcloning onto Sma I site of pBLUESCRIPT KS - sequencing restriction digestion of PCR fragment and partial MSSS I cDNA with EcoR I and X ligation Figure 2.4 Strategy of screening a cDNA library for 5’-ends via PCR 26 waoauscom com 3.823% 98 288888me Wm onE 3 Mm 56338 33 0&3; 88m 8 883% Me 28888 3 Mm #528,838 82 28.3 as 8 883.8 a 28888 0 tan ~34 mamméé gum no 38233 ”m “gunman 3 Mm E0838 :3 8m 8 8833 as 88.58 <29 we a? 88388 d e o 3835 min -— 3 Mm Eowmqua «:04 can do 38393 ”< “gamma X— w. T m ._s _ m I 3 Mm $28.3me “8.2 «6.3 «am 8 883% “m 2835 w m . 0 cl o m m 3:8 88 s_ a. an 08 h. 1 m. m m m on 23 88 W .. 527 59863? 88 5 90 8235 8-88 GEE. + 8.5 32.8.. 5.. is. I xoq W 3%.: IV 5.: 9:. MWWWWWWMMw __ __._...__._s ___ _...--.. Wm. M madam .w. mmmammmdiq_____.... .. m..- .. u... m- mmmfimmm: 27 g The amplified samples were analyzed in 1 X TBE (0.09 M Tris-borate, pH 8.0, 2 mM EDT A ) on a 1.6% agarose gel containing 0.5 ug/ml ethidium bromide. Isolation of total RNA Total RNA was prepared by the method of D.R.McCarty (35). 20 g of frozen maize B73 kernels or endosperm were thoroughly pulverized under liquid nitrogen with a mortar and pestel. The frozen powder was added to a 250-ml flask containing 60 ml of extraction bufl‘er (100 mM Tris-HCl (pH 9.0), 200 mM NaCl, 5 mM DTT, 1% (w/v) sarcosyl, and 20 mM EDTA). The frozen tissue was then dispersed and thawed by homogenization with a Polytron for 1 minute. The suspension was centrifuged at 4°C at 10 kg for 5 minutes. The supernatant was immediately extracted with an equal volume of phenol:chloroform:isoamyl alcohol(v:v:v, 50:50:1).Then the mixture was centrifuged at 4°C at 10 kg for 5 minutes. The upper aqueous phase was extracted twice with an equal volume of chlorofomi/isoamyl alcohol (vzv, 50:1), and then centrifuged as above. The upper phase was saved and 12 M LiCl was added to the final concentration of 2 M. The mixture was precipitated overnight at 4°C. The RNA was recovered by centrifugation at 4°C at 10 kg for 10 minutes, and the RNA pellet was thoroughly resuspended in 3 m1 of 2 M LiCl and centrifuged saving the pellet. This was done twice. The RNA was dissolved in 2 ml of TE (10 mM Tris-HCI (pH 7.5) and 2 mM EDTA), and centrifiiged at 4°C at 10 kg for 10 minutes to remove any insoluble 28 material. A 1/10 volume of 3 M sodium acetate (pH 5.2) was added, followed by 2.5 volumes of ethanol. The mixture was incubated overnight at -20°C, then centrifilged at 4°C at 10 kg for 10 minutes. The pellet was rinsed with 70% ethanol, then resuspended in TE buffer and stored at -20°C. Preparation of mRNA- Polyadenylated RNA ( poly(A)WA ) was purified from total RNA using Oligo(dT)- cellulose by QIAgen (.Chatsworth, CA ) or Stratagene ( La Jolla, CA ) following manufacturer’s instruction. Northern blot analysis Total RNA or mRNA was analyzed by Northern blotting as described by Sambrook et al. (43). Radioactive probes were generated by labeling the restriction fi'agments of the partial MSSSI cDNA using the E. coli DNA polymerase I large fragment (Klenow fragment) and random hexanucleotide primers. Construction of maize SSSI expression plasmid (Figure 2 .6) The cloning strategy for an expression plasmid for the partial MSSS I is outlined in Figure 2.6. For expression of MSSSI in E.coli, the PCR method was used to introduce an in- frame ATG initiation codon and a NcoI restriction site for subcloning at the 5’ end of the partial MSSSI cDNA. Primer Po (5’-aagaattccatggagattgtggttggaaagg-3’, corresponding 29 to N’-EIVVGKE-C’, Figure 2.7) was synthesized in order to introduce an ATG codon and a restriction site (NcoI), and a gene specific primer P1 was used.for 3’-amplification ( 