Biochimica etBiophysicaActa, 1161 (1993) 333-336 333 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

BBAPRO 34448 Rapid Report

Characterization of two novel subunits of the a-class glutathione S-transferases of human liver

Hassan Ahmad, Sharad S. Singhal, Manju Saxena and Yogesh C. Awasthi Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX (USA)

(Received 12 November 1992)

Key words: Glutathione S-transferase; Subunit; Amino acid sequence; (Human)

More than 85% of the complete amino-acid sequence of the a-class glutathione S-transferase to (GST to) of human liver, described for the first time in this communication, show that GST to is a heterodimer of two closely related novel a-class GST subunits. The sequences of these subunits, tol and to2, have over 97% homology between them and are also highly homologous to the two a-class subunits characterized previously. Characterization of these two novel a-class subunits described in this report would explain the molecular basis for high degree of heterogeneity observed among the a-class human GSTs.

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of dimeric proteins which are involved in the detoxification of xenobiotics, and the protection mech- anisms against endogenously generated oxidants, such as lipid hydroperoxides (see Refs. 1-4 for reviews). Various GST isozymes of human tissues are the prod- ucts of at least three different gene families designated as a,/Z and 7r [5]. A considerable heterogeneity exists among the isozymes within each of three classes. At least three genes belonging to/z-class GSTs have been identified in human tissues [6] and based upon the N-terminal amino-acid sequences of the /z-class GST isozymes of human muscle, the possibility of additional /z-class genes has been suggested [7,8]. The hetero- geneity observed among the human ~'-class [8,9] and a-class [10-17], GST isozymes is difficult to explain on the basis of available information on their genes [18- 20]. Only two genes have been characterized in humans for the a-class GSTs [20]. However, studies from our laboratory as well as from other investigators have indicated a high degree of heterogeneity among the a-class GST isozymes of human liver [10-12], lung [13], kidney [14], skin [15], bladder [16] and lens [17]. Only two a-class genes characterized so far cannot explain the molecular basis of this heterogeneity. We have previously characterized an a-class GST isozyme desig-

Correspondence to: Y.C. Awasthi, Department of Human Biological Chemistry and Genetics, 2.138 Medical Research Bldg., J-67 Univer- sity of Texas Medical Branch, Galveston, TX 77555-1067~ USA.

nated as GST to from human liver [21,22]. This isozyme cross-reacts with the antibodies raised against the a- class GSTs of liver and similar to other a-class GSTs has a blocked N-terminus. In the present communica- tion, we describe the amino-acid sequence of GST to and demonstrate that it is a dimer of two distinct but closely related novel a-class subunits. These studies provide clues for understanding the molecular basis for the high degree of heterogeneity within the a-class of human GSTs.

The purification protocol for GST to used in the present study was essentially similar to that used by us in previous studies [22]. Briefly, human liver super- natant obtained by homogenization in 10 mM potas- sium phosphate buffer containing 1.4 mM/3-mercapto- ethanol and centrifugation at 28 000 X g for 45 min was subjected to affinity chromatography over a column of GSH-linked epoxy-activated Sepharose 6B [23]. The affinity-purified total GST fraction was dialyzed against 10 mM phosphate buffer (pH 6.0) and loaded on to a DEAE (DE-52) cellulose column. The major portion of the total GST isozymes having a p l greater than pH 6.0 was not adsorbed on the column. The anionic GSTs of liver which included GST ~" and GST to were eluted from the column using a 0-200 mM NaC1 gradient. GST to was finally separated from GST rr by passing the enzyme over a column of anti-GST rr antibodies coupled to CNBr-activated Sepharose 4B which com- pletely absorbed GST ~" leaving pure preparation of GST to recovered in the flow through fraction of the column which was extensively dialyzed against 0.1% acetic acid in water.

