The Occurrence of Repetitive Glycopeptide Sequences in Bovine Submaxillary Glycoprotein

Transcription

1 Eur. J. Biochem. 32,4854 (973) The Occurrence of Repetitive Glycopeptide Sequences in Bovine Submaxillary Glycoprotein Ward PIGMAN, John MOSCHERA, Michael WEISS, and Guido TETTAMANTI Biochemistry Department, New York Medical College, New York (Received June 5/September 9, 972) Bovine submaxillary mucin, after removal of its sialic acid component, is cleaved by trypsin into two principal glycopeptide fractions ; their final yield corresponds to 70,Ilo by weight of the original mucin and 85*/, of the original amount of hexosamine in the mucin. These glycopeptides have amino acid and carbohydrate compositions that are very similar to those of the original and desialyzed mucin. The main glycopeptide contains about 28 amino acids; the second one seems to be a covalently linked trimer of the former and is resistant to further action by trypsin. The protein core of bovine submaxillary mucin is composed mainly of several hundred covalently bound sequences of these glycopeptides. These results are compatible with those reported by Downs and Pigman (969) for the products obtained by chemical cleavage of this mucin. Bovine submaxillary mucin is a glycoprotein with a molecular weight of about 4~ 06 [I]. Its protein core, which is composed of aliphatic amino acids, is particularly rich in serine, threonine, alanine, glycine and proline. About 70 /, of the dry weight of the mucin is accounted for by carbohydrates mostly disaccharides attached 0glycosidically to the hydroxyl groups of serine and threonine residues of the protein core : the major disaccharide is Osialyl 2acetamido2deoxy~galactose [2,3]. The remainder of the carbohydrate was found to occur as other di, tri, or higher oligosaccharides [4]. Earlier work in this laboratory suggested that the core protein of bovine submaxillary mucin is composed of many repetitive sequences [5]. This result was confirmed by Downs and Pigman [6] through chemical degradation. The present work which described the action of trypsin on the desialized mucin, agrees with these findings; in addition, trypsin digestion provides glycopeptides which may be used for studies of the sequence of the core protein. MATERIALS AND METHODS Bovine submaxillary mucin was prepared by the method of Tettamanti and Pigman [7], and in some of the studies wits purified further by solution in Abbreviations. Me,SO, dimethylsulfoxide; dansyl, ldimethylaminonaphthyl5sulfonyl chloride. Enzymes. Bovine pancreatic trypsin (EC ) ; porcine carboxypeptidase B (EC ). 00 /, dimethylsulfoxide (Downs, Herp & Pigman, unpublished method). Bovine pancreatic trypsin (2 x crystallized) and carboxypeptidase B were obtained from the Worthington Biochemical Corp. (Freehold, N.J.); Sephadex G25 and G50 were from Pharmacia Fine Chemicals; Dowex 50X2 ( mesh) and BioRad P4 from BioRad Laboratories. All inorganic reagents were of analytical grade. Protein was determined by the method of Lowry et al. [9] with bovine serum albumin as the standard. Its value was also calculated from the anhydro amino acids as determined by amino acid analysis. Amino acid analysis was performed on samples ( pg) hydrolyzed with 6 N HC at 0 "C for 22 h in sealed tubes using a modified Beckman Model 20R Amino Acid Analyzer. The conditions for amino acid analysis of bovine submaxillary mucin have been studied extensively by Downs and Pigman [lo]. Hexosamine was determined by the Boas [ill modification of the ElsonMorgan reaction with omission of the resin treatment. Sialic acid was analyzed by the method of Svennerholm [2]. Molecular weights and homogeneity of the glycopeptides were determined in 0.2 M NaCl using a BeckmanSpinco Model E Analytical Ultracentrifuge by the method described by Yphantis [3]. The value for the partial specific volume was taken as 0.633, as determined by Creeth and Knight [ 4 for the related human bloodgroup B substance. Typical data are given in Fig.5. The

