HS-binding Site Research Articles

Heparan sulfate promotes TRAIL-induced tumor cell apoptosis.

TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through TRAIL receptors. While it has been previously pursued as a potential anti-tumor therapy, the enthusiasm subsided due to unsuccessful clinical trials and the fact that many tumors are resistant to TRAIL. In this report, we identified heparan sulfate (HS) as an important regulator of TRAIL-induced apoptosis. TRAIL binds HS with high affinity (KD = 73 nM) and HS induces TRAIL to form higher-order oligomers. The HS-binding site of TRAIL is located at the N-terminus of soluble TRAIL, which includes three basic residues. Binding to cell surface HS plays an essential role in promoting the apoptotic activity of TRAIL in both breast cancer and myeloma cells, and this promoting effect can be blocked by heparin, which is commonly administered to cancer patients. We also quantified HS content in several lines of myeloma cells and found that the cell line showing the most resistance to TRAIL has the least expression of HS, which suggests that HS expression in tumor cells could play a role in regulating sensitivity towards TRAIL. We also discovered that death receptor 5 (DR5), TRAIL, and HS can form a ternary complex and that cell surface HS plays an active role in promoting TRAIL-induced cellular internalization of DR5. Combined, our study suggests that TRAIL-HS interactions could play multiple roles in regulating the apoptotic potency of TRAIL and might be an important point of consideration when designing future TRAIL-based anti-tumor therapy.

Targeting the heparan sulfate-binding site of RAGE with monoclonal antibodies.

Heparan sulfate (HS) plays its biological functions by interacting with hundreds of secreted extracellular and transmembrane proteins. Interaction with HS has been shown to be required for the normal function of many HS-binding proteins. Receptor for advanced glycation end-product (RAGE) is such a protein, whose activation requires HS-induced oligomerization. Using RAGE as an exemplary protein, we show here the workflow of a simple method of developing and characterizing mAbs that targets the HS-binding site. We found that HS-binding site of RAGE is quite immunogenic as 18 out of 94 anti-RAGE mAbs target various epitopes within the HS-binding site. Sequence analysis found that a common feature of anti-HS-binding site mAbs is the presence of abundant acidic residues (range between 6 to 11) in the complementarity determining region, suggesting electrostatic interaction plays an important role in promoting antigen-antibody interaction. Interestingly, mAbs targeting different epitopes within the HS-binding site blocks HS-RAGE interaction to different degrees, and the inhibitory effect is highly consistent among mAbs that target the same epitope. Functional assay revealed that anti-HS-binding site mAbs show different potency in inhibiting osteoclastogenesis, and the inhibitory potency does not have a simple correlation with the affinity and the epitope. Our study demonstrates that developing HS-binding site targeting mAbs should be applicable to most HS-binding proteins. By targeting this unique functional site, these mAbs might find therapeutic applications in treating various human diseases.

Targeting heparan sulfate-protein interactions with oligosaccharides and monoclonal antibodies.

Heparan sulfate-binding proteins (HSBPs) are structurally diverse extracellular and membrane attached proteins that interact with HS under normal physiological conditions. Interactions with HS offer an additional level of control over the localization and function of HSBPs, which enables them to behave in a more refined manner. Because all cell signaling events start at the cell membrane, and cell-cell communication relies on translocation of soluble factors across the extracellular matrix, HS occupies an apical position in cellular signal transduction by interacting with hundreds of growth factors, cytokines, chemokines, enzymes, enzyme inhibitors, receptors and adhesion molecules. These extracellular and membrane proteins can play important roles in physiological and pathological conditions. For most HS-binding proteins, the interaction with HS represents an essential element in regulating their normal physiological functions. Such dependence on HS suggests that manipulating HS-protein interactions could be explored as a therapeutic strategy to selectively antagonize/activate HS-binding proteins. In this review, we will discuss current understanding of the diverse nature of HS-HSBP interactions, and the latest advancements in targeting the HS-binding site of HSBPs using structurally-defined HS oligosaccharides and monoclonal antibodies.

Heparan sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE.

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (AgerAHA/AHA) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, AgerAHA/AHA mice phenocopied Ager-/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of AgerAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Ager-/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in AgerAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), AgerAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Ager-/- mice.

