Identification of the keratan sulfate attachment sites on bovine fibromodulin.
The small keratan sulfate-substituted proteoglycan (fibromodulin) from articular cartilage was shown to contain keratan sulfate linked to the core protein through N-glycosidic linkages to residues Asn-109, Asn-147, Asn-182, and Asn-272. Biosynthetic experiments with articular chondrocytes in the presence of tunicamycin, an inhibitor of N-linked oligosaccharide synthesis, demonstrated a specific inhibition of [35S]SO4 incorporation into fibromodulin. Under the same conditions no effect on the addition of keratan sulfate to the large aggregating proteoglycan was detected. Fibromodulin substituted with keratan sulfate was purified from bovine articular cartilage extracts by density gradient centrifugation, ion-exchange chromatography, and gel-permeation chromatography. Isolation of glycosylated peptides from tryptic digests of fibromodulin by ion-exchange chromatography and reversed-phase high performance liquid chromatography revealed four separate hexosamine-rich species, that were also immunoreactive with monoclonal antibody 5D4. Sequence analysis of these glycopeptides gave blank cycles at positions which corresponded to Asn followed by X-Ser/Thr in the sequence derived from cDNA (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegard, D. (1989) EMBO J. 8, 2601-2604). Hence, all four Asn residues in the leucine-rich region of the fibromodulin core protein can serve as acceptor sites for keratan sulfate addition.
Plaas
A H
AH
Shriners Hospital for Crippled Children, Tampa, Florida 33612.
Neame
P J
PJ
Nivens
C M
CM
Reiss
L
L
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002358:Cartilage, Articular / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D016326:Extracellular Matrix Proteins; D000071235:Fibromodulin; D006031:Glycosylation; D007632:Keratan Sulfate / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D011509:Proteoglycans; D014415:Tunicamycin / Q000494:pharmacology
D000596:Amino Acids; D002352:Carrier Proteins; D016326:Extracellular Matrix Proteins; D010446:Peptide Fragments; D011509:Proteoglycans; D014415:Tunicamycin; D000071235:Fibromodulin; D007632:Keratan Sulfate