Biochemical Genetics, Vol. 33, Nos. 1/2, 1995

Note

Glutathione S-Transferase Class ar Polymorphism in Baboons Mary Jo Aivaliotis] Teresa Cantu, 1 Ryan Gilligan, 1 and John L. VandeBerg 1,2 Received 1 Nov. 1994--Final 1 Nov. 1994

INTRODUCTION Glutathione S-transferase (GST; EC 2.5.1.18) comprises a family of isozymes with broad substrate specificities (Mannervik and Danielson, 1988; Sundberg et al., 1993). One or more GST isozymes are present in most animal tissues and function in several detoxification pathways through the conjugation of reduced glutathione with various electrophiles, thereby reducing their potential toxicity (Jakoby and Ziegler, 1990). Four soluble GST isozymes encoded by genes on different chromosomes have been identified in humans: GSTA, class alpha, chromosome 6 (Board and Webb, 1987); GSTM, class mu, chromosome 1 (DeJong et aL, 1988); GSTP, class pi, chromosome 11 (Suzuki and Board, 1984), and GSTIg, class theta, chromosome unassigned (Board et aL, 1989). The acidic class pi GST, GSTP (previously designated GST-3), is widely distributed in adult tissues and appears to be the only GST isozyme present in leucocytes and placenta (Suzuki et al., 1987). Previously reported electrophoretic analyses of erythrocyte and leucocyte extracts revealed single bands of activity, which differed slightly in mobility between the two cell types (Board, 1981; Suzuki et aL, 1987), or under other conditions, a two-banded pattern (Strange et al., 1983). To our knowledge, no genetically determined polymorphisms have previously been reported in GSTP from any species. We now report a polymorphism of GSTP in baboon leucocytes, and present family data that verifies autosomal codominant inheritance. This research was supported by N I H Grants HG00336 and HL28972, and N I H Contract HV53030. D e p a r t m e n t of Genetics, Southwest Foundation for Biomedical Research, P.O. Box 28147, San Antonio, Texas 78228-0147. 2 To w h o m correspondence should be addressed. 35 0006-2928/95/0200-0035507.50/0© 1995PlenumPublishingCorporation

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Aivaliotis et al.

MATERIALS AND M E T H O D S

The baboons used in this study comprised animals from a pedigreed breeding colony maintained at the Southwest Foundation for Biomedical Research. The founders of the colony included baboons of two subspecies: Papio hamadryas anubis and P.h. cynocephalus (nomenclature per VandeBerg and Cheng, 1986; WiUiams-Blangero et al., 1990). White blood cells (WBC) were isolated and stored in phosphate buffered saline at -80°C until assayed. The WBC were homogenized in a half volume of deionized water. Erythrocytes from blood clots stored in Tygon tubing segments at -80°C were lysed in 0.15 M dithiothreitol. Brain tissue was homogenized in one volume of deionized water. All samples were centrifuged at 12,000g for 30 min at 4°C. GSTP phenotypes were determined by isoelectric focusing (IEF) of supernatants in an agarose gel (Isogel agarose, FMC, Rockland, Maine) containing 2% Servalyte pH 3-5 and 0.5% Servalyte pH 3-10 (Serva, Paramus, New Jersey); the anolyte was 0.1 M H3PO4 and the catholyte was 0.1 N NaOH. Samples were applied at the anode and focused at 5°C using an LKB-2117 Multiphor unit; initial power was set at 10 W for 30 min, then continued for an additional 90 rain at 12 W. After IEF, proteins were passively transferred to a 0.2 ~m nitrocellulose membrane (MSI Inc., Westborough, Massachusetts) for 30 rain at room temperature. The membrane was immersed for 30 rain at 37°C in 1% Blotto [5 g instant nonfat dry milk, 5 ml liquid gelatin purchased from Norland Products Inc., New Brunswick, New Jersey, 0.02 g thimerosal, 0.002 ml antifoam A, in 1 liter borate buffered saline (BBS)]. All antibody dilutions were made using BBS and all 10 rain rinses between each incubation step were also in BBS (0.05 M boric acid, 0.15 M sodium chloride, 0.05 M potassium chloride, pH 8.0 with sodium hydroxide). The membrane was incubated overnight at 37°C with a 1:1000 dilution of sheep anti-human GST3 antibody derived from human placenta (The Binding Site, San Diego, California), then incubated for 30 min with 1:1000 dilution of biotinylated rabbit anti-goat IgG followed by the same dilution of horseradish peroxidaseavidin (Vector, Burlingame, California) for another 30 rain at 37°C. GSTP3 activity was visualized by staining the membrane with 4 mM 3-amino-9ethylcarbazole in 20% methanol, 80% BBS, and 0.03% hydrogen peroxide. A final rinse in water stopped the staining reaction.