5’-caaggaccaaaggagcctcacatg-3’, corresponding to the deduced amino acid sequence N- CEAPLVLE-C, Figure 2.7). The template for the reaction was pBS-MSSSI. The amplification reaction was carried out as described before. The resultant 450-bp PCR product was digested with NcoI and HincII, and the 300-bp restriction fragment was recovered for further subcloning. Meanwhile, the pBS-MSSSI was digested with HincII and Bsp1021 which is the isoschizomer enzyme for NotI. The 1.6-kb HincII/BsplOZI fragment as well as the 300-bp NcoI/HincII PCR fragment were subcloned into NcoI/NotI-cut expression vector pET- 23d, producing plasmid pET-MSSSI. The reconstructed gene was resequenced to ensure that no mutations have happened during the construction. Expression of maize SSS I in E. coli pET-MSSSI was transformed into E.coli BL21(DE3) and E.coli B (glgA-deficient mutant), respectively, which both contain the T7 polymerase gene under the control of lacZ promoter. An overnight culture of the transformed cells with MSSSI cDNA was diluted 1:25 in fresh LB medium containing 100 ug/ml ampicillin. The cells were grown at 37°C for 1.5-2 hours until O.D.5oo reached 0.5 - 0.6. The expression of MSSSI was induced by adding 30 0.00 coRl 0.70 incll 1.01 MSSSI cDNA 1 .9-kbp PCR amplification with pnmers P0 &. P1 Hincll/Bsp102l digestion NCOI Hincll lPo I 5'l 450—bp 1 3. 1 P Hincll Bsp102| Nco'lHind' 5' .1 .6-kb E 3' digestion VCCGG Nc‘f' Hincll 5' H 300-bp 3- ° 0° oup|o 1:4 Q; co 0 T7 promoter N00, 024 2?}. otl 017 Ncol/Notl 7 pro pET-23d digestion motor pET-23d 3.67 Kb ’ 3.67 Kb ligation restriction mapping MSSSI cDNA ‘ 1 .9-kbp Mumcmg 00’ 2.17 Figure 2.6 Construction of the expression plasmid 3 l isopropyl-B-D-thiogalactopyranoside (IPTG) to 0.5 mM. After grown at 37°C for 3 hours, cells were harvested in a refiigerated centrifilge. Cell paste was resuspended and lysed by sonication in 50 mM Tris/acetate (pH 7.5), containing 10 mM EDTA, 2.5 mM DTT and 5%(w/v) sucrose. The lysed suspension (homogenate) were separated into soluble and insoluble fractions by centrifiigation for 30 minutes at 15 kg at 4°C. The supernatant was used for enzyme activity assay. Assay of SSS activity The reaction was conducted at 37°C for 15 minutes in a mixture (0.2 ml, pH 8.5) consisting of 100 mM Bicine/NaOH, 5 mM EDTA, 2.5 mM DTT, 0.48 M sodium citrate, 5 mg/ml rabbit liver glycogen, 140 nmol ADP-[“C]-glucose (specific activity, 390 cpm/nmol, ~ 50,000 cpm), and an appropriate amount of enzyme. The reaction was terminated by addition of 75% methanol-1% KCl. The incorporation of [”C]-glucose into the methanol-insoluble material was measured using a liquid scintillation counter. One unit of enzyme activity was defined as 1 umol of ADP-[”C]-glucose transferred into the methanol-insoluble material per minute. Protein Determination Protein concentration was determined according to the method of Smith et. al. (46) using the prepared bicinchoninic acid (BCA) reagent ( Piece Chem. Co., Rockford, Illinois) and BSA as the standard. . 32 Analytical procedures Nucleotide sequence analysis was carried out by the dideoxy chain-termination method using a commercial kit from USB. Computer-aided analysis of nucleotide and protein sequences was carried out using the GCG/GAEA program (University of Wisconsin). 