334

A B

43 K-,

20.1 K--

i~ii!i!i!i!!i~i!ii!!!ili!!ii!ii~iiiiiii! !~ ~

ii! , ii ii!ii!i;iii!i i!i!!!ili!iiiiiii!!ii !i! ! 1 2 1

Fig. 1. (A), S D S - P A G E of the purified GST to. Lane 1, molecular mass markers; Lane 2, GST to. (B), Wes te rn blot of the purified GST

to using the antibodies raised against G S T a.

For cyanogen bromide cleavage, an aliquot contain- ing 4 nmol of GST to was lyophilized and resuspended in 70% formic acid and a 50-fold molar excess of CNBr over methionine was added [24]. The reaction was carried out for 48 h at 25°C in the dark. The reaction was terminated by freezing and the contents were lyophilized. For tryptic digestion, lyophilized GST to was suspended in 100 /~I of 0.1 M ammonium bicar- bonate (pH 8.0) and a 10 /xl solution of trypsin (pro- teinase-to-substrate ratio 1 : 100 (w/w)) was added. Af- ter incubation for 4 h at 37°C with occasional shaking

the reaction was terminated by quickly freezing the sample followed by lyophilization.

The tryptic and the CNBr-generated peptides of GST to were separated on a PepRPC-C 18 reverse-phase FPLC column. The column was eluted at 0.7 ml/min with a gradient of solvent A (0.1% TFA in water) and solvent B (acetonitrile containing 0.1% TFA). A 3-h quadruple gradient program was developed on a Phar- macia/LKB LCC-500 plus controller to achieve opti- mal separation of peptides. For the first 100 min the gradient was increased from 0-20% B and from 100 to 140 min the gradient was increased from 20% B to 45% B. At 160 min, the gradient was changed to increase to 70% B and finally changed to increase to 100% at 180 min. The column was monitored at 230 nm using a LKB 2141 variable wavelength detector. Fractions of 0.5 ml were collected and the peaks corre- sponding to peptides were pooled on the basis of their absorbance at 230 nm. Separately pooled peptide peaks were lyophilized and subjected to automated sequence analysis. The primary structure of each peptide was determined by microsequence analysis using an Ap- plied Biosystems Model 475A protein/peptide se- quencer with an on-line Model 120A microbore HPLC Pth analyzer and a Model 900 data processor. Com- plete details of microsequence analysis have been de- scribed elsewhere [25]. For determination of the C- terminus, GST w was suspended in 50 mM sodium citrate buffer (pH 4.0). The enzyme was incubated with carboxypeptidase P at a proteinase-to-substrate molar ratio of 1 : 50 at 37°C for varying amounts of time (0-20 min). The reaction was terminated by heat inactivation and the samples were directly loaded on to the Applied Biosystems Derivitizer Hydrolyzer Model 420H amino-acid analyzer for the determination of released amino acids.

10 20 30 4o A E K P K L H Y F N A R G R M E S T R W L L A A A G V E F E E K F I K S A E D L

50 60 70 80 D K L R N D G Y L M F Q Q V P M V E I D G M K L V Q T R A I L N Y I A S K Y N L

90 100 F S Q Q 120 Y G K D I K E R A L I D M Y I E G I A D L G E M I L L L P V C P P E E K D A K L

Q 130 140 150 ;60 A L I K E K I K N R Y F P A F E K V L K S H G Q D Y L V G N K L S R A D I H L V

170 180 190 200 E L L Y Y V E E L . . . . . . . . . . . . . . . . . I S N L P T V K . . . . . .

210 221

. . . . . . . D E K S L E E S R K I F R F 9 P

Fig. 2. S u m m a ~ of the amino-acid sequence determinat ion of G S T to. Residues identified by au tomated sequence analysis of ( ~ - - ~ ), CNBr pept ides and ( • - - - - - - • ), t~p t i c peptides. ( ~ ) indicate the residues identified by c a r b o ~ p e p t i d a s e digestion and ( • . . . . . . • ) indicate the residues identified by the amino-acid composi t ion of the CNBr pept ide which appeared to have a blocked N- te rminus during

sequence determinat ion. Blank spaces indicate the residues not determined.

