Functional characterization of the A411T (L137F) and G364A (D122N) genetic polymorphisms in human N-acetyltransferase 2

Objectives Human N-acetyltransferase 2 (NAT2) genetic polymorphisms may modify drug efficacy and toxicity and cancer susceptibility from carcinogen exposure. Two human NAT2 alleles, NAT2*51 and NAT2*12D, were identified recently. In NAT2*51, a new single nucleotide polymorphism A(411)T (L137F) was found coexisting with single nucleotide polymorphisms T 341 C (1114T), (CT)-T-481 (silent) and A(803)G (K268R). The other allele NAT2*12D consists of a new single nucleoticle polymorphism G 364 A (D122N) together with A(803)G (K268R). We undertook a study to characterize these new single nucleotide polymorphisms and NAT2 alleles to further our understanding of genotype/phenotype relationships in human populations. Methods Various human NAT2 alleles were cloned and recombinantly expressed in COS-1 cells and the effects of single nucleotide polymorphisms on NAT2 expression were determined. To further test our hypothesis that A(411)T (L137F) and G(364)A (D122N) accelerate protein degradation, various NAT2 alleles were cloned and expressed in Escherichia coli, which does not possess the ubiquitin-mediated degradation pathway. Results Both A(411)T and G(364)A reduced NAT2 immunoreactive protein to an undetectable level without causing changes in mRNA level. Missense mutants displayed different effects on sulfamethazine N-acetylation activity for both L137 (wild-type: 70.2 +/- 5.2 nmol/min/mg; L137F: 1.34 +/- 0.03 nmol/min/mg; L137W: nondetectable; L1371: 34.2 +/- 2.0 nmol/min/mg; L137G: 0.52 +/- 0.04 nmol/ min/mg) and D122 (wild-type: 70.2 +/- 5.2 nmol/min/mg; D122R: non-detectable; D122Q: non-detectable; D122E: 1.72 +/- 0.24 nmol/min/mg). In contrast to the expression in mammalian cells, recombinant NAT2 possessing either of these two single nucleotide polymorphisms showed no reduction in immuno-reactive NAT2 level when expressed in E. coli. Conclusions These findings suggest that both A(411)T ((LF)-F-137) and G(364)A (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 51 and NAT2 12D.