PH-dependent and dynamic interactions of cystatin C with heparan sulfate

Cystatin C (Cst-3) is a potent inhibitor of cysteine proteases with diverse biological functions. As a secreted protein, the potential interaction between Cst-3 and extracellular matrix components has not been well studied. Here we investigated the interaction between Cst-3 and heparan sulfate (HS), a major component of extracellular matrix. We discovered that Cst-3 is a HS-binding protein only at acidic pH. By NMR and site-directed mutagenesis, we identified two HS binding regions in Cst-3: the highly dynamic N-terminal segment and a flexible region located between residue 70-94. The composition of the HS-binding site by two highly dynamic halves is unique in known HS-binding proteins. We further discovered that HS-binding severely impairs the inhibitory activity of Cst-3 towards papain, suggesting the interaction could actively regulate Cst-3 activity. Using murine bone tissues, we showed that Cst-3 interacts with bone matrix HS at low pH, again highlighting the physiological relevance of our discovery.

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor.

SummaryCircovirus, comprising one capsid protein, is the smallest nonenveloped virus and induces lymphopenia. Circovirus can be used to explore the cell adhesion mechanism of nonenveloped viruses. We developed a single-molecule fluorescence resonance energy transfer (smFRET) assay to directly visualize the capsid's conformational feature. The capsid underwent reversible dynamic transformation between three conformations. The cell surface receptor heparan sulfate (HS) altered the dynamic equilibrium of the capsid to the high-FRET state, revealing the HS-binding region. Neutralizing antibodies restricted capsid transition to a low-FRET state, masking the HS-binding domain. The lack of positively charged amino acids in the HS-binding site reduced cell surface affinity and attenuated virus infectivity via conformational changes. These intrinsic characteristics of the capsid suggested that conformational dynamics is critical for the structural changes occurring upon cell surface receptor binding, supporting a dynamics-based mechanism of receptor binding.

Abstract P3-01-29: Impact of heparan sulphate binding domain of chemokine CCL21 to migration of breast cancer cells

Abstract Lymph node metastasis constitutes a key event in breast cancer progression. Chemokines are small proteins, which can promote metastatic spread by inducing cancer cell migration and invasion. Chemokine function is dependant upon their binding to both cell surface heparan sulphate (HS) molecules and to their specific receptor. Our group has demonstrated a significant increase in chemokine receptor CCR7 expression in cancerous breast epithelia compared to healthy controls. This study is designed to test the hypothesis that a non-HS binding forms of chemokine CCL21 can disrupt the normal response to CCL21, therefore reducing the metastasis of CCR7-expressing cancer cells. Truncated CCL21 chemokine (Δ98-134 c-terminal basic extension), was synthesised to investigate a possible linkage between chemokine binding capacity and cell activation. Wild type (WT) and mutant-CCL21 were tested for their ability to stimulate a dose-dependent increase in intracellular-free calcium in peripheral blood mononuclear cell (PBMC) and breast cancer epithelial cells MDA-MB-231. Mutant-CCL21 at concentrations 5 and 10nM showed potential to mobilise Ca2+ at levels similar to that produced by WT-CCl21. A series of experiments was performed to determine how deletion of the HS-binding site altered the ability of CCL21 to stimulate chemotaxis within a concentration gradient generated by free solute diffusion. PBMC stimulated to migrate by wild-type CCL21 was not significantly different from that stimulated by mutant (P> 0.05). Similar results were observed in assays using MDA-MB-231 cells. A further series of experiments was performed to compare the potential of WT and mutant-CCL21 to stimulate the migration of cells across endothelium. In contrast to results for trans-filter migration, it was found that the non HS-binding mutant stimulated no increased in transendothelial cell migration above the background at each of the tested concentrations, 10, 30 and 50 nM respectively (P>0.05). However, WT-CCL21 stimulated significant increased PBMC migration at each of the tested concentration (all P <0.001). Furthermore, the effect of heparin on chemotactic properties of WT and mutant- CCL21 was examined. Interestingly, heparin (250 µg/ml) completely inhibit the chemotaxis mediated by WT-CCL21 (5nM) (P < 0.001), whereas it did not inhibit the chemotaxis at concentrations 100, 250 & 500 µg/ml in response to mutant CCL21 (5nM) (P > 0.05). Similar assay will be performed using MDA-MB-231 cells. Work is ongoing to characterise the biophysical properties of mutant-CCL21 and determine its potential role for a therapeutic blockade of the migration of breast cancer cells in-vivo. Our primarily data showed that mutant CCL21 in xenograft brain tumor models showed substantial inhibition of tumour growth. Our results indicate that truncated CCL21 chemokine might be a potential preventive biofactor for human breast cancer metastasis by targeting chemokine receptor genes. Citation Format: Mohammed Imad Malki. Impact of heparan sulphate binding domain of chemokine CCL21 to migration of breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-29.

