Structural Features Of Heparin Research Articles

Structural Features of Heparin and Its Interactions With Cellular Prion Protein Measured by Surface Plasmon Resonance.

Self-propagating form of the prion protein (PrPSc) causes many neurodegenerative diseases, such as Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker syndrome (GSS). Heparin is a highly sulfated linear glycosaminoglycan (GAG) and is composed of alternating D-glucosamine and L-iduronic acid or D-glucuronic acid sugar residues. The interactions of heparin with various proteins in a domain-specific or charged-dependent manner provide key roles on many physiological and pathological processes. While GAG-PrP interactions had been previously reported, the specific glycan structures that facilitate interactions with different regions of PrP and their binding kinetics have not been systematically investigated. In this study, we performed direct binding surface plasmon resonance (SPR) assay to characterize the kinetics of heparin binding to four recombinant murine PrP constructs including full length (M23–230), a deletion mutant lacking the four histidine-containing octapeptide repeats (M23–230 Δ59–90), the isolated N-terminal domain (M23–109), and the isolated C-terminal domain (M90–230). Additionally, we found the specific structural determinants required for GAG binding to the four PrP constructs with chemically defined derivatives of heparin and other GAGs by an SPR competition assay. Our findings may be instrumental in developing designer GAGs for specific targets within the PrP to fine-tune biological and pathophysiological activities of PrP.

Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis

Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis

Decorin endocytosis: structural features of heparin and heparan sulphate oligosaccharides interfering with receptor binding and endocytosis

Receptor-mediated endocytosis of decorin depends on its core-protein-mediated interaction with a 51 kDa membrane protein, which, in addition to its core-protein-binding site, carries a binding site for glycosaminoglycan chains. Membrane-associated heparan sulphate as well as heparin are known to have an inhibitory effect on decorin endocytosis by cultured skin fibroblasts. In this study, structural features of both glycosaminoglycans required for binding to the 51 kDa protein and for inhibiting decorin endocytosis, were investigated. Upon digestion of [(3)H]glucosamine-labelled heparan sulphate with heparinase III, dodeca- and higher saccharides were able to interact with the receptor protein. In comparison with unbound fragments of the same size, bound fragments were enriched in N-sulphated disaccharides carrying one or two sulphate ester groups. Using heparinase III-generated fragments from [(35)S]sulphate-labelled heparan sulphate chains, binding of fragments as small as octasaccharides could be detected. Competition experiments between dermatan sulphate and chemically modified heparin revealed that N- and 6-O-sulphation of glucosamine residues are important structural elements for binding to the receptor, whereas iduronate-2-O-sulphate groups contribute to binding only to a limited extent. However, with respect to the inhibition of decorin endocytosis, 2-O-desulphation had a quantitatively similar effect to 6-O-desulphation. Furthermore, for maximal inhibition of decorin endocytosis, longer fragments were required than for binding to the receptor. Thus, it appears that heparin/heparan sulphate has to interact with additional component(s) for effective inhibition of decorin uptake.

Differences in the susceptibility of herpes simplex virus types 1 and 2 to modified heparin compounds suggest serotype differences in viral entry

