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.
Highlights
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
Glycosyltransferase Activities of chondroitin sulfate synthase-2 (CSS2) Variants—Previously, we demonstrated that human CSS2 has both GalNAcT-II and GlcAT-II activities [11], whereas another research group reported that it has little glycosyltransferase activity but attains polymerizing activity for disaccharide-repeating units of CS when co-expressed with soluble human chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) [14]
As the major difference of the variants was that CSS2B exhibits substantially lower GlcAT activity than CSS2A and has no polymerizing activity when co-expressed with CSS1, we examined the levels of CS biosynthesis in COS-7 cells overexpressing either variant
Summary
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. Size Exclusion Chromatography of a Multimolecular Complex of CSS—Various combinations of FLAG- and Myc-tagged CSS2A, CSS2B, and CSS1 expression plasmids were co-transfected in COS-7 cells grown in 6-well plates as described above.
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