ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 278, No. 2, May 1, pp. 39%408,199O

Primary and Secondary Structural Analyses of Glutathione S-Transferase x from Human Placenta’

Hassan Ahmad, Douglas E. Wilson, Richard R. Fritz, Shivendra V. Singh,2 Rheem D. Medh, Gregg T. Nagle,* Yogesh C. Awasthi,” and Alexander Kurosky Department of Human Biological Chemistry and Genetics and *The Marine Biomedical Institute, Department of Anatomy and Neurosciences, The University of Texas Medical Branch, Galveston, Texas 77550

Received October 23,1989, and in revised form December 11, 1989

The primary structure of glutathione S-transferase (GST) a from a single human placenta was determined. The structure was established by chemical character- ization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact en- zyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide se- quence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626- 5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleo- tide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one in- dividual placenta studied at least two GST a genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation iden- tified a hydrophobic region in GST * (residues 155- 181) that was predicted to be either a buried transmem- brane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme espe- cially in regard to the potential involvement of a histi- dine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of z, one of p, two of CY, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form. (r Iwo Academic PESS, IN.

i This investigation was supported in part by USPHS Grants CA 27967, GM 32304, EY 04396, and CA 17701. NIH support for BIONET was to IntelliGenetics, Inc. Grant 5 U41 RR 01685.

‘Present address: Department of Oncology, School of Medicine, University of Miami, Miami, FL 33136.

398

Glutathione S-transferases (GSTS;~ EC 2.5.1.18) are a family of primarily cytosolic enzymes that occur in mammalian tissues where they play an important role in cellular detoxification processes (see recent review (1)). Most of the GST isozymes characterized so far from var- ious human tissues have been grouped into three major classes on the basis of their structural, kinetic, and im- munological properties and are designated as cy, CL, and a (2). The isozymes within each class show strong struc- tural and immunological similarities indicating evolu- tionary relationships; however, the similarity of these characteristics between enzymes of other classes is much less pronounced (2).

In human tissues several isozymes from each of the three classes of GST have been characterized including the anionic isozymes of erythrocyte, placenta, lung, and kidney which are representative of the R class (2-5). Re- cently the 7r class of GST enzymes has attracted a great deal of attention because of its potential as a marker for chemical carcinogenesis (6, 7). A GST rat isozyme be- longing to the r class has been reported to be overex- pressed in preneoplastic nodules and primary hepato- mas induced by certain carcinogens (6). In humans, primary hepatic tumor (7) and metastatic tumors origi- nating from lung (8) also have elevated levels of this GST class. In addition, GST r is also overexpressed in certain drug resistant cancer cell lines (9,lO).

” To whom correspondence and reprint requests be addressed at 301 Keiller Bldg. F-20, the University of Texas Medical Branch, Galves- ton, TX 77550.

4 Abbreviations used: GSTs, glutathione S-transferases; CDNB, l- chloro-2,4-dinitrobenzene; TFA, trifluoroacetic acid; PMSF, phenyl- methylsulfonyl fluoride; DTT, dithiothreitol; HPLC, high-perfor- mance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DEPC, diethylpyrocarbonate; CNBr, cyanogen bromide; Pth, phenylthiohydantoin; CM, carboxy- methyl.

0003.9861/90 $3.00 Copyright I? 1990 by Academic Press, Inc.

All rights of reproduction in any form reserved.

STRUCTURAL ANALYSIS OF HUMAN GST ?r 399

While the structural relationship among the ?r class of GST isozymes from different human tissues has not been completely resolved, there are indications that these isoenzymes represent identical or closely related proteins (3,11,12). On the other hand, other studies sug- gest that, similar to the LY and p classes of human GST, the r class of isozymes isolated from different tissues displays some degree of heterogeneity (4,13,14). For ex- ample, it was striking that GST K isozymes character- ized from various human tissues demonstrated notice- able differences in their isoelectric points, kinetic properties, and peptide maps (4, 13, 14). These differ- ences could be explained by either differential gene ex- pression, post-translational modification, polymor- phism, or some combination(s) thereof. Evidence to establish whether such differences occur in the GST 7r class of isozymes can be obtained from primary structure determination of expressed protein.

In this communication we report the chemical charac- terization of human placental GST a, including the com- plete amino acid sequence and predicted secondary structure, and discuss its evolutionary relatedness among various classes of mammalian GST enzymes. Po- sitioning of some of the tryptic peptides was provided by published nucleotide sequence analysis of a cDNA clone (12). This is the first GST 7r primary structure estab- lished largely by protein sequence analysis.

EXPERIMENTAL PROCEDURES

Materials. 4-Vinyl pyridine was purchased from Aldrich Chemical Co. (Milwaukee, WI). TFA, triethylamine, and N-ethylmorpholine were obtained from Pierce Chemical Co. (Rockford, IL). I,-(tosylamido 2-phenyl)ethyl chloromethyl ketone-treated trypsin and carboxypep- tidase A were purchased from Worthington Biochemical Corp. (Free- hold, N,J). PMSF and DTT were obtained from Sigma Chemical Co. (St. Louis, MO). Reagents for protein and peptide sequence analysis were obtained from Applied Biosystems (Foster City, CA). For HPLC studies, a Supelcosil LC-18.DB CIR (5.Km particle size) reversed-phase column (25 cm X 4.6 mm) was purchased from Supelco, Inc. (Belle- fonte, PA). A Waters guard column was packed with pellicular Cl8 packing material obtained from Alltech Applied Science Labs (Deer- field, IL). Reagents for the purification of human placental GST were the same as reported previously (15, 16).

