.3 “an : A: - .. . ’ 9 :V {:1-I3I 19:39:11" 1. "9‘. III‘ I.”.'.I1""."'.'“.'" 1I119)1(1931|II I. 99 .3799'9 '- \th nflg. 633131 1 #99" :9 :9 1 ”31‘ "3;"2'" . ‘ “1 ‘1 A‘ ' ‘ II ‘ A ‘. . '74"? $7: - \2:§1\_5'”":f11"."}§“& Ah U' ‘ " “1%?“ I233: F1999} ‘ )‘W R' I"|I\;\I"'l - ¢ ' “:I-fy‘ .1. 1R . Etc: 11931 fiv 1I1':- Q‘!" 3‘. I V 1%. .t'1“1'- I33I1I_1::I "}i;1""‘ Ivcgl - .1. :‘V. AHA. g’ " ‘ {53:1 9'1; 3 ‘ 0“ '92:. . ” . '6'?" 9393' ":"w '1'"; '9' :.'.'. 31293113133? "'1'" ' ‘ ' 3 '3'": .1" "'"' ." 9.. 9 - {3'11” .‘I- "'13:?" 9 3, '.I 1‘1 I'v'rpI 'M . '"H‘ I"'1;1"'('.:'I'1 ‘fié'. .1 99% fi'fifiq} ” .. :1..." .‘.9:<-‘9.99 9.; 3::9'32’3 13% '3 _ ' ‘1 13%;“ .1'1‘? ’9 ‘39 V 1 A? "1.913%I 5-1.“ ' '9- " ' ' “11‘2“ """' ' ' ' ' 'I1'31"" .‘."C'.I'-‘.' ' 1'1'.h.,¢"}1 1 HII- 1 I'. 9.. ' ‘ ”"1"" '-I,1I.'3' 9 ‘.‘I"""'\y),‘1 I‘l- . . I ‘r: ‘1 IVIII" I \"1 3., II}I.‘I1 . ‘1IIYIII ;. '99 4:31 “-‘-‘ 3 '91-‘31 -.9'9'-_ 9-. I.3I - 5‘3. I O\.-'|110’QI}.1"I3 "3 3 1.31'“: ”(Suc'a 99-992 9- 3- 9 3 3.93:- ‘99" 9-9-3." 9.9- 31:32. 999-9.; :‘- 3999.9 ' I" . 1 I 3".2“;f",3" '.\'..‘.33m9 \3: ”1300‘ :333‘3 ”.3133? 3 33333 Fin-{'1I9.'I3?13I'34""9' I |'t “in“ 0‘ ' '3 ' '- 99 "9 9 ‘ !"l' " ' '1": "'.' Il'1 U 13.3: liars” ‘l q'fa‘ -fi3'1’7'9\- Q t H ' ' . ,. h ' ""3' 11.919 I ' ILL "p" PL 91 5% 'I 34:; . .I I' > 9'1! 31"‘9' "' If?“ '3 a ‘ In . ill- . 1 '. _' ."- "Lifu? :3 . 99;. 99 ..9 -«9... 1 .""""J31_' 1 I .I3. 3 .1. . "33:9" '9':"3;("."-....- '3 if”. i .5 3m '9991999'.L1‘1'.'l§\19:"7}1 Wit}: ' ' W951. " """S‘J‘M 39.931“ - «MinimumMW“mm“;iigljuu 3 1293 10 LIBRARY". ‘ Michigan State University This is to certify that the thesis entitled PARTIAL CHARACTERIZATION OF EXTENSIN BY SELECTIVE DEGRADATION OF CELL WALLS presented by Andrew J. Mort has been accepted towards fulfillment of the requirements for Ph. D . degree in Biochemistry pm fizz/”é Major professor Date]? “Md—24!? 78). 0-7 639 MSU LIBRARIES n RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped be10w. «Cm-reach; AA 0 .7- M_ W PARTIAL CHARACTERIZATION OF EXTENSIN BY SELECTIVE DEGRADATION OF CELL WALLS By Andrew J. Mort A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY l \ \ Department of Biochemistry 1978 ABSTRACT PARTIAL CHARACTERIZATION OF EXTENSIN BY SELECTIVE DEGRADATION OF CELL WALLS By Andrew J. Mort Cell walls of plants contain a glycoprotein, extensin, which is thought likely to be cross-linked to wall polysaccharides. Drastic degradative methods are necessary for the release of the protein from the cell wall. Two complementary methods for the selective degradation of cell walls have been developed in order to obtain preparations of extensin fragments for use in structural studies. The first, anhydrous HF solvolysis, cleaves neutral and acidic sugar glycosides with no peptide bond breakage. The second, NaClO2 oxidation, cleaves few pep- tide bonds, no glycosidic bonds, except to a small extent those of uronic acids, yet solubilizes large amounts of high molecular weight glycopeptides from cell walls. Treatment of tomato, tobacco, or sycamore maple cell walls with liquid HF did not release extensin from the walls, but did solubilize all the sugars, leaving a cell wall shaped residue of amino acids and some unidentified material. Thus, HF alone cannot be used to isolate intact cell wall protein in a soluble form, but can be used to determine the complete sugar composition of cell walls. Andrew J. Mort From experiments with glycoproteins containing the glycopeptide linkages, arabinose-O-hydroxyproline and galactose-O-serine (plant cell wall glycopeptides), N-acetylgalactosamine-O-serine/threonine (pig submaxillary mucin), and N-acety1glucosamine-N-asparagine (fetuin), it became apparent that anhydrous liquid HF, a reagent commonly used by synthetic peptide chemists for the complete removal of protecting groups from synthetic peptides, cleaves the O-glycosidic linkages of neutral sugars in 1 hour at 0 C, and the O-glycosidic linkages of amino sugars in 3 hours at 23 C. The N-glycosidic linkage of N-acetylglucosamine to asparagine is not cleaved by anhydrous HF under any conditions that have been tested. Sodium dodecyl sulfate polyacrylamdde gel electro- phoresis of HF treated bovine serum albumin reveals no degradation of peptide bonds. Some relatively stable enzymes (lysozyme and ribonucle- ase) have been shown by others to retain most of their enzymic activity after short treatment (1 hour at O C) in HF. Although the precise chemical nature of the oxidation reaction of NaClO with cell walls and proteins is not known, it was determined 2 that NaClO releases large quantities of glycopeptides from cell walls 2 and so is of interest. These glycopeptides have been partially char- acterized by size, charge and buoyant density. Compositional and methylation analyses showed these peptides to be highly glycosylated, containing the short arabinose side chains previously described by Lamport (l) but in addition a single galactose on almost all of the Andrew J. Mort serine residues and in some cases what appears to be a 3,6 linked galactan linked to a polymer containing galacturonic acid, i.e., pectin. The results presented give supporting evidence to the model of a primary cell wall put forward by Keegstra g£_§l, (2) and thus place the cell wall protein in a good Position to be controlling wall growth. REFERENCES 1. Lamport, D. T. A. Nature 216, 1322 (1967). 2. Keegstra, K., Talmadge, K. W., Bauer, W. D., and Albersheim, P. Plant Physiol. é}, 188 (1973). ACKNOWLEDGMENTS Thank you, Marlee, Shirley, Fernand, Jim, Bob, Mary, Frans, Dave, Debbie, Dietz, Mike, Marie-Thérése, Joyce, Anne, Ken, Anton, Lydia, Alfred, Liliana, Joan, Peter, Pam, Jane, Janet, and all the others for a very pleasant six years. Thanks also to Ma, Pa, Helen, and John and to my committee members Clarence Suelter, Paul Kindel, Willis Wood, Phil Filner and especially Derek Lamport. ii TABLE OF CONTENTS LIST OF TABLES 0 ....... 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0000000000000 0 0 0 0 LIST OF FIGURES. 0 0 . 0 0 0 . 0 0 0 0 0 ........... 0 0 0 ...... 0 0000000000000 0 0 LIST OF ABBREVIATIONS............. ...................... . ..... .. INTRODUCTION. . . . . . . . 0 . . ........... . . . 0000000000 O ......... . ...... PART I THE USE OF ANHYDROUS LIQUID HF T0 SELECTIVELY DEPOLYMERIZE POLYSACCHARIDES THUS ALLOWING DEGLYCOSYLATION OF GLYCOPROTEINS OR QUANTITATIVE SUGAR RECOVERIES FROM COMPLEX POLYSACCHARIDES A. IntrOdUCtion..00.0.0......0..0.....0... ..... . ..... 00.0....000 1. The Use of Anhydrous HF in Protein and Carbohydrate Chemistry.....0...........0.00...00........00.00....000.00 B. Materials and MethOdSoooooooooooo0.000000000000000.oooooooooo 1. Materials................... ..... ..... .......... .. ..... ... 2. Handling of Anhydrous Liquid HF........................... a. Description of HF Line................................. b. HF Solvolysis.......................................... 3. Analytical Methods........................................ a. Liquid Chromatography.................................. b. Gas Liquid Chromatography.............................. C. Mathematical Ca1cu1ations.............................. C0 Results00...0.00........0........0000.000.000.000..00 00000 .00 1. Initial Deglycosylation Experiments....................... a. Deglycosylation of Glycopeptides from Cell Walls by Periodate and by HBr in TFA............................ b. Attempted Solubilization of Intact Extensin by HBr in TFA............................................. c. Deglycosylation of Ovomucoid by HF..................... iii Page vi viii xi 10 12 l7 17 18 18 19 21 21 21 3O 31 31 31 32 32 TABLE OF CONTENTS--continued 2. Use of HF for the Depolymerization of Polysaccharides for the Quantitative Recovery of Monosaccharides............. a. Arabinogalactan....................................... b. Tomato Cell Walls..................................... c. Chitin................................................ 3. Use of HF for Deglycosylation of Glycoproteins and Glycopeptides............................................ a. Cell Wall Glycopeptides............................... b. Soluble Extracellular Hyp-Rich Glycoprotein........... c. Fetuin................................................ d. Pig Submaxillary Mucin................................ 4. Evidence for the Selectivity of HF....................... a. Gel Electrophoresis of BSA and Fetuin Before and After Treatment............................................. b. Retention of N-Acetyl Groups by N-Acetylglucosamine After HF Treatment.................................... 0 DiscuSSion..0..00.0...0..0.00.00.00.............0.00..0..0.0 PART II SOLUBILIZATION AND CHARACTERIZATION OF CELL WALL PROTEIN GLYCOPEPTIDES BY NaClO2 OXIDATION IntrOduction. ..... ...00.000.0.00......0...0......0.0....000. 1. The Use of NaClO in the Carbohydrate Chemistry of Wood.. 2. other uses Of Na 102...00.0.00..0.......0.00...0......000 B. Materials and Methods........... .......... ........ ..... ..... C. 1. General Materials and Methods............................ 2. Extraction of Pectins with Ammonium Oxalate.............. 3. NaClO Treatment of Cell Walls........................... 4. Alkaline B—Elimination................................... 5. CsCl Gradient Centrifugation........................ ..... 6. Methylation Analysis..................................... Results.....0...0......0.......0.......00.......0...0.0.000. 1. Solubilization of Hyp-Rich Material from Tomato Cell Walls by NaClOz Oxidation................................ 2. Analysis of the Residual Cell Wall....................... 3. Changes in the Peptide Portion of Proteins During NaClO2 Oxidation................................................ iv Page 37 37 37 46 46 49 51 55 57 62 62 62 68 8O 81 85 86 86 87 87 88 88 89 91 91 93 98 TABLE OF CONTENTS--continued Page a. A Model Cell Wall Glycopeptide................. ....... . 98 b. Lysozyme............................................... 100 c. Comparison of the Effects of NaC102 and N-Bromo- succinimide............................................ 100 4. Fractionation of the NaClOz Released Peptides by Size..... 103 a. Chromatography on Bio-Gel A .5 m and A 1.5 m........... 103 b. Chromatography of the High Molecular Weight Peptides after HF Treatment..................................... 107 5. Co-purification of Hyp-Containing Peptides with Uronic Acids..................................................... 107 a. Co-chromatography on DEAE Cellulose (Anion Exchange)... 110 b. Chromatography on Ultrogel AcA 22...................... 119 c. Precipitation with Alkaline DMSO....................... 119 d. Isoelectric Focusing................................... 122 e. CsCl Density Gradient Centrifugation................... 122 6. B-Elimination of Polysaccharides from Chlorite Solubilized Glycopeptides............................................. 134 7. Partial Characterization of the Lower Molecular Weight GlyCOpeptides............................................. 138 8. Methylation of Glycopeptides.............................. 140 a. Large NaClO -released Glycopeptides.................... 146 b. Smaller Molecular Weight Peptides...................... 152 D. D18cuss1°n..... .............. .00000... ..... . ...... .... ..... .0 157 BIBLIOGWHY000...00..........0................000000.00....0... 169 LIST OF TABLES TABLE 1. 10. 11. 12. 13. The Effect of HF on the Inhibitory Activity of Ovomucoid... Recovery of Sugars from 19.7 mg Larch Arabinogalactan: A Comparison of HF Solvolysis, Methanolysis and 2N TFA HydrOlYSiSooooooooooooooooooooooooooooooooooooooo00.000...- Sugar Recoveries from Tomato Cell Walls: Comparison of TFA Hydrolysis and HF Solvolysis............................... Amino Acid Composition of Tomato Cell Walls Before and After”Treatment...0.000.000.0.....0.........0...0.0..00. Sugar Recoveries from Tomato Cell Walls: Comparison of Methanolysis and HF Solvolysis in the Presence of Methanol. . Sugar Composition of Cell Wall Glycopeptides Before and After HF Solvolysis at O C. for 1 Hour..................... HF Deglycosylation of a Soluble Hydroxyproline-Rich Glyco- protein Secreted by Sycamore-Maple Cultures................ Sugar Composition of Fetuin Before and After HF Solvolysis forluour at0C.............0...0....0..00....00........0 Sugar Composition of Fetuin Before and After HF Solvolysis forBHours at 23 C0.0..000...00....000.000.000.00000..0000 Amino Acid Composition of Fetuin Before and After HF Solvolysis for 1 Hour at 0 C............................... Sugar Composition of Pig Submaxillary Mucin Before and Afterm‘SOlVOlySiSO0........0.0.........0..00.0...0.00.000 Amino Acid Composition of PSM Before and After HF $01v01y818f0r3noursat 23C.........0.....0..000...0.... HF Cleavage of Various Linkages Listed in Order of Increasingly Severe Conditions............................. vi Page 33 38 4O 43 45 50 54 56 58 59 61 63 73 LIST OF TABLES--continued TABLE 14. 15. 16. 17. 18. 19. 20. 21. Amino Acid Analysis of Cell Walls After Various sequential Treatments......000.000.00.0000000000000 ..... 0.. Sugar Composition of Tomato Cell Walls After Various sequential Treatments000000000000.00.........000000...0.0.0 Amino Acid Analysis of Fractions Obtained by HF Treatment from the Cell Wall Residue after NaClO2 0xidation.......... Amino Acid and Sugar Analysis of the NaClO2 Extract Eluting in the Void Volume of an A 1.5 m Column.................... Amino Acid Analyses of the Pooled Fractions Rich in Hyp on DEAE cellu108e0....0.0...0.....0.000000.00.00.00.0000000000 Sugar Composition of Various Fractions from Cesium Chloride Gradient8000000000000000..0.00.0.0..........0.0000 Sugar Composition of Hyp-containing Fractions Before and Mter B-Elimination000000000000000.00...0000.00.00.00000000 Comparison of Amino Acid Analyses of the Various Sized Fractions Obtained from Cell Walls by Sodium Chlorite Extraction0000...00000000000000.0000...00000000000000.0000. vii Page 94 95 99 106 117 133 139 143 LIST OF FIGURES FIGURE 1. 10. ll. 12. 13. 14. A model of the primary cell wall of dicots................. Gas liquid chromatography of amino acids as their hepta- fluorObutyry1iSObUtYlesterSoo00000000000000...ooooooooooo Amino acid analysis by liquid chromatography............... Gas liquid chromatography of sugars as their trimethylsilyl methyl glyc081de800000000000.0.0000000000000000000...000000 . Gas liquid chromatography of sugars as their alditol acetates.............0..00.0.00...0......0....00. The elution profile of ovomucoid on Sephadex G-50 before and after a 1 hour HF treatment at 0 C..................... Cell walls of suspension cultured cells before and after HF treatment0000......0.000.000.000000000000.00.00.00.00....00 Elution profile on Biogel P-2 of the fraction of HF treated crab shells which was soluble in water............. Gel filtration of a hydroxyproline-rich glycoprotein after HF solvolysis: Sephadex G-100 elution profile............. SDS gel electrophoresis of bovine serum albumin before and afterHFtreatment0000..00000.0000000000000000000000.0.000. SDS gel electrophoresis of fetuin before and after HF treatment00000........0....0....00.0.00......0............. The mass spectra of authentic and HF-treated N-acetyl glucosamine as their trimethylsilyl derivatives............ Guide to fractionation procedures.......................... Sephadex G-50 chromatography of the soluble portion of the HF treated residue of NaClOz treated tomato cell walls..... viii Page 23 25 27 29 36 42 48 53 65 67 70 92 97 LIST OF FIGURES-~continued FIGURE 15. l6. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Sephadex G-50 elution profile of NaClO2 treated lysozyme............00...0..0.................0.... ....... Chromatography of total NaC102 extracted glycopeptides on Bio-081A05m0000000.0000.00000...000.000.000.000.00000.0 Sephadex G-50 elution profile of the higher molecular weight glycopeptides off an A .5 m column after HF deglycosy18tion000000.00.0...0000000000.0.0.00000000000000 Elution profile of A .5 m void on DEAE cellulose.......... Elution profile of A .5 m void on DEAE cellulose.... ..... . Rechromatography of the Hyp-rich peak on DEAE cellulose using a shallower gradient................................ Chromatography of the Hyp-rich peak obtained from a DEAE cellulose column on Ultrogel AcA 22....................... Isoelectric focusing of NaClO2 solubilized tomato wall peptides000000..0000000.0000000000000000000000. 00000 0.0000 CsCl density gradient centrifugation of the major fraction of Hyp on a DEAE cellulose column......................... CsCl gradient centrifugation of bulk pectin extracted from tomato cell walls by boiling ammonium oxalate............. CsCl density gradient centrifugation of the trailing edge of the Hyp-rich peak on DEAE cellulose.................... Elution profile on Sephadex G-50 of the soluble portion of the B-eliminated Hyp peak from a DEAE cellulose column.... The elution profile of the NaClOz solubilized peptides retained by a Bio-Gel A .5 m column on Sephadex G-SO...... Chromatograph of the smaller glycopeptides solubilized by NaClOZ on DEAE ce11u108e0.0000000000000.00.00.00.00000000. Methylation analysis of the less dense Hyp fraction from aCS‘Cl deDSity gradient.......0....0....0.00....0.00.00... ix Page 102 105 109 112 114 116 121 124 128 130 132 137 142 145 148 LIST OF FIGURES--continued FIGURE Page 30. Methylation analysis of the denser Hyp fractions from a CSCl gradient0000...........0.00..000000.000000000.000000. 