* Cedars Sinai Medical Center, Davis Building G150, 8700 Beverly Blvd., Los Angeles, California 90048; and
Center for Ecogenetics and Environmental Health, University of Washington, 4225 Roosevelt Way NE, #100, Seattle, Washington 98105
Received June 14, 2002; accepted July 24, 2002
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ABSTRACT |
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Key Words: glutathione S-transferase, alpha class; species differences; primate; aflatoxin biotransformation; liver; clone.
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INTRODUCTION |
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Remarkably, the soluble (cytosolic) fraction of human liver has extremely low GST activity toward AFBO, although total activity towards other common GST substrates is similar to that seen in rodents (Raney et al., 1992b; Slone et al., 1995
). Consistent with the low total GST activity toward AFBO, cDNA-expressed human alpha-class GSTs (hGSTA1-1 and hGSTA2-2) have little or no AFBO detoxifying activity (Buetler et al., 1996
; Johnson et al., 1997
; Raney et al., 1992b
). However, we found that liver cytosol from the nonhuman primate, Macaca fascicularis (Mf), does have significant constitutive hepatic cytosolic GST activity toward AFBO (Wang and Eaton, 1998
). This activity is at least 10100-fold higher than observed in human liver, although it is also about 100-fold lower than that seen in mouse liver. We recently demonstrated that mu-class GSTs appear to be responsible for most of the constitutive AFBO conjugating activity in Mf liver (Wang et al., 2000
), although some contribution from alpha-class GSTs could not be ruled out. Alpha-class protein was present at relatively high levels in Mf liver, as demonstrated by Western blot analysis and activity assays (Wang et al., 2000
).
Because Mf is an important nonhuman primate species sometimes used in metabolism and disposition studies of pharmaceutical agents, we completed the characterization of the alpha-class hepatic GSTs in this species, with an emphasis on comparison to human alpha-class GSTs. The objective of this study was to clone and sequence hepatic Mf alpha-class GSTs to determine (1) sequence and catalytic similarities to human alpha-class GSTs, and (2) if they possess significant AFBO conjugating activity, with the hope of identifying a primate alpha-class GST that is orthologous to rodent alpha-class GSTs with high catalytic activity toward AFBO.
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MATERIALS AND METHODS |
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Construction of complementary DNA library.
An Mf liver cDNA library was constructed from an adult, castrated male Mf using the ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA). The liver tissue was obtained through the tissue acquisition program of the Regional Primate Research Center at the University of Washington. A castrated male Mf was chosen because of (1) availability of tissue (only castrated Mf was available) and (2) relatively high AFBO activity was observed in hepatic cytosol from this animal. Briefly, cDNA library construction was initiated with first strand cDNA synthesis using 10 µg mRNA isolated from the Mf liver with a poly(A) mRNA isolation kit (Stratagene, La Jolla, CA), in the presence of StrataScriptTM RNase H- reverse transcriptase (RT), nucleotides and a 50-base oligonucleotide; the linker-primer with the following sequence:
5-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3
"GAGA" sequence Xho IPoly(dT)
This oligonucleotide was designed with a "GAGA" sequence to protect the Xho I restriction enzyme recognition site and an 18-base poly(dT) sequence. The restriction site allowed the finished cDNA to be inserted into the Uni-ZAP XR vector in a sense orientation (EcoR I-Xho I) with respect to the lacZ promoter. The nucleotide mixture for the first strand contains normal dATP, dGTP, and dTTP plus the analog 5-methyl dCTP. The complete first strand containing a methyl group on each cytosine base protected the cDNA from restriction enzymes used in subsequent cloning steps. During second strand synthesis, RNase H was used to nick the RNA bound to the first strand cDNA to produce a multitude of fragments, which serve as primers for DNA polymerase I. DNA polymerase I was used to "nick-translate" these RNA fragments into second strand cDNA. The second strand nucleotide mixture was supplemented with dCTP to reduce the probability of 5-methyl incorporation in the second strand. This ensures that the restriction sites in the linker-primer will be susceptible to restriction enzyme digestion. The uneven termini of the double-stranded cDNA were clipped or filled in with Klenow, and EcoR I adapters were ligated to the blunt ends. The adapters have the sequence shown below.