Purification of human placental GST. A single human placenta, obtained within 1 h of delivery, was cleaned of amniotic membrane and connective tissues and washed extensively with ice-cold distilled water. A 10% (w/v) homogenate of the placental tissue (200 g) was prepared in 10 mM potassium phosphat,e buffer, pH 7.0, containing 1.4 rnM 2-mercaptoethanol (buffer A) using a PT IO-35 polytron (Kine- matica GmhH, Littau). The homogenization was performed for 5 min at 4°C and the homogenate was centrifuged at 14,OOOg for 40 min. The supernatant, following dialysis against buffer A, was subjected to affinity chromatography as reported by Simons and VanderJagt (17). A column of glutathione coupled to epoxy-activated Sepharose 6B was preequilihrated with 22 mM potassium phosphate buffer, pH 7.0, con- taining 1.4 mM 2-mercaptoethanol at a flow rate of 10 ml/h. The col- umn was washed until the absorbance at 280 nm returned to baseline and the enzyme was eluted with 10 mM glutathione in 50 rnM Tris- HCl, pH 9.6, containing 1.4 mM 2.mercaptoethanol at a flow rate of 10 ml/h. Fractions containing GST activity were pooled and dialyzed

against buffer A and subsequently subjected to isoelectric focusing us- ing an LKB 8100-l column and employing a O-50% sucrose density gradient. After focusing at 1600 V for 18 h, 0.8.ml fractions were col- lected and monitored for pH and GST activity. Throughout the puri- fication GST activity was monitored by the methodof Hahiget al. (18) using CDNB as the electrophilic substrate. Protein concentrations were determined by the method of Bradford (19) using bovine serum albumin as standard. A single peak of enzyme activity at pZ 4.5 was obtained following isoelectric focusing using an ampholyte pH range of 3.5 to 10.0. The enzyme preparations were extensively dialyzed against 0.1% acetic acid and lyophilized prior to structural analysis.

Reduction and alkylation. Purified GST r was reduced and alkyf- ated by the method described previously by Kurosky et al. (20). Briefly, the protein was resuspended in 2.2 ml of 0.5 M Tris+HCl buffer, pH 8.2, containing 6 M guanidine-HCl and 2 mM EDTA to which DTT was added to a final concentration of 30 mM. The solution was gently flushed with N, for 10 min and the reaction mixture was incubated for 2 h at 37°C. The protein was subsequently alkylated with 4-vinyl pyridine (70 mM final concentration) for 3 hat room temperature. Fol- lowing incubation, 5 ~1 of 10% DTT solution was added to each 100 ~1 of reaction mixture to stop the reaction. The reduced and alkylated protein was dialyzed against 5% triethylamine and stored at -20°C prior to use.

Trypsin digestion of GST K. A l.O-ml aliquot of the reduced and alkylated protein (40 nmol) in 5% triethylamine was adjusted to pH 8.2 with 0.1% TFA. To this solution was added 10 pl of trypsin (prote- ase:substrate ratio of 1:lOO (w/w)) in the same buffer. After incubation for 4 h at 37°C with occasional shaking, the reaction was terminated by the addition of PMSF (1.0 mM final concentration).

Purification of tryptic peptides. The tryptic peptides were applied directly to the Supelcosil LC-18-DB reversed-phase HPLC column and the column was eluted at a flow rate of 1.0 ml/min at 25°C with a gradient of Solvent A (0.1% TFA) and Solvent B (neat acetonitrile containing 0.1% TFA). A 7.5 h triple gradient was performed. For the first 120 min the gradient was increased from 0 to 20% B and from 120 min to 400 min the gradient was increased from 20% B to 40% B. At 400 min the gradient was changed to increase to 55% B at 460 min. The column eluate was monitored at 220 nm and 0.5 ml fractions were collected. Fractions were pooled on the basis of their absorbance at 220 nm and were subjected to amino acid compositiona and sequence analyses.

C’yanogen bromide hydrolysis of GST K. An aliquot containing 20 nmol of reduced and alkylated protein was dried and resuspended in water and dried again to remove residual triethylamine. The protein was subsequently resuspended in 70% formic acid, and a 50-fold molar excess of CNBr over methionine was added (21). The hydrolysis reac- tion was carried out for 24 hat 25°C in the dark. The reaction mixture was lyophilized and the peptide mixture was resuspended in 6 M guani- dine-HCI prior to HPLC analysis. HPLC conditions were the same as those employed for isolation of the tryptic peptides except that a shorter gradient time was utilized. The gradient duration was 2 h and went from 0% B to 55% B.

