MODULATION OF GLYCOSAMINOGLYCAN ADDITION IN NATURALLY EXPRESSED AND RECOMBINANT HUMAN THROMBOMODULIN

The two major glycoforms of full-length human thrombomodulin (TM), one with (TM(CS+)) and one without (TM(CS-)) chondroitin sulfate (CS) wer e analyzed on Western blots of primary and transformed cells and in ce lls expressing recombinant TM. TM on the surface of Chinese hamster ov ary and COS-7 cells is solely TM(CS-). Primary arterial endothelial ce lls (HAEC and HPAEC) express a greater fraction of TM with CS attached than venous cells (HUVEC). Human lung carcinoma cells (A549) express more TM(CS+) than primary cells and recombinant TM on human melanoma c ells (CHL-1) occurs in two very high molecular weight forms of TM(CS+) . We explored this variation in TM(CS+) with soluble recombinant TM in several cell lines and analyzed the ambiguous CS addition site in hum an TM by site-directed mutagenesis. Mutation of Ser(474) to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 ce lls which add CS to Ser(472) and Ser(474). Structure of the O-link dom ain affects partitioning into TM(CS+) since substituting with the deco rin CS addition sequence, substituting all Ser and Thr except Ser(474) with Ala, and deleting around the potential beta-turn all increase th e ratio of TM(CS+) to TM(CS-). A combination of the decorin substituti on and deletion of the remaining O-link domain yields the most TM(CS+) .