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

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

Research output: Contribution to journalArticle (journal)peer-review

24 Citations (Scopus)

Abstract

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)229-36
Number of pages8
JournalMolecular Membrane Biology
Volume18
Issue number3
DOIs
Publication statusPublished - 30 Oct 2001
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Blotting, Western
  • Cells, Cultured
  • Cystatin C
  • Cystatins/chemistry
  • Golgi Apparatus/metabolism
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Luminescent Proteins/metabolism
  • Microscopy, Fluorescence
  • Molecular Weight
  • Pigment Epithelium of Eye/cytology
  • Protein Precursors/chemistry
  • Protein Processing, Post-Translational
  • Protein Transport
  • Time Factors
  • Transfection

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