Heparan sulfate is an attachment factor for foamy virus entry

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.     

Heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5

Cell infection by adenovirus serotypes 2 and 5 (Ad2/5) initiates with the attachment of Ad fiber to the coxsackievirus and Ad receptor (CAR) followed by alpha(v) integrin-mediated entry. We recently demonstrated that heparan sulfate glycosaminoglycans (HS GAGs) expressed on cell surfaces are involved in the binding and infection of Ad2/5 (M. C. Dechecchi, A. Tamanini, A. Bonizzato, and G. Cabrini, Virology 268:382-390, 2000). The role of HS GAGs was investigated using extracellular soluble domain 1 of CAR (sCAR-D1) and heparin as soluble receptor analogues of CAR and HS GAGs in A549 and recombinant CHO cell lines with differential levels of expression of the two receptors and cultured to various densities. Complete inhibition of binding and infection was obtained by preincubating Ad2/5 with both heparin (10 microg/ml) and sCAR-D1 (200 microg/ml) in A549 cells. Partial inhibition was observed when heparin and sCAR-D1 were preincubated separately with Ad. The level of heparin-sensitive [(3)H]Ad2/5 binding doubled in sparse A549 cells (50 to 70,000 cells/cm(2)) with respect to that of cells grown to confluence (200 to 300,000 cells/cm(2)), in parallel with increased expression of HS GAGs. [(3)H]Ad2 bound to sparse CAR-negative CHO cells expressing HS GAGs (CHO K1). No [(3)H]Ad2 binding was observed in CHO K1 cells upon competitive inhibition with heparin and in HS GAG-defective CHO A745, D677, and E606 clones. HS-sensitive Ad2 infection was obtained in CAR-negative sparse CHO K1 cells but not in CHO A745 cells, which were permissive to infection only upon transfection with CAR. These results demonstrate that HS GAGs are sufficient to mediate the initial binding of Ad2/5.     

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Mechanisms underlying preferential assembly of heparan sulfate on glypican-1

Glypicans are major cell surface heparan sulfate proteoglycans, the structures of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylphosphatidylinositol membrane anchor. Despite strong evolutionary conservation of the globular domains of glypicans, no function has yet been attributed to them. By using a novel quantitative approach for assessing proteoglycan glycosylation, we show here that removal of the globular domain from rat glypican-1 converts the proteoglycan from one that bears approximately 90% heparan sulfate (HS) to one that bears approximately 90% chondroitin sulfate. Mutational analysis shows that sequences at least 70 amino acids away from the glypican-1 GAG attachment site are required for preferential HS assembly, although more nearby sequences also play a role. The effects of the glypican-1 globular domain on HS assembly could also be demonstrated by fusing this domain to sequences representing the GAG attachment sites of other proteoglycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed glypican-1 GAG attachment domain or on endogenous proteoglycans. Quantitative analysis of the effect of the globular domain on GAG addition to proteoglycan core proteins suggested that preferential HS assembly is achieved, at least in part, through the inhibition of chondroitin sulfate assembly. These data identify the glypican-1 globular domain as a structural motif that potently influences GAG class determination and suggest that an important role of glypican globular domains is to ensure a high level of HS substitution of these proteoglycans.     

Dromedary glycosaminoglycans: molecular characterization of camel lung and liver heparan sulfate

Glycosaminoglycans (GAGs) are the portion of a proteoglycan that determine its final shape and function. The molecular structure of predominant GAG species in camel liver and lung is reported for the first time. The one-humped camel survives in an extreme, arid habitat and, thus, offers a good model to study the role of glycomics on homeostasis. Heparan sulfate (HS) from the lung and liver of the one-humped camel were isolated. Characterization of these newly isolated glycosaminoglycans included (1)H NMR spectroscopy and disaccharide compositional analysis. The relative molecular weight of these GAGs was estimated by gradient polyacrylamide gel electrophoresis and their degree of sulfation was also assessed. Anticoagulant activity was determined using an anti-factor Xa assay and the HS from camel lung shows approximately 50% of heparin's activity. The structural differences of camel liver GAGs compared to human and porcine liver heparin and HS is discussed. Camel lung heparan sulfate resembles both heparin and HS in its structure and properties suggesting that it is either a highly sulfated form of HS, a mixture of heparin and HS or an undersulfated heparin.     

Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells

Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chloride (NH₄Cl), chloroquine diphosphate (CQ), and monensin were used to inhibit endosome-lysosome system acidification. NH₄Cl, CQ, or monensin increased PCV2 (Stoon-1010) infection by 726% ± 110%, 1,212% ± 34%, and 1,100% ± 179%, respectively, in porcine kidney (PK-15) cells; by 128% ± 7%, 158% ± 3%, and 142% ± 11% in swine kidney cells; by 160% ± 28%, 446% ± 50%, and 162% ± 56% in swine testicle (ST) cells; and by 313% ± 25%, 611% ± 86%, and 352% ± 44% in primary kidney epithelial cells. Similarly, increased PCV2 infection was observed with six other PCV2 strains in PK-15 cells treated with endosome-lysosome system acidification inhibitors. The mechanism behind increased PCV2 infection was further investigated in PK-15 cells using CQ. PCV2 infection of PK-15 cells was increased only when CQ was added early during PCV2 infection. CQ did not affect PCV2 virus-like particle (VLP) attachment to PK-15 cells but increased the disassembly of internalized PCV2 VLPs. In untreated PK-15 cells, internalized PCV2 VLPs localized within the endosome-lysosome system. PCV2 infection of untreated 3D4/31 and PK-15 cells and CQ-treated PK-15 cells was blocked by a serine protease inhibitor [4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride] but not by aspartyl protease (pepstatin A), cysteine protease (E-64), and metalloprotease (phosphoramidon) inhibitors. These results suggest that serine protease-mediated PCV2 disassembly is enhanced in porcine epithelial cells but inhibited in monocytic cells after inhibition of endosome-lysosome system acidification.     

The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate

Human respiratory syncytial virus (RSV) F glycoprotein (RSV-F) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. RSV-glycosaminoglycan (GAG) interactions were evaluated using heparin-agarose affinity chromatography. RSV-F from A2- and B1/cp-52 (cp-52)-infected cell lysates, RSV-F derived from a recombinant vaccinia virus, and affinity-purified F protein all bound to and were specifically eluted from heparin columns. In infectivity inhibition studies, soluble GAGs decreased the infectivity of RSV A2 and cp-52, with bovine lung heparin exhibiting the highest specific activity against both A2 (50% effective dose [ED(50)] = 0.28 +/- 0.11 microg/ml) and cp-52 (ED(50) = 0.55 +/- 0. 14 microg/ml). Furthermore, enzymatic digestion of cell surface GAGs by heparin lyase I and heparin lyase III but not chondroitinase ABC resulted in a significant reduction in cp-52 infectivity. Moreover, bovine lung heparin inhibited radiolabeled A2 and cp-52 virus binding up to 90%. Taken together, these data suggest that RSV-F independently interacts with heparin/heparan sulfate and this type of interaction facilitates virus attachment and infectivity.     

Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.     

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.     

Presence of heparan sulfate proteoglycan in thyroid tissue

Glycosaminoglycan (GAG) was extracted from the porcine thyroid gland with a buffer containing 5.3 M guanidine-HCl and proteolytic enzyme inhibitors and was fractionated by subsequent isodensity CsCl centrifugation. 60% of uronic acid positive materials was accumulated in the bottom one-fourth fraction with high buoyant density. More than 90% of this uronic acid positive material in the thyroid tissue was heparin or heparan sulfate (sensitive to nitrous acid treatment) and the rest was chondroitin sulfate or dermatan sulfate (sensitive to chondroitinase ABC treatment). When the accumulated high buoyant density GAG was analyzed on a Sepharose CL-6-B column, approximately 14% of the heparin sulfate were in the macromolecular portion as a form of proteoglycan because it was destroyed by the papain digestion or alkaline borohydride treatment which extensively digests protein or releases GAG from protein by the elimination reaction, respectively. This study demonstrates the existence of heparin sulfate proteoglycan in thyroid tissue for the first time.     

