GAG-binding Sites Research Articles

Conservation of CD44 exon v3 functional elements in mammals

BackgroundThe human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44.FindingsWe have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences.ConclusionCD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait.

Biological implications of glycosaminoglycan interactions with haemopoietic cytokines

Heparan sulphate (HS) glycosaminoglycans (GAGs) are an integral part of the signalling complex of fibroblast derived growth factor (FGF) family members, HS being regarded as a coreceptor. FGFs are also retained in the tissues by binding to HS structures. Early studies on the contribution of the bone marrow stroma to haemopoiesis suggested that cytokines with a role in haemopoiesis were similarly retained in the stroma through interactions with HS. However, the functional outcomes of these cytokines binding HS were poorly understood. Here the GAG-binding properties of cytokines of the four alpha-helical bundle family and the biological consequences of such binding are reviewed. From this analysis it is apparent that although many of these cytokines do bind GAGs, GAG binding is not a consistent feature, nor is the site of GAG binding conserved among these cytokines. The biological outcome of GAG binding depends, in part, on the location of the GAG-binding site on the cytokine. In some cases GAG binding appears to block signalling, whereas in others signalling is likely to be facilitated by binding. It is postulated that the interactions of these cytokines with their receptor complexes evolved independently of GAG binding, with GAG binding being an additional feature for a subset of this cytokine family. Nevertheless, because GAG binding localizes cytokines to sites within tissues, these interactions are likely to be critically important for the biology of these cytokines.

Matrix Metalloproteinase Processing of CXCL11/I-TAC Results in Loss of Chemoattractant Activity and Altered Glycosaminoglycan Binding

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.

The complement protein properdin recognizes glycosaminoglycan chains on apoptotic and malignant cell surfaces

The complement alternative pathway (AP) C3 convertase, a central player of the complement cascade, tags potential targets with C3 fragments that in turn direct lysis and/or clearance and inflammatory reactions. The complement protein properdin has long been known to stabilize the AP convertase. Recently we have found that properdin is also a danger‐recognition molecule: Properdin can bind malignant and apoptotic cells and certain pathogenic microbes. Once bound to a surface, properdin provides a platform for the assembly of the AP C3 convertase, leading to opsonization and clearance and/or lysis. We are interested the mechanism by which properdin can recognize such a variety of potential targets.Properdin is composed of 6 thrombospondin repeats, which in other proteins can harbor glycosaminoglycan (GAG) binding sites. Thus, we investigated the possibility that properdin attaches to mammalian cells via GAGs. Using a panel of wild type and mutant CHO lines, we observed that properdin can bind cells via heparin sulfate and chondroitin sulfate GAG chains associated with cell surface proteoglycans. We then examined properdin interactions with human cells and found that properdin binds GAGs on apoptotic and malignant cell surfaces. Experiments with soluble GAG competitors suggest that properdin harbors multiple GAG‐binding sites that differ in GAG specificity. Supported by NIHR01AI05143.

Steric Block High Affinity Oligonucleotide Analogues: A New Tool for Mapping RNA-Protein Binding Sites

Steric-block ON analogues are efficient inhibitors of RNA-protein interaction and therefore have potential to probe RNA sequences for putative protein binding sites and to investigate mechanisms of protein binding. The packaging process of HIV-1 is highly specific involving an interaction between the Gag protein and a conserved sequence that is only present on genomic viral RNA. Using oligonucleotide probes we have confirmed that the terminal purine loop is the major Gag binding site on SL3 and that a secondary Gag binding site exists at an internal purine bulge. We also demonstrate direct binding of oligonucleotide to their binding sites and confirm this interaction does not alter global RNA conformation, making them highly specific, nondisruptive probes of RNA protein interactions.

Glycosaminoglycans Facilitate Procathepsin B Activation through Disruption of Propeptide-Mature Enzyme Interactions

