Maturation of collagen fibrils in the corneal stroma results in masking of tyrosine-rich region of type V procollagen.
OBJECTIVE
To determine the molecular form of type V procollagen in collagen fibrils in mammalian corneal stromas.


METHODS
The presence of the 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 region. The antibody was generated using a glutathione S-transferase-fusion 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 exposed on the surface of striated collagen fibrils. In contrast, frozen sections of adult human and bovine corneas did not label with the polyclonal sera to the tyrosine-rich region. Immunoblot analysis of bacterial collagenase digests of human and bovine corneas, however, indicated 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 immunoblot 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 detected 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 molecules within mammalian collagen fibrils from corneal stromas and that this region becomes masked as collagen fibrils mature or the species ages.
Peters
D M
DM
Department of Pathology, University of Wisconsin Medical School Madison, USA.
Kintner
R L
RL
Steger
C
C
Bultmann
K
K
Brandt
C R
CR
D000328:Adult; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry*; D003315:Cornea / Q000737:chemistry; D003319:Corneal Stroma / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D005347:Fibroblasts / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D005982:Glutathione Transferase / Q000032:analysis; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011347:Procollagen / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis; D014443:Tyrosine / Q000032:analysis* / Q000276:immunology
D017931:DNA Primers; D011347:Procollagen; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D003094:Collagen; D005982:Glutathione Transferase