Kaji Toshiyuki, Sakurai Shigeru, Yamamoto Chika, Fujiwara Yasuyuki, Yamagishi Sho-ichi, Yamamoto Hiroshi, Kinsella Michael G, Wight Thomas N
Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan.
Biol Pharm Bull. 2004 Nov;27(11):1763-8. doi: 10.1248/bpb.27.1763.
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 [(3)H]glucosamine, [(35)S]sodium sulfate or (35)S-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 M(r) 45000 in biglycan and decorin, approximately M(r) 48000 in the small amount of HSPGs and approximately M(r) 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.
周细胞与微血管内皮细胞的外侧相连。先前的研究表明，这些细胞能合成糖胺聚糖（GAGs），但与这些GAGs相连的核心蛋白的性质尚不清楚。在本研究中，用[³H]葡糖胺、[³⁵S]硫酸钠或³⁵S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离子交换和分子筛Sepharose CL - 4B色谱法纯化这些细胞合成的蛋白聚糖。将分离的蛋白聚糖用木瓜蛋白酶、类肝素酶或软骨素ABC裂解酶消化，并用Sepharose CL - 6B色谱法对GAGs进行鉴定。在用软骨素ABC裂解酶消化前后，还通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳对蛋白聚糖进行评估。周细胞主要合成并分泌硫酸软骨素或硫酸皮肤素蛋白聚糖（CS/DS PGs），而非硫酸乙酰肝素蛋白聚糖（HSPGs）。周细胞合成了两类CS/DS PGs；一类是高Mr且电荷密度高的亚类。该亚类在Sepharose CL - 4B分子筛柱的空体积处洗脱，对软骨素ABC裂解酶敏感，且含有约550和450 kD的核心蛋白，这些核心蛋白可被多功能蛋白聚糖抗体识别。另一个主要亚类在Sepharose CL - 4B分子筛柱上的Kav约为0.45处洗脱，对软骨素ABC裂解酶敏感，且含有可被双糖链蛋白聚糖或饰胶蛋白聚糖抗体识别的核心蛋白，这些核心蛋白在SDS - PAGE中作为一条约50 kDa的宽带分离。这些细胞也合成少量的HSPG，可通过使用0.1 - 0.7 M NaCl线性梯度的DEAE - Sephacel色谱法将其与CS/DS PGs分离。蛋白酶消化释放的GAG链表明，双糖链蛋白聚糖和饰胶蛋白聚糖中GAG链的长度约为Mr 45000，少量HSPG中约为Mr 48000，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。