Carboxypeptidase digestion of GST 7. Soluhilization and activation of carhoxypeptidase A was carried out essentially according to the method of Ambler (22). After removal of triethylamine, a sample of the reduced and alkylated protein (100 fig; 4 nmol) was resuspended in 0.2 M sodium bicarbonate. The protein solution was incubated with activated carhoxypeptidase A at an enzyme:suhstrate molar ratio of 1: 70 at 37°C for 24 h. The reaction was terminated by adding 3.75% S- sulfosalicylic acid. A control reaction containing GST K was incubated with heat-inactivated carhoxypeptidase A. The samples were subse- quently subjected to amino acid analysis after filtration through a 0.45.pm filter.

Amino acid analysis. Samples were hydrolyzed with 5.7 N HCI in uucuo at 107°C for 24 h. Amino acidcompositional analysis was carried out on a Beckman 121M analyzer employing single column methodol- ogy on Beckman W-2 resin and on a Beckman 6300 analyzer (23).

400 AHMAD ET AL.

Automated amino acid sequence analysis. The primary structure of each peptide was determined by microsequence analysis using an Applied Biosystems Model 475A protein/peptide sequencer with an on-line Model 120A microbore HPLC Pth analyzer and a Model 900 data processor. The repetitive yields ranged from 90-95%. The Model 120A microbore HPLC identified and quantitated the Pth derivatives of the amino acids obtained from the sequencer using C,s reversed- phase chromatography similar to that reported by Zimmerman et al. (24). Complete details for microsequence analysis have been described elsewhere (25).

Computer assisted conformational and consensus sequence eualua- tion. These studies utilized the BIONET’ computer facilities (Intel- liGenetics, Inc., Mountain View, CA). Alignment of the available com- plete human GST enzyme structures utilized IntelliGenetics’ mainframe program GENALIGN. Other structural evaluations de- scribed utilized IntelliGenetics’ PC/GENE programs. The secondary structure of GST H was predicted using essentially the methods of Chou and Fasman (26) as modified by Novotny (program Novotny, (27)). The hydrophobicity profile of GST T was computed using pro- gram PRESIDUE (28) and the hydrophilicity profile (program ANTI- GEN) was determined as reported by Hopp and Woods (29). The methods of Rao and Argos (30) and Eisenberg et al. (31) evaluated potential transmembrane helices (program RAOARGOS) and mem- brane associated helices (program HELIXMEM), respectively. Other consensus sequence searches included predictions of membrane span- ning segments (program SOAP) (32). radial locations (program RA- DIOLOC, (33)), short half-life regions (program PESTFIND, (34)), DNA-binding regulatory regions (program REGULAT, (35)), and sig- nal peptide regions (program PSIGNAL, (36)).

RESULTS

Isolation of placental GST. Anionic GST (p14.5) was purified from human placenta in this structural study with a 35% yield. The purified enzyme, when subjected to SDS-PAGE, showed the presence of a single band corresponding to an M, value of 22,500 (data not shown), which was consistent with our previous observations (3). When this protein was immunoblotted (Western blot- ting) using antibodies raised against the 01, CL, and r classes of GST enzymes only antibodies raised against the human placental GST ir class cross-reacted with the 22,500 M, protein (data not given), indicating that the preparation was not contaminated with other classes of GST enzymes. Automated NHz-terminal sequence anal- ysis of the purified enzyme yielded only a single se- quence, providing additional evidence of homogeneity.

Purification of trypticpeptides. The elution profile of tryptic peptides obtained following reaction of reduced and alkylated placental GST R protein with trypsin and separation on a Supelcosil LC-18-DB reversed-phase HPLC column is illustrated in Fig. 1.

Primary structure of human placental GST x. Eluci- dation of the primary structure of human placental GST ?r was based mainly on amino acid sequence analysis of HPLC-purified tryptic peptides, intact GST 7r protein, and CNBr fragments. The results of quantitative micro- sequence analysis of the tryptic peptides are presented in Table I. The individual tryptic peptides were desig- nated using an alphanumeric code. The letter indicated the nature of the cleaving agent (T for trypsin) and the

number indicated the order of the peptides in the pri- mary structure of the protein beginning with the NH* terminus that resulted from cleavage of lysyl and arginyl residues. Nontryptic (chymotryptic-like) minor cleav- ages were designated alphabetically, e.g. T1, and Tlb. Complete amino acid sequences were obtained for all tryptic peptides, with the exception of T13 and Ti6. The COOH-terminal arginyl residue of fragment Ti3 was not obtained in this sequence analysis (Table I) but was demonstrated by amino acid compositional analysis (data not presented) and indicated by trypsin specificity. Similarly, the COOH-terminal glutaminyl residue of fragment Ti6 was also not established by tryptic peptide sequence analysis but was indicated by compositional results. Carboxypeptidase A treatment of the reduced and alkylated GST x subunit followed by amino acid analysis demonstrated only the release of glutamine when compared to an inactivated enzyme control. In general, the amino acid compositional results of the tryptic peptides (data not given) correlated well with the amino acids obtained by sequence analysis. A compari- son of the amino acid composition of the GST x subunit determined by amino acid analysis (3) with that deter- mined by microsequence analysis is given in Table II. The amino acid compositions determined by these two methods were in reasonable agreement.