Lysosomal cysteine cathepsin B participates in numerous diverse cellular processes. In acquiring its activity, the proregion, which blocks the substrate-binding site in the proenzyme, needs to be cleaved off. Here we demonstrate that polyanionic polysaccharides, glycosaminoglycans (GAGs), can accelerate the autocatalytic removal of the propeptide and subsequent activation of cathepsin B. We show that naturally occurring GAGs such as chondroitin sulfates and heparin, as well as the synthetic analog dextran sulfate, accelerate the processing in a concentration-dependent manner. Heparin oligosaccharides down to the size of tetrasaccharides were efficient in accelerating the procathepsin B processing, whereas disaccharides were without effect. Further, the ability of the GAGs to accelerate procathepsin B processing was sensitive to increasing NaCl concentrations, indicating that electrostatic interaction between the GAGs and procathepsin B are operative in the accelerating effect. Also the processing of the catalytic procathepsin B mutant by wild type cathepsin B was enhanced in the presence of GAGs, suggesting that GAGs induce a conformational change in procathepsin B, converting it into a better substrate. Site-directed mutagenesis showed that His(28), Lys(39), and Arg(40), located within the procathepsin B propeptide, have significant roles in the acceleration of procathepsin B activation induced by short GAGs. Because procathepsin B and GAGs often co-localize in vivo, we propose that GAGs may play a physiological role in the activation of procathepsin B.

Effects of covalently attached chondroitin sulfate on aggrecan cleavage by ADAMTS-4 and MMP-13

Aggrecan is degraded by several aggrecanase-1 (ADAMTS-4) isoforms differing in the number of sulfated glycosaminoglycan (sGAG)-binding motifs. ADAMTS-4 and MMPs cleave aggrecan more efficiently within the chondroitin sulfate (CS)-rich region than the interglobular domain (IGD). We investigated the influence of CS on aggrecan core protein cleavage by ADAMTS-4 (p68) and (p40) as well as MMP-13, which has no recognizable GAG-binding sites. Chondroitinase ABC-treated cartilage aggrecan was cleaved with ADAMTS-4 (p68) less efficiently than CS-substituted aggrecan within the CS-2 domain. Keratanase-treated aggrecan exhibited reduced IGD cleavage, but when both CS and KS were removed, the IGD cleavage was restored. This result suggests that KS in the IGD may compete with CS for ADAMTS-4 (p68) binding. In the absence of KS, however, p68 binding was shifted to the CS-2 domain. CS-deficient full-length recombinant aggrecan (rbAgg) was produced by chondroitinase ABC treatment, or by expression in the xylosyltransferase-deficient CHO-pgsA745 cell line. When digested with the ADAMTS-4 (p68), each of these preparations exhibited reduced CS-2 domain cleavage compared to CS-substituted CHO-K1 cell-derived aggrecan. Additionally, CS-deficient rbAgg showed increased IGD scission prior to cleavage within the CS-2 domain. ADAMTS-4 (p40) readily cleaved both rbAggs within the IGD, but cleaved poorly within the CS-2 domain, indicating little CS dependence. MMP-13, in contrast, cleaved the CS region and the IGD of both CS-substituted and CS-deficient rbAgg equally well. These data indicate that covalently bound CS enhances ADAMTS-4-mediated cleavage within the CS-rich region. MMP-13 also cleaves preferentially within the CS-region, but by an apparently CS-independent mechanism.

The structures of the thrombospondin-1 N-terminal domain and its complex with a synthetic pentameric heparin.

The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 A resolution is a beta sandwich with 13 antiparallel beta strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.

The Glycosaminoglycan-Binding Domain of Decoy Receptor 3 Is Essential for Induction of Monocyte Adhesion

Decoy receptor 3 (DcR3), a soluble receptor for Fas ligand, LIGHT (homologous to lymphotoxins shows inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes), and TNF-like molecule 1A, is highly expressed in cancer cells and in tissues affected by autoimmune disease. DcR3.Fc has been shown to stimulate cell adhesion and to modulate cell activation and differentiation by triggering multiple signaling cascades that are independent of its three known ligands. In this study we found that DcR3.Fc-induced cell adhesion was inhibited by heparin and heparan sulfate, and that DcR3.Fc was unable to bind Chinese hamster ovary K1 mutants defective in glycosaminoglycan (GAG) synthesis. Furthermore, the negatively charged, sulfated GAGs of cell surface proteoglycans, but not their core proteins, were identified as the binding sites for DcR3.Fc. A potential GAG-binding site was found in the C-terminal region of DcR3, and the mutation of three basic residues, i.e., K256, R258, and R259, to alanines abolished its ability to trigger cell adhesion. Moreover, a fusion protein comprising the GAG-binding region of DcR3 with an Fc fragment (DcR3_HBD.Fc) has the same effect as DcR3.Fc in activating protein kinase C and inducing cell adhesion. Compared with wild-type THP-1 cells, cell adhesion induced by DcR3.Fc was significantly reduced in both CD44v3 and syndecan-2 knockdown THP-1 cells. Therefore, we propose a model in which DcR3.Fc may bind to and cross-link proteoglycans to induce monocyte adhesion.

