Partial structural analysis of the oligosaccharide moieties of the vesicular stomatitis virus glycoprotein by sequential chemical and enzymatic degradation

VIROLOGY

78,

375-392

(1977)

Partial Structural Analysis of the Oligosaccharide Moieties of the Vesicular Stomatitis Virus Glycoprotein by Sequential Chemical and Enzymatic Degradation JAMES

R. ETCHISON,

Department

of Microbiology,

JAMES University

S. ROBERTSON, of Utah

Accepted

College

January

AND

of Medicine,

DONALD Salt

Lake

F. SUMMERS City,

Utah

84132

17,1977

The oligosaccharide moieties of the vesicular stomatitis virus glycoprotein have been characterized by sequential chemical and enzymatic degradation of the peptidyloligosaccharides obtained after Pronase digestion of the purified glycoprotein. The amino acid and carbohydrate constituents have been analyzed by gas-liquid chromatography. The number-average molecular weight of the peptidyloligosaccharides is estimated to be 3150 by gel filtration analysis and the weight-average molecular weight is estimated to be 3450 by the molar composition of the amino acid and sugar residues. Sequential removal of neuraminic acid, galactose, and the distal N-acetylglucosamine residues results in peptidyloligosaccharides with average molecular weights estimated to be 2300, 1900, and 1350-1450, respectively. The data indicate that the two major oligosaccharide moieties of the glycoprotein have similar structures. These structures are acidic oligosaccharides, each having two or three branches terminating in the sequence sialic acidgalactose-N-acetylglucosamine at the nonreducing end of the oligosaccharide chain. These terminal branch structures are attached to a tetramannosyl-di-N-acetylchitobiose core structure. Periodate oxidation indicates that the sialic acid is linked to galactose with a-2,3 bonds and the galactose toN-acetylglucosamine with p-l,3 or p-1,4 bonds. INTRODUCTION

The membraneous envelope of vesicular stomatitis virus (VSV) contains a single glycoprotein which has an apparent molecular weight of 67,000 (Mudd and Summers, 1970). The carbohydrate portion comprises approximately 10% of the mass of the glycoprotein and is present in oligosaccharide structures with apparent molecular weights in the range of 3000 to 4000 (Burge and Huang, 1970; Etchison and Holland, 1974a). These oligosaccharides have been shown to be complex structures containing fucose, mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid (McSharry and Wagner, 1971; Etchison and Holland, 1974a, b). Studies by Moyer and Summers (1974) and Moyer et al. (1976) have shown that the oligosaccharides of the VSV glycoprotein are attached to the polypeptide moiety by a linkage having the alkaline stability characteristic of a 1-N-glycosylaminide

bond of N-acetylglucosamine to the amide nitrogen of asparagine. They also showed that the VSV oligosaccharides are cleaved by an endo-/3-N-acetylglucosaminidase which cleaves a di-N-acetylchitobiosyl structure near the carbohydrate-polypeptide junction. Analysis of the cleavage products indicated that the VSV oligosaccharides contain a core structure which is similar to and common to the asparaginelinked oligosaccharides of several glycoproteins (see Lee and Scocca, 1972). The work by Moyer et al. (1976) also established that the fucose present in the VSV oligosaccharides is attached to the N-acetylglucosamine residue involved in the carbohydrate to polypeptide linkage. The present communication describes a detailed investigation of the structure of the oligosaccharide moieties of the VSV glycoprotein from virus grown in BHK21 cells. This information should facilitate and direct future studies on the synthesis

375 Copyright All rights

0 1977 by Academic Press, Inc. of reproduction in any form reserved.

ISSN

0042-6822

376

ETCHISON,

and function of the oligosaccharide tures of the VSV glycoprotein. MATERIALS

AND

ROBERTSON,

struc-

METHODS

Growth of cells and virus. The BHK 21 cells were grown as monolayers in Eagle’s MEM supplemented with 7% fetal calf serum. The Indiana serotype of VSV was grown and purified as described previously (Etchison and Holland, 1974a). Radiolabeling of virus glycoprotein. Vesicular stomatitis virus radiolabeled with 13Hlfucose, mannose, galactose, or glucosamine was prepared by incubating infected cells for 4 hr (from 2 to 6 hr postinfection) with 10 #X/ml of the radiolabeled sugar in MEM containing 0.01 the normal glucose concentration and supplemented with 2x nonessential amino acids and 2% dialyzed calf serum. At 6 hr postinfection (PI) this labeling medium was diluted twofold with normal growth medium and incubation continued until the time of harvest (16 hr PI). [14C]Glucosamine-labeled virus was prepared similarly except that the radioisotope concentration was 1 @iI ml. The radiolabeled glycoprotein was extracted from purified virus preparations and purified as described previously (Kelley et al., 1972; Etchison and Holland, 1974a). The glycoprotein was removed from the NP-40 by partitioning with 2 vol of n-butanol. After centrifuging to break the phases (e.g., 2000 g for 2-3 min), the glycoprotein was collected as a sticky precipitate at the water-butanol interface. The precipitated glycoprotein was washed with 95% ethanol to remove the residual butanol and detergent. The precipitated glycoprotein was stored as the ethanol precipitate at -2o”, lyophilized, or resuspended in an appropriate buffer for immediate use. Chemicals and isotopes. D- [6-3HlGlucosamine (5-15 Ci/mmole), L[6-3H]fucose (lo-15 Ci/mmole), n-[ l-3H]galactose (510 Ci/mmole), and n-[1-14Clglucosamine (46 mCi/mmole) were obtained from New England Nuclear Corporation (Boston, Mass.). n-[2-3H]Mannose (2 Ci/mmole) was obtained from Amersham/Searle Corporation (Arlington Heights, Ill.).

AND

SUMMERS

Bio-Gel P-6 (200-400 mesh) and Bio-Gel P-4 (-400 mesh) were purchased from BioRad Laboratories, Inc. (Richmond, Calif.). Ovalbumin (5 x crystallized) and Pronase, B grade, were purchased from Calbiochem (La Jolla, CA). Clostridium perfringens neuraminidase Type VI, was purchased from Sigma Chemical Company (St. Louis, MO.). Beef kidney /3-N-acetylglucosaminidase and jack bean cr-mannosidase were purchased from BoehringerMannheim. The a-mannosidase was also purified from jack bean meal (Sigma Chemical Company) as described by Li and Li (1972). Aspergillus niger P-N-acetylglucosaminidase and P-galactosidase were purified from Rhozyme HP-150 concentrate (a gift of Rohm and Haas Company, Philadelphia, Penn.) by a modification of the procedure described by Bahl and Agrawal (1972). Neuraminidase from Streptococcus pneumoniae was purified by a modification of the procedure described by Hughes and Jeanloz (1964). Endo-P-Nacetylglucosaminidase D was purified from S. pneumoniae as described by Koide and Muramatsu (1974). Type 1 S. pneumoniae was a gift from Dr. Paul Atkinson, Albert Einstein College of Medicine, Bronx, N. Y. Pronase digestion of glycoproteins. Peptidyloligosaccharides of the VSV glycoprotein were prepared by digestion of the purified glycoprotein with 1 mg/ml Pronase in 0.1 M Tris-HCl, pH 7.5, containing 3 mM CaCl,. A drop of toluene or chloroform was added to the digestion to prevent microbial growth. The digestion was carried out for 48 hr with an additional milligram of Pronase per milliliter added at 24 hr. Fetuin glycopeptides were prepared by digesting 50 mglml of fetuin with 1 mg/ml of Pronase as described above. The glycopeptides were isolated by gel filtration and redigested with Pronase twice. Ovalbumin glycopeptides were prepared by digesting 50 mg/ml of 5x crystallized ovalbumin as described above. After digestion the mixture was concentrated three- to fivefold by evaporation, insoluble amino acids were removed by centrifugation, and the glycopeptides were precipitated with 15 vol of 95% ethanol at -20” overnight. The glyco-

