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

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.

Keywords: homocysteine; inborn error; metabolism; methionine; missense mutation; mouse model.

Fig 1.. Phenotypes of Tg-R336C Cbs^−/− mice.

(A), Weight of age and sex matched Tg-R336C Cbs^−/− mice in comparison to sibling control Tg-R366C Cbs^+/−. Mice were aged between 2.2–5.3 months. *** is P<0.005 (paired t-test). (B), Photograph of a 4-month old male sibling pair of indicated genotypes. (C), Representative H & E stained liver sections of 4 month old sibling pair of the indicated genotypes at 40x magnification. Arrow indicates example of mitotic nuclei observed at a higher frequency in Tg-R366C Cbs^−/− mice. Also note larger size of hepatocyte nuclei throughout section.

Fig 2.. Homocysteine and methionine in Tg-R336C Cbs^−/− mice.

(A), Serum total homocysteine (tHcy) and methionine in μM in zinc-induced Tg-hCBS Cbs^−/− and Tg-R336C Cbs−/− mice. Error bar shows standard error (SE). *** indicates P<0.001. (B), tHcy, methionine, and total cysteine (tCys) in liver lysates from zinc-induced Tg-R336C Cbs^−/− and Tg-R336C Cbs^+/− mice. *** indicates P<0.001, **P<0.01, *P<0.05.

Fig 3.. Analysis of p.R336C CBS protein and mRNA.

(A), Western blot of CBS expression in liver lysates of zinc-induced Tg-hCBS Cbs^−/−, Tg-R336C Cbs^−/−, and bortezomib treated Tg-R336C Cbs^−/− mice run on denaturing (top) and native (bottom) gels. Actin was used as a loading control. Positions of molecular weight makers for each gel type is also shown. (B), CBS activity present in dialyzed crude lysates from liver of indicated genotype and treatment. Different letters at top indicate P<0.001 as judged by ANOVA followed by Tukey’s multiple comparison test. (C), Relative liver human CBS mRNA levels as determined by quantitative real time PCR using mouse actin expression as a normalizing control. (D), Serum tHcy homocysteine in indicated mice.