Characterization and engineering of S100A12-heparan sulfate interactions.

S100A12, an EF-hand calcium-binding protein, can be secreted by a variety of cell types and plays proinflammatory roles in a number of pathological conditions. Although S100A12 has been shown to interact with heparan sulfate (HS), the molecular detail of the interaction remains unclear. Here we investigate the structural basis of S100A12-HS interaction and how the interaction is regulated by the availability of divalent cations and the oligomeric states of S100A12. We discovered that S100A12-HS interaction requires calcium, while zinc can further enhance binding by inducing S100A12 hexamerization. In contrast, the apo form and zinc-induced tetramer form were unable to bind HS. Guided by the crystal structures of S100A12, we have identified the HS-binding site of S100A12 by site-directed mutagenesis. Characterization of the HS-binding site of S100A12 allowed us to convert the non-HS-binding apo and tetramer forms of S100A12 into a high affinity HS-binding variant by engineering a single-point mutation. Using a HS oligosaccharide microarray, we demonstrated that the N43K mutant displayed markedly enhanced selectivity toward longer HS oligosaccharides compared to the WT S100A12, likely due to the expanded dimension of the reengineered HS-binding site in the mutant. This unexpected finding strongly suggests that HS-binding sites of proteins might be amenable for engineering.

Heparan Sulfate Regulates the Structure and Function of Osteoprotegerin in Osteoclastogenesis

Osteoprotegerin (OPG), a decoy receptor secreted by osteoblasts, is a major negative regulator of bone resorption. It functions by neutralizing the receptor activator of nuclear factor κB ligand (RANKL), which plays a central role in promoting osteoclastogenesis. OPG is known to be a high-affinity heparan sulfate (HS)-binding protein. Presumably, HS could regulate the function of OPG and affect how it inhibits RANKL. However, the molecular detail of HS-OPG interaction remains poorly understood, which hinders our understanding of how HS functions in osteoclastogenesis. Here we report mapping of the HS-binding site of OPG. The HS-binding site, identified by mutagenesis study, consists of eight basic residues that are located mostly at the junction of the second death domain and the C-terminal domain. We further show that heparin-derived dodecasaccharide is able to induce dimerization of OPG monomers with a stoichiometry of 1:1. Small-angle X-ray scattering analysis revealed that upon binding of HS, OPG undergoes a dramatic conformational change, resulting in a more compact and less flexible structure. Importantly, we present here three lines of evidence that HS, OPG, and RANKL form a stable ternary complex. Using a HS binding-deficient OPG mutant, we further show that in an osteoblast/bone marrow macrophage co-culture system, immobilization of OPG by HS at the osteoblast cell surface substantially lowers the inhibitory threshold of OPG toward RANKL. These discoveries strongly suggest that HS plays an active role in regulating OPG-RANKL interaction and osteoclastogenesis.

Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1–VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A − ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A − vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.

Abstract 110: The Separation of Serum Amyloid A from HDL is Mediated by Heparan Sulfate Through Histidine Dependent Interactions: HDL Function is Enhanced but Amyloid-Fibrils may be the “Price to Pay”¼

Serum amyloid A (SAA) is an acute-phase protein that circulates bound to high density lipoprotein (HDL) and can influence HDL function as part of a poorly understood defense re-sponse to tissue trauma or infection. We have previously demonstrated that under mildly acidic pH the glycosaminoglycans, heparan sulfate (HS) and heparin can interact with HDL-SAA and cause SAA to dissociate from HDL. This remodeling improves HDL functionality but also predisposes SAA to form AA-amyloid fibrils. In this study we explore some potential pathophysiological conditions in vitro that could influence this HS/HDL-SAA remodeling process and the fate of SAA in vivo. SAA’s binding affinity for heparin was found to be enhanced by acidic pH and low concentrations of urea. The heparin dependent remodeling of HDL-SAA was promoted by the partial denaturation of HDL-SAA. Moreover, HDL-SAA remodeling was observed to follow a strict SAA:heparin stoichiometry and could be partially inhibited with a short heparin oligosaccharide of 8-sugar units. Evidence is also presented that once dissociated from HDL, SAA requires additional time to organize into Triton x-100 resistant amyloid-like structures. Circular dichroism spectroscopic analysis and in silico modeling of SAA’s ionizable residues highlights the importance of the histidine-36 within a highly conserved, pH-sensitive HS-binding site (HSBS-pH). A peptide containing the HSBS-pH sequence was demonstrated to have AA-amyloid seeding activity in a cell culture system. The recent determination of the 3D structure for human SAA1.1 has allowed the opportunity to re-assess and validate the HS/heparin binding sequences that had previously been identified biochemically with short synthetic peptides. We postulate that the dissociation of SAA from HDL takes place during the retro-endocytosis of HDL-SAA and is an important aspect of SAA function not previously appreciated.