Overlapping sequences to the tryptic peptides were obtained from sequence analysis of the intact protein and from CNBr-reacted protein as shown in Table III. The HPLC gradient employed to separate the CNBr peptides (data not presented) was too steep and did not separate the peptides well although it did resolve them from the 6 M guanidine-HCl. Therefore, the peptide fractions were pooled together and subjected to sequence analysis as a mixture. The CNBr sequence results shown in Table III represent a combined analysis of three pep- tides. The alignments shown for the CNBr peptides in Table III were deduced by comparing the CNBr peptide sequence results with sequence results from the intact protein and the tryptic peptides. The CNBr results were only used to establish and confirm sequences overlap- ping the tryptic peptides.

The combined protein sequence results are summa- rized in the alignment given in Fig. 2. Included in Fig. 2 are overlaps that were obtained as a result of partial hydrolysis of lysyl bonds containing the acidic residues Asp or Glu which included peptides T,-T, and Ts-Ts. Several other bonds containing either Arg-Pro, Lys- Pro, or Arg-CM-Cys cleaved partially or not at all (Fig. 2). Of the fifteen overlaps required to align the tryptic peptides, seven were established from protein sequence analysis. The remaining eight overlaps were obtained from nucleotide sequence analysis of a human placental cDNA clone reported by Kano et al. (12) during the prog- ress of this work. These overlaps were those joining T5 through T8 and T1 1 through T16.

STRUCTURAL ANALYSIS OF HUMAN GST K 401

430

0 50 100 150 200

Time (min)

FIG. 1. Separation of tryptic peptides of human GST K by reversed-phase HPLC using a CIR column. Peptides were separated using triple linear gradients of 0.1% TFA and acetonitrile containing 0.1% TFA as described under Experimental Procedures. The column eluate was monitored at 220 nm. (A) Tryptic digest; (B) Reagent blank same as tryptic digest but without GST. Blank showed no change between 225 and 460 min (results not shown).

The deduced sequence obtained by Kano et al. (12) was essentially identical to that determined in this study (Fig. 2) with the exception that we identified both isoleu- cyl and valyl residues at position 104 whereas Kano et al. found only isoleucine. The shallow gradient employed in separating the tryptic peptides (Fig. 1) allowed resolu- tion of the two allelomorphic peptides into fractions T1, (Ile- 104 containing) and T’, 1 (Val- 104 containing).

Structural comparison of human GST isozymes. A comparison of the entire subunit structures of human GST 7r with the entire primary structures of four other human GST enzymes is shown in Fig. 3. The sequence of human GST 7r compared similarly to that of human GST Hal (27% identity, 39% chemical similarity), GST Ha2 (26% identity, 37% chemical similarity), and GST Hb (26% identity, 38% chemical similarity) but was less similar to microsomal GST (10% identity, 15% chemical similarity) (Table IV). The overall degree of chemical relatedness of GST K to the four GST enzyme sequences (Fig. 3) when compared together, i.e., a residue identity was scored if present in any of the four other GSTs, indi- cated 43% residue identities and 55% chemical similari-

Consensus sequence evaluation. A number of searches were undertaken to evaluate the possible oc- currence of consensus sequences which might reveal additional insights into GST function. Of the many programs utilized, which are listed under Experimen- tal Procedures, the searches for the occurrence of transmembrane helices and signal sequence regions were noteworthy. Results of the program RAOARGOS search for a smoothed buried helix profile for the five human GST enzymes predicted the occurrence of a transmembrane segment or segments in GST ?r and microsomal GST. The region in GST r was repre- sented by residues 167 to 193 (Fig. 3). The microsomal GST contained two predicted regions, residues 11 to 35 and residues 101 to 119 (actual residue numbering) which in Fig. 3 represented regions 14 through 42 and regions 154 to 177. The region of residues 170-200 (Fig. 3) of GST 7r also satisfied the criteria for a signal sequence (program PSIGNAL). All other consensus searches listed under Experimental Procedures did not reveal any significant results.

Secondary structure. Results obtained from the ties. Chou and Fasman method of analysis (Novotny modi-

TABL

E I

Auto

mat

ed

Sequ

ence

An

alys

is

of G

ST

?r T

rypt

ic

Pept

ides

Edm

an

ReO

i*“B

(pm

ol’,

Cyc

le

TIa

Tlb

T1

TZ

T3

r4

T 34

T5

T6

T7

T8

T9

T

89

r10

T11

TIT,

T1

2 T1

3 71

4 r1

5 T,

6

L+

- l3

000,

-

(200

0,

- (1

200,

-

(150

0,

- (8

00,

- (1

000,

-

(300

0)

- (2

500,

,1

50O

l (3

000,

-

(300

0,

(250

0,

- (5

00)

- (1

500)

-

(150

0)

- (1

000,

(1

200,

-

,300

Ol

- l1

0000

, -

(100

0,

- (6

000,

1 Pr

o(13

12,

Thr

(543

, Pr

o (3

93)

Gly

(5

83)

Met

,283

1 G

lu

(344

) M

el ,4

12,

Ala,

1483

1 Ph

e (6

241

HIS

(4

81)

Thr

(784

) As

p(39

,) Th

r (1

40)

Cys

(657

) Ty

r (6

29)

Tyr

(357

, Al

a (5

06)

Thr

(789

) La

(3

949)

Le

u (2

83)

Ala

(284

0,

2 PI

0 (1

272,

“a

I ,3

59)

Pro

(364

) Ar

g 11

31)

Leu

(182

) G

lu

(327

) Le

u (6

02)

Ser

,260

) G

in

(489

) Le

u (1

622)

Le

u (1

75)

Gin

17

41)

Leu

,217

) Ly

s ,4

57)

IN

(174

) V.

