The c.797 G>A (p.R266K) cystathionine β-synthase mutation causes homocystinuria by affecting protein stability

Abstract

Mutations in the cystathionine beta-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The c.797 G>A (p.R266K) mutation in CBS was originally described in several Norwegian pyridoxine responsive CBS deficient patients, and heterologous gene expression studies have shown that the protein has near wild-type levels of enzyme activity. Here, we characterize a transgenic mouse lacking endogenous Cbs and expressing p.R266K human CBS protein from a zinc inducible metallothionein promoter (Tg-R266K Cbs^-/- ). Unlike mice expressing other mutant CBS alleles, the Tg-R266K transgene is unable to efficiently rescue neonatal lethality of Cbs^-/- on a C57BL/6J background. On a C3H/HeJ background, zinc-induced Tg-R266K Cbs^-/- mice express CBS mRNA, but have very low levels of CBS protein and enzyme activity, resulting in extreme elevations in serum total homocysteine (tHcy). Treatment with pyridoxine did not have any appreciable effect on tHcy, indicating this allele is not pyridoxine responsive in mice. However, treatment with the proteasome inhibitor bortezomib resulted in an 97% reduction in tHcy and a 2381% increase in liver CBS activity. These studies show that the p.R266K mutation causes increased proteasomal degradation in vivo, and that treatments that stabilize the protein can be used to reverse its effect.

Keywords: CBS; bortezomib; homocysteine; methionine; pyridoxine.

Figure 1. Characterization of Tg-R266K Cbs^−/− (C57BL/6J) mice

A: Pictures of 150 day old Tg-R266K Cbs^−/−, Tg⁻ Cbs^+/−, and Tg-I278T Cbs^−/− mice. B: Serum tHcy and methionine in controls (Cbs^+/+), Tg-R266K Cbs^−/−, and Tg-hCBS Cbs^−/− mice. All mice were on zinc water. Error bars show standard deviation (n=4 or 5). If letters above column are different, means are statistically significant as determined by post-hoc Tukey’s test. C: Liver CBS activity in the presence and absence of AdoMet (n=4). D: Western blot of CBS protein. All four lanes were on the same blot. At bottom is shown relative quantitation of protein (normalized to actin) and relative transcript levels as determined by qRT-PCR (normalized to actin).

Figure 2

Comparison of liver CBS expression, activity, and serum tHcy of Tg-R266K Cbs^−/− mice on C57BL/6J and C3H/HeJ backgrounds. Total Hcy in micromolar is shown below western. Bottom line shows units of CBS activity (nmoles cystathionine formed/mg total soluble liver protein/hr).

Figure 3. Response to pyridoxine

A: Dialyzed liver extracts from Zn-treated mice carrying the indicated transgene in the indicated strain (B=C57BL/6J; C=C3H/HeJ). Each reaction was done either in the absence (-PLP), or presence of 50 µM (+PLP) or 200 µM (+4PLP) PLP. Error bars show SD (n=3 or 4). B: Comparison of tHcy in Tg-R266K Cbs^−/− mice (C3H/HeJ,) treated with either regular water, zinc-water (25 mM), or zinc-water plus pyridoxine (400 mg/L). Error bars show SD (n=8 per group)

Figure 4. Bortezomib effect on Tg-R266K Cbs^−/− (C3H/HeJ) mice

A: Serum tHcy and Methionine in mice before and after bortezomib treatment (0.49/mg/kg/day, n=8). Error bars show SD. B: CBS Western blot of liver extracts from treated (B) and untreated mice (U). Control (C) is liver extract from Tg-hCBS Cbs^−/− mouse. CBS activity is expressed in nmoles cystathionine formed/mg total soluble liver protein/hr.

Figure 5. CBS activity and pyridoxine response in bortezomib-treated liver extracts

A: CBS enzyme activity in extracts from bortezomib-treated mice of the indicated genotype in the presence and absence of AdoMet. Error bar shows SD (n=4 for each group). B: Ratio of CBS enzyme activity of indicated samples in the presence and absence of added PLP in reaction mixture.