Partial Characterization of the Collagenous Component of the Basement Membrane Produced By Bovine Corneal Endothelial Cells in Vitro.

Abstract

Bovine corneal endothelial cells (BCE) are readily available, pure cultures are easily established, and the cells can be maintained in vitro for many months. Morphologically, the cultured cells closely resemble their native counterparts. Basement membrane material (BM) accumulates beneath the cells in vitro, progressively thickening with increasing time in culture. In these studies, BCE cells (3-16 wks. post-confluent) were labeled for 24 hrs. with ('3)H-Pro. Cell layers were removed with sodium deoxycholate (DOC) to obtain pure basement membrane samples. Hydrolysates of culture basement membranes contained labeled 3-OH-Pro and 4-OH-Pro (3-OH-Pro/4-OH-Pro = .08-.11) in addition to Pro, confirming the presence of collagen in these membranes. Digestion of labelled BM by protease-free bacterial collagenase showed that throughout the study period, 15%-20% of the newly deposited BM proteins were collagenous. SDS-PAGE analysis of proteins extracted from the BM by 1% SDS (90(DEGREES)C., 1hr.) under reducing conditions showed prominent collagenase-sensitive b and s with MW's of approximately 200K, 160K, and 55K when compared to type I collagen st and ards. Pepsin digestion of the BM released additional collagenous proteins that were identified on the basis of collagenase sensitivity, SDS-PAGE profiles vs. known st and ards, and solubility in salt solutions as collagen types III and V. Type III collagen was also identified in the insoluble pepsin resistant residue by a peptide mapping procedure using Staph. aureus V8 protease digestion. Types IV and V collagens were identified immunohistochemically in culture produced BM as were the noncollagenous glycoproteins laminin and fibronectin and the BM-1 basement membrane proteoglycan.