JOURNAL OF VIROLOGY, Sept. 1976, p. 871-878Copyright C 1976 American Society for Microbiology

Vol. 19, No. 3Printed in U.S.A.

Glycosylation Sites of Vesicular Stomatitis VirusGlycoprotein

JAMES S. ROBERTSON,* JAMES R. ETCHISON, AND DONALD F. SUMMERSDepartment of Microbiology, Medical Center, University of Utah, Salt Lake City, Utah 84132

Received for publication 19 April 1976

Detailed analysis on DEAE-Sephadex of the tryptic digestion products of theglycoprotein from vesicular stomatitis virus grown in HeLa suspension culturesrevealed the presence oftwo major and several minor sugar-labeled species. Theminor tryptic glycopeptides were converted to one of the two major glycopeptidespecies by treatment with neuraminidase. Thus, vesicular stomatitis virusglycoprotein contains only two oligosaccharide side chains that are heteroge-neous in their sialic acid content.

Vesicular stomatitis virus (VSV) consists ofaribonucleoprotein core surrounded by a mem-brane envelope containing two proteins, themembrane protein M and the glycoprotein G (8,16). This single viral glycoprotein can be selec-tively removed from the virion with nonionicdetergents (10). The ease of purification of theglycoprotein makes it an excellent candidatefor the chemical analysis ofa membrane-associ-ated glycoprotein.The carbohydrate moiety of the glycoprotein

constitutes 9 to 10% of the weight of the glyco-protein, and its composition suggests a struc-ture similar to that of serum-type glycoproteinoligosaccharides (5, 12). Each oligosaccharideside chain is approximately 2,000 daltons afterremoval of the terminal sialic acid residues (J.R. Etchison, J. S. Robertson, and D. F. Sum-mers, manuscript in preparation) and is at-tached to the polypeptide by a 1-N-glycosylam-inide linkage of N-acetylglucosamine to theamide nitrogen of asparagine (13). Althoughthe primary sequence of the glycoprotein isspecified by the virus (2, 3, 6), the virus genomedoes not contain sufficient information to spec-ify the various glycosyl transferases required tosynthesize the oligosaccharide side chains.Evidence based on the size of the oligosaccha-

ride side chains and carbohydrate content oftheglycoprotein indicates that there is an averageof two oligosaccharide chains per glycoproteinmolecule (5). A recent report indicates thattreatment of the VSV glycoprotein with cyano-gen bromide produces at least three peptidefragments containing glucosamine (15). Grub-man et al. (6) showed that trypsin digestion ofthe VSV glycoprotein produced several carbo-hydrate-containing peptides that were separa-ble by high-voltage electrophoresis.The objective of this report was to determine

the number of oligosaccharide side chains perVSV glycoprotein molecule by detailed analysisof tryptic digests of the purified glycoprotein.This study demonstrates that the glycoproteincontains two glycosylation sites per polypep-tide, to which greater than 95% of the carbohy-drate is attached. The data also show that thesialic acid content of each oligosaccharide chainis variable and that failure to account for sialicacid heterogeneity explains previous reports ofmore than two oligosaccharides per VSV glyco-protein.

MATERIALS AND METHODSGrowth, labeling, and purification of virus. Stock

preparations of VSV (Indiana) were grown in HeLaS3 suspension cultures, purified, and assayed asdescribed previously (7, 14). For radioactively la-beled VSV glycoprotein, 5 x 108 HeLa cells wereinfected with 10 PFU of VSV per cell in 100 ml ofglucose-free Eagle minimal essential medium sup-plemented with 2 mM glutamine. At 1 h postinfec-tion the medium was diluted 10-fold with the samemedium containing 0.2 g of glucose per liter and 5%fetal bovine serum and incubated at 32°C. At 2.5 hpostinfection 1 mCi of one of the following radioac-tive sugars wa1 added: D-[6-3H(N)]glucosamine hy-drochloride (5 to 15 Ci/mmol), L-[6-3H]fucose (10 to15 Ci/mmol), D[1-3H(N)]galactose (5 to 10 Ci/mmol)(all obtained from New England Nuclear), or -[2-3H]mannose (2 Ci/mmol) (from Amersham-Searle).At 19 h postinfection the cells were pelleted, andvirus was precipitated from the medium with poly-ethylene glycol 6000. The polyethylene glycol 6000-precipitated virus was centrifuged to equilibrium ina 20 to 50% sucrose gradient in ET buffer (10 mMTris-hydrochloride-1 mM EDTA, pH 7.4) in a Beck-man SW27 rotor at 23,000 rpm at 4°C for 16 h. Thevisible band of virus was collected with a syringeand pelleted in an angle 65 rotor at 50,000 rpm at 4°Cfor 1 h. The virus pellet was suspended in 1 ml of ETbuffer by sonic treatment and centrifuged through a5 to 20% ET-sucrose gradient in an SW27 rotor at

