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-1␣ (SDF-1␣), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nM (k on ‫؍‬ 2.16 ؋ 10 6 M ؊1 s ؊1 and k off ‫؍‬ 0.083 ؋ s ؊1 ). A modified SDF-1␣ (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys 24 , His 25 , and Lys 27 by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1␣, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1␣ 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-1␣ with HS. Importantly, the amino-terminal domain of SDF-1␣ 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 Lys 24 , His 25 , and Lys 27 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.

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.

The hepatitis C virus (HCV) genome codes for highly mannosylated envelope proteins, which are naturally retained in the endoplasmic reticulum. We found that the HCV envelope glycoprotein E2 binds the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the related liver endothelial cell lectin L-SIGN through high-mannose N-glycans. Hepatitis C virus (HCV)1 is the major causative agent of non-A, non-B hepatitis throughout the world with more than 170 million people infected (1). Contamination with infected blood by injecting drug users is the primary risk factor for acquiring HCV infection. The majority of infected patients are unable to clear the virus, and many develop chronic liver disease, cirrhosis, and hepatocellular carcinoma (2). Replication of the HCV genome could be demonstrated in vivo and in vitro in liver hepatocytes (3, 4) and hematopoietic cells including dendritic cells and B cells (5, 6). However, the molecular mechanism by which the virus targets to these sites of replication, notably in the liver, is not known.HCV is a small, enveloped, plus-strand RNA virus belonging to the family flaviviridae and genus hepacivirus. The HCV RNA genome is 9600 nucleotides in length and encodes a single polyprotein that is post-translationally cleaved into up to 10 polypeptides including three structural proteins (core, E1, and E2), located at the N terminus, and five nonstructural proteins (1,7,8). Shortly after translocation into the endoplasmic reticulum (ER), oligosaccharide transferase catalyzes addition of Glc3Man9GlcNAc2 complexes at up to 6 (E1) and 11 (E2) N-glycosylation sites (for review see Ref. 9). Glucose residues are removed by glucosidases I and II, and correctly folded proteins are released from ER chaperones calnexin and calreticulin (10 -13). The transmembrane domains of E1 and E2 are responsible for both heterodimerization (14) and retention of the glycoproteins in a high-mannose EndoH-sensitive glycoform in the ER (15)(16)(17). By analogy to other flaviviruses it is assumed that HCV capsids bud from the cytoplasm into the ER and that enveloped particles follow the secretion pathway through the Golgi. However, attempts to produce secreted HCV particles in vitro have not been successful so far (18 -20), and it is not known if E1 and E2 on mature infectious virions possess a high-mannose, complex, or mixed glycosylation.Several receptors have been proposed that could play a role in HCV entry into hepatocytes. The low density lipoprotein (LDL) receptor has been shown to mediate HCV internalization via binding to virus-associated LDL particles (21,22). A second putative HCV receptor, the tetraspanin CD81, has been identified as a high affinity binding receptor (1.8 nM) for soluble recombinant E2 from HCV genotype 1a (23, 24). CD81 and LDL receptor are expressed in most cell types and thus likely do not account for the hepatic tropism of the virus. Furthermore E2 binds to the hepatoblastoma cell line HepG2, which does not express CD81 (25). More recently tw...

No 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 NH 2 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.

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-1␣ (SDF-1␣), 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-1␣ 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-1␣ 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-and N-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1␣, 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.Chemokines are small structurally related chemo-attractant cytokines, characterized by conserved cysteine residues. Almost 40 chemokines have been identified to date which, based on the position of the first N-terminal cysteines, fall into four sub-families. Two of them have been well characterized, the CC group, which includes regulated on activation, normal T-cell expressed, and secreted, monocyte chemoattractant protein-1, and MIP-1 1 (macrophage inflammatory peptides-1), and the CXC group, the prototype of which is interleukin-8. The C chemokine (lymphotactine) and the CX 3 C chemokine (fractalkine) sub-families have been identified more recently (1-4). These proteins signal through G-protein-coupled seven transmembrane domain receptors (5) and are primarily involved in immunosurveillance, activation, and recruitment of specific cell populations during disease (1, 6 -8). Most, if not all, chemokin...

