Study On Sequence-Bioactivity Relationship Of Heparan Sulfate

Proteoglycans are a class of macromolecules that are found in all connective tissues,extracellular matrix(ECM)and on the surfaces of mammalian cells.The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan(GAG)chain(s).GAGs are highly negatively charged,linear and long chain of carbohydrate polymers.Native GAGs are devided into 4 categories:heparan sulfate(HS)and heparin,chondroitin sulfate and dermatan sulfate,keratan sulfate and hyaluronic acid.HS proteoglycans form a subgroup of proteoglycans that carry one or more HS polysaccharide chains,which are essential for their important biological functions.Heparin is an intracellular glycosaminoglycan that is found mainly in mast cell.Heparin is similar with HS in structural characteristics,they are both comprised of repeating disaccharide unit of glucuronic acid residue(GlcA)or iduronic acid residue(IdoA)1,4-linked to a glucosamine residue(GlcN),with various substitution patterns of sulfation at the 2-O-position of the hexuronic acid residue(HexA),the 3-O,the 6-O and/or the N-position of GlcN,and N-acetylation at GlcN,but HS have less sulfate groups than heparins.The binding of heparin/HS to protein ligands is primarily based on electrostatic interactions between the highly negatively charge of heparin and HS and the positive charge of the protein.It is generally believed that the interaction of heparin/HS with protein ligands is mainly dependent on the number of carboxyl and sulfate substitution groups and the position of the sulfate substitution groups in the polysaccharide chains.Since the 1930s,heparin has been successfully used as an injectable anticoagulant for the prevention and treatment of thrombotic diseases.The interaction of heparin with antithrombin(ATIII)is the most clear and representative example of heparin-protein ligand binding.The binding of heparin to ATⅢ is referred to as a "lock-and-key"binding mode that relies primarily on the specific pentasaccharide sequence(-GlcNS(or Ac)6S-GlcA-GlcNS3S±6S-IdoA2S-GlcNS6S-)in the heparin chain that contains a particular 3-0-sulfated GlcN.Heparin binds to ATIII via a specific pentasaccharide sequence,causing the comformational change of ATIII,and then the activitaed ATIII increases the inhibitory activity on factor Xa(FXa),thrombin(FIIa)and other proteases.Low molecular weight heparins(LMWHs)are manufactured by chemical or enzymatic depolymerization of heparin,which share the same monosaccharide composition,the sulfate and acetyl group substitution forms,and the oligosaccharide sequence as the parent heparin,and are a class of anticoagulants with lower molecular weight.Compared to heparin,LMWHs don’t show the pharmacokinetic limitations like heparin as anticoagulant drugs.LMWHs are overtaking the market share of heparin as new anticoagulant and antithrombotic drugs in particular due to the longer half-life,predictable anticoagulant reaction and the lower adverse reaction efficiency such as heparin-induced thrombocytopenia(HIT).LMWHs have unique structural characteristics as carbohydrate drugs compared with traditional small molecule drugs and biological macromolecular drugs.LMWHs are composed of complex oligosaccharides mixtures with different chain lengths,monosaccharide composition,the sulfate and acetyl group substitution patterns and sequences.The importance of structural characterization of heparin and LMWHs was not taken seriously until the global heparin contamination event occurred in 2008.Traditional analytical methods,including molecular weight(MW)analysis by gel permeation chromatography,monosaccharide composition analysis and bioactivity analysis,are not enough to reflect the fine structure of the polysaccharide drugs and ensure its clinical safety and efficacy.Since the approval of the first generic LMWH by the US Food and Drug Administration(FDA)in 2010,in order to ensure structural consistency and drug safety of the LMWHs generic drug,an urgent need for developing structural profiling methods has arisen for the comparison between innovator and generic LMWHs.The heterogeneity of LMWHs can be determined from the following five aspects:(1)physicochemical properties,(2)high-resolution intact chain mapping,(3)fragment mapping,(4)disaccharide building blocks and(5)the sequence of oligosaccharide species.Traditional analytical methods including high-performance liquid chromatography(HPLC),gel