Involvement of Human Natural Killer-1 (HNK-1) Sulfotransferase in the Biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl Tetrasaccharide Found in α-Thrombomodulin from Human Urine

Abstract

Thrombomodulin (TM) is an integral membrane glycoprotein, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (␤-TM) and a non-PG form without a CS chain (␣-TM) and hence is a part-time PG. An ␣-TM preparation isolated from human urine contained the glycosaminoglycan linkage region tetrasaccharide GlcUA␤1-3Gal␤1-3Gal␤1-4xylose, and the nonreducing terminal GlcUA residue is 3-Osulfated. Because the human natural killer-1 sulfotransferase (HNK-1ST) transfers a sulfate group from 3-phosphoadenosine 5-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue in the HNK-1 antigen precursor trisaccharide, GlcUA␤1-3Gal␤1-4GlcNAc, the sulfotransferase activity toward the linkage region was investigated. In fact, the activity of HNK-1ST toward the linkage region was much higher than that toward the glucuronylneolactotetraosylceramide, the precursor of the HNK-1 epitope. HNK-1ST may be responsible for regulating the sorting of ␣-and ␤-TM. Furthermore, HNK-1ST also transferred a sulfate group from 3-phosphoadenosine 5-phosphosulfate to the C-3 position of the nonreducing terminal GlcUA residue of a chondroitin chain. Intriguingly, the HNK-1 antibody recognized CS chains and the linkage region if they contained GlcUA(3-O-sulfate), suggesting that HNK-1ST not only synthesizes the HNK-1 epitope but may also be involved in the generation of part-time PGs.

A Sulfated Glycosaminoglycan Linkage Region Is a Novel Type of Human Natural Killer-1 (HNK-1) Epitope Expressed on Aggrecan in Perineuronal Nets

“…Our structural analysis suggests that GlcAT-I, the sole enzyme identified for synthesis of the GAG linkage region to date, might be involved in the linkage type HNK-1 epitope on aggrecan in PNNs. Previous findings showing overexpression of GlcAT-I evoked HNK-1 immunoreactivity [ 46 ] and the sulfated linkage region was detected in human urine [ 56 , 57 ] further supports our hypothesis that GlcAT-I is the enzyme responsible for the synthesis of the unusual HNK-1 on aggrecan. However, directly demonstrating its involvement is difficult because GlcAT-I ( B3gat3 )-knockout mice show embryonic lethality at a very early stage [ 58 ].…”

Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate

“…Actually, we and Dr. Sugahara's group demonstrated that HNK-1ST can use a glycosaminoglycan linkage tetrasaccharide on thrombomodulin as an acceptor substrate to form an unusual sulfated structure (Fig. 1B) [46,47]. The sulfated linkage region on thrombomodulin was indeed identified in human urine, SO 4 -3GlcAβ1-3Galβ1-3Galβ1-4Xyl [48], and a non-elongated form of proteoglycan is known to exist as a part-time proteoglycan [49,50].…”

LC–MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications

“…A terminal sulfated GlcUA linked to a sulfated GalNAc residue was observed the CS/DS from HCC70 cells after chondroitinase AC-I and -II degradation. It was not possible to assign the position of the sulfate group on GlcUA; however, human natural killer-1 sulfotransferase (HNK-1ST), the enzyme responsible for the 3-Osulfation of the truncated linkage region of ␣-thrombomodulin, may be involved (55). Although terminal 3-O-sulfation of GlcUA in mammals has hitherto only been observed in this context, it is noteworthy that recombinant HNK-1ST has shown activity also toward the terminal GlcUA residues in chondroitin of various lengths.…”