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872 ROBERTSON, ETCHISON, AND SUMMERS

18,000 rpm at 4°C for 50 min. The virus band wascollected as above, pelleted, and suspended in 2 ml ofET buffer.

Preparation of tryptic glycopeptides. Nonidet P-40 (Shell Oil Co.) was added to the purified virus to afinal concentration of 1% and kept at room tempera-ture for 20 min with occasional shaking. The re-leased nucleocapsids were pelleted at 50,000 rpm for60 min in an angle 65 rotor, and the glycoproteinwas extracted from the supernatant with 2 volumesof n-butanol for 5 to 10 min. The aqueous and or-ganic phases were separated by centrifugation(2,000 rpm for 5 min). The glycoprotein appeared asa sticky white precipitate at the interface and wasremoved with a Pasteur pipette, washed withethanol, and pelleted. The small white pellet wassuspended in 1 ml of 1 mM HCl containing 100 ,ug ofTPCK-trypsin (Worthington). Na2CO3 (50 mM) wasadded to pH 7 to 8, and the mixture was incubatedovernight at 37°C.

Terminal sialic acid was removed by digestion ofthe tryptic glycopeptides with 100 ,ug of Clostridiumperfringens neuraminidase (Sigma) in 0.1 M sodiumphosphate, pH 5.3, at 37°C for 16 h. Prior to diges-tion with neuraminidase the tryptic glycopeptideswere boiled for 2 min to destroy tryptic activity.

Analysis of tryptic glycopeptides. The tryptic gly-copeptides were analyzed on a column (20 by 0.9 cm)of DEAE-Sephadex A25 (Pharmacia) equilibratedwith 50 mM Tris-hydrochloride, pH 8.5. Sampleswere eluted with 50 mM Tris-hydrochloride, pH 8.5,for 20 fractions followed by a 200-ml linear gradientof 0 to 0.1 M NaCl in 50 mM Tris-hydrochloride, pH8.5. Fractions (1.8 ml) were collected and counted

I GLUCOSAMINE

0.6

0.3-Llt ,\ ,X ,

X0 ...-MANNOSE

directly in formula 950A scintillation fluid (NewEngland Nuclear). Further analyses were per-formed on a Bio-Gel P-6 (Bio-Rad) gel filtration col-umn (108 by 1.3 cm) or on a Dowex AG50W x 2 (Bio-Rad) cation-exchange column (11 by 0.6 cm). The P-6column was equilibrated with, and the sampleeluted with, 0.1 M NH4 acetate, pH 6.0. Fractions(1.3 ml) were collected and assayed for radioactivityas above. The cation-exchange column was equili-brated with 10 mM NH4 acetate, pH 3.0, and sam-ples were eluted with a linear gradient from 10 mMNH4 acetate, pH 3.0, to 0.4 M NH4 acetate, pH 5.0.Fractions (1.8 ml) were collected and assayed asabove.