Opposite role of CD44-standard and CD44-variant-3 in tubular injury and development of renal fibrosis during chronic obstructive nephropathy

Chronic kidney diseases (CKDs) are characterized by tubular atrophy and interstitial fibrosis. We previously showed that in obstructive nephropathy de novo CD44 renal expression contributes to renal fibrosis but attenuates tubular damage/apoptosis. As CD44-standard (CD44s) has been linked to TGF-β1-mediated actions and CD44-variant-3 (CD44v3) favors HGF-c-Met binding, we compared the functional properties of these CD44 isoforms in the progression of obstructive nephropathy, using specific CD44-variant knockout/knockin mice. The presence of CD44v3 diminished tubular damage during obstructive nephropathy, decreased apoptosis, and increased proliferation of tubular epithelial cells, and prevented renal fibrosis development. In contrast, expression of CD44s led to increased tubular damage and tubular epithelial cell apoptosis, and more renal fibrosis. A relative increase in renal β-catenin expression, HGF production, and HGF/c-Met signaling, together with a relative inhibition of TGF-β1 downstream signaling and TGF-β type I receptor expression, was found in CD44v3 mice compared with CD44s littermates. In line with this, Wnt3a/HGF treatment of tubular cells resulted in higher β-catenin/p-AKT levels in CD44v3(+) tubular epithelial cells, whereas TGF-β1 induced a mild collagen I upregulation in CD44v3(+) mouse embryonic fibroblasts as compared with CD44s(+) cells. Thus, CD44s and CD44v3 exert opposite roles in the progression of obstructive nephropathy, with CD44v3-v10 being the protective isoform that delays evolution of the renal pathology.

A focused sulfated glycoconjugate Ugi library for probing heparan sulfate-binding angiogenic growth factors

A library of small molecule heparan sulfate (HS) mimetics was synthesized by employing the Ugi four-component condensation of d-mannopyranoside-derived isocyanides with formaldehyde as the carbonyl component and a selection of carboxylic acids and amines, followed by sulfonation. The library was used to probe the subtle differences surrounding the ionic binding sites of three HS-binding angiogenic growth factors (FGF-1, FGF-2 and VEGF). Each compound features 3 or 4 sulfo groups which serve to anchor the ligand to the HS-binding site of the protein, with a diverse array of functionality in place extending from C-1 or C-6 to probe for adjacent favorable binding interactions. Selectivity of binding to these proteins was clearly observed and supported by molecular docking calculations.

Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate

Author SummaryThe development, homeostasis, and repair of animal tissues requires communication between cells mediated by effector proteins, which are released from source cells and must move through the surrounding extracellular matrix to reach their receptors on target cells. A major component of the extracellular matrix is the polysaccharide heparan sulfate (HS); it binds the majority of these effectors and has the crucial function of regulating their transport. The mechanism underlying this function, however, is still unknown. To understand how HS regulates the transport of effectors, in this study we labelled molecules of the effector protein fibroblast growth factor 2 (FGF2) each with a gold nanoparticle, which we could visualise and quantify by electron microscopy and by a new approach called photothermal heterodyne imaging. By imaging the gold nanoparticles, we found that the binding sites for FGF2 on HS are distributed heterogeneously in the extracellular matrix that surrounds cells in culture. Single molecule tracking indicated that these binding sites are organised into local networks that confine the FGF2 and into paths that allow its translocation over long distances (up to several micrometers). Thus, the spatial distribution of the binding sites in HS and their physicochemical properties of binding are major factors controlling the transport of effectors in extracellular matrices.

