S-adenosylhomocysteine hydrolase over-expression does not alter S-adenosylmethionine or S-adenosylhomocysteine levels in CBS deficient mice

Abstract

Elevated plasma total homocysteine (tHcy) is associated with a number of human diseases including coronary artery disease, stroke, osteoporosis and dementia. It is highly correlated with intracellular S-adenosylhomocysteine (SAH). Since SAH is a strong inhibitor of methyl-transfer reactions involving the methyl-donor S-adenosylmethionine (SAM), elevation in SAH could be an explanation for the wide association of tHcy and human disease. Here, we have created a transgenic mouse (Tg-hAHCY) that expresses human S-adenosylhomocysteine hydrolase (AHCY) from a zinc-inducible promoter in the liver and kidney. Protein analysis shows that human AHCY is expressed well in both liver and kidney, but elevated AHCY enzyme activity (131% increase) is only detected in the kidney due to the high levels of endogenous mouse AHCY expression in liver. Tg-hAHCY mice were crossed with mice lacking cystathionine β-synthase activity (Tg-I278T Cbs^-/- ) to explore the effect to AHCY overexpression in the context of elevated serum tHcy and elevated tissue SAM and SAH. Overexpression of AHCY had no significant effect on the phenotypes of Tg-I278T Cbs^-/- mice or any effect on the steady state concentrations of methionine, total homocysteine, SAM, SAH, and SAM/SAH ratio in the liver and kidney. Furthermore, enhanced AHCY activity did not lower serum and tissue tHcy or methionine levels. Our data suggests that enhancing AHCY activity does not alter the distribution of methionine recycling metabolites, even when they are greatly elevated by Cbs mutations.

Keywords: AHCY, S-adenosylhomocysteine hydrolase; CBS, cystathionine beta synthase; CMC, carboxymethylcellulose; Cbs−,  CBS knockout allele; HA, hemagglutinin; HHcy, hyperhomocysteinemia; Hcy, homocysteine; Met, methionine; Metabolism; Methionine; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; Tg-I278T, transgene human CBS containing the I278T mutation; Transgenic; Zn, zinc water; tHcy, total homocysteine.

Fig. 1

Methionine metabolism. Figure shows both the methionine recycling pathway and the transsulfuration pathway. The key enzymes for this manuscript, CBS and AHCY are shown in bold by their respective reactions.

Fig. 2

AHCY expression and activity. (A) Western blot analysis of Tg-hAHCY liver and kidney lysates from zinc-induced animals. Lanes labeled WT are from non-transgenic siblings. Top row shows level of human AHCY as determined by human specific antibody. Middle row shows total AHCY as determined by anti-body that recognizes both species. (B) Western analysis of Tg-mAhcy liver and kidney. Top row is probed with anti-HA antibody that only recognizes mAHCY expressed from the transgene, middle row shows total AHCY. (C) AHCY enzyme activity in liver (n = 7) and kidney (n = 4) from Tg-hAHCY mice and transgene negative controls. (D) AHCY enzyme activity in liver (n = 3) and kidney (n = 4) from Tg-mAhcy mice and transgene negative controls. Error bars show SEM. Asterisk indicates P < 0.05.

Fig. 3

Effect of proteasome inhibitor on AHCY protein levels. Western blot analysis of transgene negative and Tg-hAHCY liver lysates of oprozomib treated mice. Blots were probed with the indicated antibodies.

Fig. 4

Analysis of Tg-hAHCY Tg-I278T Cbs^−/− mice. (A) Serum tHcy and methionine from wild-type (n = 5), Tg-hAHCY (n = 4), Tg-I278T Cbs^−/− (n = 4) and Tg-hAHCY Tg-I278T Cbs^−/− mice (n = 7). Error bars show SEM. Asterisk indicates P < 0.0001 compared to WT. (B) Western blot analysis of liver and kidney lysates from zinc-induced Tg-hAHCY Tg-I278T Cbs^−/− and Tg-I278T Cbs^−/− mice. (C) Kidney and liver AHCY enzyme activity from Tg-I278T Cbs^−/− (n = 4) and Tg-hAHCY Tg-I278T Cbs^−/− mice (n = 8). Error bars show SEM. Asterisk indicates P < 0.003.

Fig. 5

Phenotype of zinc-treated Tg-hAHCY Tg-I278T Cbs^−/− mice. (A) Photograph of three sibling pairs of 130 day old mice. Genotype of each pair is shown above. (B) Weight gain of mice.

Fig. 6

Concentration of methionine recycling metabolites. (A) Concentration of tHcy, methionine, SAM, and SAH in kidney. The SAM/SAH ratio is also shown. Error bars show SEM. (B) Concentration of tHcy, methionine, SAM, and SAH in liver.