Hepatitis C virus attachment mediated by apolipoprotein E binding to cell surface heparan sulfate

Viruses are known to use virally encoded envelope proteins for cell attachment, which is the very first step of virus infection. In the present study, we have obtained substantial evidence demonstrating that hepatitis C virus (HCV) uses the cellular protein apolipoprotein E (apoE) for its attachment to cells. An apoE-specific monoclonal antibody was able to efficiently block HCV attachment to the hepatoma cell line Huh-7.5 as well as primary human hepatocytes. After HCV bound to cells, however, anti-apoE antibody was unable to inhibit virus infection. Conversely, the HCV E2-specific monoclonal antibody CBH5 did not affect HCV attachment but potently inhibited HCV entry. Similarly, small interfering RNA-mediated knockdown of the key HCV receptor/coreceptor molecules CD81, claudin-1, low-density lipoprotein receptor (LDLr), occludin, and SR-BI did not affect HCV attachment but efficiently suppressed HCV infection, suggesting their important roles in HCV infection at postattachment steps. Strikingly, removal of heparan sulfate from the cell surface by treatment with heparinase blocked HCV attachment. Likewise, substitutions of the positively charged amino acids with neutral or negatively charged residues in the receptor-binding region of apoE resulted in a reduction of apoE-mediating HCV infection. More importantly, mutations of the arginine and lysine to alanine or glutamic acid in the receptor-binding region ablated the heparin-binding activity of apoE, as determined by an in vitro heparin pulldown assay. HCV attachment could also be inhibited by a synthetic peptide derived from the apoE receptor-binding region. Collectively, these findings demonstrate that apoE mediates HCV attachment through specific interactions with cell surface heparan sulfate.     

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.     

Syndecan-1 Serves as the Major Receptor for Attachment of Hepatitis C Virus to the Surfaces of Hepatocytes

Our recent studies demonstrated that apolipoprotein E mediates cell attachment of hepatitis C virus (HCV) through interactions with the cell surface heparan sulfate (HS). HS is known to covalently attach to core proteins to form heparan sulfate proteoglycans (HSPGs) on the cell surface. The HSPG core proteins include the membrane-spanning syndecans (SDCs), the lycosylphosphatidylinositol-linked glypicans (GPCs), the basement membrane proteoglycan perlecan (HSPG2), and agrin. In the present study, we have profiled each of the HSPG core proteins in HCV attachment. Substantial evidence derived from our studies demonstrates that SDC1 is the major receptor protein for HCV attachment. The knockdown of SDC1 expression by small interfering RNA (siRNA)-induced gene silence resulted in a significant reduction of HCV attachment to Huh-7.5 cells and stem cell-differentiated human hepatocytes. The silence of SDC2 expression also caused a modest decrease of HCV attachment. In contrast, the siRNA-mediated knockdown of other SDCs, GPCs, HSPG2, and agrin had no effect on HCV attachment. More importantly, ectopic expression of SDC1 was able to completely restore HCV attachment to Huh-7.5 cells in which the endogenous SDC1 expression was silenced by specific siRNAs. Interestingly, mouse SDC1 is also fully functional in mediating HCV attachment when expressed in the SDC1-deficient cells, consistent with recent reports that mouse hepatocytes are also susceptible to HCV infection when expressing other key HCV receptors. Collectively, our findings demonstrate that SDC1 serves as the major receptor protein for HCV attachment to cells, providing another potential target for discovery and development of antiviral drugs against HCV.     

Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate: PROPERTIES AND ROLE IN PCPE-1 INTERACTION WITH CELLS*

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.     

Productive Hepatitis C Virus Infection of Stem Cell-Derived Hepatocytes Reveals a Critical Transition to Viral Permissiveness during Differentiation

Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancer cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. Here we report a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. RNA interference directed toward essential cellular cofactors in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. The ability to infect cultured cells directly with HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host-pathogen interactions and cell therapy.     

Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.     

Cell surface heparan sulfate proteoglycans are involved in the binding of Hsp90α and Hsp90β to the cell plasma membrane

Extracellular membrane-bound and secreted heat shock protein 90 (Hsp90) is known to be involved in cell motility and invasion. The mechanism of Hsp90 anchoring to the plasma membrane remains obscure. We showed that treatment of human glioblastoma A-172 and fibrosarcoma HT1080 cells with sodium chlorate, heparinase, and heparin causes a prominent loss of 2 Hsp90 cytosolic isoforms, Hsp90α and Hsp90β, from the cell surface and strongly inhibits the binding of exogenous Hsp90 to cells. We revealed that Hsp90α and Hsp90β are partly colocalized with heparan sulfate proteoglycans (HSPGs) on the cell surface and that this colocalization was sensitive to heparin. The results demonstrate that cell surface HSPGs are involved in the binding/anchoring of Hsp90α and Hsp90β to the plasma membrane.     

Hepatitis C Virus (HCV) Envelope Glycoproteins E1 and E2 Contain Reduced Cysteine Residues Essential for Virus Entry

The HCV envelope glycoproteins E1 and E2 contain eight and 18 highly conserved cysteine residues, respectively. Here, we examined the oxidation state of E1E2 heterodimers incorporated into retroviral pseudotyped particles (HCVpp) and investigated the significance of free sulfhydryl groups in cell culture-derived HCV (HCVcc) and HCVpp entry. Alkylation of free sulfhydryl groups on HCVcc/pp with a membrane-impermeable sulfhydryl-alkylating reagent 4-(N-maleimido)benzyl-α-trimethylammonium iodide (M135) prior to virus attachment to cells abolished infectivity in a dose-dependent manner. Labeling of HCVpp envelope proteins with EZ-Link maleimide-PEG2-biotin (maleimide-biotin) detected free thiol groups in both E1 and E2. Unlike retroviruses that employ disulfide reduction to facilitate virus entry, the infectivity of alkylated HCVcc could not be rescued by addition of exogenous reducing agents. Furthermore, the infectivity of HCVcc bound to target cells was not affected by addition of M135 indicative of a change in glycoprotein oxidation state from reduced to oxidized following virus attachment to cells. By contrast, HCVpp entry was reduced by 61% when treated with M135 immediately following attachment to cells, suggesting that the two model systems might demonstrate variations in oxidation kinetics. Glycoprotein oxidation was not altered following binding of HCVpp incorporated E1E2 to soluble heparin or recombinant CD81. These results suggest that HCV entry is dependent on the presence of free thiol groups in E1 and E2 prior to cellular attachment and reveals a new essential component of the HCV entry process.     

Lipoprotein lipase mediates hepatitis C virus (HCV) cell entry and inhibits HCV infection

The host–virus interactions leading to cell infection with hepatitis C virus (HCV) are not fully understood. The tetraspanin CD-81 and human scavenger receptor SR-BI/Cla1 are major receptors mediating virus cell entry. However, HCV in patients' sera is associated with lipoproteins and infectious potential of the virus depends on lipoproteins associated to virus particles. We show here that lipoprotein lipase (LPL), targeting triglyceride-rich lipoproteins (TRL) to the liver, mediates binding and internalization of HCV to different types of cells, acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate proteoglycans (HSPG). The dimeric structure and catalytic activity of LPL are required for LPL-mediated HCV uptake to cells. Unexpectedly, exogenous LPL significantly inhibits HCVcc infection in vitro . This effect is prevented by anti-LPL antibodies and by tetrahydrolipstatin (THL) a specific inhibitor of LPL enzymatic activity. In addition, we show that antibodies directed to apolipoprotein B (ApoB)-containing lipoproteins efficiently inhibits HCVcc infection. Our findings suggest that LPL mediates HCV cell entry by a mechanism similar to hepatic clearance of TRL from the circulation, promoting a non-productive virus uptake. These data provide new insight into mechanisms of HCV cell entry and suggest that LPL could modulate HCV infectivity in vivo .     

