Emadoldin Feyzi

Structural Requirement of Heparan Sulfate for Interaction with Herpes Simplex Virus Type 1 Virions and Isolated Glycoprotein C

Cell surface heparan sulfates mediate primary attachment of herpes simplex virus type 1, the first step in virus invasion of the cells. Removal of the host cell heparan sulfate results in a significantly diminished susceptibility of the cell to virus infection. On the virus envelope, glycoprotein C has been identified as the major binding site for heparan sulfate in the primary attachment of the virus to host cells. Using selectively desulfated heparins and metabolically labeled host cell heparan sulfate, we have analyzed the structural requirements of heparan sulfate to provide binding sites for glycoprotein C and the whole virus. Employing glycoprotein C affinity chromatography and a virus binding assay, we subfractionated oligosaccharides derived from heparan sulfate and partially desulfated heparin into selectively bound and unbound pools. These were chemically depolymerized and analyzed at the disaccharide level. The shortest glycoprotein C-binding fragment consisted of 10–12 monosaccharide units containing at least one 2-O- and one 6-O-sulfate group that have to be localized in a sequence-specific way, based on the finding that bound and unbound HS fragments do not differ in charge or composition. The binding sequence is found within N-sulfated blocks of heparan sulfate, although several N-acetyl groups can be tolerated within the minimal binding sequence. These minimal requirements for herpes simplex virus type 1 binding to heparan sulfate are clearly distinct from other identified protein binding sites.

Age-dependent Modulation of Heparan Sulfate Structure and Function

Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated l-iduronic acid (2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.

Characterization of Heparin and Heparan Sulfate Domains Binding to the Long Splice Variant of Platelet-derived Growth Factor A Chain

Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.

S5.9 Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.