151 31. A comparison of the pectinaceous fraction of a DEAE cellu- lose column effluent with the washed ammonium oxalate extract from tomato cell walls............................ 154 32. Methylation analysis of the smaller molecular weight peptideSO00000000000000......000......0...0.00.0...00 00000 156 33. The appearance and methylated sugar composition of sycamore cell wall-S000...00.000000.......0000....0000000.0 161 34. The appearance and methylated sugar composition of tomato cell wa11800..0..0..............0.......0..0.0.0.0....0... 163 LIST OF ABBREVIATIONS BSA DMSO GalU GLC Hyp Hyp-ara, Hyp-arabinoside NGNA PSM RNase SDS TFA TMS bovine serum albumin dimethylsulfoxide galacturonic acid gas liquid chromatography hydroxyproline a short chain of arabinose (1-4) on the hydroxyl group of Hyp N-Glycolylneuraminic acid pig submaxillary mucin ribonuclease sodium dodecyl sulfate trifluoroacetic acid trimethylsilyl INTRODUCTION To understand the molecular mechanism of cell wall growth we must know which polymers are present in the walls and how they are linked together. There have been a multitude of physiological experiments. showing that auxin, gibberellic acid, and even low pH (3) stimulate the elongation of stems, roots, and other organs of a wide variety of plants. There have been theories that these growth promoters are act- ing by methylation of the pectins,induction of a growth limiting protein,stimulation of glycosidases or activation of a proton pump which will acidify the cell wall and therefore activate glycosidases or cleave some acid labile bonds (4). Of these theories only the proton pump activation mechanism is generally accepted, but neither a linkage susceptible to mildly acidic conditions nor an enzyme activated by low pH capable of labilizing cell wall cross-links has been found. Until recently, these theories were all based on physiological evidence because a detailed working model of the primary cell wall (the thin, expandable wall of growing cells as opposed to the thickened, lignified, rigid secondary cell wall) was not available. Recently, from their own experiments with degradation of cell walls of sycamore suspension cultures using purified enzymes, the observation of Aspinall.g£_§l, (S) that xyloglucan polymers hydrogen bond strongly to cellulose, and the large pool of plant polysaccharide structures determined over the past century, Keegstra g£_§l. (2) have proposed a model for the cell walls of sycamore suspension cultures, which Albersheim suggests is representative of primary cell walls of all dicots (6). A diagram of the proposed structure is given in Figure 1. In this model the cellulose microfibrils are cross-linked by the matrix polymers of the wall, the direct link to the fibrils being by hydrogen bonding of a xyloglucan. The xyloglucan is linked at its reducing end to a 4-linked galactan which is then linked to the rhamnogalacturonan of the wall. From the reducing end of the rhamnogalacturonan is a 3,6-linked arabinogalactan which is glycosidically linked to serine residues of the cell wall protein. The centrally located protein has been characterized almost exclusively by Lamport and his collaborators. While studying the composition of walls prepared from suspension cultures of sycamore maple cells, Lamport found that these walls (7), and later that most plant cell walls (8), contained a protein rich in hydroxyproline (Hyp), an amino acid formed by post translational hydroxylation of proline. This amino acid has never been found in an enzyme, but is frequently found in the structural proteins of connective tissue (throughout the animal kingdom). Thus it is reasonable to propose that the Hyp- containing protein in cell walls is a structural protein, especially as it is by far the major component of some cell walls, such as those of Chlamydomonas (9). Lamport and his colleagues have partially characterized this Hyp-rich protein in the cell walls of tomato cell Figure l. A model of the primary cell wall of dicots. Reproduced with permission from Keegstra et a1. (2). cellulose elementary fibril a} loglucan wall protein with arahinosyl tetrasaccharides glchsidically attached to the hydroxyproline residues total pectic polysaccharide Figure l rhamnogalacturonan main chain of the pectic polysaccharide arabinan and d-linlted galactan side chains of the pectic polymer 3.6linked arabinogalactan attached to serine of the wall protein unsubstituted seryl residues of the wall protein suspension cultures. In tomato and other plants in which Hyp has been found there is an oligosaccharide attached glycosidically to the hydroxyl group of most of the Hyp residues (8). These Hyp-oligosaccha- rides are easily prepared by alkaline hydrolysis of intact cell walls (1). In higher plants the Hyp-oligosaccharides are almost exclusively composed of arabinose and are from 1 to 4 residues long (8). The protein has not been solubilized intact from walls of cells from suspension cultures but can be partially extracted by crude cellu- lase, pronase (10) or trypsin (11). For solubilization with trypsin the walls must be pretreated with acid, refluxing at pH 1 for 1 hour, before an appreciable fraction of the Hyp can be released. This acid treatment removes the arabinose residues from the Hyp and a fraction of the pectin, perhaps indicating that the arabinose side chains protect the protein from proteolysis by invading pathogens. The shorter pep- tides released by a combined pH 1 treatment followed by digestion with trypsin have been sequenced (11) and all contain the remarkable sequence Ser-Hyp-Hyp-Hyp-Hyp, the longer peptides (15 amino acids long) contain- ing the sequence twice. Larger tryptic peptides, although not yet fractionated and sequenced, are also extremely rich in Hyp, indicating that they too probably will contain many repeats of the sequence Ser-Hyp4. As polyproline and polyhydroxyproline each form a 3 residue- per-turn left-handed helix (12), it is possible that extensin also could take a helical conformation which would cause it to be a rigid rod and thus play a structural role in the wall. Originally Lamport suggested that primary cell wall consists of cellulose fibrils cross-linked to the cell wall protein via the hemi- cellulose and the side chains of arabinose on the Hyp residues (13). For this model to be plausible one has to postulate that the cross-links from the ends of the arabinose chains to the hemicellulose are alkali labile because nothing has ever been found attached to the arabinose- containing oligosaccharide (prepared by alkaline hydrolysis). Methyla- tion analysis of sycamore walls (a method also involving strong base) showed many terminal arabinoses consistent with there only being short arabinose chains on the Hyp or an alkali labile substituent. Glycosidic linkages to sugars are alkali stable. In the model of primary cell wall proposed by Keegstra g£_§l, an arabinogalactan cross-links the protein to the pectins of the wall. The protein—arabinogalactan linkage was supported by Lamport g£_al, (14) who found that most of the serine residues in the cell wall tryptic peptides were galactosylated, but by a single galactose. If the sugar attached to this galactose were a furanose, it would be cleaved during the pH 1 treatment used to allow trypsinization. Thus the galactose could be the start of the arabinogalactan of Keegstra.§£_al. The evi- dence (2) that the hypothetical polysaccharide is an arabinogalactan is rather indirect. The data are mostly from a protein, found in the medium bathing the cells, which was thought to be similar to the protein in the cell wall. Pope (15) found later, a polysaccharide in this extracellular protein attached to the hydroxyl group of Hyp. This is most probably the one which.was presumed to be linked to serine by Keegstra g£_§l. (2). In addition, the Hyp-arabinoside composition of the extracellular protein is unlike that of the cell wall, implying that the extracellular protein is not extensin. The role of the Hyp containing protein extensin in the cell wall still remains to be discovered, and therefore the possibility that it is involved in cell wall loosening, the prerequisite for wall extension or growth, remains reasonable. At the start of this thesis research I was hoping to determine the size of the polypeptide portion of extensin. As the protein had only been extracted from walls after extensive degradation by acid and trypsin, the longest characterized tryptic peptide consisted of only 15 amino acids. To determine the size of the polypeptide portion of the protein I wished to solubilize the cell wall selectively by cleaving glycosidic linkages, leaving the peptide bonds intact. Sodium periodate was not very effective in labilizing glycosidic linkages under conditions in which there were minimal effects on the protein. Dr. Roy Vagelos suggested in 1972 that I try using 60% hydro- fluoric acid, a reagent he had used to remove phosphopantotheine from acyl carrier protein (16). He had also used anhydrous HF to remove protecting groups from synthetic acyl carrier protein (17). As the apparatus for removing 60% aqueous HF from proteins was not readily available nor was 60% HF (commercial HF is 40%), I tried several of the reagents used by synthetic peptide chemists (18). Later, I found that 60% HF would not have worked. The first I tried was HBr gas in tri- fluoroacetic acid (TFA). It worked very well on tryptic peptides of cell wall protein, but on treatment of the entire cell wall there re- mained an insoluble residue which contained all of the protein. No non- destructive solubilizing agent solubilized the protein. As plants are notorious producers of phenolic compounds, I thought that these could be complexing with the protein to render it insoluble during the HBr solvolysis. In an effort to avoid interference by phenolics after I had used various known methods to reduce phenolic contamination with no better results, I tried to selectively oxidize the phenols with sodium chlorite, a compound often used for delignification of wood by carbo- hydrate chemists. This reagent bleached the walls from their pale or dark brown color to a brilliant white and then, in some cases, allowed total dissolution of cell walls with HBr in TFA. On analysis of the walls after NaClO2 oxidation I realized that the oxidation itself was partially solubilizing the walls and was especially effective at solu- bilizing the protein. As NaClO2 oxidation is used by carbohydrate chemists especially for its non reactivity towards sugars, I devoted much time to the study of the protein released by chlorite oxidation hoping that it would still be attached to polysaccharides. Since NaClO2 does react with proteins (it destroys tyrosine) my initial goal to solu- bilize intact extensin was not achieved, again, but a new approach was opened to the determination of how extensin is linked to the cell wall. Having been so successful in deglycosylating cell wall peptides and cell walls with HBr in TFA, I extended this chemical method to all glycoproteins using an even more effective deglycosylating agent, anhydrous liquid hydrogen fluoride. Thus from one problem, as yet unsolved, two others arose. Therefore, this thesis is in two parts: Part I--The use of anhydrous liquid HF to selectively depolymerize polysaccharides thus allowing deglycosylation of glycoproteins or quantitative sugar recoveries from complex polysaccharides, and Part II--Solubilization and characteriza- tion of cell wall protein glycopeptides by NaClO2 oxidation. PART I THE USE OF ANHYDROUS LIQUID HF TO SELECTIVELX DEPOLYMERIZE POLYSACCHARIDES THUS ALLOWING DEGLYCOSYLATION OF GLYCOPROTEINS OR QUANTITATIVE SUGAR RECOVERIES FROM COMPLEX POLYSACCHARIDES A. Introduction In the study of cell wall protein it is often inconvenient to work with glycosylated peptides. Variation in glycosylation can cause heterogeneity of a single peptide species and thus complicate its characterization. Lamport (11) found it necessary to remove the arabino- sides from the Hyp residues of cell wall protein to make the protein in_ gigg susceptible to trypsin. During this deglycosylation, a pH I treat- ment of 100°C for 1 hour, it is likely that some acid labile peptide bonds, e.g., at aspartic acid, are cleaved and about one-third of the cell wall protein is solubilized in a form which is not susceptible to trypsin. Thus an effective way to deglycosylate glycoproteins and glycopeptides was needed to simplify the characterization of extensin. If the cell wall protein were linked in the cell wall matrix by only glycosidic linkages (as proposed by Keegstra g£_gl. (2)) then a method to break glycosidic linkages specifically should allow extraction of soluble intact cell wall protein and thereby make possible determina- tion of the size of extensin. Plant cell walls contain only neutral and acidic sugars but many glycoproteins contain amino sugars and so testing the general 10 ll applicability of any deglycosylation method should also involve testing of protein containing amino sugar linkages. The classical techniques of degrading polysaccharides under con- ditions which do not affect peptide bonds have serious drawbacks. The commonly used Smith degradation (l9) involves periodate oxidation which also affects tyrosine residues and terminal serine residues. Usually periodate oxidation must be performed repetitively to remove all the sugars from a glyc0protein. In the case of the cell wall protein, extensin, the reaction with terminal arabinose residues is exceedingly slow. Enzymatic deglycosylation involves isolation of protease-free enzymes capable of hydrolyzing each particular glycosidic linkage present. Therefore, it would be advantageous to have a method which cleaves all glycosidic linkages nonspecifically but leaves peptide bonds intact. Synthetic chemists often have the need to protect sensitive func- tional groups during reactions and so many easily removable protecting groups have been developed. In peptide synthesis this approach must be carried to great sophistication. It is necessary to have many different functional groups protected yet to be able to unmask some of them selec- tively to allow continuation of the addition of amino acids to the pep- tide undergoing synthesis. At the end of the synthesis all the protect- ing groups must be removed to give a peptide indistinguishable from the biosynthesized one. HBr in TFA and anhydrous liquid hydrogen fluoride are both examples of reagents which are used to perform the final step of completely removing all the protecting groups of chemdcally synthe- sized peptides. 12 After only partially successful initial attempts to deglycosylate cell wall glycopeptides via periodate oxidation I tested the most promising synthetic peptide deprotecting agent which could be used with- out specialized equipment, HBr in TFA. This reagent was effective with both cell wall glycopeptides and intact cell walls. Anhydrous liquid hydrogen fluoride, although dangerous, is more convenient to use than HBr, given the proper equipment (see methods), is a better solubilizing agent than TFA, and can be used at lower temperatures thus giving a better possibility of retaining enzymatic activity of glycoproteins to be deglycosylated. Lysozyme and ribonuclease (20) have already been treated with HF with retention of the majority of their enzymatic activity. Consequently, a Kel-F hydrogen fluoride line (approximately $3000.00) was purchased. The effects of HF were first tested with cell walls and the glycoproteins ovomucoid and ovalbumin. Ovomucoid is not a good model glycoprotein as it is not well-characterized and ovalbumin did not redissolve after HF treatment. In later experiments, the well- characterized glycoproteins fetuin and pig submaxillary mucin, the polysaccharides arabinogalactan and chitin, and cell wall glycopeptides were used to determine the specificity and effectiveness of HF in cleav- ing various glycosidic linkages. l. The Use of Anhydrous HF in Protein and Carbohydrate Chemistry The solvating properties of anhydrous liquid HF have been known for many years. As early as 1869 Gore (21) reported that paper, gum arabic, India rubber, sealing wax, many other organic and inorganic 13 materials dissolve rapidly in liquid HF. Fredenhagen and Cadenbach (22) suggested that the dissolution of the polysaccharides of wood with liquid HF would allow quantitation and recovery of the lignin which is insoluble in HF. Fredenhagen.