Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin and integrin receptors

Heparan sulphate proteoglycans are increasingly implicated as eukaryotic cell surface receptors for bacterial pathogens. Here, we report that Neisseria gonorrhoeae adheres to proteoglycan receptors on HEp-2 epithelial cells but that internalization of the bacterium by this cell type requires the serum glycoprotein fibronectin. Fibronectin was shown to bind specifically to gonococci producing the OpaA adhesin. Binding assays with fibronectin fragments located the bacterial binding site near the N-terminal end of the molecule. However, none of the tested fibronectin fragments supported gonococcal entry into the eukaryotic cells; a 120 kDa fragment carrying the cell adhesion domain with the amino acid sequence RGD even inhibited the fibronectin-mediated uptake of MS11-OpaA. This inhibition could be mimicked by an RGD-containing hexapeptide and by alpha 5 beta 1 integrin-specific antibodies, suggesting that interaction of the central region of fibronectin with integrin receptors facilitated bacterial uptake. Fibronectin was unable to promote gonococcal entry into HEp-2 cells that had been treated with the enzyme heparinase III, which degrades the glycosaminoglycan side-chains of proteoglycan receptors. On the basis of these results, we propose a novel cellular uptake pathway for bacteria, which involves the binding of the pathogen to glycosaminoglycans that, in turn, act as co-receptors facilitating fibronectin-mediated bacterial uptake through integrin receptors. In this scenario, fibronectin would act as a molecular bridge linking to Opa-proteoglycan complex with host cell integrin receptors.     

Central nervous system abscesses due to Coccidioides species

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37 degrees C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Fig receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4 degrees C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4 degrees C and then washed, were shifted to 37 degrees C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex.     

Herpes simplex virus infection and propagation in a mouse L cell mutant lacking heparan sulfate proteoglycans

We have isolated a variant line of mouse L cells, termed gro2C, which is partially resistant to infection by herpes simplex virus type 1 (HSV-1). Characterization of the genetic defect in gro2C cells revealed that this cell line harbors a specific defect in the heparan sulfate synthesis pathway. Specifically, anion-exchange high-performance liquid chromatography of metabolically radiolabeled glycosaminoglycans indicated that chondroitin sulfate moieties were synthesized normally in the mutant cells, whereas heparin-like chains were absent. Because of these properties, we have used these cells to investigate the role of heparan sulfate proteoglycans in the HSV-1 life cycle. In this report, we demonstrate that the partial block to HSV-1 infection in gro2C cells occurs in the virus entry pathway. Virus adsorption assays using radiolabeled HSV-1 (KOS) revealed that the gro2C cell surface is a relatively poor target for HSV-1 in that virus attachment was 85% lower in the mutant cells than in the parental L cell controls. A portion of the 15% residual virus adsorption was functional, however, insofar as gro2C cells were susceptible to HSV-1 infection in plaque assays and in single-step growth experiments. Moreover, although the number of HSV-1 plaques that formed in gro2C monolayers was reduced by 85%, the plaque morphology was normal, and the virus released from the mutant cells was infectious. Taken together, these results provide strong genetic evidence that heparan sulfate proteoglycans enhance the efficiency of HSV attachment to the cell surface but are otherwise not essential at any stage of the lytic cycle in culture. Moreover, in the absence of heparan sulfate, other cell surface molecules appear to confer susceptibility to HSV, leading to a productive viral infection.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.     

The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro

Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo.     

Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding

The role of heparin or heparan sulfates in the interaction of basic fibroblast growth factor (bFGF) with its high affinity receptor were investigated using purified extracellular ligand-binding region of FGF receptor-1 (FGFR-1) and intact receptors expressed in a myeloid cell line (32D) that does not express detectable levels of heparan sulfate proteoglycans or in Chinese hamster ovary (CHO) cell mutants defective in heparan sulfate synthesis. The purified extracellular domain of FGFR-1 formed complexes with 125I-bFGF both in the presence or absence of heparin. Intact FGFR-1 expressed in 32D cells also bound the same amount of 125I-bFGF in the presence or absence of heparin when saturating concentrations of bFGF were used. Varying the concentration of 125I-bFGF showed that heparin increased the amount of 125I-bFGF bound at low bFGF concentrations and increased the affinity of bFGF for its receptor by about 3-fold. To eliminate the possibility of alteration of bFGF properties through the chemical modification reactions, bFGF was labeled biosynthetically. The binding of biosynthetically labeled bFGF to FGFR-1 also did not require heparin. When FGFR-1 or FGFR-2 were expressed in mutant CHO cells deficient in heparan sulfate synthesis, the cells also bound 125I-bFGF in the absence of heparin, and the addition of heparin increased the affinity of bFGF for its receptors 2-3-fold. Thus, heparin or heparan sulfate is not required for the binding of bFGF to its receptors but increases the binding affinity to a moderate degree. Finally, the requirement for heparin in signal transduction through the receptor was investigated. Expression of c-fos mRNA was induced by bFGF in 32D cells expressing FGFR-1 to the same extent in the presence or absence of heparin.     

