Precursor cystatin C in cultured retinal pigment epithelium cells: Evidence for processing through the secretory pathway

L. Paraoan, M. R H White, D. G. Spiller, I. Grierson, B. E H Maden

    Research output: Contribution to journalArticlepeer-review


    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.
    Original languageEnglish
    Pages (from-to)229-236
    Number of pages7
    JournalMolecular Membrane Biology
    Issue number3
    Publication statusPublished - 2001


    • Cystatin C
    • EGFP-fusion protein
    • Golgi apparatus
    • Retinal pigment epithelium
    • Secretion


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