(23) also found that casein, hemoglobin and nucleic acids are soluble in liquid HF. Katz (24) extended the solubility studies of HF to amino acids and a number of proteins. Amino acids are extremely soluble in HF, e.g., 30 gm/lOO gm for trypto- phan at -78 C, as are globular proteins, e.g., 350 mg/ml for bovine serum albumin at 0 C. After removal of the HF by evaporation under vacuum about half of the proteins tested became water insoluble. Bovine serum albumin, one of the proteins which became insoluble, remained water soluble if aspartic acid, lysine, glycine or hide collagen was added to the HF solution. Katz suggested that HF could be used as an excellent solvent for protein chemistry due to the wide range of compounds which it can dis— solve in high concentration. In subsequent experiments, Koch g£_§l. (25) found that ribonuclease retained its nuclease activity for at least 2 hours in HF at -78 C. At 0 C for 2 hours a variable proportion of the activity was lost (up to 66%). After 2 hours at 30 C ribonucle- ase had essentially lost its enzymatic activity. Lysozyme under similar conditions lost its activity somewhat more rapidly. Koch g£_§l, esti— mated the degree of peptide bond breakage under various conditions by the colorimetric determination of amino groups. After 2 hours at 0 C there was no increase in amino groups in ribonuclease and a slight increase (less than one per molecule) for lysozyme. After 2 hours at 14 30 C there was an increase of approximately 2 in ribonuclease and 1.5 in lysozyme. After 24 hours at 30 C ribonuclease had 6 additional amino groups. No disulfide interchange was found after 24 hours at 30 C. However, peptide bond breakage need not account for the appear- ance of all the new amino groups. Sakakibara §£_§l. (26) found a slow N to 0 acyl shift of the pep- tide bond to serine and threonine residues at room temperature which was about 95% complete in glycyl-DL-serine and glycyl-DL-threonine after 15 days in HF. Lenard and Hess (27) have applied the N to 0 shift to the specific cleavage of oxidized insulin A chain and a-melano- cyte stimulating hormone, since the resulting ester bond can be easily cleaved in mild alkali. The shift was accelerated by using HF at 30 C, thus the N to 0 shift was 65% complete after only 13 hours. This shift could be totally reversed by allowing the peptide to stand in sodium bicarbonate solution overnight or by the addition of pyridine. During the HF treatment internal ester formation between serine hydroxyl groups and carboxyl groups of insulin prevented the acyl shift from going to completion. This could be overcome by adding 25% methanol to the HF. The methyl esters so formed could be cleaved in aqueous piperidine at 0 C. The inclusion of methanol allowed the shift to go to 85% comple- tion in 12 hours at 30 C with negligible peptide breakage. Paper electrophoresis and amino acid analysis of insulin before and after methanolic HF treatment, piperidine reversal of N to 0 shift and methyl ester cleavage showed that the treated peptide was indistinguishable from the starting material. To cleave a rearranged peptide, the amino 15 groups must be acylated to prevent the reversal of the N to 0 shift during the alkaline treatment which specifically cleaves the amino acid ester link to serine. This is easily accomplished by formylation. After formylation the rearranged peptide is cleaved by an aqueous 1 M piperidine treatment which also cleaves the methyl esters used to allow a high yield of N to 0 shift. Eighty to 85% cleavage was reported for a-melanocyte stimulating hormone. The removal of the formyl group was not investigated, but has been reported by Sheehan and Yang (28). The only peptide bond known to be cleaved by anhydrous HF is that of methionine. Lenard g£_gl. (29) found that methionyl-glycine was completely cleaved after 36 hours at 30 C in HF, yet after oxidation of the methionine to its sulfone the dipeptide was completely stable. The cleavage of methionyl bonds in peptides and proteins is much slower. From the results described above Sakakibara and Shimonishi (30) proposed that HF would be an excellent reagent for the acidolysis of the protecting groups used during peptide synthesis. Results obtained with the fully protected nona-peptide of oxytocin proved them to be correct. After 1 hour in HF at room temperature and oxidation of the cysteine residues in air, the yield of biologically active peptide was 36-40%, a 4-fold improvement over previous deprotection methods. Lenard and Robinson (31) found that HF could also be used to cleave a protected synthetic peptide from the resin used in the Merrifield solid-phase method of peptide synthesis allowing cleavage from the resin and depro- tection in one step. l6 Hydrogen fluoride is now the method of choice for complete depro- tection of synthetic peptides and has proved its usefulness in the synthesis of many peptides. The reactions of polysaccharides in liquid HF have not been investigated to any great extent. Fredenhagen and Cadenbach (22) found that cellulose was depolymerized quantitatively to glucosyl fluoride in HF at room temperature, but that unless the HF was removed rapidly and in the cold, the product was a polyglucan. Helferich g£_al. (32) obtained similar results with starch and maltose. Thus it appears that the glucosyl fluorides formed in HF reoligomerize to a degree dependent on the concentration of sugar in the HF. Monosaccharides form glycosyl fluorides when they are dissolved in liquid HF whether or not the hydroxyl groups of the non-reducing carbon atoms are acylated. Penta-O-acetyl or -0-benzoyl glucose gives a high yield of the corresponding 2,3,4,6 tetra-O-acetyl or -0-benzoyl glucosyl fluoride in HF at room temperature for 10 minutes (33). But when the reaction is prolonged to a number of hours there is often inversion at the 2-carbon and ring contraction. The NMR spectrum of cis 1,2-diacet- oxycyclohexane in HF indicates that these side reactions may proceed via a cyclic 1,3 dioxolenium ion (34). This cyclic intermediate did not form with trans 1,2 diacetoxycyclohexane. In the absence of ester linkages to the sugars these side reactions should not take place. The reactions of sugar fluorides differ considerably from those of other glycosyl halides. The fluorides are much less reactive than the bromides or chlorides and so, are not used in glycoside synthesis. l7 Glucosyl fluoride is even stable in aqueous solution at neutral pH. Glucosyl fluoride can act as a substrate, in place of sucrose for a glycosyltransferase from Streptococcus mutans (35). When glucosyl fluorides are mixed with aromatic compounds in HF, a Friedel-Crafts reaction takes place with the C-1 of the sugar alkyl- ating the aromatic nucleus (36). B. Materials and Methods 1. Materials Ovomucoid, ovalbumin, bovine serum albumin, fetuin, and crab shells were bought from Sigma Chemical Co., St. Louis, Mo. and larch arabinogalactan from K and K Laboratories, Inc., N. Y. The cell wall tryptic glycopeptide, gei-Hyp-Hyp-Hyp-Hyp-ge: Hyp-gzi-Hyp-Hyp-Hyp-Hyp-Unk-Tyr-Lys was prepared by Katona and Lamport (11); Pig submaxillary mucin, prepared according to the method of de Salegui and Plonska (37) but without separating the major and minor fractions, was a gift from Joseph Sung (Michigan State University). Tomato cell suspension cultures were grown in a modified White's medium (38) under continuous light on a rotary shaker at 100 RPM and harvested 4 weeks after subculturing. Walls from suspension cultured tomato cells were prepared by homogenization of the cells in ice with a Super Dispax DS 45 homogenizer (Tekmar Co., Cincinnati, Ohio) for 2 minutes at maximum speed (10,000 RPM) in the presence of a small amount (N2 g/500 ml) of dithionite to 18 prevent oxidation of phenols. This was followed by a 2 minute sonica- tion of 150 ml aliquots at full power (300 watts) with a Bronwill Biosonik III (Bronwill Scientific, Rochester, N. Y.). Aliquots of about 200 ml of wall preparation were then extensively washed on a coarse fritted glass funnel with cold water until the filtrate was clear. Two or more times during the washing procedure the cell walls were removed from the funnel and the fritted glass filter unclogged by hot concen- trated sulfuric acid. The walls were then washed with 5 volumes of 1M NaCl followed by 10 volumes of distilled water. The combined washed cell wall fractions were then washed extensively with acetone and left to dry overnight in a vacuum desiccator at 60 C. Sycamore-maple suspension cultures were grown in M.E medium (39) 6 but with yeast extract which had had most of its macromolecules removed by ethanol precipitation to allow recovery of extracellular sycamore- maple polysaccharides and proteins uncontaminated by yeast polymers. GLC column support, Gas Chrom Q, and coating materials SE 30 and SP 2100 were bought from Supelco Co., Bellefonte, Pa. The alcohols used in gas chromatography were dried by refluxing over magnesium turn- ings and iodine as described by Bhatti et a1. (40). All other reagents were used without further purification. 2. Handling of Anhydrous Liquid HF a. Description of HF Line Anhydrous hydrogen fluoride can be safely handled only in a closed system. The apparatus used for the experiments, purchased from Peninsula Laboratories, Inc. (P. O. Box 205, San Carlos, Ca. 94070) was l9 originally designed for deprotection of synthetic peptides by HF after their chemical polymerization. It consists of an HF cylinder, a reservoir, two reaction chambers, another reservoir, and a trap filled with calcium oxide to neutralize the HF when it is removed from the reaction vessel. These components are connected together by Kel F tub- ing and stopcocks. The outlet from the trap is connected to a vacuum pump. A mercury manometer is provided with the apparatus to indicate the course of evacuation or filling of the reaction vessels. A more complete description and diagram of the apparatus can be found in references (18,20,41). b. HF Solvolysis HF was dried over cobalt trifluoride in the first Kel F reservoir. The dried sample (5-20 mg) and 1 ml of anisole scavenger,1 if required, were added to the reaction vessel and the entire HF line, excluding the HF containing reservoir, evacuated. The reaction vessel was cooled in a dry ice-acetone bath and ca. 10 m1 HF was allowed to distill over from the reservoir. Both vessels were stirred continuously by Teflon coated magnetic stirrers to ensure smooth distillation of the HF. After introduction of the HF, the reaction vessel was allowed to warm to the desired temperature as indicated by the vapor pressure of HF measured by the mercury manometer. The vapor pressure of HF at 0 C is 364 mm Hg 1HF solvolysis generates fluorinated compounds, e.g., glycosyl fluorides, which readily alkylate aromatic amino acids, the HF acting as a Friedel-Crafts catalyst (36). Addition of a large excess of anisole protects these amino acids, by competing for the alkylating come pounds. Thus, when studying sugar recovery, the anisole must be omitted because the adducts involve a 0-0 linkage which does not permit recovery of the original sugar. 20 and at 23 C is 850 mm Hg. Timing of the reaction was started when the vapor pressure of the HF was within 5 mm Hg of that at the required temperature, as heat transfer through Kel-F vessels is very slow. An ice bath was used to keep the reaction at 0 C ; no regulation was used for 23 C , room temperature. At the end of the reaction period the HF was evaporated quickly under reduced pressure. The pressure was dropped as rapidly as possible to keep the time for which the reactants were concentrated in HF as short as possible. Fredenhagen and Cadenbach (22) found that removal of the HF from a solution of glucosyl fluoride, the product of cellulose dissolution, led to the formation of oligomers of glucose unless the solution was evaporated rapidly and in the cold. Too rapid evacuation of the reaction vessel resulted in frothing of the HF solution out of the vessel. The line was left evacuated for at least 1 hour after there were no more visible signs of HF (the anisole changed color from light brown through red to colorless) to ensure complete removal of excess HF.2 The sample was then taken up in 0.1 N acetic acid or 50% acetic acid if the residue was difficult to redissolve and the anisole extracted into ether. The product was then dialyzed or chromatographed on an appropriate Sephadex column. 2The small amount of HF which remains after this 1 hour period should not affect the protein as HF is a weak acid (pK 3.2) at concen- trations below 5M. Above this concentration HF is a very strong acid, but rather than form H3O+ +F' as it does at low concentrations, the HF dissociates to H30+ + HFZ’, H2F3-’ etc. (42). 21 3. Analytical Methods a. Liquid Chromatography Chromatography of HF treated chitin and glycopeptides which were solubilized from cell walls by chlorite oxidation was performed on a 110 cm x 0.9 cm Biogel P-2 column in 0.1 M acetic acid. Chromatography of extracellular glycoprotein which had been treated with HF was per- formed on a 2.5 cm x 85 cm Sephadex G-100 column in 0.1 N acetic acid. Hydroxyproline was determined on an automated hydroxyproline analyzer (8) after partial hydrolysis of the sample in 5 N NaOH at 121C for 1 hour and neutralization with 5 N HCl. b. Gas Liquid Chromatography GLC was performed on a Perkin-Elmer 900 or 910 Gas Chromatograph fitted with dual columns, with the output connected to a Spectra Physics (Mountain View, California) System IV Autolab integrator. Most amino acid analyses were performed on the heptafluorobutyryl isobutyl ester derivatives as described by MacKenzie and Tenaschuk (43, Figure 2), others were performed by liquid chromatography (10, Figure 3). Sugar analyses were performed on the trimethyl silylated methyl glycosides as described by Bhatti g£_§l, (40, Figure 4), or by the alditol acetate method of Albersheim g£_§l. (44, Figure 5). Glucosamine and N-acetylglucosamine were distinguished by forma- tion of their TMS derivatives in 1:1 mixture of bistrimethylsilyltri- fluoroacetamide and pyridine at room temperature for at least 2 hours and then chromatographed on a 12 foot x 1/8" I.D. 3% SP2100 column programmed from 165-200 C at 1.5 C per minute. Figure 2. 22 Gas liquid chromatography of amino acids as their hepta- fluorobutyryl isobutyl esters. One hundred nanomoles of each amino acid plus 50 nanomoles of pipicolic acid, as internal standard, were dried in a 1 ml reactivial (Pierce Chemical Co.) in a vacuum desiccator over P205 for 3 hours. Two hundred pl of 3N HCl in dry isobutanol were added and the mixture was heated to 121 C for 20 minutes. After the vial had cooled to room temperature the isobutanol was evaporated under a stream of dry nitrogen at room tempera- ture. Twenty—five pl of heptafluorobutyric anhydride and 25 pl of ethyl acetate were then added and the vial was heated to 150 C for 10 minutes. The product was evaporated to dry- ness after it had cooled to room temperature and dissolved in 25 p1 of ethyl acetate. Two pl of this solution were injected onto a 12 foot column packed with 3% SE 30 or 3% SP 2100 on Gas ChromQ held at an initial temperature of 90 C for 4 minutes and heated at 1 C per minute to 240 C. Histidine does not give a reproducible peak by this procedure. Arginine tends to decay and so, the injection of the sample should be made immediately after the completion of the deriva- tization. Identification of peaks: 1) Ala, 2) Gly, 3) Val, 4) Thr, 5) Ser, 6) Leu, 7) Ile, 8) Pro, 9) Pipicolic Acid, 10) Hyp, 11) Met, 12) Asp, 13) Phe, 14) Glu, 15) Lys, 16) Tyr, 17) Arg, 18) His, 19) Cys. Figure 3. 24 Amino acid analysis by liquid chromatography. Fifty nanomoles of each amino acid, 20 pg of Hyp and 100 nanomoles of norleucine were placed on top of a Chromo- beads C column and eluted with a combination pH, ionic strength gradient as described by Lamport (10). The eluent from the column was fed into an automated ninhydrin analyzer and the peak areas calculated by a System IV Autolab integrator. Hyp and Pro were detected by their absorbance at 440 nm, the other amino acids at 570 nm. Identification of peaks: 1) Hyp, 2) Asp, 3) Thr, 4) Ser, 5) Glu, 6) Pro, 7) Gly, 8) Ala, 9) Val, 10) Cys, 11) Met, 12) Ile, l3) Leu, 14) Norleucine, 15) Tyr, 16) Phe, 17) NH4 , 18) Lys, 19) His, 20) Arg. Detector Response 25 234 5 L ULVLUAM .iJ U .. 