Structural Characterization Of Heparin/Heparan Sulfate And Omics Investigation Of Heparan Sulfate-Binding Proteins

Heparin and heparan sulfate(HS)are major classes of glycosaminoglycans with complex structure.The disaccharide backbones of heparin and HS consist of hexuronic acid(either glucuronic or iduronic acid)and glucosamine,connected by a β-1,4 glycosidic bond.Additionally,the disaccharides in HS can undergo various modifications such as sulfation and acetylation.The variability in the location and numbers of these modifications contributes to the structural complexity and heterogeneity of HS.Predominantly found on mammalian cell membranes and in the extracellular matrix,HS engages in interactions with various proteins to execute diverse biological functions.Heparin,a structural analog of HS,contains similar disaccharide units but features a higher degree of sulfation.Heparin is primarily located in mast cells within connective tissues and possesses anticoagulant activities.Currently,heparin and its derivative,low molecular weight heparin(LMWH),are widely employed as anticoagulants in clinical.Due to the non-template-driven biosynthesis of HS and heparin,structural elucidation is highly challenging.This complexity and heterogeneity make a detailed characterization crucial for ensuring the safety of heparin and LMWH as anticoagulants.Moreover,HS serves vital biological roles in processes such as cell proliferation,differentiation,angiogenesis,and carcinogenesis,primarily through its interactions with proteins.The heterogeneous nature of HS allows it to bind to proteins with varying affinities and mechanisms,making the structural analysis of HS oligosaccharides essential for drug development and understanding its biological roles.The structural characterization of heparin and LMWH glycan chains mainly includes two strategies,"bottom-up" and "top-down",as well as liquid chromatography,mass spectrometry,capillary electrophoresis and other techniques.For the study of interactions between HS and specific proteins,various methods are commonly employed,including hydrogen-deuterium exchange mass spectrometry,chemical cross-linking mass spectrometry,surface plasmon resonance,and other techniques.Additionally,high-throughput approaches,such as microarray and affinity proteomics,can also be used to analyze proteins that interact with HS.Despite the numerous available techniques for investigating the interactions between HS and proteins,establishing a comprehensive HS protein interaction map remains a significant challenge.The primary objectives of this work include two parts:the first part focuses on developing analytical methods for identifying and quantifying low molecular weight heparin glycans,aiding in their analysis and quality control;the second part employs proteomics to investigate the roles of HS and HS-binding proteins(HSBPs)in human diseases.Key areas of study include:1.MsPHep:An online application for low molecular weight heparin rapid characterization based on liquid chromatography-tandem mass spectrometryLow-molecular-weight heparins(LMWHs)are important anticoagulants widely used in clinic.Since they are comprised of complex and heterogenous glycan chains,high performance liquid chromatography-tandem mass spectrometry(HPLC-MS)is commonly used for structural analysis and quality control of LMWHs to ensure their safety and efficacy.Yet,the structural complexity arising from the parent heparin macromolecules,as well as the different depolymerization methods used for preparing LMWHs,makes processing and assigning the HPLC-MS data of LWMHs very tedious and challenging.We therefore developed,and here report,an open-source and easy-to-use web application,MsPHep,to facilitate the LMWH analysis based on HPLC-MS data.MsPHep is compatible with various LMWHs and chromatographic separation methods.With the HepQual function,MsPHep is capable of annotating both the LMWH compound and its isotopic distribution from mass spectra.Moreover,the HepQuant function enables automatic quantification of LMWH compositions without prior knowledge or any database generation.To demonstrate the reliability and system stability of MsPHep,we tested various types of LMWHs that were analyzed with different chromatographic methods coupled to MS.The results show that MsPHep has its own advantages compared to another public tool GlycReSoft for LMWH analysis,and it is available online under an open-source license at https://ngrc-glycan.shinyapps.io/MsPHep.2.Glycan-protein interactome reveals the ameliorative effects of heparan sulfate on atherosclerosisEndothelial dysfunction induced by oxidative stress is an early predictor of atherosclerosis,causing various cardiovascular diseases.The glycocalyx layer on the endothelial cell surface acts as a barrier to maintain endothelial biological function,and it can be impaired by oxidative stress.However,the mechanism of glycocalyx damage during the development of atherosclerosis remains largely unclear.Herein,we established a novel strategy to address these issues from the glycomics aspect that has long been neglected.Using counter-charged fluorescence protein staining and quantitative mass spectrometry,we found that heparan sulfate,a major component of glycocalyx,maintained the fuzzy structure of glycocalyx through its negative charges,but this structure collapses under oxidative stress.Comparative proteomics and protein microarray analysis reveals several new HSBPs,from which alpha-2-HeremansSchmid glycoprotein(AHSG)was identified as a critical protein.We investigated the mechanism of interaction between HS and AHSG using different techniques and examined the potential application of non-anticoagulant heparin for the treatment of atherosclerosis.The results showed non-anticoagulant heparin could relieve atherosclerosis by protecting HS from degradation during oxidative stress and by reducing the accumulation of AHSG at lesion sites.These findings provide new insights to understanding glycocalyx damage in atherosclerosis and developing corresponding therapeutics.3.Functional study of heparan sulfate in hepatocellular carcinomaLiver cancer is a global health challenge,with hepatocellular carcinoma(HCC)being the predominant type of liver cancer.Currently,HCC has been found to be associated with mutations in a variety of genes,including CTNNB1,ALB,and APOB,through genomic,phosphatomic,and proteomic analyses.And many of these proteins are HSbinding proteins reported in the literature.To understand the potential application of heparin in HCC treatment,two HCC cell lines,HepG2 and Huh7,were used in this chapter,and the inhibitory effects of heparin administration treatment on HepG2 and Huh7 were investigated in terms of changes in HS glycan chains on the cell surface after administration treatment;HSBPs,which underwent significant expression after heparin treatment,were mined through proteomes,and further mined through the HCC public database for possible HCC biomarkers.We first treated two hepatocellular carcinoma cell lines,HepG2 and Huh7,with the ati-tumor drug sorafenib,and the results showed that the HS structure on the surface of sorafenib-treated hepatocellular carcinoma cells was altered.Meanwhile,we found that heparin could inhibit the proliferation of hepatocellular carcinoma cells,and by proteomic analysis we identified HS-binding proteins that were differentially expressed after heparin treatment,including ALB,GPC3,GFA,ANXA2,AGRN,and SORT1.In this chapter,we investigated the possible targets of action of heparin to inhibit the proliferation of hepatocellular carcinoma cells from the HS oligosaccharide structure and proteomic level,and showed that the targets of action of heparin on different hepatocellular carcinomas are not consistent,and the results of the study can help to develop heparin analogs for HCC treatment,and to optimize different therapeutic regimens for different types of HCC.