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) were analyzed on Western blots of primary and transformed cells and in cells expressing recombinant TM. TM on the surface of Chinese hamster ovary and COS-7 cells is solely TM(CS-). Primary arterial endothelial cells (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 cells (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 human TM by site-directed mutagenesis. Mutation of Ser474 to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 cells which add CS to Ser472 and Ser474. Structure of the O-link domain affects partitioning into TM(CS+) since substituting with the decorin CS addition sequence, substituting all Ser and Thr except Ser474 with Ala, and deleting around the potential beta-turn all increase the ratio of TM(CS+) to TM(CS-). A combination of the decorin substitution and deletion of the remaining O-link domain yields the most TM(CS+).
Lin
J H
JH
Department of Cardiovascular Research, Berlex Biosciences, Richmond, California 94804.
McLean
K
K
Morser
J
J
Young
T A
TA
Wydro
R M
RM
Andrews
W H
WH
Light
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000032:analysis*; D006224:Cricetinae; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis; D012694:Serine / Q000032:analysis; D018180:Thrombomodulin / Q000032:analysis*
D011994:Recombinant Proteins; D018180:Thrombomodulin; D012694:Serine; D002809:Chondroitin Sulfates