Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties

Marlene Biller, Kristina Mårdberg, Helle Hassan2, Henrik Clausen2, Anders Bolmstedt, Tomas Bergström and Sigvard Olofsson1

Department of Virology, University of Göteborg, Guldhedsgatan 10B, S-413 46 Göteborg, Sweden, and 2Faculty of Health Sciences, School of Dentistry, Copenhagen, Denmark

Received on February 3, 2000; revised on May 9, 2000; accepted on August 15, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80–104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55–69 and aa 80–104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody–assisted epitope analysis of N-acetylgalactosaminidase–treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate–binding domain of gC-1.

Key words: gC-1/glycan/ldlD/conformation/heparan sulfate


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1 is highly glycosylated, containing at least eight utilized N-glycosylation sites (Rux et al., 1996Go) and many O-linked glycans, most of them in two pronase-resistant clusters of the glycoprotein (Olofsson, 1992Go). Although gC-1 seems to be dispensable for viral growth in cell culture (Spear, 1985Go), the glycoprotein is associated with two important ligand functions: the ability to bind to the C3b component of the complement system (Friedman et al., 1986Go) and the ability to bind to cell surface associated heparan sulfate (WuDunn and Spear, 1989Go; Herold et al., 1991Go; Lycke et al., 1991Go). Several lines of evidence indicate that the C3b-binding activity of gC-1 significantly contributes to the immune evasion potential of HSV-1 (Friedman et al., 1996Go), and that binding of virion-associated gC-1 to cell surface heparan sulfate represents viral attachment to the first of several consecutively acting cellular receptors (Herold et al., 1991Go; Trybala et al., 1994Go; Feyzi et al., 1997Go; Geraghty et al., 1998Go).

As for many other glycoproteins containing multiple clustered O-linked glycans (Gottschalk, 1960Go; Jentoft, 1990Go; Olofsson, 1991Go), it seems reasonable to assume that the O-linked glycans contribute to an extended conformation of gC-1, thereby increasing its accessibility as a ligand on the viral envelope or at the surface of the infected cell (Olofsson, 1992Go). Accordingly, immunogold electron microscopy data suggest that gC-1 projections of the HSV-1 envelope protrude further from the viral envelope than those of other HSV-1 specified glycoproteins (Stannard et al., 1987Go). The exact position of the clustered O-linked glycans of the N-terminal portion of gC-1 is not known, although circumstantial evidence favors a serine and threonine-rich region between amino acid 33 and 123 (Dall'Olio et al., 1985Go; Olofsson et al., 1991Go; Olofsson, 1992Go; Rux et al., 1996Go). This position is in the vicinity of domains of relevance for heparan sulfate as well as C3b-binding of gC-1 (Friedman et al., 1986Go; Trybala et al., 1994Go).

In contrast to N-glycosylation, which is initiated by one unique gene product of the human genome, that is, the oligosaccharyl transferase (Kaplan et al., 1987Go), addition of O-linked glycosylation may be initiated by at least seven different GalNAc-transferases, each expressed in a tissue-specific manner (Clausen and Bennett, 1996Go; Bennett et al., 1999Go; Hagen et al., 1999Go). Owing to the HSV-1 ability to replicate in several types of tissues from epithelium to neural cells and the large number of possible sites for O-linked glycosylation, the complement of O-linked glycans of gC-1 may vary considerably depending on the tissue origin in which the virus replicates. It is therefore of interest to determine to what extent gC-1 is able to be O-glycosylated by the different members of the GalNAc transferase family.

The aim of the present paper was to more exactly characterize gC-1 with respect to the early phases of its O-glycosylation and the significance of O-linked glycans for gC-1 maturation and conformation. Our data indicate that some regions of gC-1 serve as pan-acceptor sequences for O-linked glycosylation whereas others seem to constitute acceptors for a restricted number of GalNAc-transferases and that even small O-linked glycans may contribute to the presentation of the discontinuous epitopes overlapping the major heparan sulphate-binding site of gC-1.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Acceptor activity of synthetic gC-1 peptides for GalNAc transferases
Our previous data suggested that the two major clusters of O-linked glycans are located in the N-terminal portion of the glycoprotein. Two clusters of altogether 19 Ser or Thr residues between aa40 and aa100, with interspersed Pro residues seemed to be likely acceptor sequences for addition of the clustered O-linked glycans (Olofsson et al., 1986Go; Olofsson, 1992Go). An inspection of the amino acid sequence of gC-1, using the NetOGlyc 2.0 algorithm (Hansen et al., 1997Go, 1998), supported this notion. To more specifically analyze O-linked glycosylation of gC-1, we assayed a panel of synthetic peptides, representing domains of gC-1 likely to be O-glycosylated for their ability to serve as acceptors for various GalNAc transferases (Table I). The peptides were chosen using the strategy described by Clausen and Bennett (1996)Go. Peptide 1 (aa41–aa55) was a substrate mainly for GalNAc-T2, whereas the two peptides covering aa55–aa69 and aa90–aa104, respectively, were both efficient substrates for all tested enzymes. These results suggested that the sequences represented by the two latter peptides, should be O-glycosylated in a wide variety of cell types permissive for HSV.


