Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation.
Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002642:Chick Embryo; D002846:Chromatography, Affinity; D002854:Chromatography, Paper; D004224:Diterpenes / Q000378:metabolism*; D004286:Dolichols / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis; D006026:Glycoside Hydrolases; D005779:Immunodiffusion; D008358:Mannose / Q000378:metabolism; D009924:Organ Culture Techniques; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000302:isolation & purification; D013710:Tendons / Q000201:enzymology; D014316:Tritium
D000596:Amino Acids; D002250:Carbon Radioisotopes; D004224:Diterpenes; D004286:Dolichols; D006023:Glycoproteins; D014316:Tritium; D011393:Procollagen-Proline Dioxygenase; D006026:Glycoside Hydrolases; D005947:Glucose; D005944:Glucosamine; D008358:Mannose