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Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression
BACKGROUND: Adenosine deaminase 2 (ADA2) is a homodimeric, extracellular enzyme and putative growth factor that is produced by cells of the myeloid lineage and, catalytically, deaminates extracellular adenosine to inosine. Loss-of-(catalytic)-function variants in the ADA2 gene are associated with De...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9562767/ https://www.ncbi.nlm.nih.gov/pubmed/36248868 http://dx.doi.org/10.3389/fimmu.2022.995191 |
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author | Bowers, Sarah M. Sundqvist, Martina Dancey, Paul Cabral, David A. Brown, Kelly L. |
author_facet | Bowers, Sarah M. Sundqvist, Martina Dancey, Paul Cabral, David A. Brown, Kelly L. |
author_sort | Bowers, Sarah M. |
collection | PubMed |
description | BACKGROUND: Adenosine deaminase 2 (ADA2) is a homodimeric, extracellular enzyme and putative growth factor that is produced by cells of the myeloid lineage and, catalytically, deaminates extracellular adenosine to inosine. Loss-of-(catalytic)-function variants in the ADA2 gene are associated with Deficiency of ADA2 (DADA2), an autosomal recessive disease associated with an unusually broad range of inflammatory manifestations including vasculitis, hematological defects and cytopenia. Previous work by our group led to the identification of ADA2 variants of novel association with DADA2, among which was a unique c.1052T>A (p.Leu351Gln; herein referred to as L351Q) variant located in the catalytic domain of the protein. METHODS: Mammalian (Flp-IN CHO) cells were engineered to stably express wild-type ADA2 and ADA2 protein variants, including the pathogenic L351Q variant identified in DADA2 patients. An enzyme assay and immunoblotting were used to assess ADA2 catalytic activity and secretion, respectively, and the outcome of experimentally induced inhibition of protein processing (Golgi transport and N-linked glycosylation) was assessed. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to determine the relative expression of Type I Interferon stimulated genes (ISGs), IFIT3 and IRF7. RESULTS: In addition to abrogating catalytic activity, the L351Q variant impaired secretion of L351Q ADA2 resulting in an intracellular accumulation of L351Q ADA2 protein that was not observed in cells expressing wild-type ADA2 or other ADA2 protein variants. Retention of L351Q ADA2 was not attributable to impaired glycosylation on neighboring asparagine residues and did not impact cell growth or integrity. Constitutive expression of Type I ISGs IFIT3 and IRF7 was observed in cells expressing L351Q ADA2. CONCLUSIONS: The impaired secretion of L351Q ADA2 may be an important factor leading to the severe phenotype observed in patients with this variant further emphasizing the importance of assessing impacts beyond catalytic activity when evaluating genotype-phenotype relationships in DADA2. |
format | Online Article Text |
id | pubmed-9562767 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-95627672022-10-15 Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression Bowers, Sarah M. Sundqvist, Martina Dancey, Paul Cabral, David A. Brown, Kelly L. Front Immunol Immunology BACKGROUND: Adenosine deaminase 2 (ADA2) is a homodimeric, extracellular enzyme and putative growth factor that is produced by cells of the myeloid lineage and, catalytically, deaminates extracellular adenosine to inosine. Loss-of-(catalytic)-function variants in the ADA2 gene are associated with Deficiency of ADA2 (DADA2), an autosomal recessive disease associated with an unusually broad range of inflammatory manifestations including vasculitis, hematological defects and cytopenia. Previous work by our group led to the identification of ADA2 variants of novel association with DADA2, among which was a unique c.1052T>A (p.Leu351Gln; herein referred to as L351Q) variant located in the catalytic domain of the protein. METHODS: Mammalian (Flp-IN CHO) cells were engineered to stably express wild-type ADA2 and ADA2 protein variants, including the pathogenic L351Q variant identified in DADA2 patients. An enzyme assay and immunoblotting were used to assess ADA2 catalytic activity and secretion, respectively, and the outcome of experimentally induced inhibition of protein processing (Golgi transport and N-linked glycosylation) was assessed. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to determine the relative expression of Type I Interferon stimulated genes (ISGs), IFIT3 and IRF7. RESULTS: In addition to abrogating catalytic activity, the L351Q variant impaired secretion of L351Q ADA2 resulting in an intracellular accumulation of L351Q ADA2 protein that was not observed in cells expressing wild-type ADA2 or other ADA2 protein variants. Retention of L351Q ADA2 was not attributable to impaired glycosylation on neighboring asparagine residues and did not impact cell growth or integrity. Constitutive expression of Type I ISGs IFIT3 and IRF7 was observed in cells expressing L351Q ADA2. CONCLUSIONS: The impaired secretion of L351Q ADA2 may be an important factor leading to the severe phenotype observed in patients with this variant further emphasizing the importance of assessing impacts beyond catalytic activity when evaluating genotype-phenotype relationships in DADA2. Frontiers Media S.A. 2022-09-30 /pmc/articles/PMC9562767/ /pubmed/36248868 http://dx.doi.org/10.3389/fimmu.2022.995191 Text en Copyright © 2022 Bowers, Sundqvist, Dancey, Cabral and Brown 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 | Immunology Bowers, Sarah M. Sundqvist, Martina Dancey, Paul Cabral, David A. Brown, Kelly L. Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression |
title | Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression |
title_full | Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression |
title_fullStr | Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression |
title_full_unstemmed | Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression |
title_short | Pathogenic variant c.1052T>A (p.Leu351Gln) in adenosine deaminase 2 impairs secretion and elevates type I IFN responsive gene expression |
title_sort | pathogenic variant c.1052t>a (p.leu351gln) in adenosine deaminase 2 impairs secretion and elevates type i ifn responsive gene expression |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9562767/ https://www.ncbi.nlm.nih.gov/pubmed/36248868 http://dx.doi.org/10.3389/fimmu.2022.995191 |
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