7a 9 111214 16 17 18 19 20 'IO is 15 ' (J 09440 1 6 |_ I 1 o 10 20 Time (minutes) Figure 3 26 .Hw mmoaHomH< .uosuo sumo mo Nmtm GHSqu hHHmams mums moumoHHnan .mmHaamm us» on how mama onu some mums mpumooMum mm waoH om poow hwo> was mw>Huw>Huop omozu mo huHHHaHosvounom .o mHN ou oussHE Hon 0 H mo Hammond ououmuomeou w %n wo30HHom mousoHa c wow u ocH mo ouaumumoawu HmHuHsH am nuHs man .meom oaHEm mo mm>Hum>Humv Hhuhusn touoonmuamn can now pmnHuommv mm :EaHoo mama onu so pmuoofiaH no: :OHuSHom ago mo H: ose .aoHumuHum>Huov onu Mom cosoHHm we? “so: on HHQ: .msHvHuhmtosmHHmoHononsumanutmsmsoHHmeHksuomeo: mo musuxfia A>\>v muHuH u no H1 mm waHovm up vmumHhHHmthumaHuu away was meson m ammoH up you mon uo>o uoumoonov aooom> m sH poomHe pom woansoo mums mucoumaumaom any .Hoamnuma hat aqu moHsu cosmos can aOHummsm tHuuswo an po>oaow mm3 ovHuoHno\oumcooumo um>HHm ago mason o woum< .oovvm ovHuvhnam oHuoom mo H: 00H omnu one commoHou mos moo whoa o: HHus: mHmH> onu ou cocoa mm3 duodenumo uo>HHm .muao: NH wow 0 cm on wound: mHmH> mSu pom venom was Hosmauma haw sH Ho: 2 m.H mo Ha one .mxmmH mo comm kHucoumHmaoo oHoB mHmH> Hosuo oz .wumwom m£u mo :oHumNHHmamumo cam Ho: onu mo :oHu Imuucmocoo ou wavaoH poxme coumo mHMH> may amxmu uo: mm3 aOHuomomua mHsu NH .uon HHHum oHHaB vosmustu soon was HmH> human am no Mao: H How 0 ow ou wcHummn ho poGOHquaoo soon hHmooH>oua pm: soHns A.oo HmoHaono wouon NHQNH wav woumcHamH Hannah :onoHv umaHH :onoH m up wwomHmmw pom po>oamu mos HMH> onu mo moo oSu :H mep woman may .musoz m ammoH um How mon uo>o woumoonov aaaom> w sH AHIOHmoc oHnEHMV HMH> soup H m GH voHuv mums oumoamum HmaumuaH mm HouHacmE mo mmHoanmc OOH msHm Hmwom some mo meoEocmc pompous moo .mmemoome thuoa HhHHmHhcumaHuu “Hana mm mumwam mo mnamuwoumaouno oHovHH one .Q muame 27 on 7)! i J; e wuome AmmuacHav maHH oe on On an :33... ha 4 a. 9 32a. :9- 3 3 D ON C 5.000“. l asuodsau zoaoanaa 28 .Houemoee Am .UHo He .Hme Ac .emz Am .Hax as .mu< Am .osm Am .mam Ha "mxmom mo aOHumoHuHusovH .ouosHa use 0 H as o owH ou vmaEmuwoua mmuooHE c you 0 qu um omHHImx Nq.o can AoumcHoosm Hoosz osmthumv aHoa NN.o .Aoumnva HoohHw wslonuovaoo N~.o mo nazHoo m :o vmuomth one? H: oau ou moo .uson H How owHuomncm oHumow mo H: on sH o HNH ou wsHumma mp poNHuw>Hump smzu was mason m you mon Hm>o noumoonmv aosom> m aH voHHp was ngme maH .oHQamm mnu aouw Hosmnuwa mo soHumuoam>m pmummaou hp po>ofiwu mma dunnon wmmoxo 0:9 .mmoshuv ou vmumuomm>o swzu pom vHom oHuoom HmHome mo macho MIN :uHs vaHHmuuowa mos mHsu use: H noum< .pmppm mmB momz 2m aH «mmmz mo GOHuaHom Ha\wa om m mo H: on pom .poumnomm>m mm3 Huommu HE H m :H vaup mm3 umwow zoom mo mmHoaoso: coupon; moo .mmumumom HoquHm uHosu mm madman mo mammumoumaowno wHodHH moo .m muame 29 oq m wuame Amousswav uaHa ON ‘ d asuodsag Joaoanaq 30 c. Mathematical Calculations To allow rapid calculation and recalculation of sugar and amino acid compositions I wrote a small FORTRAN IV program for a Hewlett Packard 2100 MX computer. This program is capable of accepting any number of peaks corresponding to a particular amino acid or sugar and applying appropriate correction factors to calculate amount present in the sample by comparison with a standard. The program can also normal- ize to a particular number of residues of an amino acid to allow easy estimation of an empirical formula for a protein or peptide. Amounts of polypeptide were calculated from the sum of the number of micrograms of each amino acid present in the hydrolysate of an aliquot of the protein after 18 hours at 110 C in constant boiling HCl. As histidine, cysteine and methionine could not be determined reliably by the gas chromatographic method of MacKenzie and Tenaschuk (43), they were not included, and, consequently the result was not a true poly- peptide weight. Amounts of sugar in glycoproteins were determined by summing the number of micrograms of each sugar in an aliquot of the protein after 12 hours of methanolysis as determined by GLC of the TMS sugar derivatives (40). Both the amount of polypeptide and of sugars were corrected to exclude water added to each residue of amino acid or sugar during the hydrolysis. Carbohydrate contents of the glycoproteins fetuin and PSM are expressed as mg sugar residue per 100 mg polypeptide. Thus, final mg sugar/1004mgideglycosylated polypeptide Z deglycosylation: 1!- initial mg sugar/100 mg untreated Polypeptide 31 where weights of polypeptide exclude sugar. Note that while sugar con- tents are often expressed as a weight percent of the total, i.e., sugars + polypeptide, I prefer to use the formula given above because a weight loss of sugars expressed on the basis of polypeptide weight is directly proportional to percent deglycosylation. C. Results 1. Initial Deglycosylation Experiments a. Deglycosylation of Glycopeptides from Cell Walls by Periodate and by HBr in TFA When the cell wall tryptic peptide: Gal Gal G l Ser-Hyp4-Ser-Hyp-Ser-Hyp4-Unk-Tyr-Lys was treated with periodate (0.05M) for 3 hours, half of the galactose residues of this glycopeptide were removed but all of the unknown amino acid had disappeared. The unknown amino acid is present in some of the tryptic peptides described by Lamport (11) and is probably a derivative of tyrosine. It was detected by the appearance of a peak which was broader than normal on the amino acid analyzer. There may have been some conversion of the unknown amino acid into tyrosine as the molar ratio of tyrosine per peptide rose above one. When this peptide was treated with HBr gas in TFA in the presence of anisole for 3 hours and then chromatographed on a Biogel P2 column it contained only 5% of the original galactose as determined by GLC analysis of its TMS derivative and no bound serine-O-glycoside by the 32 hydrazinolysis method of Lamport (14). The unknown amino acid and all other amino acids were undegraded. The peptide moved slightly further than the untreated peptide on paper electrophoresis at pH 1.9 as would be expected. No smaller peptides were detected, indicating no peptide bond cleavage. b. Attempted Solubilization of Intact Extensin by HBr in TFA When tomato cell walls were treated with HBr in TFA for 3 hours most of the cell wall was soluble in water, but a residue accounting for about 10% of the initial weight of the walls remained which was insoluble in TFA, formic acid, 8 M urea, 2% SDS or water. This residue contained about 98% of the Hyp of the starting material and by agggg acid analysis about 40% of its mass was polypeptide. The other 60% has not been identified. Only a trace of sugars was present in the residue as tested by the anthrone assay. When viewed under a microscope the residue had the appearance of very thin cell walls. c. Deglycosylation of Ovomucoid by HF Although the structure of the oligosaccharides of ovomucoid are not known and the degree of glycosylation is variable, it is a highly glycosylated protein and does have an easily assayable biological activity:trypsin inhibition. After 1 hour in HF at 0 C the glucosamine content fell by 50-75%, but the treated ovomucoid was still'VBOZ as effective as the starting material in inhibiting trypsin (Table 1). The elution profile of the treated ovomucoid on a Sephadex G-50 column shows that HF treatment decreased the size of the ovomucoid (Figure 6). 33 .Ha\wa m¢.o chehuHm pHooaao>o poummuu mm pmHoon Hm Ha\wa H o.mo He~.o H: mm + eHmaauu H: ooH on aoHuomum .pHoo:Bo>o pmummuu mm NH Ha\me m.H o.m~ oaH.o H: mu + aHmesuu H: ooH vHOUHHEO>O w>HumHu OOH Ha\wa H o.me HmH.o H1 mN + eHmesuu H: ooH . . o.o 8nd «Emery H: on pouHaHeaH HouHeHsaH Ho :oHuHaHneH H=H2\eoHuo< :oHumHusmoooo unmoumm comm eHoosso>o Ho suH>Huu< enouHaHseH and no em mo uuowum one .H «Heme 34 .pmaHauouop ouo3 mGOHuowum Home pmHooa any can Am muanm oomv nadHoo onto Nopmnnmm can no on aOHuomum sH HmHumuma may no moHuH>Huom muouHoHnsH 0:3 .uoanHsoH ecu momHame mo: woop oumuunnsm oHnu on was Hoan: mH opHHHamouuHsta ochkuthntHknawAIZIU mo EM one umnu .wuowouonu .mHoxHH mH uH .oumuumnsm mnu an HoanHscH mnu mo ucoaoomHeme one ou map on on pomoeoua pom muoanHnaH :Hmnhuu mo mOHpsum :H Amqv hHm=OH>oua pouoa down was huHumoaHHtao: mHsh .oEHu ou uomnmou nuHa HowaHH uo: mp3 umuumH may omsmown Momma thuma maHusumHzcomHomosooHouta wchs chv .mMIMM moponm an pmnHwomop woo woo: MHsanoo ouoa onu omnu unnumu pom: mp3 Anqv :prosmHmm can pounds an ponHuomop Ewumhm momma may .soHumHusoosoo :uHs HmosHH on pHaos HoanHscH pmumouu can woumouuao oau mo mmoso>Huoommo m:u umsu om soHuHoHnsH Non dunno vHsoa noHns umnu ou omoHo on ou pounofipw ouo3 puma maoHuwuusmoaoo HoanHnsH one .uouoaouosaouuoonm wchuoowH ooow pHOMHHo m maHma as o~q um huHmomp HmoHuao cH owsmno o no pououHoos was coHuommw saw no omuooo 0:9 .uommoo mumsamosa :uH3 Ha h.~ ou a: opus muauxHa aoHuomou ago «0 osto> 0:» wow mumuumnsm oHooonouno mnu Ho :OHusHom HE\wE H m mo HE o.H Ou poops mp3 manuXHa can mo uo=VHHm oumHueouaem s< .auow ou onaaoo HoanHnaH wahnso onu aoHHw ou monocHa m unmoH on How .o.n we .uommop oumnemonn oH nonumwou woumoaouH who? HoanHnoH poo mahuao one .ououumnom oHauwoaouso m on mpHHHomouuHstn osHanHMIHntthaoQIZIU was: :3 53323 H... Home... .3 8:8me as 35% 25 53:2. S33 “3% 35 .Aoev huon mo ponuoa onu he sHououe wow vohmmom mono some new cmtu xopmneom so ponnmumoumaonno swan mos unapoum one .uoSuo oucH pouomwuxm mp3 wHomHsm osu paw poumuoam>o meHan mm? mm mnu uaoaummuu onu Houm< .uoon H wow u e um oHomHsm Ha H mo monomoun onu aH mm mo Ha OH :H pmuwmwu was .aaemuwoumfiouno omiu >9 unmaHamuooo uanm3 umHsomHoa HosoH m aowu poumumoom sz50H>oua some on: noHna .pHoooao>o no we mH hHoumaonuem< .0 0 up unwaumouu m: woo; H m Hmuwm paw muouon omtc xmpmsmmm co vHooaao>o mo mHHwoua aOHusHm any .0 mustm 36 @ ouame .3832 3:... 9.96m ooh 37 2. Use of HF for the Depolymerization of Polysaccharides for the Quantitative Recovery of Monosaccharides Some polysaccharides such as cellulose are notoriously difficult to hydrolyze without considerable destruction of the sugar. However, anhydrous hydrogen fluoride readily depolymerizes such polysaccharides at low temperature which raised the possibility of using this as a method to obtain good quantitative sugar analyses of "difficult" poly- saccharides. Thus, I compared the sugar recovery after HF solvolysis of (a) larch arabinogalactan and (b) tomato cell walls, with the sugar recovery after TFA hydrolysis only. a. Arabinogalactan Larch arabinogalactan (19.7 mg) was dissolved in 1 m1 H20 contain- ing 1 mg/ml mannitol. Aliquots of this solution were hydrolyzed in 2 N TFA and then analyzed as alditol acetates, or methanolyzed and then analyzed as the TMS-Odmethyl glycosides. The remaining solution of arabinogalactan was dried in the HF re- action vessel, treated with HF for 1 hour at 0 C in the absence of scavenger, and then analyzed by the two methods mentioned above. The results in Table 2 show that HF does not destroy sugar residues. b. Tomato Cell Walls Plant cell walls are difficult materials to analyze quantitatively. Cellulose is difficult to hydrolyze, polygalacturonic acid is prone to degradation during hydrolysis, protein tends to react with sugars and sugar degradation products, for example via the Maillard (50) reaction. Because of these and other problems, cell wall analyses usually involve 38 .mopHmooan HznuoEIOImzH mm pmuhHmaooom .0 mm mason w aum>ooom .o HNH moo; H unmouom Hosmnuoz uH Ho: 2 m.H unmouom mHom mm mo somHummaoo < ":muomHmwosHomu< :oHoH ma n.mH aouw mumwom mo huo>ooom .N oHomH 39 at least four separate assays: (i) cellulose, (ii) uronic acids, (iii) neutral sugars, and (iv) protein. About 90% of a cell wall preparation obtained from tomato cell suspension cultures was rendered water-soluble by treatment with anhydrous HF for 1 hour at 0 C , in the absence of scavenger. Lamport and I determined the sugar recovery by analyzing the water soluble products as alditol acetates and as TMS-O-methyl glycosides (Table 3). For maximum sugar recovery it was necessary to hydrolyze the water solu— ble HF solvolysis products in 2 N TFA or to methanolyze them with 1.5 M HCl in methanol. It seems likely that these treatments were required because of the presence of glycosyl fluoride monomers and polymers (Fredenhagen and Cadenbach (22)) stable in water. The 10% of the HF treated cell wall preparation which remained insoluble in water or buffers appeared microscoPically as very thin walls, and consisted of protein plus an unidentified component in the proportion of roughly 1:1 (Figure 7). Using the HF solvolysis we were able to account for 70-75% of the wall as sugars. The protein content was ca. 5%, the amino acid compo- sition of the untreated cell walls was almost the same as that of the insoluble residue which remained after HF treatment (Table 4). The sugar recoveries from cell walls after HF treatment, apart from the dramatically increased yield of glucose from the solubilization of the cellulose, were slightly lower than those of 2 N TFA hydrolysis alone. Since the recoveries from arabinogalactan were slightly higher after HF treatment I surmised that something in the walls was trapping 40 Table 3. Sugar Recoveries from Tomato Cell Walls: Comparison of TFA Hydrolysis and HF Solvolysisa HF Solvolysis Followed TFA Hydrolysis by TFA Hydrolysis Nanomoles pg Residues Nanomoles pg Residues Rha 39 5.7 33 5.1 Fuc - - - _ Ara 144 19.0 102 13.5 Xyl 77 10.2 60 7.9 Man 16 2.6 (16 2.6)b Cal 100 16.2 86 18.9 Glc 33 5.4 350 56.7 Total Neutral 59.1 g 97.1 pg Sugars Galacturonic n.d. 47.0 pg Percent Cell Wall Recovered as Sugars (Weight basis) 72.1% Percent Cell Wall Recovered as Amino Acids (Weight basis) 4.2% a10 mg cell walls were used for the TFA hydrolysis (2N, 1 hour, 121 C ) and 100 mg cell walls for the HF solvolysis (1 hour, 0 C ). After each treatment, the solvent was removed, water was added, and the solution centrifuged. For GC analyses, a volume of the supernatant equivalent to 200 pg cell wall was used. Neutral sugars were analyzed as alditol acetates and galacturonic acid as its TMS-O-methyl glycoside. bBoth mannitol and inositol were used as internal standards in this experiment. Therefore, the added mannitol prevented quantitation of mannose by the alditol acetate method. Figure 7. 41 Cell walls of suspension cultured cells before and after HF treatment. Aliquots of cell walls (between 20 and 100 mg) were treated in 10 to 20 ml of HF at 0 C in the absence of scavenger for 1 hour. The residue was resuspended in distilled water and washed two times. Microscopically the residue appeared cell wall-shaped. No loss of cell wall shape was observed after suspension of the residue in 90% formic acid, 8 M urea, or 6 M guanidine hydrochloride but the shape was destroyed by trypsin which also solubilized 50% of the Hyp. A Sycamore cell walls with no treatment. B Sycamore cell walls after HF treatment. C Tobacco cell walls with no treatment. D Tobacco cell walls after HF treatment. Figure 7 o "! ‘é‘ ‘5- 43 Table 4. Amino Acid Composition8 of Tomato Cell Walls Before and After HF Treatment Before After HYP 30 30 Asp 4.4 3.9 Thr 2.5 2.8 Ser 10.1 9.5 Glu 3.0 3.1 Pro n.d. 3.6 Gly 3.4 4.0 Ala 2.7 2.6 Val 4.3 4.7 Cys 0 0 Met 0 0.3 Ile 1.5 1.7 Leu 2.7 3.0 Tyr 2.6 2.3 Phe 1.3 1.6 Lys 8.4 8.9 His 1.9 1.7 Arg 1.3 1.3 aNormalized to 30 Hyp residues. For experimental details, see Table 3 and Methods. 44 the sugars in a form from which they could not be recovered as monosac- charides by acid hydrolysis. The amino acid composition of the walls did not show a great loss of aromatic amino acids, as one would expect if the sugars had been alkylating these amino acids by the reaction discussed previously (cf. footnote 1 pg. 19). There remains, however, the unidentified material in the wall residue and quite possibly more of this material in the soluble fraction. If this material were phenolic or easy to alkylate, it could have been trapping the sugars. To avoid the reoligomerization of sugars in HF reported by Fredenhagen and Cadenbach (22) and perhaps the alkylation of any phenolic or other reactive compounds which might be present in cell walls, I tried including methanol in the reaction mixture with the HF. Lenard (27) had used methanol to prevent inter- or intradmolecular ester formation in his experiments with a-melanocyte stimulating hormone by therefore causing only methyl esters to be formed (c.f. Introduction). I hoped that with a large excess of methanol present, the glycosyl fluorides formed in HF would react only with the methanol. Sugar compositions of tomato cell walls were determined by treat- ing walls (2—10 mg) in 10 ml of HF in the presence of 1 ml of dry methanol, followed by hydrolysis and derivatization discussed above. Somewhat better yields of sugar, especially pentoses, were obtained from tomato walls so treated (Table 5). The presence of methanol in the HF reaction mixture prevented browning which occurred during the reaction in the absence of methanol. 45 Table 5. Sugar Recoveries from Tomato Cell Walls: Comparison of Methanolysis and HF Solvolysis in the Presence of Methanol Weight Percent of Wall Methanolysis HF Solvolysis Followed by Methanolysis Ara 5.99 6.23 Rha 2.33 1.8 Xyl 5.22 7.8 GalU 16.63 18.95 Man 1.05 2.48 Gal 4.82 6.01 Glc 1.97 34.03 38.2% 77.4% For methanolysis alone 1.7 mg of tomato cell walls + 500 nanomoles of mannitol were heated in 2 ml 1.5 N HCl in methanol for 18 hours at 80 C and then derivatized as described in the Methods. For HF treatment 8.9 mg of cell walls + 4000 nanomoles of mannitol were treated withIV10 ml of HF and 1 m1 of dry methanol. After complete removal of the HF and methanol the sample was dissolved in water and aliquots taken for methanolysis and subsequent analysis. 46 c. Chitin HF at 0 C for 1 hour partially solubilized crab shells, a complex of chitin, calcium carbonate, protein and pigments. The solubilized material contains polymeric N-acetylglucosamine and protein which elute in the void volume of a Biogel P2 column. The more rigorous HF treat- ment of the crab shells for 3 hours at 23 C (in the absence of scavenger) solubilized nearly all the N-acetylglucosamine, and left a water insoluble residue which was considerably enriched in amino acids. The water soluble material, when chromatographed on a Biogel P2 column (Figure 8) gave three fractions: a small void peak containing protein plus a little N-acetylglucosamine, another small peak containing N-acetylglucosamine together with an unidentified compound, and a large peak containing only monomeric N-acetylglucosamine which, after deriva— tization, co-chromatographed as TMS-N-acetylglucosamine. 3. Use of HF for Deglycosylation of Glyco— Proteins and Glycopeptides Various glycoproteins and glycopeptides were used to characterize the reaction of HF with sugars bound to amdno acids. Tomato cell wall glycopeptides were used as examples containing neutral sugar-O-glyco- sidic linkages to hydroxyamino acids. Although not completely character- ized, cell wall peptides provide an excellent tool for the study of glycopeptide linkages as they contain many short sugar chains, thus allowing easy quantitation of the cleavage of glycopeptide linkages. Fetuin and pig submaxillary mucin are examples of glycoproteins contain- ing N- and O-glycopeptide linkages of amino sugars. The extracellular 47 .moHammomon Hyumomtz oHumaoaoa yHm>Hm=Hoxm pmaHmucom uH umau scam ou mpoaumz mau cH pmaHuommp mm pmaamuwoumaouao pom pmumHyHHmHyaumaHuu yHuomqu mos :oHqupm :H uoa poauma mHau ya pmNyHmam omHm was xmmn mwumH may .pHmm mHamuan pom mcHamHmHhcmam cmmauma mamma ozu ou mmHu mm>Hm muHammomaHu .mpHom ocHEm mo yammuwoumaouao pHouHH wow you pmaHuommp mm maamuwoumaouao pom GOHumuHum>Hump smau ppm 0 mOH um musoa c How Hum wcHHHoa unmumaoo aH uo=VHHm am mo mHmyHoupya ya pmuhHmsm umauusm mp3 mamma ozu umuHm may :H HmHumuma may .coHumuHuamov How umuyHmsm :Huvyach mau ouaH pmm smau paw Ho: 2 m auH3 pmuHHmuuoms mHmB muooUHHm pmnyHoupya may .m>Huommu :HupyaaHn maHEmmoman mau wcHamE mpmwom ocHEm may Eouw asoum Hyummmtz mau mm>oamu mamaummuu mHay .m>mHoouom cm sH Hooa H How aomz z m :H uH maHuyHoupya pom GOHuomum aomm Eouw uo=VHHm cm onxmu ya pmhmmmm mos waoHoo mau scum unmon may .nasHoo Ntm Hmonm m co pmaamwwoumaouao cmau mmz aoHuome mHasHom umums may .wuaoa m.m now muaumumaamu Eoou um mm :H pmummuu mums mHHmam amum we we uametyuamsy .umum3 aH mHaSHom mm? aoHa3 mHHmam ampo pmummuu mm mo aoHuomHm mau mo mum HmwOHm so mHHmoum aoHuon .w muanm 48 0' w muome 8952 men... owm 49 glycoprotein from sycamoredmaple suspension cultures is an example of a highly glycosylated protein, which can be easily purified by deglyco- sylation. a. Cell Wall Glycopeptides Because HF cleaves the glycosidic linkages of neutral sugars, I investigated the extent to which HF would deglycosylate some glyco- peptides from cell walls of suspension cultured tomato cells. These peptides were released from the walls by oxidation for 30 minutes at 75 C in a solution of 1% acetic acid and 1% sodium chlorite, a slight modification of the procedure described by Mort and Lamport (51). Hyp is over 50 mole percent of the amino acid residues in these peptides, and attached to each hydroxyl group is a short chain of 1 to 4 ara- binose residues, predominantly 4, average 3.06 (8). In addition, there are numerous serine residues many of which are attached to a galactose residue (or perhaps polysaccharide) via an O-galactosyl linkage (14). Treatment with HF for 1 hour at 0 C deglycosylated cell wall glycopeptides obtained by chlorite oxidation (Table 6). The sugars cleaved from these glycopeptides were monomers, judging from their elution in the included position on a Biogel P2 column. The 2-3% of sugars remaining linked to the peptide were not cleaved possibly because they were in a part of the reaction vial not in contact with the liquid HF. Kel F is very prone to develop a charge of static electricity; because of this it is difficult to ensure that all the sample is in the bottom of the reaction vessel. 50 Table 6. Sugar Composition8 of Cell Wall Glycopeptides Before and After HF Solvolysis at 0 C for 1 Hour Percent Sugar Before After Remaining Ara/Hyp 5.04 0.14 3 Gal/Ser 4.2 0.09 2 Galacturonic/Ser 7.0 0.04 1.3 Rha/Ser 1.7 0.02 1.2 Glc/Ser 0.84 0.05 6 aData expressed as molar ratios. Sugars were determined as their TMS-O-methyl glycosides, and amino acids as their N-heptafluorobutyryl isobutyl esters. 51 b. Soluble Extracellular Hyp-Rich Glycoprotein Suspension cultures of plant cells secrete a mixture of soluble polysaccharides, proteins, and glycoproteins into their growth medium. At least one of these components is a Hyp-rich glycoprotein in which 50% of the Hyp residues are glycosylated with arabinogalactan side chains through an O-galactosyl Hyp linkage (15). Such a highly glyco- sylated glycoprotein must be deglycosylated before the amino acid sequence can be determined. In addition, deglycosylation allows an appreciable purification of this protein as the effective size of a highly glycosylated protein is drastically reduced after its sugars have been removed. Thus a separation by size before and after HF deglycosylation should select only for those proteins whose size changes drastically. Lamport, Caughey, and Clark partially purified the crude, soluble, Hyp-rich glycoprotein of sycamore-maple cells by ultrafiltra- tion of the culture medium through an Amicon XMlOOA membrane, followed by desalting on Sephadex G-25 and freeze drying. The bulk of this crude material (both sugars and Hyp) appeared in the void volume of a Sephadex G-100 column. However, after treatment with anhydrous HF (1 hour 0 C) and extraction with 0.1 M acetic acid, soluble Hyp-rich macromolecular material was considerably retarded on a Sephadex G-100 column (Figure 9). The results of amino acid and sugar analyses (Table 7) showed a con- siderable purification of this HF-deglycosylated material. For example, before deglycosylation there were ca. 120 sugar residues for each Hyp residue; after deglycosylation there were only ca. 3.6. A comparison of amino acid analyses before and after deglycosylation (Table 7) showed a 52 H.3umHo pom hmawsmo .uuonama mo mummy .mHmyHo>Hom mm muomma GHmuoumoowa mpswm mau :H Amamuw toHoHB «NoHV ohm mau mo NHm How pmuooooom mom can mo mamuwoumHa yum vmchuoom ammo Honma may .ahm How pmymmmm mums oOHummum aomm mo muoaoHHm umuHHouoHa pmuoaoa m>Hm .pHom mHumom z H.o auHB pmumuaHHHoom cabHoo OOHIu xmpmaamm am mm x m.~ m :o mooHumoHHaem mumumamm oau ma pmumoOHuomum pom pHom oHumom z H.o HE on oH pm>Homep moo mopHmmu may .mpoaumz oH pmaHuommp mm o o um unoa H now ya HE oq auHB pmummuu mums :Hmuouaoowa mpoum ma pmupooa uame .mHHmouo GOHuon OOHtu xmpmammm ”meyHo>Hom ya umumm onuouaoowa aUHHtmoHHoummxoupAa m mo GOHumHuHHm Hmc .m mustm 53 Om m muome CE 0.3 mwméaz ZO§Q<~E 0? On q ON 0. éia 90> '— mod 0 _.oEC 8? a»: m_.O ON .0 54 Table 7. HF Deglycosylation of a Soluble Hydroxyproline-Rich Glyco- protein Secreted by SycamoreeMaple Cultures Residues per Mole of Hyp After HF and Sephadex G-100 Before HF Chromatography Rha 2 0.2 Fuc 1.2 0.1 Ara 16.8 1.4 Xyl 6.7 0.2 Gal 13.2 1.0 Glc 57.1 0.7 GalU 19.4 0 Mole Percent of Total Amino Acids Hyp 6.7 14.2 Asp 4.9 11.3 Thr 8.2 9.0 Ser 9.6 12.3 Glu 9.8 7.5 Pro n.d. n.d. Gly 10.4 7.1 Ala 9.3 12.3 Val 7.6 5.2 Cys 0 0 Met 1.1 0 Ile 4.9 3.3 Leu 7.8 5.2 Tyr 1.1 0.5 Phe 5.1 3.3 Lys 7.1 4.7 His 3.3 2.4 Arg 3.1 1.9 Source: Lamport, Caughey and Clark. 55 two-fold enrichment of Hyp compared with other amino acids. c. Fetuin Having verified that liquid HF cleaves O-glycosidic linkages of neutral and acidic sugars, I turned my attention to amino sugar N- and O-glycosidic linkages, which occur frequently as glycopeptide linkages, and are therefore especially important. Spiro and co-workers (52) showed that the fetuin molecule contains six oligosaccharides, of which three are N-glycosidically linked (type I) and three O-glycosidically linked (type II). I Man -— Man —- Man -— GlcNAc -- GlcNAc —— Asn GlcNAc GlcNAc GlcNAc Gal Gal Gal NANA NANA NANA II NANA —-—-Ga1 -——-GalNAc'-—-—- Ser (or Thr) (NANA)1/2 Treatment of fetuin with 10 ml HF for 1 hour at 0 C with 1 ml anisole as a scavenger removed most of the attached neutral sugar residues (Table 8), but substantial amounts of the N-acetylated hexosa- mines remained. The sugar composition of the treated fetuin could be explained if only the neutral sugar linkages were cleaved by the HF. From the struc- tures proposed by Spiro (52), the asparagine linked oligosaccharides would be cleaved to a short disaccharide of two N-acetyl glucosamine 56 .oHaumm mHoE\mm:pHmmu mo Hmaaso HmHuHaH muH ya pmHHmHuHoa umwsm wchHmamH mau mo uommumm "mm pmuMHamHm0a .Ammv ooumoam pom ouHam ya pmoHaHmumv mHom m: umuu< mHmyHo>Hom mm muowmm .0 o um noon H How mHmyHo>Hom mm Hmuw< pom muowmm anumm mo oOHuHmomfio0 umwom .0 mHamy 57 residues and the serine and threonine linked ones to a single N-acetyl- galactosamine. The results shown in Table 8 are all consistent with this hypothesis apart from the retention of 12% of the galactose and 8.7% of the NANA. There are at least two possibilities to explain the retention of these sugars: (1) Some of the threonine or serine linked oligomers are unavailable to the HF for reaction, and (2) some of the sugar fluorides formed during the HF cleavage reattached themselves to the protein rather than to the anisole scavenger. These linkages to the protein could be either glycosidic to serine or threonine hydroxyl groups, glycosidic to the hydroxyl groups of the amino sugars still attached to the protein, or ester links to the carboxyl groups of aspartic or glutamic acid (Lenard found that methanol in HF causes the methyl esterification of aspartic acid (27)). Harsher conditions (HF for 3 hours at 23 C ) resulted in removel of all the N-acetylgalactosamine, leaving only 2-3 residues of N-acetyl- glucosamine per mole of fetuin (Table 9). I conclude that HF at 23 C cleaves GlcNAc-GlcNAc (cf. effect of HF on chitin) and GalNAc—Ser but ‘gg£_the GlcNAc-Asn linkage. Results of amino acid analyses of fetuin were similar both before and after treatment with HF at 0 C followed by dialysis (Table 10). Thus HF did not appreciably degrade or alkylate fetuin. d. Pig Submaxillary Mucin Pig submaxillary mucin (PSM) consists of about 60% carbohydrate and 40% protein. The carbohydrate is present as many short oligosacchar- ides O-glycosidically attached to at least 85% of the threonine and 58 .0 mHamy now no mcoHumHnon0 «.5 m.o m.c NH 0.¢ Hm0 t t .Hu m m.m cm: i t .uu m m.H oHom mm umuw< mHmyHo>Hom ya muommm .0 mm um undo: m pom mHmyHo>Hom mm umum< pom muommm :Houmm mo GOHuHmogao0 umwom .m mHamy 59 Table 10. Amino Acid Composition of Fetuin Before and After HF Solvolysis for 1 Hour at 0 C. Amino Acid Residuesa Before HF After HF Ala 60 61 Gly 44 44 Valb 78 68 Thr 37 36 Ser 44 47 Leu 50 44 Ile 23 22 Pro 65 61 Hyp Met Asp 61 60 Phe 21 21 Glu 69 69 Lys 31 31 Tyr 13 14 Arg 25 25 His 15 18 Cys 0 0 aNormalized to 69 Glu residues for comparison with the published analysis (54) and to offset the inflated valine value. bGlucosamine elutes with valine on our amino acid analyzer and there- fore gives a high value for valine in the untreated fetuin. 60 serine residues (55). Carlson (56) characterized a series of PSM oligosaccharides, the largest of the series being: GalNAc Gal GalNAc Ser or Thr Fuc N-Glycolylneuraminic acid After treatment of PSM with HF (anisole as scavenger) for 1 hour at 0 C , followed by dialysis of the aqueous extract, only N-acetyl- galactosamine remained as a major sugar component attached to the peptide (Table 11). From the work of Carlson (56) we know that the presence (or absence) of terminal N-acetylgalactosamine in the PSM oligosaccharides determines the presence (or absence) of blood group A type activity. Thus PSM pooled from a number of glands contains a mix- ture of blood group types and one can infer from the data of Payza g£_al, (55) that about half the PSM from pooled glands contains two GalNAc residues/oligosaccharide while half contains only one GalNAc residue/ oligosaccharide; i.e., pooled PSM averages ca. 1.5 GalNAc residues/ oligosaccharide. HF treatment (1 hour at 0 C ) of pooled PSM actually resulted in the loss of ca. 33% N-acetylgalactosamine (Table 11) which is consistent with the data just mentioned and indicates that in PSM, as in fetuin, HF solvolysis at 0 C cleaves only neutral sugar linkages, leaving both GalNAc-Ser and GalNAc-Thr links intact. But more rigorous HF treatment of PSM for 3 hours at 23 C (anisole scavenger) almost completely stripped the protein of its sugars, only 6% of the original N-acetylgalactosamine remaining (Table 11). After only 2 hours at room temperature in HF, PSM retained 7-10% of its GalNAc. I conclude that 61 o o o o No Hom onchamm mHmNHo>Hom mHmmHo>Hom umwom unmoumm mm Hmum< Hmwnm unmoumm mm umuw< m: muommm mHmyHo>Hom mm Hmum< pom muommm :Hosz hHmHHmeaanm me mo :OHuHmoqao0 umwom .HH mHamy 62 these more rigorous conditions of HF solvolysis cleave the GalNAc-Ser and GalNAc-Thr linkages in PSM, as in fetuin. The amino acid composition of PSM did not change appreciably after HF treatment for 3 hours at 23 C followed by dialysis of the aqueous solution (Table 12). 4. Evidence for the Selectivity of HF a. Gel Electrophoresis of BSA and Fetuin Before and After Treatment Bovine serum albumin, an unglycosylated protein, was treated in HF, in the presence of anisole, at 0 C for 1 hour and subjected to SDS gel electrophoresis. No new bands appeared in the gels indicating no degradation of the protein (Figure 10). When treated and untreated fetuin were subjected to electro- phoresis in 5.6% gels in SDS and mercaptoethanol, as described by Fairbanks g£_§1, (57), it was clear that the protein was considerably lower in molecular weight after the treatment but that the fetuin remained essentially as a single band on the gel (Figure 11). The band for treated fetuin was sharper than the untreated fetuin perhaps indi- cating the removal of heterogeneity. b. Retention of N-Acetyl Groups by N-Acetyl- glucosamine After HF Treatment As most hexosaminidases require the amino group of their substrate to be acetylated (58) I thought it important to prove that HF does not deacetylate amino sugars. Thus treatment of free N—acetylglucosamine with HF at 0 C for 1 hour followed by analysis as the TMS derivative 63 Table 12. Amino Acid Composition of PSM Before and After HF Solvolysis for 3 Hours at 23 C. Amino Acid Residues (mole_percent) Before HF After HF Ala l3 l3 Gly 18 18 Val 7 7 Thr 11 11 Ser 18 18 Leu 3 3 Ile 3 3 Pro 6 6 Met 0 0 Asp 4 4 Phe 2 2 Glu 8 6 Lys 3 3 Tyr l 1 Arg 3 3 His 1 l Cys 0 0 64 Figure 10. SDS gel electrophoresis of bovine serum albumin before and after HF treatment. Bovine serum albumin was treated in HF at 0 C for 1 hour in the presence of anisole. After evaporation of the HF the residue was taken up in 50% acetic acid, dialyzed and then electrophoresed as described by Fairbanks et a1. (58). Left: Untreated Middle: Treated Right: Untreated + Treated 65 Figure 10 Figure 11. 66 SDS gel electrophoresis of fetuin before and after HF treat- ment. Fetuin was treated in HF at 0 C for 1 hour in the presence of anisole. After evaporation of the HF, the residue was dissolved in 50% acetic acid and then dialyzed against dis- tilled water. The fetuin was then electrophoresed as described for bovine serum albumin. Left: Untreated Middle: Treated Right: Untreated + Treated 67 ,_.- -~-:' ~ Figure 11 " l H 68 via combined gas chromatography and mass spectroscopy showed only TMS-N—acetylglucosamine (Figure 12). D. Discussion Anhydrous hydrogen fluoride is a reagent often used in the chem- istry of synthetic peptides because of its selective cleavage properties, but it has not been previously reported as useful for the deglycosyla- tion of glycoproteins. As summarized below, my experiments show that anhydrous HF cleaves the glycosidic linkages of neutral and acidic sugars within 1 hour at 0 C , and the O-glycosidic linkages of amino sugars within 3 hours at 23 C , but leaves intact peptide bonds and N-glycosidic linkages of amino sugars. l. Glycosidic linkages of neutral and acidic sugars are cleaved in 1 hour at 0 C in HF. a) The neutral and acidic sugars of plant cell walls were rendered completely water soluble after a 1 hour treatment in HF at 0 C. The sugars after this treatment were also soluble in methanol indicating a very low degree of polymerization. b) When glycopeptides extracted from cell walls by sodium chlorite oxidation were treated in HF for 1 hour at 0 C the degree of glycosyla- tion was reduced to an amount which could only be explained if most of the Hyp-O-arabinose and Ser-O-galactose linkages to the peptide had been broken. c) HF treatment of fetuin and pig submaxillary mucin at 0 C for 1 hour removed the mannose and galactose from the proteins. Both of Figure 12. 69 The mass spectra of authentic and HF-treated N-acetyl glucosamine as their trimethylsilyl derivatives. The trimethylsilylated samples were injected into the LKB-9000 Glc/Mass spectrometer of Dr. C. C. Sweeley (Department of Biochemistry, MSU) and the mass spectra of the appropriate peaks taken. The difference in intensity of some of the ions of the two samples was probably due to slight variations in the conditions of the machine as the spectra were taken three months apart. The fragmentation patterns of both samples clearly indi- cate an acetyl group on the amino group of the sugar and that the two samples are identical. Both mass spectra, especially that of the HF-treated N-acetyl glucosamine, are very similar to the spectrum published for trimethylsilylated N-acetyl galactosamine (59). The base peak of the mass spectra is m/e 173, which corre- sponds to . + CH—-CH I I TMSi-O NHCOCH 3 This structure only forms from N-acetylated hexosamines in the pyranose form. There is no molecular ion but a rela- tively high abundance of M+-15 ('CH )(m/e 494), a peak at m/e 404 corresponding to M+-Me°-TMSIOH, and one at m/e 314, possibly M+-Me'-2TMSiOH. Thus HF had no effect on the structure of N-acetyl glucosamine. 70 100 80‘ x10 . 60‘ D 20' b y Y T TV—V'Wr'Y—VY v—fvvt v 400 450 . .111. 11 L. JlulledL..'-,Jl...1...e 00 350 so too 150 200 fi§§ofifi a 100 80‘ 80'. 50 100 150 200 250 300 350 1000 450 Figure 12 71 these sugars were glycosidically linked to amino sugars which remained on the proteins. Therefore, the glycosidic linkages must have been broken. In the case of pig submaxillary mucin the NGNA was also removed from the protein showing that the glycosidic linkage of NGNA to GalNAc had also been cleaved. 2. O—glycosidic linkages of amino sugars are cleaved in 3 hours at room temperature in HF. a) The chitin of crab shells remained polymeric after 1 hour in HF at 0 C yet became predominantly monomeric after 3 hours in HF at room temperature. b) The GalNAc-O-Ser and GalNAc-O-Thr linkages of fetuin and pig submaxillary mucin were cleaved after 3 hours at room temperature in HF but not after 1 hour at 0 C. 3. The N-glycosidic linkage between asparagine and GlcNAc is not cleaved after 3 hours in HF at room temperature. a) The GlcNAc that remains as part of high molecular weight material after a 3 hour treatment of crab shells in HF at room tempera- ture remains associated with protein. b) After a 3 hour treatment of fetuin in HF at room temperature there remain 3 moles of GlcNAc per mole of protein, the same as the number of asparaginyl linked oligosaccharides. Aqueous hydrofluoric acid also has selective cleavage properties, although rather different ones from those of anhydrous HF. For example, 60% aqueous HF at 0 C specifically cleaves the phosphate esters of teichoic acids from Bacillus subtilis, yielding mainly glucosyl glycerol 72 (60). Table 13 shows that, depending on the reaction conditions, HF shows a surprisingly wide range of specificities for cleaving various linkages in biologically important molecules. Anhydrous HF, in particu- lar, appears to be a highly versatile reagent. We have just begun to explore its usefulness in glycoprotein and carbohydrate chemistry. I list the following applications as worthy of further study: 1. Complete suga: analysis of polysaccharides. This should be especially useful for those polysaccharides which are difficult to hydrolyze by conventional methods; such as, uronic acid-containing polymers, cellulose, and mixed polymers. 2. Identification of glycopeptide linkages. a) O-glycosidic linkages such as GalNAc-Ser For example, after HF treatment of pig submaxillary mucin at 0 C only N-acetylgalactosamine remained peptide bound. The amino acid involved in the glycopeptide linkage could have been identified by B-elimination in the presence of sulfite (53). b) N-glycosidic linkages: GlcNAc-Asn. Because treatment of glycoproteins with HF for 3 hours at 23 C cleaves all except N-glycosidic linkages, HF can be used to confirm the presence of the GlcNAc-Asn linkage. 3. Structural determination of oligosaccharides containing amino sugars. My data show that at 0 C HF cleaves glycosidic linkages of neutral sugars but leaves intact the glycosidic linkages of amino sugars. Thus HF treatment of glycoproteins (in the absence of scavenger) should 73 vmooHucoo AeHV oeHmeu Imuoma OImIOI :Hmuoum HmHuum0 Hmo< meson «H .o H HNGV meHumoeHwHe HhmommeHp + mpHummszHprTll mpHummmHmHo HymooanHp HypHumaamoam muse: em .o o muooa min .0 o A000 Houmthw HhmomwaAl Aonm mHoamHmu my mumammoaaHoumomHwaoaHymoo=H0 as; H .o o HHS meHeHuaoeoHimil Exoeonn .59. H 6 on: HHS 8.5687?! menioeoHin mmumaamoanouhm m: moom=o< N00 mHmamxm "mooHquco0 mwmxcHH mo mmhy mooHquco0 mum>mm yHmcmemuocH mo umpuo oH pmumHH mmmwmonH maoHum> mo mwm>mmH0 m: .mH mHamy 74 pmooHuooo musoa m muoumumaamu Boom pmHmHomqmo: mEHu uuoam muaumummEmu Boom use: H .o o use: H .o o moHammooanHyummmt2t0tmoHcomHay no mzHumm A....ummHo you mooHquoo0m 0 mm um musoa on pm>mmH0 0 MN um muooa m ummmH um wow mHamum Hmmv maHosHm HaeoHeumz moHammomonHyumomtz Hmonmumam< o o I H L . \ / ll. :2 :o z%\ ,flq\ x _ m mpHummm 750w m :o~zz 153.6 1 _ OHUOZII I z o moszo mmpHmoowatz umwom ooHa< mcoHqucou mHeamxm mmmaoHH mo moyy emaeHueoUiimH mHan 76 yield straight chain or branched oligosaccharide fragments with neutral sugars at the reducing terminus and amino sugars at the non-reducing terminus. In conjunction with deaminative cleavage at amino sugars (64) oligosaccharide fragments could be obtained to allow complete sequencing of the short sugar chains. 4. Facile generation of substrates for glycosyl transferases. Although the N-asparagine linked N-acetylglucosamine cannot be cleaved from glycoproteins under the conditions I have tested, natural substrates for the glycosyl transferases of O-glycosidically linked oligosaccharides can be generated easily. 5. The role of sugars ingglycoproteins. The biological activity of some glycoproteins (65) is absolutely dependent on the sugar components which often seem to act as a recogni- tion code for "topographical location" (66) of the glycoprotein. In other glycoproteins the role of sugar components is less than clear, perhaps because it is more subtle. For example, after HF treatment for 1 hour at 0 C , ovomucoid largely retained its activity as a trypsin inhibitor. This is consistent with the observation (18,20,41) that some nonglycosylated proteins such as lysozyme and RNase retain their biological activity after HF treatment at 0 C , although not after treatment at 30 C. 6. Solubilization ofgprotggglycan networks. This was my original interest, because I expected anhydrous HF to render soluble the protein of plant cell walls. However, while HF 77 solubilized the bulk of the cell wall (ca. 90%), the residue which con- tained the protein was quite insoluble in various protein solvents such as SDS and urea, indicating the presence of as yet unidentified cross links between the protein molecules of the wall. In the absence of protein-protein cross links, I predict that anhydrous HF will solubilize the cell wall proteins of yeasts, bacteria, etc. 7. As a simple method for purifying thegprotein component of some glycpproteins. Microheterogeneity caused by varying degrees of glycosylation contributes to the difficulties inherent in the purification of highly glycosylated glycoproteins, making it especially difficult to establish the presence of one unique peptide species. HF deglycosylation removes the microheterogeneity and reduces the molecular weight of the (glyco) protein. Thus fractionating by size before and after deglycosylation may yield a virtually pure apoprotein (cf. section 3 b.). 8. Assistance in amino acid sequencinggof glycoproteins. The oligosaccharide units of highly glycosylated glycoproteins present two obstacles to sequence determination: first, steric hindrance to proteolytic attack, and second, the absence of an identifi- able product after Edman degradation of a glycosylated amino acid residue. Except for the GlcNAc-Asn linkage, anhydrous HF removes these obstacles. It should now be possible to sequence previously intractable proteins such as the soluble hydroxyproline-rich glycoprotein (15). Complications may arise because of the influence of the HF on the peptide portion of the protein. HF is a very strong hydrogen bonding 78 agent and so, may denature the protein, causing it to be less soluble in aqueous solvents, or heterogenous in size. Aimoto and Shimonishi (67) found that after 1 hour at 0 C in HF, lysozyme split into three peaks on a Sephadex G-50 column; the latest eluting being native lysozyme. After 1 hour at 25 C the peak corresponding to native lysozyme had disappeared and the majority of the protein was in the second peak. They suggested that the earliest peak was a dimer. However, when fetuin treated with HF at 0 C for 1 hour was subjected to electrophoresis on a SDS gel after reduction with mercaptoethanol, I found no evidence of dimerization or degradation. Because of the extreme toxicity of HF and the expense, approxi- mately $3,000 of a Kel F line, I have considered the use of other deprotecting agents. I have obtained very good results with a short cell wall glycopeptide and intact cell walls using HBr gas in anhydrous trifluoroacetic acid as described in (18) but the conditions of the reaction (3 hours at room temperature) with HBr are not conducive to the retention of biological activity. Other promising reagents are boron tris-trifluoroacetate (68) or trifluoromethane-sulfonic acid (69) in anhydrous trifluoroacetic acid. I hope in future experiments to investigate the use of these other specific cleavage reagents. From the data presented here and that of previous workers with HF, I conclude that the use of HF will greatly assist in the determination of the structure of many glycoproteins and polysaccharides. PART II SOLUBILIZATION AND CHARACTERIZATION OF CELL WALL PROTEIN GLYCOPEPTIDES BY NaClO2 OXIDATION A. Introduction As mentioned in Part I, I was unable to solubilize cell wall protein by HBr in TFA, or HF solvolysis. One of the reasons for this could have been cross linking of the protein via phenolic compounds (70). All my attempts to avoid this possible phenolic cross linking failed. The addition of ascorbic acid or sodium dithionite to keep the phenols which had been oxidized in the hydroguinone form, or the addi- tion of soluble polyvinylpyrrolydone to complex the phenols, made no difference in the subsequent ability of HBr to solubilize cell wall pro- tein. Therefore, I attempted to destroy the phenols selectively after the wall preparation but prior to the HBr TFA solvolysis. The most selective way to destroy phenols chemically is to oxidize them.with sodium chlorite. This oxidation bleaches cell walls to a brilliant white and in one experiment allowed their complete dissolution with a combination of NaClO2 oxidation followed by HBr in TFA. When an amino acid analysis of the oxidized walls was performed I realized that a large proportion of the cell wall had been solubilized by the NaClO2 treatment alone, especially the cell wall protein. From the known non- reaction of sodium chlorite with polysaccharides this raised the possi- bility that NaClO oxidation could be used to release glycopeptides 2 80 81 from cell walls under conditions which would not remove or degrade the oligo- or polysaccharides attached to the protein. What follows is a description of my attempts to characterize the glycopeptides solubilized from cell walls by NaClO oxidation. 2 l. The Use of NaClO in the Carbohydrate Chemistry of Wood To allow the extraction of hemicelluloses from wood in good yield one must first degrade a large part of the lignin. The lignin must be removed in a way which does not change the hemicelluloses by derivatiza- tion or degradation. There is no method which removes lignin from wood without some degradation of the polysaccharides. Two methods are commonly used to prepare wood chips or powder for hemicellulose extraction: (1) chlori- nation of the lignin or (2) oxidation with sodium chlorite or chlorous acid (71). The milder more selective of these two methods is the sodium chlorite oxidation. Chlorine dioxide oxidation was known as early as 1921 (72) to selectively remove lignin from wood. Schmidt and co~workers (73) also studied the effect of chlorous acid (C10 dissolved in water) on various 2 biological compounds and found that polysaccharides were unreactive, as were most amino acids except the sulfur and aromatic amino acids exclud- ing phenylalanine. Jayme (74) and Wise g£_§1, (75) have modified the delignification procedure of Schmidt g£_§1. (76) by using sodium chlorite in hot weak acetic acid solution (instead of C102 gas), thus allowing delignifica- tion in from 3 to 6 hours as opposed to 14 hours and making the 82 procedure less cumbersome. Using chlorite oxidation, Wise g£_§l. (75) could account quantitatively for wood even after fractionation into the cellulose and hemicellulose fractions indicating that little degrada- tion and no loss of polysaccharides had resulted from the delignifica- tion process. After 6 hours of chlorite oxidation Timell g£_§1, (77) found the degree of polymerization of cellulose to have fallen to about 1/3 that of the native cellulose but purified cotton cellulose was much more readily degraded by chlorite (78) (as if the hemicelluloses and lignin protect the cellulose from oxidation). The degree of polymerization of hemicelluloses dropped to about 60% of that in untreated wood during the same treatment. In more recent experiments, Goring and codworkers have investi— gated the course of delignification of spruce by sodium chlorite oxida- tion as compared to commercial pulping processes. Ahlgren and Goring (79) found that no polysaccharide was released from the wood until at least 60% delignification was achieved using chlorite but that from the beginning of bisulfite (80), acid sulfite (81) or Kraft delignification (81), there is substantial loss of hemicelluloses. The sugar losses are reflected by the size of the lignin fragments which are solubilized during the process (82) and by the pore size of the wood (determined by solvent exclusion (83)). With chlorite treatment the average molecular weight of solubilized nondialyzable lignin is around 9,000 until at least 60% delignification (84) whereas the molecular weight of Kraft and sulfite solubilized lignin increases from 9,000 at the start of the 83 reaction to 25,000 and 75,000 respectively (82). The pore size of chlorite treated wood remains around 30 X but in Kraft and sulfite treated wood grows to 40 X and 55 8 respectively. The mechanism by which sodium chlorite oxidation delignifies wood is not fully understood. During oxidation with sodium chlorite there is generation of chlorine dioxide gas (C102) which becomes hydrated to form chlorous acid (85). It appears to be this generation of chlorous acid which is important in the delignification process as Lindgren and Nilsson (85) found that chlorite oxidation was very specific for the oxidation of aldehydes to acids if a chlorine scavenger was added to the reaction mixture. When the scavenger was effective in removing all the chlorine generated from the chlorite no chlorine dioxide gas was formed. High yields of vanillic acid were obtained from vanillin in the presence of chlorite and the scavenger, sulphamic acid. If the sulphamic acid were not present, as in experiments by Husband g£_§1, (86), the products were difficult to purify and character- ize, but there was one identifiable product in a 17% yield, chloro- vanillin. Barton (87) attempted to identify the products of slash pine lignin after chlorite oxidation but could only determine the structure of some of the ether soluble components: fumaric acid, oxalic acid and monochloroacetic acid (perhaps from chlorination of the acetic acid in the reaction mixture). The lignin fragments had varied solubility characteristics. From the absence of a peak around 280 nm in the UV spectrum of the two major ether "insoluble" fractions, Barton suggested 84 that drastic changes must have taken place in the phenolic nucleus of lignin subunits. Both fractions contained a large proportion of chlorine and oxygen. Sarkanen §£_§l. (86) used vanillin (I) as a model compound for lignin and found that chlorine dioxide oxidation of this compound resulted in a substantial amount of ring cleavage. The only product identified (a 30% yield) was B-formylmuconic acid monomethyl ester (II). If sodium chlorite was used in place of chlorine dioxide the aldehyde was also oxidized to an acid yielding B-carboxymuconic acid monomethyl ester (III). C HO cno (Ozfl c | 02 \ N 0002 fl fl on 02H 02" I II III Using vanillyl IV,oEmu EOHu thou mHasHomoH mau pom Hmums pmHHHume HE mH EH pmpommmsw mp3 mopHmmu mau mm mau mo EoHumuomm>m umum< .0 0 up munoa m.H How Aumwcm>mom mHomHom no HoomaumE oov mm mo HE ON yHmumEonummm EH mmummuu mums mHHm3 HHmo pmuomuuxm N0H0mz mo wE mmH .mHHm3 HHmo oumEou pmummuu N0H0mz mo mopHmmH pmummuu mm mau mo EOHuuom mHaaHom mau mo yammwwoumaouam omt0 xmpmammm .qH muanm 97 «H muome 832mm 1 H 59:32 83. _m 20> a _m “7. spun eMiDIes Q>I 98 in Hyp, as is the residue wall (Table 16). The insoluble fraction of the walls after HF treatment, as with the HF treated native walls, is only 40% protein again indicating the presence of some as yet unidenti- fied material. 3. Chagges in the Peptide Portion of Proteins DuringZNaClO2 Oxidation In a preliminary investigation of the effects of NaClO on pro- 2 teins, I treated a well-characterized cell wall glycopeptide and chicken egg white lysozyme, one of the best characterized proteins available. a. A Model Cell Wall Glycopeptide When a tryptic peptide from tomato cell walls isolated by Katona in this laboratory, as described by Lamport (11), with the sequence Gal Gal Gal Ser —- Hyp4 — Ser —- Hyp —- Ser — Hyp4 — Unk — Tyr — Lys was treated with NaClO2 at 75 C for 30 minutes, the tyrosine and unknown amino acid were destroyed with the appearance of about 0.6 equiv- alent of aspartic acid (c.f. effects of NaClO on lignin model come 2 pounds (87)). No destruction of serine or hydroxyproline was apparent. Fifty percent of the lysine was lost with about a 25% conversion to a new amino acid. This amino acid was identified as a-amino adipic acid as it co-chromatographed with authentic o—amino adipic acid on both the amino acid analyzer and on gas chromatography as its heptafluorobutyryl ester 0 99 Table 16. Amino Acid Analysis of Fractions Obtained by HF Treatment from the Cell Wall Residue after NaClO Oxidation 2 Void of G-50 Retarded on G-50 Insoluble Residue Mole Per Mole Per Mole Per percent 30 Hyp percent 30 Hyp percent 30 Hyp Ala .8 .5 5.1 12.8 9.4 47.4 Gly 1.2 .8 11.3 28.2 9.1 46.1 Val 3.4 2.4 5.5 13.6 6.8 34.3 Thr 3.2 2.3 4.3 10.9 5.1 25.6 Ser 13.8 9.7 10.4 25.9 7.9 40.0 Leu 2.8 2.0 6.1 15.2 9.3 46.8 Ile 1.3 .9 12.2 30.5 5.0 25.3 Pro 3.8 2.7 3.6 9.0 4.9 24.8 Hyp 42.6 30.0 12.0 30.0 5.6 30.0 Met .0 .0 .9 2.3 .0 .0 Asp 5.3 3.7 7.1 17.8 8.9 44.8 Phe 1.5 1.1 2.4 5.9 4.8 24.5 Glu 4.5 3.2 6.9 17.2 9.7 49.0 Lys 8.1 5.7 4.6 11.4 3.9 19.8 Tyr .5 .4 .9 2.3 .8 4.3 Ada 4.0 2.8 3.2 7.9 3.4 17.4 Arg 3.1 2.1 1.6 3.9 3.6 18.3 His .0 .0 .0 .0 1.2 6.2 Cys .0 .0 .0 .0 .0 .0 Amino Acid analyses determined by Glc. 100 b. Lysozyme To determine whether NaClO2 oxidation degrades protein, 1 treated chicken egg white lysozyme with NaClO at 75 C for 30 minutes and then 2 chromatographed it on a Sephadex G~50 column. During the reaction the protein first precipitated and the solution turned yellow from the C102 gas. The protein redissolved in about 20 minutes. After the addition of a few milligrams of ascorbic acid the solution went colorless and almost clear. The elution profile of the treated and untreated lysozyme is shown in Figure 15. This shows that the protein was degraded some- what but not into small pieces. More experiments would be needed to determine if NaClO2 oxidation breaks specific peptide bonds. But as NaClO2 does seem to break some peptide bonds the mechanism by which the NaClO2 is releasing the protein from the cell walls may be by peptide bond breakage, breakage of phenolic cross links or both. It seems unlikely that the peptide of cell walls is breaking at all the tyrosine residues since there are many tyrosine residues in the tryptic peptides characterized by Lamport (11). This would cause some of the peptides released by NaClO to be very short, but, as will be described later, 2 they appear to be large. c- Comparison of the Effects of NaClO2 and N-Bromosuccinimide Another chemical reagent specific for aromatic compounds, N—bromosuccinimide (104), also released cell wall glycopeptides. A treatment of sycamore maple cell walls with 50 mM N-bromosuccini- mide in 50% acetic acid for 20 hours at room temperature solubilized 50% of the Hyp (compared with 50-75% release in NaClO2 as mentioned 101 .N0H0mz mo uomwmm mau Scam ou mmmerHummom mH msyuomyH mmummpuoo mo mHHmoHa EOHuEHm may .Aqu aflMIMM yusoa mo poaumE mau ya pmoHEpmump mm3 EHmuoum .EEEHoo omt0 xmmmammm m :0 mmammuwoumEouao mmB uoEmoHQ may .uommmum umeoH on mp3 N0H0 mo MOHom 3oHHmy mau HHuoo pHom oHauoomm mo EOHuHmpm mau ya pmumEHEpmu mm: EOHuommu may .mmuscHE on you 0 my um pHmm oHumom NH EH 0H0mz auH3 vmummuu mp3 mEyNomyH mo HOEVHHm HHMEm < N .mEyNomyH pmummwu 0H0mz mo mHHmoum EOHusHm onto xmmmammm .mH wuste 102 0v Om 9:29 8385 369:5 \/\t ooaomtVddb. mH muanm 8952 many ON 0. H H H1 H1 00 Day 103 earlier). This reagent may be releasing the peptides solely by breakage of tyrosyl and histidyl bonds but may also be breaking the phenolic crosslinks that I have postulated. A study of these peptides may be undertaken fruitfully in the future. 4. Fractionation of the NaClO Released D O 2 Peptides hy Size a. Chromatography on Bio-Gel A .5 m and A 1.5 m The material solubilized by NaClO2 oxidation from tomato cell walls can be divided into two major fractions by chromatography on a Bio-Gel A .5 M or A 1.5 m column as shown in Figure 16. These columns exclude spherical molecules of molecular weight 500,000 and 1,500,000 respectively. The majority of the uronic acids elute near the void volume on either of these columns, but in some experiments, especially the ones in which the NaClO2 treatment was short, uronic acids appeared throughout the column effluent. The ratio of void to retarded Hyp peaks varied from one experiment to another, perhaps due to the variation in cell wall cross linking via phenols. Some preparations of cell walls were whiter than others. There was also variation in the loss of lysine during the NaClO treatment. This also appeared to be connected 2 to the color of the walls. The browner the walls the less the loss of lysine, as though the phenols competitively protected the amino group from oxidation. The material in the void volume is rich in Hyp, arabinose, and galacturonic acid (see Table 17); whereas, the retarded material is less enriched in Hyp and almost devoid of galacturonic acid. 104 .Eya No moEmmmHE mau you mmnyHmEm mp3 EoHuomum aomm .mEoomH> yum> mums mmpHummEoowa may EoHuEHom mmumqumoEoo EH mm EEEHom mau Ou pmHHEEm mm3 uomuuxm mau mo mHHautmEo qua< .pmNHHyanyH mEm .pmnyHmHm .mHom oHauommm auHs pmoopmu Ems oomuuxm may .mmuEEHE me How 0 My um NOH0mz auH3 pmummuu Emau mums mHHm3 pmNHEHuomEmp may .mEOumom auHB mmHHp pEm .Hmuma auH3 pmamms .EOHuEHom mummeo EEHEoEEm NEHHHoa auH3 pmuomuuxm mum: mHHm3 HHmo OumE0u pmammB mo mEmuw mmuay N .E m. E Hm0ton Eo mmmHuEmEoowa mmuomuuxm 0H0mz Hmuou mo yammuwoumEoHa0 .sH .stHe 105 On eH mustm 8220a 20> H H .5952 men... ow om 8 e __ _o suun anitoies n_>I 106 Table 17. Amino Acid and Sugar Analysis of the NaClO Extract Eluting in the Void Volume of an A 1.5 m Column 2 Mole Percent Per 30 Hyp Ala trace trace Gly trace trace Val 5.2 2.7 Thr 1.3 .7 Ser 16.2 8.5 Leu 1.4 .8 Ile .8 .4 Pro 1.7 .9 Hyp 56.9 30.0 Met n.d. n.d. Asp 4.4 2.3 Phe trace trace Glu 1.0 .6 Lys 5.7 3.0 Tyr 1.7 .9 Ada 2.8 1.5 Arg .8 .4 His n.d. n.d. Cys n.d. n.d. Ara 48.9 Rha 8.1 Fuc .0 Xyl 1.4 GalU 28.0 Man trace Gal 9.5 Glc 4.0 Note: Amino acid analysis by G.L.C. methylsilylated methyl glycosides. Sugar analysis by G.L.C. as tri- 107 Thus, there appear to be two size classes of Hyp containing peptides released by the chlorite oxidation, the high molecular weight peptides being associated with uronic acids, the low ones to a lesser extent. If the association of the uronic acid with the protein were proved covalent, then the place of the protein in the initial model of the primary cell wall of Keegstra 3&431. (2) would be verified (Figure 1). If it is only associated because of its similar chromatographic proper- ties, a revised model would be necessary. NaClO2 also solubilizes a large portion, 50% or more, of the Hyp- containing protein of tobacco and sycamore-maple cell walls. The Hyp in sycamore-maple extract behaves chromatographically the same as Hyp solubilized by NaClO2 from tomato but that of tobacco does not appear to be of as high a molecular weight. b. Chromatography of the High Molecular Weight Peptides after HF Treatment If the NaClO2 solubilized glycopeptides eluting in the void volume of an A .5 m column are deglycosylated by a 1 hour treatment in HF at 0 C the majority of the peptides elute in the void volume of a Sephadex G-50 column (Figure 17). Thus, it appears that NaClO has not severely 2 degraded the peptide backbone of the protein. 5. Co-purification of Hyp-Containing Peptides with Uronic Acids One way to test whether or not the protein and polysaccharides are covalently attached is to chromatograph the complex under conditions which would retard the protein but not the carbohydrate. As the sugars include galacturonic acid, retardation of the sugars along with the 108 .mym How pmymmmm mos uEmEHmmm EEEHoo may .EEEHoo omt0 xmmmaamm E E0 pmaamuwoumEouao mEm mHmm oHumom z H.o EH pm>Homep mHm3 mmpHuEmE mau ya mau mo EOHuEHoEm>m Hmum< .qua H How 0 0 on ma EH uEmEummwu ya mmumHymoowamm mums AwH muomev EEEHoo E m. < Em mo mEEHo> pHo> mau EH wEHuEHm mmmHuEmE mmNHHHaEHom 0H0mz may .EoHumHymoowamp mm umumm EEBHoo E m. < Em «mo mmpHummmooyHm uame3 HmHEomHoE umawHa mau mo mHHmoum EOHuEHm omt0 Empmanmm .HH musmHm 109 00 NH muEme .3832 men... On 0? On snun eAuotes .& >1 110 protein on a cation exchange column would be a strong indication of covalent attachment of the two. Chromatography of the void fraction from an A .5 m column on a CM cellex column equilibrated in 50 mM pH 4.2 sodium acetate buffer and on a cellex P column at pH 2 showed, however, that both the protein and polysaccharide were negatively charged or that protein was prevented from binding to the column because of attached uronic acids. a. Co-chromatography on DEAE Cellulose (Anion Exchange) On DEAE cellulose chromatography at pH 5.2 most of the Hyp is retained, as are most of the neutral sugars and all of the galacturonic acid. The elution profile varies from experiment to experiment but there is consistently the major peak of Hyp eluting with about 0.25 M NaCl. Often there is a peak of uronic acids following this (Figure 18), but in some experiments this was not present (Figure 19). If the Hyp- rich peak is rechromatographed on a shallower gradient, .l666-.5 M NaCl, there is still coincidence of uronic acid, neutral sugars and Hyp (Figure 20). The amino acid composition of the Hyp-rich peaks from DEAE cellu- lose columns of various experiments are given in Table 18. This frac- tion is extremely rich in Hyp (over 50 mole percent) and rich in serine (15 to 20 mole percent). This composition is very reminiscent of the tryptic peptides described by Lamport (11). The tryptic peptides of Lamport had a maximum length of 15 amino acids but the material in the peak of Hyp from the DEAE column is excluded from a A .5 m column, which only excludes molecules which act as though they are larger than 111 .Eym pEm pHom oHEoHE .mumwom pom omymmmm mums mEoHuomwm may .uEmEHm mm mmHHOHao EEHpom mo uEmHmmuw ummEHH m mEHmE mmoHEHHmo m pHo> mam EH wEHuEHm HmHumumE man no mEmeHHHHE yEMHy .mmoHEHHmo m E m. < Ho mHHwoum EOHuEHm .wH muEme 112 mm. .002 pm pt 0H muEwHy .3632 33. p0 28 2:05 a»: suun ”£40193 113 .NH muEme How mm mEoHquEoo .mmOHEHHmo m E m. < mo mHHwonm EoHuEHm .mH muome 114 mH ouamHe .3632 one... On 0? .Jow ’ . I I V . I «land. A. H H. .H H . .(r/esq 285 H. x \H .1. H 83m .8302 HH . H .H dHH\ 502 1.. 2nd- 5 a»... mmOHHHHHmO m suun aAttotaa 115 .2 m.0 ou z 0.0 Emau Hmaumu mmHuoHao EEHpom z m.o om 00H.0 Eon uEmemuw m wEHmE mmoHEHHmo mm mEau uEmemuw may «0 EOH mam Eoum EOHuEHom mau mmxmu aoHa3 .HH son HmEmn ouEm Em auH3 maEu memHqumo mau Eoum pm>0Emu mm? uEmHmmuw may .0 e um mymm m How 2mm 000.0m um pmeMHqumm mm3 mHay .qn.H mo yuHmEmm m auH3 EoHuEHom H0m0 HE N No mom Eo use mm3 .HE H EH wE m yHmumEonummm .HmHumumE mau mo EoHuuoE < .pmNHHyanmH mEm .pmNyHme .mmHooE mumz HmH mHEmev EEEHoo mmOHEHHmo moEmu mm3 uEmHmmuw may .0 e um Emu OOO.O¢ um ammo m pow mmemHqumm mma HmHHmumE mHau mo wE m yHmumEHNoHEEE .HOEmaum NON auHs pmammz mmB mumuHEHomHE may .HoEmaum EH NON EOHuEHom mau wEHamE ya pmumuHEHomHE mama mpoaumz pEm mHmemumz EH mmaHuommp mm mmummmua mumEyHoE HHm8 HHmo mHaEHom mummeo EEHEoEEm may .mumHmNo EEHEoEEm wEHHHoa ya wHHmB HHmo oumEou Eoum Omuomuuxm EHuomE xHEa mo EOHumeMHHuEmo uEmHOmww H0m0 .HH ouame 130 V; n; ma: H00\o. Hzmcmo «N muEme cm 8.08 ON H 39:52 many Om d 5550 ON H: d 1 Eco 3:95 131 .AmH mHamy .m.ov mHmyHmEm umauusm How mmHoom mama pmummHmEH mEOHu tomum may .HH 30Hm HmEmO ouEm Em wEHmE pmuomHHoo mms uEmHmme may .cN pEm mN mmeme How mmaHuommm mm uEmemuw H0m0 m EH mmemHqumo Emau pEm .pmNHHyanyH .mmNyHmHm .pmHooE mums .AOH mHEmeV mmOHEHHmo MEMO Eo ammo aha HanmE mau Emau EoHumEuEmoEoo uHmm HmawHa m um pmuEHm mums aoHa3 gym wEHEHmquo mEOHummum may .mmOHEHHmu moamu mmB mumuHEHomEE may .EEEom> HmpEE mmmmmwmm pEm pHom oHumom HmHome auHB pmNHHmEuEmE mma mEEuxHE EoHu tommE mau mEan 0 man Hmum< .Eoow 0 NM m EH mEan 0 How wEHEEHum ummH Emau pEm 1Vmmmz EH 2 H mmmE mm3 EOHmEmamEm may .mmEEom mumuHEHomEE memH m momz mau Ho EoHuHmpm mau Eon: .aomz z N mo HE N.O pEm “mama mo HE 0.0 .HoEmaum «0 HE N.O pmmmm mm? aoHa3 ou HmH>oEoHE HE m m EH Oman mo HE H EH 0m>HommHm mum? EEEHoo mmoHEHHmo HEMO m Eoum HmHEmumE ammE gym may no wE EmmumHm ou Emy .EEEHoo mmOHEHHmo mmmn m Eoum amma Ohm pmumEHEHHmtm mau mo EoHquE mHaEHom mau mo Omt0 Empmammm Eo mHHmoum EOHuEHm .ON mEEwHy 137 Oh aw ..awHe H xmmn. H 59:32 many 00 d O? 20> suun Wines 1 >. I 138 The peptide must, therefore, have been degraded by the alkaline treat- ment, as before alkali treatment the totally deglycosylated peptides were excluded by a G-50 column (Figure 17). The sugar and Hyp composi- tion of the larger of the two peaks (Table 20) shows that much of the galactose, rhamnose, and arabinose that are associated with the peptides no longer co-chromatographs with the Hyp. In the void volume of the G-50 column there is a very high ratio of arabinose to Hyp, 21 ara/Hyp indicating the presence of polysac- charides not linked to peptides. The material insoluble in DMSO B-elimination reaction mixture eluted in the void volume of a Biogel A .5 m column and had a composi- tion much enriched in sugars as compared with the starting material but still contained some Hyp, probably due to incomplete B-elimination. It is unlikely that the reaction would go to completion as the highly glycosylated Hyp-containing peptides are insoluble in the B-elimination reaction mixture. Since the alkaline conditions used to bring about B-elimination of the glycopeptides lead to some peptide bond breakage the results of the experiment are not conclusive. They are, however, consistent with the B-elimination of a polysaccharide from the glycopeptides. 7. Partial Characterization of the Lower Molecular Weight Glycopeptides The ratio of Hyp retarded by the A .5 m column to that excluded by the column varies from about 1:1 to 2:1. Thus, these smaller pep- tides are a significant portion of the chlorite extract. They do not 139 Table 20. Sugar Composition of Hyp-containing Fractions Before and After B-Elimination Total Soluble Peak 1 on Before in Reaction Sephadex G-50 Elimination Mixture (Figure 26) Ara/Hyp 6-8 5.1 4.9 Glc/Ser l trace 0.3 Rha/Ser 0.6 trace - 140 appear to arise from the larger peptides as the ratio of large to small was smaller when the chlorite treatment was carried out for only 8 minutes. The larger peptides after this short treatment did not peak as sharply in the void of an A 1.5 m column as the 30 or 45 minute chlorite extracts. The majority of the Hyp retarded on the A 1.5 m column eluted in the void volume of a Sephadex G-50 column (Figure 27). The amino acid composition of the peptides eluted in the void volume of the G-50 column was different from that of larger peptides discussed previously (Table 21), the smaller peptides being richer in lysine and valine and a little poorer in serine. When the peptides eluted in the void volume of a Sephadex G-50 column were chromatographed in the same way as those in the void volume of the A .5 m column on a DEAE cellulose column the peptides split into two fractions, one which was not adsorbed to the column and a broad adsorbed peak (Figure 28). Again, there is co-chromatography of uronic acids and Hyp although there is a lower proportion of uronic acid to Hyp than for the larger peptides. Little of either of the two Hyp-rich fractions was retarded on a Biogel-P100 column. 8. Methylation of Glycopeptides If a unique oligo- or polysaccharide is responsible for linking extensin to the hemicelluloses or pectins of the cell wall, methylation analysis of NaClOz-released glycopeptides should reveal its presence, if not its complete identity. As mentioned previously, the data put forward by Keegstra g£_§l. (2) to support their proposal that a 3,6-linked arabinogalactan links extensin to the pectin of the wall was 141 .Eym How mmymmmm mum? mEoHuomEH may .mEEHo> mmOEHoEH mau EH pmuEHm AmuoEmoEm EoHummeo muHEoHaov HmHEmumE mmHmHuEmmHEE anua mo ammo < .EoHummEH mamHmz EmHEomHoE EmSoH mau EH yuHmoowH> aqu EmHaoue oE mm3 mumay .Ont0 Emomammm Eo pmammuwoumEouao mEm OHom oHumom z H.O mo HE N EH a: memu Emau pEm mmNHHHanyH HuEm HomHooHH mumz SH 93me mmmv E m. a Eo ammo my: mEommm mau Eowm mEOHuomEm may .Omt0 xmmmaemm Eo EEEHoo E m. < NOH0mz mam mo mHHHoEE EoHuEHm may Hm0ton m ya meHmumE mmOHuamE omNHHHaEHom .HH mustE 5N mEEme 142 8.08 H J .3632 Bay Om 0v Om H H H l . T. .. N a m. w m. n m. I v a EH: ..0 Table 21. 143 Comparison of Amino Acid Analyses of the Various Sized Frac- tions Obtained from Cell Walls by Sodium Chlorite Extraction Excluded by A .5 m Excluded hy G-50 Retained by G-50 Mole Per 30 Mole Per 30 Mole Per 30 A.A. Percent Hyp Percent Hyp Percent Hyp Ala .0 . .0 .0 3.8 6.0 Gly .0 . .0 .0 6.3 10.0 Val 5.2 2.7 6.6 3.7 4.8 7.6 Thr 1.3 . 2.5 1.4 4.8 7.8 Ser 16.2 8.5 12.4 6.8 12.5 20.0 Leu 1.4 . .8 .5 4.4 7.0 Ile .8 . .6 .3 2.8 4.4 Pro 1.7 . 