335

GST to was isolated from the liver of a single individual using the protocol reported by us previously [22]. The purified enzyme showed a single band in SDS-denaturing gels (Fig. 1A). In Western blots, this band recognized only the antibodies raised against the a-class GSTs of humans (Fig. 1B) and did not cross react with the antibodies raised against either the tz or 7r class GSTs. The N-terminal analysis of the enzyme did not yield any sequence and the unreacted protein was quantitatively recovered, indicating that the N- terminus of GST to was blocked. These results were consistent with the results of our previous studies [22].

Tryptic digest fragments, as well as CNBr digest fragments of GST to were prepared and separated by FPLC as described above and each of the peptide fragments were subjected separately to sequence analy- ses. The integrated sequence of GST to obtained through the overlapping sequences of CNBr and tryptic digests, along with the results of carboxy-terminal anal- yses, and amino-acid analyses is presented in Fig. 2. The sequence showed two residues in a 1 : 1 ratio at 5 different positions, indicating that GST to was most probably a dimer of two subunits (designated in pre- sent study as tol and to2) having more than 97% homology. As indicated in Fig. 2 we were not able to determine the complete sequences of tol and to2. The dotted line in these sequences (Fig. 2) represents those residues which could not be determined during the present studies. From the tryptic digest, we were un-

able to isolate any peptide fragment(s) corresponding to the unsequenced region (Fig. 2) of GST to. We suspect that, due to hydrophobic nature, these frag- ments precipitated from the reaction mixture and, therefore, could not be isolated. The carboxy-terminal analyses of GST to indicated the presence of phenyl- alanine residue in both the subunits which was consis- tent with the carboxy-terminal residues of phenyl- alanine in the sequences of a-class GSTs Ha subunit 1 (Hal) and Ha subunit 2 (Ha2) described previously [20].

A high degree of sequence homology has been ob- served among the mammalian a-class GSTs [4]. To date, two a-class genes designated as Hal and Ha2 have been characterized in humans and about 95% homology has been observed between the deduced amino-acid sequences of these subunits [20]. Compari- son of the sequences of tol and to2 subunits with the sequences of Hal and Ha2 derived from the sequences of their cDNA (Fig. 3) indicated a high degree of homology among all these four a-class subunits. Maxi- mum homology was observed between tol and Hal with a single amino-acid difference at residue 215. At this position, alanine in Hal was substituted by serine in tol. These results raise the possibility that tol and Hal are allelic. Subunits of 7r and /z class human GSTs differing only in one amino-acid residue in their sequences have been characterized previously [25,26]. The results of present studies demonstrated the exis-

10

H u m a n GST H a l ARE K pK LHY['~N['~RGRME S Human GBT ~1 A K P L YIPJS]l&Jn G R M g S HUman GBT e 2 A m K P K L H ~IP{N~A~R g R N u j Human GST Ha2 A E K P K L H Y s~JNiIIR G R M E S

Human GST Human GBT n u a t a n GBT Human GST

Human GST H ~ GBT H u m a n GBT Human GST

Human GST Human GriT Human GBT Human GST

Human GST HuJ~ .n GBT H u l a n GBT Human GST

20 30 40

~ W i L A A A G V E FEE F I KS i DL

II L I ~ A A G V I I F B B K F I l I B l i D T. WLLAAAGVE FEEKFIKSAEDL

50 60 70 80 Hal DKLRN DG¥ LMFQQVPMVE I DGMKLVQTRAI LNY IAS KYHL el D K L R N D G Y L N F Q Q V P M V B I DGMKLVQT RA I LNY I A J KYIIL ~2 D K L R N D G Y L N F Q Q V P M V E I D G M K L V Q T R A I L H Y I A B K T N L Ha2 DKLRN DGY LMFQQVPMVE I DGMKLVQTRAI LNY IAS KYNL

