Human antibodies to herpes simplex virus type 1 glycoprotein C are neutralizing and target the heparan sulfate-binding domain

Human antibodies specific for glycoprotein C (gC1) of herpes simplex virus type 1 (HSV-1) neutralized the virus infectivity and efficiently inhibited attachment of HSV-1 to human HaCaT keratinocytes and to murine mutant L cells expressing either heparan sulfate or chondroitin sulfate at the cell surface. Similar activities were observed with anti-gC1 monoclonal antibody B1C1. In addition to HaCaT and L cells, B1C1 antibody neutralized HSV-1 infectivity in simian GMK AH1 cells mildly pre-treated with heparinase III. Human anti-gC1 antibodies efficiently competed with the binding of gC1 to B1C1 antibody whose epitope overlaps a part of the attachment domain of gC1. Human anti-gC1 and B1C1 antibodies extended survival time of mice experimentally infected with HSV-1. We conclude that in HaCaT cells and in cell systems showing restricted expression of glycosaminoglycans, human and some monoclonal anti-gC1 antibodies can target the cell-binding domain of this protein and neutralize viral infectivity.     

Developmental changes of proteoglycan synthesis in rat liver and isolated hepatocytes

The synthesis of sulfated proteoglycans in late fetal (19th to 22nd day of intrauterine life), early postnatal, and adult liver tissue as well as in hepatocytes and their distribution in plasma membranes were studied. Overall proteoglycan production is enhanced two-fold in fetal as compared with adult liver tissue. In contrast to slices from adult liver, in which the synthesis of heparan [35S]-sulfate comprises more than 80% and chondroitin sulfate less than 5% of total glycosaminoglycans, chondroitin [35S]sulfate is the major type of glycosaminoglycans synthesized in fetal liver representing about 50% of total sulfated glycosaminoglycans. Thus, the synthesis of chondroitin sulfate is elevated nearly 30-fold in fetal liver as compared with the adult counterpart. Immediately after birth chondroitin sulfate formation decreases rapidly reaching adult levels between the 10th and 15th day of postnatal life. The production of heparan sulfate is almost unchanged during perinatal liver development due to a relatively low fractional synthesis of heparan [35S]sulfate in fetal liver. Hepatocytes were identified as the cell type responsible for elevated chondroitin sulfate production in fetal liver. Erythroblasts, which synthesize chondroitin sulfate, contribute less than 10% to total glycosaminoglycan synthesis in embryonic liver. Plasma membranes of adult liver contain exclusively heparan sulfate whereas in neonatal liver cell membranes 25% of labeled glycosaminoglycans is represented by chondroitin sulfate, a fraction which decreases rapidly after birth. In parallel to the postnatal shut down of chondroitin sulfate synthesis the activity of the UDPxylose:coreprotein xylosyltransferase (EC. 2.4.2.26) decreases from 4.8 +/- 0.5 dpm/h per microgram protein to 0.3 +/- 0.1 dpm/h per microgram protein suggesting a regulatory function of the enzyme for proteochondroitin sulfate synthesis in developing liver. The formation of both heparan sulfate and chondroitin sulfate is dependent on functioning protein synthesis, which indicates, together with double labeling experiments using [3H]serine and [14C]glucosamine as isotopic precursors, their synthesis as proteoglycans. The positive correlation (r = 0.949) between the incorporation of [3H]thymidine into DNA and chondroitin [35S]sulfate production supports the assumption of a cell growth promoting activity of chondroitin sulfate and points to a significant role of the glycosaminoglycan in the process of cellular proliferation and tissue differentiation.     

Structural requirements of glycosaminoglycans for their interaction with HIV-1 envelope glycoprotein gp120

Heparan sulfate proteoglycans are known to assist HIV-1 entry into host cells, mediated by the viral envelope glycoprotein gp120. We aimed to determine the general structural features of glycosaminoglycans that enable their binding to gp120, by surface plasmon resonance. Binding was found to be dependent on sequence type, size and sulfation patterns. HIV-1 gp120 prefers heparin and heparan sulfate (with at least 16 monomers in length) over chondroitin and dermatan. Sulfate groups were essential to promote this interaction. These results advance the understanding of the molecular-level requirements for virus attachment and cell entry.     

Identification and characterization of a Candida albicans-binding proteoglycan secreted from rat submandibular salivary glands.

