Novel Human N -Acetyltransferase 2 Alleles That Differ in Mechanism for Slow Acetylator Phenotype*

Three novel human NAT2 alleles ( NAT2*5D , NAT2*6D , and NAT2*14G ) were identified and characterized in a yeast expression system. The common rapid ( NAT2*4 ) and slow ( NAT2*5B ) acetylator human NAT2 alleles were also characterized for comparison. The novel recombinant NAT2 allozymes catalyzed both N - and O acetyltransferase activities at levels comparable with NAT2 5B and significantly below NAT2 4, suggesting that they confer slow acetylation phenotype. In order to investigate the molecular mechanism of slow acetylation in the novel NAT2 alleles, we assessed mRNA and protein expression levels and protein stability. No differences were observed in NAT2 mRNA expression among the novel alleles, NAT2*4 and NAT2*5B . However NAT2 5B and NAT2 5D, but not NAT2 6D and NAT2 14G protein expression were significantly lower than NAT2 4. In contrast, NAT2 6D was slightly (3.4-fold) and NAT2 14G was substantially (29-fold) less stable than NAT2 4. These results suggest that the 341T 3 C (Ile 114 3 Thr) common to the NAT2*5 cluster is sufficient for reduction in NAT2 protein expression, but that mechanisms for slow acetylator phenotype differ for NAT2 alleles that do not contain 341T 3 C, such as the NAT2*6 and NAT2*14 clusters. Different mechanisms for slow acetylator phenotype in humans are consistent with multiple slow acetylator phenotypes.

Genetic variation in N-acetyltransferase 2 (NAT2) 1 predisposes individuals to environmentally induced cancers (reviewed in Ref. 1). Over 50% of most non-Asian populations are slow acetylator phenotype(s) who experience higher incidences of toxicity from many aromatic amine and hydrazine drugs (2).
Slow acetylators also are predisposed to urinary bladder cancer from aromatic amine carcinogens (3,4). One study (3) reported that the slowest acetylator phenotype had the highest incidence of urinary bladder cancer.
Initial studies suggested that slow acetylator phenotype was due to decreased or absent hepatic N-acetyltransferase (5). Further studies reported that the combination 341T 3 C/481C 3 T polymorphism and the 590G 3 A polymorphism in the NAT2 coding region confer reduced expression of recombinant human NAT2 protein in COS-1 cells (6). Moreover, studies showed the 590G 3 A and 857G 3 A polymorphisms in the NAT2 coding region were associated with reduced expression of NAT2 protein in human liver (7) and reduced expression of recombinant NAT2 protein in Chinese hamster ovary cells (8). Recombinant expression studies in prokaryotic systems, however, did not show a reduction in NAT2 protein associated with slow acetylator alleles (9 -12). Thus, the molecular mechanism(s) responsible for the slow acetylator phenotype(s) remain incompletely understood (reviewed in Ref. 13).
In the course of molecular epidemiological investigations into the relationship between NAT2 genotype and cancer, we identified three novel NAT2 alleles. The novel NAT2 alleles, together with the most common rapid (NAT2*4) and slow (NAT2*5B) acetylator NAT2 alleles were characterized by recombinant expression in yeast (Schizosaccharomyces pombe). Our findings suggest that the three novel human NAT2 alleles confer slow acetylator phenotype and that the mechanism for slow acetylator phenotype differs among the NAT2 alleles.

EXPERIMENTAL PROCEDURES
NAT2 Genotype Assay-NAT2 genotype was determined using a modification of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay (14). This assay was modified in order to distinguish between all known human NAT2 alleles (23 when the study began). NAT2 was amplified by PCR using 250 ng of genomic DNA in a 50-l reaction containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 0.5 g of primer 5Ј-GGCTATAA-GAACTCTAGGAAC-3Ј, 0.7 g of 5Ј-AAGGGTTTATTTTGTTCCTTAT-TCTAAAT-3Ј, and 1.25 units of Taq DNA polymerase. The mixture was subjected to a 5-min pretreatment at 94°C followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, 1 min at 72°C, and concluded with a 5-min extension step at 72°C. Nucleotide substitutions 191G 3 A, 434A 3 C, and 481C 3 T were detected by digestion with MspI and KpnI. The 590G 3 A, 759C 3 T, and 857G 3 A nucleotide substitutions were distinguished following digestion with TaqI and BamHI. The 590G 3 A and 759C 3 T substitutions were detected by digestions with TaqI. Nucleotide substitution 857G 3 A was detected by digestion with BamHI. Nucleotide substitutions 282C 3 T and 845A 3 C were detected by digestion with FokI and DraIII. The 341T 3 C and 803A 3 G nucleotide substitutions were detected following nested PCR. One l of amplified NAT2 was used as the template in a 20-l reaction containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 220 ng of primer 5Ј-CACCTTCTCCTGCAGGTGACCG-3Ј and 5Ј-TGTCAAGCAGAAAATG-CAAGGC-3Ј (note: bold indicates the nucleotide change made in the primer sequence to generate a partial AciI restriction site that is underlined) or 240 ng of primer 5Ј-TGAGGAAGAGGTTGAAGAAGT-GCT-3Ј and 290 ng of 5Ј-AAGGGTTTATTTTGTTCCTTATTCTAAAT-3Ј, respectively, and 0.5 unit of Taq DNA polymerase. The mixture was pretreated at 94°C for 5 min followed by 30 cycles of 30 s at 94°C, 30 s at 58°C, 30 s at 72°C, and concluded with a 5-min extension step at 72°C. Twenty l of the nested PCR product was digested with AciI and DdeI to detect 341T 3 C and 803A 3 G, respectively.
Cloning, Sequencing, and Recombinant Expression-Human NAT2 alleles were amplified with human NAT2 specific primers 5Ј-TGGAAT-TCCATATGGACATTGAAGCAT-3Ј and 5Ј-AAGGCGCGCCCTAAAT-* This work was supported by United States Public Health Service Grant CA-34627 from the NCI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Portions of this work were in partial fulfillment for the Ph.D. in pharmacology and toxicology at the University of Louisville.
Southern Blot Analysis-DNA was isolated from yeast cells using the QIAmp tissue kit (Qiagen, Valencia, CA) and was quantified by absorbance at 260 nm. The DNA (10 g) was digested with NdeI (10 units) and AscI (10 units) at 37°C overnight, and run out on a 1% agarose gel. DNA was transferred to Zeta-Probe membrane (Bio-Rad). Equal loading was verified by ethidium bromide staining of DNA. The membrane was hybridized with human NAT2 PCR product labeled with [ 32 P]deoxy-CTP (NEN Life Science Products) using the random primer method of Feinberg and Vogelstein (16). Hybridization and wash procedures were conducted using the methods of Church and Gilbert (17). Southern blots were quantitated by densitometry.
Northern Blot Analysis-Total RNA was isolated from yeast cells and extracted via the RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified by absorbance at 260 nm. RNA (10 g/lane) was denatured in formaldehyde, subjected to electrophoresis on 1% agarose gels, and transferred to Zeta-Probe membranes (Bio-Rad). Equal loading per lane was verified by ethidium bromide staining of rRNA. For evaluation of NAT2 mRNA, the membrane was hybridized, washed, and quantitated as described above for Southern blot analysis.
Western Blot Analysis-Yeast cytosols (10 g of total protein) containing NAT2 protein were mixed with SDS-polyacrylamide gel sample buffer containing 5% (final) 2-mercaptoethanol and boiled for 5 min. Protein samples were separated on 7.5% SDS-polyacrylamide gels, transferred electrophoretically to Immuno-Lite membranes (Bio-Rad), and reacted to a human NAT2 antibody (kindly provided by Dr. Dennis Grant, University of Toronto). Chemiluminescent detection was achieved with an Immuno-Lite kit following manufacturer's instructions. Western blots were quantitated by densitometry.