90 100 110 120 Hal Y G K D I i KE~ A L I MY I E U I A DiG EiI LL L P ~ P E E~ DA K'L ~1 Y G K D KE l& L I DMY I E G I AD G B I L LL P PEg DAKL e 2 yG KD I KB ALI DMY I E G I AD GB I LLL P PZ B DI&KL Ha2 ¥GKD KE ALI DMYIEGIADLGEMI LLLP PEE DAKL

130 Hal A L I~i K~K N S Y F PA ~I ALI K KMMY F P]& e 2 ALI K KNRY F PA Ha2 ALI K KNR¥ FPA

140 150 160 FEKVLKSHGQD¥ LVGNKLS:ADIIHLV F E K V L K H G ~ D Y L V G N r 1 , 8 A D H L V F , K V L K SS f i G . D Y L v G N r t 8 R A D I H L V FEKVLK HGQDY LVGNK S RAD I H LV

170 180 190 200 Hal ELLYYVEELDS S LISSFPLLKALKTRI SNLPTVKKFLQPG 6)1 IB LLY If VE I~ L ................. I 8 HLPTVK ...... ~2 ELL Y YVE K L ................. I 8 NLPTVK ...... Ha2 ELLYYVEELDS SLIS S F P L L K A L K T R I S N L P T V K K F L Q P G

2 1 0 2 2 1

]l£uman GBT (J l . . . . . . . D B K a L B E K I ; ] l lUmLn GBT e2 . . . . . . . D ! E B I". B E X ][ F Human GST Ha2 S P R K P P M D E K S L E E K I F R

Fig. 3. Comparison of the primary structures of human liver GST subunits ~I and to 2 with human liver GST subunits Ha] and Ha2. Boxed residues indicate the region in which observed differences among the four subunits are confined. Blank spaces indicate the residues not

determined. Sequences for Hal and Ha2 are from Rhoad et al. [20].

336

tence of such closely related subunits within the a-class GSTs also. The sequences of o~1 and ~o2 differ at five positions. It is interesting to note that these differences were confined between the residues 110-124, a region which showed maximum differences in the sequences of Hal and Ha2 [20]. The difference between the amino-acid sequences of wl and Ha2 was relatively more. Of the total number of amino-acid residues which have been sequenced, col subunit differed from Ha2 at 10 positions. As shown in Fig. 3, these differ- ences were observed at positions 9, 11, 18, 88, 110, 111, 112, 116, 124 and 127. These amino-acid residue substi- tutions in all the positions, except at positions 11 and 111, could be explained on the basis of a single base substitution in the codons of Ha2. Comparison of GST o)2 with Hal revealed six amino-acid residue differ- ences at positions 110, 111, 112, 116, 124 and 215. GST w2 sequences also showed six amino-acid residue dif- ferences from that of Ha2. However, these differences were at positions 9, 11, 18, 88, 111 and 127. All but two (at positions 11 and 111) amino-acid differences of GST w2 with Hal and Ha2 subunits could be ex- plained by a single base substitution in the codons of Hal and Ha2, indicating very high degree of homolo- gies among the cDNA of these genes. The homologies between Hal, Ha2 and ~o subunits in the region whose sequence has not been determined in present studies need to be investigated. With known differences in the p l values of GST oJ and other a-class GSTs, se- quences of GST w and Hal /Ha2 may be expected to have some differences in this region also. Present stud- ies also provide insight into the molecular basis of the high degree of heterogeneity observed among the hu- man a-class GSTs [10-12]. It has been shown that GST subunits within each class can hybridize to yield various homo- and hetero-dimeric combinations [27]. Homo- and hetero-dimeric combinations of 4 subunits could give rise to ten possible holoenzymes. Differ- ences in the primary structure and kinetic properties of these subunits would give rise to structural and func- tional diversity [10-12] observed among the o~-class GSTs.

This investigation was supported in part of USPHS grant CA27967 awarded by National Cancer Institute.

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