Apolipoprotein E receptor binding versus heparan sulfate proteoglycan binding in its regulation of smooth muscle cell migration and proliferation

This study showed that synthetic peptides containing either a single copy or tandem repeat of the receptor binding domain sequence of apolipoprotein (apo) E, or a peptide containing its C-terminal heparin binding domain, apoE-(211-243), were all effective inhibitors of platelet-derived growth factor (PDGF)-stimulated smooth muscle cell proliferation. In contrast, only the peptide containing a tandem repeating unit of the receptor binding domain sequence of apoE, apoE-(141-155)(2), was capable of inhibiting PDGF-directed smooth muscle cell migration. Peptide containing only a single unit of this sequence, apoE-(141-155), or the apoE-(211-243) peptide were ineffective in inhibiting PDGF-directed smooth muscle cell migration. Additional experiments showed that reductively methylated apoE, which is incapable of receptor binding yet retains its heparin binding capability, was equally effective as apoE in inhibiting PDGF-stimulated smooth muscle cell proliferation. However, reductively methylated apoE was unable to inhibit smooth muscle cell migration toward PDGF. Additionally, the receptor binding domain-specific apoE antibody 1D7 also mitigated the anti-migratory properties of apoE on smooth muscle cells. Finally, pretreatment of cells with heparinase failed to abolish apoE inhibition of smooth muscle cell migration. Taken together, these data documented that apoE inhibition of PDGF-stimulated smooth muscle cell proliferation is mediated by its binding to heparan sulfate proteoglycans, while its inhibition of cell migration is mediated through apoE binding to cell surface receptors.     

Heparin increases the infectivity of Human Papillomavirus Type 16 independent of cell surface proteoglycans and induces L1 epitope exposure

Human Papillomaviruses (HPVs) are the etiological agents of cervical cancer, and HPV‐16 is the most prevalent type. Several HPVs require heparan sulfate proteoglycans (HSPGs) for cell binding. Here, we analyse the phenomenon that preincubation of HPV‐16 with increasing concentrations of heparin results in partial restoration rather than more efficient inhibition of infection. While corroborating that the HSPGs are cell‐binding receptors for HPV‐16, heparin‐preincubated virus bound to the extracellular matrix (ECM) via laminin‐332. Furthermore, the interaction of virions with heparin, a representative of the highly sulfated S‐domains of heparan sulfate (HS) chains of HSPGs, allowed HPV‐16 infection in the absence of cell surface HSPGs. Therefore, we concluded that specific glycan moieties but not specific HSPG protein backbones are required for infection. The increased binding of an epitope‐specific antibody to the viral capsid after heparin binding suggested that initial conformational changes in the HPV‐16 virion occur during infection by interaction with‘heparin‐like’ domains of cellular HSPGs. We propose that HS sequences with specific sulfation patterns are required to facilitate HPV‐16 infection.     

Optimal inhibition of X4 HIV isolates by the CXC chemokine stromal cell-derived factor 1 alpha requires interaction with cell surface heparan sulfate proteoglycans

The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1 alpha interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first beta-strand of SDF-1 alpha as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1 alpha to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1 alpha mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1 alpha when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1 alpha to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1 alpha and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1 alpha to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1 alpha by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor.     

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC₅₀ values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.     