N-Acetyltransferase Assay--N-Acetyltransferase activities were measured with the human NAT2-selective substrate sulfamethazine (SMZ) using high performance liquid chromatography (HPLC) to separate N-acetyl-SMZ product from SMZ substrate as described previously (18). Reactions (300 l) containing yeast cytosol, SMZ (300 M), and acetyl coenzyme A (1 mM) were carried out at 37°C for 5 min, terminated by the addition of 30 l of perchloric acid, and neutralized with 22 l of 1 M NaOH. SMZ and N-acetyl-SMZ were quantitated by their absorbance at 260 nm.
NAT2 Stability-Yeast cytosols (2 mg/ml) expressing recombinant human NAT2 allozymes were incubated at 50°C for up to 32 min. Following incubation, SMZ N-acetyltransferase activities were measured as described above.
Statistical Analysis-Differences observed between recombinant NAT2 alleles were tested for significance by one-way analysis of variance followed by Dunnett's multiple comparison test.

RESULTS
Three new human NAT2 alleles were identified via PCR-RFLP analysis and sequenced to verify specific nucleotide substitutions. Each novel allele was amplified in two independent PCR reactions, cloned, and sequenced separately. Two of the NAT2 alleles contained new combinations of previously identi-fied nucleotide substitutions and were designated NAT2*5D (GenBank TM accession number AF042740) and NAT2*14G (GenBank TM accession number AF055874) according to the consensus rules of NAT nomenclature (19,20). The NAT2*5D allele contained the 341T 3 C substitution alone, while the NAT2*14G allele contained the 191G 3 A, 282C 3 T, and the 803A 3 G substitutions. The third novel NAT2 allele identified a 111T 3 C (silent) nucleotide substitution (Fig. 1) that has not been reported. This allele also contained 282C 3 T and 590G 3 A nucleotide substitutions and was designated NAT2*6D (Gen-Bank TM accession number AF055875) according to the consensus rules for NAT nomenclature (19,20). The 111T 3 C nucleotide substitution created a novel TaqI restriction site that was confirmed by PCR-RFLP.
The three human NAT2 alleles, as well as the most common NAT2*4 (rapid) and NAT2*5B (slow) acetylator alleles, were recombinantly expressed in yeast. Southern blot analysis showed no differences in transformation efficiency (data not shown). The three novel NAT2 allozymes as well as the common slow acetylator NAT2 5B catalyzed SMZ N-acetyltransferase ( Fig. 2A) and N-OH-PhIP O-acetyltransferase (Fig. 2B) activities at levels significantly (p Ͻ 0.01) lower than NAT2 4. Thus, the novel NAT2 alleles were designated slow acetylator alleles.
Western blot analysis was carried out to assess levels of NAT2 protein expression of the recombinant NAT2 allozymes. As shown in Fig. 3, NAT2 5B and NAT2 5D expressed significantly (p Ͻ 0.01) lower levels of immunoreactive protein compared with NAT2 4. NAT2 6D and NAT2 14G protein levels were not significantly lower than NAT2 4, even though these allozymes exhibited significantly lower N-and O-acetyltransferase activities (Fig. 2, A and B). Protein expression of NAT2 14G actually was slightly higher than NAT2 4 (Fig. 3).
To investigate possible pretranslational mechanisms for altered protein expression, we analyzed mRNA levels expressed for each of the NAT2 alleles. Levels of NAT2 mRNA did not differ between the NAT2 alleles as determined by Northern blot analysis (Fig. 4).
