Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis.
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 (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) 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 co-immunoprecipitated 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.
Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences.
Yasuhiko
Y
Kizuka
Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
Takahiro
T
Matsui
Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
Hiromu
H
Takematsu
Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan.
Yasunori
Y
Kozutsumi
Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences; Research Center for Glycobiotechnology, Ritsumeikan University, Shiga 525-8577, Japan.
Toshisuke
T
Kawasaki
Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences. Electronic address: shogo@pharm.kyoto-u.ac.jp.
Shogo
S
Oka
D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000096:biosynthesis*; D006224:Cricetinae; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D051381:Rats; D015238:Sulfotransferases / Q000737:chemistry / Q000378:metabolism*; D000097811:Carbohydrate Sulfotransferases
D002241:Carbohydrates; D014453:Glucuronosyltransferase; D015238:Sulfotransferases; C499547:Chst10 protein, rat; D000097811:Carbohydrate Sulfotransferases