A previously identified Candida albicans-binding glycoprotein secreted from rat submandibular glands (RSMG) has been further purified from an aqueous RSMG extract by ion-exchange chromatography and gel filtration. Biochemical analysis of the glycoprotein revealed high levels of uronic acid and sulfate, suggesting that it was a proteoglycan. Its amino acid and carbohydrate compositions were similar to those observed for other proteoglycans and differed significantly from those of RSMG mucin, the major secretory glycoprotein of RSMG. In addition, the apparent molecular weight of the glycoprotein was reduced following treatment with either chondroitinase ABC or heparitinase, demonstrating the presence of chondroitin sulfate and heparan sulfate. On the basis of its structure and anatomical source, the glycoprotein is referred to as submandibular gland secreted proteoglycan 1 (SGSP1). SGSP1 also binds monoclonal antibody 1F9, which recognizes the human blood group A carbohydrate epitope found on RSMG mucin. Hence, SGSP1 appears to be a hybrid molecule with carbohydrate structures found in both proteoglycans and RSMG mucin. Enzymatic digestion of SGSP1, followed by its interaction with a radiolabelled C. albicans strain in a filter-binding assay, demonstrated that binding to this strain appears to be mediated primarily via the heparan sulfate side chains of SGSP1 and not via the blood group A oligosaccharide.     

Sulfated polymers inhibit the interaction of human cytomegalovirus with cell surface heparan sulfate.

Several sulfated polysaccharides (dextran sulfate, pentosan polysulfate, heparin) and copolymers of acrylic acid with vinylalcohol sulfate have proved to be potent inhibitors of human cytomegalovirus (CMV) infectivity in vitro. Sulfated alpha-cyclodextrins are only weak inhibitors of CMV. A close correlation was found between the 50% inhibitory concentrations of the sulfated polymers for CMV cytopathogenicity, virus-cell binding, and expression of immediate early antigens (IEA) in human embryonic lung (HEL) cells. CMV particles bound specifically to heparin-Sepharose. Sulfated polymers specifically eluted the virus particles from this matrix. Enzymatic digestion of cell surface heparan sulfate, but not of chondroitin sulfate, prevented the cells from being infected with CMV. Moreover, radiolabeled CMV bound efficiently to, and were infective for wild-type Chinese hamster ovary (CHO) cells, whereas virus binding to, and infection of, mutant CHO cell lines that were deficient in either all glycosaminoglycans or heparan sulfate only was significantly impaired. The mechanism of action of the sulfated polymers can be attributed to an inhibitory effect on the binding of CMV particles to the host cells. Presumably, the sulfated polymers interact with the viral envelope site(s) involved in the attachment of the CMV virions to cell surface heparan sulfate.     

Glycosaminoglycan-binding ability is a feature of wild-type strains of herpes simplex virus type 1

Adaptation of some viruses to replication in cultured cells selects variants that due to alterations in the viral attachment proteins convert to using heparan sulfate (HS) as initial receptor. We report that the nucleotide sequence of herpes simplex virus type 1 (HSV-1) glycoprotein C (gC), a principal attachment component of the virus, remained unchanged during adaptation of wild-type strains to cultured cells. Likewise, amino acid residues critical for binding of gC to HS were conserved in viral strains that replicated in vivo in different human tissues. Moreover wild-type HSV-1 strains derived directly from clinical specimens were, similar to their cell culture propagated progeny viruses and common laboratory strains, sensitive to heparin and demonstrated impairment in their ability to infect HS/chondroitin sulfate deficient cells. These results demonstrate that the HS-binding ability is a feature of wild-type strains of HSV-1.     

Cellular invasion of Orientia tsutsugamushi requires initial interaction with cell surface heparan sulfate

Role of transmembrane heparan sulfate proteoglycans on invasion of Orientia tsutsugamushi into host cells was investigated. Pretreatment with heparan sulfate and heparin inhibited the infection of O. tsutsugamushi for L cell, mouse fibroblast, whereas other glycosaminoglycans had little effect. These same treatments were also shown to reduce the infection in a dose-dependent manner, and enzymatic treatment of cells with heparitinase, but not chondroitinase ABC, inhibited the infection. In addition, mutant cell lines of Chinese hamster ovarian cell defective in heparan sulfate synthesis but not chondrotin sulfate synthesis and defective in all glycosaminoglycan synthesis showed marked reduction in susceptibility to infection by O. tsutsugamushi. Also mutant cell lines, which express heparan sulfate proteoglycans at low level, showed intermediate level of infectivity. Finally O. tsutsugamushi bind to(35)S-labelled heparin. Collectively, these findings provide strong evidence that heparan sulfate proteoglycans contribute to the attachment of O. tsutsugamushi to the cells.     

Initial characterization of small proteoglycans synthesized by embryonic chick leg muscle-associated connective tissues

Muscle development in the embryonic chick involves the interfacing of two different cell types: myogenic cells forming the contractile network and connective tissue cells which wrap this network into discrete morphologies. Previous reports from this laboratory detailed the characterization of large extracellular matrix proteoglycans synthesized by skeletal muscle. This report describes the initial characterization of small muscle-associated connective tissue proteoglycans synthesized in embryonic chick leg in ovo and by embryonic leg muscle-associated secondary fibroblasts in vitro by CsCl equilibrium density gradient centrifugation. Analysis of the D1 gradient fractions from the in ovo and in vitro material revealed that the proteoglycans were of relatively small size. These molecules eluted from Sepharose CL-2B through a Kav range of 0.6-0.8. Analysis of alkaline borohydride-released glycosaminoglycan chains by Sepharose CL-6B, Sephadex G-25, and thin layer chromatography demonstrated a mixture of three proteoglycan families composed of chondroitin sulfate, dermatan sulfate, and heparan sulfate glycosaminoglycans.     

Change in synthesis of sulfated glycoconjugates during muscle development, maturation and aging in embryonic to senescent CBF-1 mouse

Previous biochemical and morphological studies have demonstrated a change in the synthetic pattern of sulfated proteoglycans during skeletal musculogenesis in the embryonic chick. These studies revealed that a transition occurs in both composition and deposition of sulfated glycoconjugates that parallels the developmental state of the tissue. The current study was undertaken to ascertain whether this transition in the embryonic chick is a conserved developmental process during musculogenesis in the mouse. Leg musculature from embryonic, newborn, juvenile, adolescent, young adult, mature adult and senescent mice, radiolabeled in vivo with [35S]sulfate, was analyzed for relative size and composition of newly synthesized sulfated macromolecules. The data reveal a transition in the synthesis of sulfated proteoglycans and glycoproteins that parallels the myogenic differentiative state of the mouse leg muscle. Embryonic mouse leg musculature synthesizes relatively large proteoglycans consisting of large chondroitin sulfate glycosaminoglycan chains. Subsequently, these major newly synthesized proteoglycans are replaced synthetically by smaller molecules composed of mixtures of dermatan sulfate, chondroitin sulfate and heparan sulfate glycosaminoglycans (newborn through 2 weeks); dermatan sulfate, heparan sulfate and chondroitin sulfate glycosaminoglycans (13 months) and heparan sulfate and dermatan sulfate glycosaminoglycans (25-26 months). The sulfated glycoproteins demonstrate a reciprocal synthetic pattern. Early in development sulfated glycoproteins form a small proportion of the newly synthesized sulfated material. With increasing developmental and maturational age, the proportion of sulfated glycoproteins increases. This continues until they become the predominant sulfated moieties synthesized by senescent mouse muscle. The results from this study thus extend observations initially made in chick to muscle development in the mouse and, therefore, suggest that the transition in synthesis of sulfated glycoconjugates is a conserved developmental process during musculogenesis.     

Interaction of an uuter membrane protein of enterotoxigenic Escherichia coli with cell surface heparan sulfate proteoglycans

We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.     

Chondroitin sulfate proteoglycan and heparan sulfate proteoglycan production by cultured pigeon peritoneal macrophages.

Proteoglycan production was examined in cultures of thioglycollate-elicited peritoneal macrophages obtained from White Carneau and Show Racer pigeons. Following a 24-h incubation in the presence of [35S]sulfate and [3H]serine, total production and distribution of 35S-labeled proteoglycan into media (60-65%), pericellular (21-27%), and intracellular (13-14%) compartments was similar in White Carneau and Show Racer macrophage cultures. Media proteoglycans consisted of high-molecular-weight chondroitin sulfate proteoglycan, low-molecular-weight chondroitin sulfate proteoglycan, and heparan sulfate proteoglycan. High-molecular-weight chondroitin sulfate proteoglycan was predominantly 6-sulfated (80%) and contained a core protein larger than 200 kd, whereas low-molecular-weight chondroitin sulfate proteoglycan was 4-sulfated and contained a 28-kd core protein. Pericellular proteoglycan was similar in size to low-molecular-weight proteoglycan and consisted of a predominantly 6-sulfated (75%) chondroitin sulfate proteoglycan and heparan sulfate proteoglycan. Intracellular 35S-labeled chondroitin sulfate and heparan sulfate were smaller than media and pericellular proteoglycans, suggestive of intracellular degradative processing.     