3 (9

3,

Le”

(509

) Ph

e (1

204)

Se

r (9

22)

Lys

(24)

Ph

e (1

989)

3 Ty

r (5

01)

“a,

(349

) Ty

r (3

37)

Cys

(3

30)

Le”

(155

) Va

i (2

101

Leu

(436

, C

YS

(324

) As

p (1

28)

Giy

(1

445)

G

iy

(135

) G

in

(713

) G

iy

(7,)

SW

,110

, se

r (4

6,

Pro

,281

) ,,e

,4

29,

Ala

(317

4)

L.2”

(1

653,

4 Ty

r (8

70,

Thr

(134

) Al

a ,6

35)

Ala

,149

) “a

l (2

19)

Ala

(448

) Le

u (6

19,

Gly

(3

09)

Arg

(163

) Le

u (9

0)

Gl”

(626

) La

(5

4)

Le”

(447

) Le

u ,2

06,

Gly

(1

47)

Val

(790

) Ar

g (7

63)

Ala

(231

,)

5 Ph

e 66

0)

Val

(274

) Al

a (5

31)

Asp

(57)

Th

r (2

02)

Asp

(194

) Ty

r (5

94)

Asp

(122

) Ty

r (1

44)

Ala

(818

) TV

(8

5)

Iwe

(128

) Ile

W

I G

in

(150

) G

ly

(956

) Pr

o (2

504)

se

r (3

65)

6 Pr

o (6

85)

Val

(236

) Le

u (4

03)

Gin

(9

9)

“al

(154

) G

in

(239

) G

ly

(437

) Le

u (3

80)

Gly

(8

5,

Ala

(790

) G

ly

(35)

Ty

r (3

70)

Tyr

(249

) Le

u (2

07)

Asp

(656

) Ly

s (1

241)

Pr

o (1

378)

7 Va

t (2

84)

Tyr

(215

) Ar

g (3

0)

Gly

(6

3,

Glu

(1

35)

Gly

(8

1)

Gin

(5

9,)

Thr

(43,

) LY

S (4

4)

LW

(335

) LY

S (1

61

Thr

(324

) Th

r (9

8)

Lys

(97)

G

in

(105

6)

Glu

(9

45)

8 Ar

g (5

0)

Phe

,216

) G

in

(92)

Th

r (1

28)

Gin

(1

56,

Leu

(568

, Lw

(3

59)

“ai

,769

) N

DC

-

Asn

,212

) As

n (6

8)

Pm

(155

) lie

,2

81,

Tyr

(104

7,

9 Pr

o (1

94)

Ser

(22,

Tr

p (9

8)

Ser

(71)

Pr

o (4

97)

Tyr

(435

) As

p (1

79)

Gin

(1

4)

Tyr

(390

) Ty

r (1

28)

Phe

(147

) Se

r (3

78)

“al

(535

,

10

va,

,177

) Tr

p (4

4)

Gin

(1

25)

Trp

(90)

Ly

s 18

8,

Gin

(3

48)

Met

(2

75,

Gin

(1

5)

Glu

,2

47)

Glu

(1

16,

Gi”

(82)

Ph

e (6

47)

Asn

(546

)

1,

Arg

(16)

Ly

s (5

11

Glu

(8

5)

Lys

(84)

se

r ,1

31,

“aI

e25,

G

lu

(16)

Al

a (3

23,

Ala

(146

) Th

r (6

2)

Ala

(928

) Le

u (8

09,

12

Gly

(9

01

Glu

(9

0,

As”

(110

) As

,. (1

97,

Aia

(14)

G

ly

(202

) G

ly

(77,

Le

u (9

1)

Asp

(474

) Pr

o (9

67)

13

Ser

(16)

G

lu

,110

, Th

r (5

9,

Asp

(102

) Al

a (8

1 Ly

s (1

29)

Lys

(36,

Le

” (9

3)

Tyr

(679

) k

(287

1 14

LW

(7

1)

vat

(63)

lie

(1

49)

Giy

,1

11,

Leu

(18)

AS

P (6

0)

Asp

(58)

Se

r (1

9)

Asn

(622

) AW

W

I

15

Lys

113)

Va

l (1

22)

Le”

(145

) va

i 14

4)

“aI

(IQ)

Asp

(108

) AS

P (4

4)

Gin

(3

3)

Leu

(534

) G

UY

161

16

Thr

(36)

Ar

g (9

1 G

lu

(100

, N

DC

-

Tyr

(166

) Ty

r (7

1,

Asn

(7,

Leu

(450

) AS

n (1

1

17

“a,

(26)

