cDNA cloning of chick cartilage chondroitin sulfate (aggrecan) core protein and identification of a stop codon in the aggrecan gene associated with the chondrodystrophy, nanomelia.

We previously reported the cloning and sequencing of a 1.5-kilobase cDNA which encoded a portion of the chondroitin sulfate domain from the chick cartilage proteoglycan core protein and the localization of a species-specific monoclonal antibody epitope. Using polymerase chain reaction amplification and primer extension, cDNA clones which code for the entire proteoglycan core protein have now been obtained from a 10-day chick embryo cDNA library. The composite sequence is 6464 nucleotides long, coding for a protein of 2109 amino acid residues with a calculated M(r) = 223,500. The overall arrangement of globular and carbohydrate-attachment domains is similar to human and rat chondrosarcoma aggrecan, but there are significant differences in detailed homology between chick and mammalian core proteins. Most significantly a highly repetitive region (19 repeat units of 20 residues each), not found in either human or rat, enlarges one of the characteristic serine-glycine containing regions (designated CS-2) while the other serine-glycine containing domain (designated CS-1) is approximately one-fourth the length of the mammalian CS-1. Analysis of a polymerase chain reaction-amplified fragment encoding the chick-specific repeat region revealed a single base mutation at position 4553 (G to T transversion) that converted the codon GAA for glutamate at amino acid 1513 to TAA, a stop codon, in nanomelic chondrocytes. Genomic DNA from nanomelic liver was also digested with restriction enzyme BsaBI to verify the G to T transversion. This single mutation leads to a shortened core protein precursor with a calculated M(r) = 158,300. The resulting phenotype, nanomelia, arises because the truncated core protein is neither processed to a mature proteoglycan, nor secreted from the chondrocyte.

Stable heparin-producing cell lines derived from the Furth murine mastocytoma.

Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.

Biosynthetic precursors of cartilage chondroitin sulfate proteoglycan.

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.