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Bridgette L. Berryhill, Marianne P. Beales, John R. Hassell; Production of Prostaglandin D Synthase as a Keratan Sulfate Proteoglycan by Cultured Bovine Keratocytes. Invest. Ophthalmol. Vis. Sci. 2001;42(6):1201-1207.
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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-β-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-β-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-β-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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