Agric. Biol. Chem., 43 (3), , Carbohydrate Sequence of a Soybean 7S Protein. Fumio YAMAUCHI and Tatsunori YAMAGISHI

Agric. Biol. Chem., 43 (3), 505-510, 1979 505 Carbohydrate Sequence of a Soybean 7S Protein Fumio YAMAUCHI and Tatsunori YAMAGISHI Department of Food Chemistry, Faculty of Agriculture, Tohoku University Received July 26, 1978 The sequences of three Asn-carbohydrates, IIa, Asn(GlcNAc)2-(Man)e; IIb, Asn(GlcNAc)2 (Man)e; and IIc, Asn(GlcNAc)2(Man), isolated from the pronase digest of a 7S soybean protein (ƒà-conglycinin) were investigated. In each case, methylation analysis of the Asncarbohydrates gave 3,6-di-O-methyl derivative from the N-acetyl-glucosamine residue, and 2,3,4,6-tetra-O-methyl, 3,4,6-tri-O-methyl and 2,4-di-O-methyl derivatives from the mannose residues. The first Smith degradation of all the Asn-carbohydrates gave Asn(GlcNAc) 2- (Man)2 and the second degradation afforded Asn(GlcNAc)2. Partial acetolysis of the Asncarbohydrates followed by deacetylation yielded mannobiose and mannotriose from, Ila, and mannose and mannobiose from lib and IIc. From these results, their possible structures are discussed. A previous paper" reported the separation of three L-ƒÀ-aspartamido-carbohydrates (Asn-car bohydrates) from the pronase digest of a 7S soybean protein (ƒà-conglycinin). Their com ponents were Asn-(G1cNAc)2(Man)9 for IIa, Asn(G1cNAc)2(Man)8 for IIb, and Asn(Glc- NAc)2(Man)7 for IIc. Furthermore, 1-L-ƒÀaspartamido-2-acetamido-l,2-dideoxy-ƒÀ-gluco se, which corresponds to the protein-carbo hydrate linkage, was separated by partial hy drolysis of the Asn-carbohydrates. Glycopeptides from the pronase digest of the 7S were also separated into five fractions," four of which were tripeptide-carbohydrates. Two kinds of tripeptides, Asn-Gly-Thr and Asn- Ala-Thr, were characterized by amino acid sequence analysis. All of the fractions had asparagine as the N-terminal amino acid residue to which the carbohydrate moiety was attached. However, nothing has been known on the chemical structure of the carbohydrate moiety of the Asn-carbohydrates from the 7S soybean protein. This paper deals with the sequence analysis of the carbohydrate moiety by using the 'methods of methylation analysis, partial acetolysis and Smith degradation. MATERIALS AND METHODS Asn-carbohydrates. A 7S protein was isolated from soy bean (Clycine max var. Raiden) by the method of Thanh and Shibasaki.'> Glycopeptides from the pro nase digest of the 7S protein were prepared by the same method as described in the previous paper.') Asncarbohydrates (IIa, IIb, and IIc) were separated on a column of Dowex 50W-X2 with 1mM sodium-acetate buffer (ph 2.6). Sugar standards for methylation study. By partial methylation of methyl ƒ -mannoside, 2,3,4,6-tetra-; 3,4,6-, 2,4,6-, 2,3,6-, and 2,3,4-tri-; and 2,4- and 3,4-di- O-methyl ethers of mannose were prepared according to the procedure of Handa and Montgomery.') Chito biose was prepared by partial acetolysis of chitin.5) After methylation followed by acetolysis and hydrolysis of the chitobiose, 3,4,6-tri- and 3,6-di-O-methyl-Nmethyl-acetylglucosamine were prepared. The methyl derivatives in the Asn-carbohydrates were identified by comparison of the relative retention time of the above standard materials and of reported values of the mannose4,6) and glucosamine7) derivatives. Analytical methods. Hexose was analyzed by the phenol-sulfuric acid method.') Glucosamine and as partic acid were determined with a Hitachi KLB-3B amino acid analyzer after hydrolysis with 4 N hydrochloric acid at 105 C for 4 hr. N-Acetylation of Asn-carbohydrates. Prior to methy lation, the Asn-carbohydrates, were N-acetylated9) in order to facilitate the extraction of the methylated Asncarbohydrates with chloroform. Asn-carbohydrates (5mg of IIa, IIb, and IIc) were dissolved in Iml of 4.5M sodium acetate. Acetic anhydride (100,ul) was added to the solution at room temperature in five equal portions over a period of 1 hr. The reaction was terminated by dilution of the sample with 20ml of water