Although heparan sulfate (HS) serves as an initial receptor for the binding of both herpes simplex virus type 1 (HSV-1) and HSV-2 to cell surfaces, the two serotypes differ in epidemiology, cell tropism, and ability to compete for viral receptors in vitro. These observations are not necessarily contradictory and can be explained if the two serotypes recognize different structural features of HS. To compare the specific features of HS important for the binding and infection of HSV-1 and HSV-2, we took advantage of structural similarities between heparin and cell surface HS and compared the abilities of chemically modified heparin compounds to inhibit plaque formation. We found that the antiviral activity of heparin for both serotypes was independent of anticoagulant activity. Moreover, specific negatively charged regions of the polysaccharide, including N sulfations and the carboxyl groups, are key structural features for interactions of both HSV-1 and HSV-2 with cell surfaces since N desulfation or carboxyl reduction abolished heparin's antiviral activity. In contrast, 6-O sulfations and 2-,3-O sulfations are important determinants primarily for HSV- 1 infection. The O-desulfated heparins had little or no inhibitory effect on HSV-1 infection but inhibited HSV-2 infection. Using a series of intertypic recombinant mutant viruses, we found that susceptibility to O-desulfated heparins can be transferred to HSV-1 by the gene for glycoprotein C of HSV-2 (gC-2). This supports the notion that the envelope glycoproteins of HSV-1 and HSV-2 interact with different affinities for different structural features of heparin. To determine if the modified heparin compounds inhibited plaque formation by competing with cell surface HS for viral attachment, binding studies were also performed. As anticipated, most compounds inhibited binding and plaque formation in parallel. However, several compounds inhibited the binding of HSV-1 to cells during the initial attachment period at 4 degrees C; this inhibitory effect was reversed when the cells and inoculum were shifted to 37 degrees C. This temperature-dependent differential response to modified heparin compounds was evident primarily when glycoprotein C of HSV-1 (gC-1) was present in the virion envelope. Minimal temperature-dependent differences were seen for HSV-1 with gC-1 deleted and for HSV-2. These results suggest differences in the interactions of HSV-1 and HSV-2 with cell surface HS that may influence cell tropism.

Structural characterization of heparin's binding domain for human platelets

The structural features of heparin that are involved in binding to human platelets were investigated by a competitive binding approach. A range of heparin-derived glycosaminoglycans (GAGs) with relatively defined structure were prepared by different methods of depolymerization of pharmaceutical heparin, followed by fractionation according to molecular weight and net charge. Competitive binding to platelets was dependent on molecular weight but not on the net charge of the GAGs. The method for depolymerization significantly affected the binding activity of the resulting GAG. Heparinase I and nitrous acid depolymerization produced GAGs with lower binding affinity for platelets than those GAGs derived from the treatment with periodate followed by alkali. The IC 20 (concentration producing 20% inhibition of binding) was 0.05 μM for unfractionated heparin, 0.11 μM for a periodate treated GAG, and 2 μM for comparably sized GAGs (M r ∼ 6,000 – 8,000) derived by heparinase I or nitrous acid treatment. Thus, the disaccharide units GlcNSO 3-6S-IdoA-2S or GlcNSO 3--IdoA-2S [(2-deoxy-2-sulfoamido-6-O-sulfo-α-D-glycopyranosyl)-(1-4)-O-(2-O-sulfo-α-L-idopyranosyluronic acid) or (2-deoxy-2-sulfoamido-α-D-glycopyranosyl)-(1-4)-O-(2-O-sulfo-a-L-idopyranosyluronic acid)] may be crucial elements for binding to the platelet, because these are known to be preserved during periodate/alkali treatment, but readily decomposed by heparinase I and nitrous acid. Understanding this structural specificity for platelet binding may be useful for the development of heparins with high or low platelet reactivity.

Structural features of heparin and their effect on heparin cofactor II mediated inhibition of thrombin

Heparins from different species and tissues show similar levels of ATIII and HCII mediated anti-IIa activities. On fractionation, chains containing predominantly ATIII or HCII activities could not be separated. Oligosaccharide mapping demonstrates that the concentration of an oligosaccharide comprising a portion of heparin's ATIII binding site in a particular heparin fraction correlates with ATIII mediated anti-IIa activity, but does not correlate with HCII mediated anti-IIa activity. These results suggest that ATIII and HCII do not share a common binding site. Partial enzymatic depolymerization of heparin resulted in large oligosaccharides which could be purified and partially characterized. Although oligosaccharides of degree of polymerization (dp) 18 and 20 showed significant ATIII and HCII mediated anti-IIa activities no separation of these activities resulted. These data suggest however that a minimum chain length of dp18 was required for HCII mediated anti-IIa activity.