OLIGOSACCHARIDE

peptides were redigested and isolated for two more cycles. Monodisperse asparaginyloligosaccharides were purified from the ovalbumin glycopeptide population by the fractionation procedure of Huang et al. (1970). Gel filtration of peptidyloligosaccharides. Gel filtration analyses were carried out through a 1.3 x llO-cm column of BioGel P-6 (flow rate, 8 ml/hr; fractions, 1.3 ml) and a 0.9 x 115cm column of Bio-Gel P-4 (flow rate, 4.5 ml/hr; fractions, 0.7 ml). The column eluant was 0.1 M NH,OCOCH,, pH 6, containing 0.5 mM NaN,. Radiolabeled samples were located by liquid scintillation spectroscopy. Sialic acid-containing oligosaccharides were located by hydrolyzing aliquots of each fraction in 0.1 N H&SO, for 60 min at 80” and assaying released sialic acid by the thiobarbituric acid procedure (Warren, 1959). Peptidyl- and asparaginyloligosaccharides and neutral oligosaccharides were located by assaying aliquots of each fraction for neutral hexose by the phloroglucinolH,SO, procedure, a modification of the phenol-H&SO, procedure of Dubois et al. (1956) having increased sensitivity (Etchison et al., in preparation). The excluded volume of the gel filtration columns was determined using bovine serum albumin (BSA) and the totally included volume using mannose. Glycosidase digestion of peptidyloligosaccharides. Vesicular stomatitis virus peptidyloligosaccharides were digested with 0.1 unit of Cl. perfringens neuraminidase (1.5 units/mg of protein) in 0.1 M phosphate buffer, pH 5.3, for 24 hr at 37”. Digestion with A. niger P-galactosidase (78 IU/mg of protein), A. niger p-N-acetylglucosaminidase (48 IU/mg protein), beef kidney p-N-acetylglucosaminidase, and jack bean a-mannosidase were carried out in 0.05 M citrate-phosphate buffer, pH 4.2, using 5-10 units/ml of enzyme and incubating at 37” for 24-48 hr. Digestion with a partially purified mixture of S. pneumoniae glycosidases containing the endo-pN-acetylglucosaminidase D, p-galactosidase, p-N-acetylglucosaminidase, and neuraminidase was carried out in 0.05 M citrate-phosphate buffer, pH 6.5, using 100

STRUCTURE

OF

377

VSV

pg of the enzyme mixture and incubating at 37” for 24 hr. Amino acid and carbohydrate analyses. Carbohydrate analyses were performed by gas-liquid chromatography of the trimethylsilyl ethers of the methyl glycosides as described previously (Etchison and Holland, 1974a, 1975). Amino acids were analyzed by gas-liquid chromatography of the N-heptafluorobutyryl-isoamyl esters as described by Zanetta and Vincendon (1.973)) using pipecolic acid as an internal standard. Smith degradation of the peptidyloligosaccharides. Vesicular stomatitis virus peptidyloligosaccharides were oxidized in 0..03 M NaIO, buffered at pH 4.5 with 0.05 hf sodium acetate. The oxidation was carried out at 0” in the dark for either 2 hr or 210 to 24 hr. At the end of the oxidation period glycerol was added to a final concentration of 0.05 M (a twofold molar excess over the periodate added). After a 30min incubation at room temperature, an equal volume of freshly prepared 1 M NaBH, in 0.04 M NaOH was added and the solution was buffered at pH 9.0 by the addition of 1 M sodium borate pH 9.0 (prepared by titration of boric acid with NaOH) to give a final concentration of 0.1 i%f. The borohydride reduction was allowed to proceed at room temperature for 2 hr. The excess borohydride was destroyed by the careful addition of 1 drop of 10 N H,SO,. The pH of the solution was then adjusted to pH 1 with 1 N H&SO, and an equal volume of 0.2 N H,SO, was added. The sample was then hydrolyzed at 100 for 1 hr. The hydrolyzed sample was neutralized and analyzed by gel filtration. RESULTS

Isolation and Composition of the Peptidyloligosaccharides Obtained after Pronase Digestion of the VSV Glycoprotein Digestion of glycoproteins with Pronase yields the intact oligosaccharide structures attached to a small peptide usually containing only a few amino acid residues (Marshall and Neuberger, 1972b). Figure 1 shows the gel filtration profile of the pepti-

378

ETCHISON,

30

50 FRACTION

70

ROBERTSON,

I

90

NUMBER

FIG. 1. Gel filtration of the VSV peptidyloligosaccharides. (A) Approximately 8 mg of the VSV glycoprotein was digested with a total of 1.5 mg of Pronase (1 mg initially and 0.5 mg after 24 hr) for 48 hr. The digestion products were analyzed by gel filtration through a 1.3 x loo-cm column of Bio-Gel P-6. Aliquots of each fraction were assayed for sialic acid (0-a) by the thiobarbituric acid procedure and for neutral hexose (O- - -0) by the phoroglucinol-H,SO, procedure. Fractions 26-35 and 40-60 were pooled for analysis of amino acid and sugar components. (B) Similar analysis of 13HlGlcN-labeled VSV peptidyloligosaccharides on Bio-Gel P-6. The column excluded volume (V,) was located with BSA and the included volume (V,) with mannose.

dyloligosaccharides obtained after Pronase digestion of the purified VSV glycoprotein. Figure 1A shows the distributional analysis of sialic acid and neutral hexose detected by the thiobarbituric acid assay (Warren, 1959) and the phloroglucinol-sulfuric acid assay (Etchison et al., in preparation), respectively. Figure 1B shows the distribution of the peptidyloligosaccharides radiolabeled with 114Clglucosamine. In Fig. 1A some of the carbohydrate elutes in the void volume whereas none of the radiolabeled oligosaccharide material is excluded (Fig. 1B). Analysis of the carbo-

AND

SUMMERS

hydrate by gas-liquid chromatography indicated that the same sugars were present in similar ratios in both the excluded and included carbohydrate-containing species with the exception that the excluded material contained glucose while the included material did not (data not shown). The excluded material is considered to be artifactual and due to extreme differences in the ratio of glycoprotein to Pronase in the digestion mixtures. The ratio of glycoprotein to Pronase in Fig. 1A was approximately 5 to 1, whereas the Pronase was present in large excess in Fig. 1B. As will be apparent later, since the peptidyloligosaccharides of Fig. 1A were destined for analysis of their carbohydrate and amino acid constituents, it was necessary to keep the Pronase concentration low in order to avoid contamination of the peptidyloligosaccharides with autodigestion products of the Pronase. The included peptidyloligosaccharides shown in Fig. 1A (fractions 40-60) were pooled and divided into two portions. One portion was used for analysis of the carbohydrate constituents and the other analyzed for amino acid content. The results are shown in Table 1. Since the carbohydrate of the VSV glycoprotein is attached to the polypeptide by a linkage with the alkaline stability characteristics of the lN-glycosylaminide bond of N-acetylglucosamine to asparagine (Moyer et al., 19761, the data are tabulated as moles of sugar or amino acid per mole aspartic acid. The relative proportions of the different sugars are similar to that obtained with the intact glycoprotein (Etchison and Holland, 1974a) except for the absence of glucose. It was suggested previously that the glucose probably arose from cellulosic contamination. The molar ratios of the sugars to aspartic acid are approximately one-half the values obtained for moles of sugar per mole of glycoprotein (Etchison and Holland, 1974a). These data further substantiate previous data which indicated that there are an average of two oligosaccharides per VSV glycoprotein and agree with recent data which show that there are only two major carbohydrate-containing tryptic peptides on the VSV glycoprotein (Robert-