RESULTSThe carbohydrate moiety of VSV glycoprotein

was radiolabeled with either [3H]glucosamine,[3H]fucose, [3H]mannose, or [3H]galactose, andthe glycoprotein was digested extensively withTPCK-trypsin. The labeled tryptic glycopep-tides were fractionated on a column of DEAE-Sephadex (Fig. 1). A similar elution profileconsisting of two major and three minor peakswas obtained with all four radioactive sugars.In addition, a small but detectable peak wasusually observed in the region of fractions35-45.The oligosaccharide side chains of viral glyco-

proteins have been shown to exist in differentforms containing different numbers of sialicacid residues (9; Etchison et al., manuscript in

I?9x

:E

20 40 60 80 100 120 20 40 60 80 100 120FRACTION NUMBER

FIG. 1. Analysis of tryptic glycopeptides on DEAE-Sephadex. 3H-sugar-labeled glycoprotein was digestedextensively with trypsin and analyzed on DEAE-Sephadex A25 as described in Materials and Methods.

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GLYCOSYLATION SITES OF VSV GLYCOPROTEIN 873

preparation). The negative charge of the sialicacid residues might produce multiple peakswhen the oligosaccharide-peptides were ana-lyzed by DEAE-Sephadex chromatography.The extent to which the sialic acid was affectingthe analysis was determined by digesting thetryptic glycopeptides with neuraminidase priorto DEAE-Sephadex chromatography. The re-sults are shown in Fig. 2. In all cases the morecomplex pattern seen in Fig. 1 was reduced to apattern containing only two major species.Peaks I and II were apparently unaltered byneuraminidase treatment (a double-label ex-periment indicated that peak II eluted at ex-actly the same position before and after neura-minidase treatment; data not shown), whereaspeaks Ia, Tb, and hIa were completely removed,presumably converted into one or both of thetwo major glycopeptide species.

After neuraminidase digestion of [3H]gluco-samine tryptic glycopeptides, a small peakeluted at approximately the position of peak Ib(Fig. 2, upper left). When virus is grown in thepresence of [3H]glucosamine, some of the radio-activity is incorporated as sialic acid (4, 12).The presence of this peak, seen only in glucosa-mine-labeled, neuraminidase-treated trypticglycopeptides, suggested that it may be sialicacid that had been removed from the trypticglycopeptides. This was tested by analyzingneuraminidase-treated, [3H]glucosamine-la-

beled tryptic glycopeptides, along with a smallamount of authentic sialic acid (Fig. 3a). In ad-dition to determining the radioactivity in eachfraction, a 100-l1A sample was assayed chemi-cally for the presence of sialic acid (17). Thesialic acid co-eluted with the small peak ofradioactivity at fractions 45 to 50. In a separateexperiment, desialated [3H]glucosamine-labeledtryptic glycopeptides were separated on aDEAE-Sephadex column, and the small peakwas further analyzed on a Bio-Gel P-6 gel fil-tration column (Fig. 3b). The radioactivity co-eluted with sialic acid from the column as asingle homogeneous species.The small peak that was observed in the

analysis of tryptic glycopeptides in the region offractions 35-45 (Fig. 1) was more readily de-tected at the same position after neuraminidasetreatment (Fig. 2). It is probably a minor glyco-peptide and will be discussed below.The above results indicated that glycopep-

tides Ia, Ib, and IIa contained sialic acid andwere more "complete" forms of glycopeptides Iand II that lacked sialic acid. It is likely thatthe presence of sialic acid caused stronger bind-ing of glycopeptides Ia, Ib, and Ila to theDEAE-Sephadex, and after neuraminidasedigestion peaks Ia and lb were converted toglycopeptide I and peak Ila was converted toglycopeptide II.

The effect ofneuraminidase on the individual

8.0-

4.0-

If,

0.

2o-

1.0-

2.0

1.0

20.

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-2.0

GLUCOSAMINE FUCOSE

MAN NOSE GALACTOSE

! . L2040608.0020 20406080. ~ ~.

- I'.20 40 60 80 1000 120 20 40 60 80 100 120

FRACTION NUMBERFIG. 2. Analysis of neuraminidase-treated tryptic glycopeptides on DEAE-Sephadex. 3H-sugar-labeled

tryptic glycopeptides were digested with neuraminidase and analyzed on DEAE-Sephadex A25 as describedin Materials and Methods.

