Host cell-induced differences in O-glycosylation of herpes simplex virus gC-1

VIROLOGY161,385-394(1987)

Host Cell-Induced

Differences

I. Structures MARITA LUNDSTROM,

in 0-Glycosylation

of Nonsialylated

of Herpes Simplex Virus gC-1

HPA- and PNA-Binding

Carbohydrates

SIGVARD OLOFSSON,’ STIG JEANSSON, ERIK LYCKE, ROELF DATEMA,* AND JAN-ERIC MANSSONt

Department of Clinical Virology, University of Gbteborg, Guldhedsgatan 106, S-4 13 46 Gbteborg, *Department of Antiviral Chemotherapy, Research and Development Laboratories, Astra Alab AB, S- 151 85 S0dertaQe. and fDepartment of Psychiatry and Neurochemistry, University of Gbteborg, S-422 03 Hisings Backa, Sweden Received April 17, 1987; accepted July 17, 1987 Lectins with narrow oligosaccharide specificities were established as probes to study the host cell influence on the biosynthesis of O-linked oligosaccharides of the herpes simplex virus type 1 (HSV-l)-specified glycoprotein C (gC-I). We found that only gC-1 and no other glycoprotein bound to the peanut lectin (PNA), with main specificity for Gal(@l-3)GalNAc. Previously, we have shown that only gC-1 binds to the He/ix porn&a lectin (HPA), with main specificity for terminal GalNAc. The O-linked oligosaccharides binding to PNA and HPA were released by alkaline borohydride treatment and characterized. A structural determination of these oligosaccharides showed that the HPA-binding carbohydrates were monosaccharides (GalNAc), and that the PNA-binding oligosaccharides were disaccharides with the structure Gal-GalNAc. The PNA- and HPA-binding oligosaccharides were arranged as Pronase-resistant clusters on gC-1, consisting of about seven individual, adjacent oligosaccharides. In addition to these disaccharides, Pronase-resistant PNA-binding glycopeptides of gC-1 also contained neutral trisaccharides. Larger O-linked oligosaccharides, binding to the wheat germ lectin, were found in gC-I, but not in proximity to the PNA-binding ones. It was concluded that the lectins mentioned should be useful probes in screening HSV-infected cells of different lineages for differences in processing of O-linked oligosaccharides. 0 1937 Academic Press, Inc.

INTRODUCTION

1985). Some evidence for this conclusion stems from studies with HSV-1 -infected, mutant cell lines deficient in glycosyltransferases engaged in the biosynthesis of IV-glycosyl-oligosaccharides (Campadelli-Fiume et a/., 1982). Glycoprotein C-l contains in addition to N-linked oligosaccharides different 0-glycosyl-oligosaccharides, which may be recognized by their different lectin-binding properties (Olofsson er al., 1981 a, b, 1983, 1985; Johnson and Spear, 1983; Dall’Olio et al., 1985). One such class of oligosaccharides, binding to the wheat germ lectin, contains terminal sialic acid, probably attached to galactose (Johnson and Spear, 1983; Olofsson et al., 1983, 1985; Dall’Olio et a/., 1985), while another class contains 0-glycosyl-oligosaccharides binding to the Helix pomatia lectin (HPA) (Olofsson et al., 198 1a, 1983), specific for terminal A/-acetylgalactosamine (HammarstrCim et a/., 1977; Goldstein and Hayes, 1978). Probably, a large proportion of the Oglycosyl-oligosaccharides of gC-1 are arranged in Pronase-resistant clusters on the polypeptide (Dall’Olio el al., 1985), as was repot-ted for some other membrane glycoproteins, for example leukosialin (Carlsson and Fukuda, 1986) and the low density lipoprotein receptor (Cummings et al., 1983). Some of the 0-glycosyl-oligosaccharides of gC-1 produced in

The herpes simplex virus type 1 (HSV-I)-specified glycoprotein C (SC-l) has been implicated in a number of phenomena of biological interest. Thus, gC-1 may act as a virus adsorption protein (Fuller and Spear, 1985) and it has also been shown to serve as a receptor for the C3b factor of the complement system (Friedman et a/., 1984). The protein is heavily glycosylated and contains a number of both N-glycosyl and 0-glycosyl-oligosaccharides (Olofsson et a/., 198 1 a, b; Wenske et a/., 1982; Campadelli-Fiume et a/., 1982; Johnson and Spear, 1983; Campadelli-Fiume and Serafini-Cessi, 1985; Dall’Olio et a/., 1985; Serafini-Cessi et al., 1983). The oligosaccharides of gC-1 must be of at least indirect importance for the biological activity of gC-1 as both expression of C3b-receptor activity and maintenance of a number of antigenic determinants are dependent on the degree of glycosylation of the protein (Smiley and Friedman, 1985; Sjijblom et a/., 1987). It is generally assumed that N-glycosyl-oligosaccharides from HSV-1 glycoproteins largely, if not totally, are assembled by host cell-coded glycosyltransferases (Campadelli-Fiume and Serafini-Cessi, ’ To whom requests for reprints should be addressed. 385

