Endo-beta-N-acetylglucosaminidase H sensitivity of precursors to herpes simplex virus type 1 glycoproteins gB and gC.

The endoglycosidase endo-beta-N-acetylglucominidase H (endo H) was used to examine the nature of the oligosaccharides associated with the herpes simplex virus type 1 glycoproteins gA, gB, and gC. Immunoprecipitates from detergent extracts of infected cells, using monospecific antisera to gAB and gC, were treated with endo H. The low-molecular-weight precursor to gC, pgC(105), was found to be sensitive to endo H. Removal of the endo H-sensitive oligosaccharide chains from pgC(105) resulted in a protein with an apparent molecular weight of 75,000. In contrast, the fully glycosylated gC was not sensitive to endo H treatment. These results suggested that the oligosaccharide chains of pgC(105) were primarily of the simple high-mannose type. Both gA and gB were sensitive to endo H treatment; however, gB appeared to be only partially susceptible, whereas [3H]mannose-labeled gA was not detectable after endo H treatment. These results that gB contained both complex- and simple-type oligosaccharides, and gA contained only simple-type oligosaccharides. An accumulation of the high-mannose glycoproteins pgC(105) and gA was observed in monensin-treated infected cells with a concomitant inhibition of gB and gC. Glycoproteins gA and pgC(105) synthesized in the presence of monensin were also sensitive to endo H treatment.     

Expression of HSV-1 glycoproteins in tunicamycin-treated monkey kidney cells

The role of glycosylation in transport and expression of HSV-1 glycoproteins on the surface of HSV-1-infected African green monkey kidney cells was investigated by using tunicamycin (TM). A concentration of 0.05 microgram/ml of TM inhibited the replication of HSV-1 by greater than 99%. Immunoblot analysis of TM-treated and virus-infected cells indicated that 0.05 microgram/ml of TM blocked the addition of N-linked oligosaccharides into glycoproteins B, C and D. An immunofluorescence assay of TM-treated (0.05 and 0.1 microgram/ml) and virus-infected cells demonstrated the presence of nonglycosylated gC, gD and a reduced amount of gB on the surface of infected cells. The results suggest that the addition of N-linked oligosaccharides on the studied HSV-1 glycoproteins was not necessary for their transport and expression on the virus-infected cell surface.     

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.     

Quantitation of the beta-elimination reaction as used on glycoproteins

The carbohydrate side chains of mucus-type glycoproteins are O-glycosidic bonds between N-acetylgalactosamine to the hydroxyl groups of serine and threonine in the protein core. The alkaline catalyzed beta-elimination reaction, in the presence of sodium borohydride, is used for determining the number of side chains. The present paper presents a study of the quantitativeness of the alkaline borohydride procedure, using four parameters: the loss of seryl and threonyl residues, the formation of alanine and 2-aminobutanoic acid; the decrease in N-acetylhexosamine and the recovery of the amino sugar alcohols. Bovine, ovine and porcine submandibular glycoproteins were studied. Evidence is presented for the existence of N-acetylglucosamine involvement in O-glycosidic linkages to serine and threonine. Results for the relative rates of beta-elimination indicate that serine-linked glycosides are released more rapidly than threonine-linked glycosides.     

D-galactofuranose in the N-linked sugar chain of a glycopeptide from Ascobolus furfuraceus

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced beta-elimination of a small oligosaccharide. Evidence for the O-glycosidic linkage was provided by increase in absorbance at 240 nm, decrease in threonine and serine content after the alkaline treatment and detection of tritiated oligosaccharide following alkaline NaB3H4 reduction. Mannose is the sugar involved in the O-glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N-glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo-beta-N-acetyl-glucosaminidase. Sequential NaB3H4 reduction and acid hydrolysis gave [3H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis.     

Carbohydrates of influenza virus. III. Nature of oligosaccharide-protein linkage in viral glycoproteins.

Four different glycopeptides can be distinguished after pronase digestion of influenza A virus glycoproteins: Ia and Ib, containing N-acetylglucosamine, mannose, galactose, and fucose, and IIa and IIb, containing mannose and N-acetylglucosamine. All glycopeptides yielded N-acetylglucosaminyl-asparagine after mild acid hydrolysis. There was no evidence for O-glycosidic bonds. Thus, the carbohydrate complement is linked to the polypeptide exclusively by N-glycosidic linkages between N-acetylglucosamine and asparagine.     

Herpesvirus glycoprotein synthesis and insertion into plasma membranes.

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.     

Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface.

Herpes simplex virus specifies five glycoproteins which have been found on the surface of both the intact, infected cells and the virion envelope. In the presence of the drug tunicamycin, glycosylation of the herpes simplex virus type 1 glycoproteins is inhibited. We present in this report evidence that the immunologically specificity of the glycoproteins designated gA, gB, and gD resides mainly in the underglycosylated "core" proteins, as demonstrated by the immunoblotting technique. We showed also that tunicamycin prevented exposure of the viral glycoproteins on the cell surface, as the individual glycoproteins lost their ability to participate as targets for the specific antibodies applied in the antibody-dependent, cell-mediated cytotoxicity test. Immunocytolysis was reduced between 73 and 97%, depending on the specificity of the antibodies used. The intracellular processing of the herpes simplex virus type 1-specific glycoprotein designated gC differed from the processing of gA, gB, and GD, as evidenced by the identification of an underglycosylated but immunochemically modified form of gC on the surface of infected cells grown in the presence of tunicamycin.     