2 Vol.32, No., 973 W. PIQMAN, J. MOSCHERA, M. WEISS, and G. TETTAMANTI 49 continuous straight lines are a good indication of homogeneity of molecular weights. Aminoterminal analysis was performed using the dansylation procedure of Gray [5]. The dansyl derivatives were identified by thinlayer chromatography on silica gel G plates [6]. Purification of the dansyl derivative of the glycopeptide (D502, see Fig.) was achieved on a BioRad P4 column ( x 0 cm). Elution with water was monitored by fluorescence under ultraviolet light. The glycopeptide was then subjected to twodimensional thinlayer chromatography using the system of Atherson and Thompson [ 7. The aminoterminal amino acid of the glycopeptide (D502) was also determined by using the subtractive Edman degradation [IS]. Cterminal analysis was performed by the carboxypeptidase B method of Guidotti et al. [9]. Glycopeptides D50 and D502 were submitted to highvoltage paper electrophoresis. A pyridineacetate buffer at ph 6.4 (200 ml pyridine, 8 ml acetic acid) was employed. Samples containing 60 pg of each glycopeptide were spotted on Whatman 3MM paper. After completion of the run, the paper was dried, and the spots were visualized by the ninhydrin and benzidine periodate stains as described by Block et al. [20]. Other solvent systems used for electrophoresis were a lo/, borate buffer at ph 8.6 and 6.80/, formic acid at ph.9. PREPARATION AND SEPARATION OF GLYCOPEPTIDES The procedures used for the separation of glycopeptides are outlined in the flow diagram in Fig. and described in more detail below. Removal of Sialic Acid Sialic acid was removed by incubating a 0.5O/, solution of bovine submaxillary mucin in H,SO,, ph 2.0, at 80 "C for 90 min. The solution was dialyzed at 4 "C against 0. M NaCl for 4 h, and against distilled water for another 48 h, with frequent changes of water. The undialyzable material contained 2 to 50, sialic acid and was lyophilized. Trypsin Digest The desialyzcd bovine submaxillary mucin was treated with trypsin as follows. Samples of 0.35 to.2 g at a concentration of lo/, by weight in 0. M phosphat,e buffer ph 7.8 were incubated with trypsin at a final concentration of 0.02O/, for 5 h at 37 "C, and the hydrolysis was followed by the ninhydrin method of Moore and Stein [2]. The digest was then ultrafiltered in 0.25inch cellophane tubing by the procedure of Craig [22]. The nonfilterable residue was taken up in phosphate buffer, and digestion and ultrafiltration were repeated. Three successive Mucin (80 "C for 90 min) (H,SO,, ph 2.0) J I Desialized mucin Trypsin f Ultrafiltration residue SephadLx G25t I J Excluded glycopeptide fraction Sephadex 650 I 2 x Retarded fraction J Glycopeptide Glycopeptide G50 w G502 Dowex50 Dome;50 Glycopeptide D50 Glycopeptide D502 Fig.. Flow sheet for separation of glycopeptides obtained from bovine submaxillary mucin by treatment with trypsin digestions were required before all of the desialyzed mucin became ultrafilterable. Recovery, based on the hexosamine content of the combined ultrafiltrate, amounted to 97O/, of that in the starting material. The combined ultrafiltrates were lyophilized and dissolved in mm phosphate buffer ph 8.8. About 50 mg in 3 ml was applied to column (.5 x 00 cm) of Sephadex G25 (fine) which had been preequilibrated with the above buffer. A flow rate of 25 ml/h was