0042.6822187 Copyright All rights

$3.00

0 1987 by Academic Press. Inc of reproduction I” any form reserved

386

LUNDSTRdM ET AL

HSV-1 -infected BHK cells have been isolated as monoor disialylated tri- and tetrasaccharides, respectively (Dall’Olio et a/., 1985). Evidence that at least the innermost sugar of the 0-glycosyl-oligosaccarides of gC-1 is added by host cell enzymes has been presented (Serafini-Cessi et al., 1983) but no information is available so far regarding the engagement of the host cell or virus-specified factors in the addition of the peripheral sugars of these oligosaccharides. The large coding capacity of HSV-1 should be sufficient to influence glycosylation by introduction of virus-specified glycosyltransferases. If the O-linked oligosaccharides of gC-1 are synthesized largely or totally by host cellencoded enzymes it could be expected that they could vary in both size and structure, dependent on the cell line used for HSV-1 production (Carlsson and Fukuda, 1986; Datema et al., 1987). The aim of this and an accompanying paper was to determine if the structure and biosynthesis of the Oglycosyl-oligosaccharides of gC-1 were under the control of the host cell. In order to obtain additional probes for such studies we investigated if gC-1, in addition to the oligosaccharides mentioned above, also contained 0-glycosyl-oligosaccharides binding to the peanut lectin (PNA), with a narrow specificity for the terminal disaccharide sequence Gal@1 -3)GalNAc (Pereira et a/., 1976; Reisner et a/., 1976; Goldstein and Hayes, 1978; Momoi et a/., 1982; Mansson and Olofsson, 1983). This lectin was chosen because a disaccharide sequence with maximal affinity for PNA is generated if the immediate precursor to the WGAbinding 0-glycosyl-oligosaccharides, mentioned above, is formed by the addition of one single galactose to terminal GalNAc of the HPA-binding oligosaccharides. In the present paper it is shown that HPA-binding carbohydrates of gC-1 are precursors to O-linked PNA-binding disaccharides, and that these latter oligosaccharides may act as precursors to the WGA-binding 0-glycosyl-oligosaccharides with terminal sialic acid (Peters et a/., 1979) and, possibly, also to neutral trisaccharides. MATERIALS

AND METHODS

Virus and cells The HSV-1 strain F was used throughout the study. In addition, vesicular stomatitis virus (VSV), Indiana strain, was used in some experiments. For production of viral glycoproteins green monkey kidney (GMK) cells were used (GOnalp, 1965). The cells were grown in 50-mm petri dishes, and the details for propagation and maintenance have been published elsewhere (Olofsson et a/., 1981 a, 1983).

Preparation

of gC-1 from infected

cells

Cells were infected with HSV-1, strain F, at a multiplicity of 10. Four hours postinfection, the cells were radiolabeled with 50 pCi/ml of [3H]glucosamine (Amersham, 30 Ci/mmol). Monosaccharides and their derivatives are abbreviated as follows: GlcNAc (Nacetylglucosamine); GalNAc (N-acetylgalactosamine); GlcN (glucosamine); GalN (galactosamine); Gal (galactose). Sugar alditols are indicated by the suffix-o/, and all carbohydrates are of the D configuration. The procedures for preparation of a purified gC-1 fraction and subsequent protease digestion have been published previously (Olofsson et a/., 1983). Briefly, the cells were harvested at 18 hr postinfection by scraping, and solubilized in Tris-buffered saline (TBS), pH 7.4, containing 1% Triton X-l 00. A soluble fraction was obtained after centrifugation at 100,000 g for 1 hr. This radiolabeled Triton extract was applied to an immunosorbent column, containing protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden), saturated with a polyclonal rabbit antiserum, monospecific for gC-1. This antiserum, designated K642, has been characterized previously (Olofsson et al., 1983). The IgG-SC-1 complexes were eluted with 1 M acetic acid, which was removed by gel filtration on Sephadex G-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) against TBS. The purified complexes were subjected to Pronase digestion, as previously described (Olofsson et a/., 1983). Similar experiments were carried out in the presence of tunicamycin (2 pg/ml), added at 1 hr postinfection. Chromatographic

procedures

(i) Affinity chromatography. Lectin affinity chromatography of glycopeptides from purified gC-1 was carried out as previously described (Olofsson et al., 1983). The pronase digest was inactivated by a brief heating to 100” and applied to 6-mm wide minicolumns containing 200 ~1 of gel-bound lectins. The following lectins were used: HPA and Vicia villosa lectin B4 isolectin (WA B4) specificity GalNAc, lentil lectin; cy-mannosides, peanut lectin (PNA); Gal@1 -3)GalNAc (Pereira et al., 1976; Hammarstrijm et a/., 1977; Goldstein and Hayes, 1978; Kornfeld eta/., 1981; Tollefsen and Kornfeld, 1983, 1984). The glycopeptides were allowed to adsorb to the columns and subsequently the columns were washed thoroughly with TBS and, finally, eluted with solutions of appropriate sugars as indicated in the legends to the figures and in Table 1. (ii) Gel filtration. Molecular weight estimations were carried out in 0.9 X 120-cm columns, equilibrated with 0.1 n/l pyridine-acetate, pH 7.8, as previously described (Olofsson et al., 1983). Pronase-resistant gly-

HOST CELL-INDUCED

copeptideswereseparatedon Sephadex G-50(Phar-

0-GLYCOSYLATION

387

clonalantibody, coupled to CNBr-activated Sepharose

maciaFineChemicals), andtheliberated oligosaccha- at a concentrationof 2 mg IgGlml.The boundSC-1

rides fromalkaline borohydride treatment (seebelow) waseluted with Obl Mglycine-HCI, pH23;containing

onBio-Gel P2(Eio-Rad laboratories,0.5%(w/v\Tween wereseparated 80aspreviously described. The Richmond, CA). Chemical

degradation

Alkaline borohydride treatments were carried out by slight modifications of the methods by Carlson (1968) or Spiro (1966). Radiolabeled glycopeptides were desalted against HZ0 on short Sephadex G-25 columns, and NaBH, and NaOH were added from a fresh stock solution to give a final concentration of 1 M NaBH, and 0.05 M NaOH. The samples were deaerated by a stream of N2 and incubated for 16 hr at 45”. The method by Spiro (0.1 M NaOH, 1 M NaBH, for 48 hr at 37”) gave similar results. After incubation the samples were neutralized with 1 M acetic acid and dried under a stream of nitrogen and excess borate was evaporated as methylborates by repeated dissolving and evaporation with methanol in the presence of acetic acid. Oligosaccharide pools in a pyridine-acetate buffer from Bio-Gel P2 gel filtrations were dried by evaporation under a stream of nitrogen and hydrolyzed in 3 M HCI at 100” as previously described (Olofsson et a/., 1981 b). After incubation and drying under a stream of nitrogen, the samples were analyzed by thin layer chromatography on silica (Merck Alufolien, Darmstadt), developed with ethanol/pyridine/l -butanol/acetic acid/water (100: 10: 10:3:30), containing 1O/O dipotassium tetraborate, as described by Dall’Olio et a/. (1985). The plates were cut into 5-mm strips, and the radioactivity was assayed by liquid scintillation counting. Localization of relevant amino sugar derivative markers was done using the ninhydrin reagent. SDS-polyacrylamide

gel electrophoresis

Detergent extracts from glucosamine-labeled, HSV-1 -infected cells were immunoprecipitated with gC-1 specific monoclonal or polyclonal antibodies, as previously described (Olofsson et al., 1983). The precipitates were solubilized and electrophoresed in 9.25% polyacrylamide gels according to the method by Morse et a/. (1978). The bands were visualized by fluorography, as previously described (Olofsson et a/., 1981 a). Specificity

of the binding

between

gC-1 and PNA

The specificity of the binding between PNA and gC-1 was analyzed by an enzyme-linked assay (Sjdblom et a/., 1987). Nonlabeled gC-1 from HSV-linfected cells was purified by a gC-l-specific mono-

purity of the gC-1 preparation was ascertained by SDS-polyacrylamide gel electrophoresis and by its lack of reactivity toward monoclonal antibodies specific for gB-1 and gE-1. Polystyrene microtiter plates were coated with gC-1 (concentration about 0.25 pg/ml in 0.5 M Tris buffer (pH 8.2) by incubation with 100-~1 volumes overnight at 4”. Inhibiting sugars (50 bl/cavity) were added in twofold dilutions, starting from 25 pM. Thereafter, biotinylated PNA (Vector, Inc.) was added to a final concentration of 5 pg/ml (reaction volume 100 ~1) and the plates were incubated at 22” for 1 hr. Subsequently, the plates were washed with the coating buffer and 50 /II of horseradish peroxidase-conjugated avidin was added. After 30 min incubation at 22” a paraphylendiamine-based substrate was added, and the plates were read at 498 nm in a Flow Multiscan photometer. After 5-10 min incubation with the substrate, PNA without competing sugars gave an absorbance value of about 1.O. The disaccharide Gal@1 -3)GalNAc was prepared by hydrolysis of ganglioside GM 1 in 0.10 M aqueous HCI for 90 min. After neutralization with NaOH, chloroform and methanol were added to give a ratio of C/M/W 4:2:1 (v/v/v). The appearing upper phase, containing the disaccharide, was evaporated to dryness, drssolved in water, and applied to a column of charcoal/ celite 1: 1 (w/v). After washing the column with water, the disaccharide was eluted with 7.5% ethanol in water. The purity of the fraction was checked by thinlayer chromatography in 1-butanol/acetic acid/water 2:2:1 (v/v/v) and the sugar composition was determined by gas-liquid chromatography ot the corresponding alditol acetates (Helm et a/., 1972). RESULTS 1.

