title: Biosynthetic precursors of cartilage chondroitin sulfate proteoglycan.
abstract: Early steps in the biosynthesis of chondroitin sulfate proteoglycan (CSPG) and collagenous cartilage matrix molecules were examined by the comparison of products translated in mRNA-directed cell-free reactions and those synthesized by intact cartilage cells. RNA isolated from embryonic chicken sterna was used to direct cell-free translation reactions. Chicken sternal chondrocytes in culture were pulse-labeled with [35S]-methionine. The CSPG core protein was identified by immunoprecipitation. The Mr of the cartilage cell-synthetized core protein was determined to be 370K, approximately 10-15K greater than that of the comparable cell-free translation product. Experimental results strongly support the view that the observed difference in Mr reflects the cotranslational addition of mannose-rich, N-asparagine-linked oligosaccharides to the cell-synthesized core protein: 1) the cell-synthesized product was labeled with [3H]-mannose and precipitated by concanavalin A-sepharose beads; 2) the incorporated [3H]-mannose could be subsequently removed by digestion with endoglycosidase H (Endo H); 3) the Mr of the cell-synthesized core protein was reduced by Endo H digestion to that of the comparable cell-free translation product; 4) the core protein synthesized by tunicamycin-treated chondrocytes (inhibited in their ability to add N-asparagine-linked mannose-rich oligosaccharides to proteins) was comparable in electrophoretic mobility to that of the core protein cell-free translation product; and 5) the core protein translated in microsome-coupled cell-free reactions had an Mr 8-10K greater than that of the core protein translated in the absence of microsomes. For the purpose of examining biosynthetic intermediates, chondrocytes were labeled continuously or pulse-chase labeled for varying times. No biosynthetic CSPG intermediates migrating between the core protein and the CSPG monomer were detected. However, a band of 355Kdal appeared to share certain characteristics with the 307Kdal core protein (including its immunoprecipitability with CSPG antibodies), and a 340Kdal band was noted. Type II procollagen and other collagenase-sensitive products of 205Kdal and 110Kdal were observed among translation and chondrocyte-synthesized products. In chondrocytes, all three products exhibited labeling or chase time-dependent increases in Mr which were accelerated by ascorbate supplements and inhibited by the addition of alpha, alpha'-dipyridyl. These results suggest that the observed time-dependent increases in Mr are a consequence of collagen hydroxylation. The 110Kdal and 205Kdal collagenous proteins may be related to the minor collagens recently described in cartilage.
authors.forename: B M
authors.initials: BM
authors.lastname: Vertel
authors.forename: Y
authors.initials: Y
authors.lastname: Hitti
mesh_terms: D053674:Aggrecans; D000818:Animals; D002356:Cartilage / Q000196:embryology / Q000378:metabolism*; D002474:Cell-Free System; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics; D004285:Dogs; D016326:Extracellular Matrix Proteins; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D037181:Lectins, C-Type; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D011509:Proteoglycans / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*
chemical_list: D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D003094:Collagen