RESULTS AND DISCUSSION

Figure i illustrates GSTP phenotypes of baboon WBC; these were clear and easily typeable. The alleles were designated as GSTP*A, GSTP*B, and

GSTP Polymorphism in Baboons

37

+

m

IHUMANI[ 1

BABOON 2

3

4

I 5

Fig. 1. GSTP phenotypesin white blood cell extracts. Lane 1: Human. Lanes 2-5: Baboon GSTP AB, GSTP B, GSTP BC, and GSTP C, respectively. GSTP*C. Homozygotes exhibit a single band, whereas heterozygotes exhibit a 3-banded pattern characteristic for a dimeric protein. The human WBC GSTP band migrated slightly cathodal to the most anodal baboon isoform, GSTP A. The same clear phenotypic patterns were also characteristic of baboon brain extracts (Fig. 2). A report of GST enzymes isolated from human brain found the most anionic form (pI 4.6) to contain the major portion of GST activity (Theodore et al., 1985). Baboon hemolysates gave a more complex multi-banded pattern (Fig. 2) and each phenotype corresponded with one of the WBC phenotypes. However, the multiplicity of bands made it difficult in some instances to distinguish heterozygotes from homozygotes (e.g., see the RBC lanes in Fig. 2). The results suggest the GSTP isozymes expressed in baboon white cells, erythrocytes, and brain are the products of the same gene locus. In Western blots, these bands were recognized only by antibodies raised against the pi-class GST from human placenta and did not bind antibodies raised against the alpha-class GST isolated from liver. Table I shows the distributions of GSTP phenotypes and gene frequencies determined by direct gene counting in a panel of 118 randomly chosen baboons. The family data obtained from 231 triads (parents and offspring)

38

Aivaliotis et al.

+

~3

[13

u3 m z

< f13

"z~ r~

m

v

u

-~.=

h-w

W..= o

O'z~

Oa::

O

"~

GSTP Polymorphism in Baboons

39

Table I. A. Distribution of GSTP Phenotypes and G e n e Frequencies in 118 Unrelated Baboons Phenotypes

Observed Expected

G e n e frequency

A

AB

B

AC

a

b

c

56 55.57

49 50.07

12 11.28

1 0.67

0.686

0.309

0.004

B. Family Data for Baboon GSTP Progeny type b Mating type a

A

AB

B

AC

AxA A x AB AB x AB AxB B x AB B×B Ax AC

6 45 12 0 0 0 0

0 50 22 32 21 0 0

0 0 11 0 26 5 0

0 0 0 0 0 0 1

~Reciprocal crosses were combined because their results did not differ significantly in any instance. bGenotypic frequencies did not differ significantly between sexes. Segregation ratios did not differ significantly from those expected under the hypothesis of autosomal codominant inheritance.

were consistent with autosomal codominant inheritance. The genotypic frequencies were in Hardy Weinberg equilibrium. Baboons are used as animal models for a variety of human diseases including some to which susceptibility is strongly influenced by genetic factors (VandeBerg and Cheng, 1986). Development of a baboon linkage map is in progress for the purpose of locating and identifying genes that affect susceptibility to dyslipoproteinemias, atherosclerosis, hypertension, osteoporosis, and other common multifactorial diseases (Rogers et al., 1994). Although microsatellite polymorphism will comprise most of the mapped markers, polymorphic blood protein genes will serve as important anchor loci, particularly for relating the baboon gene map to the human and mouse maps. The high heterozygosity (0.461) of GSTP in baboons makes it a useful marker for pedigree verification, quantitative trait linkage analysis, and as a potential marker for research on the protective role that GSTP plays in carcinogenesis and against toxic substances.

