Hugues Lortat-Jacob

Human Cytomegalovirus Binding to DC-SIGN Is Required for Dendritic Cell Infection and Target Cell trans-Infection

Cytomegalovirus (CMV) infection is characterized by host immunosuppression and multiorganic involvement. CMV-infected dendritic cells (DC) were recently shown to display reduced immune functions, but their role in virus dissemination is not clear. In this report, we demonstrated that CMV could be captured by DC through binding on DC-SIGN and subsequently transmitted to permissive cells. Moreover, blocking DC-SIGN by specific antibodies inhibited DC infection by primary CMV isolates and expression of DC-SIGN or its homolog DC-SIGNR rendered susceptible cells permissive to CMV infection. We demonstrated that CMV envelope glycoprotein B is a viral ligand for DC-SIGN and DC-SIGNR. These results provide new insights into the molecular interactions contributing to cell infection by CMV and extend DC-SIGN implication in virus propagation.

STROMAL CELL-DERIVED FACTOR-1ALPHA ASSOCIATES WITH HEPARAN SULFATES THROUGH THE FIRST BETA -STRAND OF THE CHEMOKINE

Biological properties of chemokines are believed to be influenced by their association with glycosaminoglycans. Surface plasmon resonance kinetic analysis shows that the CXC chemokine stromal cell-derived factor-1α (SDF-1α), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nm (k on = 2.16 × 106 m −1 s−1 andk off = 0.083 × s−1). A modified SDF-1α (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys24, His25, and Lys27 by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1α, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1α was unable to bind in a CXCR4-independent manner on epithelial cells that were treated with heparan sulfate (HS)-degrading enzymes or constitutively lack HS expression. The inability of SDF-1 3/6 to bind to cells underlines the importance of the identified basic cluster for the physiological interactions of SDF-1α with HS. Importantly, the amino-terminal domain of SDF-1α which is required for binding to, and activation of, CXCR4 remains exposed after binding to HS and is recognized by a neutralizing monoclonal antibody directed against the first residues of the chemokine. Overall, these findings indicate that the Lys24, His25, and Lys27 cluster of residues forms, or is an essential part of, the HS-binding site which is distinct from that required for binding to, and signaling through, CXCR4.

Selective Interactions of Polyanions with Basic Surfaces on Human Immunodeficiency Virus Type 1 gp120

ABSTRACT It is well established that the gp120 V3 loop of T-cell-line-adapted human immunodeficiency virus type 1 (HIV-1) binds both cell-associated and soluble polyanions. Virus infectivity is increased by interactions between HIV-1 and heparan sulfate proteoglycans on some cell types, and soluble polyanions such as heparin and dextran sulfate neutralize HIV-1 in vitro. However, the analysis of gp120-polyanion interactions has been limited to T-cell-line-adapted, CXCR4-using virus and virus-derived gp120, and the polyanion binding ability of gp120 regions other than the V3 loop has not been addressed. Here we demonstrate by monoclonal-antibody inhibition, labeled heparin binding, and surface plasmon resonance studies that a second site, most probably corresponding to the newly defined, highly conserved coreceptor binding region on gp120, forms part of the polyanion binding surface. Consistent with the binding of polyanions to the coreceptor binding surface, dextran sulfate interfered with the gp120-CXCR4 association while having no detectable effect on the gp120-CD4 interaction. The interaction between polyanions and X4 or R5X4 gp120 was readily detectable, whereas weak or undetectable binding was observed with R5 gp120. Analysis of mutated forms of X4 gp120 demonstrated that the V3 loop is the major determinant for polyanion binding whereas other regions, including the V1/V2 loop structure and the NH2 and COOH termini, exert a more subtle influence. A molecular model of the electrostatic potential of the conserved coreceptor binding region confirmed that it is basic but that the overall charge on this surface is dominated by the V3 loop. These results demonstrate a selective interaction of gp120 with polyanions and suggest that the conserved coreceptor binding surface may present a novel and conserved target for therapeutic intervention.

Structural diversity of heparan sulfate binding domains in chemokines

Heparan sulfate (HS) molecules are ubiquitous in animal tissues where they function as ligands that are dramatically involved in the regulation of the proteins they bind. Of these, chemokines are a family of small proteins with many biological functions. Their well-conserved monomeric structure can associate in various oligomeric forms especially in the presence of HS. Application of protein surface analysis and energy calculations to all known chemokine structures leads to the proposal that four different binding modes are created by the folding and oligomerization of these proteins. So, based on the present state of our knowledge, four different clusters of amino acids should be involved in the recognition process. Our results help to rationalize how unique sequences of HS specifically bind any given chemokine. The conclusions open the route for a rational design of compounds of therapeutical interest that could influence chemokine activity.

Characterization of the stromal cell-derived factor-1alpha-heparin complex.