33 2.3 Results and Discussion 2.3.1 Cloning ofMSSS I Two positive partial clones were identified by screening approximately 3.5 X 105 plaques fi'om the maize endosperm cDNA library in UNI-ZAPTM XR vector, using restriction fragments of RSSSI cDNA as a probe (Figure 2.3). The phage DNA was purified and analyzed by restriction enzyme digestion. Different overlapping restriction fragments were blunt-ended and subcloned on pBluescriptSK(-) and subjected to sequence analysis. The restriction map and sequencing strategy used are given in Figure 2.5. PCR screening of the cDNA library resulted in 250-bp more 5’-ends, using a gene specific primer (P1) designed on the basis of the sequence known fiom the partial cDNA clone which was obtained by plaque hybridization. The PCR cloning strategy is outlined in Figure 2.4. 2.3.2 Sequence analysis of MSSS I The composite nucleotide and the deduced amino acid sequences of the partial cDNA insert along with its 3’ flanking nucleotide sequence are shown in Figure 2.7. The nucleotide sequence shares very high identity with that of rice soluble starch synthase I cDNA (86% identity on GAP). The partial cDNA clone (l913-bp) contains a 1515- nucleotide open reading frame that is flanked by 3’ untranslated region of 398 nucleotides (Figure 2.7). The partial open-reading-frame encodes a polypeptide of 505 amino acid residues, with a calculated molecular weight of 56 KDa (Figure 2.7). 34 Since SSSI of maize endosperm is about 72 KDa based on sucrose density ultracentrifugation (23), the partial cDNA sequence encoding MSSSI probably lacks a sequence of 450 to 500 nucleotides at the 5’ end, which could make the final molecular weight of the putative MSSSI up to 72-KDa. The difliculty of cloning a full-length cDNA from a cDNA library could be caused by the first-strand synthesis. Rare transcripts or premature termination of the reverse transcription, in particular with large transcript or if extended secondary Structures are present, could result in partial cDNA. Therefore, work with RNA or preparation of a new cDNA library is very usefill for this purpose. The consensus sequence of ADP-glucose binding site for E. coli glycogen synthase is found at residues 29 to 33 (Lys-Ser-Gly-Gly) (Figure 2.7), therefore, it is reasonable to consider that the isolated cDNA clone encodes one isoform of soluble starch synthase. Sequence comparison between SSS, GBSS, and glgA A comparison of the deduced amino acid sequence of MSSSI and several other known sequences is shown in Figure 2.8. As shown in Figure 2.8, The MSSS I sequence is very similar to that of rice soluble starch synthase I (RSSSI, 94.9% similarity, 89.3% identity on GAP; gap weight 3.0, length weight 0.1; 11), but has a low sequence identity with the waxy protein of maize (MGBSSI, 61.7% similarity, 40.5% identity), and E. coli glycogen synthase (glgA, 55.8% similarity, 32.0% identity), respectively. However, the three homologous boxes, which have been found in the sequence comparison between E. coli glycogen synthase and plant GBSS ( 2, 12, 27, 40, 41, 46, 50 ), are well—conserved in the 35 10 30 50 60 gagattgtggttggaaaggagcaagctcgagctaaagtaacacaaaacattgtctttgta ctctaacaccaacctttcctcgttcgagctcgatttcattgtgttttgtaacagaaacat E I V 'V G K E Q A R .A K V T Q N I V F V 70 90 110 120 actggcgaagcttctccttatgcaaagtctgggggtctaggagatgtttgtggttcattg tgaccgcttcgaagaggaatacgtttcagacccccagatcctctacaaacaccaagtaac T G E A. S P Y A. K S G G L G D V C G S L 130 150 170 180 ccagttgctcttgctgctcgtggtcaccgtgtgatggttgtaatgcccagatatttaaat ggtcaacgagaacgacgagcaccagtggcacactaccaacattacgggtctataaattta P V .A L A. A. R G H R V M V V M P R Y L N 190 210 230 240 ggtacctccgataagaattatgcaaatgcattttacacagaaaaacacattcggattcca ccatggaggctattcttaatacgtttacgtaaaatgtgtctttttgtgtaagcctaaggt G T S D K N Y A N .A F Y T E K H I R I P 250 270 290 300 tgctttggcggtgaacatgaagttaccttcttccatgagtatagagattcagttgactgg acgaaaccgccacttgtacttcaatggaagaaggtactcatatctctaagtcaactgacc C F G G E H E V T F F H E Y R D S V D W 310 330 350 360 gtgtttgttgatcatccctcatatcacagacctggaaatttatatggagataagtttggt