Heparin binding sites on Ross River virus revealed by electron cryo-microscopy

Cell surface glycosaminoglycans play important roles in cell adhesion and viral entry. Laboratory strains of two alphaviruses, Sindbis and Semliki Forest virus, have been shown to utilize heparan sulfate as an attachment receptor, whereas Ross River virus (RRV) does not significantly interact with it. However, a single amino acid substitution at residue 218 in the RRV E2 glycoprotein adapts the virus to heparan sulfate binding and expands the host range of the virus into chicken embryo fibroblasts. Structures of the RRV mutant, E2 N218R, and its complex with heparin were determined through the use of electron cryo-microscopy and image reconstruction methods. Heparin was found to bind at the distal end of the RRV spikes, in a region of the E2 glycoprotein that has been previously implicated in cell-receptor recognition and antibody binding.     

A role for glycoprotein C in pseudorabies virus entry that is independent of virus attachment to heparan sulfate and which involves the actin cytoskeleton

Glycoprotein C (gC) of pseudorabies virus, a swine herpesvirus, initiates virus attachment by binding to heparan sulfate (HS) linked to proteoglycans (HSPGs) on the cell surface. This interaction facilitates a required step in virus entry, the binding to a non-HS coreceptor, likely by another viral glycoprotein, gD. We demonstrate that gC has an even more direct role in virus entry than simply promoting adhesion strengthening. A porcine cell line expressing gC trans-complemented the penetration, but not attachment, defect of gC null mutants. In addition, gC promoted the colocalization of cell surface HSPGs and the actin cytoskeleton, suggesting a role for filamentous actin in virus entry. This was supported by results showing that both the engagement of a non-HS coreceptor and entry events subsequent to coreceptor binding were impaired if cells were first treated with an actin depolymerizing agent, cytochalasin D. Our results suggest a model in which gC-HS interactions promote not only virus attachment but also virus entry by usurping the normal properties of HSPGs.     

Heparin-dependent attachment ofrespiratory syncytial virus (RSV) to host cells

Summary.  In this study we could demonstrate that heparin (), but not heparan sulphate or chondroitin sulphate C is able to inhibit in vitro infection of cells by respiratory syncytial virus (RSV). In addition, this protective effect of heparin could only be observed, when heparin was present at the time of inoculation. Enzymatic digestion of cell surface glycosaminoglycans with heparinase and heparitinase, but not chondroitin sulphate ABC lyase reduced the effectiveness of RSV-infection. Affinity chromatography experiments, using immobilised heparin further demonstated that RSV attachment protein G was able to bind specifically to heparin. Therefore heparin-like proteoglycans showed properties required for attachment of RSV to host cells. Received September 16, 1996 Accepted January 9, 1997     

Host Glycosaminoglycan Confers Susceptibility to Bacterial Infection in Drosophila melanogaster

Many pathogens engage host cell surface glycosaminoglycans, but redundancy in pathogen adhesins and host glycosaminoglycan-anchoring proteins (heparan sulfate proteoglycans) has limited the understanding of the importance of glycosaminoglycan binding during infection. The alpha C protein of group B streptococcus, a virulence determinant for this neonatal human pathogen, binds to host glycosaminoglycan and mediates the entry of bacteria into human cells. We studied alpha C protein-glycosaminoglycan binding in Drosophila melanogaster, whose glycosaminoglycan repertoire resembles that of humans but whose genome includes only three characterized membrane heparan sulfate proteoglycan genes. The knockdown of glycosaminoglycan polymerases or of heparan sulfate proteoglycans reduced the cellular binding of alpha C protein. The interruption of alpha C protein-glycosaminoglycan binding was associated with longer host survival and a lower bacterial burden. These data indicate that the glycosaminoglycan-alpha C protein interaction involves multiple heparan sulfate proteoglycans and impairs bacterial killing. Host glycosaminoglycans, anchored by multiple proteoglycans, thereby determine susceptibility to infection. Because there is homology between Drosophila and human glycosaminoglycan/proteoglycan structures and many pathogens express glycosaminoglycan-binding structures, our data suggest that interfering with glycosaminoglycan binding may protect against infections in humans.     

Infectivity of Chlamydia trachomatis serovar LGV but not E is dependent on host cell heparan sulfate

The ability of heparan sulfate, heparin, and other glycosaminoglycans to inhibit the infectivity of Chlamydia trachomatis serovars E and LGV was examined using a simple competitive inhibition assay with three cell types from the human female reproductive tract, including primary human endosalpingeal cells. With the majority of the glycosaminoglycans tested, LGV was more significantly inhibited than serovar E. We have compared chlamydial infectivity between a wild-type Chinese hamster ovary cell line and two glycosaminoglycan-deficient cell lines. LGV was shown to be unable to infect heparan sulfate-deficient and GAG-deficient Chinese hamster ovary cell lines, whereas the E serovar infected these cells as efficiently as the control (nondeficient) cells. These two sets of experiments confirmed that serovar LGV is more dependent on a heparan sulfate-related mechanism of infectivity than is serovar E. This is further supported by the fact that attempts to purify a heparan sulfate-like molecule from either serovar cultured in glycosaminoglycan-deficient cell lines were nonproductive. Previous reports have suggested that chlamydia are able to produce a heparan sulfate-like molecule that is important for attachment and infectivity. We have attempted to detect possible binding of a specific heparan sulfate antibody to C. trachomatis by flow cytometry. Results showed no binding of the heparan sulfate antibody to C. trachomatis serovar LGV or E. Our results strongly indicate that chlamydiae do not produce a heparan sulfate-like molecule but rather use host cell heparan sulfate in order to infect cells.     

Early interactions of human herpesvirus 6 with lymphoid cells: role of membrane protein components and glycosaminoglycans in virus binding

A microassay was developed to detect human herpesvirus 6 (HHV-6) binding to its cellular receptor using flow cytometry. Comparable results were obtained either by using HHV-6 preparations conjugated with fluorescein isothiocyanate or by indirect immunofluorescent labeling of membrane-bound virus using as primary antibody a monoclonal antibody specific for the HHV-6 gp60/110 envelope glycoprotein. Virus attachment to the plasma membrane was specific and saturable. As expected, among cell lines of various origin, maximum binding was detected on human T-lymphoid cells (HSB-2). Papain digestion of HSB-2 cells prevented HHV-6 attachment and reduced significantly virus infection, indicating the involvement of a protein-based receptor in the attachment step. After removal of the protease, virus receptors were resynthesized and their regeneration was prevented partially by cycloheximide, an inhibitor of protein synthesis. Unexpectedly, only high concentrations (mg/ml) of soluble heparan sulfate and heparin inhibited HHV-6 binding and infection. Under the same conditions, few micrograms (per ml) of heparin suppressed completely herpes simplex type 1 (HSV-1) attachment to the same cell line. Treatment of HSB-2 cells with heparitinase and heparinase, at doses that reduced significantly HSV-1 attachment, had little effect on HHV-6 binding to the cell membrane, indicating a different requirement of heparan sulfate-containing glycosaminoglycans for the two herpesviruses. These data suggest that protein components of the cellular membrane play an essential role in HHV-6 binding and infection while heparan sulfate-glycos-aminoglycans appear to be involved only partially in virus-receptor interaction.     

Inhibition of herpes simplex virus infection by lactoferrin is dependent on interference with the virus binding to glycosaminoglycans

Previous reports have indicated that lactoferrin inhibits herpes simplex virus (HSV) infection during the very early phases of the viral replicative cycle. In the present work we investigated the mechanism of the antiviral activity of lactoferrin in mutant glycosaminoglycan (GAG)-deficient cells. Bovine lactoferrin (BLf) was a strong inhibitor of HSV-1 infection in cells expressing either heparan sulfate (HS) or chondroitin sulfate (CS) or both, but was ineffective or less efficient in GAG-deficient cells or in cells treated with GAG-degrading enzymes. In contrast to wild-type HSV-1, virus mutants devoid of glycoprotein C (gC) were significantly less inhibited by lactoferrin in GAG-expressing cells, indicating that lactoferrin interfered with the binding of viral gC to cell surface HS and/or CS. Finally, we demonstrated that lactoferrin bound directly to both HS and CS isolated from surfaces of the studied cells, as well as to commercial preparations of GAG chains. The results support the hypothesis that the inhibition of HSV-1 infectivity by lactoferrin is dependent on its interaction with cell surface GAG chains of HS and CS.