HNK-1 (human natural killer-1) carbohydrate epitope (HSO(3)-3GlcA beta 1-3Ga beta 1-4GlcNAc-) recognized by a HNK-1 monoclonal antibody is highly expressed in the nervous system and biosynthesized by a glucuronyltransferase (GIcAT-P or GlcAT-S), and sulfotransferase (HNK-1ST). A similar oligosaccharide (HSO(3)-3GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl) also recognized by the HNK-1 antibody had been found in a glycosaminoglycan (GAG)-protein linkage region of alpha-thrombomodulin (TM) from human urine. However, which sulfotransferase is involved in sulfation of the terminal GlcA in the GAG-protein linkage region remains unclear. In this study, using CHO-K1 cells in which neither GIcAT-P nor GlcAT-S is endogenously expressed, we found that HNK-1ST has the ability to produce HNK-1 immunoreactivity on alpha-TM. We also demonstrated that HNK-1ST caused the suppression of chondroitin sulfate (CS) synthesis on TM and a reduction of its anti-coagulant activity. Moreover, using an in vitro enzyme assay system, the HNK-1-positive TM was found not to be utilized as a substrate for CS-polymerizing enzymes (chondroitin synthase (ChSy) and chondroitin polymerizing factor (ChPF)). These results suggest that HNK-1ST is involved in 3-O-sulfation of the terminal GlcA of the linkage tetrasaccharide which acts as an inhibitory signal for the initiation of CS biosynthesis on TM. (C) 2011 Elsevier Inc. All rights reserved.