Identification of chick corneal keratan sulfate proteoglycan precursor protein in whole corneas and in cultured corneal fibroblasts.
The precursor protein to the chick corneal keratan sulfate proteoglycan was identified by immunoprecipitation with antiserum to its core protein from lysates of [35S]methionine-pulsed corneas and corneal fibroblasts in cell culture. Antiserum to the keratan sulfate proteoglycan immunoprecipitated a doublet of Mr 52,000 and 50,000 and minor amounts of a Mr 40,000 protein from pulsed corneas. Pulse-chase experiments, which permitted the conversion of the precursor proteins to proteoglycans and digestion of the glycosaminoglycans on immunoprecipitated proteoglycans with keratanase or chondroitinase ABC, showed that the Mr 52,000-50,000 doublet was converted to a keratan sulfate proteoglycan and the Mr 40,000 protein was converted to a chondroitin sulfate proteoglycan. Chick corneal fibroblasts in cell culture primarily produced the smaller (Mr50,000) precursor protein, and in the presence of tunicamycin the precursor protein size was reduced to Mr35,000, which indicates that the core protein contains approximately five N-linked oligosaccharides. Pulse-chase experiments with corneal fibroblasts in culture showed that the precursor protein was processed and secreted into the medium. However, its sensitivity to endo-beta-galactosidase and resistance to keratanase indicate that the precursor protein was converted to a glycoprotein with large oligosaccharides and not to a proteoglycan. This suggests that, although the precursor protein for the proteoglycan is produced in cultured corneal fibroblasts, the sulfation enzymes for keratan sulfate may be absent.
Schrecengost
P K
PK
Department of Ophthalmology, University of Pittsburgh School of Medicine, Pennsylvania.
Blochberger
T C
TC
Hassell
J R
JR
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis* / Q000096:biosynthesis; D003315:Cornea / Q000737:chemistry*; D005347:Fibroblasts / Q000737:chemistry*; D007106:Immune Sera / Q000737:chemistry; D007632:Keratan Sulfate / Q000032:analysis* / Q000096:biosynthesis; D000071234:Lumican; D008970:Molecular Weight; D011498:Protein Precursors / Q000032:analysis* / Q000096:biosynthesis
D011508:Chondroitin Sulfate Proteoglycans; D007106:Immune Sera; D000071234:Lumican; D011498:Protein Precursors; D007632:Keratan Sulfate