To investigate if differences in NAT2 protein stability mediate reduced levels of N-and O-acetyltransferase activity, we determined heat inactivation rate constants for each recombinant NAT2 allozyme. NAT2 6D was slightly (3.4-fold) less stable than NAT2 4, whereas NAT2 14G was substantially (29-fold) less stable than NAT2 4 (Fig. 5). DISCUSSION Novel human NAT2 alleles were identified and characterized in a yeast expression system. We observed significant reductions in protein expression levels for the NAT2*5B and NAT2*5D alleles. The 341T 3 C single nucleotide polymorphism present in NAT2*5D was sufficient to decrease NAT2 Identification and Characterization of Novel NAT2 Alleles 34520 immunoreactive protein levels. This finding clarifies previous results in human liver (5, 7) and recombinant expression of human NAT2 alleles in COS-1 (6) and Chinese hamster ovary (8) cells.
Both the 341T 3 C and the 481C 3 T polymorphisms were reported to be necessary to reduce recombinant human NAT2 expression in COS-1 cells (6). The 341T 3 C polymorphism changes the amino acid sequence, Ile 114 3 Thr, while the 481C 3 T polymorphism does not alter an amino acid. Our data (Fig.  3) clearly demonstrate that the 341T 3 C polymorphism alone was sufficient to reduce immunoreactive protein levels compared with NAT2 4, a result consistent with previous studies in Chinese hamster ovary cells (8). These data suggest the 341T 3 C and not the 481C 3 T is the important polymorphism that confers slow acetylator status in human populations. This is significant because most NAT2 genotyping procedures are designed to detect 481C 3 T but not 341T 3 C. Our findings as well as those of others (6 -8) suggest post-transcriptional mechanisms as mRNA levels did not vary between rapid and slow acetylator alleles.
NAT2 6D and NAT2 14G catalyzed SMZ N-acetyltransferase and N-OH-PhIP O-acetyltransferase activities at levels significantly (p Ͻ 0.01) lower than NAT2 4 (Fig. 2, A and B), but immunoreactive protein levels were similar to that of NAT2 4. Thus, mechanisms for slow acetylator phenotypes encoded by alleles not containing the 341T 3 C polymorphism may include deficiencies in protein stability. NAT2 6D and NAT2 14G allozymes were less stable than NAT2 4, 5B, and 5D. These results are consistent with previous reports in a prokaryotic system (9,21).
In previous studies, NAT2 possessing the 590G 3 A polymorphism exhibited lower protein levels in human liver (7) and lower levels of recombinant NAT2 expression in COS-1 cells (6). However, NAT2*6D, which contains the 590G 3 A polymorphism expressed similar recombinant NAT2 protein levels as NAT2*4 in our study. A decrease in stability may be the mechanism by which the NAT2*6D confers slow acetylator status in humans. Our data suggests that NAT2*6D may yield a different phenotype than NAT2*5B and NAT2*5D or NAT2*14G. Greater quantities of protein were expressed for NAT2*6D and NAT2*14G than for NAT2*5B and NAT2*5D, but the NAT2 5B and NAT2 5D proteins were more stable.
In conclusion, these data identify and characterize three novel human NAT2 slow acetylator alleles. One of these alleles (NAT2*6D) contained a 111T 3 C single nucleotide polymorphism not reported previously. This polymorphism creates a novel TaqI restriction site that is detectable by PCR-RFLP. The 341T 3 C single base substitution present in NAT2*5D was sufficient to decrease NAT2 immunoreactive protein levels. However, the mechanism for slow acetylator phenotype of the NAT2*6D and NAT2*14G alleles did not involve reduction in protein expression, but rather reductions in protein stability. These results suggest at least two mechanisms for slow acetylator phenotypes in humans, which are of high significance for Relative levels of recombinant NAT2 protein (shown on abscissa) are shown in arbitrary units following densitometry (NAT2 4 was set to 1). **, levels of NAT2 5B and NAT2 5D protein were significantly (p Ͻ 0.01) lower than NAT2 4. *, in contrast, levels of NAT2 14G protein were significantly (p Ͻ 0.05) greater than NAT2 4. Each bar represents mean Ϯ S.E. for four separate determinations. A representative Western blot is illustrated below (B).  Identification and Characterization of Novel NAT2 Alleles 34521