506 F. YAMAUCHI and T. YAMAGISHI followed by heating in a boiling water bath for 10 min. The reaction mixture was passed through a column (1 x 5 cm) of Dowex 50W-X8 to remove sodium ions. The column was then washed with 30ml of water. The effluent and washings were combined and lyo philized. The yield was 4.5-5 mg. Methylation of N-acetylated Asn-carbohydrates. According to the method of Hakomori10) as described by Spiro,9) the N-acetylated sample (4mg) was stirred in 2ml of dimethylsulfoxide in a glass tube (1.5x15cm) under nitrogen stream. After complete dissolution of the sample, methylsulfinylcarbanion (20ml) was added and the mixture was stirred for 1hr at room tempera ture, followed by the addition of 2.Oml of methyl iodide. After 2 hr, the reaction mixture was put on a column (2x25cm) of Sepahdex LH-20 (Pharmacia Fine Chemi cals) in chloroform. The column was then eluted with chloroform in 2ml fractions. The fractions containing sugar were checked by the phenol-sulfuric acid method and evaporated. The residue was methylated again in a similar manner. The product showed only a slight infrared absorption at 3200 3700cm-1 of free hydroxyls. hydrochloric acid or sulfuric acid followed by neutrali zation with IR-45 or Dowex-1 (carbonate or acetate form) resulted in poor detection of methylated mannose derivatives, especially of tetra-o-methyl mannose. Hydrolysis with 90% formic acid followed by 0.3 N hydrogen chloride6) as described above gave a good result. Gas-liquid chromatography of methylated glucosamine residue. Alditol acetates of the methylated gluco samine were prepared and subjected to gas-liquid chromatography according to the method of Stellner et al.7) The permethylated Asn-carbohydrate (1 mg) was acetolyzed with 0.6ml of 0.5 N sulfuric acid in 95 acetic acid at 80 C overnight. The reaction mixture was then hydrolyzed by addition of water (0.6ml) at 80 C for 5hr. After passing through a column of anion exchanger IR-45 (acetate form), the hydrolyzate in 0.2ml of water was reduced with 10 mg of sodium borohydride for several hours. The product was then acetylated with 0.5ml of acetic anhydride at 100 C for 2hr. Gas chromatography was carried out on a glass column (3mmx2m) packed with 3 % ECNSS-M on Gaschrom-Q (100 `120mesh) at a constant temperature, of 190 C. The second glass column (2.5mmx1m) Gas-liquid chromatography of methylated mannose residue. The methylated mannose residue was determined, at first, in the derivative of methyl mannoside.4) The permethylated Asn-carbohydrate (1mg) was methanolyzed with I N methanolic hydrogen chloride in a boiling water bath for 1hr. After removal of the hydrogen chloride with anion exchanger IR-45 (carbonate form), the methanolyzate was injected to a Yanagimoto chromatograph, model GL-4A PTF fitted with a flame ionization detector. A column of stainless tube (3mm x 1m) was packed with 5% NPGS on Chromosorb W (AW, 60 `80 mesh) and operated at an initial temperature of 160 C, increasing at the rate of 6 C/min to 210 C. The methylated mannose residue was also determined in the derivative of alditol acetate.6) The permethy lated Asn-carbohydrate (5 mg) was hydrolyzed with 90% formic acid at 100 C for 2hr. After rotary evaporation under reduced pressure at 40 C to remove the formic acid, the residue was hydrolyzed with 0.3 N hydrochloric acid at 100 C for 12 hr. The hydrolyzate was evaporated to dryness under reduced pressure at 40 C and dried in a vacuum desicator. The residue was dissolved in 0.5 ml of water and reduced with 5 mg of sodium borohydride for 1 hr. The reduction product was then acetylated with 0.5ml of pyridine at 100 C for 10min. Gas chromatography was carried out on a column of glass tube (2.5mmx1m) packed with 3 ECNSS-M on Gaschrom-Q (100 120 mesh) at a con stant temperature of 185 C or at an initial temperature of 170 C, increasing at the rate of 4 C/min to 210 C. Hydrolysis of the methylated sample with 1 `2N was packed with 5% OV-17 on Chromosorb G (AW- DMCS, 100 mesh) and operated at an initial tempera ture of 180 C, increasing at the rate of 10 C/min to 260 C. Partial acetolysis of Asn-carbohydrate. According to the method described by Kocourek and Ballou,11) the Asn-carbohydrates (8 mg) were acetylated with 0.4ml of pyridine and 0.4ml of acetic anhydride at 100 C for 8hr. The solvents were removed in vacuo at 50 C and to the syrupy residue were added 0.4ml of acetic acid, 0.4ml of acetic anhydride and 40 µl of conc. sulfuric acid. The solution was kept at 40 C for 13hr. The mixture was then neutralized by adding 1.6ml of pyridine, and solvents were evaporated. The residue was dissolved in 3ml of chloroform and shaken with 3ml of water. The water layer was washed once with chloroform. The combined chloroform solution was dried with anhydrous sodium sulfate and evaporated to dryness. The acetolyzate was dissolved in 1 ml of methanol and deacetylated with 50ƒÊl of 1N methanolic sodium methoxide solution for about 20min at room temperature. The reaction mixture was diluted with 9ml of water and neutralized with 0.1N hydrochloric acid and the methanol was removed by evaporation. Gel filtration on Sephadex G-15. The deacetylated acetolyzate was applied to a column (1.0x145cm) of Sephadex G-15. The column was eluted with water at a rate of 20ml/hr in 1.5ml fractions. The neutral sugar in the effluent was determined by the phenolsulfuric acid method.

