Study On The Specific Domains In Heparin Responsible For Binding To Crucial Proteins In Diseases

Glycosaminoglycans are a family of linear and negatively charged polysaccharides that exist ubiquitously on the human cell surface as well as in the extracellular matrix.Glycosaminoglycans interact with a wide range of proteins,including proteases,growth factors,cytokines,chemokines and adhesion molecules,enabling them to mediate many physiological processes,such as protein function,cellular adhesion and signaling.Glycosaminoglycanprotein interactions participate in and intervene in a variety of human diseases,including cardiovascular disease,infectious disease,neurodegenerative diseases and tumors.These interactions have been studied for decades but still lag behind the study of protein-protein and protein-nucleic acid interactions due to the structural complexity of glycosaminoglycans and limitations of analytical tools.In this dissertation,the analytical methods and molecular mechanisms of the interactions between glycosaminoglycans(such as heparin)and crucial proteins in diseases were’ studied.The main contents and conclusions of this dissertation are as follows:(1)Many glycosaminoglycan-protein interactions remain undiscovered since there is insufficient structural information on the interacting glycosaminoglycans.We used heparin,the typical glycosaminoglycan,to develop a method and system for sequencing oligosaccharide mixtures,and we also designed a sequencing kit and a Seq-GAG software.The method,kit,and software are based on the basic building blocks of heparin chains obtained through mass spectrometry,and then all the theoretical sequences and relative content can be calculated and provided.This system can be used to analyze complex heparin oligosaccharide mixtures and achieve rapid analysis of heparin drugs.(2)Due to the inherent heterogeneity of glycosaminoglycans,multiple oligosaccharides,containing certain common sequence domains,often can interact with clusters of basic amino acid residues on a target protein.We established a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity glycosaminoglycan oligosaccharides leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein.As a proof of concept,antithrombin Ⅲ(AT)-binding oligosaccharides were examined,resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence with extended reducing end and nonreducing end.Repeating this approach,a new highly sulfated symmetric pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-y(IFNy).The molecular mechanism of the interaction between heparin and IFNy was also elucidated.Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel glycosaminoglycan-based therapeutics.(3)In addition to biological activities,glycosaminoglycan can also cause severe adverse clinical reactions by binding to some proteins,and the typical case is the interaction between heparin and platelet factor 4(PF4).The ultra-large complexes formed by PF4 with heparin or low molecular weight heparins(LMWHs)are important participants in inducing the immune response and heparin induced thrombocytopenia(HIT).Nonetheless,adequate detail about the interaction of PF4 with long-chain heparin oligosaccharides and robust analytical methods are still currently lacking.The characteristics of PF4-enoxaparin complexes were analyzed by multiple analytical methods,especially liquid chromatographymass spectrometry(LC-MS)and LC-MS/MS multiple reaction monitoring(MRM)were developed to qualitatively and quantitatively monitor heparin oligosaccharides and PF4 in HITinducing complexes.A binding model of PF4 and enoxaparin in ultra-large complexes is proposed for the first time with one heparin oligosaccharide chain(～dp 18)bound to two PF4 tetramers in different morphologies to form ultra-large complexes,while PF4 tetramer is surrounded by multiple heparin chains in smaller complexes.Our study provides new insights into the structural mechanism of PF4-LMWH interaction,which help to further understand the mechanism of LMWH immunogenicity and develop safer heparin products.(4)Heparan sulfate,located on the surface of host cells,plays an important role for virushost cell interaction.The monkeypox virus and SARS-CoV-2,which caused the global epidemic in recent years,were taken as examples to study the important roles of glycosaminoglycans in virus invasion.The first case is the interaction between monkeypox virus and glycosaminoglycans.We demonstrated that monkeypox virus A29 protein bound to glycosaminoglycans including heparin and chondroitin sulfate/dermatan sulfate using surface plasmon resonance.The negative charges on glycosaminoglycans are important for glycosaminoglycan-monkeypox virus A29 interaction.Glycosaminoglycan analogs,pentosan polysulfate and mucopolysaccharide polysulfate,show strong inhibition of monkeypox virus A29-heparin interaction.Another case is the interaction between SARS-CoV-2 and glycosaminoglycans.Phylogenetic tree of variants and mutations in Spike protein receptorbinding domain of Omicron BA.2.12.1,BA.4 and BA.5 were described.Solution competition studies by surface plasmon resonance revealed that different Omicron sub-lineages tend to bind heparin with different chain lengths and sulfation patterns.Furthermore,blind docking experiments showed the contribution of basic amino acid residues in receptor-binding domain and sulfo groups and carboxyl groups on heparin to the interaction.Finally,pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and Spike protein receptor-binding domain of Omicron BA.2.12.1,BA.4/BA.5,and both showed much stronger inhibition than heparin.In conclusion,through the analysis of typical cases,this dissertation solved the key problems involved in the study of heparin-protein interaction,enriched the understanding of the structure and function of glycosaminoglycan,and is of great significance for the development of glycosaminoglycan drugs.