5-AATTCGGCACGAG-3
3- GCCGTGCTC-5
These adapters are composed of 9- and 13-mer complementary oligonucleotides, with an EcoR I cohesive end. The 9-mer oligonucleotide was phosphorylated to allow ligation to other blunt termini available in the form of cDNA and other adapters. The 13-mer oligonucleotide was maintained in a dephosphorylated state to prevent ligation to other cohesive ends. Following completion of adapter ligation, heat inactivation of ligase, the 13-mer oligonucleotide was phosphorylated to enable ligation into the dephosphorylated vector arms. Then digestion was completed with Xho I to release the EcoR I adapter and residual linker-primer from the 3 end of the cDNA, and the two fragments were separated on a Sephacryl S-400 spin column. The size-fractionated cDNAs were then precipitated and ligated to the Uni-ZAP XR vector arms (Stratagene, La Jolla, CA). The lambda library was packaged in a high-efficiency system, Gigapack II Gold packaging extract (Stratagene, La Jolla, CA), and plated on the E. coli strain XL1-Blue MRF (Stratagene, La Jolla, CA). Quality of the synthesized cDNA library was determined by both plaque-forming efficiency and positive actin clones, with mouse ß-actin as a probe. The constructed cDNA library had an efficiency of 1010 plaque-forming units/ml, and 0.04% of actin when checked using mouse ß-actin as a probe. Typically, highly expressed cDNA clones can be found in libraries yielding greater than 0.01% actin (Hagen et al., 1988).
Isolation and sequencing of Mf alpha-class GST clones.
Human alpha-class GST cDNA (hGSTA1, a gift from Dr. Chen-Pei D. Tu from the Pennsylvania State University) was used as a probe to screen the Mf hepatic cDNA library using a protocol provided by the manufacturer (Stratagene, La Jolla, CA). Positive clones from the primary screening were subjected to secondary and tertiary screenings with a higher stringency. The plasmids (pBluescripts) containing GST inserts were prepared, and sequence was obtained with an ABI 377 automatic sequencer, using T3, T7, and nested primers. Sequencing reactions were done on a thermal cycler using 0.5 µg of plasmid DNA and 8 µl of dye-terminator mix purchased from Perkin-Elmer (Foster City, CA). All sequences were analyzed with DNAide (Laboratoire de Biochimie, 91128 Palaiseau cedex, France), and DNA Strider (Service de Biochimie, Cedex, France) software.
Subcloning.
Because all cDNA clones isolated from tertiary cloning were not in a proper reading frame for GST protein expression, one of the three GST cDNAs was subcloned into an expression vector, pET 17b (Novagen, Madison, WI), for recombinant protein expression. mfaGSTA1 full-length cDNA has 992 bases including a 19-base polyA tail. Two primers, (forward primer [GAAGACTGCTCATATGGCAGAGAAA] and reverse primer [TACCGGGCCCCCCCTCGAGT]), were designed to subclone mfaGSTA1 cDNA into the pET 17b expression vector. A Nde I cloning site was introduced in the forward primer, and there was an Xho I cloning site in the reverse primer. The cDNAs generated with the above primers were digested with Nde I and Acc I (fragment A), whereas pBluescript plasmids containing mfaGSTA1 inserts were digested with Acc I and Xho I (fragment B). Both fragments A and B were then cloned into pET 17b, which had been digested with both Nde I and Xho I. This cloning strategy was used because the cDNA contained an internal Nde I site and digesting the cDNA with Acc I first facilitated directional cloning. This allowed for highly efficient expression of recombinant mfaGSTA1-1, because the starting codon (ATG) of mfaGSTA1 was aligned in optimal proximity to the upstream ribosome binding site in the expression vector (Fig. 1). The protein was expressed in Escherichia coli, BL21 (DE3; Novagen), and purified by glutathione-affinity chromatography as previously described (McHugh et al., 1996
).
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Enzymatic assays.
AFBO conjugating activity was determined using an HPLC method as previously described (van Ness et al., 1998) in which mouse microsomes were used to generate AFBO, with an initial AFB1 substrate concentration of 128 µM. AFBO hydrolyzes rapidly in aqueous solution with a half-life of approximately 1 s (Johnson et al., 1997
). AFBO endo- and exo-activities were determined using a method as previously described by Raney et al. (1992a)
, with the following modifications: (1) mouse microsomes were used to generate AFBO, with an initial AFB1 substrate concentration of 128 µM; (2) 1 mM NADPH was used in place of an NADPH regenerating system. Other general enzymatic glutathione S-transferases activities were assayed using CDNB, DCNB, CHP, and ECA as substrates according to standard procedures (Habig and Jakoby, 1981
).