Carbohydrate Sequence of a Soybean 7S Protein 507 Fractions of each peak in the elution curve on Se phadex G-15 were evaporated and subjected to thinlayer chromatography on a microcrystalline cellulose (10x10cm) using a solvent of n-butanol-pyridinewater (10:8:8, by volume). Sugars were detected with aniline hydrogen-phthalate. The degree of polymeri zation of the fraction was measured by a modified method of Peat et at.") Reduction of the sugar (5 `35ƒÊg) was carried out by treating 0.4ml of the sugar solution with 0.1ml of 0.8% sodium borohydride in 0.001N sodium hydroxide solution for 1 hr at room temperature. The reduced solution was mixed with 0.5ml of 5% phenol and 2.5ml of conc. sulfuric acid, and measured at 490nm. Smith degradation of Asn-carbohydrates. The Asncarbohydrates were oxidized with 0.05 M sodium meta periodate (5-fold excess) at 4 C for 72hr in the dark. The oxidized sample was then reduced with 0.3M sodium borohydride at 4 C for 24hr. Excess sodium RESULTS Characterization of the mannose residues by methylation analysis The Asn-carbohydrates were N-acetylated, fully methylated, and subjected to fragmen tation. The resulting methylated mannose re sidues were analysed as methyl glycosides by gas-liquid chromatography on a column of NPGS. As shown in Fig. 1, three peaks were detected and identified as the derivatives of 2,3,4,6-tetra-O-methyl-mannose, 2,3,4-tri- or 3, 4, 6-tri-O-methyl-mannose and 2, 4-di-Omethyl-mannose for the methylated Asncarbohydrate IIa. lib and IIc also showed borohydride was decomposed by adding glacial acetic acid, and the preparation was evaporated to dryness. The residue was hydrolyzed in 0.1N hydrochloric acid at 30 C for 16hr. The hydrolysate was dried and subjected to gel filtration on a column (1x95cm) of Sephadex G-10 to separate the degradation products. This was subjected to the second Smith degradation in a similar manner. FIG. 1. Gas-liquid Chromatogram of Derivatives of Methyl O-Methyl-mannoside Obtained from the Methanolyzate of Methylated Asn-carbohydrate (IIa). A, methyl 2,3,4,6-tetra-O-methyl-ƒ -mannoside; B, methyl 2,3,4-tri-O-methyl-ƒ -mannoside or 3,4,6-tri-Omethyl-ƒ -mannoside; C, methyl 2,4-di-O-methyl-ƒ - mannoside. Column, 5% NPGS on Chromosorb W (160 C to 210 C, 6 C/min). FIG. 2. Gas-liquid Chromatogram of the Alditol Acetates of the Methylated Mannose Obtained from the Hydrolyzate of Methylated Asn-carbohydrates. A, 2,3,4,6-tetra-O-methyl-l,5-di-O-acetylmannitol; B, 3,4,6-tri-O-methyl-1,2,5-tri-O-acetylmannitol; C, 2,4- di-o-methyl-1,3,5,6-tetra-o-acetylmannitol. Column, 3% ECNSS-M on Gaschrom-Q (170 Ž to 210 Ž, 4 Ž/min).