Further evidence that papillomavirus capsids exist in two distinct conformations

Cell surface heparan sulfate proteoglycans (HSPGs) serve as primary attachment receptors for human papillomaviruses (HPVs). To demonstrate that a biologically functional HPV-receptor interaction is restricted to a specific subset of HSPGs, we first explored the role of HSPG glucosaminoglycan side chain modifications. We demonstrate that HSPG O sulfation is essential for HPV binding and infection, whereas de-N-sulfated heparin interfered with VLP binding but not with HPV pseudoinfection. This points to differences in VLP-HSPG and pseudovirion-HSPG interactions. Interestingly, internalization kinetics of VLPs and pseudovirions, as measured by fluorescence-activated cell sorting analysis, also differ significantly with approximate half times of 3.5 and 7.5 h, respectively. These data suggest that differences in HSPG binding significantly influence postbinding events. We also present evidence that pseudovirions undergo a conformational change after cell attachment. A monoclonal antibody (H33.J3), which displays negligible effectiveness in preattachment neutralization assays, efficiently neutralizes cell-bound virions. However, no difference in H33.J3 binding to pseudovirions and VLPs was observed in enzyme-linked immunosorbent assay and virus capture assays. In contrast to antibody H33.B6, which displays equal efficiencies in pre- and postattachment neutralization assays, H33.J3 does not block VLP binding to heparin, demonstrating that it interferes with steps subsequent to virus binding. Our data strongly suggest that H33.J3 recognizes a conformation-dependent epitope in capsid protein L1, which undergoes a structural change after cell attachment.     

Dynamic and stable pools of apoE differ functionally at the HepG2 cell surface

Synthesis of apolipoprotein (apo)E in hepatocytes leads to both secretion and retention in cell surface pools. Inclusion of Brefeldin A to HepG2 cells prompted a rapid decrease of cell surface apoE to about 37% of control values after a 3-h incubation. The t(1/2) for this dynamic pool was estimated to be 15 min. In contrast, a stable fraction of apoE (t(1/2) > 20 h) was found in association with the extracellular matrix (ECM). Increased content of apoE on the ECM correlated with decreased binding of VLDL. Decreased apoE on the cell surface correlated with increased binding of VLDL to cells. Collectively, this suggests that glycosaminoglycan-bound apoE can occlude binding sites for apoE-containing lipoproteins on glycosaminoglycans. In solid-phase assays, heparin, suramin, and chondroitin sulfates A and B efficiently inhibited the binding of apoE to heparan sulfate proteoglycans, but were unable to displace apoE from this glycosaminoglycan. Finally, decreasing cell surface apoE with suramin subsequently decreased the apoE content on secreted apoB-containing lipoproteins without affecting the overall secretion of apoE or apoB to the extracellular medium.In summary, cell surface apoE comprises both dynamic fractions, which can be donated to newly secreted lipoproteins, and stable fractions, which may act to minimize the unproductive binding of lipoproteins to the ECM.     

TnBP?Triton X-45 Treatment of Plasma for Transfusion Efficiently Inactivates Hepatitis C Virus

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products.     

Interaction between mouse adenovirus type 1 and cell surface heparan sulfate proteoglycans

Application of human adenovirus type 5 (Ad5) derived vectors for cancer gene therapy has been limited by the poor cell surface expression, on some tumor cell types, of the primary Ad5 receptor, the coxsackie-adenovirus-receptor (CAR), as well as the accumulation of Ad5 in the liver following interaction with blood coagulation factor X (FX) and subsequent tethering of the FX-Ad5 complex to heparan sulfate proteoglycan (HSPG) on liver cells. As an alternative vector, mouse adenovirus type 1 (MAV-1) is particularly attractive, since this non-human adenovirus displays pronounced endothelial cell tropism and does not use CAR as a cellular attachment receptor. We here demonstrate that MAV-1 uses cell surface heparan sulfate proteoglycans (HSPGs) as primary cellular attachment receptor. Direct binding of MAV-1 to heparan sulfate-coated plates proved to be markedly more efficient compared to that of Ad5. Experiments with modified heparins revealed that the interaction of MAV-1 to HSPGs depends on their N-sulfation and, to a lesser extent, 6-O-sulfation rate. Whereas the interaction between Ad5 and HSPGs was enhanced by FX, this was not the case for MAV-1. A slot blot assay demonstrated the ability of MAV-1 to directly interact with FX, although the amount of FX complexed to MAV-1 was much lower than observed for Ad5. Analysis of the binding of MAV-1 and Ad5 to the NCI-60 panel of different human tumor cell lines revealed the preference of MAV-1 for ovarian carcinoma cells. Together, the data presented here enlarge our insight into the HSPG receptor usage of MAV-1 and support the development of an MAV-1-derived gene vector for human cancer therapy.     

Characterization of the recognition of tumor cells by the natural cytotoxicity receptor, NKp44

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.     

LTBP-2 has multiple heparin/heparan sulfate binding sites

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of poorly understood function associated with fibrillin-1-containing microfibrils during elastinogenesis. In this study we investigated the molecular interactions of LTBP-2 with heparin and heparan sulfate proteoglycans (HSPGs) since unidentified cell surface HSPGs are critical for normal fiber assembly. In solid phase assays, heparin conjugated to albumin (HAC) bound strongly to recombinant full-length human LTBP-2. This interaction was completely blocked by addition of excess heparin, but not chondroitin sulfate, confirming specificity. Analysis of binding to LTBP-2 fragments showed that HAC bound strongly to N-terminal fragment LTBP-2 NT(H) and more weakly to central fragment LTBP-2 C(H). No binding was detected to C-terminal fragment LTBP-2 CT(H). Kds for heparin binding were calculated for full-length LTBP-2, LTBP-2 NT(H) and LTBP-2 C(H) as 0.9 nM, 0.7 nM and 80 nM respectively. HAC interaction with fragment LTBP-2 NT(H) was not sensitive to EDTA or EGTA indicating that binding had no requirement for Ca²⁺ ions whereas HAC binding to fragment LTBP-2 C(H) was markedly reduced by these chelating agents indicating a degree of Ca²⁺ dependence. Inhibition studies with synthetic peptides identified three major heparin binding sequences in fragment LTBP-2 NT(H), including sequence LTEKIKKIKIV in the first large cysteine-free domain of LTBP-2, adjacent to the previously identified fibulin-5 binding site. LTBP-2 was found to interact strongly in a heparin-inhibitable manner with cell surface HSPG syndecan-4, but showed no interaction with recombinant syndecan-2. LTBP-2 also showed strong interaction with the heparan sulfate chains of basement membrane HSPG, perlecan. The potential importance of HSPG–LTBP-2 interactions in elastic fiber assembly and microfibril attachment to basement membranes is discussed.     

Membrane anchoring of heparan sulfate proteoglycans by phosphatidylinositol and kinetics of synthesis of peripheral and detergent-solubilized proteoglycans in Schwann cells

Previous studies have shown that Schwann cells synthesize both peripheral and integral hydrophobic cell surface heparan sulfate proteoglycans (HSPGs). The experiments reported here were undertaken to investigate the mode of attachment of these proteins to the cell surface and their potential interrelationship. The binding of the hydrophobic HSPGs to membranes appears to be via covalently linked phosphatidylinositol based on the observation that incubation of the detergent-solubilized protein with purified phosphatidylinositol-specific phospholipase C significantly reduces the ability of the HSPGs to associate with phospholipid vesicles in a reconstitution assay. The peripherally associated HSPGs were released from the cells by incubation in the presence of heparin (10 mg/ml), 10 mM phytic acid (inositol hexaphosphate), or 2 M NaCl. These treatments also solubilized basement membrane HSPGs synthesized by the Schwann cells. These data suggest that the peripheral HSPGs are bound to the surface by electrostatic interactions. The peripheral and hydrophobic HSPGs were identical in overall size, net charge, length of glycosaminoglycan chains, and patterns of N-sulfation. To determine whether the peripheral HSPGs were derived from the membrane-bound form by cleavage of the membrane anchor, we examined the kinetics of synthesis and degradation of the two forms of HSPGs. The results obtained indicated the existence of two pools of detergent-solubilized HSPG with fast (t1/2 = 6 h) and slow (t1/2 = 55 h) turnover kinetics. The data were consistent with a model in which the peripheral HSPGs were derived from the slowly turning over pool of detergent-solubilized HSPGs.