Cell-free translation and characterization of corneal keratan sulfate proteoglycan core proteins.
Bovine corneal keratan sulfate proteoglycan (KSPG) contains two core proteins, 37 and 25 kDa, if fully deglycosylated, but 47 and 35 kDa, respectively, after endo-beta-galactosidase (Funderburgh, J. L., and Conrad, G. W. (1990) J. Biol Chem. 265, 8297-8303). Chicken corneal KSPG released a single core protein of 47 kDa after endo-beta-galactosidase, and of 35 and 36 kDa, if deglycosylated with N-glycanase or trifluoromethanesulfonic acid. Affinity purified rabbit antibodies against each KSPG recognized only the intact proteoglycan or its core proteins in immunoblots of unfractionated guanidine-HCl extracts of whole cornea after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Affinity purified antibody to a synthetic peptide duplicating the NH2-terminal sequence of the 37-kDa bovine core protein showed little reactivity with untreated corneal extract but reacted with the 47-kDa bovine protein in endo-beta-galactosidase-treated extracts. RNA was isolated from bovine and chick corneal stromas and used for in vitro translation. Antibody against bovine KSPG immunoprecipitated two proteins of 56-53 kDa and a protein of 41 kDa after translation of bovine RNA. Translation of chick RNA produced a double band of 38-39 kDa and a single band of 25 kDa precipitating with antibody against chicken KSPG. Homologous unlabeled KSPG competed for binding of antibodies to these translation products. These data suggest that in vertebrate corneas, the multiple KSPG core protein isoforms may arise as products of separate mRNAs, rather than from proteolytic processing of a large polypeptide precursor.
Jost
C J
CJ
Division of Biology, Kansas State University, Manhattan 66506.
Funderburgh
J L
JL
Mann
M
M
Hassell
J R
JR
Conrad
G W
GW
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D002645:Chickens; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics* / Q000302:isolation & purification; D003315:Cornea / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007632:Keratan Sulfate / Q000235:genetics* / Q000302:isolation & purification; D000071234:Lumican; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification
D011508:Chondroitin Sulfate Proteoglycans; D000071234:Lumican; D010455:Peptides; D012313:RNA; D007632:Keratan Sulfate