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874 ROBERTSON, ETCHISON, AND SUMMERS

a

2.0-

200A 0.31.0 03

_ 20 40 60 80 100 120b

V0 V10-

5-

40 60 80 100FRACTION NUMBER

FIG. 3. Identification of sialic acid. (a) Neura-minidase-treated [3H]glucosamine-labeled trypticglycopeptides were co-chromatographed on DEAE-Sephadex with a few milligrams of sialic acid. Theposition of sialic acid was determined chemically bythe method of Warren (17). The presence of sialicacid was indicated by a bright pink color. Symbols:

*, [3H]glucosamine; ---, optical density at 550nm. (b) The small peak of radioactivity at fraction45-50 in (a) was analyzed by gel filtration on a Bio-Gel P-6 column as described in Materials and Meth-ods. The radioactivity eluted as a low-molecular-weight species at the position ofsialic acid. VO and V,indicate the void volume and the totally includedvolume, respectively.

tryptic glycopeptides is shown in Fig. 4. Byusing [3H]glucosamine-labeled tryptic glyco-peptides, any sialic acid removed should havebeen detected as a small peak in the region offraction 50. Tryptic glycopeptide I did not alterits elution position on treatment with neura-minidase, and no sialic acid was detected (Fig.4a). Neuraminidase treatment of glycopeptideIa altered its elution position to correspond toglycopeptide I, and a small peak of sialic acidwas detected (Fig. 4b). Glycopeptide lb wassimilarly converted to glycopeptide I, and asmall amount of sialic acid was released (thispeak was proportionately larger than the oneobtained with glycopeptide la) (Fig. 4c).

Tryptic glycopeptide II remained predomi-nantly unaltered by neuraminidase treatment(Fig. 4d). However, several small additionalpeaks were detected. First, a peak coincidentwith glycopeptide I was observed. This peakwas no doubt a form of glycopeptide I contain-ing sialic acid such that before neuraminidase

treatment it co-eluted with glycopeptide II. Itwill be referred to hereafter as glycopeptide Ic.This was followed by a peak at fraction 35 thatcorresponds to the minor glycopeptide men-tioned above (Fig. 2), indicating that it existspartially in a form containing sialic acid (thisform co-eluting with glycopeptide II beforeneuraminidase treatment). There was a smallpeak at fraction 50 corresponding to sialic acidthat was no doubt derived from peak Ic andfrom the minor glycopeptide at fraction 35. Twoadditional peaks were observed in the region offractions 90-100 and fractions 115-125. Theyrepresent less than 5% of the tryptic glycopep-tides and are at present of unknown composi-tion. However, these two peaks, and also thesialic acid peak, were absent when [3H]fucose-labeled glycopeptide II was treated with neura-minidase and analyzed (Fig. 4f). Neuramini-dase treatment of glycopeptide IIa produced onepredominant peak corresponding to glycopep-tide II plus three smaller peaks (Fig. 4e). First,a small peak corresponding to glycopeptide Iwas observed. Whether this represents glyco-peptide I or not is unknown. A peak correspond-ing to the minor glycopeptide was also detected,possibly indicating an additional form of thisglycopeptide that co-eluted with glycopeptideIIa before neuraminidase treatment. The thirdsmall peak represented sialic acid, most ofwhich was derived from glycopeptide Ila itself.The above results indicate that there are only

two major tryptic glycopeptides. However, gly-copeptide I did not bind to the DEAE-Sephadexcolumn and eluted with the equilibratingbuffer. It was necessary to establish that it wasa homogeneous species and not a mixture oftwoor more glycopeptides that did not bind toDEAE-Sephadex. Neuraminidase-treated [3H]-fucose-labeled tryptic glycopeptides were sep-arated on DEAE-Sephadex, and glycopeptide Iwas analyzed further on a Dowex AG50W x 2cation-exchange column (Fig. 5). GlycopeptideI bound to the cation exchanger and was elutedas a single homogeneous species.The two major tryptic glycopeptides, I and II,

were analyzed further on a Bio-Gel P-6 gelfiltration column calibrated for molecularweight determination (Etchison et al., manu-script in preparation). Desialated tryptic glyco-peptides I and II eluted from the column withmolecular weights of 2,800 to 2,900 and 2,600 to2,700, respectively (Fig. 6). The carbohydratemoiety of the desialized Pronase glycopeptidesfrom VSV grown in BHK cells has a molecularweight of approximately 2,000 (Etchison et al.,manuscript in preparation). The difference be-tween the molecular weights of the tryptic gly-copeptides shown here and the carbohydrate

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GLYCOSYLATION SITES OF VSV GLYCOPROTEIN

ano1 l b)Ila

2.0-

c) lb d)I

2.0-

.0.4 IEela f)11

OA-~~~~

20 40 60 80 100 120 20 40 60 80 100 120FRACTION NUMBER

FIG. 4. Analysis of individual tryptic glycopeptide species after treatment with neuraminidase. Theglycoprotein was digested extensively with trypsin and chromatographed on DEAE-Sephadex (Fig. 1). Peakfractions were pooled, lyophilized, and desalted on a column of Sephadex G-15. The radioactive trypticglycopeptides were lyophilized, resuspended in 0.1 M sodium phosphate buffer, pH 5.3, and analyzed on

DEAE-Sephadex after digestion with neuraminidase. (a) Peak I + neuraminidase; (b) peak Ia + neuramini-dase; (c) peak Ib + neuraminidase; (d) peak II + neuraminidase; (e) peak IIa + neuraminidase; and (f) peakII + neuraminidase. Symbols: - , [3H]glucosamine; -- -, [3H]fucose.

moiety is due to the amino acid sequence of thetryptic peptides and suggests that tryptic glyco-peptide I contains in the region of eight aminoacid residues and that tryptic glycopeptide IIcontains in the region of six amino acid resi-dues.

Quantitative analysis of the distribution ofradioactivity in the peaks in Fig. 1 and 2 isshown in Table 1. The results are approximateand are averages of the different sugar labels.Before neuraminidase treatment, the [3H]glu-cosamine results were biased in favor of sialicacid containing glycopeptides and thereforewere not included in determining the aver-ages. Approximately 50% of glycopeptide spe-cies I contained sialic acid, whereas less than20% of glycopeptide species II contained sialicacid. This means that only one-third ofthe totaloligosaccharide side chains contained sialicacid when the virus was grown in HeLa suspen-sion cells. Before removal of sialic acid withneuraminidase, radiolabel in glycopeptide spe-cies I and II occurred in a 1:1 ratio. However,after desialation this ratio was 3:2. This change

in the ratio was due to the existence of glyco-peptide Ic that co-eluted with glycopeptide IIbefore neuraminidase treatment, resulting in a

distribution of radioactivity in favor of glyco-peptide species II. The results indicate only theproportion of oligosaccharide side chains thatcontained sialic acid and not the number ofsialic acid residues present on any particularglycopeptide. However, the proportion of sialicacid that was removed from glycopeptide Ib isdouble that removed from glycopeptide Ia (Fig.4b and c), suggesting that glycopeptide Ia con-tained one residue of sialic acid and glycopep-tide Ib contained two.The minor glycopeptide (fractions 35-40, Fig.

2) represented only a few percent of the totalcarbohydrate. It was labeled more stronglywith galactose than with glucosamine, fucose,or mannose (see Fig. 1 and 2).

DISCUSSIONAs part of our investigations into the struc-

ture of the glycoprotein of VSV, we set out todetermine the number of oligosaccharide side

2.0

-1.0

-2.0

-1.0

-0.4

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876 ROBERTSON, ETCHISON, AND SUMMERS

.0-,°1.0 (il0.5-\~

i I , , .20 40 60 80 100

FRACTION NUMBERFIG. 5. Analysis of tryptic glycopeptide I on a cat-

ion exchanger. [3H]fucose-labeledi tryptic glycopep-tides were treated with neuraminidase and chromat-ographed on DEAE-Sephadex. Peak I was pooled,lyophilized, and desalted on Sephadex G-15. Theglycopeptide was again lyophilized, suspended in 10mM NH4 acetate, pH 3.0, and analyzed on a columnof Dowex AG50W x 2, a cation exchanger, as de-scribed in Materials and Methods.