electrophoresis,capillary electrophoresis(CE)and nuclear magnetic resonance spectroscopy(NMR)are not sufficient to provide precise structural information on the individual components in LMWHs.Mass spectrometry(MS)techniques,especially electrospray ionization(ESI)-MS linked to LC or CE(LC/CE-ESI-MS),are therefore indispensable in the characterization of LMWHs,especially for various chain mapping analyses and sequence analysis methods.Two analysis strategies such as "top-down" and "bottom-up",are similar to proteomic analysis,have been successfully used for the structural characterization of LMWHs."Top-down" and "bottom-up"provide the basic component information of LMWHs such as chain mapping,fragment mapping and basic building blocks,furthermore,the structural information obtained from each individual oligosaccharide including the number of uronic acid(HexA)and GlcN,the terminal structure(reducing end and non-reducing end)and the number of sulfate and acetyl group substitutions.The biological activity of HS and heparin are particularly dependant on the specific binding sites provided by unique oligosaccharide sequences and sulphation pattern in polysaccharides chain that can interact with various protein ligands.The sequence analysis of these specific oligosaccharide structure is indispensable and important for understanding the interaction machanisms of GAG-protein.The most notable example to date is the antithrombin-binding pentasaccharide sequence containing a unique 3-O-sulphated glucosamine.In recent years,studies on the fibroblast growth factor-2(FGF-2)have shown that IdoA2S and GlcNS of HS are essential for recognition of FGF-2,and its activation by HS also relies on the presence of 6-O-sulphate groups.The binding of HS and heparin to other growth factors,such as FGF-1,FGF-4,hepatocyte growth factor and vascular epithelial cell growth factor,has also been shown to be dependant on the specific structural features of HS/heparin,indicating the existence of distintic ligand sites within HS chains.In addition,during the depolymerization of LMWHs from heparin,the majority of antithrombin Ⅲ-binding pentasaccharide domains are retained,while the longer factor Ⅱa binding domains are more likely destroyed.Heparin-induced side effects,HIT,also proved to be closely related to the oligosaccharide sequence in heparin polysaccharide chains.In order to understand the mechanism of GAGs-protein ligand interaction and ensure the drug safety and clinical efficacy of LMWHs,there is an urgent need to develop desice analysis techniques to sequence the specific oligosaccharide domains in the polysaccharide chains.However,the exact structural information of the interaction between HS/heparin and protein ligands are not available due to a lack of analytical methods for the precise structure chracterization of GAGs.One of the objectives of this paper is to develop an analytical method to characterize the sequence of the shorter oligosaccharides of LMWHs,and further lay the foundation for the sequence characterization of HS involving in GAG-protein interaction.HS and heparin isolated from natural sources are highly heterogeneous mixtures,which are composed of oligosaccharides with various chain lengths and substitution patterns of sulfation.The availability of structurally defined HS and heparin oligosaccharides provides the opportunity to investigate the casual relationship between saccharide sulfation/conformation and biological functions,a major step forward in dissecting the structure and function relationship of HS.The development of HS-based drugs with certain size and number of sulfation patterns remains a challenge.A number of oligosaccharides are synthesized via a purely organic synthetic approach,a pentasaccharide with anticoagulant activity,fondaparinux sodium(Arixtra)has been synthesized by chemical synthesis,however,it is still very difficult to synthesize oligosaccharides larger than hexasaccharides with complex sulfation patterns because the compolicated group protection and deprotection steps during chemical synthesis process.Therefore,the chemical synthesis of HS oligosaccharides is only performed by skilled synthetic chemists in a few highly specialized laboratories.In recent years,a chemoenzymatic synthesis method,using HS biosynthetic enzymes,including glycosyltransferases,C5-epimerase,and sulfotransferases to synthesize HS oligosaccharides has emerged.Chemoenzymatic synthesis method