MATURATION OF COLLAGEN FIBRILS IN THE CORNEAL STROMA RESULTS IN MASKING OF TYROSINE-RICH REGION OF TYPE-V PROCOLLAGEN

Purpose. To determine the molecular form of type V procollagen in coll agen fibrils in mammalian corneal stromas, Methods, The presence of th e tyrosine-rich region in the NH2-propeptide of type V procollagen in collagen fibrils was examined in human, bovine, and mouse corneas and human corneal fibroblast cultures by immunofluorescence microscopy and immunoblot analysis using a polyclonal antibody specific for this reg ion. The antibody was generated using a glutathione S-transferase-fusi on peptide. Results, The tyrosine-rich region was detected readily in frozen sections of 5- to 6-month-old mouse corneal stromas without the need for any unmasking techniques, indicating that this domain is exp osed on the surface of striated collagen fibrils. In contrast, frozen sections of adult human and bovine corneas did not label with the poly clonal sera to the tyrosine-rich region. Immunoblot analysis of bacter ial collagenase digests of human and bovine corneas, however, indicate d that peptide fragments containing the tyrosine-rich region of type V procollagen and of the expected molecular weight of 70 to 85 kDa were present. Further immunofluorescence microscopic studies and immunoblo t analysis of mouse corneas at different ages and of collagen fibrils formed in human corneal fibroblast cultures over time indicated that, initially, the tyrosine-rich region of type V procollagen could be det ected in all these collagen fibrils; however, as the age of the mouse and the culture increased, the ability to detect this region decreased . Conclusions. These results suggest that, in vivo, the tyrosine-rich region of type V procollagen is retained on type V procollagen molecul es within mammalian collagen fibrils from corneal stromas and that thi s region becomes masked as collagen fibrils mature or the species ages .