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Table I. Ser/Thr-rich peptides of gC-1: Substrate specificity (mU/mla) of purified recombinant GalNAc-transferases
 
Analysis of a gC-1 mutant, lacking Thr residues in a putative O-linked glycosylation signal
An HSV-1 mutant, designated HSV-18995+, with three changed threonines, one changed proline, and three changed lysines in the suspected O-linked glycosylation signal between aa 75 and 95, was generated (Figure 1). This mutant was fully infectious in cell culture. To determine if the induced mutation, which eliminated three possible acceptor threonines, had any effect on the size of the O-glycan clusters we digested [3H]-GlcN-labeled gC-18995+ and wild type gC-1 (gC-1Rescue2) with pronase, and isolated pronase-resistant glycopeptides by HPA chromatography for subsequent separation on Sephadex G75 (Figure 2). We have previously shown that these HPA-binding glycopeptides constitute N-glycan free peptide stretches, corresponding in size to linear polysaccharides of 4000 and 7500 Da, respectively, and that these peptides contain multiple O-linked GalNAc residues, but no N-linked glycans (Lundström et al., 1987Go). We found that both wild type gC-1 and gC-18995+ appeared as one major and one minor peak glycopeptide peak, but the major peak from gC-18995+ eluted significantly earlier (fraction 72) than the corresponding peak from wild type gC-1 (fraction 82) (Figure 2). The minor peak from gC-18995+ as well as wild type gC-1 both eluted at about fraction 105. These data indicated that the mutation induced affected the larger but not the smaller of the two gC-1-associated clusters of O-linked glycans.



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Fig. 1. Positions in the gC-1 amino acid sequence for the substitutions (indicated by arrows) caused by nucleotide-directed mutagenesis of the two gC-1 mutants studied. The lollipops indicate the positions of an N-linked glycan. Solid symbols, glycans eliminated by mutations in the present study; gray symbols, glycans not affected in the present study. The arrows denote the amino acid substitutions introduced into gC-18995+. The upper part of the figure consists of a structural representation of the entire gC-1. The peptide stretch of clustered serine and threonine residues, suspected to contain O-linked glycans is marked as a box. Data used for mapping reviewed in Olofsson (1992)Go. The positions of synthetic peptides P1-P3 (see Table II) are indicated.

 


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Fig. 2. Sephadex G75 gel filtration of pronase-resistant [3H]-GlcN-labeled glycopeptides from gC-1N148, gC-18995+, and gC-1Rescue2. The positions of the void volume (V) and the totally included (T) volume are indicated.

 
This result appeared initially intriguing because it implied that elimination of three candidate acceptor Thr residues for O-glycosylation in fact resulted in larger pronase-resistant clusters of O-linked glycans, suggesting that the threonines eliminated were of less importance for the O-glycosylation signal. However, one of the threonines eliminated in gC-18995+ also resulted in elimination of the N-glycosylation site of N73 and in addition, there were three substitutions of hydrophobic amino acids for basic ones (Figure 1). Thus, one possibility could be that the increased size of the pronase-resistant O-glycan cluster of gC-18995+ was caused by the induced absence of the N73 oligosaccharide, owing to the presumed ability of a large complex type glycan to block enzyme access to clustered serine and threonine in the close vicinity of the glycan. There was no size difference in the O-linked glycan clusters between wild type gC-1 and gC-1N148 (Figure 2), demonstrating that an N-linked glycan situated at some distance did not affect the accessibility of the O-linked glycosylation signal (Figure 1). The alternative explanation for the difference observed between gC-18995+ and wild type gC-1 was that the introduced hydrophobic amino acids in place of the wild type positively charged amino acids in the O-glycosylation signal of gC-18995+ resulted in a better acceptor sequence for GalNAc transferases.

Mechanism behind the improved O-linked glycosylation of gC-18995+ compared with wild type glycoprotein
Two experimental strategies were used to determine which of the two explanations mentioned above was responsible of the increased ability of gC-18995+ to be O-glycosylated. First, we introduced tunicamycin treatment of HSV-infected cells to exclude the influence of N-linked glycans on O-linked glycosylation. Radioimmunoprecipitation (RIPA) and SDS–PAGE showed that wild type and mutant gC-1, produced in tunicamycin-treated [3H]-GlcN-labeled cells, had a similar but significantly higher electrophoretic mobility than the corresponding normally N-glycosylated gC-1 (Figure 3). This is in accordance with previous results where we showed that gC-1 produced under these conditions has an apparent molecular weight slightly above 100K, completely devoid of N-linked glycans, but equipped with equally large pronase-resistant O-glycan clusters as fully glycosylated gC-1 (Lundström et al., 1987Go).



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Fig. 3. RIPA and SDS-PAGE of [3H]-GlcN-labeled glycoproteins from tunicamycin-treated (TM) and untreated (N) HSV-1 infected cells. The cellular extracts were precipitated with a polyclonal rabbit serum, specific for gC-1. The positions of molecular weight markers are indicated.

 
Next, pronase-resistant peptides containing clustered O-linked glycans, were prepared from gC-18995+, and gC-1Rescue2 of tunicamycin-treated cells, and subjected to gel filtration on Sephadex G75 (Figure 4). This is feasible because (1) the processing of gC-1 and its intracellular transport as well as incorporation into enveloped virus particles is not dependent on addition of N-linked glycans and takes place also in the presence of tunicamycin (reviewed in Olofsson, 1992Go), and (2) the elution positions of the major and minor peak of HPA-binding O-glycan clusters of gC-1 are not altered by tunicamycin treatment of HSV-infected cells (Olofsson et al., 1983bGo; Lundström et al., 1987Go). The results showed that the major peak, containing the large O-glycan cluster derived from gC-18995+ as well as gC-1Rescue2 produced in tunicamycin-treated cells had the same elution position, that is, a difference of less than 1–2 fractions. This was consistent for several runs with gC-1 from different batches of tunicamycin-treated cells and should be compared with a difference of the least 10 fractions difference noted for the same clusters, prepared without any involvement of tunicamycin (Figure 2). The finding that blocking of N-linked glycosylation almost totally reduced the mobility difference between the wild type and the gC-18995+ large fragment peak strongly suggested that that the N-linked glycan of N73 blocked access for GalNAc-transferases to the large peptide signal for addition of clustered O-linked glycans, and that this phenomenon accounted for most of the size difference between the large O-linked glycan cluster of gC-18995+ and gC-1Rescue2, respectively.



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Fig. 4. Sephadex G75 gel filtration of pronase-resistant [3H]-GlcN-labeled glycopeptides from gC-18995+ (solid line) and gC-1Rescue2 (dotted line) derived from tunicamycin-treated HSV-infected cells. The positions of the void volume (V) and the totally included (T) volume are indicated.

 
We also determined if there was any difference between the large cluster signals of gC-18995+ and gC-1Rescue2 as substrates for GalNAc transferases. Two sets of synthetic peptides, representing the peptide stretch with the gC-18995+ mutations, were analyzed with the GalNAc transferase assay (Table II). It was found that peptides 6 (wild type) and 7 (mutant), covering the region with the most distal mutations of gC-18995+ in relation to the deleted N-glycosylation site, were readily glycosylated by GalNAc-T2 and GalNAc-T3 with only moderate differences between peptides 6 and 7. In contrast, the capacity of mutant peptide 7 to serve as a substrate for GalNAc T1 was three times higher than that of wild type peptide 6. The high rate of GalNAc incorporation into peptides 6 and 7 by GalNAc-T2 and GalNAc-T3 was of the same magnitude as observed for the two control peptides, representing strong O-glycosylation signals of Muc1b and AHG21. These data further underline the conclusion that the shielding capacity of the N-linked glycan at N73 rather than possible quality differences in the O-glycosylation signal between gC-18995+ and gC-1Rescue2 was responsible for the larger O-glycan cluster size of the latter, at least in HSV-infected cells not expressing GalNAc-T1.


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Table II. Differences between wild type and mutant Ser/Thr-rich peptides of gC-1 as substrates for purified recombinant GalNAc transferases (mU/mla)
 
An opposite pattern was observed for the peptides, representing the mutations adjacent to the eliminated glycosylation site, where wild type peptide 4 was a far more efficient substrate for GalNAc-T2 as well as GalNAc-T3 than the corresponding mutant peptide. This was expected because three of the four threonine residues in the wild type peptide stretch were absent in peptide 5. However, it should be kept in mind that the most prominent difference in this region between wild type and mutant gC-1 was that the wild type but not the mutant protein contained a complex type glycan. This difference between the wild type and mutant sequence was not possible to mimic with commercially available procedures for preparation of synthetic peptides.

Glycoprotein C-1 is exposed on the surface in O-glycosylation-deficient ldlD cells
To analyze the significance of the early steps in O-linked glycosylation for the biological properties of gC-1 we used the CHO cell variant ldlD, deficient in O-glycosylation due to a defective galactose-4-epimerase (Kingsley et al., 1986Go; Kozarsky et al., 1988aGo), which has been used for analysis of other enveloped viruses (Kozarsky et al., 1989Go; Wertz et al., 1989Go). However, CHO cells are not permissive for HSV (Shieh et al., 1992Go), and therefore CHO ldlD and wild type cells were transfected with the expression vector pCMV3gC, where the coding region of the gC-1 gene is situated downstream from a CMV promoter (Hung et al., 1992Go). The cells were solubilized at 70 h post-transfection, and the proteins were separated on SDS–PAGE, and further analyzed by Western blot, using C2H12, recognizing a linear epitope in gC-1 antigenic site II (Sjöblom et al., 1992Go) (Figure 5, left panel). We found that the gC-1 band produced in ldlD cells migrated considerably faster (an apparent molecular weight of about 115K) than the corresponding band of wild type CHO cells (125K). The experiment was carried out in the presence of 1 mM galactose to avoid effects on the processing of N-linked glycans (Kingsley et al., 1986Go; Kozarsky et al., 1988bGo). Although molecular weight estimates of the complement of O-linked glycans, based on mobility shifts observed in SDS–PAGE, are associated with some uncertainty (Smith et al., 1978Go), the results would indicate that the gC-1 O-linked glycans totally may contain as many as about 50 monosaccharide units. The conclusion that the O-glycan complement of gC-1 is large is further supported by the finding that 12–14% of its incorporated GlcN label is released by N-acetylgalactosaminidase or O-glycanase, despite that these enzymes release only a subfraction of the possible known O-glycan structures of viral glycoproteins (Table III; see below).



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Fig. 5. Expression of SDS-PAGE gC-1 produced in O-glycosylation-defective ldlD cells and corresponding wild type CHO cells. Left panel, immunoblot analysis of gC-1 from ldlD and wild type CHO cells. The positions of molecular weight markers are indicated. Right panel, surface localization of gC-1 demonstrated by indirect immunofluorescence of ldlD and wild type CHO cells, transfected with pCMV3gC. Cells analyzed at 18 h (exposed for 30 s) and 70 h (exposed for 8 s) post-transfection were analyzed.

 

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Table III. Susceptibility of gC-1 to glycosidases releasing O-linked glycans
 
Next, we used indirect immunofluorescence to determine whether O-linked glycan-deficient gC-1, produced in ldlD cells was transported to the plasma membrane. O-Glycosylation defective ldlD cells and wild type CHO cells were transfected with pCMV3gC and subjected to indirect immunofluorescence, using monoclonal antibody C4H11 (Figure 5, right panel). There was a significant cytoplasmic and cell membrane fluorescence at 16 h whereas at 40 and 70 h post-transfection, most of the gC-1 fluorescence was localized to the cell surface with no detectable difference between CHO and ldlD cells. This indicated that O-glycosylation of gC-1 was not a prerequisite for transport to the plasma membrane, in contrast to several other highly O-glycosylated membrane proteins.