40

Aivaliotis et aL

ACKNOWLEDGMENTS W e t h a n k B e n n e t t D y k e for d e v e l o p i n g t h e c o m p u t e r p r o g r a m s for d a t a m a n a g e m e n t , J i m B r i d g e s for e n t e r i n g t h e d a t a , a n d P a u l S a m o l l o w for critically r e v i e w i n g t h e m a n u s c r i p t .

REFERENCES Board, P. G. (1981). Biochemical genetics of glutathione-S-transferase in man. Am. J. Hum. Genet. 33:36. Board, P. G. and Webb, G. C. (1987). Isolation of a eDNA clone and localization of human glutathione S-transferase 2 genes to chromosome band 6p12. Proc. Natl. Acad. Sci. USA 84:2377. Board, P. G., Webb, (3. C, and Coggan, M. (1989). Isolation of a cDNA clone and localization of the human glutathione S-transferase 3 genes to chromosome bands 11q13 and 12q13-14. Ann. Hum. Genet. 53:205. DeJong, J. L., Chang, C.-M., Whang-Peng, J., Knutsen, T., and Tu, C.-P. D. (1988). The human liver gtutathione S-transferase gene superfarnily: Expression and chromosome mapping of an Hb subunit cDNA. Nucleic Acids Res. 16:8541. Jakoby, W. B., and Ziegler, D. M. (1990). The enzymes of detoxication. J. Biol. Chem. 265:20715. Mannervik, B., and Danielson, U. H. (1988). Glutathione transferases: Structure and catalytic activity. CRC Crit. Rev. Biochem. 23:283. Rogers, J., Witte, S. M., Karnmerer, C. M., Hixson, J. E., and MacCluer, J. W. (1994). Gene mapping in Papio baboons using hypervariable microsatellite and RFLP markers: Results for chromosome l q.Am. J. Phys. Anthropol. 18(Suppl.):172. Strange, R. C., Hirrell, P. H., Kitley, G. A., Hopkinson, D. A., and Cotton, W. (1983). Erythrocyte glutathione S-transferase. Biochem. J. 215:213. Sundberg, A. G. M., Nilsson, R., Appelkvist, E-L., and Daliner, G. (1993). Irnmunohistochemical localization of a and "rr class glutathione transferases in normal human tissues. Pharrnacol. Toxicol. 72:321. Suzuki, T., and Board, P. (1984). Glutathione-S-transferase gene mapped to chromosome 11 is GST3 not GST1. Somatic CellMol. Genet. 10:319. Suzuki, T., Coggan, M., Shaw, D. C., and Board, P. G. (1987). Electrophoretic and immunological analysis of human glutathione S-transferase isozymes. Ann. Hum. Genet. 51:95. Theodore, C., Singh, S. V., Hong, T. D., and Awasthi, Y. C. (1985). Glutathione S-transferases of human brain: Evidence for two immunologically distinct types of 26,500 Mr subunits. Biochem. Z 225:375. VandeBerg, J. L., and Cheng, M.-L. (1986). The genetics of baboons in biomedical research. In Else, J. G., and Lee, P. C. (eds.), Primate Evolution (Selected Proceedings of the Tenth Congress of the International Prirnatological Society), Cambridge University Press, Cambridge, p. 317. Williarns-Blangero, S., VandeBerg, J. L., Blangero, J., Konigsberg, L., and Dyke, B. (1990). Genetic differentiation between baboon subspecies: Relevance for biomedical research. Am. J. Primatol. 20:67.