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 o f 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-UT M) in C127 cells. The glycosylation sites were determined bq amino aci d sequence analysis of peptides digested with trypsin after S-carboxym ethylation. The structures of N-linked oligosaccharides were estimated by two-dimensional sugar mapping of pyridylaminated oligosaccharides that were treated with exoglycosidase. The disaccharide composition an alysis of the GAG chain was performed by HPLC using digestion with cho ndroitinase 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 triantennar y and 2-4 branched triantennary complex type oligosaccharides that wer e 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-1-sulfate and dermatan sulfate, which were repeated approximately 30 times. In this paper, the GAG attachment site and structural characteristics of GAG-U TM. were confirmed. Moreover, structures of the N-linked oligosacchari des of GAG-UTM are described for the first time. (C) 1998 Elsevier Sci ence Ltd. All rights reserved.