title: Chondroitin sulfate synthase-2/chondroitin polymerizing factor has two variants with distinct function.
abstract: Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.
authors: Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, USA.
authors: Hiroyasu
authors: H
authors: Ogawa
authors: Masafumi
authors: M
authors: Shionyu
authors: Nobuo
authors: N
authors: Sugiura
authors: Sonoko
authors: S
authors: Hatano
authors: Naoko
authors: N
authors: Nagai
authors: Yukihiko
authors: Y
authors: Kubota
authors: Kiyoji
authors: K
authors: Nishiwaki
authors: Takashi
authors: T
authors: Sato
authors: Masanori
authors: M
authors: Gotoh
authors: Hisashi
authors: H
authors: Narimatsu
authors: Katsuji
authors: K
authors: Shimizu
authors: Koji
authors: K
authors: Kimata
authors: Hideto
authors: H
authors: Watanabe
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006025:Glycosaminoglycans / Q000737:chemistry; D006602:Hexosyltransferases / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry*; D041681:NIH 3T3 Cells; D017386:Sequence Homology, Amino Acid
chemical_list: D006025:Glycosaminoglycans; D006602:Hexosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C477294:chondroitin sulfate synthase-2; C412725:chondroitin synthase