OLIGOSACCHARIDE TABLE CARBOHYDRATE AND AMINO THE PRONASE-DIGESTED

Sugar or amino acid

Fucose Mannose Galactose GlcNAc NeuNAc GalNAc Glucose Total

Aspartic acid Glycine Serine Tyrosine Threonine Glutamic acid Leucine Alanine Proline Valine Isoleucine Total

STRUCTURE

1 ACID

COMPOSITION GLYCOPEPTIDES”

OF

VSV Moles ner mole of Molecular asp&tic acid weight equivalents per mole of asp+& 0.42 4.39 2.98 4.50 2.63 0.74 Not detected 15.7 1.00

0.82 0.45 0.41 0.32 0.26 0.23 0.21 0.12 0.11 0.05 3.98

61.3 711.2 482.8 913.2 765.3 150.2 3084.3 133.1 47.6 39.2 66.9 32.3 33.6 26.0 14.9 11.7 10.0 5.7 374.2

a Carbohydrate composition was determined by gas-liquid chromatography (GLC) of the trimethylsilyl ethers of the methyl glycosides; amino acid composition was determined by GLC of the N-heptafluorobutyryl-isoamyl esters. b Values are corrected for water of glycoside or peptide bond formation.

son et al., 1976). The pooled fractions from Fig. 1A contained approximately 550 nmole of amino acids and 465 pg of carbohydrate. Approximately 35% of each fraction was used in the assay for sialic acid and neutral hexose (starting material was approximately 8 mg of glycoprotein). Therefore, approximately 715 pg of carbohydrate is accounted for and this indicates that virtually all (85-90%) of the carbohydrate is represented in these analyses. The major amino acids other than aspartic acid present in the peptidyloligosaccharides are glycine, serine, tyrosine, and threonine. The column on the right in Table 1 lists the molecular weight equivalents for each residue per mole of aspartic acid. Adding these values gives a value of

OF VSV

379

approximately 3450 for the average molecular weight of the peptidyloligosaccharides. This method of computing an average molecular weight, which is independent of gel filtration, is based on the assumption that aspartic acid occurs only once in the peptide moieties and that the carbohydrate is attached to that residue. It is possible that a fraction of the aspartic acid does not satisfy both of these requirements in which case the above value would tend to be an underestimation of the actual average molecular weight. Heterogeneity rides

of the Peptidyloligosaccha-

From the gel filtration profiles shown in Fig. 1 it is apparent that the peptidyloligosaccharide population is heterogeneous. This heterogeneity is more readily demonstrated when the population is subjected to gel filtration through Bio-Gel P-4. Figure 2A shows the gel filtration of the peptidyloligosaccharides before and after treatment with neuraminidase. This separation is similar to that obtained by Sefton (1976) except that there is a prominent peak larger than S, (designated So), less Sz, and no S,. Treatment with neuraminidase converts So, S1, and S, to a single peak which migrates in the same way as S, (Sefton, 1976; Fig. 2A) and a small peak of sialic acid. When the peptidyloligosaccharides were treated with neuraminidase at room temperature for only a few minutes in order to obtain a partial digestion, the result shown in Fig. 2B was obtained. In this experiment only 20-30% of the sialic acid has been released (4-5% of the glucosamine label), only a small amount of peak S:, has been formed, and some of the label originally present in peaks S, and S, has been shifted to peak S,. As suggested by Sefton (19761, it appears that the structures designated So, S1, Sz, and S, are related structures having different amounts of’sialic acid (probably three, two, one, and zero residues, respectively). Analysis of the carbohydrate-containing tryptic peptides of the VSV glycoprotein by ion-exchange chromatography, before and after neuraminidase treatment, has shown a similar sialic acid heterogeneity for the

380

ETCHISON,

_ 50

70

90 FRACTION

.A.& I HO NUMBER

I 130

ROBERTSON,

1 150

I

FIG. 2. Sialic acid heterogeneity of the VSV peptidyloligosaccharides. (A) [3H]GlcN-labeled VSV peptidyloligosaccharides obtained by digestion of the radiolabeled glycoprotein with Pronase were further digested with Cl. perfiingens neuraminidase for 24 hr. The neuraminidase was inactivated at 100” for 2 min and the digest was mixed with [WlGlcNlabeled peptidyloligosaccharides which had not been neuraminidase-treated. The mixture was analyzed on a 0.9 x 115 cm column of Bio-Gel P-4 (-400 mesh). The arrows mark the elution position of N-acetylneuraminic acid (NeuNAc), pentamannosyl-N-acetylglucosamine (Man,GlcNAc) and stachyose determined in a separate analysis. (B) [WlGlcN-labeled peptidyloligosaccharides treated with neuraminidase for only 5 min at room temperature before analysis on Bio-Gel P-4 column. O-O, 13H]GlcN; 0- - -0, [14ClGlcN.

individual oligosaccharide son et al., 1976).

chains

(Robert-

Gel Filtration Analysis of the Molecular Weight of the VSV Glycoprotein Oligosaccharides Previous results have demonstrated that the peptidyloligosaccharides obtained after Pronase digestion of the VSV glycoprotein have an average molecular weight in the range of 3000 to 4000 (Burge and Huang 1970; Etchison and Holland, 1974a; Moyer and Summers, 1974). In order to obtain a more quantitative estimate, the

AND

SUMMERS

peptidyloligosaccharides were sized by gel filtration relative to several peptidyloligosaccharides, peptides, and oligosaccharides with known molecular weights. During these studies it became apparent, as suggested by Burge and Huang (19701, that gel filtration on small pore polyacrylamide gels involves a charge exclusion effect as well as a size exclusion. This charge exclusion is evident in Fig. 2 where it can be seen that free sialic acid (NeuNAc) is excluded four to five times more than would be expected by size alone. This charge exclusion almost certainly accounts for a greater than expected resolution of the VSV peptidyloligosaccharides containing different amounts of sialic acid (see Fig. 2). This dramatic charge exclusion effect is not seen on small pore Sephadex gels (L. A. Hunt and D. F. Summers, in press), and decreases with increasing pore size in the polyacrylamide Bio-Gel series (J.R.E., unpublished observations). Therefore, any gel filtration study using the small pore Bio-Gels must take into account this charge exclusion effect. Table 2 lists the gel filtration characteristics of several oligosaccharides, peptides, and peptidyloligosaccharides according to their relative elution (K,) and molecular weight. These data are displayed in a semilogarithmic plot in Fig. 3. The points which deviate most from the curve are Nacetylneuraminic acid (r) and N-acetylneuraminyllactose (s). These two compounds are excluded more than neutral oligosaccharides of similar molecular weight. Bacitracin (k), a small oligopeptide, is not excluded as much as asparaginyloligosaccharides of similar molecular weight. Several asparaginyloligosaccharides (h, i, and l-o) are shown which represent a family of related, branched oligosaccharide structures. Their relative elution is linear with log molecular weight. After acetylation of the a-NH, group of asparagine in these structures, which results in a change from a net charge of 0 to -1, these compounds are excluded 10 to 15% more than expected by size alone relative to the nonacetylated structures (data not shown). In view of the above results, it would