Enzymatic O-glycan removal and its effects on gC-1 topology
The high content of O-linked glycans of gC-1 was further analyzed using glycosidases known to liberate O-linked glycans, that is, N-acetylgalactosaminidase, releasing the O-linked monosaccharide GalNAc, or O-glycanase, releasing the O-linked disaccharide Gal(ß1–3)GalNAc, respectively, from serines or threonines of the polypeptide backbone. HSV-1 infected cell cultures were radio labeled with [3H]-GlcN and differently glycosylated fractions of gC-1 were purified. It was pertinent for these experiments was that (1) O-linked glycans in HSV-infected cells are radiolabeled in their GalNAc residues by addition of [3H]-GlcN to infected cell cultures (Olofsson et al., 1981aGo; Varkit, 1994Go), and (2) the broad gC-1 band (see Figure 3 and Figure 5, left panel) containing complex type N-linked glycans contains two partly overlapping subfractions of gC-1: one high mobility HPA-binding band, containing clustered O-linked GalNAc units, and one slower PNA-binding, containing preferentially Gal(ß1–3)GalNAc units with or without terminal sialic acid (Olofsson et al., 1983aGo; Lundström et al., 1987Go; Olofsson and Bolmstedt, 1998Go). Therefore, we isolated the HPA- and PNA-binding fractions of gC-1 after sialidase treatment by lectin chromatography and subjected each isolated fraction to glycosidase treatment. The released small saccharides were separated from gC-1 by gel filtration on small Sephadex G25 columns (Table III). The amount of radiolabel released from PNA-binding gC-1 by O-glycanase was more than four times larger than the radiolabel released by N-acetylgalactosaminidase treatment. This corroborated that the predominant O-linked glycan in the PNA-binding fraction was Gal-GalNAc, which was expected from the specificity of this lectin. Analogously, N-acetylgalactosaminidase–sensitive glycans were predominant in the HPA-binding gC-1 fraction. These results confirmed that both enzymes each had the assigned specificity during the experimental conditions and indicated that a considerable portion, ~15%, of the [3H]-GlcN label of gC-1 was found in short O-linked glycans.

To determine if O-linked glycans of gC-1 influenced the topology of the glycoprotein we analyzed the effect of the two glycosidases on the ability of gC-1 to react with gC-1-specific monoclonal antibodies. The antibodies chosen, B1C1 and C4H11, react with conformation-dependent epitopes in the vicinity of the clustered O-linked glycans (Sjöblom et al., 1987Go; Trybala et al., 1994Go). We treated gC-1, coated onto 96-well microplates with O-glycanase or N-acetylgalactosaminidase, and assayed the reactivity of gC-1 in an ELISA with both monoclonal antibodies (Figure 6). O-Glycanase treatment of gC-1 did not alter the reactivity significantly with any of the antibodies, whereas N-acetylgalactosaminidase treatment of gC-1 caused a prominent reduction of its ability to bind to B1C1. N-Acetylgalactosaminidase had no significant effect on the gC-1 binding to C4H11. This result suggested that the B1C1 epitope of gC-1 is less available to B1C1 in the absence of O-linked GalNAc.



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Fig. 6. Antibody binding of gC-1 digested by N-acetylgalactosaminidase and O-glycanase, as assayed in ELISA. Mean values and SEM are given (n = 4). Immunosorbent-purified gC-1 was coated onto 96-well microplates and treated with 0.1 U or 0.01 U of N-acetylgalactosaminidase (GalNAcase; a and c) or 0.6 mU of O-glycanase (b and d), respectively, in 100 µl per well. Monoclonal antibodies B1C1 and C4H11 were analyzed in ELISA (see Materials and methods) and the absorbance values at 405 nm are indicated on the ordinate.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The biologically active region of gC-1 delimited by amino acid residues 50 and 150 is highly glycosylated, containing six complex type N-linked glycans and numerous clustered O-linked glycans (Olofsson, 1992Go). The present data narrowed the position for the clustered O-linked glycans further. First, two synthetic peptides representing amino acids 55–69 and 80–104, respectively, were excellent substrates for a number of GalNAc-transferases capable of initiating O-linked glycosylation. Second, a gC-1 mutant assumed to be defective in O-linked and N-linked glycosylation in a gC-1 region (delimited by amino acids 73 and 95) contained a larger pronase-resistant cluster of O-linked glycans than did the corresponding wild type gC-1. These data strongly suggest that the large pronase-resistant cluster of O-linked glycans is localized to the gC-1 stretch, delimited by amino acids 75 and 104 (essentially peptide 3), whereas the small cluster is localized within a stretch represented by peptide 2.

Elimination of three threonines in mutant gC-18995+, each constituting a possible acceptor for O-linked glycosylation would be expected to give rise to a smaller cluster of O-linked glycans than wild type gC-1 after pronase digestion. Indeed, the results obtained for peptide 4 and 5, showed that the induced amino acid changes (see peptide 5) almost completely abolished the ability of this peptide to be O-glycosylated. However, we noted a totally opposite effect of the induced mutation, that is, a large pronase-resistant O-glycan cluster of gC-18995+ with a significantly increased molecular size compared with normal gC-1. One explanation solving this paradox is that the large N-linked glycan, coeliminated by the induced mutation of T75, in fact blocked access for the GalNAc-transferases to the three threonines situated adjacent to this N-glycosylation at N73. This would be possible because the N-linked glycans are added at the RER level (Kornfeld and Kornfeld, 1985Go) and O-linked glycosylation is not initiated until the glycoprotein has reached the cis Golgi region (Roth et al., 1994Go; Clausen and Bennett, 1996Go). The finding that the relatively large size difference of 10 fractions between the mutant and wild type large O-glycan cluster is reduced to only one to two fractions for wild type and mutant gC-1, produced in tunicamycin-treated cells, would imply that also other serine or threonine residues than T75, T76, and T78, which are the only hydroxy amino acids eliminated in mutant gC-1, are blocked for GalNAc transferases in the presence of the N73 glycan. To our knowledge this is the first report, suggesting this type of N-glycan dependent blocking of initiation of O-linked glycosylation.