stimulates HS biosynthesis pathways in vivo to synthsize the HS oligosaccharides with desired biological activity.However,the synthesis of certain oligosaccharide sequences is not yet possible due to a lack of understanding of the substrate specificities of HS biosynthetic enzymes.Another important goal of this dissertation is to explore the substrate specificity of 3-O-sulfotransferase(3-OST-1 and 3-OST-3)during HS biosynthesis and to verify the biological activity of its modified substrate.The main research results of this thesis are as follows:1.An offline SAX-ESI-MS/MS method for the sequence analysis of dalteparin short oligosaccharide was established.We combined an offline strong anion exchange(SAX)-high performance liquid chromatography(HPLC)with ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin.The oligosaccharide mixtures were separated from dalteparin using size exclusion chromatography(SEC).And then,the obtained oligosaccharides with sizes equal or smaller than octasaccharides were further separated using high resolution SAX-HPLC.Finally,the individual chains were sequenced using ESI-MS/MS.With the help of in-house developed MS/MS interpretation software,the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained.Interestingly,we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis.This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization,and further lay the foundation for the sequence characterization of HS involving in GAG-protein interaction.2.Chemoenzymatic synthesis and structural characterization of 3-O-sulfated HS oligosaccharidesAs a rare modification present in HS,the availability of 3-O-sulfated oligosaccharides is very limited.In this study,we investigated the substrate specificity of 3-OST-1 and 3-OST-3 using structurally defined HS oligosaccharides.According to the substrate specificity difference between 3-OST-1 and 3-OST-3,we rearranged enzymatic modification sequences to accommodate the substrate specificity of 30ST-3 and synthesize novel 3-O-sulfated oligosaccharides,including three hexasaccharides and three octasaccharides.And then,the synthesized HS oligosaccharides were separated and characterized by HPLC,high resolution MS and nuclear magnetic structure verification(NMR).In this study,we further explored the effect of 3-0-sulfation on the conformation of IdoA2S using synthetic 3-O-sulfated HS oligosaccharides,which provides a basis for understanding the molecular mechanism of HS interaction with protein ligands.In this study,we demonstrated that different chemoenzymatic schemes are required for 3-OST-1 and 3-OST-3 to prepare different 3-O-sulfated oligosaccharide sequences,witch further expand 3-O-sulfated oligosaccharide library.And we investigated the impact of 3-O-sulfation on the conformation of the pyranose ring of IdoA2S(1C4 and 2So)within HS chains.3.The biological activities of 3-OST-3 modified HS oligosaccharides were studiedThe biological activities of 3-OST-3 modified HS oligosaccharides were studied in vivo and vitro,respectively.The biological activities of synthesized HS oligosaccharides were evaluated using anticoagulant activity(anti-FXa activity).The anticoagulant activity of oligosaccharides was confirmed in an in vivo experiment using a rat model.Each group was intravenously administered with either endotoxin-free fondaparinux,synthesized oligosaccharides or saline.Blood was then drawn at specified time points after the compounds had been administered,and the treated blood samples were subjected to FXa activity analysis.We also performed the anti-FXa activity assay of synthesized oligosaccharides in vitro.According to the study of biological activity,we identified a novel 3-OST-3 modified octasaccharide that interacts with antithrombin and displays anti-factor Xa activity.The drug concentrations in the blood sample were also obtained to determine the clearance rate of each compound in vivo.The results suggest that 3-OST-3 modified HS octasaccharide with anticoagulant activity shows a faster clearance in vivo than fondaparinux in the rat model,making this octasaccharide a potential short-acting anticoagulant drug candidate that could reduce bleeding risk.Having access to a set of critically important 3-O-sulfated oligosaccharides provides a foundation for understanding specificity of interaction between the HS and proteins,and further offers the potential to develop new heparan sulfate-based therapeutics.