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Precursor cystatin C Ala25Ser variant displays an intermediary phenotype between wild–type and AMD–associated cystatin C

Precursor cystatin C Ala25Ser variant displays an intermediary phenotype between wild–type and AMD–associated cystatin C
Precursor cystatin C Ala25Ser variant displays an intermediary phenotype between wild–type and AMD–associated cystatin C
Purpose: To investigate intracellular trafficking of a signal sequence variant of precursor cystatin C with biochemical properties intermediary between those of the wild–type and of AMD–associated variant cystatin C.

Methods: The penultimate amino acid of the wild–type cystatin C signal sequence (Ala) was mutated to serine by in vitro mutagenesis using the QuikChange Site–directed mutagenesis kit. The resulting construct (Ala25Ser cysC–EGFP) encoded the full–length mutated pre cystatin C fused in–frame to the N–terminus of pEGFP–N3 vector. Primary RPE cells in culture and HeLa cells were transiently transfected with an endotoxin–free plasmid preparation of the construct using FUGENE 6 transfection reagent. The localization and trafficking of the newly synthesized Ala25Ser pre cystatin C were studied in living cells by confocal microscopy. MitoTracker Red CM–H2Xros was used to stain active mitochondria. Bleaching experiments of various sub–cellular organelles and regions of the cells were performed in order to assess the specificity of the localization. In addition, precursor cystatin C was analyzed qualitatively and semi–quantitatively in cell lysates and conditioned media of transfected cells by Western blotting.

Results: Ala25Ser cysC–EGFP was distributed between a perinuclear localization identified as ER/Golgi, and the mitochondria, the latter indicated by co–localization with MitoTracker Red. Some diffuse staining was observed in the nucleus but not in the nucleolus, and in the cytoplasm of transiently transfected cells. Following bleaching, rapid re–fluorescence of the ER/Golgi body was observed while bleaching of the nucleus removed the minimal nuclear fluorescence which was not restored readily. Endogenous cystatin C and cystatin C–EGFP fusion protein were detected in similar proportions in lysates of cells transfected with wild–type and mutant precursor cystatin C constructs. Secretion of the Ala25Ser cystatin C fusion protein was however reduced compared with the wild–type cystatin C fusion protein while secretion of endogenous cystatin C was not affected.

Conclusions: Variant cystatin C investigated displays an intermediary phenotype which helps elucidate the mechanism of intracellular mis–localization previously determined for the AMD–associated variant Ala25Thr cystatin C. The intracellular pattern of localization, together with the partial secretion impairment suggests that the mutant precursor cystatin C fails to be properly translocated to the ER.
Retinal Pigment Epithelium (RPE), Age-related Macular Degeneration (AMD), enzymes/enzyme inhibitors
0146-0404
1753
Ratnayaka, J.A.
002499b8-1a9f-45b6-9539-5ac145799dfd
Paraoan, L.
252bc6c9-e9b1-4bbb-a2d3-d7fb91826b0e
Hiscott, P.
c5f18179-36ab-4466-98a3-906e6638e10a
Spiller, D.G.
7e3d0b1f-86a8-4fb5-b9f5-2cd4c2f64b44
White, M.R.H.
d0d97c08-20c6-4452-a7f4-db92ae05109c
Grierson, I.
b7edfcbe-f96e-4fe9-a5f1-b235300d011a
Ratnayaka, J.A.
002499b8-1a9f-45b6-9539-5ac145799dfd
Paraoan, L.
252bc6c9-e9b1-4bbb-a2d3-d7fb91826b0e
Hiscott, P.
c5f18179-36ab-4466-98a3-906e6638e10a
Spiller, D.G.
7e3d0b1f-86a8-4fb5-b9f5-2cd4c2f64b44
White, M.R.H.
d0d97c08-20c6-4452-a7f4-db92ae05109c
Grierson, I.
b7edfcbe-f96e-4fe9-a5f1-b235300d011a

Ratnayaka, J.A., Paraoan, L., Hiscott, P., Spiller, D.G., White, M.R.H. and Grierson, I. (2005) Precursor cystatin C Ala25Ser variant displays an intermediary phenotype between wild–type and AMD–associated cystatin C. Investigative Ophthalmology & Visual Science, 46 (13), 1753.

Record type: Meeting abstract

Abstract

Purpose: To investigate intracellular trafficking of a signal sequence variant of precursor cystatin C with biochemical properties intermediary between those of the wild–type and of AMD–associated variant cystatin C.

Methods: The penultimate amino acid of the wild–type cystatin C signal sequence (Ala) was mutated to serine by in vitro mutagenesis using the QuikChange Site–directed mutagenesis kit. The resulting construct (Ala25Ser cysC–EGFP) encoded the full–length mutated pre cystatin C fused in–frame to the N–terminus of pEGFP–N3 vector. Primary RPE cells in culture and HeLa cells were transiently transfected with an endotoxin–free plasmid preparation of the construct using FUGENE 6 transfection reagent. The localization and trafficking of the newly synthesized Ala25Ser pre cystatin C were studied in living cells by confocal microscopy. MitoTracker Red CM–H2Xros was used to stain active mitochondria. Bleaching experiments of various sub–cellular organelles and regions of the cells were performed in order to assess the specificity of the localization. In addition, precursor cystatin C was analyzed qualitatively and semi–quantitatively in cell lysates and conditioned media of transfected cells by Western blotting.

Results: Ala25Ser cysC–EGFP was distributed between a perinuclear localization identified as ER/Golgi, and the mitochondria, the latter indicated by co–localization with MitoTracker Red. Some diffuse staining was observed in the nucleus but not in the nucleolus, and in the cytoplasm of transiently transfected cells. Following bleaching, rapid re–fluorescence of the ER/Golgi body was observed while bleaching of the nucleus removed the minimal nuclear fluorescence which was not restored readily. Endogenous cystatin C and cystatin C–EGFP fusion protein were detected in similar proportions in lysates of cells transfected with wild–type and mutant precursor cystatin C constructs. Secretion of the Ala25Ser cystatin C fusion protein was however reduced compared with the wild–type cystatin C fusion protein while secretion of endogenous cystatin C was not affected.

Conclusions: Variant cystatin C investigated displays an intermediary phenotype which helps elucidate the mechanism of intracellular mis–localization previously determined for the AMD–associated variant Ala25Thr cystatin C. The intracellular pattern of localization, together with the partial secretion impairment suggests that the mutant precursor cystatin C fails to be properly translocated to the ER.

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More information

Published date: 1 May 2005
Keywords: Retinal Pigment Epithelium (RPE), Age-related Macular Degeneration (AMD), enzymes/enzyme inhibitors

Identifiers

Local EPrints ID: 423095
URI: http://eprints.soton.ac.uk/id/eprint/423095
ISSN: 0146-0404
PURE UUID: 9a62af21-caa1-4b3c-9055-ca41c4acac9c
ORCID for J.A. Ratnayaka: ORCID iD orcid.org/0000-0002-1027-6938

Catalogue record

Date deposited: 14 Aug 2018 16:30
Last modified: 10 Nov 2021 03:34

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Contributors

Author: J.A. Ratnayaka ORCID iD
Author: L. Paraoan
Author: P. Hiscott
Author: D.G. Spiller
Author: M.R.H. White
Author: I. Grierson

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