STUDIES ON BIOSYNTHESIS OF THE LINKAGE REGION OF CHONDROITIN SULFATE-PROTEIN COMPLEX

Abstract

In the native state, chondroitin-4-sulfate is covalently bound to protein.Since the original studies of M\uir,' which implicated serine in this linkage, Rod6n et al. in this laboratory have demonstrated that the linkage region between protein and polysaccharide in heparin,' chondroitin-4-sulfate,3' 4 and dermatan sulfate5 has the following structure: GlcUA-Gal-Gal-Xyl-serine.Other studies6'7 have indicated that mucopolysaccharides of a wide variety of tissues of different species contain a similar linkage region.Previous publications have shown that a cell-free particulate enzyme derived from embryonic chick cartilage catalyzes the synthesis of chondroitin sulfate from the appropriate uridine nucleotide sugars.8-'0Since the CS isolated from embryonic chick cartilage was found to contain serine, xylose, and galactose with molar ratios of 1.00: 1.32: 2.06," it seemed logical to use the same enzyme preparation for studies on the biosynthesis of the linkage region.The transfer of xylose-C'4 from UDP-Xyl-Cl4 to protein has been previously reported from this laboratory.'2Bdolah and Feingold'3 have found that UDP-GlcUA can be converted to UDP-Xyl by a crude enzyme preparation from hen oviduct.Grebner et al.'4' 1' reported the transfer of xylose from UDP-Xyl to protein using cell-free preparations derived from hen oviduct and mouse mastocytoma.The linkage of xylose to serine in the protein was demonstrated.The incorporation of galactose from UDP-Gal into glycoproteins, catalyzed by cell-free preparations, has been reported by a number of laboratories.16'8This paper presents evidence for the transfer of both xylose and galactose from the corresponding uridine diphosphate sugars to protein by a cell-free preparation from embryonic chick cartilage.Materials and Methods.-UDP-GlcUA-U-C'4 (125mc/mmole) and UDP-Gal-U-C'4 (120 mc/ mmole) were purchased from the New England Nuclear Corp. UDP-GlcUA, UDP-Xyl, UMP- morpholidate, and pronase were purchased from the California Biochemical Corp. UDP-Gal was prepared as described by Roseman et al.'9 UDP-Xyl-U-Cl4 (104 mc/mmole) was kindly prepared by Drs.H. Ankel and D. S. Feingold from UDP-GlcUA-C'4 with purified UDP- glucuronate carboxy-lyase from Cryptococcus laurentii.200-,%D-xylosyl-L-serine, 4O-3-D- galactosyl-D-xylose, and fl-galactosidase were gifts from Dr. L. Roden.