Role of the N- and C-terminal domains in binding of apolipoprotein E isoforms to heparan sulfate and dermatan sulfate: a surface plasmon resonance study

The ability of apolipoprotein E (apoE) to bind to cell-surface glycosaminoglycans (GAGs) is important for lipoprotein remnant catabolism. Using surface plasmon resonance, we previously showed that the binding of apoE to heparin is a two-step process; the initial binding involves fast electrostatic interaction, followed by a slower hydrophobic interaction. Here we examined the contributions of the N- and C-terminal domains to each step of the binding of apoE isoforms to heparan sulfate (HS) and dermatan sulfate (DS). ApoE3 bound to less sulfated HS and DS with a decreased favorable free energy of binding in the first step compared to heparin, indicating that the degree of sulfation has a major effect on the electrostatic interaction of GAGs with apoE. Mutation of a key Lys residue in the N-terminal heparin binding site of apoE significantly affected this electrostatic interaction. Progressive truncation of the C-terminal alpha-helical regions which favors the monomeric form of apoE3 greatly weakened the ability of apoE3 to bind to HS, with a much reduced favorable free energy of binding of the first step, suggesting that the C-terminal domain contributes to the GAG binding of apoE by the oligomerization effect. In agreement with this, dimerization of the apoE3 N-terminal fragment via disulfide linkage restored the electrostatic interaction of apoE with HS. Significantly, apoE4 exhibited much stronger binding to HS and DS than apoE2 or apoE3 in both lipid-free and lipidated states, perhaps resulting from enhanced electrostatic interaction through the N-terminal domain. This isoform difference in GAG binding of apoE may be physiologically significant such as in the retention of apoE-containing lipoproteins in the arterial wall.     

Autoantibodies from patients with celiac disease inhibit transglutaminase 2 binding to heparin/heparan sulfate and interfere with intestinal epithelial cell adhesion

Autoantibodies from patients with celiac disease (CD) can influence transglutaminase 2 (TG2) activity and its cellular functions, but the exact mechanisms have remained unknown. Our objective was to study whether autoantibodies could modulate TG2 binding to heparin/heparan sulfate (HS) and intestinal epithelial cell attachment to fibronectin-TG2 matrix. Anti-TG2 antibodies were purified by TG2 affinity chromatography from sera of patients with active CD. Serum and antibody effects on TG2 binding to heparin/HS, on transamidase activity of TG2, as well as on Caco-2 cell attachment to fibronectin-TG2 matrix were assessed using microplate assays. Both sera and purified anti-TG2 antibodies from CD patients with high anti-TG2 IgA levels reduced TG2 binding to heparin/HS as compared with those with low anti-TG2 IgA or controls. There was a negative correlation between anti-TG2 IgA levels and TG2 binding to heparin/HS. Treatment of fibronectin-TG2 coated wells with CD patients' sera or purified anti-TG2 antibodies reduced attachment of Caco-2 cells onto the plate as compared with the control samples. The effect of CD patients' antibodies on Caco-2 cell attachment to fibronectin-TG2 matrix occurred independently of the inhibition of cell adhesion by Arg-Gly-Asp sequence containing peptides. Anti-TG2 autoantibodies had no effect on transamidase activity of TG2 in vitro. We suggest that modulation of adhesion function of TG2 by autoantibodies from patients with CD could be related to the inhibition of TG2 binding to HS residues of cell surface proteoglycans and could have possible implications for CD pathogenesis.     

Inhibition of transfer to secondary receptors by heparan sulfate-binding drug or antibody induces noninfectious uptake of human papillomavirus

Infection with various human papillomaviruses (HPVs) induces cervical cancers. Cell surface heparan sulfates (HS) have been shown to serve as primary attachment receptors, and molecules with structural similarity to cell surface HS, like heparin, function as competitive inhibitors of HPV infection. Here we demonstrate that the N,N'-bisheteryl derivative of dispirotripiperazine, DSTP27, efficiently blocks papillomavirus infection by binding to HS moieties, with 50% inhibitory doses of up to 0.4 mug/ml. In contrast to short-term inhibitory effects of heparin, pretreatment of cells with DSTP27 significantly reduced HPV infection for more than 30 h. Using DSTP27 and heparinase, we furthermore demonstrate that HS moieties, rather than laminin 5, present in the extracellular matrix (ECM) secreted by keratinocytes are essential for infectious transfer of ECM-bound virions to cells. Prior binding to ECM components, especially HS, partially alleviated the requirement for cell surface HS. DSTP27 blocks infection by cell-bound virions by feeding into a noninfectious entry pathway. Under these conditions, virus colocalized with HS moieties in endocytic vesicles. Similarly, postattachment treatment of cells with heparinase, cytochalasin D, or neutralizing antibodies resulted in uptake of virions without infection, indicating that deviation into a noninfectious entry pathway is a major mode of postattachment neutralization. In untreated cells, initial colocalization of virions with HS on the cell surface and in endocytic vesicles was lost with time. Our data suggest that initial attachment of HPV to HS proteoglycans (HSPGs) must be followed by secondary interaction with additional HS side chains and transfer to a non-HSPG receptor for successful infection.     