maintained using a polystaltic pump, and 5ml fractions were collected. The elution, which was monitored by protein and hexosamine determinations, gave the pattern shown in Fig. 2. A trypsin blank was also run, and its ultrafilterable fraction was found to be retarded on the Sephadex column. The excluded material was pooled, lyophilized, and applied in a 3O/, aqueous solution to a Sephadex G50 (fine) column (2.0 ~ 9cm); 0 the sample was eluted with water, at a flow rate of 25 ml/h and 5ml fractions were collected. The elution pattern was monitored as before and is shown in Pig.3. Each of the glycopeptide fractions (650 and 2) was rechromatographed until single peaks were obtained. The two major glycopeptides (G50, G502) were each dissolved in a minimum volume of 2.5O/, acetic acid (ph 3.5) and applied in 00mg

3 50 Tryptic Peptides from Bovine Submaxillary Glycoprotein Eur. J. Biochem. Excluded I_ Retained A Void volume Volume (rnl) Fig.2. Gel filtration of bovine submaxillary mucin tryptic glycopeptides on Sephadex 625. nu, Lowry protein; 0, hexosamine 0'4 volume A Vqid O 4p IIO lo ' 60 ' 80 ' 260 ' 220 ' Volume (ml) Fig.3. Gel filtration of bovine submaxillary muck tryptic glycopeptides on Sephadex G50, using the Lowry protein method. The horizontal lines represent the two fractions collected (G50,2) portions to a Dowex 50X2 column (.5 x 00 cm) that had previously been equilibrated with 20//, acetic acid. The column was eluted with a 0.6 M pyridineacetate buffer ph 5.5. The elution pattern for peptide G502 is given in Fig.4, and is similar, except for position, to that found for G50. Fractions corresponding to peaks D50 and 2 were pooled and lyophilized for further study. RESULTS AND DISCUSSION After removal of more than 9B0/, of its sialic acid by mild acid hydrolysis, bovine submaxillary mucin was cleaved by trypsin into two major ultrafilterable fractions which were separated and purified " Volume (rnl ) Fig.4. Elution pattern for glycopeptide 6502 on Dowex50, using the Lowry protein method. The horizontal line represents fraction D502 by the procedures shown in Fig. I. The glycopeptide fractions were separately recycled through a column of Sephadex 650 until complete resolution was achieved. This treatment was followed by fractionation on Dowex 50X2. Glycopeptide D50 accounted for approximately 30 /, of the protein in the starting material, and glycopeptide D502 for about 40 /,; thus, the combined recovery, as based on protein, amounted to around 700/, of the original mucin. The recovery of hexosamine was in the order of 800/, for the combined products. The Sephadex and Dowex 50 glycopeptides when subjected to equilibrium ultracentrifugation gave linear Yphantis plots of In dc vus (x2 m2 ) as shown in Fig.5. The linearity indicates homogeneity with respect to molecular weight. The molecular weights are recorded in Table 3. The smallest glycopeptides had molecular weights of 5200 and The larger, G50 and D50, appear to be trimers, which are, however, resistant to the action of trypsin. Application of the Edman degradation [23] to D502 showed glycine as the sole aminoterminal amino acid, and analysis of the resulting glycopeptide showed the loss of one glycine unit per arginine residue. The molecular weight on this basis was All of the glycopeptides, when subjected to the dansylation method of Gray [I5 followed by acid hydrolysis and thinlayer chromatography, showed glycine as the only aminoterminal amino acid. From the known action of trypsin, arginine would be expected to be the carboxyterminal amino acid. Treatment of the D50 and 2 glycopeptides with carboxypeptidaseb yielded arginine in yields of 70 to Boo/,.

4 Vol.32, No., 973 W. PIGMAN, J. MOSCHERA, M. WEISS, and G. TETTAMANTI 5.6 o.2l. OO (x rn*) Fig. 5. Yphantis plot of sedimentation equilibrium data for glycopeptides G50 (o), G502 (e), 050 (0) and D502 (m) Further tests of homogeneity were carried out by highvoltage electrophoresis of the Dowex 50 glycopeptides. At ph 6.4 and 8.6, both materials showed only one sharp spot. D502, examined at ph.8, moved as one spot. Both materials moved as single bands in polyacrylamide disc electrophoresis. In earlier work from this laboratory [5], bovine submaxillary glycoprotein was treated with several proteolytic enzymes. Although its viscosity was greatly decreased and dialyzable material found, the composition of the resulting products, except for those obtained from the pronasetreated glycoprotein, remained essentially unchanged. The enzymes acted more extensively on desialyzed bovine submaxillary mucin and for this reason, desialyzed material was used in the present work. The above observations suggested that repetitive sequences of amino acids and carbohydrates might be present in bovine submaxillary mucin and that, for this reason, despite its large molecular weight, sequence studies might be feasible. Downs and Pigman [B] found that bovine submaxillary mucin could be cleaved into small units by mild acid hydrolysis of the dehydroserine residues which result from alkaline /Ielimination of its carbohydrate side chains. The main product obtained by this treatment contained about 25 amino acids, an amino terminal unit of pyruvic acid, and one disaccharide sidechain. When the chemical changes involved in these reactions are taken into account, the glycopeptide was shown to have an amino acid composition closely similar to that of the original mucin. Since then, we have found that.5 to 2 dehydroseryl residues were removed as pyruvic acid in the acid hydrolysis step ; hence, the core protein was apparently cleaved at positions comprised of Table. Carbohydrate and protein compositions of bovine submaxillary mucin and glycopeptides obtained after treatment with trypsin The products before trypsin treatment also contained about.5 to 2.O0/, each of fucose and galactose. The figures in parenthesis indicate the number of independent analyses made, usually on different samples. The glycopeptide analyses are for single samples, analyzed several times. All analyses are percentages on a moisture, ashfree basis. The hexosamine and sialic acid values are for the anhydro residue. The Lowry method was used for protein determination. The preparation and purification of most of the products in Tables and 2 were carried out independently by three of the authors over a period of 4 years. The Dowex 50 treatment was carried out by only one of the investigators Product Native (major) (6) Me,SO fraction (3) Desialyzed (3) G50 G502 D50 D502 NAcetylhexosaniine N,ODiacetylneurarninic acid OIo two or three contiguous seryl residues. Thus, the principal glycopeptide contained about 28 amino acids. The present work provides a new and more direct method for the isolation of glycopeptides which correspond to the principal repeating sequence of bovine submaxillary mucin. In conjunction with the glycopeptides produced by chemical degradation, the new glycopeptides should greatly facilitate amino acid sequencing by providing overlap regions. In Table, the compositions of the original bovine submaxillary mucin products are compared with those of the tryptic glycopeptides that were purified on Sephadex G50 (G50 and 2) and with those subsequently purified on Dowex50 (D50 and 2). The Me,SO fraction is that fraction of native bovine submaxillary mucin (major) which is soluble in i0oo/, dimethylsulfoxide. Its preparation will be described elsewhere. This treatment removes all but trace amounts of the amino acids usually found in small amounts in bovine submaxillary mucin (major) [7]. Since most of the sialic acid was removed, the composition of the products derived by the trypsin treatment must be compared with that of the desialyzed mucin. The composition of the glycopeptides is fairly similar to that of the desialyzed mucin. However, a slight but significant increase in hexosamine content seems to occur (Tables and 4). Apparently, products of lower hexosamine content remained in the unrecovered fractions. This result suggests some

