The preparation and characterization of a heparin-derived oligosaccharide that binds to herpes simplex virus type 1 glycoprotein D.

摘要

Heparan sulfate (HS) is a structurally diverse and highly sulfated polysaccharide that has been found to exist on the surface of mammalian cells in substantial quantities. Unique saccharide sequences of HS have been shown to bind specifically to a number of biologically relevant proteins, thus allowing HS to play a role in numerous biological processes including regulation of blood coagulation, inflammation, cancer cell growth and viral infections. Understanding the structure-function relationship of HS will aid in the development of novel anti-viral and/or anti-cancer therapeutics. Previous studies have shown that 3-O-sulfotransferase isoform 3 (3-OST-3) generates 3-O-sulfated HS that can bind to glycoprotein D (gD) and facilitate HSV viral entry into target cells, thus implicating 3-O-sulfated HS as a HSV entry receptor. The goal of this work is to provide additional structural information concerning HS ability to assist in the HSV viral infection mechanism, while providing evidence to suggest that HSV infections may be inhibited by disrupting the interactions with its polysaccharide based cellular receptors. The use of 3-O-sulfated heparin (HP) oligosaccharides, along with high expression levels of gD purified from E. coli., allowed for the investigation of the gD binding of various sized HP oligosaccharides. Results obtained from immunoprecipitation and affinity co-electrophoresis experiments suggested that the 3-O-sulfated HP octasaccharide was of the minimal required length for gD binding with a Kd value of 19 muM. Structural characterization using chemical and enzymatic approaches suggested the gD binding 3-O-sulfated HP octasaccharide had a structure of DeltaUA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS6S 3S-IdoUA2S-GlcNS6S (3-O-sulfation site is underlined). Coupling a sulfo donor regeneration system with 3-OST-3 modification, sufficient amounts of the gD binding 3-O-sulfated HP octasaccharide was generated for cell based viral entry assays. The characterization of a novel gD binding octasaccharide as described herein, provides additional structural information concerning HS/HP ability to assist in the HSV viral entry mechanism. Specifically, it allows for further investigations to be conducted as the characterized 3-O-sulfated gD binding HP octasaccharide may serve as a good lead compound for the inhibition of HSV infections. The further development of this project could uncover a new way to treat diseases related to HSV infections.