However, our data suggest that the shielding effect of the N-linked glycan may not be the sole virus-induced factor of importance for regulation of O-linked glycosylation in this region. Thus, the analysis of the synthetic peptides, derived from the wild type and the mutant sequence surrounding the positions of the amino acid substitutions most distal from the N73 glycan indicated that the mutant sequence is in fact a 3-fold better substrate for GalNAc-T1 than the wild type sequence. There are two immediate implications of these results. First, the substitution of two alanines for the basic lysine residues at position 89 and 95, respectively, results in a dramatic increase of peptide 6 capacity as a substrate to GalNAc-T1, suggesting that the activity of this enzyme is hampered by the presence of basic amino acids in its peptide substrate. Second, most HSV-1 strains contain lysine residues at those particular positions (Bergström et al., unpublished observations), suggesting that despite the high degree of O-linked glycosylation of the gC-1 region, the peptide stretch delimited by amino acids 80 and 104 is still not fully optimized as a substrate for GalNAc-T1. One possible explanation is that these lysines are important for other viral functions. We have previously shown that some of the basic amino acids are critical for gC-1 to bind to heparan sulfate (Trybala et al., 1994Go), but no such function has hitherto been assigned to the lysine residues at the particular positions mentioned above.

The finding that the two gC-1 peptides 2 and 3 constituted excellent acceptor structures for all of the GalNAc transferases investigated is intriguing. In contrast to GalNAc-T1 and GalNAc-T2, the expression of GalNAc-T3 and GalNAc-T6 is restricted to a few types of tissues (Suijkerbuijk et al., 1991Go; Bennett et al., 1996Go; Mandel et al., 1999Go). In addition, GalNAc-T3 seems to have similar requirements on the acceptor peptides as has the recently discovered GalNAc-T6, which is expressed in the brain (Bennett et al., 1999Go). Thus, there are reasons to assume that the peptide stretch delimited by amino acids Asp55 and Asn69, is heavily O-glycosylated irrespective of the cell in which HSV-1 replicates. As HSV-1 normally infects a wide variety of cells (Roizman, 1978Go), including neurons, it is tempting to speculate that the multi-enzyme substrate function of the peptide stretch delimited by Ala55 and Pro104 reflects a part of a viral strategy to ensure sufficient O-linked glycosylation, in a wide variety of cell types permissible for HSV-1. On the other hand, the clear quantitative differences documented here, between the performances of the isoenzymes on some of the peptide stretches allow for a fine-tuned variation of the O-linked glycosylation of gC-1 during the complex life cycle of this virus.

Although several functions have been attributed to mucin-type O-linked glycans no specific function of the O-linked glycans of gC-1 has been found so far. Thus, our data show that gC-1 synthesized in ldlD cells, deficient in O-linked glycosylation appeared to be transported to the plasma membrane as efficiently as in the O-glycosylation-competent wild type cells. This is in contrast to many O-glycosylated proteins of the normal cell such as the ldlD receptor, being degraded during transport in the absence of O-linked glycans (Kingsley et al., 1986Go; Kozarsky et al., 1988aGo), and the IL-2 receptors, failing to be correctly sorted in ldlD cells (Kozarsky et al., 1988bGo). Another function of O-linked glycans which might turn out to be of relevance for the biology of HSV-1 is the finding that the complement of O-linked glycans may modify the binding of CD44 to glycosaminoglycans in a cell-dependent manner (Dasgupta et al., 1996Go). The O-linked glycans of gC-1 are situated adjacent to the region binding to the primary HSV-1 receptor, heparan sulfate (Trybala et al., 1994Go). The presence of a pan O-glycosylation signal in the proximity of this region, likely to be utilized in most if not all types of human cells is intriguing. Finally, the O-linked glycan influence of the B1C1 epitope seems to be in analogy with a newly recognized O-glycan–dependent epitope of CD43, where intramolecular interactions between the specific O-glycan determinants and the polypeptide backbone regulate the exposure of this epitope (Carlow et al., 1999Go). However, the B1C1 epitope is strictly dependent on a threonine residue at position 150 (Olofsson et al., 1999Go), and the N-acetylgalactosaminidase data are therefore compatible with a model where a GalNAc residue at this position is an active part of the B1C1 epitope, and this possibility is now under investigation.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Cells and monoclonal antibodies
African green monkey kidney (GMK-AH1; Günalp, 1965Go) cells were grown in Eagle’s minimum essential medium (MEM) supplemented with 2% calf serum, 100 U of penicillin per ml, and 100 mg of streptomycin per milliliter. The UDP-Gal/UDP-GalNAc 4-epimerase deficient cell mutant (Kingsley et al., 1986Go; Kozarsky et al., 1988aGo) and its parent CHO cell mutant was a kind gift of Dr. Monty Krieger.

