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 AT (L137F) was found coexisting with single nucleotide polymorphisms TC (I114T), CT (silent) and AG (K268R). The other allele NAT2*12D consists of a new single nucleotide polymorphism GA (D122N) together with AG (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 AT (L137F) and GA (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 AT and GA 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 AT (L137F) and GA (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 5I and NAT2 12D.