Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway.
Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.
Paraoan
L
L
School of Biological Sciences, University of Liverpool, UK. lparaoan@liverpool.ac.uk
White
M R
MR
Spiller
D G
DG
Grierson
I
I
Maden
B E
BE
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D013997:Time Factors; D014162:Transfection
C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D008164:Luminescent Proteins; D011498:Protein Precursors; D049452:Green Fluorescent Proteins