Cargando…

Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases

Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogen...

Descripción completa

Detalles Bibliográficos
Autores principales: Sumya, Farhana Taher, Pokrovskaya, Irina D., Lupashin, Vladimir
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8484713/
https://www.ncbi.nlm.nih.gov/pubmed/34603392
http://dx.doi.org/10.3389/fgene.2021.733048
_version_ 1784577379081912320
author Sumya, Farhana Taher
Pokrovskaya, Irina D.
Lupashin, Vladimir
author_facet Sumya, Farhana Taher
Pokrovskaya, Irina D.
Lupashin, Vladimir
author_sort Sumya, Farhana Taher
collection PubMed
description Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs.
format Online
Article
Text
id pubmed-8484713
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-84847132021-10-02 Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases Sumya, Farhana Taher Pokrovskaya, Irina D. Lupashin, Vladimir Front Genet Genetics Conserved Oligomeric Golgi (COG) is an octameric protein complex that orchestrates intra-Golgi trafficking of glycosylation enzymes. Over a hundred individuals with 31 different COG mutations have been identified until now. The cellular phenotypes and clinical presentations of COG-CDGs are heterogeneous, and patients primarily represent neurological, skeletal, and hepatic abnormalities. The establishment of a cellular COG disease model will benefit the molecular study of the disease, explaining the detailed sequence of the interplay between the COG complex and the trafficking machinery. Moreover, patient fibroblasts are not a good representative of all the organ systems and cell types that are affected by COG mutations. We developed and characterized cellular models for human COG4 mutations, specifically in RPE1 and HEK293T cell lines. Using a combination of CRISPR/Cas9 and lentiviral transduction technologies, both myc-tagged wild-type and mutant (G516R and R729W) COG4 proteins were expressed under the endogenous COG4 promoter. Constructed isogenic cell lines were comprehensively characterized using biochemical, microscopy (superresolution and electron), and proteomics approaches. The analysis revealed similar stability and localization of COG complex subunits, wild-type cell growth, and normal Golgi morphology in all three cell lines. Importantly, COG4-G516R cells demonstrated increased HPA-647 binding to the plasma membrane glycoconjugates, while COG4-R729W cells revealed high GNL-647 binding, indicating specific defects in O- and N-glycosylation. Both mutant cell lines express an elevated level of heparin sulfate proteoglycans. Moreover, a quantitative mass-spectrometry analysis of proteins secreted by COG-deficient cell lines revealed abnormal secretion of SIL1 and ERGIC-53 proteins by COG4-G516R cells. Interestingly, the clinical phenotype of patients with congenital mutations in the SIL1 gene (Marinesco-Sjogren syndrome) overlaps with the phenotype of COG4-G516R patients (Saul-Wilson syndrome). Our work is the first compressive study involving the creation of different COG mutations in different cell lines other than the patient’s fibroblast. It may help to address the underlying cause of the phenotypic defects leading to the discovery of a proper treatment guideline for COG-CDGs. Frontiers Media S.A. 2021-09-17 /pmc/articles/PMC8484713/ /pubmed/34603392 http://dx.doi.org/10.3389/fgene.2021.733048 Text en Copyright © 2021 Sumya, Pokrovskaya and Lupashin. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Sumya, Farhana Taher
Pokrovskaya, Irina D.
Lupashin, Vladimir
Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases
title Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases
title_full Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases
title_fullStr Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases
title_full_unstemmed Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases
title_short Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases
title_sort development and initial characterization of cellular models for cog complex-related cdg-ii diseases
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8484713/
https://www.ncbi.nlm.nih.gov/pubmed/34603392
http://dx.doi.org/10.3389/fgene.2021.733048
work_keys_str_mv AT sumyafarhanataher developmentandinitialcharacterizationofcellularmodelsforcogcomplexrelatedcdgiidiseases
AT pokrovskayairinad developmentandinitialcharacterizationofcellularmodelsforcogcomplexrelatedcdgiidiseases
AT lupashinvladimir developmentandinitialcharacterizationofcellularmodelsforcogcomplexrelatedcdgiidiseases