Underwood P A, Bennett F A, Mott M R, Strike P
CSIRO Division of Biomolecular Engineering, Sydney Laboratory, North Ryde, NSW, Australia.
Exp Eye Res. 1994 Feb;58(2):139-53. doi: 10.1006/exer.1994.1002.
This paper describes the biochemical characteristics and immunostaining properties of four monoclonal antibodies (MAbs) raised to the extracellular matrix of bovine corneal endothelial cells in culture. All four antibodies labelled molecules in frozen sections of bovine cornea. MAb A15 labelled the collagen lamellae of the stroma. Immuno-electron microscopy located the label between rather than over the collagen fibrils. Antibody label was removed by collagenase treatment of the stroma. This antibody bound to several denatured collagen types in ELISAs and Western blots, the common epitope being located close to the collagenase binding site. A15 immunoprecipitated native polypeptides of 173 kDa and 150 kDa from the conditioned medium of corneal endothelial cells. Amino acid analysis of the 150 kDa molecule showed broad similarity to bovine type VI collagen although there was no immunological cross reactivity. The binding of this antibody in the corneal stroma may be to a collagen type VI-like molecule. MAb A70 bound to a collagenase sensitive molecule in corneal endothelial cell extracellular matrix in vitro, and to basement membranes in vivo. It showed staining typical of type IV collagen in frozen sections of cornea. MAbs A67 and A49 both labelled flexuous fibre-like structures in the cornea, which appeared to wrap around the fibrillar collagen lamellae. The molecule labelled by A67, of 86 kDa was sensitive to collagenase treatment of endothelial cell conditioned medium, yet totally resistant to collagenase treatment of the cornea, which completely removed the fibrillar collagen from the stroma. In the endothelial extracellular matrix, this antigen was resistant to all proteases tested and required severe denaturing conditions to remove antigenic activity. Amino acid analysis did not yield the high proportion of glycine typical of collagens, so if collagenous sequences occur they must be relatively short. The molecule labelled by A49 was a 51-kDa protein, of similar amino acid composition to antigen 67, and showed a similar distribution in frozen sections of bovine cornea. There was, however, no immunological cross reactivity between the two. Unlike antigen 67, antigen 49 was not sensitive to collagenase under any conditions and was very sensitive to protease treatment of the endothelial extracellular matrix. Immuno-electron microscopy showed labelling with both these antibodies between the collagen fibrils in the stroma. We postulate that these two molecules may be involved in stabilising the lamella structure of the corneal stroma.
本文描述了针对培养的牛角膜内皮细胞细胞外基质产生的四种单克隆抗体（MAb）的生化特性和免疫染色特性。所有四种抗体均标记了牛角膜冰冻切片中的分子。单克隆抗体A15标记了基质的胶原板层。免疫电子显微镜将标记定位在胶原纤维之间而非之上。通过对基质进行胶原酶处理可去除抗体标记。该抗体在酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法（Western blot）中与几种变性胶原类型结合，共同表位位于靠近胶原酶结合位点处。A15从角膜内皮细胞条件培养基中免疫沉淀出173 kDa和150 kDa的天然多肽。对150 kDa分子的氨基酸分析显示与牛VI型胶原具有广泛相似性，尽管不存在免疫交叉反应。该抗体在角膜基质中的结合可能是针对一种VI型胶原样分子。单克隆抗体A70在体外与角膜内皮细胞细胞外基质中对胶原酶敏感的分子结合，在体内与基底膜结合。它在角膜冰冻切片中显示出典型的IV型胶原染色。单克隆抗体A67和A49均标记了角膜中弯曲的纤维状结构，这些结构似乎包裹着纤维状胶原板层。A67标记的86 kDa分子对内皮细胞条件培养基进行胶原酶处理敏感，但对角膜进行胶原酶处理完全耐受，后者可从基质中完全去除纤维状胶原。在内皮细胞外基质中，该抗原对所有测试的蛋白酶均耐受，并且需要严苛的变性条件才能去除抗原活性。氨基酸分析未产生胶原典型的高比例甘氨酸，因此如果存在胶原序列，它们必定相对较短。A49标记的分子是一种51 kDa的蛋白质，其氨基酸组成与抗原67相似，并且在牛角膜冰冻切片中显示出相似的分布。然而，两者之间不存在免疫交叉反应。与抗原67不同，抗原49在任何条件下对胶原酶均不敏感，并且对内皮细胞外基质进行蛋白酶处理非常敏感。免疫电子显微镜显示，这两种抗体均在基质中的胶原纤维之间产生标记。我们推测这两种分子可能参与稳定角膜基质的板层结构。
