Biosynthesis of proteoglycans by rat embryo parietal yolk sacs in organ culture.
The embryonic rat parietal yolk sac has been previously shown to synthesize a number of basement membrane glycoconjugates including type IV procollagen, laminin, and entactin. In this study, parietal yolk sacs were isolated from 14.5-day rat embryos and incubated in organ culture for 4-7 h with [35S]sulfate, [3H] glucosamine, and/or 3H-labeled amino acids, and the newly synthesized proteoglycans were characterized. The major [35S]sulfate-labeled macromolecule represented approximately 90% of the medium and 80% of the tissue radioactivity. It also represented nearly 80% of the total [3H]glucosamine-labeled glycosaminoglycans. After purification by sequential ion-exchange chromatography and isopycnic CsCI density gradient ultracentrifugation, size-exclusion high-performance liquid chromatography showed a single species with an estimated Mr of 8-9 X 10(5). The intact proteoglycan did not form aggregates in the presence of exogenous hyaluronic acid or cartilage aggregates. Alkaline borohydride treatment released glycosaminoglycan chains with Mr of 2.0 X 10(4) which were susceptible to chondroitinase AC II and chondroitinase ABC digestion. Analysis by high-performance liquid chromatography of the disaccharides generated by chondroitinase ABC digestion revealed that chondroitin 6-sulfate was the predominant isomer. The uronic acid content of the glycosaminoglycans was 92% glucuronic acid and 8% iduronic acid, and the hexosamine content was 96% galactosamine and 4% glucosamine. No significant amounts of N- or O-linked oligosaccharides were detected. Deglycosylation of the proteoglycan with chondroitinase ABC in the presence of protease inhibitors revealed a protein core with an estimated Mr of 1.25-1.35 X 10(5). These results indicated that the major proteoglycan synthesized by the 14.5-day rat embryo parietal yolk sac is a high-density chondroitin sulfate containing small amounts of copolymeric dermatan sulfate. Hyaluronic acid and minor amounts of heparan sulfate proteoglycan were also detected.
D000818:Animals; D005260:Female; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D009924:Organ Culture Techniques; D010446:Peptide Fragments / Q000032:analysis; D011247:Pregnancy; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D012694:Serine / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium; D015017:Yolk Sac / Q000166:cytology / Q000378:metabolism*
D010446:Peptide Fragments; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; D012694:Serine; D008715:Methionine; D007930:Leucine; D008358:Mannose