Consensus Heparin-binding Sequences Research Articles

Glycosaminoglycans are involved in bacterial adherence to lung cells

BackgroundLower respiratory infections are among the top ten causes of death worldwide. Since pathogen to cell adhesion is a crucial step in the infection progress, blocking the interaction between eukaryotic receptors and bacterial ligands may enable the pathogenesis process to be stopped. Cell surface glycosaminoglycans (GAGs) are known to be mediators in the adhesion of diverse bacteria to different cell types, making it of interest to examine their involvement in the attachment of various pathogenic bacteria to lung cells, including epithelial cells and fibroblasts.MethodsThe function of cell surface GAGs in bacterial adhesion was studied by reducing their levels through inhibiting their biosynthesis and enzymatic degradation, as well as in binding competition experiments with various species of GAGs. The participation of the different bacterial adhesins in attachment was evaluated through competition with two peptides, both containing consensus heparin binding sequences. Blocking inhibition assays using anti-syndecans and the enzymatic removal of glypicans were conducted to test their involvement in bacterial adhesion. The importance of the fine structure of GAGs in the interaction with pathogens was investigated in competition experiments with specifically desulfated heparins.ResultsThe binding of all bacteria tested decreased when GAG levels in cell surface of both lung cells were diminished. Competition experiments with different types of GAGs showed that heparan sulfate chains are the main species involved. Blocking or removal of cell surface proteoglycans evidenced that syndecans play a more important role than glypicans. The binding was partially inhibited by peptides including heparin binding sequences. Desulfated heparins also reduced bacterial adhesion to different extents depending on the bacterium and the sulfated residue, especially in fibroblast cells.ConclusionsTaken together, these data demonstrate that the GAG chains of the cell surface are involved in the adhesion of bacterial adhesins to lung cells. Heparan sulfate seems to be the main species implicated, and binding is dependent on the sulfation pattern of the molecule. These data could facilitate the development of new anti-infective strategies, enabling the development of new procedures for blocking the interaction between pathogens and lung cells more effectively.

Peptide amphiphile nanostructure–heparin interactions and their relationship to bioactivity

Heparin–protein interactions are important in many physiological processes including angiogenesis, the growth of new blood vessels from existing ones. We have previously developed a highly angiogenic self-assembling gel, wherein the self-assembly process is triggered by the interactions between heparin and peptide amphiphiles (PAs) with a consensus heparin binding sequence. In this report, this consensus sequence was scrambled and incorporated into a new peptide amphiphile in order to study its importance in heparin interaction and bioactivity. Heparin was able to trigger gel formation of the scrambled peptide amphiphile (SPA). Furthermore, the affinity of the scrambled molecule for heparin was unchanged as shown by isothermal titration calorimetry and high Förster resonance emission transfer efficiency. However, both the mobile fraction and the dissociation rate constant of heparin, using fluorescence recovery after photobleaching, were markedly higher in its interaction with the scrambled molecule implying a weaker association. Importantly, the scrambled peptide amphiphile–heparin gel had significantly less angiogenic bioactivity as shown by decreased tubule formation of sandwiched endothelial cells. Hence, we believe that the presence of the consensus sequence stabilizes the interaction with heparin and is important for the bioactivity of these new materials.

Histidine‐rich glycoprotein exerts antibacterial activity

Histidine-rich glycoprotein (HRGP), an abundant heparin-binding protein found in plasma and thrombocytes, exerts antibacterial effects against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Fluorescence studies and electron microscopy to assess membrane permeation showed that HRGP induces lysis of E. faecalisbacteria in the presence of Zn2+ or at low pH. Heparin blocked binding of the protein to E. faecalis and abolished antibacterial activity. Furthermore, truncated HRGP, devoid of the heparin-binding and histidine-rich domain, was not antibacterial. It has previously been shown that peptides containing consensus heparin-binding sequences (Cardin and Weintraub motifs) are antibacterial. Thus, the peptide (GHHPH)4, derived from the histidine-rich region of HRGP and containing such a heparin-binding motif, was antibacterial for E. faecalis in the presence of Zn2+ or at low pH. The results show a previously undisclosed antibacterial activity of HRGP and suggest that the histidine-rich and heparin-binding domain of HRGP mediates the antibacterial activity of the protein.

Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III

Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). The dissociation constant (K(d)), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. The conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K(d) values were 17 nM and 100 nM respectively. The CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K(d) values. The synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-d-glucosamine; G, beta-d-glucuronic acid; A*, N,3,6-O-sulphated alpha-d-glucosamine; I, 2-O-sulphated alpha-l-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500 Da heparin was displaced by protamine. In conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.

