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

Abstract

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*5I and NAT2*12D, were identified recently. In NAT2*5I, a new single nucleotide polymorphism A⁴¹¹T (L137F) was found coexisting with single nucleotide polymorphisms T³⁴¹C (I114T), C⁴⁸¹T (silent) and A⁸⁰³G (K268R). The other allele NAT2*12D consists of a new single nucleotide polymorphism G³⁶⁴A (D122N) together with A⁸⁰³G (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 A⁴¹¹T (L137F) and G³⁶⁴A (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 A⁴¹¹T and G³⁶⁴A reduced NAT2 immuno-reactive 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; L137I: 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 A⁴¹¹T (L137F) and G³⁶⁴A (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 5I and NAT2 12D.