Identification, Partial Characterization, and Distribution of Versican and Link Protein in Bovine Dental Pulp
Identification, Partial Characterization, and Distribution of Versican and Link Protein in Bovine Dental Pulp
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DOI：
10.1177/00220345970760110301
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发表时间：
1997-11
影响因子：
7.6
通讯作者：
S. Yamauchi;Hui Cheng;Peter J. Neame;Bruce Caterson;Mitsuo Yamauchi
中科院分区：
文献类型：
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作者：
S. Yamauchi;Hui Cheng;Peter J. Neame;Bruce Caterson;Mitsuo Yamauchi
在牙齿发育和成熟过程中，牙髓细胞结构和细胞外基质组成的变化动态变化很大。在本文中，我们研究了基质蛋白聚糖，假设它们在牙髓发育和维护的结构、空间和运输方面发挥着重要作用。从部分萌出的牛门牙中收集牙髓组织，粉碎，然后用6M盐酸胍提取。提取物进行阴离子柱层析（DEAE-8HR），收集的级分通过斑点印迹免疫分析法，通过分别针对4-和6-硫酸化硫酸软骨素异构体以及硫酸角质素、2-B-6、3-B-3和5-D-4产生的单克隆抗体进行筛选。 6-硫酸软骨素是主要的糖胺聚糖种类，并且以大分子量蛋白聚糖（> 500 kDa）的形式出现。进一步纯化后，进行琼脂糖/丙烯酰胺复合凝胶电泳，用Stains-All染色，迁移为单条带。通过蛋白质印迹分析，条带针对抗体 3-B-3 呈免疫阳性。核心蛋白的部分氨基酸序列分析清楚地表明该分子是多功能蛋白聚糖。使用抗连接蛋白单克隆抗体 8-A-4 进行蛋白质印迹分析也证实了连接蛋白的存在。此外，免疫组织化学研究表明，多功能蛋白聚糖和连接蛋白在牙髓中的分布一致，并且在成牙本质细胞层下方的组织外围区域富集。由于牙髓含有透明质酸，多功能蛋白聚糖可以通过其透明质酸结合结构域与透明质酸结合，其中这种结合通过连接蛋白稳定。然后，这种复合物可以形成大型水合蛋白聚糖聚集体，填充细胞外空间，支持成牙本质细胞，和/或促进组织内代谢物和营养物质的运输功能。
The dynamics of changes in the cellularity and extracellular matrix composition of dental pulp varies considerably during tooth development and maturation. In this paper, we studied matrix proteoglycans where we hypothesized that they played important roles in structural, spatial, and transport aspects of pulpal development and maintenance. The pulpal tissue was collected from partially erupted bovine incisors, pulverized, and then extracted with 6 M guanidine-HCl. The extract was subjected to anion column chromatography (DEAE-8HR), and the fractions collected were screened by dot-blot immunoassay by means of monoclonal antibodies generated against 4- and 6-sulfated chondroitin sulfate isomers, and keratan sulfate, 2-B-6, 3-B-3, and 5-D-4, respectively. The chondroitin-6-sulfate was the major glycosaminoglycan species and occurred as a large-molecular-weight proteoglycan (> 500 kDa). After further purification, it was subjected to agarose/acrylamide composite gel electrophoresis, and it migrated as a single band stained with Stains-All. The band was immunopositive against antibody 3-B-3 by Western blot analysis. The partial amino acid sequence analyses of the core protein clearly indicated this molecule to be versican. The presence of link protein was also confirmed by Western blot analysis with an anti-link protein monoclonal antibody, 8-A-4. Furthermore, immunohistochemical study indicated that the distributions of versican and link protein coincide in the dental pulp and are enriched in the peripheral area of the tissue just beneath the odontoblast layer. Since the dental pulp contains hyaluronan, versican may bind to hyaluronan via its hyaluronan-binding domain, where this association is stabilized by link protein. This complex, then, could form large hydrated proteoglycan aggregates that fill the extracellular space, support odontoblasts, and/or facilitate the transport function of metabolites and nutrients within the tissue.