Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1

Abstract

Genetic variants of human N-acetyltransferase 1 (NAT1) are associated with cancer and birth defects. N- and O-acetyltransferase catalytic activities, Michaelis-Menten kinetic constants (K(m) and V(max)) and steady-state expression levels of NAT1-specific mRNA and protein were determined for the reference NAT1*4 and variant human NAT1 haplotypes possessing single nucleotide polymorphisms (SNPs) in the open reading frame. Although none of the SNPs caused a significant effect on steady-state levels of NAT1-specific mRNA, C97T(R33stop), C190T(R64W), C559T (R187stop) and A752T(D251V) each reduced NAT1 protein level and/or N- and O-acetyltransferase catalytic activities to levels below detection. G560A(R187Q) substantially reduced NAT1 protein level and catalytic activities and increased substrate K(m). The G445A(V149I), G459A(synonymous) and T640G(S214A) haplotype present in NAT1*11 significantly (P<0.05) increased NAT1 protein level and catalytic activity. Neither T21G(synonymous), T402C(synonymous), A613G(M205V), T777C(synonymous), G781A(E261K) nor A787G(I263V) significantly affected K(m), catalytic activity, mRNA or protein level. These results suggest heterogeneity among slow NAT1 acetylator phenotypes.

Figure 1

Effects of SNPs in the NAT1 coding region on PABA N-acetyltransferase maximum velocities. The upper panel illustrates PABA N-acetyltransferase maximum velocities determined with PABA concentrations of 25 to 1000 μM in the presence of 1 mM AcCoA. The bottom panel illustrates PABA N-acetyltransferase maximum velocities determined with AcCoA concentrations of 25 to 3000 μM in the presence of 750 μM PABA. PABA N-acetyltransferase activities were normalized to β-galactosidase activity. Each bar represents Mean ± S.E.M from three transfections in duplicate. Characters under bars indicate SNP(s) in corresponding haplotypes: REF, no SNP (reference NAT1*4); CD11: SNPs in NAT1*11 coding region (G445A, G459A, T640G). Activity for each variant allozymes was compared to reference NAT1 4 catalytic activity by one way ANOVA followed by Dunnett’s post-test. * significantly lower than NAT1 4 (p<0.01). ** significantly greater than NAT1 4 (p<0.05). PABA N-acetyltransferase catalytic activities for C97T, C190T, C559T and A752T were too low (<0.05 nmol/min/mg) to determine Vmax.

Figure 2

Effects of SNPs in the NAT1 coding region on PABA (top) and AcCoA (bottom) Km. The upper panel illustrates PABA Km determined with PABA concentrations of 25 to 1000 μM in the presence of 1 mM AcCoA. The bottom panel illustrates AcCoA Km determined with AcCoA concentrations of 25 to 3000 μM in the presence of 750 μM PABA. Each bar represents Mean ± S.E.M from three transfections in duplicate. Characters under bars indicate SNP(s) in corresponding haplotypes: REF, no SNP (reference NAT1*4); CD11: SNPs in NAT1*11 coding region (G445A, G459A, T640G). Km for each variant allozyme was compared to NAT1 4 by one way ANOVA followed by Dunnett’s post-test. * significantly greater than NAT1 4 (p<0.01). PABA N-acetyltransferase catalytic activities for C97T, C190T, C559T and A752T were too low (<0.05 nmol/min/mg) to determine Km.

Figure 3

Effect of SNPs in the NAT1 coding region on steady state levels of recombinant human NAT1 mRNA. NAT1 mRNA levels were determined by quantitative real-time RT-PCR. The results are expressed as relative amount of allelic mRNA as a percentage to reference haplotype NAT1*4. Each bar represents Mean ± S.E.M from three transfections in duplicate. Characters under bars indicate SNP (s) in corresponding haplotypes: REF, no SNP (reference NAT1*4); CD11: SNPs in NAT1*11 coding region (G445A, G459A, T640G). mRNA levels were tested for significant differences from reference (NAT1*4) by one way ANOVA. No significant differences in mRNA level were observed.

Figure 4

Effects of SNPs in the NAT1 coding region on recombinant NAT1 protein level.COS-1 cell lysate for each NAT1 variant was loaded onto 12% SDS-PAGE gel, and NAT1 protein and endogenous control α-tubulin levels were visualized by western blots. Numbers represent the corresponding SNP(s) in the NAT1 coding region: REF, reference NAT1 4; CD11, three SNPs (G445A, G459A, T640G) in coding region. C+: recombinant NAT1 expressed in yeast (positive control); C-: recombinant NAT2 expressed in yeast (negative control). The two upper panels represent separate expressions. The western data was quantitated by densitometry and presented in the lower panel. Each bar represents Mean ± S.E.M from three transfections in duplicate. Characters under bars indicate SNP(s) in corresponding haplotypes: REF, no substitution (reference NAT1*4); CD11: SNPs in NAT1*11 coding region (G445A, G459A, T640G). NAT1-specific protein levels for the variant allozymes was compared to NAT1 4 by one way ANOVA followed by Dunnett’s post-test. C97T and C559T decreased the protein to non-detectable (ND) levels. The NAT1 antibody did not detect the truncated NAT1 proteins from C97T(R33stop) and C559T(R187stop). **C190T and A752T significantly (p<0.01) reduced NAT1 protein level over 40-fold and G560A significantly (p<0.01) reduced NAT1 protein level 4-fold. *CD11 significantly (p<0.05) increased the level of recombinant NAT1 protein level about 2-fold.

Figure 5

Effects of SNPs in the NAT1 coding region on N-OH-PhIP O-acetyltransferase activities. Results were normalized to β-galactosidase activity. Each bar represents Mean ± S.E.M from three transfections in duplicate. Characters under bars indicate SNP(s) in corresponding haplotypes: REF, no substitution (reference NAT1*4); CD11: SNPs in NAT1*11 coding region (G445A, G459A, T640G). Activity for each variant allozymes was compared to activity of NAT1 4 by one way ANOVA followed by Dunnett’s post-test. *significantly lower than NAT1 4 reference (p<0.01). ND, N-OH-PhIP O-acetyltransferase catalytic activities for C97T, C190T, C559T and A752T were not detected (<0.01 nmol/min/mg).