Differential interaction of molecular chaperones with procollagen I and type IV collagen in corneal endothelial cells.

PURPOSE: Procollagen I is synthesized and intracellularly degraded in corneal endothelial cells (CEC), whereas type IV and VIII collagens are secreted into Descemet's membrane. In our previous study, we demonstrated that procollagen I synthesized by CEC is improperly folded and that the molecule was largely colocalized with protein disulfide isomerase (PDI) within the endoplasmic reticulum (ER). In the present study, we further investigated whether the alpha-subunit of prolyl 4-hydroxylase (P4Halpha) and glucose regulatory protein/immunoglobulin heavy chain binding protein (Grp78/BiP) were also involved in ER retention of procollagen I in CEC.
METHODS: Immunocytochemical analysis was performed to determine the colocalization of procollagen I with molecular chaprones. Protein synthesis was measured by immunoblot analysis and the association between proteins was determined by coimmunoprecipitation followed by immunoblot analysis. mRNA was quantitated using RT-PCR.
RESULTS: To study the interaction of procollagen I with certain molecular chaperones involved in the collagen biosynthetic pathway, we determined whether procollagen I colocalized with P4Halpha and Grp78/Bip, and then compared this molecular chaperone colocalization with their association with type IV collagen. Procollagen I was colocalized with either P4Halpha or Grp78/Bip to a much lesser degree than type IV collagen was colocalized with these same ER proteins. Colocalization between the molecular chaperones demonstrated that P4Halpha and Grp78/Bip were largely colocalized in the peripheral region of the ER, whereas colocalization of P4Halpha and PDI was mostly limited to a small region of the ER. When cells were treated with alpha,alpha-dipyridyl, the inhibitor did not affect the colocalization profiles of collagens with the molecular chaperones. However, the inhibitor markedly increased colocalization of P4Halpha and PDI, but it significantly decreased colocalization between P4Halpha and Grp78/Bip. When synthesis of the molecular chaperones was compared between CEC and corneal stromal fibroblasts (CSF), more Grp78/Bip and PDI were produced by CEC than by CSF. On the other hand, expression of Hsp47 was lower in CEC than it was in CSF. Coimmunoprecipitation was used to compare the association of P4Halpha or Grp78/Bip with collagens in CEC and CSF. The association of collagens (regardless of type) with P4Halpha or Grp78/Bip was much higher in CEC than in CSF. When the association of collagen molecules with respective molecular chaperones was compared in CEC, the degree of association between Grp78/Bip and procollagen I was similar to that between the molecular chaperone and type IV collagen. On the other hand, the degree of association between P4Halpha and type IV collagen was much higher than that between P4Halpha and procollagen I.
CONCLUSIONS: These data suggest that procollagen I and type IV collagen may use different molecular chaperones in the ER, thus targeting their distinctive destinations.