Demonstration

of PNA-binding

gC-1

Radiolabeled extracts from uninfected, VSV-, or HSV-1 -infected GMK cells were subjected to PNA affinity chromatography (Fig. 1). About 6% of the [3H]GlcN-labeled glycoprotein extract from HSV-1 -infected cells bound to PNA but only about 1% of the corresponding material from uninfected cells was found in the PNA-binding fraction. To determine if Nlinked oligosaccharides acquired PNA-binding activity during biosynthesis, we included a Triton extract from VSV-infected GMK cells, which contained only one [3H]glucosamine-labeled peptide of about 67K (Fig. 2,

LUNDSTROM ETAL

388

TABLE1

ClPW 75000

0

7

HSV

50000

25000

0

i 5

10

-

vsv

r-ii-l FRACTION NUMBER

FIG. 1. PNA chromatography of solubilized, [3H]glucosamine-labeled glycoproteins from uninfected cells and HSV-l- and VSV-infected cells. Eluting sugar (o-galactosamine) was added after fraction 10.

lane E). The VSV glycoprotein, which is known to contain two complex-type-N-linked oligosaccharides, was synthesized completely by the host cell enzymes (Etchison et a/., 1977; Tabas and Kornfeld, 1978) and did not bind to PNA (Fig. 1C). The PNA-binding HSV-1 glycoproteins were identified by SDS-PAGE and the fraction eluted from the PNA chromatography of HSV-l-infected cells appeared as one band, situated in the region containing gB-1 and gC-1 (Fig. 2, lane B). No band was observed in the electrophoretic regions, containing gG-1, gE-1, and gD-1, even after extended fluorography of the gel. lmmunoprecipitation and subsequent electrophoresis showed, however, that most of the PNA-binding activity was located in gC-1, and that only trace amounts of gB-1 were found in the PNA-binding fraction (Fig. 2, lanes C and D). Whether this latter phenomenon was due to the fact that gB-1 could contain PNA-binding oligosaccharides or that trace amounts of gB were associated with the PNA-binding gC-1 was not further ABCD

E

gc gB PElC

gE gD

FIG. 2. SDS-polyacrylamide gel electrophoresis of (A) solubilized HSV-1 -specified glycoproteins, used for PNA chromatography; (B) the eluted HSV-1 glycoprotein fraction from the chromatography depicted above; (C) the same fraction, precipitated with a gC-1 -specific antibody or(D) precipitated with a gB-specific antibody; (E) the VSV glycoprotein fraction used in the chromatography depicted above.

SPECIFICITYOF THE BINDING BETWEENgC-1 AND PNA

Inhibiting sugar Gal@1 -3)GalNAc GalN Methyl-a-galactopyranoside Galactose GalNAc GlcNAc

Inhibitory concentration (mM) 0.1 2.1 2.9 5.4 >25 >25

a Determined by the enzyme-linked lectin assay to be the sugar concentration leading to absorbance values twice the background value obtained with total inhibition. Figures given are mean values interpolated from duplicate experiments.

