The structural study of newly synthesized proteoglycans from Swarm rat chondrosarcoma cell culture

Long term cell cultures of Swarm rat chondrosarcoma were labeled with ({dollar}sp{lcub}35{rcub}{dollar}S) cysteine, ({dollar}sp3{dollar}H) serine, {dollar}sp{lcub}35{rcub}{dollar}SO{dollar}sb4{dollar}, or ({dollar}sp3{dollar}H) glucosamine and aggrecan was isolated by dissociative CsCl density gradients followed by rate zonal sedimentation separation from small proteoglycans. The purified proteoglycans were cleaved at methionine residues with cyanogen bromide, and the chondroitin sulfate containing fragments were isolated by Q-Sepharose. Cleavage produced one large fragment containing 855 amino acids (residues 596 to 1450) from the N-terminal side and several smaller fragments from the C-terminal side of the chondroitin sulfate attachment region. These fragments were separated by molecular sieve chromatography on Sepharose CL-4B. Chondroitin sulfate chains were released by alkali/borohydride treatment and the liberated chains were size fractionated on a calibrated Superose 6 column. When chains from the C-terminal fragments were examined, they were shorter by two disaccharides on average than those from the N-terminal fragment. Further, it appeared that the O-linked oligosaccharides were proportionately distributed in the chondroitin sulfate attachment region since 58% of the O-linked oligosaccharides were in the N-terminal portion of the chondroitin sulfate attachment region which contained 59% of the protein of this region. Similarly, the distribution of chondroitin suflate chains also closely correlated with the content of Ser-Gly in the N- and C-terminal portions of the region. These data are consistent with the substitution of the available glycosylation sites. The results further suggest that the lengths of chondroitin sulfate chains on aggrecan are not simply dependent on factors such as the length of time the molecule spends in the chain polymerizing compartment or the nutritional state of the cells but is also affected by positional factors such as location on the core protein. The results are consistent with the suggestion by others that the chain polymerizing enzymes are spatially arranged in the Golgi to provide nearly simultaneous assembly of all the chondroitin sulfate chains on the molecule.; During previous work for identification of disulfide bond patterns within the newly synthesized aggrecan from rat chondrosarcoma cells, the newly synthesized hyaluronic acid binding region was successfully purified, and the synthesis biglycan by Swarm rat chondrosarcoma cells was identified. This project was discontinued because the disulfide bond pattern was published by another laboratory.