Three monoclonal antibodies against gC-1 were used: monoclonal antibody (Mab) B1C1, defining an carbohydrate-modulated antigenic site II epitope (Olofsson et al., 1983bGo; Sjöblom et al., 1987Go), and an efficient blocker of gC-1 interaction with cell surface heparan sulfate (Trybala et al., 1994Go), Mab C4H11, defining an carbohydrate-independent epitope of site II (Bergström et al., 1992Go), and Mab C2H12, defining a linear epitope in close vicinity to antigenic site II (Sjöblom et al., 1992Go).

Construction and identification of HSV gC-1 mutants
Site directed mutagenesis was performed using Promegas Altered Sites II in vitro Mutagenesis System as described previously (Hansen et al., 1996Go). The mutated gC-1 gene was inserted into infectious clones of HSV as described by DeLuca et al. (1985)Go. The genotype of mutant viruses was confirmed by sequencing as described elsewhere (Olofsson et al., 1999Go). One mutant, designated HSV-18995+ (a structural representation in Figure 1), and the corresponding wild type rescue virus HSV-1Rescue2 were used in further experimentation. As a control a mutant HSV-1 strain was used obtained in a similar manner and characterized elsewhere (Olofsson et al., 1999Go), designated HSV-1gCN148, which lacks an occupied glycosylation site at N148.

Metabolic labeling of HSV-1 glycoproteins and analysis of clustered O-linked glycans
Confluent layers of GMK cells in 6-well culture plates were washed two times with serum-free MEM and infected with HSV-18995 or HSV-1Rescue2 at a m.o.i. of 3. The virus was allowed to adsorb to the cells for 1 h at 37°C, followed by addition of MEM, containing antibiotics. After incubation in CO2-incubator for 4 h, 50°Ci/ml D-[6-3H]-glucosamine hydrochloride ( [3H]-GlcN; Amersham, 30 Ci/mmol) was added. In some experiments tunicamycin (2 µg/ml) Sigma-Aldrich, Stockholm, Sweden) was added to interfere with N-linked glycosylation. The cells were radiolabeled for 20 h until harvest. The labeled culture supernatant was removed from cells and supplemented with 1% (v/v) Triton X-100 and 1 mM AEBSF (4-(2-aminoethyl)-benzesulfonyl, HCl:p-aminoethylbenzenesulfonyl fluoride, HCl; Calbiochem/Novabiochem). Cells were detached from culture vessels using a rubber policeman, washed twice with TBS (tris-buffered saline; 150 mM NaCl, 50 mM tris HCl, pH 7.5), and resuspended in 1 ml TBS containing, 1% (v/v) Triton X-100 and 1 mM AEBSF. Cell suspensions were sonicated on ice followed by centrifugation to remove cell debris. Radiolabeled gC-1 was purified using HPA affinity chromatography (Olofsson et al., 1981bGo) or by an immunosorbent technique (Olofsson et al., 1983bGo).

Pronase-resistant clusters of radiolabeled O-linked glycans were prepared and analyzed by gel filtration as described previously (Lundström et al., 1987Go; Olofsson and Bolmstedt, 1998Go).

