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Analysis of the Qatari R336C cystathionine β-synthase protein in mice


Gupta, Sapna; Gallego-Villar, Lorena; Wang, Liqun; Lee, Hyung-Ok; Nasrallah, Gheyath; Al-Dewik, Nader; Häberle, Johannes; Thöny, Beat; Blom, Henk J; Ben-Omran, Tawfeg; Kruger, Warren D (2019). Analysis of the Qatari R336C cystathionine β-synthase protein in mice. Journal of Inherited Metabolic Disease, 42(5):831-838.

Abstract

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs $^{-/-}$ ). Zinc-treated Tg-R336C Cbs $^{-/-}$ mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs $^{-/-}$ mice are significantly reduced compared to that found in Tg-hCBS Cbs $^{-/-}$ mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs $^{-/-}$ mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs $^{-/-}$ wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

Abstract

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs $^{-/-}$ ). Zinc-treated Tg-R336C Cbs $^{-/-}$ mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs $^{-/-}$ mice are significantly reduced compared to that found in Tg-hCBS Cbs $^{-/-}$ mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs $^{-/-}$ mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs $^{-/-}$ wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Children's Hospital Zurich > Medical Clinic
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:September 2019
Deposited On:07 Feb 2020 11:36
Last Modified:07 Feb 2020 11:49
Publisher:Wiley-Blackwell Publishing, Inc.
ISSN:0141-8955
OA Status:Closed
Publisher DOI:https://doi.org/10.1002/jimd.12140
PubMed ID:31240737

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