4.0-

T

0 -

2.0-

-1.0

-0.5

40O 60 80 100FRACTION NUMBER

FIG. 6. Gel filtration chromatography of trypticglycopeptides I and II. [3H]fucose-labeled tryptic gly-copeptides were digested with neuraminidase andchromatographed on DEAE-Sephadex. Peak frac-tions were pooled, lyophilized, and desalted on Seph-adex G-15. The glycopeptides were lyophilized, sus-

pended in 0.1 M NH4 acetate, pH 6.0, and analyzedon columns ofBio-Gel P-6. The analyses ofglycopep-tides I and II were performed separately but havebeen superimposed for ease of comparison.

chains per glycoprotein molecule. Trypsindigestion of the glycoprotein of VSV grown inHeLa suspension cells produced two major andseveral minor glycopeptides when analyzed on

DEAE-Sephadex.

TABLE 1. Quantitative analysis of the trypticglycopeptidesa

Radioactivity (%)

Glycopeptide species Before Afterneuramin- neuramin-

idase idaseI 25 58Ia 13lb 11

II 43 39IIa 8Minor 3

% of I with sialic acid 49% of II with sialic acid 16

Total with sialic acid 32Total without sialic acid 68

I + Ia + Ib 49 58II + IIa (+ Ic) 51 39

a The data for the glycopeptides before neuramin-idase treatment are averages of analyses of[3H]fucose-, [3H]mannose-, and [3H]galactose-la-beled glycopeptides only. Due to the incorporation ofradioactivity into sialic acid with [3H]glucosaminelabel, these data are omitted. After neuraminidasetreatment, the values represent averages of all foursugar labels. Radioactivity in sialic acid represented7% of the total [3H]glucosamine radioactivity.

Three of the minor glycopeptides (Ia, Ib, andIc) were all forms of tryptic glycopeptide I, con-taining additional residues of sialic acid (pre-sumably one, two and three residues, respec-tively). Another of the minor glycopeptides(IIa) was a form of tryptic glycopeptide II, con-taining presumably one residue of sialic acid.Recent separation of the tryptic glycopeptideson Whatman DE 52 anion-exchange cellulose(unpublished observations of J. S. R.) has re-sulted in a sharper elution of all species and hasallowed the detection of an additional minorspecies, Ilb, which corresponds to peak II withtwo sialic acid residues. A minor tryptic glyco-peptide (fractions 35-40, Fig. 2) also existed indifferent forms that contain additional sialicacid residues. Approximately one-third of thetotal oligosaccharide chains contains sialic acid.The number of sialic acid residues will probablyvary for VSV glycoprotein derived from virusgrown in other cell lines (9), and preliminarystudies have demonstrated this to be true forVSV grown in BHK cells compared with HeLasuspension cells (unpublished observations ofJ. S. R. and J. R. E.). Asialo-tryptic glycopep-tides I and II are homogeneous species whenanalyzed on DEAE-Sephadex and by gel filtra-tion. In addition, tryptic glycopeptide I elutedfrom a cation exchanger as a single species.

,I

VO Vti i

I on

II IIII?

IloI I"0 .

- ----

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GLYCOSYLATION SITES OF VSV GLYCOPROTEIN 877

Thus, there are two major tryptic gly(tides plus a minor species that representthan 5% of the carbohydrate content cglycoprotein. The existence of only two tVglycopeptides per glycoprotein moleculecates that VSV glycoprotein contains onlglycosylation sites. The presence of moretwo glycosylation sites with similar aminsequences, although unlikely, cannot beout. Although our results do not in(whether or not these sites are fully glycos3on each glycoprotein molecule, the unequ;tribution of label between tryptic glycope]I and II may be due to a difference in the eto which the two sites are glycosylatedthe virus is grown in different host cells.is likely, the initial glycosylating enzy:host specific, then the enzyme must be coIto the different cell lines and must recog-specific amino acid sequence or structurature of the glycoprotein polypeptide back

Determination of the molecular weig]glycopeptide I and II by gel filtration indithat they contain approximately eight aramino acid residues, respectively. Thesestimates based on size alone and are the]tentative. Since glycopeptide I is largerand contains more of the carbohydratethan glycopeptide II, it is possible that tholigosaccharide chains, whereas similarnonidentical. Studies are in progress to clterize both the carbohydrate and the p-portions of these two glycopeptides. Thesaccharide chains probably have a strusimilar to that of serum-type glycoproteiishown in Fig. 7 (5, 11-13).