OLIGOSACCHARIDE TABLE GEL FILTRATION

a b c d e f g h i k 1 m n o p q r s

2

OF MOLECULAR WEIGHT ON BIO-GEL P-6

Marker Mannose N-Acetylglucosamine Cellobiose Raftinose Stachyose Man,GlcNA@ Man,GlcNAcb Man,G1cNAc2Asnc Man,GlcNAc,Asnc Bacitracin Man,GlcNAc2AsnC Man,GlcNAc,AsnC Man,GlcNAc,Asn’ Man,GlcNAc,Asn’ Fetuin glycopeptides Bio-Rad Laboratories erence pointd N-Acetylneuraminic Neuraminyllactose

Molecular weight

refacid

STRTJCTURE

MARKERS Kde

180 221 342 504 666 869 1031 1187 1349 1411 1511 1714 1917 2120 (3800) 4600

1.00 0.96 0.98 0.92 0.85 0.80 0.74 0.65 0.59 0.65 0.55 0.50 0.45 0.42 0.17 0.10

309 633

0.83 0.78

OF

381

VSV

ligosaccharides were shown to be slightly smaller than AsnGlcNAc,Man, (MW = 2120) and nearly the same size as AsnGlcNAc,Man, (MW = 1917). From these studies we conclude that the molecular weight of the asialoagalacto structures is approximately 1900. IJsing the above value of 1900 daltons and working with the data in Table 1 which show three residues of galactose per residue of aspartic acid, a value of 2386 daltons is calculated for the asialopeptidyloligosaccharides. This number is within 10% of the value estimated from Fig. 4A. Assuming that the VSV peptidyloligosaccharides resolved as So, S1, and S, have three, two, and one residues of sialic acid, respectively, the calculated values for their molecular weights are 3259, 2968, and 2677, respectively. The molecular

a Relative elution defined by Kd = (V, - V,)/(V, - V,) where V, is the elution volume, Vu is the excluded volume, and V, is the totally included volume (Bio-Rad Laboratories, 1972). h Markers f and g isolated after endoglycosidase digestion of h and i, respectively. c Isolated from Pronase digests of ovalbumin by the procedure of Huang et al. (1970). d Manufacturer’s statement that a polypeptide of 4600 daltons has an R, of 0.9 (K,, of 0.1) on Bio-Gel P6.

appear that analyzing the molecular weight of the VSV peptidyloligosaccharides would be more reliable after removing the sialic acid residues to eliminate charge and charge heterogeneity. Figure 4A shows a comparison of the VSV asialopeptidyloligosaccharides with AsnGlcNAc,Man, (MW = 2120). As can be seen they are just slightly larger (- 200 daltons) than AsnGlcNAc,Man,. Removal of both sialic acid and galactose (see following sections) converts the VSV peptidyloligosaccharides to compositional homologues of the asparaginyloligosaccharide molecular weight markers. The gel filtration of the resulting asialoagalacto structures relative to AsnGlcNAcaMan, (MW = 1717) is shown in Fig. 4B. In other comparisons (not shown) asialoagalactopeptidylo-

I

0

I

I

1

I

I

.2

.4

.6

.8

I.0

Kd FIG. 3. Molecular weight calibration curve for Bio-Gel P-6 column. The relative elution (expressed as Kd; see Table 2) of several compounds of known molecular weight was determined, the logarithm of the molecular weight was plotted as a function of their Kd. The lettered points are identified in Table 2. S,,, S,, S, refer to the VSV peptidyloligosaccharides (Gpeps) containing three, two, and one residues of sialic acid, respectively. The “asialo” arrow indicates the Kd for the VSV Gpeps after treatment with neuraminidase.

382

ETCHISON,

ROBERTSON,

6

,4

2 Y

0

x I 6:

4

2

FRACTION

NUMBER

FIG. 4. Comparison of neuraminidase- and P-galactosidase-treated VSV peptidyloligosaccharides with monodisperse asparaginyloligosaccharides from ovalbumin. (A) VSV peptidyloligosaceharides were digested with neuraminidase, mixed with hexamannosyl-penta-N-acetylglucosaminyl-asparagine (AsnGlcNAc,Man,; MW = 2120), and analyzed by gel filtration on Bio-Gel P-6. (B) The VSV peptidyloligosaccharides were digested with neuraminidase and P-galactosidase and cochromatographed with hexamannosyl-tri-iV-acetylglucosaminyl asparagine (AsnGlcNAc,Man,; MW = 1714). The VSV peptidyloligosaccharides were radiolabeled with [3H1GlcN (O- - -0); the asparaginyloligosaccharides were located by assaying aliquots of each fraction for neutral hexose by the phloroglucinol-H,SO, assay (0-O). The small peak of neuraminic acid (NeuNAc) was radiolabeled by the metabolism of 13HlGlcN during the in uiuo incorporation.

weights of So, S,, and Sp estimated by gel filtration using the calibration curve in Fig. 3 are 3600, 3100, and 2700, respectively. The values for SZ arrived at by the two methods are nearly identical and the values for S,,, and S, differ by only lo-15%. At least part of this difference is due to the charge exclusion discussed earlier. The distribution of GlcN label in peaks S,,, S,, and SZ was approximately 25%, 60%, and 15% respectively (see Fig. 2A). Using this distribution and the molecular

AND SUMMERS

weights given above, an average molecular weight of 3165 daltons is obtained using the gel filtration estimates while a value of 3000 daltons is obtained when the backcalculated numbers are used. These values are within approximately lo-15% of the value of 3450 daltons arrived at independently from the data in Table 1. It should be noted, however, that the latter value is a weight-average molecular weight while the former values are number-average molecular weights (Tanford, 1961). With a distribution which favors S,, over &, a weight-average molecular weight would be expected to be slightly larger than a number-average molecular weight. Removal of Sialic Acid from the VSV Peptidyloligosaccharides

Since previous results showed that the VSV glycoprotein contained substantial amounts of neuraminic acid (Burge and Huang, 1970; McSharry and Wagner, 1971; Etchison and Holland, 1974a, b), we analyzed the conditions for selective removal of these sialic acid residues. Figure 5A shows that [3H]GlcNand [14ClGlcN-labeled peptidyloligosaccharides prepared by separate digestions of the purified VSV glycoprotein with Pronase coelute on BioGel P-6. This was generally true, and premixing of similar samples prior to Pronase digestion (limit digests) appears to be unnecessary. Two methods were used to remove the sialic acid residues. First, the sialic acid residues were released by dilute acid hydrolysis using conditions which leave other glycoside bonds intact (Marshall and Neuberger, 1972a). Figures 5B and 5C show the elution of the desialyzed VSV peptidyloligosaccharides prepared by hydrolysis with 0.1 N H,SO, at 80” for 30 and 60 min, respectively, relative to the untreated peptidyloligosaccharides. Figure 5D shows a similar experiment except that the peptidyloligosaccharides were treated with 0.1 unit of Cl. perfiingens neuraminidase to remove the sialic acid. The results were identical for both methods of removing the neuraminic acid residues. Approximately 15% of the GlcN label was released from the structures by all

