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Molecular mechanisms governing impaired secretion of the AMD-associated variant B precursor cystatin C in RPE cells

Molecular mechanisms governing impaired secretion of the AMD-associated variant B precursor cystatin C in RPE cells
Molecular mechanisms governing impaired secretion of the AMD-associated variant B precursor cystatin C in RPE cells
Purpose: : To characterize the secretion of mature cysteine proteinase inhibitor cystatin C by RPE cells and to determine the molecular basis of decreased secretion of precursor variant B cystatin C associated with increased risk of developing exudative age-related macular degeneration (AMD).

Methods: : Monolayers of ARPE19 cells established on Matrigel-covered membranes, with and without AGE (advanced glycation endproducts)-treatment, were used to assess the efficiency and directionality of cystatin C secretion. Fusion constructs of green fluorescent protein (GFP) with full length wild type precursor, mature, variant B (Ala25 Thr) and biochemically related variant Ala25Ser cystatin C were engineered to be expressed by RPE cells in culture. Confocal fluorescence microscopy was used to determine the subcellular localization of the fusion protein and to monitor the fate of newly synthesized cystatin C-GFP in real-time and living transfected cells. Western blot analysis was used to confirm the expression and stability of the cystatin C-GFP fusion proteins and to assess the levels of mature cystatin C secreted in the culture medium.

Results: : The level of mature cystatin C-GFP fusion protein was significantly reduced in the media of RPE cells transfected with the variant B cystatin C construct, compared with the level in media of wild-type cystatin C-expressing cells. Constructs lacking the 26 amino acids N-terminal signal sequence of cystatin C were not processed through the secretory pathway of RPE or control cells. The biochemically intermediate variant Ala25Ser was processed for secretion more efficiently than variant B, indicating that the hydrophobicity of the C-terminal region of the signal sequence determines the efficiency of processing through the endoplasmic reticulum, most likely at the level of interaction with the translocon. Secretion of mature cystatin C appeared polarized basally in RPE cells and its efficiency decreased in AGE-ing cells.

Conclusions: : In addition to effects caused by inappropriate intracellular processing, the diminished secretion of active, mature cystatin C by RPE cells expressing variant B precursor cystatin C may contribute to the development of AMD most likely through an imbalance in proteolytic activities involved in RPE basement membrane homeostasis.
Retinal Pigment Epithelium (RPE), Proteolysis, age-related macular degeneration
0146-0404
2345
Paraoan, Luminita
252bc6c9-e9b1-4bbb-a2d3-d7fb91826b0e
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Spiller, D.M.
67e058fd-5428-4918-9cec-43a45a449516
White, Michael R.H.
d0d97c08-20c6-4452-a7f4-db92ae05109c
Hiscott, Paul
c5f18179-36ab-4466-98a3-906e6638e10a
Grierson, Ian
b7edfcbe-f96e-4fe9-a5f1-b235300d011a
Paraoan, Luminita
252bc6c9-e9b1-4bbb-a2d3-d7fb91826b0e
Ratnayaka, J. Arjuna
002499b8-1a9f-45b6-9539-5ac145799dfd
Spiller, D.M.
67e058fd-5428-4918-9cec-43a45a449516
White, Michael R.H.
d0d97c08-20c6-4452-a7f4-db92ae05109c
Hiscott, Paul
c5f18179-36ab-4466-98a3-906e6638e10a
Grierson, Ian
b7edfcbe-f96e-4fe9-a5f1-b235300d011a

Paraoan, Luminita, Ratnayaka, J. Arjuna, Spiller, D.M., White, Michael R.H., Hiscott, Paul and Grierson, Ian (2009) Molecular mechanisms governing impaired secretion of the AMD-associated variant B precursor cystatin C in RPE cells. Investigative Ophthalmology & Visual Science, 50 (13), 2345.

Record type: Meeting abstract

Abstract

Purpose: : To characterize the secretion of mature cysteine proteinase inhibitor cystatin C by RPE cells and to determine the molecular basis of decreased secretion of precursor variant B cystatin C associated with increased risk of developing exudative age-related macular degeneration (AMD).

Methods: : Monolayers of ARPE19 cells established on Matrigel-covered membranes, with and without AGE (advanced glycation endproducts)-treatment, were used to assess the efficiency and directionality of cystatin C secretion. Fusion constructs of green fluorescent protein (GFP) with full length wild type precursor, mature, variant B (Ala25 Thr) and biochemically related variant Ala25Ser cystatin C were engineered to be expressed by RPE cells in culture. Confocal fluorescence microscopy was used to determine the subcellular localization of the fusion protein and to monitor the fate of newly synthesized cystatin C-GFP in real-time and living transfected cells. Western blot analysis was used to confirm the expression and stability of the cystatin C-GFP fusion proteins and to assess the levels of mature cystatin C secreted in the culture medium.

Results: : The level of mature cystatin C-GFP fusion protein was significantly reduced in the media of RPE cells transfected with the variant B cystatin C construct, compared with the level in media of wild-type cystatin C-expressing cells. Constructs lacking the 26 amino acids N-terminal signal sequence of cystatin C were not processed through the secretory pathway of RPE or control cells. The biochemically intermediate variant Ala25Ser was processed for secretion more efficiently than variant B, indicating that the hydrophobicity of the C-terminal region of the signal sequence determines the efficiency of processing through the endoplasmic reticulum, most likely at the level of interaction with the translocon. Secretion of mature cystatin C appeared polarized basally in RPE cells and its efficiency decreased in AGE-ing cells.

Conclusions: : In addition to effects caused by inappropriate intracellular processing, the diminished secretion of active, mature cystatin C by RPE cells expressing variant B precursor cystatin C may contribute to the development of AMD most likely through an imbalance in proteolytic activities involved in RPE basement membrane homeostasis.

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More information

Published date: 1 April 2009
Keywords: Retinal Pigment Epithelium (RPE), Proteolysis, age-related macular degeneration

Identifiers

Local EPrints ID: 423096
URI: http://eprints.soton.ac.uk/id/eprint/423096
ISSN: 0146-0404
PURE UUID: 552b98f5-5c27-4fbf-9f4c-7f2d2b1a17f3
ORCID for J. Arjuna 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: Luminita Paraoan
Author: D.M. Spiller
Author: Michael R.H. White
Author: Paul Hiscott
Author: Ian Grierson

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