Secretion-capture role for apolipoprotein E in remnant lipoprotein metabolism involving cell surface heparan sulfate proteoglycans

To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.     

Heparan sulfate proteoglycans control intracellular processing of bFGF in vascular smooth muscle cells

Basic fibroblast growth factor (bFGF) is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated in a number of vascular disorders. bFGF interacts with high-affinity receptors and heparan sulfate proteoglycans (HSPG) at the cell surface. HSPG have been demonstrated to enhance bFGF binding to its receptors, yet no known role for HSPG in modulating postbinding events has been identified. In the present study, we analyzed bFGF internalization, intracellular distribution, degradation, and stimulation of DNA synthesis within native and HSPG-deficient VSMC. HSPG-deficient VSMC were generated by treating cells with sodium chlorate to inhibit the sulfation of HSPG. We found that stimulation of DNA synthesis by bFGF in chlorate-treated VSMC was markedly reduced as compared with native cells, even at doses of bFGF where receptor binding was similar in the two conditions. This was not a general lack of mitogenic potential, as the addition of calf serum, or epidermal growth factor, stimulated DNA synthesis to a similar extent in native and chlorate-treated cells. Analysis of the accumulation of internalized bFGF within cytoplasmic and nuclear fractions of native and HSPG-deficient VSMC showed striking differences. At early time points (0-2 h), nearly identical amounts of bFGF were observed in the cytoplasmic fractions under both conditions, yet significant amounts of bFGF were only found in the nuclear fractions of native cells. At later time points (2-48 h), the amount of cytoplasmic bFGF was significantly greater in the native compared to HSPG-deficient cells, and nuclear deposition of bFGF began to reach similar levels under both conditions. Furthermore, the intracellular half-life of bFGF was dramatically prolonged in native compared to HSPG-deficient cells, in part, due to decreased bFGF degradation in native cells. Thus, HSPG appears to accelerate nuclear localization, increase cytoplasmic capacity, and inhibit intracellular degradation of bFGF in VSMC. Modulation of intracellular processing of bFGF by HSPG might control the biological activity of bFGF in VSMC.     

The genome organization of the broad bean necrosis virus (BBNV)

In this study the effect of lipoprotein lipase (LPL) on the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by hepatic cells was investigated. Human HDL3 (d 1.125-1.21 g/ml) was radiolabeled with 125I in the protein moiety and with 3H in the CE moiety. LPL was prepared from bovine milk. Human hepatocytes in primary culture and human Hep3B hepatoma cells were incubated in medium containing doubly radiolabeled HDL3 with or without LPL. Without LPL, apparent HDL3 particle uptake according to the lipid tracer (3H) was in excess of that due to the protein label (125I) indicating selective CE uptake from HDL3. Addition of LPL increased selective CE uptake up to 7-fold. This stimulation of HDL3 selective CE uptake was independent of the lipolytic activity of LPL as suggested by several experimental approaches. Cell surface heparan sulfate proteoglycan deficiency decreased the LPL-mediated increase in selective CE uptake suggesting an important role for these molecules. In low density lipoprotein (LDL) receptor- or LDL receptor-related protein-(LRP)-deficient cells, LPL increased selective CE uptake as it did in normal cells yielding no evidence that these receptors play a role in the LPL effect on selective CE uptake. In summary, lipoprotein lipase increases the selective uptake of high density lipoprotein-associated cholesteryl ester by hepatic cells in culture. This effect is dependent on cell surface heparan sulfate proteoglycans but independent of lipolysis and of endocytosis mediated by low density lipoprotein receptor-related or low density lipoprotein receptors.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo

The factors controlling cationic liposome-DNA complex (CLDC)-based gene transfer in cells and in animals are poorly understood. We found that cell surface heparin/heparan sulfate-bearing proteoglycans mediate CLDC-based gene transfer and expression both in cultured cells and following intravenous gene delivery into animals. CLDC did not transfect Raji cells, which lack proteoglycans, but did efficiently transfect Raji cells stably transfected with the proteoglycan, syndecan-1. Fucoidan, heparin, or dextran sulfate, all of which are highly anionic polysaccharides, each blocked CLDC-mediated transfection both in cultured cells and following intravenous injection into mice, but had no effect on transfection by either recombinant adenovirus infection or electroporation. Intravenous pretreatment of mice with heparinases, which specifically cleave heparan sulfate molecules from cell surface proteoglycans, blocked intravenous, CLDC-mediated transfection in mice, confirming that proteoglycans mediate CLDC gene delivery in vivo. Modulation of proteoglycan expression may prove useful in controlling the efficiency of, as well as targeting the sites of, CLDC-based gene transfer in animals.     

In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.     

On the putative mechanistic basis for intraoperative propofol-induced penile erections

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection.     

The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae

Confocal and immunoelectron microscopic analysis of urethral exudates from 12 men with gonococcal urethritis showed that Neisseria gonorrhoeae can invade urethral epithelial cells. Studies with acridine orange stain demonstrated that the majority of organisms within urethral epithelial cells were viable at the time of fixation. Three-dimensional modeling of an infected epithelial cell using image analysis of 21 digitized confocal sections stained with YOYO-1 and DiIC 18(3) revealed that gonococcal invasion of these cells occurred in a polar fashion, most likely at the epithelial luminal surface. Serial immunoelectron micrographs showed evidence of membrane fusion with pedestal formation between the gonococcus and the epithelial cell, gonococci within vacuoles, and occasional gonococci free in the cytoplasm. Immunoelectron microscopy studies showed ruptured vacuoles at the cell surface releasing organisms. These studies demonstrate that urethral epithelial cells are invaded by gonococci during the course of infection in males.     

Multiple heparan sulfate chains are required for optimal syndecan-1 function

Syndecans have three highly conserved sites available for heparan sulfate attachment. To determine if all three sites are required for normal function, a series of mutated syndecans having two, one, or no heparan sulfate chains were expressed in ARH-77 cells. Previously, we demonstrated that expression of wild-type syndecan-1 on these myeloma cells mediates cell-matrix and cell-cell adhesion and inhibits cell invasion into collagen gels. Here we show that to optimally mediate each of these activities, all three sites of heparan sulfate attachment are required. Generally, an increasing loss of syndecan-1 function occurs as the number of heparan sulfate attachment sites decreases. This loss of function is not the result of a decrease in either the total amount of cell surface heparan sulfate or syndecan-1 core protein. In regard to cell invasion, cells expressing syndecan-1 bearing a single heparan sulfate attachment site exhibit a hierarchy of function based upon the position of the site within the core protein; the presence of an available attachment site at serine 47 confers the greatest level of activity, while serine 37 contributes little to syndecan-1 function. However, when all three heparan sulfate chains are present, significantly greater biological activity is observed than is predicted by the sum of the activities occurring when the chains act individually. This synergy provides a functional basis for the evolutionary conservation of the three heparan sulfate attachment sites on syndecans and supports the idea that molecular heterogeneity, which is characteristic of proteoglycans, contributes to their functional diversity.     

A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans

The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.     

Receptor-binding properties of a soluble form of human cytomegalovirus glycoprotein B

The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (Kd): Kd1, 4.96 x 10(-6) M; Kd2, 3.07 x 10(-7) M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (Kd1, 9.85 x 10(-6) M; Kd2, 4.03 x 10(-8) M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (Kd, 7.46 x 10(-6) M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.     

Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro

The malaria circumsporozoite (CS) protein binds to glycosaminoglycan chains from heparan sulfate proteoglycans present on the basolateral surface of hepatocytes and hepatoma cells in vitro. When injected into mice, CS protein is rapidly cleared from the blood circulation by hepatocytes. The binding region for the HSPGs is the evolutionarily conserved region II-plus of the CS protein. Here we have asked whether the presence of glycosaminoglycans on the plasma membrane of target cells is required for sporozoite invasion in vitro. Two types of target cells were used: HepG2 cells, which are permissive for Plasmodium berghei sporozoite development into mature exoerythrocytic forms, and CHO cells, in which the intracellular development of the parasites is arrested early after penetration. The invasion of mutant CHO cells expressing undersulfated glycosaminoglycans or no glycosaminoglycans was only inhibited 41-49% or 24-32%, respectively, in comparison to invasion of CHO-K1 cells. Previous cleavage of HepG2 surface membrane glycosaminoglycans with heparinase or heparitinase had no significant inhibitory effect on subsequent P. berghei sporozoite invasion and EEF development in these cells, although the glycosaminoglycan lyase treatments removed over 80% of CS binding sites from the cell surface. These results suggest that although the presence of glycosaminoglycans on the target cell surface enhances sporozoite invasion, glycosaminoglycans are not required for sporozoite penetration or the development of exoerythrocytic forms in vitro.     

Expression patterns of transmembrane and released forms of neuregulin during spinal cord and neuromuscular synapse development

We mapped the distribution of neuregulin and its transmembrane precursor in developing, embryonic chick and mouse spinal cord. Neuregulin mRNA and protein were expressed in motor and sensory neurons shortly after their birth and levels steadily increased during development. Expression of the neuregulin precursor was highest in motor and sensory neuron cell bodies and axons, while soluble, released neuregulin accumulated along early motor and sensory axons, radial glia, spinal axonal tracts and neuroepithelial cells through associations with heparan sulfate proteoglycans. Neuregulin accumulation in the synaptic basal lamina of neuromuscular junctions occurred significantly later, coincident with a reorganization of muscle extracellular matrix resulting in a relative concentration of heparan sulfate proteoglycans at endplates. These results demonstrate an early axonal presence of neuregulin and its transmembrane precursor at developing synapses and a role for heparan sulfate proteoglycans in regulating the temporal and spatial sites of soluble neuregulin accumulation during development.