OLIGOSACCHARIDE

30

50 70 FRACTION

FRACTION

90 NUMBER

STRUCTURE

383

OF VSV

110

NUMBER

FRACTION

FRACTION

NUMBER

NUMBER

FIG. 5. Removal of sialic acid from the VSV peptidyloligosaccharides by dilute acid hydrolysis and by neuraminidase. (A) 13H]GlcN- and [WlGlcN-labeled peptidyloligosaccharides (Gpeps) were mixed and chromatographed on Bio-Gel P-6; (B) and (C) the [3H]GlcN-labeled Gpeps were hydrolyzed with 0.1 N H&SO, at 80” for 30 and 60 min, respectively, before neutralizing and cochromatographing with untreated [14C]GlcNlabeled Gpeps; (D) the 13H]GlcN-labeled Gpeps were digested with 0.1 unit of C1. perfringens neuraminidase for 24 hr before cochromatogranhins -- with untreated [*4C1GlcN-labeled Gpeps. O----e, [3H]GlcN; 0- - -0, WlGlcN.

conditions. The released radioactivity represents label derived from GlcN metabolized into neuraminic acid during incorporation in the infected cell. Similar levels of metabolism of GlcN label into sialic acid have been reported by others (Burge and Huang, 1970). The radioactivity released by both dilute acid hydrolysis and neuraminidase had a relative elution (Kd) of 0.82. This is the same as the elution of authentic N-acetylneuraminic acid and different from that of N-acetylglucosamine. Removal of Galactose from the VW Peptidyloligosaccharides By analogy with the oligosaccharide structures of serum glycoproteins, we surmised that the galactose residues present in the VSV peptidyloligosaccharides would be located penultimately in the oligosaccharide structure. In order to demonstrate this, we digested the VSV peptidyloligosaccharides with highly purified p-galactosidase from A. niger in the presence and

absence of neuraminidase. Figure 6A shows the gel filtration profile of the untreated peptidyloligosaccharides radiolabeled with 13H]Gal and [14ClGlcN. Figure 6B shows that treatment of the same peptidyloligosaccharide mixture with neuraminidase removes approximately 15% of the glucosamine label as sialic acid, does not remove any of the galactose label, and converts the mixture to smaller structures which coelute with an apparent molecular weight of 2300. Figure 6C shows that treatment with P-galactosidase alone removes only a few percent of the galactose label and does not alter the gel filtration profile very much from that seen in Fig. 6A. The S, peak is slightly smaller and more distinct, however, suggesting that the small amount of galactose label released may have come from these structures. When the peptidyloligosaccharides are incubated in the presence of both neuraminidase and &galactosidase, as shown in Fig. 6D, the galactose label is quantitatively released from the peptidyloligosaccharide struc-

384

ETCHISON,

ROBERTSON,

50 FRACTION

AND SUMMERS

50

70

90

110

NUMBER

FIG. 6. Removal of galactose from the VSV peptidyloligosaccharides by p-galactosidase. The VSV peptidyloligosaccharides (Gpeps) were prepared from [3H]Gal- and [%!]GlcN-labeled VSV glycoprotein by extensive digestion with pronase. (A) The cochromatography on Bio-Gel P-6 of the [3H]Gal- (0-O) and the WlGlcN-labeled (0- - -0) Gpeps before glycosidase treatment. (B) The same mixture after treatment with neuraminidase only. (Cl A similar chromatogram after treatment of the mixture with P-galactosidase alone. (D) The chromatogram after treatment with both neuraminidase and p-galactosidase.

tures. The resulting glucosamine-labeled structures are smaller than those in Fig. 6B and have an apparent molecular weight of 1900. No glucosamine label other than that present as sialic acid was released. To show that no other sugars were released by treatment with these enzymes, 13HlMan- and [3HlFuc-labeled peptidyloligosaccharides were digested under the same conditions with the neuraminidase and /3-galactosidase. Figure 7 shows that neither mannose nor fucose was released from the peptidyloligosaccharides by this treatment. We conclude that the galactose present in the VSV oligosaccharides is located penultimate to the sialic acid residues. The only alternative explanation is that the sialic acid residues inhibit the action of the /3-galactosidase by steric hindrance. This possibility is ruled out later by the periodate oxidation studies. In Fig. 7B it can be seen that the fucose-labeled peptidyloligosaccharides do not completely coelute with the glucosamine-labeled structures but rather elute with the leading edge and peak one fraction earlier. The most reasonable explanation for this is as follows: The VSV glycoprotein has an average of

only one fucose residue but has two oligosaccharide side chains (Etchison and Holland, 1974a; Robertson et al., 1976). However, we have recently shown (Robertson et al., 1976) that both oligosaccharide side chains contain about equal amounts of fucase. Therefore, each population of the oligosaccharide side chains has an average of approximately 0.5 residues of fucose. Hence, the oligosaccharide structures are heterogeneous in fucose content as well as sialic acid. It is clear, then, that that portion of the peptidyloligosaccharides having a fucose attached will be larger than the remainder having none, and the one fraction difference seen in Fig. 7B is of the magnitude expected for a difference of one sugar residue. Release of N-Acetylglucosamine and Mannose from the VSV Peptidytoligosaccharides by exo-/3-N-Acetylglucosaminidase, a-Mannosidase, P-Mannosidase, and endo- /3-N-Acetylglucosaminidase D Figure 8A shows the gel filtration of untreated [3Hlmannose-labeled versus l’4ClGlcN-labeled peptidyloligosaccharides. The elution profiles are virtually

OLIGOSACCHARIDE

STRUCTURE

A 3 4 6 2 3 :

N h

0-

x

x

5 E

I %

6 Y

$2

I

30

50 FRACTION

70

90

110

NUMBER

FIG. 7. Treatment of [3H]mannoseand [3H]fucase-labeled VSV peptidyloligosaccharides with neuraminidase plus P-galactosidase. To show that no sugars other than sialic acid and galactose were released by the neuraminidase and p-galactosidase digestion, 13HlMan(Al or 13HlFuc- (Bl labeled VSV peptidyloligosaccharides were mixed with [‘%IGlcN-labeled peptidyloligosaccharides and digested with neuraminidase and p-galactosidase as in Fig. 6D, and analyzed on Bio-Gel P-6 column. O-O, 13HlManor 13H]Fuc-; 0- - -0, [WlGlcN.

superimposible. This result is identical to that reported by Sefton (1976) for VSV and contrasts with the result obtained for Sindbis virus (Sefton and Keegstra, 1974; Sefton, 1976). Sefton and Keegstra (1974) and Sefton (1976) have shown that one of the two oligosaccharide chains present on each of the Sindbis virus glycoproteins (E, and E,) contains mannose and glucosamine as the only sugar components. To show that the VSV glycoprotein does not contain such a structure, we digested the mannose- and glucosamine-labeled peptidyloligosaccharides with a mixture of beef kidney exo-/3-N-acetylglucosaminidase and jack bean a-mannosidase. Figure 8B shows that neither mannose nor glucosamine can be hydrolyzed from the struc-