As

p (8

8)

Met

(9

) Va

l (5

2)

Val

(25)

G

in

(14,

As

p (3

62)

GlY

(5

)

18

Glu

(4

0)

Le”

(98)

Va

l (2

) LY

S (2

5)

Lys

(IO,

Gly

(9

) Le

” (3

69)

LYS

,251

19

Thr

(22)

Ar

g (2

5,

As”

(1)

Gly

(1

4)

Leu

(362

) G

in

ND

d

20

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STRUCTURAL ANALYSIS OF HUMAN GST ?r 403

TABLE II DISCUSSION

Amino Acid Composition of Human Placental GST r

Residues/m01 protein

Amino acid

Aspartic acid Asparagine Threonine Serine Glutamic acid Glutamine Proline Glycine Alanine Valine Cysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

Total

” From Dao et al. (3).

Composition”

21

9 11 24

11 19 14 13

2 1 6

32 10

8 11

4 8 3

207

Sequenceh

13 8 9

10 10 13 11 18 15

x/14 4 2

617’ 32 12

7 12

2 8 2

209

In view of the fact that the GST isozymes show con- siderable genetic variation particularly in regard to tis- sue specificity, and the fact that even within a class of isozymes there is evidence of molecular differences (4, 13, 14), it is reasonable to inquire about how well re- ported cDNA nucleotide sequences of GSTs correlate with the primary structures of their expressed gene

products. In the case of human GST X, the placental cDNA sequence of an isolated clone agreed exactly with the amino acid sequence analysis determined with puri- fied placental enzyme, with one exception. Position 104 contained both isoleucine and valine whereas the re- ported cDNA sequence indicated isoleucine at this posi-

TABLE III

Automated Sequence Analysis of CNBr Peptides of GST*

Edman cycle GSTrb

h Residues established by sequence analysis described herein. ’ Variable residue number due to a Val/Ile allelomorphism at posi-

tion 104.

fied, (27)) of polypeptide conformation for human pla- cental ?r are summarized in Fig. 4. The analysis predicted that the polypeptide chain was significantly (Y helical with predominant helices represented by residues 13-22, 27-34, 81-93, 155-165, and 188195. The polypeptide chain possessed significant P-sheet potential between residues 2 and 8,31 and 35,99 and 111,175 and 183, and 204 and 207. Considerable P-turn potential was pre- dicted throughout the molecule with major turn poten- tials at residues 23-26, 114-117, 136-139, and 166-169. These predicted values indicated that the a-helix, p- sheet, and P-turn constituted about 37%, 16%, and 47% of the polypeptide chain, respectively. Like the rat GST structures (37) the human r structure typically alter- nated cu-helices and P-pleated sheets. The hydrophobic- ity, hydrophilicity, and charge distribution profiles of the GST x polypeptide are also summarized in Fig. 4. Clearly, the COOH-terminal portion of the molecule (148209) was significantly hydrophobic. The hydrophi- licity profile of the polypeptide revealed that there were three regions that were predominantly hydrophilic. The segments of amino acids in decreasing order of hydrophi- licity were in the NH2-terminal portion of the molecule in regions of residues 112-117,80-85, and 97-102.

0’ 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 21 28 29 30

Pro Pro Tyr Thr Val Val Tyr Phe Pro Val Arg Gly Arg CYS Ala Ala Leu Are Met Leu Leu Ala Asp Gln Gly Gin Ser Trp LYS GIU

a Results obtained with gas phase microsequencer. Pth amino acids were quantitated by HPLC.

’ From Dao et al. (3). Residue 28 was reported as Arg (3) and herein has been corrected as shown from tryptic peptides results. Also, posi- tion 27, which was previously undetermined (3), was established to be Ser from tryptic peptides.

’ Initial pmol applied to the sequencer. d ND, not determined, established from tryptic peptides.

Residue (pmol) a

CNBr, CNBr, CNBr,

- (1500) Pro (710) Pro (694) Tyr (577) Thr (345) Val (341) Val (148) Tyr (109) Phe (143) Pro (96) Val (63) Arg (41) Gly (51) Arg (47) Cys (ND) Ala (46) Ala (47) Leu (31) Arg (16)

- (1500) - (1500) Leu (488) Val (563) Leu (492) Asn (534) Ala (484) Asp (389) Asp (386) Gly (368) Gln (310) Val (340) Gly (78) Glu (95) Gin (84) Asp (101) Ser (69) Leu (117) Trp (ND)d Arg (66) Lys (33) Cys (ND) Glu (32) Lys (33) Glu (34) Tyr (48) Val (44) Ile (24) Val (35) Ser (50) Thr (19) Leu (28) Val (23) Ile (23) Glu (16) Tyr (20) Thr (11) Thr (11) Trp (ND) Asn (24) Gln (13) Tyr (14) Glu (8) Glu (9) ‘21~ (5) Ala (9) Ser (2) GIY (7) Leu (10) LYS (3) Lys (I) Asp (8) Ala (4) Asp (10) Ser (1) Tyr (4) CYS (ND) Val (4) Leu (ND) Lys (1) Tyr (1) Ala (4)

404 AHMAD ET AL.

1 20 40 PPYTVVYFPVRGRCAALRMLLADQGQSWKEEVVTVETWQE cT,ae - T,,--------, -TT,- 4 T3 . . . T,

T4 * 4

----- _____ CNBr, ____ -----+ +----------->-? CNBr z----------- e ___________.________-------------------.-. GCJn .__-- - --______ - _--_-______-_-____._-----.----. l

41 60 80 GSLKASCLYGQLPKFQDGDLTLYQSNTILRHLGRTLGLYG -T,- e-------T5- -

---- CNBr,-----

T6 w c-T,- -TT,- - T,, -

81 100 V 120 KDQQEAALVDMVNDGVEDLRCKY ISL IYTNYEAGKDDYVK *. T9 * *Tv* * T,, .

