Biosynthesis of O-linked oligosaccharides on proteoglycans by chondrocytes from the swarm rat chondrosarcoma.

The core protein of proteoglycans from cartilage is substituted with glycosaminoglycans as well as N- and O-glycosidically linked oligosaccharides. We have taken advantage of the long intracellular half-life of the core protein precursor to the rat chondrosarcoma proteoglycan to study the temporal relationship between the addition of the chondroitin sulfate chains and the O-linked oligosaccharides onto the core protein during the formation of the completed proteoglycan molecule. Chondrocyte cultures were pulsed on day 2 with [6-3H]glucosamine for times ranging from 30-420 min. Media and corresponding 4% zwittergent, 4 M guanidine HCl extracts were then pooled and subjected to dissociative density gradient ultracentrifugation to yield purified proteoglycan monomers which were then subjected to alkaline borohydride treatment. The released chondroitin sulfate chains were then purified by precipitation with 50% (v/v) ethanol. The O-linked oligosaccharide-alditols in the supernatant fractions were purified by molecular sieve chromatography on Bio-Gel P-6, and analyzed after digestion with alpha-neuraminidase and subsequent chromatography on Bio-Gel P-2. The different O-linked oligosaccharide-alditols were identified from their hexosamine and hexosaminitol contents. The kinetics of entry of 3H label into N-acetylgalactosamine of chondroitin sulfate was indistinguishable from that into either N-acetylglucosamine or N-acetylgalactosaminitol residues of the oligosaccharide-alditols, with half-times to linear incorporation of 10-17 min. These results show that initiation as well as completion of the O-linked oligosaccharides on the core protein occurs essentially at the same time that chondroitin sulfate chains are added. The results suggest that these biosynthetic processes occur in the Golgi apparatus during the last few minutes of the total intracellular dwell time (half-time of about 90 min) of the core protein acceptor.