508 F. YAMAUCHI and T. YAMAGISHI identical three peaks. Furthermore the me thylated mannose residues were also determined as their alditol acetates by gasliquid chromatography on a column of ECNSS-M as shown in Fig. 2. The trimethyl-mannose was shown to be the 3,4,6-tri-O-methylmannose derivative. Consequently, all of the methylated Asn-carbohydrates (IIa, IIb, and IIc) were shown to be composed of 2,3,4,6- tetra-o-methyl-mannose, 3,4,6-tri-O-methylmannose and 2,4-di-O-methyl-mannose re sidues. The ratios of the peak areas of the three components in Fig. 2 were calculated as pre sented in Table I. Characterization of the glucosamine residue by methylation analysis The fully methylated Asn-carbohydrates were acetolyzed, reduced and acetylated. The resulting methyl derivatives of N-methyl-acetylglucosaminitol acetate were analyzed by gas- TABLE I. RATIOS OF METHYL DERIVATIVES OF MANNOSE IN THE MET,HYLATEA ASn-CARBOHYDRATES Ratios were calculated from the peak area. liquid chromatography on the column of both ECNSS-M and OV-17. As shown in Fig. 3, a major peak was detected in each case, and identified as 3,6-di-O-methyl-1,4,5-tri-O-acetyl- N-methyl-N-acetylgucosaminitol indicating the presence of the C1- and C4-substituted N- acetylglucosamine residue. The 3,6-di-Omethyl derivative of glucosamine residue was also confirmed on the column of ECNSS-M, Partial acetolysis of the Asn-carbohydrates The Asn-carbohydrates were subjected to FIG. 3. Gas-liquid Chromatogram of the Alditol Acetates of Methylated N-Methyl-acetylglucosamine Obtained from the Hydrolyzate of Methylated Asncarbohydrates. Arrow A shows the position of 3,4,6-tri-O-methyl-l,5- di-o-acetyl-n-methyl-n-acetylglucosaminitol; B, 3,6- di-o-methyl-1,4, 5-tri-O-acetyl-N: methyl-n-acetylglucosaminitol. Column, 5% OV-17 on Chromosorb G (180 C to 260 C, 10 C/min). FIG. 4. Gel Filtration of the Deacetylated Acetolysis Products of Asn-carbohydrates on Sephadex G-15. The deacetylated acetolysis products were applied to a column (1.0x145 cm) of Sephadex G-15 and eluted with 0.1N acetic acid in 1.5ml fractions. Aliquots of the fractions were analyzed by the phenol-sulfuric acid method.

Carbohydrate Sequence of a Soybean 7S Protein 509 partial acetolysis which is known to cleave the 1 6 mannosidic linkage selectively."' As shown in Fig. 4, the deacetylated acetolysis products of IIa, IIb, and lie were fractionated into four components, M-1, M-2, M-3, and M-4 by gel filtration on a column of Sephadex G-15. M-1 showed the same mobility as Methylation analysis of the N-acetylgluco samine residues gave 3,6-di-O-methyl deriva tive (Fig. 3) indicating the presence of the Ciand C4-linked residues of N-acetylglucosamine. From these results, it is possible to assign the following partial structure to all the Asncarbohydrates. mannose (Rf, 0.35), on thin-layer chromato graphy and M-2 and M-3 showed Rf values of 0.41 and 0.30, respectively. M-1, M-2 and M-3 showed degrees of polymerization of 1-1.1, 2.0-2.1, and 3.1, respectively. Smith degradation of Asn-carbohydrates Smith degradation products were obtained by oxidation of the sample with sodium meta periodate followed by reduction with sodium borohydride, hydrolysis under a mild con dition and fractionation by gel filtration. The first and the second degradation products were analyzed for the components. As shown in Table II, the first product of Ha was composed of one mole of aspartic acid and two moles each of glucosamine and mannose. After the second degradation, the product contained one mole of aspartic acid and two moles of gluco The first Smith degradation of the Asn-carbo hydrates gave the product consisting of one mole of asparagine, two moles of glucosamine and two moles of mannose. The presence of the two moles of mannose indicates the sub stitution at the C3, or C,,, and C,.4 of mannose residue, and also indicates that the two branching mannose residues are linked next to the N-acetylglucosamine residue. Methylation a nalysis of mannose residues showed the C,,, substitution because of the appearance of the 2,4-di-O-methyl derivative (Fig. 1). From these results, it is possible to assign the two partial core structures to all the Asn-carbohydrates as follows. samine. However, the mannose residue was almost decomposed. The same results were obtained with l 1b and IIc. TABLE II. COMPOSITION OF FIRST AND SECOND SMITH DEGRADATION PRODUCTS IN ASn-CARBOHYDRATES Values are expressed as molar ratios. Most of glycoproteins, composed of both mannose and N-acetylglucosamine residues, have the core structure"' of (A), such as oval bumin,14) Taka-amylase A,15> Rhizopus sac charogenic amylase,"' tyroglobulin,17) and im DISCUSSION As shown in Table II, the second Smith degradation of Asn-carbohydrates gave the product consisting of 1 mole of aspartic acid and two moles of glucosamine. This indicates that the two N-acetylglucosamine residues are situated close to the aspartic acid residue. munoglobulin.18) On the other hand, recently, the core structure of (B) was suggested in heamagglutinin.19) For the purpose of determining the number of mannose residues attached to X or Y and Z position in the above partial structure, partial acetolysis which cleaves the 1 6 mannosidic linkage selectively was applied. Since each

510 F. YAMAUCHI and T. YAMAGISHI Asn-carbohydrate contained two 1-6 mannosidic linkages, two kinds of sugar are theo retically expected to appear. As shown in Fig.4, mannobiose and mannotriose were detected from IIa together with a small amount of mannose, which seemed to be yielded by non-selective acetolysis. This phenomenon is consistent with a slight overyield of mannose compared with the theoretical one in lib and IIc. At positions X and Y in the cases of both (A) and (B), the above experiments can not distinguish the position to which mannobiose and mannotriose are attached in IIa. The other mannose residue must be attached at Z. Methylation analysis afforded only 3,4,6-tri-Omethyl-mannose as the trimethyl derivative, and so the linear portion was composed of 1-*2 mannosidic linkages. From IIb and IIc, mannose and mannobiose were detected. At positions X and Y in (A) structure, mannose and mannobiose must be attached at X and Y, respectively. However, in (B) structure, the above experiments cannot distinguish the position of the attachment in IIb and IIc. From these results, it is possible IIa, x=1, y=1 and z=2, or x=2, y=0 and z=2 IIb, x-y=0, z=3 IIc, x=y=0, z=2 IIa, x=1, y=2 and z=1, or x=2, y=1 and z=1 IIb, x=0, y=l and z=2, or x=1, y=0 and z=2 He, x=0, y=l and z=1, or x=1, y=0 and z=1 to postulate the following structures for IIa, IIb, and IIc. Studies on the complete sequence and the configuration of the sugar residue in Asncarbohydrates are under investigation. REFERENCES 1) F. Yamauchi, M. Kawase, M. Kanbe and K. Shibasaki, Agric. Biol. Chem., 39, 873 (1975). 2) F. Yamauchi, V. H. Thanh, M. Kawase and K. Shibasaki, ibid., 40, 691 (1976). 3) V. H. Thanh and K. Shibasaki, J. Agric. Food Chem., 24,1117 (1976). 4) N. Handa and R. Montgomery, Carbohydr. Res., 11, 467 (1969). 5) S. A. Barker, A. B. Foster, M. Stacey and J. M. Webber, J. Chem. Soc., 1958, 2218. 6) B. Lindberg, "Methods in Enzymology," Vol. XXVIII, ed. by V. Ginsburg, Academic Press Inc., New York, 1972, p. 178. 7) K. Stellner, H. Saito and S. Hakomori, Arch. Biochem. Biophys., 155, 464 (1973). 8) J. E. Hodge and B. E. Hofreiter, "Method in Carbohydrate Chemistry," Vol. I, ed. by R. L. Whistler and M. L. Wolfrom, Academic Press Inc., New York, 1962, p. 388. 9) R. G. Spiro, "Methods in Enzymology," Vol. XXVIII, ed. by V. Ginsburg, Academic Press Inc., New York, 1972, p. 3. 10) S. Hakomori, J. Biochem., 55, 205 (1964). 11) J. Kocourek and C. E. Ballou, J. Bacterial., 100, 1175 (1969). 12) S. Peat, W. J. Whelan and J. G. Roberts, J. Chem. Soc., 1954, 2258. 13) R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem., 45, 217 (1976). 14) T. Tai, K. Yamashita, M. Ogata, N. Koide, T. Muramatsu, S. Iwashita, Y. Inoue and A. Kobata, J. Biol. Chem., 250, 8569 (1975). 15) H. Yamaguchi, T. Ikenaka and Y. Matsushita, J. Biochem., 70, 587 (1971). 16) K. Watanabe and T. Fukinbara, Agric. Biol. Chem., 38, 1973 (1974). 17) S. Ito, K. Yamashita, R. G. Spiro and A. Kobata, J. Biochem., 81, 1621 (1977). 18) S. Hickman, R. Kornfeld, C. K. Osterland and S. Kornfeld, J. Biol. Chem., 247, 2156 (1972). 19) H. Lis and N. Sharon, ibid., 253, 3468 (1978).