T 8-9 c t------------------CNBr~-----------------

121 140 160

ALPGQLKPFETLLSQNQGGKTF IVGDQ ISFA DYNLLDLLL * T,, .+ T 13

161 180 200 IHEVLAPGCLDAFPLLSAYVGRLSARPKLKAFLASP EYVN

T 13 . - T,,- +Tls+ -TT,,

201 209

LPINGNGKQ -T * 16 C

FIG. 2. Summary of amino acid sequence determination of human placental GST n. Position 104 was allelomorphic and was represented by valine as well as isoleucine. Carboxypeptidase digestion of intact GST revealed only Gln (-I. Residues identified by automated sequence analysis of intact GST * (Table III) (4 - - --- b), tryptic peptides (Table I) (4 - b), and CNBr peptides (Table III) (4 - - F) are as shown.

tion. These results demonstrated the likelihood that, in the placenta studied, there was allelomorphism at the GST x locus and that at least two genes occurred at this locus. Whether this allelomorphism represents a rare ge- netic variant or a polymorphism is presently an open question. Also, it should be mentioned that there was no evidence of post-translational modifications observed during the course of the protein structural studies. The structural methods employed, however, could not rule out all possibilities; partial phosphorylation of seryl resi- dues, for example, would not have been detected.

Although the observed allelomorphism was a small variation, it does point to the possible expression of GST isoenzymes with minor structural differences that might well be responsible for some of the heterogeneity re- ported (4,13,14). A previous report (38) has also shown an amino acid variation at position 14 in the sequence of

rat placental GST P and that of an otherwise identical protein expressed in preneoplastic nodules in rat liver. It is possible that reported differences in GST isozymes are due in part to genetic allelomorphisms, such as that observed in the placental enzyme; however, some of these differences could be related also to post-transla- tional modifications.

Comparison of GST isozymes of the same class be- tween species demonstrated about 70-80% residue iden- tity in their NHa-terminal region (data not shown). Sim- ilarly, a comparison of complete sequences of GST r between human and rat showed 86% residue identity (12). Comparison of GST ?r with the complete primary structures of different classes of human GST indicated a significantly lower degree of chemical relatedness with typically about lo-27% residue identity and 1539% chemical similarity (Table IV). Clearly, human GST ?r

STRUCTURAL ANALYSIS OF HUMAN GST rr 405

1 20 40

* l * * * * * *

x (Placenta) a (Hal, Liver) a (Ha2. Liver) p (t-lb, Liver) Micro (Liver)

41

I LRH I LNY 1 I LNY I LCY

WOE&-- - - - - - D L T[ (Placenta) a (Hal, Liver) a (Ha2, Liver) p (lib, Lwer) Micro (Liver)

- - - - - D L DYDRDQW AFYR---

---i

81 . . .

LENQTM -----fq

A (Placenta) a (Hal, Liver) a (Ha2, Liver) p (Hb, Lwer) Micro (Liver)

121 * *

-NDLENI IP-----

t- -----

n (Placenta) a (Hal, Liver) a (HaP, Lwer) p (Hb, Liver) Micro (Lwer)

161 t t * * l l * l

K (Placenta) a (Hal, Lwer) a (Ha2, Lwer) p (Hb, Ltver) Micro (Lwer)

El- - R - R F -

II F R --

- - F L V Y - L F V GARI I AYLT

--I m-w

201 220 229

x (Placenta) a (Hal, Liver) a (Ha2, Liver) p (Hb. her) Micro (Lwer)

u - . EEiRK 1%; .

FIG. 3. Alignment and comparison of complete primary structures of human GST enzymes. The primary structures of GST Hal, GST Ha& GST Hb, and the microsomal GST were taken from (43). (44), (45), and {46), respectively. Boxed residues indicate identities when compared with GST K. Residues marked by an asterisk indicate one or more identities at that position when compared with the microsomal enzyme. Predicted transmemhrane helices for GST B ( / - / ) and the microsomal GST ( ) -- -- 1) histidyl residues.

are indicated. Arrows identify putative active site

is evolutionarily related to the other human GSTs com- pared in Fig. 3 and demonstrated an overall combined

of the cysteinyl residues even among the more homolo- gous GST classes (Fig. 3).

comparison of 43% residue identity and 55% chemical The evolutionary relatedness of the microsomal GST similarity. It was striking that there was poor alignment to the cytosolic enzymes has been somewhat question-

