EL TEMPLO AL EXTERIOR

structures between the rat GSTM3-3 and the pig GSTP1-1

rat GSTM 3-3 pig GSTP1-1

structural elem ent

no. o f residues and range subunit subunit A B no. o f residues and range structural elem ent Dom ain 1 p i 6 ( 2 - 7 ) 6 (2 - 7 ) 5 ( 3 - 7 ) p i p -tu rn ( 1 0 - 1 3 ) ( 1 0 - 1 3 ) ( 1 1 - 1 4 ) p-turn a1 9 ( 1 4 - 2 2 ) 9 ( 1 4 - 2 2 ) 9 ( 1 5 - 2 3 ) aA p2 6 ( 2 7 - 3 2 ) 6 ( 2 7 - 3 2 ) 4 ( 2 9 - 3 2 ) p2 c/s-Pro bend ( 3 6 - 3 9 ) ( 3 6 - 3 9 ) ( 3 4 - 3 7 ) p-turn a 2 4 ( 4 3 - 4 6 ) 4 ( 4 3 - 4 6 ) 4 ( 3 8 - 4 1 ) aB

3 1 0 ( 4 9 - 5 1 ) ( 4 2 - 4 4 ) 3 10

p-turn ( 4 8 - 5 1 )

bend ( 5 4 - 5 7 ) ( 5 4 - 5 7 ) ( 4 5 - 4 8 ) p-turn

c/s-Pro bend ( 5 8 - 6 1 ) ( 5 8 - 6 1 ) ( 4 9 - 5 2 ) c/s-Pro bend p3 4 ( 6 1 - 6 4 ) 4 ( 6 1 - 6 4 ) 4 ( 5 2 - 5 5 ) p3 p4 4 ( 6 7 - 7 0 ) 4 ( 6 7 - 7 0 ) 4 ( 5 8 - 6 1 ) P4 a 3 1 1 ( 7 2 - 8 2 ) 12 ( 7 2 - 8 3 ) 12 ( 6 3 - 7 4 ) aC Dom ain II a 4 2 5 ( 9 0 - 1 1 4 ) 2 4 ( 9 0 - 1 1 3 ) 2 7 ( 8 1 - 1 0 7 ) aD p-turn ( 1 1 6 - 1 1 9 ) ( 1 1 6 - 1 1 9 ) aSa 9 ( 1 1 9 - 1 2 7 ) 8 ( 1 1 9 - 1 2 6 ) 2 4 ( 1 0 9 - 1 3 2 ) aE aS b 12 ( 1 3 0 - 1 4 1 ) 12 ( 1 3 0 - 1 4 1 ) p-turn ( 1 4 1 - 1 4 4 ) ( 1 4 1 - 1 4 4 ) ( 1 3 5 - 1 3 7 ) 3 10 bend ( 1 4 6 - 1 4 9 ) ( 1 4 6 - 1 4 9 ) ( 1 4 0 - 1 4 3 ) p-turn a 6 16 ( 1 5 4 - 1 6 9 ) 16 ( 1 5 4 - 1 6 9 ) 16 ( 1 4 8 - 1 6 3 ) aF p-turn ( 1 7 0 - 1 7 3 ) ( 1 7 0 - 1 7 3 ) ( 1 6 4 - 1 6 7 ) p-turn p-turn ( 1 7 4 - 1 7 7 ) ( 1 7 4 - 1 7 7 ) ( 1 6 8 - 1 7 1 ) p-turn a 7 1 2 ( 1 7 8 - 1 8 9 ) 1 0 ( 1 7 8 - 1 8 7 ) 1 1 ( 1 7 2 - 1 8 2 ) aG a 8 7 ( 1 9 1 - 1 9 7 ) 7 ( 1 9 1 - 1 9 7 ) 8 ( 1 8 5 - 1 9 2 ) aH p-turn ( 1 9 9 - 2 0 2 ) ( 1 9 9 - 2 0 2 ) ( 1 9 6 - 1 9 9 ) p-turn c/s-Pro turn ( 2 0 4 - 2 0 7 ) ( 2 0 4 - 2 0 7 ) p-turn ( 2 0 9 - 2 1 2 ) ( 2 0 9 - 2 1 2 )