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. C hemokines, derived from chemoattractant cytokine and now comprising more than 50 members, represent a recently identified family of small proteins (8 to 12 kDa in their monomeric form). Depending on the structure of a conserved cysteine-containing motif in the amino-terminal region of the molecule, four subgroups have been characterized and named C, CC, CXC, or CX 3 C according to the number and spacing of these cysteine residues. Based on the physiological features of these proteins, chemokines also have been classified as inflammatory (or inducible) or homeostatic (or constitutive) (1, 2).Although the chemotactic effect on leukocytes represents an important activity of the chemokines, it appears that these proteins also control a range of other functions that extend well beyond the regulation of leukocyte migration, including development, angiogenesis, neuronal patterning, hematopoiesis, viral infection, wound healing, and metastasis. The chemokine receptors, 20 of which have been identified, and through which these effects are transduced, are G protein-coupled, seven-helix transmembrane receptors (2). The observation that most chemokines bind to several different receptors and that most chemokine receptors exhibit overlapping specificity raises the question of the specificity of this system. Glycosaminoglycans (GAGs) are linear polysaccharides present on all animal cell surfaces and in the extracellular matrix, where they are usually found to be attached covalently to core proteins to form the proteoglycan family (3). Each tissue produces specific repertoires of GAGs (4), some of which are known to bind and regulate chemokine activity (5, 6). Several lines of evidence point to the importance of one particular GAG, heparan sulfate (HS), in promoting chemokine activity. First, in vitro, almost all chemokines studied to date appear to bind HS, suggesting that this represents a fundamental aspect of these proteins. Second, the finding that, in vivo, T lymphocytes secrete CC chemokine as a complex with proteoglycans indicates that this form is physiologically relevant (7)....

It was previously reported that treatment with the sulfated polysaccharide fucoidan or the structurally similar dextran sulfate increased circulating mature white blood cells and hematopoietic progenitor/stem cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that the SDF-1 increase is due to its competitive displacement from heparan sulfate proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other tissues. Although moderately increased levels of interleukin-8, MCP1, or MMP9 were also present after fucoidan treatment, studies in gene-ablated mice (GCSFR ؊/؊ , MCP1 ؊/؊ , or MMP9 ؊/؊ ) and the use of metalloprotease inhibitors do not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan-mobilizing treatments and not with several other mobilizing agents tested, are likely responsible. To the authors' knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and peripheral blood through a physiologically relevant mechanism, resulting in mobilization with kinetics similar to other mobilizing CXC chemokines. The study further underscores the importance of the biological roles of carbohydrates. IntroductionStromal-derived factor 1 (SDF-1) is a highly potent chemoattractant both in vitro and in vivo for mature leukocytes and hematopoietic progenitor/stem cells (HPCs), which carry its receptor CXCR4. [1][2][3][4][5][6][7] This highly conserved chemokine is constitutively expressed by virtually all tissues, 8 including bone marrow (BM). 3 It is expressed as 2 alternatively spliced isoforms, the predominant ␣ form and the ␤ form containing 4 additional amino acids at the C terminus, each possessing a heparin-binding domain. 9,10 The SDF-1-CXCR4 interaction plays a dominant role in hematopoiesis, and mice deficient in either gene die in utero exhibiting defects in B-cell lymphopoiesis and BM myelopoiesis. 4,5 Additionally, a critical role for CXCR4 on human cells in engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice 6,7 has been shown. Although involvement of SDF-1 in mobilization-the egress of HPCs from the BM to the peripheral blood (PB)-has also been speculated, direct evidence has only recently been obtained in mice using a synthetic SDF-1 analog 11 or following injection with an adenovirus expressing human SDF-1. 12 Previously, we reported that the sulfated polysaccharide fucoidan (FucS) and the structurally similar dextran sulfate (DexS) can elevate circulating white blood cells (WBCs) and mobilize HPCs within hours in a selectin-independent manner in mice and nonhuman primates. 13...