406 AHMAD ET AL.

TABLE IV

Comparison of Complete Primary Structures of Human GST Enzymes”

Identities (%)

= (Placenta) a (Hal, liver) a (Ha& liver) g (Hb, liver) Microsomal (liver)

;;

5 15 16 17

Chemical similarities (%)*

a See Fig. 3 for references to sequences. b Chemical similarity defined as: Ile = Val = Leu, Ala = Gly, Asp

= Glu, Asn = Gin, Phe = Tyr = Trp, Arg = Lys, Asp = Am, Glu = Gln, Ser = Thr.

able and, heretofore, current thinking (1) has been that the microsomal enzyme represents a separate class of GST that probably occurred as a result of a convergent form of evolution. It is also important to note that the microsomal isozyme is considerably smaller (153 resi- dues) when compared to the other four listed human en- zymes (209-221 residues). This difference in residues was not localized to one or two regions when aligned to the other GST classes (Fig. 3) but was due to deletions throughout the alignment. However, a combined com- parison of the microsomal GST to the other four GSTs (Fig. 3) revealed a significant 27% residue identity and 34% chemical similarity. The microsomal enzyme com- pared best with GST Hb (18% residue identity) but con- siderably less with each of the other three GSTs (Table IV). These results, together with the occurrence of a hy- drophobic region in the microsomal GST similar to that found in GST K (Fig. 3), argue strongly that the micro- somal enzyme is a homolog of the cytosolic enzymes and that these two groups have diverged considerably with time.

A sequence region of significant hydrophobic charac- ter among the five human GSTs compared in Fig. 3 is the region of residues around 165 through 192 of which 52-62% of the residues were hydrophobic in the case of each enzyme. Strikingly, in the case of GST 7r (residues 167-193, Fig. 3) and the microsomal GST (residues 154- 177, Fig. 3), this region satisfied the prediction criteria for transmembrane helices using the PC/GENE pro- gram RAOARGOS. In the case of the microsomal GST this region was shifted slightly toward the NH2 terminus (residues 154-177, Fig. 3). Interestingly, a second region in the microsomal GST (residues 14-42, Fig. 3) also sat- isfied this criteria and may represent the actual mem- brane interacting site of this enzyme. Of the five com- plete human primary structures of GST compared in Fig. 3 all are cytosolic except for the microsomal enzyme.

Therefore, the occurrence of a predicted transmembrane helix in GST ir is curious. It could be that this region is a vestigial region that once functioned either as a trans- membrane helix or as a signal sequence, since, in the case of GST x, this approximate region (residues 170- 200, Fig. 3) was also predicted to be a signal sequence region using the program PSIGNAL. Taken together, these results point to the fact that, in the very least, this region of GST, especially in GST x and microsomal GST, demonstrates significant hydrophobicity that may

.

-2------J 1 40 80 120 160 200

ResidueNumber

FIG. 4. Summary of computer analyses of predicted structures for human GST ?r. (A) predicted secondary structure, (B) charged residue distribution, (C) hydrophobicity profile, and (D) hydrophilicity pro- file.

STRUCTURAL ANALYSIS OF HUMAN GST II 407

well be involved in the biological activity of the enzyme. This hydrophobic region is a reasonable candidate for the binding of substrate, for subunit interaction, or for some other potential hydrophobic interaction. It is also remarkable that this region in GST a is devoid of posi- tive charge potential at physiologic pH (Fig. 4B).

Although a definitive structure of the active site of GSTs is presently unknown, it has been suggested that the active site of GST has a glutathione binding site and a hydrophobic site for the binding of electrophilic sub- strates (39). GSTs typically bind lipophilic compounds including bile acids, steroid hormones, thyroid hormone, and a wide variety of xenobiotics (40). It has also been demonstrated that the glutathione binding site of GST is highly specific for the sulfhydryl groups of glutathione. It is therefore logical to assume that the glutathione binding site should be conserved. Recent studies from our laboratory have demonstrated (41) that modification of the histidyl residue of human GST $ (cl class) by DEPC caused inactivation of the enzyme activity. In ad- dition, preincubation of the enzyme with glutathione protected the enzyme from inactivation by DEPC. These results indicated that a histidyl residue is likely to be involved in the active site of the enzyme. In this regard, it is important to note that all of the complete structures of human GST which have been reported show the occurrence of a histidine residue (positions 166 and 174) within the above discussed hydrophobic region (Fig. 3). A histidine residue in this region is also con- served in the rat GST enzymes that have been character- ized from all three different classes (37) and the only known sequence of a mouse GST belonging to the a class also showed a histidine residue at position 158 (42). Since the imidazole group of histidine could provide a base for the protonation of the sulfhydryl group of gluta- thione and could therefore facilitate the conjugation re- action, it is reasonable to suggest that the histidine resi- due conserved within the hydrophobic region might be an essential residue at the glutathione-binding site of the enzyme.

Hopefully, these observations will provide the basis for further inquiry into the active site mechanism of this unique and ubiquitous family of transferase enzymes.

ACKNOWLEDGMENTS

The authors thank Horace D. Kelso for expert technical assistance and Ms. Angelina Mouton for preparation of the manuscript.

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