Cargando…

Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

Alteration in cellular prion protein (PrP(C)) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrP(Sc)). A patient with Gerstmann-Sträussler-Scheinke...

Descripción completa

Detalles Bibliográficos
Autores principales: Shen, Pingping, Dang, Johnny, Wang, Zerui, Zhang, Weiguanliu, Yuan, Jue, Lang, Yue, Ding, Mingxuan, Mitchell, Marcus, Kong, Qingzhong, Feng, Jiachun, Rozemuller, Annemiek J. M., Cui, Li, Petersen, Robert B., Zou, Wen-Quan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695670/
https://www.ncbi.nlm.nih.gov/pubmed/32889654
http://dx.doi.org/10.1007/s12035-020-02098-8
_version_ 1783615240138653696
author Shen, Pingping
Dang, Johnny
Wang, Zerui
Zhang, Weiguanliu
Yuan, Jue
Lang, Yue
Ding, Mingxuan
Mitchell, Marcus
Kong, Qingzhong
Feng, Jiachun
Rozemuller, Annemiek J. M.
Cui, Li
Petersen, Robert B.
Zou, Wen-Quan
author_facet Shen, Pingping
Dang, Johnny
Wang, Zerui
Zhang, Weiguanliu
Yuan, Jue
Lang, Yue
Ding, Mingxuan
Mitchell, Marcus
Kong, Qingzhong
Feng, Jiachun
Rozemuller, Annemiek J. M.
Cui, Li
Petersen, Robert B.
Zou, Wen-Quan
author_sort Shen, Pingping
collection PubMed
description Alteration in cellular prion protein (PrP(C)) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrP(Sc)). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrP(Sc) and cellular trafficking of PrP(Q227X) remain to be determined. Here, we show that PrP(Sc) in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrP(Wt)), suggesting pathological PrP(Q227X) is incapable of recruiting PrP(Wt) in vivo. This mutant anchorless protein, however, is able to recruit PrP(Wt) from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrP(Sc)-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrP(Q227X) or PrP(Wt) were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrP(Wt) is recovered in the cell lysate fraction, while most of the mutant PrP(Q227X) is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrP(Q227X) spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.
format Online
Article
Text
id pubmed-7695670
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Springer US
record_format MEDLINE/PubMed
spelling pubmed-76956702020-12-09 Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro Shen, Pingping Dang, Johnny Wang, Zerui Zhang, Weiguanliu Yuan, Jue Lang, Yue Ding, Mingxuan Mitchell, Marcus Kong, Qingzhong Feng, Jiachun Rozemuller, Annemiek J. M. Cui, Li Petersen, Robert B. Zou, Wen-Quan Mol Neurobiol Article Alteration in cellular prion protein (PrP(C)) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrP(Sc)). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrP(Sc) and cellular trafficking of PrP(Q227X) remain to be determined. Here, we show that PrP(Sc) in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrP(Wt)), suggesting pathological PrP(Q227X) is incapable of recruiting PrP(Wt) in vivo. This mutant anchorless protein, however, is able to recruit PrP(Wt) from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrP(Sc)-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrP(Q227X) or PrP(Wt) were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrP(Wt) is recovered in the cell lysate fraction, while most of the mutant PrP(Q227X) is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrP(Q227X) spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein. Springer US 2020-09-05 2021 /pmc/articles/PMC7695670/ /pubmed/32889654 http://dx.doi.org/10.1007/s12035-020-02098-8 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Shen, Pingping
Dang, Johnny
Wang, Zerui
Zhang, Weiguanliu
Yuan, Jue
Lang, Yue
Ding, Mingxuan
Mitchell, Marcus
Kong, Qingzhong
Feng, Jiachun
Rozemuller, Annemiek J. M.
Cui, Li
Petersen, Robert B.
Zou, Wen-Quan
Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro
title Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro
title_full Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro
title_fullStr Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro
title_full_unstemmed Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro
title_short Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro
title_sort characterization of anchorless human prp with q227x stop mutation linked to gerstmann-sträussler-scheinker syndrome in vivo and in vitro
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695670/
https://www.ncbi.nlm.nih.gov/pubmed/32889654
http://dx.doi.org/10.1007/s12035-020-02098-8
work_keys_str_mv AT shenpingping characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT dangjohnny characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT wangzerui characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT zhangweiguanliu characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT yuanjue characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT langyue characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT dingmingxuan characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT mitchellmarcus characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT kongqingzhong characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT fengjiachun characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT rozemullerannemiekjm characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT cuili characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT petersenrobertb characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro
AT zouwenquan characterizationofanchorlesshumanprpwithq227xstopmutationlinkedtogerstmannstrausslerscheinkersyndromeinvivoandinvitro