Characterization of chondroitin/dermatan sulfate proteoglycans synthesized by bovine retinal pericytes in culture
Characterization of chondroitin/dermatan sulfate proteoglycans synthesized by bovine retinal pericytes in culture
复制标题
DOI：
10.1248/bpb.27.1763
复制
发表时间：
2004-11-01
影响因子：
2
通讯作者：
Wight, TN
中科院分区：
文献类型：
--
作者：
Kaji, T;Sakurai, S;Wight, TN
周细胞与微血管中的内皮细胞外部相关联。以前的研究表明，这些细胞合成糖胺聚糖（GAGS），但这些GAGS所连接的核心蛋白质的性质是未知的。用[H-3]氨基葡萄糖、[S-35]硫酸钠或S-35标记的氨基酸对培养的牛视网膜周细胞进行代谢标记，并用DEAE-Sephacel离子交换层析和SepharoseCL-4 B分子筛层析纯化这些细胞合成的蛋白多糖。分离的蛋白多糖用木瓜蛋白酶、肝素酶或软骨素ABC裂解酶消化，并通过Sepharose CL-6 B层析表征GAGS。蛋白聚糖也进行了评估，十二烷基硫酸钠聚丙烯酰胺凝胶电泳前后与软骨素ABC裂解酶消化。周细胞主要合成和分泌软骨素或硫酸皮肤素蛋白聚糖（CS/DS PG），而不是硫酸乙酰肝素蛋白聚糖（HSPG）。CS/DS PG的两个亚类由周细胞合成;一个是具有高电荷密度的高M-r亚类。该亚类在Sepharose CL-4 B分子筛柱的空隙体积中洗脱，对软骨素ABC裂解酶敏感，并含有ca. 550和450 kD的蛋白质被多功能蛋白聚糖抗体识别。其他主要亚类在K-av ca处洗脱。0.45在SepharoseCL-4 B分子筛柱上，对软骨素ABC裂解酶敏感，并且含有被双糖链蛋白聚糖或核心蛋白聚糖的抗体识别的核心蛋白，其分离为宽的ca条带。SDS-PAGE中50 kDa。少量的HSPG也由这些细胞合成，并且可以通过使用0.1-0.7 m NaCl的线性梯度的DEAE-Sephacel层析从CS/DS PG分离。通过蛋白酶消化释放的GAG链表明，GAG链的长度在双糖蛋白聚糖和核心蛋白聚糖中约为Mr 45000，在少量的HSPG中约为Mr 48000，在多功能蛋白聚糖中约为Mr 66000。这些蛋白聚糖类似于血管平滑肌细胞合成的蛋白聚糖，但与血管内皮细胞合成的蛋白聚糖明显不同。
Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGS) but the nature of the core proteins to which these GAGS are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [H-3]glucosamine, [S-35] sodium sulfate or S-35-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGS characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high M-r subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a K-av ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1-0.7 m NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately Mr 45000 in biglycan and decorin, approximately Mr 48000 in the small amount of HSPGs and approximately Mr 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.