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Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency

Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures...

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Autores principales: Staklinski, Stephen J., Chang, Mario C., Yu, Fang, Collins Ruff, Kathleen, Franz, David N., Qian, Zhijian, Bloom, Linda B., Merritt, Matthew E., McKenna, Robert, Kilberg, Michael S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9478401/
https://www.ncbi.nlm.nih.gov/pubmed/35985424
http://dx.doi.org/10.1016/j.jbc.2022.102385
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author Staklinski, Stephen J.
Chang, Mario C.
Yu, Fang
Collins Ruff, Kathleen
Franz, David N.
Qian, Zhijian
Bloom, Linda B.
Merritt, Matthew E.
McKenna, Robert
Kilberg, Michael S.
author_facet Staklinski, Stephen J.
Chang, Mario C.
Yu, Fang
Collins Ruff, Kathleen
Franz, David N.
Qian, Zhijian
Bloom, Linda B.
Merritt, Matthew E.
McKenna, Robert
Kilberg, Michael S.
author_sort Staklinski, Stephen J.
collection PubMed
description Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child’s cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child’s cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype.
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spelling pubmed-94784012022-09-22 Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency Staklinski, Stephen J. Chang, Mario C. Yu, Fang Collins Ruff, Kathleen Franz, David N. Qian, Zhijian Bloom, Linda B. Merritt, Matthew E. McKenna, Robert Kilberg, Michael S. J Biol Chem Research Article Asparagine synthetase (ASNS) catalyzes synthesis of asparagine (Asn) and Glu from Asp and Gln in an ATP-dependent reaction. Asparagine synthetase deficiency (ASNSD) results from biallelic mutations in the ASNS gene. Affected children exhibit congenital microcephaly, continued brain atrophy, seizures, and often premature mortality. However, the underlying mechanisms are unclear. This report describes a compound heterozygotic ASNSD child with two novel mutations in the ASNS gene, c.1118G>T (paternal) and c.1556G>A (maternal), that lead to G373V or R519H ASNS variants. Structural mapping suggested that neither variant participates directly in catalysis. Growth of cultured fibroblasts from either parent was unaffected in Asn-free medium, whereas growth of the child’s cells was suppressed by about 50%. Analysis of Asn levels unexpectedly revealed that extracellular rather than intracellular Asn correlated with the reduced proliferation during incubation of the child’s cells in Asn-free medium. Our attempts to ectopically express the G373V variant in either HEK293T or JRS cells resulted in minimal protein production, suggesting instability. Protein expression and purification from HEK293T cells revealed reduced activity for the R519H variant relative to WT ASNS. Expression of WT ASNS in ASNS-null JRS cells resulted in nearly complete rescue of growth in Asn-free medium, whereas we observed no proliferation for the cells expressing either the G373V or R519H variant. These results support the conclusion that the coexpression of the G373V and R519H ASNS variants leads to significantly reduced Asn synthesis, which negatively impacts cellular growth. These observations are consistent with the ASNSD phenotype. American Society for Biochemistry and Molecular Biology 2022-08-17 /pmc/articles/PMC9478401/ /pubmed/35985424 http://dx.doi.org/10.1016/j.jbc.2022.102385 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Article
Staklinski, Stephen J.
Chang, Mario C.
Yu, Fang
Collins Ruff, Kathleen
Franz, David N.
Qian, Zhijian
Bloom, Linda B.
Merritt, Matthew E.
McKenna, Robert
Kilberg, Michael S.
Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
title Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
title_full Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
title_fullStr Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
title_full_unstemmed Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
title_short Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
title_sort cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to asparagine synthetase deficiency
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9478401/
https://www.ncbi.nlm.nih.gov/pubmed/35985424
http://dx.doi.org/10.1016/j.jbc.2022.102385
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