Functional characterization of single-nucleotide polymorphisms and haplotypes of human N-acetyltransferase 2

Abstract

Human N-acetyltransferase 2 (NAT2) is polymorphic in humans and may associate with cancer risk by modifying individual susceptibility to cancers from carcinogen exposure. Since molecular epidemiological studies investigating these associations usually include determining NAT2 single-nucleotide polymorphisms (SNPs), haplotypes or genotypes, their conclusions can be compromised by the uncertainty of genotype-phenotype relationships. We characterized NAT2 SNPs and haplotypes by cloning and expressing recombinant NAT2 allozymes in mammalian cells. The reference and variant recombinant NAT2 allozymes were characterized for arylamine N-acetylation and O-acetylation of N-hydroxy-arylamines. SNPs and haplotypes that conferred reduced enzymatic activity did so by reducing NAT2 protein without changing NAT2 mRNA levels. Among SNPs that reduced catalytic activity, G191A (R64Q), G590A (R197Q) and G857A (G286E) reduced protein half-life but T341C (I114T), G499A (E167K) and A411T (L137F) did not. G857A (G286E) and the major haplotype possessing this SNP (NAT2 7B) altered the affinity to both substrate and cofactor acetyl coenzyme A, resulting in reduced catalytic activity toward some substrates but not others. Our results suggest that coding region SNPs confer slow acetylator phenotype by multiple mechanisms that also may vary with arylamine exposures.

Figure 1

SMZ N-acetylation activities in COS-1 cells expressing NAT2 alleles possessing different SNPs (top) and haplotypes (bottom). Each error bar represents the standard deviation for transfections performed in triplicate on different days. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4. Activity in COS-1 cells transfected with NAT2 allele possessing G364A was below the detection limit (0.3 nmol/min/mg).

Figure 1

SMZ N-acetylation activities in COS-1 cells expressing NAT2 alleles possessing different SNPs (top) and haplotypes (bottom). Each error bar represents the standard deviation for transfections performed in triplicate on different days. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4. Activity in COS-1 cells transfected with NAT2 allele possessing G364A was below the detection limit (0.3 nmol/min/mg).

Figure 2

Effects of NAT2 SNPs and haplotypes on the O-acetylation of N-OH-ABP (open bars) and N-OH-PhIP (solid bars). Each error bar represents the standard deviation for transfections performed in triplicate. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4. Activity in COS-1 cells transfected with NAT2 allele possessing G364A was below the detection limit (0.05 nmol/min/mg).

Figure 2

Effects of NAT2 SNPs and haplotypes on the O-acetylation of N-OH-ABP (open bars) and N-OH-PhIP (solid bars). Each error bar represents the standard deviation for transfections performed in triplicate. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4. Activity in COS-1 cells transfected with NAT2 allele possessing G364A was below the detection limit (0.05 nmol/min/mg).

Figure 2

Effects of NAT2 SNPs and haplotypes on the O-acetylation of N-OH-ABP (open bars) and N-OH-PhIP (solid bars). Each error bar represents the standard deviation for transfections performed in triplicate. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4. Activity in COS-1 cells transfected with NAT2 allele possessing G364A was below the detection limit (0.05 nmol/min/mg).

Figure 2

Effects of NAT2 SNPs and haplotypes on the O-acetylation of N-OH-ABP (open bars) and N-OH-PhIP (solid bars). Each error bar represents the standard deviation for transfections performed in triplicate. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4. Activity in COS-1 cells transfected with NAT2 allele possessing G364A was below the detection limit (0.05 nmol/min/mg).

Figure 3

Steady state mRNA levels of NAT2 (solid bars) and β-actin (open bars) in COS-1 cells transfected with NAT2 alleles possessing different SNPs. NAT2 mRNA was first normalized to internal β-actin mRNA and then expressed relative to the NAT2 mRNA level in COS-1 cells transfected with NAT2*4 (defined as 1.0). Each bar shows the mean and the standard deviation of the relative level based on three independent transfections. None of the NAT2 mRNA levels differed significantly (p>0.05) from NAT2*4.

Figure 4

Thermostability of NAT2 allozymes encoded by alleles possessing different SNPs. Heat inactivation assays were performed at 37°C (top) and 50°C (bottom). Due to the extremely low catalytic activity of A411T (Figure 1), thermostability for this variant was not determined at 50°C. Each bar represents the mean and standard deviation of three calculated half-lives using lysates of COS-1 cells transfected separately. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4.

Figure 4

Thermostability of NAT2 allozymes encoded by alleles possessing different SNPs. Heat inactivation assays were performed at 37°C (top) and 50°C (bottom). Due to the extremely low catalytic activity of A411T (Figure 1), thermostability for this variant was not determined at 50°C. Each bar represents the mean and standard deviation of three calculated half-lives using lysates of COS-1 cells transfected separately. *: Significantly (p<0.01) lower than the reference (Ref) NAT2 4.

Figure 5

Western blot of NAT2 allozymes expressed in COS-1 cells transfected with plasmids containing NAT2*4 and variant alleles possessing different SNPs (top) and haplotypes (bottom). Immunoblots of NAT2 and α-tubulin are indicated by arrows. Mock: COS-1 cells transfected with the mock pcDNA5/FRT plasmid.