Partial characterization of proteoglycans synthesized by human gingival epithelial cells in Culture

摘要

Proteoglycans (PGs) were extracted from the 35S‐sulfate labelled medium and cell layer of proliferating human ginigival epithelial cells and anlyzed by ion exchanged and molecular sieve chromatorgraphy, and by SDS‐PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sephartose CL‐6B contained two spieces – a quantitatively major peak at Kav= 0.30 (Mt≃ 235 000 on SDS‐PAGE) and a qantitatively major peak at Kav= 0.39. The major peak was sensitive to alkaline borohydride, shifting to Kav= 0.45, and nitrous acid degradtion, indicating the presence of heparan sulfate PG with glyscosaminoglycan chins with Mt≃ 26 000. The minor peak is chondroitin/dermatan sulfateP with glycpsminoglycan chains of Mt= 22 200 as indicted by sensitivity to alkaline borohydride (shifting to Kav= 0.48) and chondroitin ABC lyase digestion. The 35S‐sulfate labelled material from the cell layer eluted in a broad peak between 0–0.50 M NaCL from DEAE Sephacel. Chromatography of this material on Sepharose CL‐6B revealed the presence of three peaks at Kav=0.20, 0.31, and 0.75. The largest peak (Kav=0.20 and Mr≃ 245 000 on SDS‐PAGE) shifted elution position to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of Mt≃ 20000. Two peaks containing 35S‐sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL‐6B with Kavs=0.42 (Mr≃ 30 500) and 0.75 (Mr≃ 5300). The larger peak was predominately chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulati