Chondroitin Sulfate Synthase-2 Is Necessary for Chain Extension of Chondroitin Sulfate but Not Critical for Skeletal Development

Hiroyasu Ogawa,Katsuji Shimizu,Takashi Sato,Sonoko Hatano,Naoko Nagai,Koji Kimata,Hisashi Narimatsu,Nobuo Sugiura,Hideto Watanabe,Nikos K Karamanos

doi:10.1371/journal.pone.0043806

Abstract

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid residues, modified with sulfated residues at various positions. Based on its structural diversity in chain length and sulfation patterns, CS provides specific biological functions in cell adhesion, morphogenesis, neural network formation, and cell division. To date, six glycosyltransferases are known to be involved in the biosynthesis of chondroitin saccharide chains, and a hetero-oligomer complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is known to have the strongest polymerizing activity. Here, we generated and analyzed CSS2−/− mice. Although they were viable and fertile, exhibiting no overt morphological abnormalities or osteoarthritis, their cartilage contained CS chains with a shorter length and at a similar number to wild type. Further analysis using CSS2−/− chondrocyte culture systems, together with siRNA of CSS1, revealed the presence of two CS chain species in length, suggesting two steps of CS chain polymerization; i.e., elongation from the linkage region up to Mr ∼10,000, and further extension. There, CSS2 mainly participated in the extension, whereas CSS1 participated in both the extension and the initiation. Our study demonstrates the distinct function of CSS1 and CSS2, providing a clue in the elucidation of the mechanism of CS biosynthesis.

Highlights

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcUA) residues, modified with sulfated residues at various positions [1,2,3,4]
Six glycosyltransferases involved in mammalian CS biosynthesis have been identified: chondroitin sulfate synthase-1 (CSS1)/ chondroitin synthase-1 (ChSy-1) [11,12], chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor (ChPF) [12,13], chondroitin sulfate synthase-3 (CSS3)/chondroitin synthase-2 (ChSy-2) [14,15], chondroitin sulfate glucuronyltransferase (CSGlcAT)/chondroitin synthase-3 (ChSy-3) [16,17], and chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT) -1 [18] and -2 [19]
Generation of CSS22/2 Mice We designed a conditional targeting vector for CSS2, in which exon 1 was flanked with the loxP sequence, while a neomycin selection (Neo) cassette flanked with the FRT sequence was inserted in intron 1 (Fig. 1A)

Summary

Introduction

Chondroitin sulfate (CS) is a linear polysaccharide consisting of repeating disaccharide units of N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid (GlcUA) residues, modified with sulfated residues at various positions [1,2,3,4]. CS biosynthesis is initiated by transfer of GalNAc to the linkage region of a glucuronic acid- galactose-galactose-xylose (GlcUAGal-Gal-Xyl) tetrasaccharide primer that is attached to a serine residue of a core protein. Following this step termed chain initiation, chain polymerization occurs by the alternate addition of GalNAc and GlcUA residues. CSS1, CSS2, and CSS3 contain two glycosyltransferase domains: b-3 domain at the N-terminal region and b-4 domain at the C-terminal region They exhibit dual enzymatic activities of N-acetylgalactosaminyltransferase-II (GalNAcT-II) and glucuronyltransferase-II (GlcAT-II); another research group failed to find glycosyltransferase activity in CSS2, thereby naming chondroitin polymerizing factor (ChPF). CSGalNAcT-1 and -2 contain a b-4 glycosyltransferase domain, and exhibit both GalNAcT-I and -II activities responsible for chain initiation and polymerization, respectively [18,19]

Methods

Results

Conclusion