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From 1 The Center for Research in Skeletal Development and Pediatric Orthopaedics, Shriners Hospital for Children, Tampa, Florida; and the 2 Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa.
PURPOSE. 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 (35S-Express; Dupont NEN Life Science Products, Boston, MA) and digested with chondroitinase ABC, keratanase, and endo-ß-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 
50 kDa core proteins
that immunoreacted with antisera to lumican, keratocan, and
osteoglycin-mimecan. Chondroitinase ABC digestion produced a 55-kDa
core protein that immunoreacted with antisera to decorin. A 28-kDa band
generated by keratanase or endo-ß-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-ß-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.
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