investigated. As reported for the HPA-binding fraction of gC-1 (Olofsson et a/., 1983) the PNA-binding fraction constituted 20 to 309/o of the total [3H]GlcN-labeled gC-1 produced in GMK cells. Most of the remainder of gC-1 was found to be heavily sialylated gC-1, with affinity for the wheat germ lectin (Olofsson et a/., 1983). The HPA-binding fraction had a significantly higher electrophoretic mobility than the remainder of gC-1 produced in GMK cells. The specificity of the binding between immunosorbent-purified gC-1 and PNA was investigated in the biotin-avidin based solid-phase assay, where inhibitory concentrations of relevant low molecular weight saccharides were determined (Table 1). The binding between gC-1 and PNA was characterized by a high specificity as demonstrated by the ranking of different low molecular weight saccharides with respect to their inhibiting capacities. The disaccharide Gal@1-3)GalNAc was the most efficient inhibitor, followed by galactosamine, methyl-a-galactopyranoside, and galactose. The acetamido sugars demonstrated little if any inhibitory effect when used within the concentration interval studied. These data are in accordance with previous reports on the specificity of PNA (Pereira et a/., 1976; Goldstein and Hayes, 1978) suggesting that a terminal Gal@1-3)GalNAc is responsible for the binding to PNA. 2. O-Linked PNA-binding oligosaccharides of gC-1 To determine if the PNA-binding oligosaccharides of gC-1 were of the 0-glycosyl or ALglycosyl type, we analyzed gC-1 produced in the presence of tunicamytin (TM), which is an efficient inhibitor of glycosylation (Elbein, 1985). Underglycosylated gC-1 produced in the presence of TM, is totally devoid of N-linked oligosaccharides but contains several O-linked oligosaccharides (Olofsson eta/., 1983; Wenske and Courtney, 1983). In SDS-PAGE, this underglycosylated SC-1 had

HOST CELL-INDUCED

an apparent molecular weight of about lOOK, compared to about 130K observed for fully glycosylated gC-1 (Fig. 3). When gC-1, produced in the presence of TM and radiolabeled with [3H]GlcN, was subjected to Pronase digestion and subsequent lectin chromatography, it was found that a large proportion of the Pronase-resistant glycopeptides specifically bound to PNA (Table 2). No glycopeptides binding to the lentil lectin, specific for mannose residues in moderately branched N-glycosyl oligosaccharides (Kornfeld et al., 1981) were found. The results demonstrated that PNA-binding oligosaccharides were added to gC-1 under conditions where N-glycosylation was blocked.

3. Organization of HPA-binding and PNA-binding oligosaccharides in Pronase-resistant clusters The sizes of the HPA-binding and PNA-binding glycopeptides from gC-1 were determined by gel filtration on Sephadex G-50 (Fig. 4). As previously reported (Olofsson et a/., 1983), the HPA-binding glycopeptides appeared as two distinct peaks, corresponding to molecular weights of 7000 and 4000, respectively. The PNA-binding glycopeptides were more heterogeneous and larger, and found predominantly in the void volumn of Sephadex G-50, corresponding to a molecular weight of 7500 or more. When these HPA-binding and PNA-binding glycopeptides were subjected to alkaline borohydride treatment, known to release O-glycosyloligosaccharides from the polypeptide backbone, they were fragmented into considerably smaller units, as determined by Bio-Gel P2 filtration. From HPA-binding glycopeptides mainly material corresponding in size to monosaccharides was obtained and from the PNAbinding glycopeptides most of the material corre-

a

b

0GLYCOSYLATION

389 TABLE 2

DEMONSTRATION

OF TM-RESISTANT PNA-BINDING

OLIGOSACCHARIDES

IN gC-1 a

-Sialidase

Lectinlspecificityb

+Sialidase

14.1 26.9 34.0 0.3

HPA/GalNAc PNA/Gal(Pl-3)GalNAc WGAisialrc acid Lentilk-mannose

14.3 44.2 3.1 0.3

’ Percentage binding to lectin and elutlng with adequate sugars: 0.05 M GalNAc (HPA); 0.1 M GalN (PNA); 0.1 M GlcNAc (WGA); and 0.5 M a-methylmannoside (lentil lectin). Eluting conditions were according to Goldstein and Hayes (1978). Underglycosylated gC-1, produced in the presence of TM, was purified by an rmmunosorbent and a polyclonal gC-1 -specific antiserum and subsequently digested by pronase, as previously described (Olofsson et al., 1983). In each chromatography experiment 12,000 cpm was analyzed. * References for lectin specificities are in the text.

sponded in size to disaccharides and larger structures (Fig. 5; results presented in detail below). These data indicated that the HPA-binding and PNA-binding oligosaccharides were arranged in Pronase-resistant clusters on the polypeptide. Our data demonstrated that a significant proportion of the O-linked carbohydrates were sialylated in the presence of tunicamycin (Table 2). As sialylation normally takes place in the Vans-region of the Golgi apparatus (Schachter, 1986) our data indicate that TM did

CPM

c

gccmPgC-

gEgD-

FIG. 3. Electrophoretic characterization of the radiolabeled gC-1 preparations used in the present paper. Radiolabeled gC-1 was isolated from tunicamycin-treated cells (b), and untreated cells(c). Lane a, an SDS-polyacrylamide gel electrophoresis of the total glycoprotein extract from HSV-1 -infected cells used in the present investigation. Positions of glycoproteins gC-1, precursor gC-1 (pgC-1), gE, and gD are indicated. All samples were run on the same gel but lanes a and b were taken from a 24.hr fluorographic exposure, white lane b was from a 48-hr exposure.

40

60

80

Fractim

FIG. 4. Sephadex G-50 of pronase-resistant glycopeptides, binding to PNA (A) or HPA (B), from gC-1, produced in TM-treated cells. Void volume (V), the position of a complex-type oligosaccharide with a molecular weight of 3100 (C), and the position of GlcN (I) are indicated.

LUNDSTROM

390 CPM

a0

so Fraction

loo

120

No

FIG. 5. Bio-Gel P2 gel filtration of oligosaccharides released by the alkaline borohydride treatment of PNA-binding (A) and HPA-binding (B) glycopeptides. Void volume is indicated by an arrow and positions of mono-, di-, and trisaccharides are indicated as m, d, and t, respectively.

not significantly affect the processing of O-linked oligosaccharides during the transport of gC-1 through the Golgi region. Similar findings have been reported for other glycoproteins with clustered O-linked oligosaccharides, including the sialoglycoprotein of human reticulocytes (Gahmberg et al., 1980).

4. Structural characterization of HPA-binding and PNA-binding O-linked oligosaccharides Several lines of evidence indicated that the HPAbinding and PNA-binding glycans of gC-1 constituted structurally different oligosaccharides. One such difference was that sialidase treatment of O-linked glycopeptides produced PNA-binding glycopeptides but not HPA-binding ones. (Table 2). To produce individual O-linked oligosaccharides and evaluate possible structural differences we subjected HPA-binding and PNA-binding Pronase-resistant gC-1 glycopeptides, produced in the presence of TM and labeled with [3H]GlcN, to alkaline borohydride treatment. A flow sheet for the experimental procedures is found in Fig. 6. The released oligosaccharides from HPA-binding and PNA-binding glycopeptides were separated on Bio-Gel P2 (Fig. 5). The majority of the material from HPA-binding glycopeptides was eluted as one single peak at the position of an acetamido sugar, suggesting that the HPA-binding carbohydrate was a monosaccharide. The only labeled product from this monosaccharide peak was GalN-ol (Fig. 7) as

ET AL

shown by TLC after strong acid hydrolysis, indicating that the HPA-binding glycopeptide contained several O-linked GalNAc monosaccharide units attached to the peptides. This was confirmed by lectin affinity chromatography of the HPA-binding glycopeptides on the vicia villosa B4 isolectin (Fig. 8) which has a high specificity for such clustered GalNAc residues (Tollefsen and Kornfeld, 1983). It was found that the majority (more than 60%) of the glycopeptides bound to this lectin. These data confirmed that HPA-binding Olinked carbohydrates were multiple GalNAc monosaccharides concentrated to two peptide stretches of gC-1, with apparent molecular weights (including carbohydrate complement) of about 7000 and 4000, respectively (Figs. 4 and 6). From the PNA-binding glycopeptides a different set of oligosaccharides was obtained. Thus, the fragments from PNA-binding glycopeptides yielded a number of peaks, where one major component eluted together with a disaccharide marker and one minor component behaved as a monosaccharide. In addition, a considerable amount of the radioactivity eluted as trisaccharides or larger components. To further analyze the monosaccharide composition of these fragments, material from each peak was subjected to hydrolysis with 4 IVI HCI at 100” for 4 hr to result in radiolabeled

O-linked

oligosaccharides

of gc-1

[-V] Pronase dig&ion

r-----l Chromatography Eluted glycopeptides

HGA

>7.500

FIG. 6. Flow sheet for degradation and gel filtration of [3H]GlcN-labeled gC-1. The data for the WGA-binding gycopeptides were obtained from Olofsson et al. (1983).

HOST CELL-INDUCED

0-GLYCOSYLATION

391

loo0

1000

r

500 so0

I

i

a4

1500 , -

ti 1000 / -

10

5

500

1ooc I-

5013-

FIG. 7. Thin-layer chromatography of strong HCI hydrolysates of oligosaccharides obtained after alkaline borohydride treatment (see Fig. 5). The hydrolysates were applied to cellulose plates, developed with ethanol/pyridine/l -butanol/acetic acid/water (100: 10: 10:3:30), containing 1% (w/v) potassium tetraborate. The plates were processed for liquid scintillation counting as indicated under Materials and Methods. Disaccharide (A) and trisaccharide (B) from alkaline borohydride-treated PNA-binding glycopeptides, and monosaccharides from HPA-binding glycopeptides (C) were analyzed. Positions of GalN-ol (a), GalN (b) and GlcN (c) are indicated.

