Collagen-associated molecules in the cornea: localisation with monoclonal antibodies

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.