The binding of chemokines to glycosaminoglycans is thought to play a crucial role in chemokine functions. It has recently been shown that stromal cell-derived factor-1α (SDF-1α), a CXC chemokine with potent anti-human immunodeficiency virus activity, binds to heparan sulfate through a typical consensus sequence for heparin recognition (BBXB, where B is a basic residue KHLK, amino acids 24–27). Calculation of the accessible surface, together with the electrostatic potential of the SDF-1α dimer, revealed that other amino acids (Arg-41 and Lys-43) are found in the same surface area and contribute to the creation of a positively charged crevice, located at the dimer interface. GRID calculations confirmed that this binding site will be the most energetically favored area for the interaction with sulfate groups. Site-directed mutagenesis and surface plasmon resonance-based binding assays were used to investigate the structural basis for SDF-1α binding to heparin. Among the residues clustered in this basic surface area, Lys-24 and Lys-27 have dominant roles and are essential for interaction with heparin. Amino acids Arg-41 and Lys-43 participate in the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in complex formation. Binding studies with selectively desulfated heparin, heparin oligosaccharides, and heparitinase-resistant heparan sulfate fragments showed that a minimum size of 12–14 monosaccharide units is required for efficient binding and that 2-O- andN-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1α, and a docking study was undertaken. During the energy minimization process, heparin lost its perfect ribbon shape and fitted the protein surface perfectly. In the model, Lys-1, Lys-24, Lys-27, and Arg-41 were found to have the major role in binding a polysaccharide fragment consisting of 13 monosaccharide units.

Heparan Sulfate/Heparin Oligosaccharides Protect Stromal Cell-derived Factor-1 (SDF-1)/CXCL12 against Proteolysis Induced by CD26/Dipeptidyl Peptidase IV

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that is constitutively expressed in most tissues and displayed on the cell surface in association with heparan sulfate (HS). Its numerous biological effects are mediated by a specific G protein-coupled receptor, CXCR4. A number of cells inactivate SDF-1 by specific processing of the N-terminal domain of the chemokine. In particular, CD26/dipeptidyl peptidase IV (DPP IV), a serine protease that co-distributes with CXCR4 at the cell surface, mediates the selective removal of the N-terminal dipeptide of SDF-1. We report here that heparin and HS specifically prevent the processing of SDF-1 by DPP IV expressed by Caco-2 cells. The level of processing increases with the level of differentiation of these cells, which correlates with an increase of DPP IV activity. A mutant SDF-1 that does not interact with HS is readily cleaved by DPP IV, a process that is not inhibited by HS, demonstrating that a productive interaction between HS and SDF-1 is required for the protection to take place. Moreover, we found that protection depends on the degree of polymerization of the HS sulfated S-domains. Finally a structural model of SDF-1, in complex with HS oligosaccharides of defined length, rationalizes the experimental data. The mechanisms by which HS regulates SDF-1 may thus include, in addition to its ability to locally concentrate the chemokine at the cell surface, a control of selective protease cleavage events that directly affect the chemokine activity.

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations.

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (KD ∼ 2 μm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg47 and Arg66, participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl2 to endostatin enhanced its binding to heparan sulfate by ∼40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.

Heparan Sulfate Targets the HIV-1 Envelope Glycoprotein gp120 Coreceptor Binding Site

Human immunodeficiency virus (HIV) attachment to host cells is a multi-step process that involves interaction of the viral envelope gp120 with the primary receptor CD4 and coreceptors. HIV gp120 also binds to other cell surface components, including heparan sulfate (HS), a sulfated polysaccharide whose wide interactive properties are exploited by many pathogens for attachment and concentration at the cell surface. To analyze the structural features of gp120 binding to HS, we used soluble CD4 to constrain gp120 in a specific conformation. We first found that CD4 induced conformational change of gp120, dramatically increasing binding to HS. We then showed that HS binding interface on gp120 comprised, in addition to the well characterized V3 loop, a CD4-induced epitope. This epitope is efficiently targeted by nanomolar concentrations of size-defined heparin/HS-derived oligosaccharides. Because this domain of the protein also constitutes the binding site for the viral coreceptors, these results support an implication of HS at late stages of the virus-cell attachment process and suggest potential therapeutic applications.

A synthetic CD4–heparan sulfate glycoconjugate inhibits CCR5 and CXCR4 HIV-1 attachment and entry

The HIV-1 envelope, gp120, which features the binding determinants for both CD4 and coreceptor recognition, is key for virus entry and represents an attractive pharmacological target. However, critical domains for entry (coreceptor and CD4 binding sites) are either cryptic or located in partially occluded cavities. Here we developed a chemical approach to synthesize a CD4-mimetic peptide linked to a heparan sulfate dodecasaccharide. This molecule binds to gp120, induces the exposure of the coreceptor binding domain and renders it available for interaction with the oligosaccharide. The linkage between the CD4 mimetic and the heparan sulfate derivative provides strong cooperative effects, resulting in low-nanomolar antiviral activity toward both CCR5- and CXCR4-tropic HIV-1 strains. This compound, which has the unique ability to simultaneously target two critical and highly conserved regions of gp120, establishes a new type of inhibitor and suggests a general concept for the inhibition of numerous other biological systems.

The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models.