Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

Abstract

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were coimmunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.Glycosylation is one of the major post-translational protein modifications, especially for cell surface proteins, that play important roles in a variety of cellular functions, including recognition and adhesion. Among them, human natural killer-1 (HNK-1) 2 carbohydrate, which is recognized by HNK-1 monoclonal antibody, is predominantly expressed in the nervous system (1, 2). HNK-1 carbohydrate is expressed on several glycoproteins and glycolipids and is considered to be involved in cell migration, recognition, and adhesion. The unique structural feature of this carbohydrate is a sulfated trisaccharide (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc) (3, 4). Recently, we cloned two glucuronyltransferases (GlcAT-P and GlcAT-S) (5-9) and one sulfotransferase (HNK-1ST) (10) that are involved in the biosynthesis of HNK-1 carbohydrate. To investigate the biological function of HNK-1 carbohydrate in vivo, we generated GlcAT-P gene-deficient mice and revealed that HNK-1 carbohydrate is involved in synaptic plasticity (11). More recently, we found a nonsulfated form of HNK-1 carbohydrate in mouse kidney (12), where only GlcAT-S was expressed and not HNK-1ST. In the nervous system, however, all the HNK-1 carbohydrate structures identified so far are sulfated forms because of the presence of glucuronyltransferases (GlcAT-P and GlcAT-S) and HNK-1ST (13, 14). The fact that only the existence of HNK-1ST is sufficient for the sulfation of HNK-1 carbo...

Mechanism of Regulation and Suppression of Melanoma Invasiveness by Novel Retinoic Acid Receptor-γ Target Gene Carbohydrate Sulfotransferase 10

“…CHST10, together with two glucuronyltransferases (GlcAT-P and GlcAT-S), directs the biosynthesis of HNK-1 glycan on neural cell adhesion proteins such as NCAM and glycolipids (47). Whereas little else is known about CHST10 other than its enzymatic activities, HNK-1 has been more studied.…”

“…Expression of the HNK-1 epitope is highly regulated during fetal development and is found on migrating neural crest cells, cerebellum, and Schwann cells in motor neurons (20). HNK-1 has been implicated in a diverse array of biological activities such as cell migration, recognition, and particularly adhesion (33,35,47,48), but unfortunately, the effects of HNK-1 on tumor cell properties still remain unclear. Confounding issues are that different anti-HNK-1 antibodies can recognize different HNK-1 epitopes on different proteins (18), and whereas some epitopes are important for cell adhesion, others are not (35).…”

Specific Enzyme Complex of β-1,4-Galactosyltransferase-II and Glucuronyltransferase-P Facilitates Biosynthesis of N-linked Human Natural Killer-1 (HNK-1) Carbohydrate

“…Seko and Yamashita (5) showed that a complex of ␤3GnT2 and ␤3GnT8 produced more poly-N-acetyllactosamine. Our previous study demonstrated that GlcAT-P interacts with HNK-1ST, and the interaction enhances the enzymatic activity of HNK-1ST (37). Also, it has been reported that the activities of some glycosyltransferases are regulated through interaction with nonglycosyltransferases.…”

“…6A) (36). We previously reported that GlcAT-P and HNK-1ST formed an enzyme complex through their catalytic domains in the Golgi lumen (37). Thus, we examined whether GlcAT-P and ␤4GalT-II also form a complex via their Golgi luminal domains.…”

Development and Application of High Performance Liquid Chromatography Map of Glucuronyl N-glycans

“…COS7 cells grown in a 10-cm-diameter tissue culture dish (Coring) were transfected with 4 μg of the relevant plasmid, pEF-BOS-HNK-1sulfotransferase (ST) [19] using LipofectAMINE PLUS (Invitrogen) according to manufacturer instructions. After 24 h of culture in Dulbecco's modified Eagle's medium containing 10 % (v/v) fetal calf serum, the medium was replaced with Dulbecco's modified Eagle's medium containing 2 % (v/v) IgG-free fetal calf serum.…”