OF

VSV

385

tures by glucosaminidase and mannosidase alone. In contrast, if sialic acid and galactose residues are first removed by treatment with neuraminidase and P-galactosidase, then approximately 50-60% of the glucosamine and at least 90% of the mannose labels are released as the monosaccharides by the p-glucosaminidase and cY-mannosidase (Fig. 8E). These results demonstrate that the glucosamine and ma.nnose are located internally in the oligosaccharide structures. (See also periodate oxidation section). Figure 8C shows that treatment of the peptidyloligosaccharides with neuraminidase, galactosidase, and exo-glucosaminidase in the absence of mannosidase released approximately 50-60% of the glucosamine label as monomeric N-acetylglucosamine without removing any of the mannose label. The resulting peptidyloligosaccharides have an average molecular weight of 1350-1450. In contrast, if the peptidyloligosaccharides are incubated with neuraminidase, galactosidase, and a-mannosidase without adding the exo-glucosaminidase, none of the mannose label is released (Fig. 8D). These results demonstrate that the mannose residues are blocked by a portion of the N-acetylglucosamine residues and that the glucosamine residues are located internally to the termmal sialylgalactose structures. As mentioned above, Fig. 8E shows that treatment of the peptidyloligosaccharides with neuraminidase, P-galactosidase, /3glucosaminidase, and a-mannosidase releases approximately 60% of the glucosamine label and about 90% of the mannose la‘bel. Yet a small glycopeptide which contains approximately 40% of the glucosamine label remains (fractions 70-80). This structure is resistant to further digestion with p-glucosaminidase. However, if the digestion is carried out in the presence of a partially purified /3-mannosidase (ex A. ni,ger), additional digestion of this peak occurs which results in the disappearance of the peak and the release of more glucosamine label as is shown in Fig. 8F. The only other glycosidase activities detected in this partially purified /3-mannosidase were CY-and /3-galactosidase. Note that

386

ETCHISON.

ROBERTSON,

AND SUMMERS

30

FRACTION

50

m

90

110

NUMBER

Fro. 8. Removal of N-acetylglucosamine and mannose residues from the VSV peptidyloligosaccharides by exo+N-acetylglucosaminidase, cY-mannosidase, and p-mannosidase. [3HlMan- and [WGlcN-labeled VSV peptidyloligosaccharides were prepared by Pronase digestion of the radiolabeled VSV glycoprotein, mixed, treated with different combinations of exo-glycosidases, and analyzed on a Bio-Gel P-6 column. (A) Untreated; (B) treated with p-N-acetylglucosaminidase and cY-mannosidase in the absence of neuraminidase and /3-galactosidase; (C) treated with neuraminidase, /3-galactosidase, and p-N-acetylglucosaminidase; (D) treated with neuraminidase, p-galactosidase, and a-mannosidase; (E) treated with neuraminidase, /3and a-mannosidase; (F) same as (E) plus p-mannosidase. galactosidase, p-N-acetylglucosaminidase, O-O, 13H1Man; 0- - -0, [WlGlcN.

there is a small amount of the mannose label which elutes under (but is slightly larger than) the glucosamine peak in Fig. 8E. However, this label does not appear to have been released by the p-mannosidase (Fig. 8F). We suspect, but have not shown, that this may be fucose labeled by the metabolism of GDP-mannose into GDPfucose during incorporation in the infected cells. To summarize this result, shown in Figs. 8E and 8F, there appears to be a structure in the oligosaccharides which blocks the release of some of the glucosamine residues by p-glucosaminidase, is not released by a-mannosidase, is released by a partially purified p-mannosidase, and is not labeled with [3H]mannose when this precursor is supplied to the infected cells. The simplest interpretation of this result is that the oligosaccharide moieties of the

VSV glycoprotein contain a p-linked mannose residue analogous to that which has been described for the oligosaccharides of ovalbumin (Tai et al., 1975) but which is not labeled with exogenous mannose added during the synthesis of the oligosaccharides in the infected cells. Alternative explanations are possible and a chemical characterization of this structure and its digestion products using a more highly purified /3-mannosidase will be needed to substantiate the data presented here which have been interpreted as indicative of a p-mannose linkage. If the interpretation of these results is essentially correct, significant difference in the biosynthetic pathways of (Y-and p-linked mannose residues may be inferred. Figure 9A shows the gel filtration analysis of the products of the digestion of

OLIGOSACCHARIDE

STRUCTURE

[3Hlmannose- and [14C]GlcN-labeled peptidyloligosaccharides with neuraminidase, galactosidase, exo-glucosaminidase, and endo-/3-N-acetylglucosaminidase D (all isolated from S. pneumoniae). This enzyme mixture lacks a-mannosidase and, as can be seen in Fig. 9A, none of the mannose label is released as the monosaccharide. The products of a similar digestion of the VSV oligosaccharides have been analyzed previously by Moyer and Summers (1974). The mannose was shown to be released as a neutral oligosaccharide containing mannose and glucosamine. From the known substrate specificity of the endo-glucosaminidase (Tai et al., 1975), this oligosaccharide is almost certainly an oligomannosyl-l\‘-acetylglucosamine. Figure 9B shows that this oligomannosyl-glucosamine structure is slightly smaller than the pentamannosyl-N-acetylglucosamine product of the endo-glucosaminidase digestion of AsnGlcNAczMan,, but is larger than stachyose, a tetrasaccharide. Based on this comparison and the data in Table 1 which indicate an average of approximately four residues of mannose per residue of aspartic acid, the structure released from the VSV oligosaccharides is tentatively identified as tetramannosyl-Nacetylglucosamine. Proof that this is indeed the structure will ultimately rely on further chemical analysis.

30

50

70

90

387

VSV

Periodate Oxidation ligosaccharides

of the VSV Peptidylo-

In order to obtain further information on the nature of the VSV oligosaccharide structures, the peptidyloligosaccharides were subjected to periodate oxidation followed by borohydride reduction and dilute acid hydrolysis (Smith degradation, see review by Marshall and Neuberger, 1972b). The products were then neutralized and analyzed by gel filtration. YFigure 10 shows the gel filtration analysis of the products of the Smith degradation of [3H]galactoseand [14Clglucosamine-labeled VSV peptidyloligosaccharides before and after treatment with neuraminidase. Figures 10A and 10B show the results obtained when the oxidation is carried out for only 2 hr, while Figs. 1OC and 10D show the results after a 20-hr periodate oxidation. A primary purpose of this experiment was to determine if the linkage of sialic acid to galactose renders the galactose residues periodate resistant or not. If the galactose is resistant to perioda.te oxidation before removal of the sialic acid, the linkage of sialic acid to galactose must be an a-2,3 linkage. If it is not resistant, then the linkage may theoretically be a-2,2; (r-2,4; or a-2,6. Of these latter linkages only the a-2,6 linkage has ever been observed for sialic acid linked to galactose

110

FRACTION

OF

60

SO

100

120

140

160

NUMEER

FIG. 9. Digestion of the VSV peptidyloligosaccharides with endo-P-N-acetylglucosaminidase D. (A) A mixture of 13H]Manand [WlGlcN-labeled VSV peptidyloligosaccharides was digested with endo-6-Nacetylglucosaminidase D in the presence of neuraminidase, P-galactosidase, and exo-6-N-acetylglucosaminidase. The digestion products were analyzed by gel filtration through Bio-Gel P-6. O-O, 13H]Man; 0- - -0, 114C]GlcN. (B) 13HlMan-labeled VSV peptidyloligosaccharides were mixed with asparaginylGlcNAc,Man,, digested with the endo-glycosidase mixture as in (A), and chromatographed on Bio-Gel P-4. The mannose-containing digestion product of AsnGlcNAc2Man, is GlcNAciMan, (Tai et al., 1975) and was located by assaying aliquots of each fraction for neutral hexose (O- - -0). The elution position of stachyose was determined in a separate run and is indicated by an arrow. O-O, t3H]Man.