Analysis of gC-1, produced in O-glycosylation-deficient cells
An expression vector, designated pCMV3gC (Hung et al., 1992Go), where the open reading frame encoding gC-1 is inserted downstream from a CMV promoter, was used to express gC-1 in CHO wild type and ldlD cells. Briefly, 6 µg plasmid DNA was suspended in 100 µl Ham’s F10 medium, containing 6 µl Fugene Transfection Agent (Roche). This mixture was added to the CHO cells in a 6-well plastic cluster dish (2 ml Ham’s F10, supplemented with 5 mM galactose to prevent effects on the formation of N-linked glycans (Kingsley et al., 1986Go; Kozarsky et al., 1988bGo), 5% dialyzed fetal calf serum, and antibiotics). The transfected cultures were incubated at 37°C in a CO2 incubator and harvested with a rubber policeman in the presence of TBS, containing 25 mM octylglucoside and the AEBSF protease inhibitor (Sigma) at 72 h post-transfection. The lysate was sonicated at 0°C and clarified by centrifugation. The supernatant was subjected to SDS–PAGE, transferred to PVDF membranes, and subjected to Western blotting. The gC-derived bands were detected by Mab C2H12. The reaction was visualized by addition of a secondary HRP-conjugated anti-Mouse-Ig antibody (DAKO P260) and subsequent incubation with ECL chemoluminiscence reagents as recommended by the manufacturer (Amersham Life Science).

For indirect immunofluorescence subconfluent CHO and ldlD cells in 6-well cluster dish plates were transfected with 4 µg of pCMV as described above. At 18, 40, or 70 h post-transfection the cells were transferred in 0.1% fetal calf serum in TBS to Teflon-coated object glasses, using Costar Cell Lifters (Costar, Cambridge, MA). The cells were fixed in methanol and processed for indirect immunofluorescence as described previously (Bolmstedt et al., 1991Go), using Mab C4H11.

Polypeptide GalNAc-transferase assay
The assay was carried out essentially as described using soluble, secreted, purified recombinant GalNAc-T1, -T2, -T3, and -T6, expressed in insect cells (Bennett et al., 1996Go; Wandall et al., 1997Go). Acceptor peptides (for structure, see Tables II and III) derived from the N-terminal region of gC-1, which contain clusters of potential O-glycosylation sites (Olofsson, 1992Go), were synthesized by Chiron Mimotopes, and purified by the manufacturer to 99% purity. Briefly, the assays were performed in 50 µl total reaction mixture containing 25 mM cacodylate (pH 7.4), 10 mM MnCl2, 0.25 Triton X-100, 50 µM UDP- [14C]-GalNAc (2000 c.p.m./nmol) (Amersham), 25 µg acceptor peptide, and purified GalNAc-transferase at the concentrations indicated in the tables. Soluble, secreted forms of human GalNAc-T1, -T2, -T3, and T6 were expressed in Sf9 cells and purified to near homogeneity with specific activities of 0.6 U/mg for GalNAc-T1, 0.5 U/mg for GalNAc-T2, 0.5 U/mg for GalNAc-T3, and 2.35 U/mg for GalNAc-T6 measured using peptides derived from Muc2, Muc1, and Muc7 tandem repeats (Suijkerbuijk et al., 1991Go; Wandall et al., 1997Go). Products were quantified by scintillation counting after chromatography on Dowex-1, octadecyl silica cartridges (Bakerbond), or HPLC (PC3.2/3 or mRPC C2/C18 SC2.1/10 Pharmacia, Smart System).

Glycosidase digestion of gC-1
Radiolabeled, purified gC-1 was separated in an HPA-binding and a PNA-binding fraction as described (Lundström et al., 1987Go), and subjected to digestion with {alpha}-N-acetylgalactosaminidase (from chicken liver, Sigma), according to Mark and Mangkornkanok (1989)Go, or O-glycanase (from D.pneumonie, Roche), according to Pinter and Honnen (1988)Go. Released glycans were separated from the glycoprotein fraction by gel filtration on Sephadex PD-10 columns (Olofsson et al., 1993Go). Alternatively, nonlabeled, immunosorbent-purified gC-1 (Olofsson et al., 1983bGo; Sjöblom et al., 1987Go) was coated onto 96-well microplates and treated with 0.1 U or 0.01 U of N-acetylgalactosaminidase or 0.6 mU of O-glycanase, respectively, in 100 µl per well. The plates were incubated at 37°C for 2 h. Subsequently, the enzyme-treated gC-1 was analyzed in ELISA for reactivity to anti-gC-1 monoclonal antibodies as described previously (Sjöblom et al., 1987Go, 1992).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
This work was supported by grants from, The Swedish Medical Research Council, (Grants 9083, 9483, and 11225), The Medical Faculty, University of Göteborg, and The Danish Research Council. The skillful technical assistance of Richard Lymer is gratefully acknowledged.


    Footnotes
 
1 To whom correspondence should be addressed at: Department of Virology, University of Göteborg, Guldhedsgatan 10B, S-413 46 Göteborg, Sweden Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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