Toxicological Sciences 70, 20-26 (2002)
Copyright © 2002 by the Society of Toxicology
BIOTRANSFORMATION AND TOXICOKINETICS |
Complementary DNA Cloning, Protein Expression, and Characterization of Alpha-Class GSTs from Macaca fascicularis Liver
,1
* 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
Large species differences exist in sensitivity to aflatoxin B1 (AFB1)-induced liver cancer. Mice are resistant to AFB1-induced liver cancer because they express an alpha-class GST (mGSTA3-3) that has high activity toward the reactive intermediate aflatoxin B1-8,9-epoxide (AFBO). Rats constitutively express only small amounts of a GST with high AFBO activity (rGSTA5-5) and thus are sensitive to AFB1-induced hepatocarcinogenesis, although induction of rGSTA5-5 can confer resistance in rats. In contrast to rodents, constitutively expressed human hepatic alpha-class GSTs have little or no AFBO detoxifying activity. Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), has significant constitutive hepatic GST activity toward AFBO and most of this activity belongs to mu-class GSTs. To determine if any alpha-class GSTs in Mf liver have AFBO activity, a cDNA library from a male Mf liver was constructed and screened using the human alpha-class GstA1 cDNA as a probe. Three different cDNA clones with full-length open reading frames were identified from the Mf hepatic cDNA library. Analyses of the cDNA deduced protein sequences indicated that these three alpha-class cDNA clones were 97–98% homologous with each other, and shared 93, 95, and 95% identity with human GSTA1, and were named mfaGSTA1, mfaGSTA2, and mfaGSTA3, respectively. Bacterially expressed mfaGSTA1-1 recombinant protein had similar activities toward classic GST substrates such as DCNB, CHP, and ECA, but slightly lower CDNB conjugating activity relative to human GSTA1-1. However, similar to hGSTA1-1, mfaGSTA1-1 had no AFBO conjugating activity. In addition, similar to human GSTA1 gene, cDNA-derived amino acid sequence analyses demonstrated that all of these Mf alpha-class GSTs genes (mfaGSTA1, mfaGSTA2, and mfaGSTA3) had none of the six critical residues that were identified previously to confer high AFBO activity in mouse alpha-class GSTA3-3. Thus, in contrast to rodents but similar to humans, alpha-class GSTs from the nonhuman primate, Mf, have little conjugating activity toward AFBO.
Key Words: glutathione S-transferase, alpha class; species differences; primate; aflatoxin biotransformation; liver; clone.