The glycosylation sites and structural characteristics of oligosaccharides on recombinant human thrombomodulin.
Thrombomodulin (TM) is an anticoagulant glycoprotein on the surface of endothelial cell that directly inhibits the procoagulant activities of thrombin, and the TM-thrombin complex accelerates thrombin-catalyzed activation of protein C. Soluble TM in urine has no glycosaminoglycan (GAG) chain which accelerates the anticoagulant activities. Therefore, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C127 cells. The glycosylation sites were determined by amino acid sequence analysis of peptides digested with trypsin after S-carboxymethylation. The structures of N-linked oligosaccharides were estimated by two-dimensional sugar mapping of pyridylaminated oligosaccharides that were treated with exoglycosidase. The disaccharide composition analysis of the GAG chain was performed by HPLC using digestion with chondroitinase ABC, ACII and B. Consequently, it was revealed that the N-linked oligosaccharides were assigned to Asn29, Asn98, Asn364, Asn391; those structures were estimated biantennary, 2-6 branched triantennary and 2-4 branched triantennary complex type oligosaccharides that were linked by fucose at the ratio of 1.0:0.5:0.1, respectively. Moreover, the attachment site of the GAG chain was assigned to Ser472. It was then estimated that the GAG chain contained chondroitin-4-sulfate and dermatan sulfate, which were repeated approximately 30 times. In this paper, the GAG attachment site and structural characteristics of GAG-UTM, were confirmed. Moreover, structures of the N-linked oligosaccharides of GAG-UTM are described for the first time.
Edano
T
T
Tokyo Research Laboratories, Kowa Co. Ltd, Japan.
Kumai
N
N
Mizoguchi
T
T
Ohkuchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002240:Carbohydrate Sequence; D002470:Cell Survival / Q000187:drug effects; D002477:Cells / Q000378:metabolism; D019765:Chondroitin ABC Lyase / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D008040:Genetic Linkage; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000235:genetics / Q000652:urine; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000652:urine; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000652:urine; D018180:Thrombomodulin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014357:Trypsin / Q000737:chemistry
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D018180:Thrombomodulin; D006026:Glycoside Hydrolases; D014357:Trypsin; D019765:Chondroitin ABC Lyase