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RESULTS |
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DISCUSSION |
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In the present study, three different cDNA clones (pBluescript-SK(-)-mfaGSTA1, pBluescript-SK(-)-mfaGSTA2, pBluescript-SK(-)-mfaGSTA3) were isolated from a Mf hepatic cDNA library and found to have high sequence homology (> 96%, Table 1). One of these clones, mfaGSTA1, was subcloned into an expression vector, pET17b, for recombinant GST protein expression in bacteria. Consistent with what was found in human alpha-class GSTA1-1, bacterially expressed recombinant mfaGSTA1-1 had no detectable AFBO conjugating activity (< 2 pmol/min/mg), and had a very similar activity profile to the human GSTA1-1 toward other substrates such as CDNB, DCNB, CHP, and ECA (Table 2
). These results confirm our previous studies that suggested constitutively expressed alpha-class GSTs in Mf liver do not contribute substantially to overall hepatic cytosolic GST activity toward AFBO observed in this species (Wang and Eaton, 1998
; Wang et al., 2000
).
Whether humans (or other primate species) possess a nonconstitutively expressed, but inducible, alpha-class GST that is orthologous to the rodent GSTs with high catalytic activity toward AFBO is an important question relevant to dietary and/or chemointervention strategies to reduce AFB carcinogenesis in humans. Van Ness et al. (1998) identified six amino acid residues as being critical in conferring high AFBO activity to the rodent alpha-class GSTs, mGSTA3-3 and rGSTA5-5. Primary sequence analysis revealed that neither mfaGSTA1, mfaGSTA2 nor mfaGSTA3 shared any of those six residues with mGSTA3 and rGSTA5 (Fig. 2
). Therefore, it is unlikely that the other two Mf alpha-class GST genes (mfaGSTA2 and mfaGSTA3) will result in proteins with any significant AFBO activity, given that (1) they share high similarity with human GSTA1, based on cDNA-derived amino acid sequence analyses, and (2) as is the case for hGSTA1subunit, they have none of the six critical amino acid residues associated with high exo-AFBO activity of rodent alpha-class GSTs (mGSTA3-3, rGSTA5-5; Fig. 2
).
Thus, these results further suggest that strategies for chemoprevention against AFB1-induced human hepatocarcinogenesis should not rely upon induction of alpha-class GSTs. However, induction of other GSTs may be of some value. Oltipraz, a model chemopreventive agent that can induce an alpha-class GST, rGSTA5-5, with high activity toward the exo-AFBO in rats (Buetler et al., 1995, 1996
; Hayes et al., 1996
; Kensler, 1997
; Kensler et al., 1992
; Primiano et al., 1995
), has been used on a phase IIa clinical trial in China (Kensler et al., 1998
; Wang et al., 1999
, 2001
). Daily intervention with 125 mg oltipraz led to a 2.6-fold increase in median aflatoxin-mercapturic acid excretion (Kwak et al., 2001
; Wang et al., 1999
), suggesting that the treatment did induce GSTs with activity toward AFBO. Studies in isolated human hepatocytes demonstrated that oltipraz can induce human alpha-class GSTs, hGSTA1-1, and hGSTA2-2 (Morel et al., 1993
). However, like Mf alpha-class GSTs (A1-1, A2-2, and A3-3), these two human alpha-class GSTs have no activity toward the exo-AFBO isomer and thus would not be expected to provide significant protection from AFB1-induced hepatocarcinogenesis (Buetler et al., 1996
; Johnson et al., 1997
; Raney et al., 1992b
). Although the possibility of induction of certain unidentified nonconstitutively expressed human hepatic alpha-class GSTs possessing high activity toward exo-AFBO by oltipraz can not be excluded, evidence is mounting to suggest that induction of mu-class GSTs might be responsible, at least in part, for the increase in formation of AFB-GSH conjugates in humans (Raney et al., 1992b
; Slone et al., 1995
; Wang et al., 2000
). Recent molecular epidemiology studies in populations at high risk for AFB-related liver cancer provide additional circumstantial evidence that human GSTM1 might contribute to hepatic detoxification of AFBO (Chen et al., 1996
; Sun et al., 2001
).
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ACKNOWLEDGMENTS |
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NOTES |
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