Characterization of Heparin-binding Site of Tissue Transglutaminase: ITS IMPORTANCE IN CELL SURFACE TARGETING, MATRIX DEPOSITION, AND CELL SIGNALING

Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 (202)KFLKNAGRDCSRRSSPVYVGR(222). We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Amino acid substitutions in the E2 glycoprotein of Sindbis-like virus XJ-160 confer the ability to undergo heparan sulfate-dependent infection of mouse embryonic fibroblasts

We have recently demonstrated an essential role of the domain of 145-150 amino acid in the E2 glycoprotein of Sindbis virus in the interaction with cellular heparan sulfate (HS) and in the infection of mouse embryonic fibroblasts (MEF) cells. In this study, we constructed and characterized the mutants of Sindbis-like virus XJ-160 in which Tyr-146 and/or Asn-149 in the E2 glycoprotein had been substituted with His and Arg, respectively. Unlike parental virus XJ-160, mutants with either or both substitutions were able to infect wild-type mouse embryonic fibroblasts (MEF-wt) or MEF-Epi-/- cells which produce mutant HS. Significantly more infectious particles were released from MEF-wt than from MEF-Epi-/- cells. The mutant virus with both substitutions release was inhibited by pre-incubation of virus with heparin or pre-treatment of BHK-21 cells with HS-degrading enzyme. Both XJ-160 and the mutant viruses retained substantial neurovirulence in suckling mice. Our findings provide further support to the importance of positively charged residues in the HS-binding site of E2 in mediating Sindbis virus infection of MEF cells.

Mutations in the Heparan-Sulfate Proteoglycan Glypican 6 (GPC6) Impair Endochondral Ossification and Cause Recessive Omodysplasia

Glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored, membrane-bound heparan sulfate (HS) proteoglycans. Their biological roles are only partly understood, although it is assumed that they modulate the activity of HS-binding growth factors. The involvement of glypicans in developmental morphogenesis and growth regulation has been highlighted by Drosophila mutants and by a human overgrowth syndrome with multiple malformations caused by glypican 3 mutations (Simpson-Golabi-Behmel syndrome). We now report that autosomal-recessive omodysplasia, a genetic condition characterized by short-limbed short stature, craniofacial dysmorphism, and variable developmental delay, maps to chromosome 13 (13q31.1-q32.2) and is caused by point mutations or by larger genomic rearrangements in glypican 6 (GPC6). All mutations cause truncation of the GPC6 protein and abolish both the HS-binding site and the GPI-bearing membrane-associated domain, and thus loss of function is predicted. Expression studies in microdissected mouse growth plate revealed expression of Gpc6 in proliferative chondrocytes. Thus, GPC6 seems to have a previously unsuspected role in endochondral ossification and skeletal growth, and its functional abrogation results in a short-limb phenotype.

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.

Isolation and Characterization of Low Sulfated Heparan Sulfate Sequences with Affinity for Lipoprotein Lipase

Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains.

Structural and functional features of the polycationic peptide required for inhibition of herpes simplex virus invasion of cells

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) mediates initial virus contact with cells by binding to heparan sulfate (HS) chains. The synthetic peptide 137 GSRVQIRCRFRNSTR 151 overlapping a major part of the HS-binding site of gC inhibited HSV-1 infection and, to some extent, HSV-2 infection of cells. Experiments on mutant, glycosaminoglycan-deficient cells as well as the binding assays involving peptide and purified cell surface components identified HS, and, to a lesser degree, chondroitin sulfate as sites of peptide activity. Anti-HSV-1 activity of the peptide was due to (i) partial inhibition of virus binding to cells and (ii) arresting the virions, which managed to attach to the cells in the presence of peptide, at a step of initial relatively weak binding. Analysis of the ionic-strength dependence of the peptide–HS and the virus–HS interactions revealed that the more efficient inhibition by the peptide of HSV-1 than HSV-2 infectivity was due to a relatively high affinity of HSV-2 for HS, a feature of importance in overcoming the peptide block. Mutational analysis of viral gC and peptide variants identified, apart from basic amino acids, two hydrophobic residues Ile 142 and Phe 146 as important in maintaining the specific affinity of peptide for HS and, hence, its anti-HSV activity. These results could contribute to the development of anti-HSV compounds that target initial events in the virus–cell interaction.