Study Of The Interaction Between Heparin Oligosaccharides And Interferon ?

Interferon ?(IFN-?)is a pleiotropic cytokine with antiviral,antitumor activity and immunomodulatory effects.In recent years,more and more studies have shown that IFN-? plays a dual role in the development of inflammation,atherosclerosis and tumors.For example,IFN-? can promote tumorigenesis and angiogenesis leading to tumor immune escape.Under certain circumstances,effective inhibition of IFN-?activity may provide new strategy for the treatment of certain diseases.Studies have shown that heparin can effectively inhibit the activity of IFN-? and become the potential active inhibitors of IFN-?.Heparan sulfate(HS),a highly sulfated linear polysaccharide,is ubiquitous on the mammalian cell surface and extracellular matrix.Electrostatic interactions can be generated between the highly sulfated HS region and specific basic amino acid residues in the IFN-? structure,thereby detaining IFN-y on the cell surface,and the concentration of IFN-? on the cell surface is thus,changed.IFN-? retained on the cell surface will optimize the binding of IFN-? to the transmembrane receptor resulting in high efficiency signalling.Studies have shown that heparin can interact with IFN-? in the extracellular matrix,effectively inhibit the activity of IFN-? by block the above process,and mediate many diseases related to IFN-?.However,the activity of this inhibitory effect is strongly related to the heterogeneity of the fine structure of heparin.Therefore,studying the structure-activity relationship of the interaction between heparin and IFN-? is the basis for a comprehensive understanding of the mechanism of this inhibitory effect.At present,most of the research results are focus on the structure of IFN-?,there are few reports on the influence of heparin structural differences on the interaction between IFN-? and heparin,especially the lack of molecular mechanisms of the influence of the fine structure of the heparin sugar chain on the interaction between the two.Studying the structure-activity relationship of the interaction between heparin oligosaccharides and IFN-? at the molecular level can be the basis for synthesizing heparin oligosaccharides with certain structure and achieve the purpose of controlling the inhibition of certain activities of IFN-? by heparin.This cannot only provide new treatment strategies for diseases such as inflammatory,autoimmune diseases,allograft rejection and cancer,but also explore other potential therapeutic effects of the classic anticoagulant—heparin.The purpose of this project is to study the effect of differences in heparin structure on the interaction between heparin and IFN-y,and to analyze the structure-activity relationship of the interaction between heparin oligosaccharides and IFN-? at the molecular level,so as to provide a theoretical basis for the development of related inhibitors.Gel mobility shift assay(GMSA)/Isothermal titration calorimetry(ITC)/Molecular docking(MD)were combined for the first time to study the effect of the fine structure of heparin chain on its interaction with IFN-? at the molecular level.Heparin oligosaccharides were prepared by enzymatic hydrolysis.And three heparin tetrasaccharides and four heparin hexose were obtained by a series of separation and purification steps,including gel permeation chromatography(GPC),strong anion exchange-high performance Liquid chromatography(SAX-HPLC)and G10 desalination.And the structures of the oligosaccharides were characterized by nuclear magnetic resonance spectroscopy(NMR)and mass spectrum(MS).The interaction and binding ratio between heparin oligosaccharides and IFN-y were preliminarily determined by GMSA.The differences in affinity of the interaction between IFN-y and heparin oligosaccharides with different chain length and sulfation pattern were determined by ITC,comparing the structural differences of the heparin oligosaccharides.ITC data indicate that in the oligosaccharides with the same degree of polymerization,the 6S position of the reducing terminal GlcNS6S play the most important role in the interaction,while the 2S group of the terminal IdoA2S can further enhance the binding ability of 6S.The 6S of GlcNS6S in the intermediate disaccharide unit is still the key group to enhance the binding ability as the degree of polymerization increases.In the interaction between heparin and C-IFN-y,the sulfation pattern of certain oligosaccharide sequence is more important than the sulfation degree.Simply increasing the degree of polymerization of sugar chains does not necessarily enhance the binding effect.Through molecular docking(MD)simulation,the interaction between heparin and IFN-y is more intuitively explained,and the important role of GlcNS6S in the interaction between heparin and IFN-? is further verified.