Functional characterization and mechanistic studies on single nucleotide polymorphisms of human N-acetyltransferase 2.

摘要

Human N-acetyltransferase 2 (NAT2) catalyzes the biotransformation of various substrates, including many drugs and carcinogens. Since the genetic polymorphisms of the human NAT2 gene result in different levels of catalytic capacities, NAT2 status is considered a genetic factor that potentially modifies individual cancer susceptibility to carcinogen exposure. In molecular epidemiological studies, the interpretation of the results could be compromised by the uncertainty of genotype-phenotype relationships of NAT2 alleles and the consequent misgrouping of study subjects. In this study, a comprehensive characterization of naturally occurring NAT2 SNPs and haplotypes was carried out by cloning and expressing different NAT2 allozymes in mammalian cells. NAT2*4 reference allele was first inserted into pcDNA5/FRT vector. SNPs G191A, C282T, T341C, G364A, A411T, C481T, G499A, G590A, A803G, G857A were then each introduced into the pcDNA-NAT2*4 plasmid by site-directed mutagenesis. Plasmids containing common NAT2 alleles or haplotypes, NAT2*5B, NAT2*6A, NAT2*7B, or NAT2*14B were similarly constructed for mammalian expression. NAT2 variant alleles were functionally characterized by measuring the sulfamethazine (SMZ) N-acetylation activity catalyzed by the allozymes they encoded. The bioactivation of N-hydroxy-aromatic and heterocyclic amines (via O-acetylation) by these allozymes was also measured. The results showed that G191A(R64Q), T341C(I114T), G364A(D122N), A411T(L137F), G499A(E167K), G590(R197Q) and G857A(G286E) significantly reduced the SMZ N-acetylation activity in transfected cells and therefore are associated with slow acetylator phenotype. N- and O-acetylation activities correlated very well, suggesting that rapid acetylators also have higher capacity to activate aromatic and heterocyclic amines. Further mechanistic studies showed that SNPs and SNP combinations causing slow acetylator phenotypes reduced cytosolic NAT2 protein levels without changing NAT2 mRNA levels. It was also observed that G191A(R64Q), G590(R197Q), G857A(G286E), but not T341C(I114T), G499A(E167K) and A411T(L137F), decreased NAT2 half-life in heat inactivation assays. Alleles possessing G857A encoded variant proteins with changed affinity for their substrates. A homology-based NAT2 structural model was constructed to help explain the mechanism for loss of protein, which is the most likely to be protein degradation. This study established a comprehensive genotype-phenotype relationship for NAT2 SNPs and also suggests that coding region SNPs confer slow acetylator phenotype by multiple mechanisms.