Sequence Analysis Of Decorin Glycosaminoglycan Chain From Cartilage

Decorin is a family member of small leucine rich proteoglycans.It is composed of a core protein and glycosaminoglycan(GAG)chains.Decorin GAG chain is a weak acidic linear polysaccharide composed of a repeating disaccharide unit.It is a hybrid chain,containing chondroitin sulfate(CS)and dermatan sulfate(DS).Decorin participates in many physiological and pathological processes.Many previous reports showed that decorin from different sources have different biological functions or activities.With the rapid development of proteomics,the sequence and structure of core protein have been known to be the same.However,glycomics study is still in the growth stage.Determining GAG structure is a formidable analytical challenge because of their structural complexity.In addition,GAG is post translational modification products with no definite template.GAG from different individuals and tissues have different structure.Our laboratory had preliminarily studied that the anticancer activities of decorins from porcine skin and cartilage are different in previous work.Therefore,clear sequence of GAG is necessary to understand the glycomics of decorin better.In this work,we choose the decorin from porcine cartilage as the research subject to sequence its GAG chains.At first,we optimize the traditional extraction process of Decorin from animal tissues.The purity and recovery have been greatly improved.The preparation period has been shorted twice.Next,we investigated the specificity of tool enzymes,Chondroitinase AC,Hyaluronidase and Chondroitinase B,which could be digest the specific domains in decrorin GAG chains.Though both Chondroitinase AC and Hyaluronidase could digest hyaluronic acid,only Chondroitinase AC digests CS domains specifically.In addition,Chondroitinase B digests DS domains specifically.The digested products with chrondroitinase AC and B were analyzed with carbohydrate electrophoresis and HILIC-ESI-Q/TOF.It is suggested that CS is the main backbone of Decorin GAG,some DS domains occurs in the main chain randomly.Finally,we use Orbitrap Fusion with high energy collision dissociation(HCD)as the best dissociation mode to sequence the enzymatically digested oligosaccharides.The results were listed as below:1.It was observed that the in-source dissociation order of sulfo group in diverse positions of oligosaccharides is different.The dissociation of 2 sulfo group is easiest,while the dissociation of 4 sulfo group is the hardest.2.We successfully established a method to determine the number and position of sulfo groups using HCD mode MS/MS and sulfo group dissociation pattern in ion source.The sequences of oligosaccharides in the digested products were exactly characterized by this method.3.Based on the sequencing information of oligosaccharides,the action patterns of two enzymes involved in decorin GAG biosynthesis were estimated.The C5 epimerase prefers to act on GlcA connected with GalNA4S/6S,but not non-sulfated GalNAc.The 2-O sulfotransferase always transfers activated sulfates to GlcA linked to GalNA4S/6S and that adjacent to DS domains.4.Decorin GAG from porcine cartilage was roughly sequenced.It's a hybrid chain with main chain of CS and some DS domains occurs in the main chain randomly.Most of DS domains are composed of IdoA-GalNAc4 S homogeneously.Most CS domains are composed of GlcA-GalNAc,GlcA-GalNAc6 S,GlcA-GalNAc4 S,GlcA2S-GalNAc4 S and small amount of GlcA2S-GalNAc.The 2-O-sulfation is always occurs on CS domains adjacent to DS domain.