Fucose is usually found attached to tacetylglucosamine involved in the peptidcosidic linkage (1). Etchison and Hollanusing gas-liquid chromatography, deterthat the VSV glycoprotein contains onlresidue of fucose. The presence of fucose irtryptic glycopeptides in this study indthat only some (-50%) of either tryptic Ipeptide species have fucose attached. Thuwould infer that the oligosaccharides are ]ogeneous in their fucose content as well as

a.a. + Fuc

Asn-G1cNAc-GlcNAc-(Man)n

I

sialic acid content. This heterogeneity was notdetectable by the ion-exchange separations em-ployed here.The prominence of [3H]galactose label in the

minor tryptic glycopeptide species suggeststhat it may be a minor galactosamine-contain-ing glycopeptide with a structure similar tomucin-type oligosaccharides characterized byan alkali-labile bond between galactosamineand serine or threonine. The presence of such aminor oligosaccharide has been previously sus-pected (5) and is currently under investigation.From the studies presented here it should be

clear that failure to account for the heterogene-ity of the oligosaccharide moieties of glycopro-teins (sialic acid heterogeneity in particular)can give misleading results when classical tech-niques of protein chemistry are applied to theanalysis of carbohydrate-containing peptides.

ACKNOWLEDGMENTSThis research was supported by Public Health Service

grant no. 1 R01 AI12316-01 from the National Institute ofAllergy and Infectious Diseases to D. F. S. and by NationalScience Foundation grant no. BMS 74-21128 A01 to D. F. S.J. S. R. is in receipt of a NATO postdoctoral fellowship fromthe Science Research Council, London, U.K. J. R. E. is apostdoctoral fellow of the American Cancer Society. D. F. S.is a recipient of an American Cancer Society FacultyAward.

LITERATURE CITED1. Atkinson, P. H. 1975. Synthesis and assembly of HeLa

cell plasma membrane glycoproteins and proteins. J.Biol. Chem. 250:2123-2134.

2. Both, G. W., S. A. Moyer, and A. K. Banerjee. 1975.Translation and identification of the mRNA speciessynthesized in vitro by the virion-associated RNApolymerase of vesicular stomatitis virions. Proc.Natl. Acad. Sci. U.S.A. 72:274-278.

3. Briendi, M., and J. J. Holland. 1975. Coupled in vitrotranscription and translation of vesicular stomatitisvirus messenger RNA. Proc. Natl. Acad. Sci. U.S.A.72:2545-2549.

4. Burge, B. W., and A. S. Huang. 1970. Comparison ofmembrane protein glycopeptides of Sindbis virus andvesicular stomatitis virus. J. Virol. 6:176-182.

5. Etchison, J. R., and J. J. Holland. 1974. Carbohydratecomposition of the membrane glycoprotein of vesicu-lar stomatitis virus grown in four mammalian celllines. Proc. Natl. Acad. Sci. U.S.A. 71:4011-4014.

6. Grubman, M. J., E. Ehrenfeld, and D. F. Summers.1974. In vitro synthesis of proteins by membrane-

GlcNAc - Gal

GlcNAc - Gal , + Sialic Acid

GlcNAc - GalJ

FIG. 7. Proposed oligosaccharide chain structure.

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878 ROBERTSON, ETCHISON, AND SUMMERS

bound polyribosomes from vesicular stomatitis virusinfected HeLa cells. J. Virol. 14:560-571.

7. Grubman, M. J., and D. F. Summers. 1973. In vitroprotein-synthesizing activity of vesicular stomatitisvirus-infected cell extracts. J. Virol. 12:265-274.

8. Howatson, A. F., and G. F. Whitmore. 1962. The devel-opment and structure of vesicular stomatitis virus.Virology 16:466-478.

9. Keegptra, K., B. Sefton, and B. W. Burge. 1975. Sind-bid virus glycoproteins: effect of the host cell on theoligosaccharides. J. Virol. 16:613-620.

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