Human betaine-homocysteine methyltransferase (BHMT) and BHMT2: common gene sequence variation and functional characterization

Abstract

Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT2 encodes a protein 73% identical in amino acid sequence to BHMT, but the function of BHMT2 remains unclear. We set out to identify and functionally characterize common genetic variation in BHMT and BHMT2. Specifically, we sequenced exons, exon-intron splice junctions and the 5'-flanking regions (5'-FRs) of BHMT and BHMT2 using 240 DNA samples from four ethnic groups. Twenty-five single nucleotide polymorphisms (SNPs), including 4 nonsynonymous SNPs, and 39 SNPs, including 4 nonsynonymous, were observed in BHMT and BHMT2, respectively. BHMT wild type (WT) and variant allozymes were expressed in COS-1 cells. Variant allozymes showed no significant differences from WT in levels of enzyme activity or immunoreactive protein, but there were statistically significant differences in apparent K(m) values. Luciferase reporter gene constructs were created for the three most common BHMT 5'-FR haplotypes, and significant variation was observed in the ability of these constructs to drive transcription. Although BHMT2 mRNA has been observed in human liver and kidney, expression of the protein has not been reported. We were unable to express BHMT2 in mammalian cells, and the protein aggregated after bacterial expression. Furthermore, BHMT2 was rapidly degraded in a rabbit reticulocyte lysate, but it could be stabilized by cotransfection of COS-1 cells with BHMT and, after cotransfection, it coprecipitated with BHMT. These studies have defined common genetic variation in BHMT and BHMT2 and functionally characterized BHMT SNPs. They may also help to explain why BHMT2 has not previously been defined functionally.

Figure 1

The methionine and folate cycles – showing the location of BHMT.

Figure 2

Human BHMT and BHMT2 genetic polymorphisms. The figure shows a schematic representation of the structures of (A) BHMT and (B) BHMT2, with arrow indicating the locations of polymorphisms. Exons encoding the open-reading frame (ORF) are represented as dark rectangles and portions of exons that encode untranslated region sequences are indicated by open rectangles. “Indel” represents an insertion-deletion. Colors of arrows indicate minor allele frequencies for polymorphisms.

Figure 3

BHMT allozyme functional genomics. (A) BHMT allozyme activity levels assayed with 400 μM L-homocysteine and 64 μM betaine as substrates. Activities are expressed as percentages of that of the WT. (B) BHMT allozyme immunoreactive protein levels. Protein levels are expressed as percentages of that of the WT. Values for (A) and (B) represent mean ± SEM. (C) Locations in the human BHMT crystal structure of residues altered by nonsynonymous SNPs. Human BHMT crystallizes as a dimer of dimers [27]. The monomers in the upper dimer are depicted as light blue and purple ribbon structures, and monomers in the lower dimer have lighter shades of the same colors. Bound in the active site of each monomer are a zinc ion (green) and the bi-substrate analog S(δ-carboxybutyl)-L-homocysteine (orange), both shown as space-filling structures. The Cα atoms of the four residues altered by SNPs (Arg16 red; Pro197, Gly199, and Arg239 blue) are drawn as spheres.

Figure 4

Human BHMT 5′-FR reporter gene studies. Luciferase activity levels for reporter gene constructs containing BHMT 5′-FR haplotypes transfected into (A) HepG2 or (B) HEK293T cells are shown as percentages of values for the WT haplotype. See Table 4 for the locations of SNPs. Each bar represents the average of six independent experiments (mean ± SEM); * p < 0.01.

Figure 5

BHMT2 translation, degradation and interaction with BHMT. (A) Human BHMT and BHMT2 in vitro translation in a rabbit reticulocyte lysate (RRL) is shown in the top panel. Representative autoradiographs for ³⁵S-methionine radioactively-labeled BHMT and BHMT2 at different time points in degradation experiments are shown in the bottom panel. The data shown are representative of three independent experiments. (B) BHMT and BHMT2 protein remaining at each time point in the RRL degradation study, expressed as percentages of the basal value. Each point is the mean ± SEM for 3 independent experiments. BHMT2 differed significantly from BHMT at each point (p < 0.0001). (C) Immunoblot analysis of HA-tagged BHMT2 expressed in COS-1 and HepG2 cells either together with BHMT or alone. Western blotting was performed with anti-HA antibody. Samples were loaded on the SDS-PAGE gels based on cotransfected β-galactosidase activity to correct for possible variation in transfection efficiency. (D) BHMT2 and BHMT interaction. HA-tagged BHMT2 was transfected or cotransfected with BHMT into COS-1 and HepG2 cells. IP was performed with equal quantities of total protein and anti-HA antibody, followed by immunoblot analysis with anti-BHMT antibody. Results shown are representative of 3 independent experiments. (E) HA-tagged BHMT2 expressed in HEK293T cells was stabilized in the presence of 1 mM homocysteine. Cells expressing HA-tagged BHMT2 were cultured with 1 mM homocysteine (Hcy), 1 mM betaine or water for 24 h. Western blot analysis was performed with anti-HA antibody, and actin was used as a control for loading.

Figure 6

BHMT and BHMT2 bacterial expression. (A) BHMT and BHMT2 were expressed as GST fusion proteins in BL21 E. coli. The GST-tags were removed by PreScission Protease after protein purification. (B) Recombinant BHMT and BHMT2 enzyme activity. Equal quantities of bacterially expressed BHMT and BHMT2 were used to perform enzyme assays with L-homocysteine and betaine as substrates. (C) Size-exclusion chromatography of purified bacterially expressed recombinant GST-BHMT and GST-BHMT2 proteins. Protein profiles are expressed as percentages of peak milliabsorbance units (mAu). The initial peaks for both preparations eluted with the void volume.