GalN, GlcN, and GalN-ol, because the sialic acid will be destroyed and GalNAc and GlcNAc deacetylated (Montreuil, 1980). The products of acid hydrolysis were resolved in a TLC system (Fig. 7; see Materials and Methods). The major fraction after alkaline borohydride treatment of the PNA-binding glycopeptide, which appeared as a disaccharide on Bio-Gel P2 filtration, was hydrolyzed to one labeled component, namely GalN-ol. Because this disaccharide was readily labeled with [3H]galactose we conclude that the disaccharide sequence released by alkaline borohydride treatment is Gal-GalN-o/. The minor component of the borohydride-treated PNA-binding glycopeptides, appearing as a monosaccharide on Bio-Gel P2 also yielded GalN-ol on TLC after acid hydrolysis. This finding indicated presence of O-linked monosaccharides also in the PNA-binding glycopeptides. Hydrolysis of the trisaccharide peak of PNA-binding glycopeptides yielded upon TLC two major peaks, GalN-ol and GlcN, and a minor peak migrating as GalN (molar ratio

FIG. 8. VVA B, affinity chromatography of HPA-binding [3H]-GlcNlabeled glycopeptides from gC-1, produced in the presence of TM. Prior to chromatography on VVA, the preparation was desalted on a short column of Sephadex G-25 to remove eluting sugar from the HPA chromatography. The column was eluted with 0.05 IL1GalNAc.

10:9:1). This ratio suggested that the trisaccharide peak in fact contained a mixture of two structurally different trisaccharides containing either terminal GlcNac or terminal GalNAc (structures c and e in Fig. 9), where the trisaccharide with terminal GlcNAc must be the predominant one. Control experiments demonstrated similar results also after alkaline borohydride treatment and subsequent acid hydrolysis of PNAbinding glycopeptides from gC-1, produced in the absence of TM. For the larger fragments obtained after alkaline borohydride treatment of PNA-binding glycopeptides only traces of radiolabeled GalN-ol were detected after acid hydrolysis (data not shown), indicating that these fragments represented residual glycopeptides with unreleased O-linked oligosaccharides. A similar incomplete release of oligosaccharides after alkaline borohydride treatment was reported for both gC-1 and other glycoproteins (Dall’Olio et al., 1985; Niemann et a/., 1984; Spiro and Boyroo, 1974). In some experic

e

0 -Ser

HPA

-

ii

-Seri PNA

@‘A)

e

-

i-

-fWGA

@GA)

FIG. 9. Tentative molecular representations of O-linked oligosaccharides of gC-1, as characterized in the present study. Most probable structures of gC-1 glycopeptides, responsible for binding to HPA, PNA, and WGA are indicated. Designations within brackets denote structures with potential lectin affinity, although no binding was demonstrated in the present investigation. The following symbols were used: GalNAc (0) GlcNAc (o), galactose (cl), and sialic acid (v). References for the lectin specificites are given in the text. Oligosaccharides may be attached to a serine or threonine residue.

LUNDSTROM ET AL

392

ments, lower levels of such larger peptides and higher levels of the trisaccharide peak were found, suggesting that the relative amount of trisaccharides could be larger than indicated in Fig. 5. The data, thus, indicated that the larger fragments of alkaline borohydride treament represented arrays of trisaccharides not released by the treatment. As gC-1 obviously contained sialic acid (Table 2) we subjected the PNA-binding glycopeptides to treatment with excess sialidase (10 u/pg) or treatment with 0.05 M H,SO, to investigate if they contained sialic acid. Possible released radiolabeled sialic acid was isolated in the included volume in Sephadex G-25 gel filtration and detected by liquid scintillation counting (limit of detection 1% of total radiolabel in glycopeptides). No radioactivity was found in the included volume, indicating that PNA-binding glycopeptides contained no sialic acid. DISCUSSION In the present paper we report that gC-1 contains oligosaccharides binding to PNA, a lectin with main specificity for the terminal sequence Gal@1-3)GalNAc (Pereira et al., 1976; Goldstein and Hayes, 1978). A high specificity in the binding between gC-1 and PNA was indicated by the competition experiments in which the disaccharide indicated above was the best inhibitor. It can be concluded that the PNA-binding oligosaccharides were 0-glycosidically linked to gC-1: (i) they were synthesized in the presence of tunicamytin, (ii) they were released from glycopeptides by alkaline borohydride treatment under very mild conditions and with excess NaBH,, and (iii) the detection of GalN-ol in the released fragments consituted a direct chemical proof for the 0-glycosidic linkage between GalNAc and a hydroxy amino acid (Montreuil, 1980). This is the first report of a viral glycoprotein with specific affinity for PNA, a lectin previously known to differentiate between functional classes of T lymphocytes (Reisner era/., 1976). From the present data it is possible to obtain structural information about the HPA-binding as well as the PNA-binding oligosaccharides (see Fig. 9). The HPAbinding O-linked carbohydrates are GalNAc monosaccharides situated very close to each other on the polypeptide stretch, making the peptide resistant to the action of pronase. These monosaccharides most likely are immediate precursors to the PNA-binding oligosaccharides, with the most probable structure Gal@13)GalNAc. Although no proof is available on the particular carbon atom of GalNAc engaged in the glycosidic linkage it is reasonable to assume that the indicated one is correct because (i) this disaccharide is by far the most common inner core structure in O-glycosyl-oligo-