Novel Design of Peptides to Reverse the Anticoagulant Activities of Heparin and other Glycosaminoglycans

Patients undergoing anticoagulation with unfractionated heparin, low molecular weight heparin, or danaparoid may experience excess bleeding which requires reversal of the anticoagulant agent. Protamine is at present the only agent available for reversal of unfractionated heparin. Protamine is not effective in patients who have received low molecular weight heparin or danaparoid. We have developed a series of peptides based on consensus heparin binding sequences (Verrecchio et al., J Biol Chem 2000; 275: 7701-7707) that are capable of neutralizing the anti-thrombin activity of unfractionated heparin in vitro, the antifactor Xa activity of unfractionated heparin, Enoxaparin (Lovenox) and danaparoid (Orgaran) in vitro and the anti-Factor Xa activity of Enoxaparin in vivo in rats. These peptides may serve as alternatives for Protamine reversal of UFH and may be useful for neutralization of enoxaparin and danaparoid in humans.

Binding of a de novo designed peptide to specific glycosaminoglycans

The binding of glycosaminoglycans to a synthetic peptide (SKAQKAQAKQAKQAQKAQKAQAKQAKQW–CONH 2), consisting of a hybrid consensus heparin binding sequence, is studied using circular dichroism, fluorescence anisotropy and nuclear magnetic resonance techniques. The results unveil certain novel features, most importantly, the peptide binds preferentially to iduronic acid containing glycosaminoglycans and the dissociation constant for the peptide–heparin complex was found to be 30 nM. Interestingly, higher order intermolecular association(s)/aggregation was not observed, especially at saturating concentrations of the ligand. The helical structure of the peptide backbone, induced upon binding to a particular glycosaminoglycan is directly related to their binding affinity. In our opinion, studies on such unconventional hybrid peptide sequences containing low density basic amino acid residues would lead to the design of sequence specific glycosaminoglycan binding peptides.

Heparin-binding property of human prolactin: a novel aspect of prolactin biology.

Prolactin (PRL) shares several characteristics with growth factors and cytokines, many of which are known to bind to heparan sulfate proteoglycans. In this study we examined the heparin-binding properties of selected members of the PRL/GH family, using heparin affinity columns followed by gel electrophoresis/Western blotting. Purified human PRL and its cleaved 16K fragment, but not human GH or placental lactogen, were retained on the heparin column and were displaced by 0.5 M NaCl. Native PRL in human pituitary extracts and amniotic fluid showed a similar binding affinity to heparin as the purified hormone. None of the other hormones tested, e.g., rat, ovine and bovine PRL, glycosylated ovine PRL or rat GH, bound to heparin. Two consensus heparin-binding sequences are present in human PRL but not in the other hormones included in this study. We postulate that the heparin-binding capability of PRL affects its biological activity as a growth factor and the angiostatic actions of its 16K fragment.

Loss of a consensus heparin binding site by alternative splicing of latent transforming growth factor-β binding protein-1

Latent transforming growth factor-β binding protein-1 (LTBP-1), plays an important role in controlling localisation and activation of transforming growth factor-β (TGF-β). We show that alternative splicing generates a form of mRNA which lacks bases 1277–1435 (termed LTBP-1Δ53). The 53 amino acids encoded by these bases include the eighth cysteine of the first cysteine repeat and a consensus heparin binding sequence. Sequencing of genomic clones showed that alternative splicing resulted from the use of an intra-exonic 3′ splice acceptor site. The loss of the heparin binding site implies that LTBP-1Δ53 will bind to the extracellular matrix less efficiently than LTBP-1.

Analysis of Binding of Cobra Cardiotoxins to Heparin Reveals a New β-Sheet Heparin-binding Structural Motif

Heparin and heparan sulfate have recently been shown to bind to snake cardiotoxin (CTX) and to potentiate its penetration into phospholipid monolayer under physiological ionic conditions. Herein we analyze the heparin-binding domain of CTX using 10 CTXs from Taiwan and African cobra venom. We also performed computer modeling to obtain more information of the binding at molecular level. The results provide a molecular model for interaction of CTX-heparin complex where the cationic belt of the conserved residues on the concave surface of three finger beta-sheet polypeptides initiates ionic interaction with heparin-like molecules followed by specific binding of Lys residues near the tip of loop 2 of CTX. The dissociation constants of CTXs differ by as much as 4 orders of magnitude, ranging from approximately 140 microM for toxin gamma to approximately 20 nM for CTX M3, depending on the presence of Lys residues near the tip of loop 2. High affinity heparin binding becomes possible due to the presence of Arg-28, Lys-33, or the so-called consensus heparin binding sequence of XKKXXXKRX near the tip of the loop. The well defined three-finger loop structure of CTX provides an interesting template for the design of high affinity heparin-binding polypeptides with beta-sheet structure. The finding that several cobra CTXs and phospholipase A2 bind to heparin with different affinity may provide information on the synergistic action of the two venom proteins.