| Glycosaminoglycans(GAGs)are a class of biological macromolecules with extremely high heterogeneity and complex structure,which are widely present on the cell surface and in extracellular matrix of mammals.The backbone of GAGs is mainly composed of repeated disaccharide units linked by hexuronic acid or galactose and hexosylamine.GAGs can be divided into four types,including heparin/heparan sulfate(HS),chondroitin sulfate/dermatan sulfate(DS),keratan sulfate and hyaluronic acid according to the glycosidic bond connection form,the degree of sulfation and the position of sulfation.Compared with other GAGs,heparin and HS have more variable sulfation sites and high negative charge density,and participate in the regulation of various biological processes in the organism,making them the most complex and important polysaccharides in GAGs family.Due to the large molecular weight(MW)and complex structure of GAGs chains,it is very difficult to characterize the structure of polysaccharides.At present,most analysis methods are focused on the analysis of polysaccharide components or oligosaccharide fragments.For high MW components of GAGs,it is difficult to be specifically characterized due to the low resolution of chromatogram techniques.However,the interaction between GAGs and their target proteins usually requires a specific chain length.Therefore,the first goal of this research is to develop a structural characterization method for the GAGs chains with high MW,which can provide a powerful tool for directly elucidating the structure of these relatively large GAGs chains with more important biological significance.GAGs play an important role in many biological processes,including cell signal transduction,recognition,migration and adhesion.GAGs are also involved in the occurrence and development of a variety of diseases in the human body,such as tumors,inflammations and infectious diseases.In addition,GAGs have a wide range of biological activities,including anti-inflammatory,anticoagulant,anti-tumor metastasis and control of angiogenesis.Besides being an important bioactive substance,GAGs have also been widely used in the clinical practice for decades.In addition,efforts to develop novel GAG drugs for the treatment of various acute or chronic diseases are also continuously ongoing worldwide.These important functions of GAGs mainly depend on the interaction between GAGs and proteins.The interaction between GAGs and proteins is achieved through the specific recognition of the characteristic sequences in their structures.For example,the anticoagulant effect of heparin is achieved by the specific binding of a pentasaccharide sequence containing 3-O-S in the chain with antithrombin III.Therefore,elucidating the key sequences and molecular mechanisms in the interaction between GAGs and proteins is essential for finding new therapies that specifically target GAGs-protein interactions.The second goal of this research is to develop a set of strategies for the molecular mechanism of interaction between GAGs and proteins using mass spectrometry(MS).We chose heparin and its interacting protein interferon-y(IFN-y)as the model research object to clarify the molecular mechanism of the interaction between heparin and IFN-y in terms of chain length and the characteristic sequence of heparin.This research provides a fast and accurate analysis strategy for more research on the interaction between GAGs and proteins.The main research results of this thesis are as follows:1.A polyacrylamide gel electrophoresis(PAGE)combined with MS method for the analysis of GAGs chain was established.PAGE is one of the powerful tools to characterize the structure and properties of GAGs.Compared with liquid chromatography,it has a wider range of chain MW distribution.However,the application of PAGE is very limited due to the failure to provide chain structure information.Therefore,we applied the in-gel protein digestion strategy to the analysis of GAGs.A new GAGs extraction method was established by using the characteristics of polyacrylamide gel that can syneresis and swell with water,which combines PAGE and ultra-sensitive LC-MS/MS to characterize the GAGs chains.Through this method,we carried out a comprehensive structural analysis of heparin.The results of silver staining showed that the distribution of heparin by PAGE analysis was continuous,and there was no obvious boundary between the bands.The heparin band was divided into 20 equal segments for in-gel enzymatic hydrolysis and LC-MS/MS analysis respectively.Among the 12 segments of the gel,8 disaccharides derived from the backbone structure and 3 saturated disaccharides derived from the non-reducing end(NRE)were detected.According to the MW and ratio of saturated disaccharides and backbone disaccharides,the MW of polysaccharides was calculated.In PAGE analysis,the MWs of heparin ranges from 3.9 kDa to 46.8 kDa,with an average MW of 14.4 kDa,and the migration distance was inversely proportional to the logarithm of the MW.The disaccharide composition of heparin oligosaccharides was basically the same in all 12 gel zones,indicating that the separation of heparin oligosaccharides in PAGE is not affected by the negative charge density,and distribute evenly and continuously according to the MW.We also compared the disaccharides obtained by direct enzymatic hydrolysis and in-gel digestion methods.The composition and content of disaccharides obtained by these two methods are basically the same,indicating that the PAGE-MS method can ensure the structural information of GAGs chains is complete.In addition,the method validation showed that the PAGE-MS method also has good reliability and repeatability.The PAGE-MS method combines the high separation ability of PAGE with the high sensitivity and high information content of MS,which provides a powerful tool support for the characterization of heparin/HS polysaccharides from different sources and the structural consistency evaluation of heparin drugs.2.An agarose-gel electrophoresis(AGE)combined with MS method for the analysis of GAGs chain was established.AGE is another electrophoresis method that is often used for direct separation and analysis of mixed GAGs samples.However,due to the different gel properties,it is difficult for heparinase to enter the agarose gel,and the in-gel digestion method is not suitable for AGE.Therefore,we developed a sample extraction method for AGE.This method is based on the characteristics of agarose gel that can be melted at relatively low temperature.The AGE strips were heated and melted into liquid,and then recovered by the strong anion exchange(SAX)spin column and treated with GAGs enzyme.The obtained components were analyzed qualitatively and quantitatively by LC-MS/MS,and the