388

ETCHISON,

ROBERTSON,

AND

SUMMERS

FRACTION NUMBER FIG. 10. Periodate oxidation of the VSV peptidyloligosaccharides, [3H]Gal(0-O) and [“‘C]GlcN(O- - -0) labeled VSV peptidyloligosaccharides were mixed. The mixture, with and without prior treatment with neuraminidase, was oxidized with sodium periodate for either 2 or 20 hr, reduced with sodium borohydride, hydrolyzed with 0.1 N H,SO, at loo”, neutralized, and the products analyzed by gel filtration through Bio-Gel P-6. (A) A 2-hr oxidative degradation; (B) 2-hr oxidation after neuraminidase treatment; (0 20-hr oxidation; and (D) 20-hr oxidation after neuraminidase treatment.

(Tuppy and Gottschalk, 1972). Thus, if the galactose is sensitive to periodate oxidation before removal of the sialic acid, we may infer an a-2,6 linkage. The results in Fig. 10 show that the sialic acid is joined by an (u-2,3 link to the galactose residues. The interpretation of the gel filtration data in Fig. 10 which gives this conclusion is as follows: Fig. 10A shows that only approximately 10% of the galactose label elutes in the totally included volume after a 2-hr periodate oxidation-smith degradation. Figure 1OC shows that very little or no additional galactose label elutes in the totally included volume even after a 20-hr oxidation step. Thus, only approximately 10% of the galactose label is liberated as either monomeric galactose or degradation products of galactose even after a prolonged oxidation. It is apparent in Fig. 1OC that internal oxidations have occurred since a peak which is partially resolved from the peptidyloligosaccharides has appeared and a smaller peak (fractions 80-90) has appeared in the tri- to pentasaccharide region of elution. Figures 10B and 10D show that removal of sialic acid before the Smith degradation results in approximately 60% and 90% of the galactose label being released and eluting in

the totally included volume of the column after periodate oxidation steps of 2 and 20 l-n-, respectively. Hence, at least 90% of the galactose residues are resistant to periodate oxidation before, but not after, removal of the sialic acid residues. It should be noted that the amount of galactose label released by the Smith degradation before removal of sialic acid is approximately the same as that released by P-galactosidase before removal of the sialic acid residues (Fig. 60. Additional information concerning the general structure of the VSV oligosaccharides can be obtained from the data in Fig. 10. As can be seen in Figs. 1OC and lOD, none of the glucosamine label is released from the intact oligosaccharide structures as either monomeric N-acetylglucosamine or its degradation products which would peak between fractions 91 and 97. (A portion is released as a short oligosaccharide.) This indicates that the glucosamine residues are substituted at carbons 3 or 4 and confirms the enzymatic degradation studies (see Fig. 8B) by showing that none of the glucosamine residues are located in terminal positions. As is evident in Figs. 1OC and lOD, there is at least one internal linkage in the oligosaccharides which is

OLIGOSACCHARIDE

STRUCTURE

periodate sensitive. Figure 1OC suggests that the small oligosaccharide (apparent MW approximately 600-700) released by the internal oxidation contains galactose as well as glucosamine. This, in conjunction with the enzymatic degradation, indicates that the released structure contains a portion of the oligosaccharide from the “nonreducing” end of the peptidyloligosaccharide structures. Structure of the moieties

VSV

Oligosaccharide

Figure 11 shows the structure of the VSV glycoprotein oligosaccharide moieties consistent with the results of the sequential degradation studies presented here. Sites of heterogeneity in this structure are indicated by a ” 2” symbol. This is a generalized structure and applies to both of the oligosaccharide chains of the G protein of VSV grown in BHK21 cells. It is apparent from the uniform behavior of the oligosaccharide population at each step in the sequential enzymatic degradation that both side chains have very similar structures. It would not be detectable, however, if one had only two branches at the “nonreducing” end of the oligosaccharide while the other had three. Therefore, this uncertainty is reflected in Fig. 11 by the “+?” where one of the branch chain GlcNAc residues is attached to the oligosaccharide core structure. It should be noted that both the quantitative data in Table 1 and the stoichiometry of the sequential degradation as determined by the decrease in molecular weight of the peptidyloligosaccharides is consistent, within the limits of experimental error, either with such a mixture or with both containing three branches. NeuNAc~Gal”*30’4 (*I

GlcNAc

OF

389

VSV

The branch chain /I-GlcNAc residues are shown connected to three a-mannose residues in the core structure. The nature of these linkages will require further studies by periodate oxidation and methylation analysis. These mannose residues are shown connected in an unspecified manner to a fourth mannose residue. This mannose residue is most likely in the p conformation as discussed earlier in the results. The “reducing” end of the oligosaccharide structure has two residues of GlcNAc as indicated by cleavage at this point with endo-p-N-acetylglucosaminidase D. The GlcNAc residue proximal to the peptide moiety is substituted with fucose (Moyer and Summers, 1974), but heterogeneity exists at this point since there is only one fucose residue per two oligosaccharide chains and both contain approximately equal amounts of the fucose (Etchison and Holland, 1974a; Robertson et al., 1976). DISCUSSION

The results presented here show that the VSV peptidyloligosaccharides obtained after Pronase digestion of the purified VSV glycoprotein are heterogeneous and have a weight-average molecular weight of 3450 and a number-average molecular weight of 3150. Analysis of the arnino acid and carbohydrate constituents indicated an average of 4 amino acid residues and 16 sugar residues. These data also show that the values for the residues of sugar per residue aspartic acid are approximately one-half the values obtained for residues of sugar per glycoprotein (Etchison and Holland, 1974a). A high-resolution gel filtration column (Bio-Gel P-6) has been used to monitor sequential degradation of the VSV oligo-

P

\ NeuNAce

(*I

Gal~GIcNAc~

[*?)

’ (Man)3

*Man-GlcNAdGlcNAc-Asn

/ NeuNAca FIG. 11. Structure acidic oligosaccharide neity in these structures.

(4 Fkse

_/”

Gal”‘.3Q4GlcNAc (4 of the VSV glycoprotein oligosaccharides. structures attached to the VSV glycoprotein.

The above The “?”

structure applies to both of the signs indicate sites of heteroge-

390

ETCHISON,

ROBERTSON,

saccharides. This column was calibrated for molecular weight estimation using several oligosaccharides and peptidyloligosaccharides. Removal of sialic acid residues by either neuraminidase or dilute acid hydrolysis results in peptidyloligosaccharides having an average molecular weight of approximately 2300. The galactose residues can be hydrolyzed by /3-galactosidase only after removal of the sialic acid residues. The peptidyloligosaccharides remaining after the removal of the galactose residues have an average molecular weight of approximately 1900. This decrease in molecular weight is consistant with the loss of two to three residues of galactose. After removal of the sialic acid and galactose, approximately 50-60% of the glucosamine label can be released by /?-glucosaminidase, and the resulting peptidyloligosaccharides have an average molecular weight of 1350-1450. This decrease in molecular weight corresponds to the removal of two to three residues of N-acetylglucosamine. The remainder of the glucosamine residues can be released only after removal of the mannose residues. None of the mannose can be released by cY-mannosidase without first treating the intact peptidyloligosaccharides with neuraminidase, P-galactosidase, and /3-N-acetylglucosaminidase. All of the peptidyloligosaccharides from VSV grown in BHK21 cells can be cleaved by the endo-/3-N-acetylglucosaminidase from S. pneumoniae in the presence of neuraminidase, p-galactosidase, and exop-N-acetylglucosaminidase. One of the products of this digestion is a mannosecontaining oligosaccharide having the properties of tetramannosyl-N-acetylglucosamine. Sequential digestion of the peptidyloligosaccharides with specific exoglycosidases has given preliminary evidence that three of the mannose residues are in the (Yconformation and one has a ,8 linkage to the N-acetylglucosamine. The periodate oxidation studies show that the sialic acid residues are linked (Y2,3 to the galactose residues. Since none of the glucosamine label was released as monomeric glucosamine or degradation products, the galactose residues must pro-

