Burton-Wurster N, Lust G
New York State College of Veterinary Medicine, Cornell University, Ithaca 14853.
Arch Biochem Biophys. 1989 Feb 15;269(1):32-45. doi: 10.1016/0003-9861(89)90084-2.
Two new monoclonal antibodies (Mabs) which reacted with canine fibronectin were produced and characterized. Data supported the conclusion that the epitope recognized by Mab 1H9A4 is within the first three Type III homology repeats of the Hep 2 domain and that the epitope for Mab 13G3B7 is within the last Type III homology repeat of fibronectin. These antibodies, along with three others, Mabs IST-2, IST-7, and IST-9, produced and characterized in the laboratories of L. Zardi of Genoa, Italy, were used to characterize canine cartilage and plasma fibronectin. In addition, cartilage explants were labeled with [35S]methionine in order to characterize newly synthesized cartilage fibronectin. The following observations were made. (i) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NaDodSO4-PAGE) of reduced canine plasma fibronectin revealed a characteristic doublet; reduced cartilage fibronectin revealed two major bands and one minor band. The lower molecular weight band was 10 kDa less than the beta subunit of plasma fibronectin. In Western blots, this band stained with Mab 1H9A4 but failed to react with Mab 13G3B7. (ii) Western blots of thermolysin and trypsin digests of cartilage fibronectin revealed cleavage patterns which differed from those obtained from digestions of plasma fibronectin. (iii) The ED-A sequence, detected by Mab IST-9, was present in less than 2% of the cartilage fibronectins. (iv) NaDodSO4-PAGE of purified and reduced 35S-labeled fibronectin revealed two major radioactive bands and one minor radioactive band which comigrated with the fibronectin from the cartilage but not with plasma fibronectin. We concluded that like "cellular" fibronectin, the ratio of alpha-type subunits to beta subunits was greater than 4 to 1 in cartilage fibronectin compared to 1.25 to 1 for plasma fibronectin; however, cartilage fibronectin was not a cellular fibronectin by the criterion of the presence of the ED-A sequence. Another difference between plasma and cartilage fibronectin was the presence in cartilage fibronectin of a subpopulation of subunits on which the last Type III homology repeat could not be detected. Biosynthetic data were consistent with the concept that cartilage fibronectin originates from local synthesis by the chondrocyte.
制备并鉴定了两种与犬纤连蛋白发生反应的新型单克隆抗体（Mab）。数据支持以下结论：Mab 1H9A4识别的表位位于Hep 2结构域的前三个III型同源重复序列内，而Mab 13G3B7的表位位于纤连蛋白的最后一个III型同源重复序列内。这些抗体与另外三种在意大利热那亚的L. Zardi实验室制备并鉴定的抗体Mab IST-2、IST-7和IST-9一起，用于鉴定犬软骨和血浆纤连蛋白。此外，用[35S]甲硫氨酸标记软骨外植体，以鉴定新合成的软骨纤连蛋白。得到了以下观察结果。（i）还原后的犬血浆纤连蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（NaDodSO4-PAGE）显示出特征性的双峰；还原后的软骨纤连蛋白显示出两条主要条带和一条次要条带。分子量较低的条带比血浆纤连蛋白的β亚基小10 kDa。在蛋白质免疫印迹中，这条带与Mab 1H9A4发生反应，但不与Mab 13G3B7反应。（ii）软骨纤连蛋白的嗜热菌蛋白酶和胰蛋白酶消化产物的蛋白质免疫印迹显示出与血浆纤连蛋白消化产物不同的裂解模式。（iii）由Mab IST-9检测到的ED-A序列存在于不到2%的软骨纤连蛋白中。（iv）纯化并还原的35S标记的纤连蛋白的NaDodSO4-PAGE显示出两条主要放射性条带和一条次要放射性条带，它们与软骨中的纤连蛋白迁移位置相同，但与血浆纤连蛋白不同。我们得出结论，与“细胞型”纤连蛋白一样，软骨纤连蛋白中α型亚基与β型亚基的比例大于4比1，而血浆纤连蛋白为1.25比1；然而，根据ED-A序列的存在标准，软骨纤连蛋白不是细胞型纤连蛋白。血浆纤连蛋白和软骨纤连蛋白之间的另一个差异是软骨纤连蛋白中存在一个亚群的亚基，在其上无法检测到最后一个III型同源重复序列。生物合成数据与软骨纤连蛋白起源于软骨细胞的局部合成这一概念一致。