structure of each component was characterized.Using this method,we selected a representative multi-component GAG drug sulodexide for structural characterization.Sulodexide has three distinct bands in the agarose gel,including two main components of fast-moving heparin(FMH)and DS,and a very small amount of slow-moving heparin(SMH).The three bands were extracted by SAX and degraded by heparinase or chondroitinase respectively,and finally analyzed by the optimized LC-MS/MS method.In FMH,a total of 17 basic components were characterized and quantified,which come from the structure of NRE,middle backbone,core pentasaccharide and reducing end(RE).The average MW of FMH was 10.7 kDa according to the ratio of terminal structures to backbone structures.In SMH,13 structures were quantified with an average MW of 15.4 kDa.According to the degree of sulfation,SMH has a more typical heparin structure,while FMH can be regarded as a low sulfated heparin with a lower MW.The structure of DS was relatively simple,where a total of 13 basic components were identified with an average MW of 19.9 kDa.The three composition ratios of sulodexide quantified by LC-MS/MS were basically consistent with the results obtained by direct staining of AGE gel.In addition,we also applied a diffusion-ordered spectroscopy(DOSY)of nuclear magnetic technology to analyze sulodexide for the first time.The characteristic signals of heparin/HS and DS were assigned at different dififusion coefficients of the spectrum.The ratio of these two peaks is about 3:1,which is equivalent to the composition of AGE-MS,AGE,MS and DOSY methods provide new ideas for the direct characterization of mixed GAGs samples.3.The interaction between different polymerization degree of heparin and IFN-y was studied by hydrogen deuterium exchange-mass spectrometry(HDX-MS).The chain length of polysaccharide is one of the key factors for the interaction between GAGs and proteins.We applied the HDX-MS method to investigate the interaction between different polymerization degrees of heparin and IFN-y.The binding mode of IFN-y and heparin was elucidated by analyzing the deuteriation rate of different peptides and the three-dimensional conformation of IFN-y.Firstly,10 peptides with suitable length and covering the entire amino acid sequence were selected from the IFN-y digestion products for subsequent analysis,including two known heparin binding domains D1(KTGKRKR)and D2(RGRR)reported in the literature.Then,the deuteriation time and the binding ratio of heparin to IFN-y were optimized.Heparin with different chain length and IFN-y were analyzed by HDX-MS,and the deuteriation rate curves of each peptide were calculated.According to the variation of deuteriation rate,peptides canbe divided into four groups:stable type,continuous descent type,platform stable type and multi-platform descent type.According to the combination of various deuteration rate changes and protein spatial position,basic amino acid distribution and molecular length,it is inferred that when the length of heparin chain is short,the chain and the basic amino acid clusters on the surface of IFN-y attract each other.As the length increases,the chain begins to interact with D1,and extends to the other side of the IFN-y dimer.When the length of the oligosaccharides reaches about dp20,it can simultaneously coveres the two D1 regions of the dimer.And at dp28,the conformation of the sugar chain and the dimer becomes stable,and the deuteration rate of some peptides does not change.After that,the heparin chain begins to further fix the D2 region.During this process,the local conformation of IFN-y also changes due to the interaction.The HDX-MS analysis of GAGs and proteins can not only obtain the characteristic binding domains and conformational changes of the protein,but also clarify the binding modes of heparin with different polymerization degrees and proteins,which provides a strong technical support for the study of interaction between GAGs and proteins.4.The sequence of heparin domain interacting with IFN-? was characterized.The special sequences contained in GAGs chains are very essential for the binding of polysaccharides to proteins.Based on affinity chromatography,MS and computer technology,the characteristic binding sequence interacted with IFN-y in heparin chains was characterized.The affinity of heparin chains with different polymerization degrees was first enriched by IFN-y affinity column.The length and sulfation degree of the affinity components were higher than unenriched sample by LC-MS/MS analysis.The affinity dp8 was isolated for sequencing analysis.In order to solve the problem of MS/MS determining the sequencing of complex mixed oligosaccharides,we developed a computer-simulated sequencing software.The software is based on permutation and probability calculation of disaccharide composition of mixed heparin chains with the same degree of polymerization to obtain all possible chain sequences and their relative contents.The affinity dp8 was calculated by computer software and sequenced by MS/MS,and 5 confirmed sequences were obtained finally.These structures all contain a pentasaccharide sequence "-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-".This structure satisfies the interaction with any D1 region of IFN-y dimer regardless of the number of monosaccharides,the degree of sulfation,and the sequence symmetry.Moreover,in affinity dp24,the content of sugar chain containing two pentasaccharide sequences that can respectively interact with IFN-y dimer is 1.7 times higher than that of unenriched chains.This result also illustrates that the interaction between the heparin chain and the two C-terminal ends of IFN-y requires at least two pentasaccharide structures with total sulfuric acid.In addition,the results of molecular docking between IFN-? and pentasaccharide shows that the oligosaccharide is mainly bound to D1 region,and is more easily attracted by the basic amino acids in the two C-terminal central regions of IFN-y.The binding is dominated by electrostatic force,and four basic amino acid residues in D1 domain and almost all acidic groups in pentasaccharide are involved in the interaction.The computer docking results further illustrate that the pentasaccharide structure in heparin chain plays an important role in the combination of heparin and IFN-?.The discovery of this sequence provides an important theoretical basis for the design of targeted drugs for artificial intervention of IFN-y.At the same time,the combination of affinity chromatography,computer-assisted sequencing,MS/MS sequencing,and molecular docking analysis strategies can also be used for the researches of more GAGs sequence characterization. |
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