cacaaacaactagtagggagtatagtgtctggacctttaaatatacctctattcaaacca V F ‘V D H P S Y H R P G N L Y G D K F G 370 390 410 420 gcttttggtgataatcagttcagatacacactcctttgctatgctgcatgtgaggctcct cgaaaaccactattagtcaagtctatgtgtgaggaaacgatacgacgtacactccgagga A F G D N Q F R Y T L L C Y A A C E .A P 430 450 470 480 ttggtccttgaattgggaggatatatttatggacagaattgcatgtttgttgtcaatgat aaccaggaacttaaccctcctatataaatacctgtcttaacgtacaaacaacagttacta L V L E L G G Y I Y G Q N C M F V’ V N D 490 510 530 540 tggcatgccagtctagtgccagtccttcttgctgcaaaatatagaccatatggtgtttat accgtacggtcagatcacggtcaggaagaacgacgttttatatctggtataccacaaata W H A S L V P V L L A .A K Y R P Y G V Y 550 570 590 600 aaagactcccgcagcattcttgtaatacataatttagcacatcagggtgtagagcctgca tttctgagggcgtcgtaagaacattatgtattaaatcgtgtagtcccacatctcggacgt K D S R S I L V I H N L A. H Q G V E P A 610 630 650 660 agcacatatcctgaccttgggttgccacctgaatggtatggagctctggagtgggtattc tcgtgtataggactggaacccaacggtggacttaccatacctcgagacctcacccataag S T Y P D L G L P P E W Y G A. L E W V F 670 690 710 720 cctgaatgggcgaggaggcatgcccttgacaagggtgaggcagttaattttttgaaaggt ggacttacccgctcctccgtacgggaactgttcccactccgtcaattaaaaaactttcca P E W A R R H A L D K G E A V N F L K G Figure 2.7 Nucleotide sequence and deduced amino acid sequence of the partial MSSSI cDNA (the three conserved regions underlined) 36 730 750 770 780 gcagttgtgacagcagatcgaatcgtgactgtcagtaagggttattcatgggaggtcaca cgtcaacactgtcgtctagcttagcactgacagtcattcccaataagtaccctccagtgt A. V’ V T .A D R I V T V S K G Y S W E V T 790 810 830 840 actgctgaaggtggacagggcctcaatgagctcttaagctccagaaagagtgtattaaac tgacgacttccacctgtcccggagttactcgagaattcgaggtctttctcacataatttg T .A E G G Q G L N E L L S S R K S V L N 850 870 890 900 ggaattgtaaatggaattgacattaatgattggaaccctgccacagacaaatgtatcccc ccttaacatttaccttaactgtaattactaaccttgggacggtgtctgtttacatagggg G I V N G I D I N D W N P A T D K C I P 910 930 950 960 tgtcattattctgttgatgacctctctggaaaggccaaatgtaaaggtgcattgcagaag acagtaataagacaactactggagagacctttccggtttacatttccacgtaacgtcttc C H Y S V D D L S G K A K C K G A L Q K 970 990 1010 1020 gagctgggtttacctataaggcctgatgttcctctgattggctttattggaagattggat ctcgacccaaatggatattccggactacaaggagactaaccgaaataaccttctaaccta E L G L P I R P D V P L I G F I G R L D 1030 1050 1070 1080 tatcagaaaggcattgatctcattcaacttatcataccagatctcatgcgggaagatgtt atagtctttccgtaactagagtaagttgaatagtatggtctagagtacgcccttctacaa Y Q K G I D L I Q L I I P D L M R E D V 1090 1110 1130 1140 caatttgtcatgcttggatctggtgacccagagcttgaagattggatgagatctacagag gttaaacagtacgaacctagaccactgggtctcgaacttctaacctactctagatgtctc Q F V M L G S G D P E L E D W M R S T E 1150 1170 1190 1200 tcggtcttcaaggataaatttcgtggatgggttggatttagtgttccagtttcccaccga agccagaagttcctatttaaagcacctacccaacctaaatcacaaggtcaaagggtggct S V F K D K F R G W V G F S V P V S H R 1210 1230 1250 1260 ataactgccggctgcgatatattgttaatgccatccagattcgaaccttgtggtctcaat tattgacggccgacgctatataacaattacggtaggtctaagcttggaacaccagagtta I T A. G C D I L L M P S R F E P C G L N 1270 1290 1310 1320 cagctatatgctatgcagtatggcacagttcctgttgtccatgcaactgggggccttaga gtcgatatacgatacgtcataccgtgtcaaggacaacaggtacgttgacccccggaatct Q L Y A M Q Y G T V P V V H .A T G G L R 1330 1350 1370 1380 gataccgtggagaacttcaaccctttcggtgagaatggagagcagggtacagggtgggca ctatggcacctcttgaagttgggaaagccactcttacctctcgtcccatgtcccacccgt D T V E N F N P F G E N G E Q G T G W A 1390 1410 1430 1440 ttcgcaccccctaaccacagaaaacatgttgtggacattgcgaactgcaatatctacata aagcgtgggggattggtgtcttttgtacaacacctgtaacgcttgacgttatagatgtat F A. P P N H R K H V V D I A N C N I Y I 1450 1470 1490 1500 cagggaacacaagtcctcctgggaaggtctaatgaagcgaggcatgtcaaaagacttcac gtcccttgtgttcaggaggacccttccagattacttcgctccgtacagttttctgaagtg ,Q G T Q V L L G R S N E .A R H V K R L H Figure 2.7 (cont’d) 37 1510 