Analysis of the Qatari R336C Cystathionine -Synthase Protein in Mice

• Published in: Journal of inherited metabolic disease Vol. 42; no. 5; pp. 831 - 838
• Main Authors: 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.
• Format: Journal Article
• Language: English
• Published: 10-07-2019
• ISSN: 0141-8955; 1573-2665
• DOI: 10.1002/jimd.12140

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 tHcy and liver tHcy compared to 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 seven-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.