Production of prostaglandin D synthase as a keratan sulfate proteoglycan by cultured bovine keratocytes.
OBJECTIVE
To characterize the major proteoglycans produced and secreted by collagenase-isolated bovine keratocytes in culture.


METHODS
Freshly isolated keratocytes from mature bovine corneas were cultured in serum-free Dulbecco's modified Eagle's medium/ F12. Secreted proteoglycans were radiolabeled with protein labeling mix ((35)S-Express; Dupont NEN Life Science Products, Boston, MA) and digested with chondroitinase ABC, keratanase, and endo-beta-galactosidase to remove glycosaminoglycan chains, and core proteins were analyzed by autoradiography and Western blot analysis. An unidentified keratan sulfate proteoglycan (KSPG) was purified by gel filtration (Superose 6; Amersham Pharmacia, Piscataway, NJ) and anion-exchange chromatography (Resource Q; Amersham Pharmacia) and subjected to amino acid sequencing.


RESULTS
Keratanase digestion of proteoglycans produced approximately 50 kDa core proteins that immunoreacted with antisera to lumican, keratocan, and osteoglycin-mimecan. Chondroitinase ABC digestion produced a approximately 55-kDa core protein that immunoreacted with antisera to decorin. A 28-kDa band generated by keratanase or endo-beta-galactosidase digestion did not react with these antibodies. Chromatographic purification and amino acid sequencing revealed that the protein was prostaglandin D synthase (PGDS). Identity was confirmed by Western blot analysis using antisera to recombinant PGDS. PGDS isolated from corneal extracts was not keratanase sensitive but was susceptible to endo-beta-galactosidase, suggesting that it contains unsulfated polylactosamine chains in native tissue and is therefore present as a glycoprotein.


CONCLUSIONS
These results indicate that bovine keratocytes, when cultured under serum-free conditions, produce the four known leucine-rich proteoglycans decorin, keratocan, lumican, and osteoglycin/mimecan and maintain a phenotype that is comparable to that of in situ keratocytes. Additionally, these cells produce PGDS, a known retinoid transporter, as a KSPG.
Berryhill
B L
BL
The Center for Research in Skeletal Development and Pediatric Orthopaedics, Shriners Hospital for Children, 12502 North Pine Drive, Tampa, FL 33612-9499, USA. bberryhill@shctampa.usf.edu
Beales
M P
MP
Hassell
J R
JR
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D002850:Chromatography, Gel; D003319:Corneal Stroma / Q000166:cytology / Q000201:enzymology*; D016895:Culture Media, Serum-Free; D058575:Decorin; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000201:enzymology*; D006023:Glycoproteins / Q000096:biosynthesis; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis*; D007632:Keratan Sulfate / Q000096:biosynthesis*; D054834:Lipocalins; D000071234:Lumican; D011509:Proteoglycans / Q000096:biosynthesis
D011508:Chondroitin Sulfate Proteoglycans; D016895:Culture Media, Serum-Free; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C490949:KERA protein, Bos taurus; D054834:Lipocalins; D000071234:Lumican; D011509:Proteoglycans; D007632:Keratan Sulfate; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase