Cystathionine beta-synthase p.S466L mutation causes hyperhomocysteinemia in mice

Abstract

Missense mutations in the cystathionine beta-synthase (CBS) gene are the most common cause of clinical homocystinuria in humans. The p.S466L mutation was identified in a homocystinuric patient, but enzymatic studies with recombinant protein show this mutant to be highly active. To understand how this mutation causes disease in vivo, we have created mice lacking endogenous mouse CBS and expressing either wild-type (Tg-hCBS) or p.S466L (Tg-S466L) human CBS under control of zinc inducible metallothionein promoter. In the presence of zinc, we found that the mean serum total homocysteine (tHcy) of Tg-S466L mice was 142+/-55 microM compared to 16+/-13 microM for hCBS mice. Tg-S466L mice also had significantly higher levels of total free homocysteine and S-adenosylhomocysteine in liver and kidney. Only 48% of Tg-S466L mice had detectable CBS protein in the liver, whereas all the Tg-hCBS animals had detectable protein. Surprisingly, CBS mRNA was significantly elevated in Tg-S466L animals compared to Tg-hCBS, implying that the reduction in p.S466L protein was occurring due to posttranscriptional mechanisms. In Tg-S466L animals with detectable liver CBS, the enzyme formed tetramers and was active, but lacked inducibility by S-adenosylmethionine (AdoMet). However, even in Tg-S466L animals that had in vitro liver CBS activity equivalent to Tg-hCBS animals there was significant elevation of serum tHcy. Our results show that p.S466L causes homocystinuria by affecting both the steady state level of CBS protein and by reducing the efficiency of the enzyme in vivo.

FIGURE 1

Serum tHcy and methionine inTg-S466L and Tg-hCBS mice. Mice lacking mouse CBS containing either the p.S466L or wild-type human CBS transgene were induced with zinc water for 10 days. Serum was then collected and analyzed for tHcy and methionine. A: Distribution of tHcy measurements. Dots indicate individual animals. Lines indicate average and standard deviation. The difference between the distributions is highly significant (P < 0.0001). B: Distribution of serum methionine concentrations.

FIGURE 2

Total free homocysteine and AdoHcy levels in tissues. Total free homocysteine and AdoHcy were measured in liver, kidney, and brain from Tg-hCBS and Tg-S466L animals as described in Materials and Methods. A:Tissue homocysteine content. Mean and standard deviation are shown. B:Tissue AdoHcy content. **P < 0.005; *P < 0.05.

FIGURE 3

CBS protein levels in the liver. Mice carrying the indicated transgene were induced with zinc water for 10 days and liver extracts were assessed for CBS by Western blot. A: Representative Western blots showing the variable expression of CBS in Tg-S466L mice and in comparison with Tg-hCBS mice. Actin loading control is shown below. B: Bar graph summarizing Western data for all 29 Tg-S466 mice assessed for liver CBS protein.

FIGURE 4

Examination of p.S466L and wild-type CBS enzyme activity and multimers formation. A: Enzyme activity and AdoMet response of wild-type and p.S466L CBS. The first set of columns shows CBS activity in liver extracts derived from Tg-hCBS mice in the absence and presence of AdoMet (n = 3). The second set of columns shows the same for Tg-S466L mice (n = 6). The third set shows the relative Tg-S466L activity when adjusted for total p.S466L protein as judged by Western blot analysis. B: Comparison of the CBS protein in Tg-hCBS and expressing Tg-S466L mice livers using native gels followed by Western blot analysis.

FIGURE 5

Correlation between CBS activity, tetramer formation, and serum tHcy. A: Correlation between CBS tetramer density and CBS activity (in presence of AdoMet) in the livers of mice of respective genotype. Black dots represent Tg-hCBS mice and white dots represent Tg-S466L mice. B: Correlation between liver CBS activity (in presence of AdoMet) and serum tHcy in Tg-hCBS (n = 8) and Tg-S466L (n = 9) mice. CBS activity has been measured in the terms of micromoles (µmol) of cystathionine formed per milligram of protein per hr.