Potter-Perigo S, Prather P, Baker C, Altman L C, Wight T N
Department of Pathology, School of Medicine, University of Washington, Seattle.
J Periodontal Res. 1993 Mar;28(2):81-91. doi: 10.1111/j.1600-0765.1993.tb01054.x.
Proteoglycans (PGs) were extracted from the [35S]-sulfate labelled medium and cell layer of proliferating human gingival epithelial cells and analyzed by ion exchange and molecular sieve chromatography, 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 Sepharose CL-6B contained two species--a quantitatively major peak at K(av) = 0.30 (M(r) congruent to 235,000 on SDS-PAGE) and a quantitatively minor peak at K(av) = 0.39. The major peak was sensitive to alkaline borohydride, shifting to K(av) = 0.45, and nitrous acid degradation, indicating the presence of heparan sulfate PG with glycosaminoglycan chains with M(r) congruent to 26,000. The minor peak is chondroitin/dermatan sulfate PG with glycosaminoglycan chains of M(r) = 22,200 as indicated by sensitivity to alkaline borohydride (shifting to K(av) = 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 K(av) = 0.20, 0.31, and 0.75. The largest peak (K(av) = 0.20 and M(r) congruent to 245,000 on SDS-PAGE) shifted elution position to K(av) = 0.50 after alkaline borohydride treatment and was completely sensitive to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M(r) congruent to 20,000. Two peaks containing [35S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K(av)S = 0.42 (M(r) congruent to 30,500) and 0.75 (M(r) congruent to 5300). The larger peak was predominantly chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase while the chains at K(av) = 0.75 were predominantly heparan sulfate with 83% 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 regulation of PG synthesis by these cells.
从[35S] - 硫酸盐标记的增殖期人牙龈上皮细胞培养基和细胞层中提取蛋白聚糖（PGs），并通过离子交换、分子筛色谱和SDS - PAGE进行分析。分泌到培养基中的大部分掺入放射性物质从DEAE Sephacel离子交换柱上以单一峰形式在0.44M NaCl处洗脱，在0.52M NaCl处有一个小肩峰。该物质在Sepharose CL - 6B上进行色谱分析时包含两种成分——在K(av) = 0.30处有一个定量上主要的峰（在SDS - PAGE上Mr约为235,000）和在K(av) = 0.39处有一个定量上次要的峰。主要峰对碱性硼氢化钠敏感，洗脱位置移至K(av) = 0.45，并且对亚硝酸降解敏感，表明存在具有Mr约为26,000的糖胺聚糖链的硫酸乙酰肝素PG。次要峰是硫酸软骨素/硫酸皮肤素PG，其糖胺聚糖链的Mr = 22,200，这通过对碱性硼氢化钠的敏感性（洗脱位置移至K(av) = 0.48）和硫酸软骨素ABC裂解酶消化得以表明。来自细胞层的[35S] - 硫酸盐标记物质在DEAE Sephacel上于0 - 0.50M NaCl之间以宽峰形式洗脱。该物质在Sepharose CL - 6B上进行色谱分析时在K(av) = 0.20、0.31和0.75处显示有三个峰。最大的峰（K(av) = 0.20且在SDS - PAGE上Mr约为245,000）在碱性硼氢化钠处理后洗脱位置移至K(av) = 0.50，并且对亚硝酸降解完全敏感。这些结果表明该峰包含具有Mr约为20,000的糖胺聚糖链的硫酸乙酰肝素PG。通过对细胞层提取物在Sepharose CL - 6B上进行色谱分析检测到两个含有[35S] - 硫酸盐标记糖胺聚糖链的峰，其K(av)分别为0.42（Mr约为30,500）和0.75（Mr约为5300）。较大的峰主要是硫酸软骨素/硫酸皮肤素糖胺聚糖，这通过对硫酸软骨素ABC裂解酶的敏感性得以表明，而在K(av) = 0.75处的链主要是硫酸乙酰肝素，对亚硝酸的敏感性为83%。这些结果表明培养的人牙龈上皮细胞主要合成和分泌硫酸乙酰肝素PGs以及少量的硫酸软骨素/硫酸皮肤素PGs。这项工作将作为未来研究的基础，这些研究旨在检查参与这些细胞PG合成调节的那些因素。
