Abstract
Abstract: :
Purpose: To investigate the role of protein disulfide isomerase (PDI) in intracellular degradation of procollagen I and to determine the degradation site of procollagen I in corneal endothelial cells (CEC). Methods: Protein-protein interaction was determined by co-immunoprecipitation and immunoblotting. Colocalization of the proteins was determined by immunocytochemical analysis. Subcellular fractionation was performed using a self-generating gradient. Overexpression of PDI and PDI minus KDEL (PDI-KDEL) was performed by transfection, using Effectene. Ribostamycin was used to block the chaperone activity of PDI. Lactacystin (proteasome inhibitor) and chloroquine (lysosome inhibitor) were used to determine the degradation site of procollagen I. Results: The immune complex precipitated with anti-procollagen I carboxyl propeptide (PICP) antibody contained more PDI than those precipitated with either anti-pro α1(I) or pro α2(I) antibodies. PDI has a different sedimentation profile in CEC when compared to that of corneal stromal fibroblasts. Cells transfected with PDI showed a coincidental ER localization of PDI and procollagen I, whereas cells transfected with PDI-KDEL showed a colocalization of the two proteins in the Golgi complex. CEC transfected with PDI-KDEL secreted the protein complex of PDI and procollagen I into the medium, whereas cells transfected with PDI vector did not. Ribostamycin completely blocked colocalization of PDI and procollagen I in the ER. Lactacystin facilitates the colocalization of procollagen I and ubiquitin in the cytosol. On the other hand, chloroquine failed to recruit procollagen I into the Golgi complex. Conclusions: The present study suggests that PDI facilitates ER retention of procollagen I and that the undesirable procollagen I is degraded via ubiquitin-proteasome.
Keywords: cornea: endothelium • chaperones • protein modifications-post translational