Register T C, Wagner W D
Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27103.
Connect Tissue Res. 1990;25(1):35-48. doi: 10.3109/03008209009009811.
Small proteoglycans were dissociatively extracted from normal human thoracic aorta with 4 M guanidine hydrochloride containing protease inhibitors, and purified by Sepharose CL-4B chromatography, dissociative cesium chloride density gradient centrifugation, and diethylaminoethyl cellulose chromatography. The intact proteoglycans migrated in the 270,000-340,000 range on 4-20% sodium dodecyl sulfate polyacrylamide gradient gels. Core proteins prepared following digestion of the intact proteoglycan monomer with chondroitinase ABC consisted of a major Coomassie blue-staining protein band of 50,000 along with a minor band of 44,000. Subsequent studies using endoglycosidases H, F, and N-glycanase demonstrated that mainly complex type N-linked glycans were present on the 50,000 cores while the 44,000 cores appeared to be devoid of N-linked glycans. Western blotting demonstrated that both of these cores were recognized by the monoclonal antibody 2-B-6, indicating the presence of the terminal 4-sulfated unsaturated disaccharide (delta Di-4S) remaining on the linkage region following chondroitinase ABC digestion. In contrast, a diffuse pattern of delta Di-4S epitopes ranging from 50,000 to approximately 60,000 was observed following chondroitinase AC II digestion of the dermatan sulfate proteoglycan, suggesting the presence of iduronate residues in close proximity to the glycosaminoglycan-linkage region. Conversely, the large chondroitin sulfate-proteoglycan core proteins from aorta (Mr 200,000-400,000) did not react with either monoclonal antibody 3-B-3 (recognizing the terminal delta DI-6S) or 2-B-6 following chondroitinase AC II digestion, although both delta DI-4S and delta DI-6S were present on these cores following chondroitinase ABC digestion. Additional studies using antisera against synthetic peptides derived from sequences of the core proteins of human bone small PG I and PG II indicated the presence of both gene products in PG isolated from human thoracic aorta. The Mr approximately 44,000 and 50,000 core proteins represent small PG I type cores while a closely spaced doublet (Mr 49,000 and 51,000) represented small PG II type cores. The results demonstrate that the core proteins of dermatan sulfate proteoglycan from human aorta are heterogeneous in primary structure and in the content of N-linked glycans.
用含有蛋白酶抑制剂的4M盐酸胍从正常人胸主动脉中解离提取小分子蛋白聚糖，然后通过琼脂糖CL - 4B柱层析、解离性氯化铯密度梯度离心和二乙氨基乙基纤维素柱层析进行纯化。完整的蛋白聚糖在4 - 20%十二烷基硫酸钠聚丙烯酰胺梯度凝胶上的迁移范围为270,000 - 340,000。用软骨素酶ABC消化完整的蛋白聚糖单体后制备的核心蛋白由一条主要的考马斯亮蓝染色的50,000蛋白带和一条次要的44,000蛋白带组成。随后使用内切糖苷酶H、F和N - 聚糖酶进行的研究表明，50,000核心上主要存在复合型N - 连接聚糖，而44,000核心似乎没有N - 连接聚糖。蛋白质印迹法表明，这两种核心都能被单克隆抗体2 - B - 6识别，表明在软骨素酶ABC消化后，连接区域上仍存在末端4 - 硫酸化不饱和二糖（δDi - 4S）。相比之下，硫酸皮肤素蛋白聚糖经软骨素酶AC II消化后，观察到δDi - 4S表位呈现出从50,000到约60,000的弥散模式，这表明在糖胺聚糖连接区域附近存在艾杜糖醛酸残基。相反，主动脉中的大硫酸软骨素蛋白聚糖核心蛋白（Mr 200,000 - 400,000）在软骨素酶AC II消化后，与单克隆抗体3 - B - 3（识别末端δDI - 6S）或2 - B - 6均无反应，尽管在软骨素酶ABC消化后，这些核心上同时存在δDI - 4S和δDI - 6S。使用针对源自人骨小分子PG I和PG II核心蛋白序列的合成肽的抗血清进行的进一步研究表明，在从人胸主动脉分离的蛋白聚糖中存在这两种基因产物。Mr约为44,000和50,000的核心蛋白代表小分子PG I型核心，而紧密间隔的双峰（Mr 49,000和51,000）代表小分子PG II型核心。结果表明，人主动脉硫酸皮肤素蛋白聚糖的核心蛋白在一级结构和N - 连接聚糖含量上是异质的。