5 ~ ~ ~~ ~ 52 Tryptic Peptides froni Bovine Submaxillary Glycoproteiii Eur. J. Biochem. Table 2. Comparison of aminoacid composition of bovinesubmaxillurymucin preparations and glycopeptides derived by action of trypsin or by chemical degradation All values are moles per 00 moles of total amino acids. Values for the bovine submaxillary mucin materials are averages of several analyses on the same basis as Table. The glycopeptides marked DownsA and B were derived by alkaline 8elimination followed by mild acid hydrolysis[lo]. The DownsA and B products were corrected by addition of the serine and threonine residues lost during the,&elimination reaction; the arginine values were corrected for the loss arising from alkaline degradation which produces some ornithine, appearing on the recording trace as apparent lysine Amino acid Native Me,SO treated DeGalyzed G50 D50 Dou?ls A G502 D502 Downs B Lysine Arginine Aspartic/ asparagine Threonine Serine C lutamic/ glutamine Proline Glycine Alanine Valine Isoleucine Leucine Phenylalanine mo/00 mol Table 3. Molecular weights of bovine submaxillary mucin and of glycopeptides Prodiict, Molccular weight Measured * CalCulatd Calculatrd (carbohydrate frce) Native 4 X lo6 (2590), b n = D OOc fi D d Downs B ~. 2560d a By equilibrium ultracentrifugation except for the native mucin for which lightscattering data were used. The partial specific volunie vas taken as b Calculated from the data in Table2 on the basis of a 28;tniinoacid repeating sequence and for a mol. wt of thc core protein as.3 x 0. C Calculated from the 5300 value as a trinier. $ Culeolated as the snni of anhydro amino acids corresponding to a. 28aminoacid peptide, with and without Nacetylhexosamine residiies. heterogeneity in the distribution of hexosamines attached along the protein core of bovine submaxillary mucin. Indeed, several types of carbohydrate side chains are known to be present [4] in the native material. A comparison of the molar content of hexosamine and the serine and threonine is made in Table 4. Although the sum of serine and threonine in the native mucin is very close to that for total hexosamine, a small proportion of the serine and threonine is probably unsubstituted since some of the minor oligosaccharide side chains contain several hexosamine units [4]. Table 4. Comparison of serine, threonine and hexosamine content in native bovine submaxilla,ry mucin and glycopeptides Constituent Native G50. G502 D50 D502 mmol/g Serine Threonine Total(SerfThr) Hexosamine GalNAc/GluNAc The amino acid composition of the original products and of the glycopeptides obtained after trypsin treatment is given in Table 2. The table also includes data for the glycopeptides A and B derived by pelimination followed by mild acid hydrolysis [6]; these products were put on a comparable basis by using the calculations explained in Table 2. Since the values are expressed as moles per 00 moles of amino acids, all products can be compared directly. From this table as well as from Table I it can be seen that while the various products show definite similarities, they also exhibit some differences. The G50 and D50 glycopeptides, which appear to be trimers of glycopeptides G502 and D502, respectively, seem to have slightly less arginine, threonine, glutamic acid and proline and slightly more glycine, alanine, and valine than the native and desialized preparations. The smaller glycopeptides (2502 and D502 may have slightly less aspartic acid, proline and valine ; they seem to contain slightly higher

6 Vol.32, No., 973 W. RQWAN, J. MOSCHERA, M. WEISS, and G. TETTAMANTI 53 proportions of threonine, serine and glutamic acid as compared to the untreated bovine submaxillary mucin preparations. An even closer agreement with the latter preparations is observed for the corrected Downs A and B glycopeptides obtained by chemical treatment. Downs A material may have slightly less arginine and alanine and more threonine and serine. However, the significance of these variations is questionable. The observed and calculated molecular weights of the various products studied are given in Table 3. This table also includes corresponding data for the glycopeptide B of Downs and Pigman. The measured molecular weights are in close agreement with those calculated on the basis of a repeating sequence of 28 amino acids and for its trimer. Also, if for the calculation of the molecular weight, arginine is set equal to i mole, closely similar values are obtained for glycopeptides 6502 and D502. This calculations is justifiable since trypsin cleaves peptide chains and leaves arginine as the carboxyl terminal amino acid ; thus, each glycopeptide should contain at least one arginine residue. For all of the trypsin glycopeptides, glycine was the sole aminoterminal group. The data in the last column of Table 3 report the contribution of the carbohydratefree protein portion of the total molecular weight for the various products. According to these data, the basic repeating peptide sequence as obtained by enzymic treatment of bovine submaxillary mucin, seems to have a