Cargando…

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism

Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interes...

Descripción completa

Detalles Bibliográficos
Autores principales: Nguyen, Annie, Hulleman, John D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741414/
https://www.ncbi.nlm.nih.gov/pubmed/26845025
http://dx.doi.org/10.1371/journal.pone.0147684
_version_ 1782413985771945984
author Nguyen, Annie
Hulleman, John D.
author_facet Nguyen, Annie
Hulleman, John D.
author_sort Nguyen, Annie
collection PubMed
description Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively). Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as ‘site 2’ cleavage). However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K) mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 (‘site 1’) or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C.
format Online
Article
Text
id pubmed-4741414
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-47414142016-02-11 Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism Nguyen, Annie Hulleman, John D. PLoS One Research Article Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively). Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as ‘site 2’ cleavage). However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K) mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 (‘site 1’) or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C. Public Library of Science 2016-02-04 /pmc/articles/PMC4741414/ /pubmed/26845025 http://dx.doi.org/10.1371/journal.pone.0147684 Text en © 2016 Nguyen, Hulleman http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Nguyen, Annie
Hulleman, John D.
Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
title Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
title_full Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
title_fullStr Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
title_full_unstemmed Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
title_short Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
title_sort evidence of alternative cystatin c signal sequence cleavage which is influenced by the a25t polymorphism
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741414/
https://www.ncbi.nlm.nih.gov/pubmed/26845025
http://dx.doi.org/10.1371/journal.pone.0147684
work_keys_str_mv AT nguyenannie evidenceofalternativecystatincsignalsequencecleavagewhichisinfluencedbythea25tpolymorphism
AT hullemanjohnd evidenceofalternativecystatincsignalsequencecleavagewhichisinfluencedbythea25tpolymorphism