1530 1550 1560 gtgggaccatgccgctgacaatacgaacaaatcttccagtgggccttcatcggatctacc caccctggtacggcgactgttatgcttgtttagaaggtcacccggaagtagcctagatgg V G P C R 1570 1590 1610 1620 ctatgtcatgtaaaaaaggaccaaaaaagtggtggttccttgaagatcatcagttcatca gatacagtacattttttcctggttttttcaccaccaaggaacttctagtagtcaagtagt 1630 1650 1670 1680 tcctatagtaagctgaatgatgaaagaaaaccccctgtacattacatggaaggcagaccg aggatatcattcgacttactactttcttttgggggacatgtaatgtaccttccgtctggc 1690 1710 1730 1740 gctattggctccattgctccaatgtctgctttggctgccttgcctcgatggaccggatgc cgataaccgaggtaacgaggttacagacgaaaccgacggaacggagctacctggcctacg 1750 1770 1790 1800 agtgaggaatccagccgaacgacagttttgaaggataggaaggggagctggaagcagtca tcactccttaggtcggcttgctgtcaaaacttcctatccttcccctcgaccttcgtcagt 1810 1830 1850 1860 cgcaggcagcctcgccgtgattcatatggaacaagctggagtcagtttctgctgtgccac gcgtccgtcggagcggcactaagtataccttgttcgacctcagtcaaagacgacacggtg 1870 1890 1910 tcactgtttaccttaagattattacctgtgttgttgaaaaaaaaaaaaaaaaa agtgacaaatggaattctaataatggacacaacaacttttttttttttttttt Figure 2.7 (cont’d) 38 sequence of the partial MSSSI. The roles of these boxes for the enzymatic filnction are not certain at the present time, except that the Lys-X-Gly-Gly sequence of box I serves as the ADP-glucose (substrate) binding site (16,17,18) in E.coli. The other two homologous boxes are found at the C-terminus whose filnctions are unclear. But interestingly, boxIII (XGGLXD) is similar to the domain around the conserved polyphosphate binding site (KS/TGGLXD), although it lacks the lysyl residue that is thought to interact with the polyphosphate group of ADP-glucose/ADP via ionic interaction. The filnction of these two regions could be involved in chain elongation or primer preference (preference of transfer of glucose to A or B chains or preference for branched or less branched polysaccharides). Although there are also several regional sequence similarities, the significance is not clear. Glycogen synthase from E.coli has been reported to require sulfliydryl group(s) for the activity (25). The partial MSSS I contains 11 Cys residues (Figure 2.7). The location of Cys’s is not well conserved between MSSS I and MGBSS; only 2 Cys’s, at residues 133 and 417 in the MSSSI enzyme are conserved (Figure 2.8). Only one of the four Cys’s in the sequence of E.coli glycogen synthase is located at the same position in those of SSS and GBSS (Figure 2.8). Thus, this Cys residue may be correlated with the activity of both plant and bacterial enzymes. Dry et al. (1992) (12) characterized cDNA clones encoding two isoforms of GBSS (GBSSI and GBSSII) from pea embryos and potato tubers. 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H mmmz ooH om om or om on H xom om om oH H 40 pea embryos is thought to be SSSII, since it has been localized both in the soluble fraction and tightly bound to the starch granules, as well as the region at the N-terminus is very hydrophilic and Ser-rich which could be involved in determining the partitioning of the GBSSII protein between soluble and granule-bound phases or it could represent a domain subject to phosphorylation through its high Ser content. However, this may not be true in maize endosperm, since each of the starch synthase isoforms is distinguished by its physical and kinetic properties, and immunochemical studies indicate that the two SSS and at least the major GBSS in maize endosperm are not related (40, Table 1.1) Sequence comparison between SSS and mammalian GS No significant sequence similarity is found between soluble starch synthase and mammalian glycogen synthase(6). In mammalian species, two forms of glycogen synthase have been characterized in several tissues; one form of the enzyme shows no activity except in