molecular weight of 2500 to 2600 and is the same as the corrected values for that derived by chemical cleavage. When the protein core is considered to consist of repeating peptide sequences of 28 amino acids in length, as calculated on the basis of its molar amino acid composition, the molecular weight, for the repeating sequence is From the molecular weight of the protein core, it is concluded that about 500 of such sequences would be present in the native bovine submaxillary mucin. However, a considerable portion of the core protein contains the trimer, which is resistant to trypsin. A value of 4 x lo6 has been used for the molecular weight as determined by light scattering [l]. Various preparations have given different values, especially by the equilibrium sedimentation method. In this method the salt concentration of the solvent is important [24]. Current work is in progress to determine whether different samples have different molecular weights. When the amino acid composition on a molar basis is calculated from Table 2 for a 28aminoacid peptide, fractional molar values are obtained for some of the amino acids. This result suggests that the basic repetitive sequences are not identical, and some homologous substitutions of amino acids are probably present. The glycopeptides are similar enough to behave very much the same way in the several systems studied. However, further work is in progress using more refined separation techniques. The sequence of the amino acids in the glycopeptides is under current study. Some of the implications of the existence of such repetitive sequences for the mechanisms of biosynthesis of glycoproteins have been discussed elsewhere [25]. Because of its possible importance, the present work has been repeated in most of its aspects by three independent investigators. We also have found that ovine submaxillary mucin, whether treated by the chemical procedure used by Downs and Pigman or by trypsin as outlined in the present work, gave Sephadex G50 fractions closely resembling those for bovine submaxillary mucin. Repeating sequences of amino acids in proteins have been previously found in some proteins but not to the extent observed here. The subject has been reviewed by Nolan and Margoliash [26]. Recently, small serum glycoproteins of Antartic fishes have been found to have the sequence AlaAlaThr as a repeating structural unit with each threonine carrying a disaccharide side chain [27]. We have also shown that various mucus glycoproteins including the bloodgroup substances probably have similar repeating sequences to those reported here. This work was supported by a grant from the National Institutes of Health (AM0469). Bovine and ovine submaxillary glycoproteins were prepared by courtesy of the New England Enzyme Center, Tufts University School of Medicine, Boston, Mass. REFERENCES. Bettelheim, F. A., Hashimoto, Y. & Pigman, W. (962) Biochim. Bwphys. Acta, 63, Gottschalk, A. & Graham, E. R. B. (959) Biochkm. Biophys. A&, 34, Murtv. V. L. N. & Horowitz. M. I. (968) Carbohud. \ I Y Re;. 6, Bertolini. M. & Pieman. W. (970) Curbohud. Res Hashimoto, Y., Tsuiki, S., Nisizawa, K. & Pigman, W. (963) Ann. N.Y. A d. Sci. 06, Downs, F. & Pigman, W. (969) Biochemistry, 8, 760 to Tettarnanti, G. & Pigman, W. (968) Arch. Biochem. Biophys. 24, Reference deleted. 9. Lowry, O., Rosebrough, N. J., Farr, A. W. & Randall, R. J. (95) J. Biol. Chem. 93, Downs, F. & Pigman, W. (970) Int. J. Protein Res. 2, Boas,N. P. (953) J. Bbl. Chem. 204, Svennerholm, L. (957) Biochim. Bbphys. Actu, 24, Yphantis, D. A. (964) Biochemistry, 3, Creeth, J. M. & Knight, C. G. (967) Biochem. J. 05, Gray, W. (967) Methods Enzymol., Morse, D. & Horecker, B. (966) AnaE. Biochern. 4,

7 54 W. PIGMAN et al.: Tryptic Peptides from Bovine Submaxillary Glycoprotein Eur. J. Biochem. 7. Atherson, R. & Thompson, A, (969) Biochem. J., Konigsberg, W. (967) Methods Enzymol., Guidotti, G., Hill, R. & Konigsberg, W. (962) J. Biol. Chem. 237, Block, R., Durrum, E. & Zweig, 0. (958) Paper Chromatography and Paper Electrophoresis, Academic Press, New York. 2. Moore, S. & Stein, W. (954) J. Biol. Chem. 2, 907 to Craig, L. C. (967) Methods Enzymol., Edman, P. (956) Acta Chem. Scund. 0, Payza, N., Robert, M. & Herp, A. (970) Int. J. Protein Res. 2, Pigman, W., Downs, F., Moschera, J. & Weiss, M. (970) Blood and Tissue Antigens (Aminoff, D., ed.) pp , Academic Press, New York. 26. Nolan, C. & Margoliash, E. (968) Ann. Rev. Bwchem. 37, De Vries, A. L., Komatsu, S. K. & Feeny, R. E. (970) J. Biol. Chem. 245, W. Pigman, J. Moschera, M. Weiss, and G. Tettsmanti Department of Biochemistry, New York Medical College Flower and Fifth Avenue Hospitals 5th Avenue at 06th Street, New York 0029, U.S.A.