Chondroitin Sulfate N-Acetylgalactosaminyltransferase 1 Is Necessary for Normal Endochondral Ossification and Aggrecan Metabolism

Takashi Sato,Takashi Kudo,Yuzuru Ikehara,Hiroyasu Ogawa,Tomoko Hirano,Katsue Kiyohara,Kozue Hagiwara,Akira Togayachi,Masatsugu Ema,Satoru Takahashi,Koji Kimata,Hideto Watanabe,Hisashi Narimatsu

doi:10.1074/jbc.m110.159244

Abstract

Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.

Highlights

Chondroitin sulfate (CS) biosynthesis is initiated by the transfer of a GalNAc residue to the linkage region of a glucuronic acid (GlcUA)-␤1,3-galactose (Gal)␤1,3-Gal-␤1,4-Xyl tetrasaccharide primer that is attached to a serine residue of the core protein (Fig. 1A)
A targeting vector was constructed in which exon 1, containing the 5Ј-untranslational region (UTR) and encoding a translational start site followed by the transmembrane domain, was flanked with loxP sequences (Fig. 1B)
Chimeric mice were obtained by microinjecting Csgalnact1flox/ϩ embryonic stem (ES) cells into mouse blastocyst, and the resulting animals were crossed with C57BL/6J mice to achieve germ line transmission of the floxed allele

Summary

Introduction

CS biosynthesis is initiated by the transfer of a GalNAc residue to the linkage region of a GlcUA-␤1,3-galactose (Gal)␤1,3-Gal-␤1,4-Xyl tetrasaccharide primer that is attached to a serine residue of the core protein (Fig. 1A). These observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan. The tibial growth plate of 2-week-old Csgalnact1Ϫ/Ϫ mice exhibited a slight disorganization of chondrocyte columns and a decrease in proliferative chondrocytes.

Results

Conclusion