AND SUMMERS

tect the distal or branch chain GlcNAc residues from oxidation. This means that the galactose must be linked p-1,3 or p-1,4 to these GlcNAc residues. The structural studies described here pertain only to the two major oligosaccharide moieties of the VSV glycoprotein. These two structures make up at least 90% of the total carbohydrate of the VSV glycoprotein. These structures are the complex, acidic type of oligosaccharides frequently found on serum glycoproteins. The VSV glycoprotein does not contain any simple oligosaccharides terminating in mannose or glucosamine such as are found in ovalbumin or ribonuclease. We do suspect, however, the presence of a third, minor oligosaccharide containing only a few sugar residues which may be similar to the short oligosaccharides found attached to submaxillary mucins. Several studies have indicated that glycosylation of viral polypeptides is a function of the host cell and requires the action of the glycosyl transferases of the host cell (Burge and Huang, 1970; Etchison and Holland, 1974b; Moyer and Summers, 1974; Schloemer and Wagner, 1975b; Schlessinger et al., 1976). In addition, studies by Sefton (1976) have shown that the size and size distribution of the viral glycopeptides are different for different viruses grown in the same types of cells. Thus, while the host cell glycosyl transferases are clearly necessary and essential for the synthesis of the viral glycoprotein oligosaccharides, they may not be sufficient to specify, in toto, the overall oligosaccharide composition and structures which are attached to different viral glycoproteins. The structure of the viral polypeptide may be expected to determine the number of oligosaccharides which the enzymes of a given cell type may add to the glycoprotein. In addition, if a host cell is capable of attaching more than one type of oligosaccharide structure to polypeptides, then different viral glycoproteins may acquire different oligosaccharide structures as a function of their respective polypeptide structures. Using the detailed structural information presented here and the techniques

OLIGOSACCHARIDE

employed here, a direct analysis of any differences in the structure of the viral glycoprotein oligosaccharides acquired as a function of the host cell may be made and the differences defined. The function of the oligosaccharides on the VSV glycoprotein is not clear. Studies by Schloemer and Wagner (1975a) indicate that the sialic acid residues are required for viral infectivity whereas studies by Schlessinger et al. (1976) indicate that they are not. While it has been shown that the VSV glycoprotein is the virion component which induces and binds anti-VSV neutralizing antibodies (Kelley et al., 1972; Dietzschold et al., 19741, any role which the oligosaccharides may have in these processes has not been studied. The influence of the oligosaccharides on virion stability has not been studied. However, Zavada (1972) has shown that mutants of VSV selected for altered glycoprotein structure are thermolabile. ACKNOWLEDGMENTS This research was supported by Public Health Service Grant No. l-ROl-AI-12316-01 from the National Institute of Allergy and Infectious Diseases and by National Science Foundation Grant No. BMS 74-2112%A01 to Donald F. Summers. James S. Robertson is the recipient of a NATO postdoctoral fellowship from the Science Research Council, London, United Kingdom; James R. Etchison is a postdoctoral fellow of the American Cancer Society. REFERENCES BAHL, 0. P., and AGRAWAL, K. M. L. (1972). pGalactosidase, P-galactosidase, and P-N-acetylglucosaminidase from Aspergillus niger. In “Methods in Enzymology” (V. Ginsburg, ed.) Vol. 28, pp. 728-734, Academic Press, New York. BIG-RAD LABORATORIES (19721. “Gel Chromatography,” Bio-Rad Laboratories, Richmond, Calif. BURGE, B. W., and HUANG, A. S. (1970). Comparison of membrane protein glycopeptides of Sindbis virus and vesicular stomatitis virus. J. Viral. 6, 176-182. DIETZSCHOLD, B., SCHNEIDER, L. G., and Cox, J. H. (1974). Serological characterization of the three major proteins of vesicular stomatitis virus. J. Viral. 14, l-7. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., and SMITH, F. (1956). Calorimetric method for determination of sugars and related substances. AnaZ. Chem. 28. 350-356. ETCHISON, J. R., and HOLLAND, J. J. (1974a). Carbo-

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VSV

391

hydrate composition of the membrane glycoprotein of vesicular stomatitis virus. Virology 60, 217-229. ETCHISON, J. R., and HOLLAND, J. J. (1974b). Carbohydrate composition of the membrane glycoprotein of vesicular stomatitis virus grown in four mammalian cell lines. Proc. Nat. Acad. Sci. USA 71, 4011-4014. ETCHISON, J. R., and HOLLAND, J. J. (1975). A procedure for the rapid, quantitative N-acetylation of amino sugar methyl glycosides. Anal. Biochem. 66, 87-92. HUANG, C.-C., MAYER, H. E., JR., and MONTGOMERY, R. (1970). Microheterogeneity and paucidispersity of glycoproteins. Part I. The carbohydrate of chicken albumin. Carbohyd. Res. 13,127-137. HUGHES, R. C., and JEANLOZ, R. W. (19641. The extracellular glycosidases of DipZococcus pneumoniae, I. Purification and properties of a neuraminidase and a P-galactosidase. Action on the alacid glycoprotein of human plasma. Biochemistry 3, 1535-1543. KELLEY, J. M., EMERSON, S. U., and WAGNER, R. R. (19721. The glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody. J. Viral. 10, 12311235. KOIDE, N., and MURAMATSU, T. (1974). Endo-/3-Nacetylglucosaminidase acting on carbohydrate moieties of glycoproteins. Purification and properties of the enzyme from Diplococcus pneumoniae. J. Biol. Chem. 249, 4897-4904. LEE, Y. C., and SCOCCA, J. R. (1972). A common structural unit in asparagine-oligosaccharides of several glycoproteins from different sources. J. Biol. Chem. 247, 5733-5758. LI, Y.-T., and LI, S.-C. (1972). a-Mannosidase, p-Nacetylhexosaminidase, and p-galactosidase from jack bean meal. In “Methods in Enzymology” (V. Ginsburg, ed.) Vol. 28, pp. 702-713, Academic Press, New York. MARSHALL, R. D., and NEUBERGER, A. (1972a). In “Glycoproteins. Their Synthesis, -Structure, and Function” (A. Gottschalk, ed.1, pp. 224-299, Elsevier, Amsterdam. MARSHALL, R. D., and NEUBERGER, A. (1972b). In “Glycoproteins. Their Synthesis, Structure, and Function” (A. Gottschalk, ed.), pp. 322-380, Elsevier, Amsterdam. MCSHARRY, J. J., and WAGNER, R. R. (1972). Carbohydrate composition of vesicular stomatitis virus. J. Viral. 7, 412-415. MOYER, S. A., and SUMMERS, D. F. (1974). Vesicular stomatitis virus envelope glycoprotein alterations induced by host cell transformation. Cell 2, 63-70. MOYER, S. A., TSANG, J. M., ATKINSON, P. H., and SUMMERS, D. F. (1976). Oligosaccharide moieties of the glycoprotein of vesicular stomatitis virus. J. Virol. 18, 167-175.

ETCHISON,

ROBERTSON,

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