saccharides of mammalian cells (Montreuil, 1980) and (ii) this linkage shows maximal affinity for PNA (Pereira e2 a/., 1976; Goldstein and Hayes, 1978). Also the PNA-binding oligosaccharides were clustered in Pronase-resistant peptide stretches, although these arrays seemed to be more heterogeneous with respect to the carbohydrate composition, compared with the HPAbinding ones, which contained almost exclusively GalNat. In addition to the predominant disaccharide mentioned above, we also detected GalNAc and two neutral trisaccharides. Our data suggest the presence of two structurally different O-linked trisaccharides, present in the PNA-binding glycopeptide (Fig. 9) but probably the one with terminal GalNAc is present in very small amounts. It is reasonable to assume that neither the trisaccharides nor the GalNAc is engaged in the binding to PNA, as terminal acetamido sugars do not bind to this lectin at all (Goldstein and Hayes, 1978). Our data suggested the presence of a trisaccharide with a terminal GalNAc (structure c in Fig. 9) possibly expressing affinity for HPA. However, this oligosaccharide seems not to be engaged in the binding of gC-1 to HPA, because this trisaccharide was not found after alkaline borohydride treatment of the HPAbinding glycopeptides. One explanation for this could be that the multiple terminal galactose units of the PNA-binding peptides are able to quench the potential HPA-binding capacity of the, obviously very few, trisaccharides with terminal GalNac. Our data clearly indicated that sialic acid is not a constituent of the PNA-binding oligosaccharide arrays. However, as PNA-binding glycopeptides are generated after sialidase treatment of WGA-binding glycopeptides from gC-1, produced in the presence of tunicamycin, our data suggest that structure d in Fig. 9 is an important constituent of gC-1, produced in GMK cells. A similar O-linked trisaccharide was described by Dall’Olio et al. (1985) for gC-1, produced in BHK cells. It cannot be excluded that also the neutral trisaccharides, found in the PNA-binding glycopeptides, are precursors to larger sialylated structures. Taking into consideration the possibility that the structures of O-linked oligosaccharides are influenced by the host cell (see accompanying paper, Lundstrom et a/., 1987) the structures of O-linked oligosaccharides of gC-1 are compatible with those of Dall’Olio et a/., (1985) and those reported for glycoprotein El of the mouse hepatitis virus (Niemann et al., 1984) demonstrating the monosialylated trisaccharide and neutral disaccharide. However, they did not find any counterpart to the neutral trisaccharides reported in the present study. This discrepancy may be due either to the use of different methods for preparation and

HOST CELL-INDUCED 0-GLYCOSYLATION

selection of glycopeptides or to the fact that the oligosaccharides are differently processed in the cells used in their studies. The size determination of the HPA-binding glycopeptides, containing monosaccharide units only, enables a rough estimate of the number of O-linked oligosaccharides in the pronase-resistant arrays. Assuming an average molecular weight of 115 each of serine, threonine, and proline, which are reported to be the major components in peptide stretches (for a review, see Montreuil, 1980), containing O-linked oligosaccharides and an average of three amino acids per monosaccharide, the number of GalNAc residues in the smaller HPA-binding glycopeptide would be approximately seven. This is in accordance with the report of Dall’Olio et a/. (1985) describing an array of O-linked oligosaccharides in a glycopeptide lacking affinity for ConA. VVA B, and HPA, together with PNA, constitute lectins with relatively narrow specificities for structural determinants in O-linked oligosaccharides. Moreover, the clustered organization of such oligosaccharides in gC-1 increases the affinity constants between the lectins mentioned and gC-1. In accordance, the data of the present paper emphasize that HPA, VVA B,, and PNA are vet-y specific probes for detection of structural differences in O-linked oligosaccharides of gC-1. In a following paper (Lundstrijm et a/., 1987), this system is utilized to present evidence that O-linked oligosaccharides of gC-1 are produced by host cell-specified glycosyltransferases rather than virus-encoded ones. ACKNOWLEDGMENTS We thank lnger Sjdblom for her skillful technical assistance. This work was supported by grants from the Swedish Medical Research Council (Grant 4514) and the Medical Faculty, University of Gateborg.

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