Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells.
To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.
Doheny Eye Institute, 1450 San Pablo Street, DVRC 203, Los Angeles, California 90033, USA.
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D000818:Animals; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000737:chemistry* / Q000166:cytology; D011347:Procollagen / Q000032:analysis* / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis* / Q000096:biosynthesis; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000032:analysis / Q000096:biosynthesis; D011817:Rabbits; D013347:Subcellular Fractions
D011347:Procollagen; D011393:Procollagen-Proline Dioxygenase; D019704:Protein Disulfide-Isomerases