Heparin-binding Sites in Granulocyte-Macrophage Colony-stimulating Factor: LOCALIZATION AND REGULATION BY HISTIDINE IONIZATION

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.

Site-Specific Polymer Attachment to a CCL-5 (RANTES) Analogue by Oxime Exchange

A synthetic strategy that allows for the site-specific attachment of polymers such as poly(ethylene glycol) (PEG) to protein pharmaceuticals is described. PEG was attached to a 67-amino acid fully synthetic CCL-5 (RANTES) analogue at its GAG binding site both to reduce aggregation and to increase the circulating lifetime. Effective protection of an Aoaa chemoselective linker during peptide assembly, total chemical protein synthesis, and protein folding was achieved with an isopropylidene group. Mild deprotection of the resulting folded synthetic protein and subsequent polymer attachment occur without interference with the native folded structure and activity.

Autocatalytic Cleavage of ADAMTS-4 (Aggrecanase-1) Reveals Multiple Glycosaminoglycan-binding Sites

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.

Identification of the glycosaminoglycan-binding site on the glycoprotein E(rns) of bovine viral diarrhoea virus by site-directed mutagenesis.

Bovine viral diarrhoea virus (BVDV) envelope glycoprotein E(rns) interacts with highly sulphated heparin-like glycosaminoglycans (GAGs) located on the cell surface as an early step in virus infection of cells. Site-directed mutagenesis of recombinant E(rns) was undertaken and analysis of mutants by heparin-affinity chromatography and cell surface binding showed that a cluster of basic amino acids (480KKLENKSK487) near the C terminus of E(rns) was essential for binding. Mutants with amino acid substitutions of lysine residues 481 and 485 in E(rns) reduced the binding of E(rns) to immobilized heparin and cellular GAGs but retained ribonuclease activity. In contrast to normal E(rns), E(rns) that was unable to bind to cells also failed to inhibit BVDV infection of cells when the cells were pre-incubated with E(rns). It is proposed that the cluster of basic residues (480KKLENKSK487) localized at the C-terminal end of E(rns) constitutes a GAG-binding site.

Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins.

Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 A spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection.

HIV-1 Gag binds specifically to RNA stem-loops in the 5′ leader sequence

GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1–406, in vitro, with a K d of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Gag in an RNA containing nucleotides 325–362. SL4 is predicted by both biochemical studies and computer folding to be located between nucleotides 335 and 358. An RNA transcript ending at nucleotide 335 does not bind Gag. The deletion of nucleotides 334–358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleotides 297–300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of nucleotides 348–351 in SL4 by RNAse V1 is enhanced by Gag binding. At least two Gag binding sites are therefore located in the leader RNA. Those located 5′ of nucleotide 335 require the presence of additional 3′ sequences.

Interaction and co-encapsidation of human immunodeficiency virus type 1 Gag and Vif recombinant proteins.

Human immunodeficiency virus type 1 (HIV-1) wild-type (WT) virion infectivity factor (Vif) protein (Vifwt) and full-length Gag precursor (Pr55Gag) were found to be co-encapsidated into extracellular, membrane-enveloped virus-like particles released by budding from Sf9 cells co-expressing the two recombinant proteins in trans, with an average copy number of 3.5+/-0.6 Vifwt per 100 Pr55Gag molecules. No preferential localization at the plasma membrane was observed for recombinant Vif in the absence of Gag expression, and a significant proportion of Vif accumulated within the nucleus. Two conserved motifs, W89RKRRY94 and P156KKIKP161, seemed to act as nuclear addressing signals. The Pr55Gag and Vifwt interacting domains were analysed by biopanning of a phage-displayed hexapeptide library. The Vif-binding domain, which spanned residues H421-T470 in Pr55Gag, corresponded to the C-terminal region of nucleocapsid (NC), including the second zinc finger, the intermediate spacer peptide sp2 and the N-terminal half of the p6 domain. Deletions in these Gag domains significantly decreased the Vif encapsidation efficiency, and complete deletion of NC abolished Vif encapsidation. In Vif, four discrete Gag-binding sites were identified, within residues T68-L81 (site I) and W89-P100 (site II) in the central domain, and within residues P162-R173 (III) and P177-M189 (IV) at the C terminus. Substitutions in site I and deletion of site IV were detrimental to Vif encapsidation, whereas substitution of basic residues for alanine in sites III and IV had a positive effect. The data suggest a direct intracellular Gag-Vif interaction and the occurrence of a Pr55Gag-mediated membrane-targeting pathway for Vif in Sf9 cells.

Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation.

Retrovirus particles each contain two copies of the viral genome in the form of a noncovalently linked RNA dimer. Earlier studies have mapped a cis-acting region near the 5' end of the human immunodeficiency virus type 1 (HIV-1) genome, termed the psi locus, which appears essential for initiation of genomic dimerization, as well as for interactions with the HIV-1 Gag protein that are thought to target the RNA into nascent virions. This HIV-1 psi locus is proposed to be organized in four independent RNA stem-loops; at least three (SL1, SL3, and SL4) contain binding sites for Gag, and one of these (SL1) is implicated in dimer initiation through a kissing-loop mechanism. In this study, we have created HIV-1 proviruses containing psi mutations that affect in vitro Gag binding, RNA dimerization, or both, and we have characterized the effects of these mutations on viral assembly and infectivity by using a single-step infectious assay. We find that various mutations which eliminate the Gag binding sites in SL1 or SL3 produce marked defects in genomic RNA packaging and viral infectivity. In each case, the reduced genomic content of the mutant virions is associated with an increased content of spliced viral transcripts, suggesting that both SL1 and SL3 contribute to the discrimination between spliced and unspliced RNAs. The structures, but not the specific sequences, of the SL1 and SL3 stems appear critical for RNA packaging. Disruption of the stem or deletion of SL1 also results in abnormal genomic dimerization, as assessed by nondenaturing gel electrophoresis of virion-derived RNA. Virions carrying less extensive mutations in the SL1 loop that are known to prevent in vitro dimerization have impaired infectivity despite normal virion RNA content. This suggests that RNA dimerization is not a prerequisite for genomic packaging but instead serves an independent function in the retroviral infectious cycle.

RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1.

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.

Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin.

Lactoferrin is an iron-binding protein which is synthesized by mucosal epithelium and neutrophils and released by these cells in response to inflammatory stimuli. It promotes neutrophil aggregation and manifests iron-dependent and -independent antimicrobial properties in vitro. Since lactoferrin binds to glycosaminoglycans (GAGs) and sulfated polysaccharides can inhibit its clearance in vivo and in vitro, we sought to examine its interaction with the GAGs chondroitin sulfate and heparin. Amino-terminal sequencing of proteolytic fragments of human lactoferrin that were fractionated by GAG chromatography suggested that the amino-terminal 6 kDa of the secreted protein mediates its interaction with GAGs. Synthetic peptides were used to show that the first 33 residues of human lactoferrin can bind well to solid-phase or solution-phase GAGs. The first 33 residues bound fluoresceinamine-labeled heparin with an IC50 (611 nM) which approximated that of the intact protein (124 nM). In contrast, when the first six residues (GRRRRS) were removed from this peptide, it then bound poorly to heparin (IC50 = 49 microM). Our results suggest that the GRRRRS sequence at the amino terminus of human lactoferrin acts synergistically with an RKVR sequence at positions 28-31 to form the predominate functional GAG-binding site of human lactoferrin. Molecular modeling of the crystalline structure of lactoferrin supports a synergistic activity between these two sites since it shows that they juxtapose each other on the surface of the folded protein. Solid docking calculations indicate that they can form a cationic cradle as a binding site for chondroitin sulfate.

Characterization of a synthetic peptide from the glycosaminoglycan binding site of heparin cofactor II

We characterized a synthetic peptide based on the glycosaminoglycan (GAG)-binding site of the serine proteinase inhibitor (serpin) heparin cofactor II (HCII): HCII165–195, K165DFVNASSKYEITTIHNLFRKLTHRLFRRNF195. HCII165–195 negated acceleration of the HCII/thrombin inhibition reaction (IC50 for the peptide shown in parentheses) by heparin (∼250 nM) and dermatan sulfate (∼500 nM). Circular dichroism spectra of HCII165–195 showed that GAGs increase the α-helical content of the peptide (percentage α-helix of the peptide/GAG complex given in parentheses): no GAG (7%) < low-molecular-weight heparin (32%) < heparin (42%) < dermatan sulfate (55%). A molecular model of HCII predicts that this region is 48% α-helix. Our results suggest: (i) HCII165–195 binds to GAGs; (ii) an α-helical conformation is preferable in the presence of GAGs; and (iii) GAGs may help stabilize a specific protein conformation in the HCII GAG-binding site, important for serpin function.