Kapoor R, Sakai L Y, Funk S, Roux E, Bornstein P, Sage E H
Department of Biological Structure, University of Washington, Seattle 98195.
J Cell Biol. 1988 Aug;107(2):721-30. doi: 10.1083/jcb.107.2.721.
A pepsin-resistant triple helical domain (chain 50,000 Mr) of type VIII collagen was isolated from bovine corneal Descemet's membrane and used as an immunogen for the production of mAbs. An antibody was selected for biochemical and tissue immunofluorescence studies which reacted both with Descemet's membrane and with type VIII collagen 50,000-Mr polypeptides by competition ELISA and immunoblotting. This antibody exhibited no crossreactivity with collagen types I-VI by competition ELISA. The mAb specifically precipitated a high molecular mass component of type VIII collagen (EC2, of chain 125,000 Mr) from the culture medium of subconfluent bovine corneal endothelial cells metabolically labeled for 24 h. In contrast, confluent cells in the presence of FCS and isotope for 7 d secreted a collagenous component of chain 60,000 Mr that did not react with the anti-type VIII collagen IgG. Type VIII collagen therefore appears to be synthesized as a discontinuous triple helical molecule with a predominant chain 125,000 Mr by subconfluent, proliferating cells in culture. Immunofluorescence studies with the mAb showed that type VIII collagen was deposited as fibrils in the extracellular matrix of corneal endothelial cells. In the fetal calf, type VIII collagen was absent from basement membranes and was found in a limited number of tissues. In addition to the linear staining pattern observed in the Descemet's membrane, type VIII collagen was found in highly fibrillar arrays in the ocular sclera, in the meninges surrounding brain, spinal cord, and optic nerve, and in periosteum and perichondrium. Fine fibrils were evident in the white matter of spinal cord, whereas a more generalized staining was apparent in the matrices of cartilage and bone. Despite attempts to unmask the epitope, type VIII collagen was not found in aorta, kidney, lung, liver, skin, and ligament. We conclude that this unusual collagen is a component of certain specialized extracellular matrices, several of which are derived from the neural crest.
从牛眼角膜后弹力层分离出一种抗胃蛋白酶的VIII型胶原三螺旋结构域（分子量50,000），并将其用作制备单克隆抗体的免疫原。选择一种抗体用于生化和组织免疫荧光研究，通过竞争ELISA和免疫印迹法，该抗体与后弹力层以及VIII型胶原50,000分子量的多肽均发生反应。通过竞争ELISA，该抗体与I-VI型胶原无交叉反应。该单克隆抗体从代谢标记24小时的亚汇合牛角膜内皮细胞培养基中特异性沉淀出VIII型胶原的高分子量成分（EC2，分子量125,000）。相反，在含有胎牛血清和同位素的情况下培养7天的汇合细胞分泌出一种分子量60,000的胶原成分，该成分与抗VIII型胶原IgG不发生反应。因此，VIII型胶原似乎是由培养中的亚汇合增殖细胞合成的一种不连续三螺旋分子，主要链分子量为125,000。用该单克隆抗体进行的免疫荧光研究表明，VIII型胶原以纤维形式沉积在角膜内皮细胞的细胞外基质中。在胎牛中，VIII型胶原不存在于基底膜中，仅在少数组织中发现。除了在后弹力层观察到的线性染色模式外，VIII型胶原还存在于眼巩膜的高度纤维状排列中、围绕脑、脊髓和视神经的脑膜中以及骨膜和软骨膜中。在脊髓白质中可见细纤维，而在软骨和骨基质中则呈现更广泛的染色。尽管尝试揭示该表位，但在主动脉、肾脏、肺、肝脏、皮肤和韧带中未发现VIII型胶原。我们得出结论，这种不寻常的胶原是某些特殊细胞外基质的组成成分，其中几种源自神经嵴。