the presence of glucose-6-phosphate, while the other is fully active in the absence of this allosteric activator. This enzyme possesses the phosphorylation sites, which are located in the sequences of Arg-X-X—Ser and Ser-X-X-X-Ser in the rabbit muscle enzyme (6). The interconversion between the two forms of glycogen synthase is dependent on specific protein kinases and protein phosphatases. Whether or not soluble starch synthases in plants are regulated by phosphorylation and dephosphorylation is an interesting problem. A sequence, Arg-Ile-Val-Thr, similar to the above Arg-X—X-Ser is present at residues 248 to 251 in the sequence of the partial MSSSI 41 (Figure 2.7). However, the Thr residue seems not to be phosphorylated in vivo, because the phosphorylation sites. of mammalian glycogen synthase are located in the very negatively charged regions at the amino and carboxyl termini (6). However, the amino- terrninal 12-residue sequence of the 57-KDa RSSSI, which is rich in acidic amino acids, includes a Ser-Glu-Gln-Glu—Ser sequence (2). It is possible that one or both of the Ser residues "are capable of being phosphorylated (2). Even if so, the regulatory mechanism of SSS through phosphorylation like mammalian glycogen synthase remains to be established. Sequence comparison between SSS and BE It has been shown that branching enzyme, which is able to catalyze the formation of branches with or-l,6-glucosidic linkages, conserves 4 consensus sequences of the catalytic sites of amylolytic hydrolases, even though the branching enzyme belongs to a family of transferases (2,41). SSS, GBSS, and GS, however, possess no conserved sequence of the four catalytic sites in the amylolytic enzymes. Thus, SSS, GBSS and GS are functionally and structurally different from the amylolytic enzymes. Sequence comparison between starch synthase and starch phosphorylase Starch synthase catalyzes the transfer of the glucosyl residue from ADP-glucose to the nonreducing end of or-l,4-glucan. Phosphorylase can catalyze the reversible transfer of the glucose moiety of glucose-l—phosphate to give ot(1->4)-or-D-glucan and inorganic phosphate. In terms of physiology, the starch synthase-catalyzed reaction can be thought 42 of as the complement of the reaction carried out by starch phosphorylase, although the chemistry of the two reactions is not equivalent. The complementarity of regulation is not known at present. The complementarity of fimction might be represented in terms of structural similarity, though the primary structures of starch synthase and starch phosphorylase are unrelated. Although much effort has been directed towards understanding the enzymatic mechanism of starch synthesis, the role of each type of soluble starch synthase is still unclear because of the lack of the information on these enzymes. One of the possible reasons is that the enzyme is unstable and apparently present at low levels in plant tissues (2,40,41,46). Purification of the starch synthase in large amounts from plant sources and to a high specific activity has proven to be difiicult, and partly for this reason, it has not been possible so far to find out how the enzymes interact to produce amylose and amylopectin in starch granules. So, the isolation of the cDNA coding for SSS will facilitate informative studies of the structure-function relationship of this protein and also the more detailed examination of its role in plant starch synthesis. 2.3.3 Expression of the partial MSSSI cDNA in E.coli The partial open—reading-frame of the MSSS I cDNA encodes a polypeptide of 505 amino acid residues, with a calculated molecular weight of 56 KDa, less than the molecular 43 weight determined by SDS-PAGE and sucrose density untracentrafilgation. However, since it contains the three highly conserved regions, it is of interest to examine whether it encodes an active starch synthase. On the other hand, the sequence comparison (Figure 2.1, 2.8) shows that E.coli glycogen synthase, mature forms of granule-bound starch synthase and rice soluble starch synthase I all have 14-34 amino acid residues at the N- tenninus to the Lys-Ser/Thr-Gly-Gly motif. In the case of the partial MSSS I, there are 28 amino acid residues N-terminal to the Lys-Ser-Gly-Gly sequence. The N-terminus of the partial MSSS I was modified to introduce a Met codon by PCR method. The 1.9-kb MSSS I cDNA was ligated onto the expression vector pET-23d to yield the expression plasmid pET-MSSSI, which was subsequently transformed into E. coli BL21 (DE3), and glgA—deficient E. coli B, respectively. In the preliminary expression experiments, starch synthase activity was observed in the crude extract of both the wild-type and glgA—mutant cells transformed with pET-MSSSI, but not in the cells transformed with control plasmid pET-23d containing no insert (data not shown). Extracts of E.coli glgA—mutant cells expressing the partial MSSSI had readily detectable levels of starch synthase activity (approximately 0.05 units/mg proteins), whereas activity was undetectable in the comtrol cells transformed with the vector alone and induced in exactly the same way. The expression in glgA-mutant E.coli cells was much lower than that in wild type cells 44 (E.coli BL21 (DE3)). The probable reason for the low expression in the lysogen may be due to other characteristics of the strain genotype. For example, if the strain is wild type for the [on and amp proteases, either the target protein or the T7 RNA polymerase may be degraded in the cells. E. coli BL21(DE3) is deficient in the [on and amp proteases, which is the possible reason for the higher expression. The preliminary expression experiments indicate that the partial MSSS I cDNA could encode an active starch synthase. Growth conditions can be optimized to improve the expression in glgA-mutant E.coli cells. And immunological methods can be used to distinguish the expressed starch synthase from E. coli glycogen synthase. Chapter 3 Summary And Perspective This study demonstrates that maize endosperm has an isoform of starch synthase that is very similar in sequence to the 57-KDa rice seed soluble starch synthase 1. A partial cDNA clone of soluble starch synthase I from maize endosperm has been isolated from a cDNA library. It encodes a protein of 505 amino acid residues. The deduced amino acid sequence is very similar to that of rice seed soluble starch synthase 1 (89 % identity over much of its length), and shares limited similarity to maize waxy protein (41% identity) and E. coli glycogen synthase (32 % identity), respectively. Sequence alignment of the partial MSSS I, RSSS I, MGBSS, and E.coli GS demonstrates several conserved regions, including the homologous boxes I, II, and III. The roles of these boxes for the enzymatic filnction are not clear at present, except that the Lys- Ser/Thr-Gly-Gly sequence of box I acts as the ADP-glucose binding site in E. coli GS. The other two homologous regions at the C-terminus could be involved in binding of primer and primer preference ( preference of transfer of glucose to A or B chains or preference for branched or less branched polysaccharides) in chain elongation process. The sequence 45 46 comparison of starch synthase from different sources could help us to study the structure- function relationships of starch synthase using site-directed mutagenesis. Crystallization of starch synthase will certainly provide exclusive evidence on the three-dimensional structure. Northern analysis indicated that the specific mRNA of maize endosperm soluble starch synthase I is around 3.0 kb. To clone the full-length cDNA of MSSS I, work with mRNA or preparation of a new cDNA library is worth consideration. Expression of this partial cDNA in glgA—mutant E.coli cells produced active starch synthase (about 0.05 units/mg proteins in crude extracts compared to control), though further work needs to be done to improve the expression in glgA-mutant E. coli cells or distinguish the expressed starch synthase kinetically and immunologically from E.coli glycogen synthase in wild-type E.coli cells. These results suggest that the partial cDNA codes for an active starch synthase, since it contains the highly conserved regions which imply their important roles in substrate binding and catalysis. Expression of MSSS I in E.coli will not only pemtit the availability of a large amount of MSSS I for filrther structure-function studies, but also confirm that the MSSS I cDNA encodes an active isoform of starch synthase. The earlier results suggested that SSS I and SSS 11 may play different roles in the synthesis of amylopectin in vivo, i.e., SSS I could prefer the short A chains, while SSS II 47 could be involved mainly in the synthesis of the longer chains (B chains). Starch synthase could be responsible in establishing a particular size distribution of glucan chains (29).The production of characteristic glucan lengths is an intrinsic property of the starch synthase. Or, granule-bound and soluble starch synthases all have distinct abilities to interact directly or indirectly with the starch branching enzymes. Direct interaction could be mediated through physical contact in the building of a multi-subunit complex. Indirect specific interaction could be achieved either by both the selective use ( as primers) or the production of structural motives which are, respectively, products or selective substrates of distinct starch branching enzymes (29). Knowledge of the structure-fimction relationships in terms of the apparent specificity for primer may lead to modification of the gene. Subsequent transformation of the modified gene into plants could produce altered starch granules suitable for specific industrial uses. Transformation of potato tubers with E. coli glycogen synthase resulted in a highly branched starch (44), which provides evidence for the feasibility. BIBLIOGRAPHY Bibliography [1] Baba, T. et al. (1990). Properties of primer-dependent starch synthesis catalyzed by starch synthase fi'om potato tubers. Phytochemistry, 29: 719-723. [2] Baba, T. et al. (1993). Identification, cDNA Cloning, Gene Expression of Soluble Starch Synthase in Rice ( OIyza sativa L.) Immature Seeds. Plant Physiol, 1103: 565-573. [3] Ball, S. et al. (1990). Physiology of Starch Storage in the Monocellular Alga Chlamydomonas reinhardtii. Plant Science, 66: 1-9. [4] Blanshard, J.M.V. (1987). 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The discovery of glycogenin and the primimg mechanism for glycogen biogenesis. Eur. J.Biochem., 200:625-631. [48] Takeda, Y., Guan, HP, and Preiss, J. (1993). Branching of amylose by the branching isoenzymes of maize endosperm. Carbohydrate Res, 240: 253-263. [49] Tanaka, K. et al. (1995). Structure, Organization, and Chromosomal Location of the Gene Encoding a Form of Rice Soluble Starch Synthase. Plant Physiol, 108: 677-683. [50] Van der Leij et al. (1991). Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele. Mol. Gen. Genet, 228:240-248. HIG srn E UN IE3 “illumiilllini liliiilliiijii.l 812930157063 5