3.9 2.2 5.0 8.0 Hyp 56.9 30.0 54.4 30.0 18.8 30.0 Met .0 . .0 .0 .0 .0 Asp 4.4 2.3 2.9 1.6 8.2 13.2 Phe .0 .8 .4 1.9 3.0 Glu 1.0 .7 .4 7.3 11.7 Lys 5.7 3.0 9.0 5.0 6.6 10.6 Tyr 1.7 . 1.2 .7 .8 1.3 Ada 2.8 1.5 4.1 2.3 4.1 6.6 Arg .8 . .0 .0 1.7 2.8 His .0 . .0 .0 .0 .0 Cys .0 . .0 .0 .0 .0 Amino acid analyses were performed by gas liquid chromatography as described in the Methods. 144 .mmHom oHEOHE pEm mumwom .Eym How pmymmmm mmz mumEHm may .2 m.OtO .H0mz mo uEmHomEm EmmEHH m auHB omuEHm pEm EEEHoo mEOHEHHmo m0(8)’ one can estimate that there is more than enough T-galactose to account for one T-gal for all the seryl hydroxyl groups present. This concurs with the result that hydrazinolysis of the glycopeptides(l4) 147 .HouHmoeH .HH .oHo mmum Ham .Hmo mmum Hem .Hmoim.m Hm~ .oHoie.e ANN .Hmoie.e HHN .emHmHuemeHas How .emHmHuemeHe: HHH .Hmoie HHH .muaim.m.~ HHH .UHoie HeH .Hmeie HmH .emHHHuameHaa HHH .HmHHHuemeHea HmH .Hmoim HHH .mnmie.~ HHH .emHmHHameHes HOH .Haxie no N Ha .Hmoie Hm .muO Nm.O moon 0 m Eo mmuomfiEH mums H: th EoHumHyumom Emuw< .mmoaumz mau EH mmaHEommo mm mmumHyaumE pEm .mmNyHmHo .mmHooE mums mN mEEme EH gym uawHH pmumeHmmp mEoHuomEm may .uEmHmme yuHmEmm H0m0 m EoEH EOHuomEm mym mmEmo mmmH may 00 mHmyHmEm EOHumHyaumz .HN muawHH 148 ON mHEwHy AmmuEEHEv mEHy Oe ON [4| 3 E 33 a. H. H. o v asuodsag xoaoenaq 149 causes 80% destruction of the serines, indicating 80% glycosylation of the serines. I had expected that the galactosyl-serine linkage would be broken in a B-elimination reaction caused by the strongly alkaline conditions of the methylation procedure. Lindberg (97) reported that O-acetyl groups are cleaved during the methylation reaction. But Baenziger and Kornfeld (116) found no evidence of B-elimination when working with O-glycosidically linked oligosaccharides of IgA, and Lindberg (97) finds no evidence of the destruction of methyl esterified uronic acids which are known to be susceptible to B-elimination induced by alkali. Present in only slightly lesser quantities than T-galactose are 3,6-; 3-; and 6-linked galactose (Figure 29), the sugars characteristic of the oligosaccharide Keegstra g£_§l, hypothesized links extensin to the pectins of cell walls. There are in addition small amounts of the sugars characteristic of pectins: 4-linked galactose, 5-linked arabinose and 2,4-1inked rhamnose, but only a trace if any of 2-linked rhamnose. There is also the unexplained presence of 4-1inked glucose. The denser fractions (see Figure 23) off the CsCl gradient of the Hyp-rich peak on DEAE cellulose are much richer in sugars and poorer in Hyp. These fractions also contain 3,6-; 3-; and 6-1inked galactose, but lesser amounts of 2- and 3-1inked arabinose, consistent with the lower percentage Hyp (Figure 30). There is a much increased presence of 4-linked galactose, 5-linked arabinose, 2,4-linked rhamnose, and the appearance of a small amount of 2-linked rhamnose, indicating a greater proportion of pectin. Again, there is the unexplained presence of 150 .aN muEme mmm mmHuHuEmOH Home Mom .mooaumz mau EH pmaHEommp mm pmumHyaumE pEm .OmNyHmHO .omHooa muma MN mEEwHy EH my: mmEmo omumeHmmO mEOHuome may .uEmHmme H0m0 m Eoum mEOHuomEH gym EmmEmo mau mo mHmyHmEm EOHumHyaumz .om orste 151 on «pawHE H8335 3:. OO OHH ON O 1 «H SH 822 :29: H. :9. us on v an n n 3 o 1 m d o u s a 152 4-linked glucose, suggesting that either tomato walls are a little dif- ferent from those of sycamore-maple, or that the amylase treatment used by Talmadge.g£_§1, (99) was removing something other than just starch from the walls. The dense fraction off the CsCl gradient of the pectinaceous frac- tion on DEAE cellulose (see Figure 25) contains little 3,6; 6-; and 3-1inked galactose but a large amount of 2-linked and 2,4-linked rhamnose, T- and 4-linked galactose, and again 4-linked glucose (Figure 31). The GLC profile for this fraction resembles that of methylated pectin released from the cell walls by boiling ammonium oxalate (Figure 31). b. Smaller Molecular Weight Peptides The smaller molecular weight peptides (in the void of Sephadex G-50, retained on A .5 m), which were adsorbed on DEAE cellulose and purified from any contamination by free polysaccharides by CsCl centri- fugation, were methylated and found to contain almost exclusively the T-, 2-, and 3-1inked arabinose expected for Hyp-arabinosides and T-galactose adequate to account for the hydrazinolysis data indicating 80% glycosylation of the serine residues of the peptides. Only traces of the other linkages present in the large molecular weight peptides were present (Figure 32). 153 .ummuuxm mumHmNo EEHanEm pmammz "momma Eouuom .mN mEEme EH omummeEH mm pmHoom mEOHummum mmEmo may ”momuu Eoy .mHHmB HHmo oumEou EoEH ummuuxm mummeo EEHEoEEm pmammz mau auHB uEmEmem EEEHoo mmoHEHHmo mmmn m mo EOHuomEm mEomomEHuomm mau mo EomHEmEEoo E .Hm mustE 154 O0 Hm mustE AmmuEEHEV mEHy Oc nu «a ‘2 Op hp 9 up my = O» 0y asuodsag 10338180 155 .aN mEEwHy mmm mmHuHuEmmH ammo pom .mmmHuEmE uames EmHEomHoE umHHmEm mau mo mHmyHmEm EOHumHyaumz .Nm mEEwHy 156 Nm mEEth AmmuEEHEV mEHy om cc ON ‘ OR a a ha: 0‘ I C C asuodsau ionoanaq 157 D. Discussion The experiments in Section II-C indicate that NaClO2 oxidation solubilizes large fragments of extensin from cell walls. These frag- ments are for the most part extremely rich in Hyp, over 50 mole percent, and contain the amino acids found in the short tryptic peptides described and sequenced earlier by Lamport (11). The peptide portion of the glycopeptides solubilized by NaClO cannot have been degraded to 2 a great extent, since the size of lysozyme treated with NaClO2 under the same conditions is not drastically reduced. However, lysine is changed to a—amino adipic acid, and tyrosine and the unidentified amino acid are destroyed or changed to aspartic acid. From the literature (see Section II—A) one would not expect NaClO oxidation to change the 2 polysaccharides attached to the peptides and so the oligosaccharides of the glycopeptides released should represent those present in un- treated walls. Unfortunately, the conditions used for the NaClO2 oxida— tion are such that pectins whether or not linked to protein would be ' extracted from the walls along with the glycopeptides. Thus, strong evidence for the covalent linkage of any oligosaccharide to the peptides must be provided before any conclusions-can be made about the linkages of extensin to the wall polysaccharides. In addition to the problem of co-extraction of free polysaccha- rides and glycopeptides, there is the problem of random breakage of the polygalacturonic acid chains. Thus, if a peptide is linked directly or indirectly to the pectic polymers the size of its oligosaccharide side chains will be very variable. 158 Some evidence that the peptides are linked to a polymer containing galacturonic acid was given in Section II-C-5. The CsCl density grad— ient centrifugation experiments provide the strongest evidence for the attachment of pectins to extensin: 1. When the major peak fractions of hydroxyproline from a DEAE cellulose column (Figure 18) are centrifuged in CsCl, there is galac- turonic acid associated with the resulting major Hyp peak (see Figure 23) at a density much lower than 1.6 g/ml. 2. There is Hyp which behaves as though it has a greater density than this major peak, indicating more highly glycosylated peptides (Figure 23). 3. The more tightly adsorbed pectinaceous material on a DEAE cellulose column contains a lesser amount of Hyp which floats away from the majority of the pectin on CsCl centrifugation but this Hyp also is associated with galacturonic acid (Figure 25). Methylation analysis of the Hyp peaks (Figure 29) from the CsCl gradients show that they contain the sugar linkages of the 3,6-linked arabinogalactan proposed to link extensin to the pectins. These sugar linkages are present in only trace amounts in the pectic fractions not associated with Hyp (Figure 31). The Hyp-containing peptides also con- tain T-, 2-, and 3-linked arabinose and enough T-galactose to account for a single galactose residue on every serine residue (Figure 29). These results strengthen the possibility, suggested by W. D. Bauer (personal communication) that extensin may allow cell wall growth by a transglycosylation mechanism. Growth would be mediated by an 159 enzyme which can either be glycosylated at its active site by a single galactose or a 3,6-linked galactan. Such an enzyme would be similar to dextran synthetase which uses sucrose as its substrate to make dextran and fructose, thereby dispensing with the need for a sugar phosphate intermediate. The interchange of galactose for the 3,6-linked galactan would make or break cell wall cross links thus allowing extension. There are still quite a few questions to be answered before we can truly describe the structure of extensin in cell walls. It has become apparent from the work described here that some type of linkage in addition to glycosidic links are holding the protein in the wall. What are these linkages? The amino acid composition of the residue from cell walls after they have been treated with liquid HF shows that although there is very little sugar detectable there is only 40% of the residue which can be accounted for by amino acids. The rest is unidenti- fied. When sycamore-maple or tomato cell walls were methylated and extracted with chloroform:methanol the residue again contained most of the cell wall protein along with some sugars, but surprisingly (99) there was relatively little glucose present. Most of the sugars were arabinose (T, 2 and 3-1inked as in the Hyp—arabinoside). This residue appeared microscopically to be cell wall shaped (Figures 33 and 34), but from the sugar linkage analysis contained very little cellulose, again indicating that the protein holds together in the shape of a cell wall in the absence of polysaccharides. This suggests to me that there is an unidentified cross link between the protein molecules of the wall. 160 .mN mHEme EH mm mmHuHuEmpH ammm .mHHmz HHmo mEoEmmym pmumHyaumE Ho mEmHmmE mHaEHomEH mau mo moEmEmmEEm oHEoomouoHE may HuawHE Eouuom .mHHm3 HHmm mEoEmoym mmumHyaumE mo mEOHmmE mHaEHomEH may uuHmH Eouuom .mHHm3 HHmo muoEmoym mmummEuEE mo moEmEmmEEm oHEoomouoHE may HuawHE moy .mHHmB HHmo muoEmoym mo mumwom omumHyaumE.mHaEHom HoEmaumE\EEomouoHao may HuHmH eoy .mHHm3 HHmo muoamoym mo EoHuHmeroo Emwom mmumHyaumE pEm moEmEmmEEm may .mm mEEwHy H.325 «a: OO Ov ON on a O" In: On .- ”. u a O 1"} .0 on v. n"— ."n O..— n—l’n—E 2. 0 an on C an o a... asuodsau 10393330 162 .mmHuHuEmmH ammm How ON mEEme mmm .mHHm3 HHmo oumEOE mmumHyaumE mo mEpHmmE mHaEHomEH mau mo moEmEmmEEm oHeoomoEoHE may .mHHm3 HHmo oumEou pmumHyaumE mo mEmHmmE mHaEHomEH may .mHHm3 HHmo oumEou mmuomuuxm NOH0mz mo mHmyHmEm EOHumHyaumz .mHHm3 HHmm oumEou mmummEuEE mo moEmEmmEEm oHEoomouoHE may .mHHm3 HHmo oumEou mo mumem pmumHyaumE mHaEHom HOEmaumE\EEoonoHao may "uawHE Ecuuom uume Eouuom HummH EmuEm0 "uawHE Eoy "ummH eoy .mHHm3 HHmo oumE0u mo EoHuHmoEEoo Emwam mmumHyaumE mEm moEmEmmEEm may .Hm mustm ...... .HH 8&2 33:55 2.: S .H . 8 HH 3 HH ness—-..: an... H. H H On .0 On “a. .HONEIh-l I... on c. I a u H asuodsaa 10:; oaaaa 164 The resistance of these cross links to both HF and the strongly basic conditions of methylation analysis excludes the possibility that the cross links are either glycosidic or ester linkages. If the cross links were direct condensations between amino acids of adjacent pep- tides as in collagen (117) there is still the presence of the unidenfi- fied material in the HF residue of cell walls. My failure to account for all of the cell walls as sugar and amino acids, points to a firmly attached additional component. Are these cross links artifacts of the procedures used to isolate or treat the walls or are they naturally occurring? This question remains unanswered. Plants produce an abundance of secondary products including phenolic compounds which are compartmentalized in the intact cell but upon cell breakage are released and can interact with proteins. In the case of suspension cultures, the cells are surrounded by the same medium for anywhere up to 1 month. Therefore, any excreted prod- ucts or released products from dead cells are in contact with the cell wall for a long period of time. The reactions of proteins in HF, especially in the presence of large amounts of sugars, has not been well-characterized but could by some unknown mechanism cross link the protein. This would not explain the retention of cell wall shape after removal of most of the wall poly- saccharides by methylation. A different unexplained mechanism would have to be involved for the generation of cross links under alkaline conditions. Thus, it is unlikely that the cross links are artifacts of the cell wall treatments (HF or methylation). The possibility that the 165 cross links are generated during the cell wall isolation cannot be excluded, although, as mentioned earlier, all my precautions to prevent interaction of soluble cell contents with the walls during their isola- tion failed to prevent the cross linking. This leaves the possibility that the cross links are formed be- cause the cells, being in suspension culture, were surrounded by their own by-products or that the cross links are normally present in the walls of the cells that these cultured cells represent in the whole plant. Whitmore (118) has investigated the occurrence of lignin in wheat coleoptiles and its effect on cell elongation. Coleoptiles are one of the most popular plant parts for the study of the plant hormone auxin since they are very responsive to the hormone yet are one of the simplest tissues. Whitmore fed radioactive phenylalanine (a known lignin precursor) to coleoptiles and then partially characterized the labelled cell wall products by their extractability in various solvents. About one-fourth of the radioactivity was found in lignin-like molecules or precursors to lignin. These lignin-like molecules were only extract- able in alkali, not in hot water, indicating that my ammonium oxalate extractions would not remove them (c.f. the low percentage of sugar in depectinized cell walls-~Tab1e 15). Whitmore found a residue which he called lignin that was insoluble in all his solvents including 6 N HCl at 100 C., 72% H2804, and 24% KOH. Almost all of the counts in the intact walls could be removed by a prolonged (4 hours) sodium chlorite treatment alone. Thus, the presence of lignin and compounds of a pheno- lic nature in the walls of growing tissue has been reported. 166 CSté g£_gl. (119) have used very concentrated aqueous (up to 80%) HF, as Fredenhagen and Cadenbach suggested (22) to remove the sugars from wood to allow electron microscopic observation of the deposition of lignin. Their electron micrographs show that the cell walls, even though devoid of sugars, retain their original shape. Plants and plant tissue cultures are renowned for their production of phenols. There have been many reports of phenol production by suspension cultures (120,121,122). The presence of phenols in walls of suspension cultured cells has not been reported specifically, but Nimz g£_§l, (123) have detected hardwood-like lignin in washed, dried soybean suspension cultures by 13NMR spectroscopy. Another indication that there could be phenolic compounds in cell walls comes from the work of Burke g£_§l, (124). Their estimate of the protein content of cell walls of rye grass cultures by the Lowry assay was three and a half times that of Smith and Stone (125) who determined total nitrogen. The sugar analysis of both groups agreed well, thus the protein estimates should be comparable. It is likely that the Lowry assay was measuring phenolic compounds extracted from the walls by the alkali of Lowry reagent A (46). In the same report (124), Burke g£_§l, state that they could account for between 18 and 107% of the extracellu- lar polymers precipitated by 70% ethanol from the culture medium of a range of plant suspension cultures as polysaccharides and protein. For all but two cultures they accounted for less than 60% of the polymers. What is the identity of this unidentified polymeric material? Again, they assayed for protein using the Lowry assay and found high protein 167 contents as they did in cell walls. Yet, in the only case in which protein was determined by total nitrogen, sycamore-maple, the protein content of the analyzed extracellular polysaccharides was less than 1%. In all the cultures tested by the Lowry assay, the protein content of the extracellular material accounted for 20—40% by weight. Since the extracellular polymers of suspension cultured cells are in most cases very similar to those in the cell walls (124,126), it is likely that the unidentified polymers are also present in cell walls. Phenols are readily oxidized to quinones which Morrison and others have found (127) readily form 1,4 addition products with amdno groups and thiols, and an uncharacterized addition product with tyro- sine or other phenols. In plants there are enzymes which oxidize phenols to orthoquinones which like para-quinones are very reactive with amino groups (128). 0 on. o R 0"l-ll‘hlli'l2 H. on _°Z_. a O a " :in tt’u H, :21 .. 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