Primary attachment of murine leukaemia virus vector mediated by particle-associated heparan sulfate proteoglycan

Target cell entry of murine leukaemia virus vectors proceeds via primary attachment, independent of the viral envelope protein and subsequent envelope-receptor interaction. Although much attention has been paid to modifying the latter for target cell specificity, the initial binding interaction has been overlooked, despite its opposing involvement both in providing the virus available for receptor binding and in depleting free virus. As a first step towards modifying primary attachment, both to provide specificity and to enhance vector availability, we sought to determine the nature of this interaction. Following an initial screen of GAGs (glycosaminoglycans) for their ability to inhibit virus binding and transduction, we have shown that production of virus from cells in which GAG sulfation is inhibited, or treatment of virus with heparinase III, reduces both particle attachment and infection. Detection in purified virus preparations of a neo-epitope generated by heparinase III confirmed the presence of virus-associated HSPG [HS (heparan sulfate) proteoglycan], acquired from the producer cell. We propose that host-acquired cell-surface HSPG (potentially including syndecan-2) provides a means of virus attachment to target cells that precedes specific receptor interaction and membrane fusion. Inhibition of HS biosynthesis may provide a sufficiently reduced background of primary binding such that novel mechanisms of attachment, ideally with appropriate target cell specificity, can be introduced.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate. Implications for dynamic assembly of a ternary signaling complex

Heparan sulfate (HS) regulates the kinetics of fibroblast growth factor 2 (FGF2)-stimulated intracellular signaling and differentially activates cell proliferation of cells expressing different FGF receptors (FGFRs). Evidence suggests that HS interacts with both FGFs and FGFRs to form active ternary signaling complexes. Here we compare the interactions of two FGFRs with HS. We show that the ectodomains of FGFR1 IIIc and FGFR2 IIIc exhibit specific interactions with different characteristics for both heparin and porcine mucosal HS. These glycans are both known to activate FGF signaling via these receptors. FGFR2 interacts with a higher apparent affinity than FGFR1 despite both involving 6-O-, 2-O-, and N-sulfates. FGFR1 and FGFR2 bind heparin with mean association rate constants of 1.9 x 10(5) and 2.1 x 10(6) m(-1)s(-1), respectively, and dissociation rate constants of 1.2 x 10(-2) and 2.7 x 10(-2) s(-1), respectively. These produced calculated affinities of 63 and 13 nm, respectively. Hence, FGFR1 and FGFR2 bind to heparin chains with markedly different kinetics and affinities. We propose a mechanistic model where the kinetic parameters of the HS/FGFR interaction are a key element regulating the formation of ternary complexes and the resulting FGF signaling outcomes.     

Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.     

Passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein E(rns)

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E(rns) and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E(rns) purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E(rns) with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E(rns) genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E(rns) (amino acid 476 in the polyprotein of CSFV). Replacement of the E(rns) gene of an infectious DNA copy of C1.1.1 with the E(rns) genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E(rns) receptor.     

A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein: significance for malarial sporozoite attachment to hepatocytes

Circumsporozoite protein (CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate (HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site(s) of CSP. We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity. Only one of these peptides (Pf 2), which contains a motif we have named region I-plus, demonstrated both high affinity heparin/HS binding activity and the ability to block the binding of recombinant CSP to heparin-Sepharose 4B. Analysis by isothermal titration calorimetry revealed that region I-plus has a binding constant of K(d) = 5.0 microm and a stoichiometry of n = 7.8 binding sites/heparin chain. Heparin binding was dependent on the amino acid sequence of region I-plus, and the binding sites on heparin/HS are contained within a decasaccharide. Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content (heparin-like) are required for efficient binding. Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I-plus/HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes. Finally, the region I-plus heparin/HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N-terminal portion of CSP.