Production of prostaglandin D synthase as a keratan sulfate proteoglycan by cultured bovine keratocytes.

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 ((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.