Developmental and evolutionary analysis of betaine homocysteine methyltransferase genes

Methionine (Met) is an essential amino acid because mammals cannot synthesize homocysteine. Betaine-homocysteine methyltransferase (BHMT) and BHMT-2 methylate homocysteine to form methionine using betaine and S-methylmethionine, respectively. Betaine is produced de novo from choline and is also found in the diet, whereas S-methylmethionine is only made in plants and fungi and so must be obtained from the diet. These enzyme activities are only be detected in the liver of adult rodents, but in adult humans and pigs, these activities are found in both the liver and kidney cortex. Since both pigs and humans are omnivores and share the same pattern of BHMT and BHMT-2 expression, the pig represents an excellent model for studying the physiological roles of these enzymes in human biology. As a prelude to investigating the influence of diet and development on the expression of porcine BHMT and BHMT-2, their full-length cDNAs were cloned and sequenced, and their corresponding genes were characterized. The genes are adjacent to each other on the same chromosome, and to study the evolutionary relationship between them, all the available sequences from 37 species of deuterostomes were analyzed. Unlike BHMT, the BHMT-2 gene is not found in sea urchins, amphibians, reptiles and birds, indicating it was derived from BHMT following a gene duplication event in mammals. These findings imply that the BHMT-2 gene may offer an advantage to mammals in scavenging Met from the environment. As expected based on enzyme activity data from humans and pigs, BHMT and BHMT-2 mRNAs were observed to be highly expressed in liver and kidney cortex, whereas there is comparatively very little expression in other organs. The BHMT mRNA was higher in liver than kidney cortex (3:2 ratio), but the BHMT-2 mRNA was more abundant in kidney cortex than liver (3:1 ratio). The expression of BHMT mRNA was studied further. A total of ten different BHMT splice variants were observed in adult liver, kidney cortex, kidney medulla, lungs, heart, brain and fetal lungs. These included two variants that if translated would encode a truncated form of BHMT. BHMT mRNA expression was quantified during development at gestational week 30 (G30), G45, G90, and adult tissues. BHMT was low in G30 whole embryos, but was found to be easily quantifiable and progressively increased in the liver and kidney at and after G45. BHMT activity also progressively increased with age in both organs. The truncated transcripts represented ~10% of the total BHMT mRNA in the G30 fetus, the G45 liver and adult liver and kidney cortex. A computer-generated model of the truncated BHMT protein revealed a horseshoe fold structure of the protein, but the function of this putative protein is unknown. Using bisulfite sequencing, three CpG sites and the promoter region of the BHMT gene were identified that were more methylated in adult lungs compared to adult liver, suggesting that DNA methylation may be an important factor in the regulation of BHMT expression during development