Virus-specific glycoproteins associated with the nuclear fraction of herpes simplex virus type 1-infected cells.

Monospecific antisera to herpes simplex virus type 1 (HSV-1) glycoproteins gB, gC, and gD were used to identify the HSV-1-specific glycoproteins associated with the nuclear fraction as compared with those associated with cytoplasmic fraction, whole-cell lysates, and purified virions. The results indicate that a predominance of HSV glycoprotein precursors pgC(105), pgB(110), and pgD(52) is associated with the nuclear fraction. Treatment of the nuclear fraction with the enzyme endo-beta-N-acetylglucosaminidase H indicated that the lower-molecular-weight glycoproteins are sensitive to this endoglycosidase. These results suggest that in the nuclear fraction of HSV-1-infected cells virus-specific glycoproteins gB, gC, and gD are predominately in the high-mannose precursor form; however, detectable amounts of the fully glycosylated forms of gC and gD were also found.     

Differential rates of processing and transport of herpes simplex virus type 1 glycoproteins gB and gC.

The kinetics of processing and transport of herpes simplex virus type 1 (HSV-1) glycoproteins gB and gC was investigated. The conversion of precursor to mature forms and the appearance of the glycoproteins at the infected-cell surface at different times postinfection (p.i.) were studied. gB, synthesized at 4 h p.i., was converted to the mature form with a half-time (t1/2) of 120 min and appeared at the plasma membrane with a t1/2 of 270 min. The gB synthesized at later times p.i. (6, 8, and 10.5 h) was transported less efficiently. Less than 50% of gB synthesized at later times p.i. was processed and transported to the cell surface. gB synthesized in transfected cells was transported to the plasma membrane with kinetics similar to that for gB synthesized at early times p.i. gC was processed efficiently when synthesized at both 8 and 10.5 h p.i., with t1/2 of conversion of pgC to gC of 40 and 60 min, respectively. Approximately 90 to 95% of the gC synthesized was converted to the mature form. The gC synthesized at 8 h p.i. was also transported rapidly to the cell surface, compared with the transport of gB synthesized at the same time, with a t1/2 of 240 min. Greater than 70% of the gC synthesized at 8 h p.i. appeared at the cell surface. The gC synthesized at 10.5 h was transported less efficiently to the cells surface during a 6-h chase.     

Extensive homology between the herpes simplex virus type 2 glycoprotein F gene and the herpes simplex virus type 1 glycoprotein C gene.

The region of the herpes simplex virus type 2 (HSV-2) genome which maps colinearly with the HSV-1 glycoprotein C (gC) gene has been cloned, and the DNA sequence of a 2.29-kilobase region has been determined. Contained within this sequence is a major open reading frame of 479 amino acids. The carboxyterminal three-fourths of the derived HSV-2 protein sequence showed a high degree of sequence homology to the HSV-1 gC amino acid sequence reported by Frink et al. (J. Virol. 45:634-647, 1983). The amino-terminal region of the HSV-2 sequence, however, showed very little sequence homology to HSV-1 gC. In addition, the HSV-1 gC sequence contained 27 amino acids in the amino-terminal region which were missing from the HSV-2 protein. Computer-assisted analysis of the hydrophilic and hydrophobic properties of the derived HSV-2 sequence demonstrated that the protein contained structures characteristic of membrane-bound glycoproteins, including an amino-terminal signal sequence and carboxy-terminal hydrophobic transmembrane domain and charged cytoplasmic anchor. The HSV-2 protein sequence also contained seven putative N-linked glycosylation sites. These data, in conjunction with mapping studies of Para et al. (J. Virol. 45:1223-1227, 1983) and Zezulak and Spear (J. Virol. 49:741-747, 1984), suggest that the protein sequence derived from the HSV-2 genome corresponds to gF, the HSV-2 homolog of HSV-1 gC.     

Herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate

Cell surface heparan sulfate (HS) serves as an initial receptor for many different viruses, including herpes simplex virus types 1 and 2 (HSV-1 and 2, respectively). Glycoproteins C and B (gC and gB) are the major components of the viral envelope that mediate binding to HS. In this study, purified gB and gC homologous proteins as well as purified HSV-1 and HSV-2 virions were compared for the ability to bind isolated HS receptor molecules. HSV-1 gC and HSV-2 gC bound comparable amounts of HS. Similarly, HSV-1 gB and its HSV-2 counterpart showed no difference in the HS-binding capabilities. Despite the similar HS-binding potentials of gB and gC homologs, HSV-1 virions bound more HS than HSV-2 particles. Purified gC and gB proteins differed with respect to sensitivity of their interaction with HS to increased concentrations of sodium chloride in the order gB-2 > gB-1 > gC-1 > gC-2. The corresponding pattern for binding of whole HSV virions to cells in the presence of increased ionic strength of the medium was HSV-2 gC-neg1 > HSV-1 gC(-)39 > HSV-1 KOS 321 > HSV-2 333. These results relate the HS-binding activities of individual glycoproteins with the cell-binding abilities of whole virus particles. In addition, these data suggest a greater contribution of electrostatic forces for binding of gB proteins and gC-negative mutants compared with binding of gC homologs and wild-type HSV strains. Binding of wild-type HSV-2 virions was the least sensitive to increased ionic strength of the medium, suggesting that the less extensive binding of HS molecules by HSV-2 than by HSV-1 can be compensated for by a relatively weak contribution of electrostatic forces to the binding. Furthermore, gB and gC homologs exhibited different patterns of sensitivity of binding to cells to inhibition with selectively N-, 2-O-, and 6-O-desulfated heparin compounds. The O-sulfate groups of heparin were found to be more important for interaction with gB-1 than gB-2. These results indicate that HSV-1 and HSV-2 differ in their interaction with HS.     

Expression of seven herpes simplex virus type 1 glycoproteins (gB, gC, gD, gE, gG, gH, and gI): comparative protection against lethal challenge in mice.

We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100% survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.     

Binding of complement component C3b to glycoprotein C is modulated by sialic acid on herpes simplex virus type 1-infected cells.

Neuraminidase treatment of cells infected with herpes simplex virus type 1 (HSV-1) markedly enhanced the binding of complement component C3b to HSV 1 glycoprotein C (gC). When HSV-1 was grown in BHK RicR14 cells in which glycoproteins had reduced amounts of N-linked complex oligosaccharides, including sialic acid, the binding of C3b to gC was markedly enhanced. We used neuraminidase treatment to demonstrate that cloning the gC gene from the HSV-1 F strain into an HSV-1 mutant which fails to express gC converted the mutant virus from C3b receptor negative to receptor positive. These results further support a role for gC as a C3b receptor and indicate that sialic acid modifies receptor activity.     

Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma alpha(1)-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1-->4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2-->2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl-->N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas-liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.     

Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells

Hyperimmune antisera to purified Sindbis (SIN) or Semliki Forest (SF) virus were used to identify alphavirus-specific and cross-reactive proteins in virions and infected cells. The hyperimmune sera participated in homologous and cross-cytolysis of alphavirus-infected cells, and the use of monospecific antisera to SIN structural proteins suggested that E1 and E2 could serve as target proteins in cytolysis. Proteins from purified virions or infected cells were extracted with Nonidet P-40, denatured by procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose solid supports, and reacted with hyperimmune sera and 125I-labeled protein A (immunoblotting on denatured proteins). Alternatively, native proteins extracted by mild Nonidet P-40 treatment were precipitated with hyperimmune sera before denaturation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After immunoblotting, homologous antiserum reacted with the virus structural proteins E1, E2, capsid extracted from purified virions, and the counterparts of these proteins extracted from infected cells. In addition, PE2 and a 92,000-molecular-weight protein from infected cells reacted with homologous antiserum. These proteins were also immunoprecipitated with homologous antiserum. After immunoblotting, the Sindbis capsid protein was shown to be cross-reactive whether derived from purified virions or from infected cells; no cross-reactivity was observed with PE2 or E2 from either source, and the E1 glycoprotein was shown to be cross-reactive only when obtained from virions. However, the E1 glycoprotein could be cross-immunoprecipitated from infected cells (as well as from disrupted virions), and, in addition, capsid and a 92,000-molecular-weight protein were cross-immunoprecipitated from infected cells. These results suggest that a native conformation of the cell-associated E1 glycoproteins may be required for immunological cross-reactivity (immune precipitation), whereas virion but not cell-associated E1 retains immunological cross-reactivity after denaturation (immunoblot technique). The findings extend our previously published evidence which suggested that alphavirus maturation is accompanied by a change in immunological cross-reactivity with respect to E1.     

Incorporation of CD4 into virions by a recombinant herpes simplex virus.

Two herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the human CD4 gene into the HSV-1 genome between the gC promoter and the gC structural gene. These viruses, designated K delta T/CD4 and K082/CD4, synthesized a significant quantity of CD4. CD4 was expressed on the surface of infected cells at levels substantially higher than on the surface of HUT78 cells, a CD4+ cell line. Most significantly, a small but detectable quantity of CD4 was incorporated into virions produced by the recombinant viruses. This was demonstrated both by immunoprecipitation of CD4 from purified virions and by neutralization of the recombinant virions by OKT4 and complement. These results suggest that specific virion incorporation signals are not strictly required for inclusion of glycoproteins into HSV-1 virions. It may be possible to utilize this ability to alter the host range or tissue specificity of HSV-1.     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies.

Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.     

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.