A pepsin-resistant triple helical domain (chain 50,000 M(r)) 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-M(r) 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 M(r)) from the culture medium of subconfluent bovine corneal endothelial cels 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 M(r) 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 M(r) by subconfluent, proliferating cells in culture. Immunofluorescence studies with the mAb showed the type VIII collagen was deposited as fibrils in the extracellular matrix of corneal endothelial cells. In the fetal calf, type VII 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.
ASJC Scopus subject areas
- Cell Biology