Data from Reinemer e t a i, 1 991 and Ji a t a i, 1 9 9 2 . a i : a-helix 1 ; oA: a-helix A; p i : p-strand 1; etc. 310: 310-helix. Boldfaced numbers indicate differences. Numbers in parentheses are sequence numbers.

specific m utagenesis, and the results are consistent with the crystallographic data, which suggest that the tyrosine plays an essential role in stabilising the thiolate anion of G SH through hydrogen bonding (S tenberg et al., 1 9 9 1 a ) (M an o h aran et a!., 1992) (Liu et a!., 1992) (W ang e t a!., 1992) (Kolm e t a!., 1 9 9 2 ) (Kong et a!., 1992a) (Kong e t a!., 1 9 9 2 b ). Tyrosine 115 which is located in domain II of the protein was found to play a direct role of catalysis in rat G S T M 3 -3 (Johnson e ta !., 1993).

1 . 4

GST gene structure and regulation

As mentioned above, the cytosolic G S Ts have been grouped into four classes. Although they all have the basic capability of catalysing nucleophilic attack of G SH on electrophilic compounds, different isoenzymes in each class have m arked d iffe re n c e s in substrate sp e c ific ity and h a v e d is tin g u is h a b le physicochem ical and immunological properties that reflect the differences in their primary structures. The cytosolic G S Ts are inducible by a num ber of drugs, xenobiotics, food additives, and natural d ietary com ponents. The compounds which have been shown to increase hepatic levels of G S T include p hénobarbital, 3-m ethylcholanthrene, propylthiouracil, fran s-stilb en e oxide, and antioxidants such as butylated hydroxyanisole (B H A ). The increase in specific G STs is due to increases in levels of m RNA. The mechanisms that are responsible for transcriptional activation of the G S T s are currently being addressed (Pickett, 1987) (Pickett and Lu, 1 9 8 9 ) (R ushm ore et al., 1991) (D a n ie l, 1 9 9 3 ).

M am m alian GSTs have been investigated most extensively. At least eight full-length cD N A (m RNA) and three genom ic DMA for human G S T subunits, twelve cDNAs and six genomic DMAs for rat subunits, seven cDNAs for mouse subunits, two cDNAs for rabbit subunits, one genomic DMA for hamster subunit.

one cDNA for pig subunit and one cDNA for chicken subunit have been isolated and sequenced, respectively (Pickett, 1987) (Pickett and Lu, 1989) (Tsuchida and S ato, 1992) (D aniel, 1993).

1 .4 .1 Alpha class GST genes

Although multiple forms of human alpha class G S T enzym es have been purified, only two cDNAs and their corresponding genes, GSTA1 and G STA2, have been cloned and sequenced (Tu and Qian, 1986) (Board and W ebb, 1987a) (R hoads et al., 1987) (Chow et al., 1988) (R ozen e t al., 1992) (KIone et al., 1992) (Rohrdanz et al., 1992). The GSTA1 gene spans a region of about 12 kb and the GSTA2 gene is about 13 kb in length. Both of the genes have seven exons encoding polypeptides containing 2 22 am ino acids with a M r of 2 6 ,0 0 0 . A comparison of the structure of these two genes shows substantial sequence identity and deduced amino acid sequences identity (95% ). A 209 bp S'-flanking region of the GSTA1 gene has been found to contain a TA TA box (TATAAA) at position -26 to -21 and two potential C A T boxes at positions -4 8 to -44 (C T A T T ) and at -8 0 to -75 (C C T A A C T ). In addition. An G-rich sequence (TG G G A G G G A A C ) upstream of the TATA box at position -5 7 to -48 has been identified as a potential binding site for the transcription factor 1 ( S p l) . Upstream of this region, a potential binding site for the nuclear factor 1 (N F 1), and a hepatocyte nuclear factor 1 (H N F1) which is a liver-specific transcription factor w ere also found (Klone et al., 1992). The preferential expression of alpha class G S T in liver may be due to the presence of these regulatory elem ents, presumably involved in the constitutive and hepatocyte-specific expression. In the mouse GSTA1 (Ya) gene, a xenobiotic responsive elem ent (X R E ), also referred to as electrophilic-responsive elem ent (E p R E ), are located betw een

contains two core sequences (T G A C — G C ) of an activator protein-1 (A P -1) binding site. This regulatory element was found to be responsive to both planar arom atic compounds, e.g. p-naphthoflavone, 3 -m e th y lc h o la n th re n e , 2 ,3 ,7 ,8 - tetra c h lo ro d ib e n zo -p -d io x in (T C D D ), and electrophilic com pounds, e.g. tert- b u tylh yd ro q u in o n e, d ie th y lfu m a ra te , and f r a n s - 4 - p h e n y l- 3 - b u t e n - 2 - o n e (Friling et al., 1990). Sim ilar regulatory elem en t was also found in the S'- flanking region of rat GSTA1 (Ya) gene and defined as an antioxidant-responsive elem ent (ARE), because of its activation by a phenolic antioxidant such as tert- butylhydroquinone (Figure 1.4) (Rushm ore et a!., 1990).

1 .4 .2 Mu class GST genes

Four human cDNA from the mu class G S T have been cloned and sequenced: G S T M Ia (DeJong e t a ! ., 1988a), G S T M Ib (S eidegârd e t a ! ., 1 9 88), G S T M 2 (Vorachek e ta !., 1991),and G S T M 3 (Cam pbell e t a ! ., 1990). The G S T M Ia and G S T M Ib cDNAs have been identified from liver and lymphocytes and both have an open reading frame of 654 bp,^encode a polypeptide of 2 17 amino acids with a Mr of 26,600. The G S T M Ia and G S T M Ib were found to differ by a single base pair in the protein coding region to encode at residue 172, a lysine in G S T M Ia or asparagine in G S T M Ib . The G STM 2 cDNA clone was isolated from myoblasts and also encodes a polypeptide of 217 amino acids. G S T M Ia /b and G S T M 2 share 8 4 .8% amino acid sequence? identity. The G S T M 3 -3 isoenzyme was identified in brain and testes and cDNA clones for this form w ere sequenced. The G S T M 3 subunit comprises 225 amino acids with a Mr of 26,3 0 0 and shows 72% deduced amino acid sequence homology with the predicted am ino acid sequence of G S T M la /b . There is no information about the structure and regulation of expression of the human mu class gene. The rat mu class G S T M 2 gene spans about 4.4 kb and contains eight exons. A clone containing 4 0 4 bp of the 5'-flanking

Figure 1.4 Regulatory elements of the rat GSTA1 gene. -908 -899 -867 -857 XRE Ah receptor TCDD Basal expression Ah receptor transcription initiation -722 -682 ARE Ah receptor P 4 5 0 la-1 p-naphthoflavone t-butylhydroquinone

XRE, xenobiotic-response elem ent-like sequence; ARE, antioxidant-response elem ent;

region has been found to contain two C CAAT boxes (CCAAT) at nucleotide positions -84 to-80 and -76 to -72, as well as a putative TA TA box (TATCA) at nucleotide position -2 8 to -2 4 that is com m only found in prom oters recognized by eukaryotic RNA polymerase II (Lai et al., 1988). The hamster mu class GSTM1 gene contains nine exons dispersed over a 6 .3 kb region. It has been found to possess a typical eukaryotic promoter structure in the S'-flanking region of the gene. Within 4400 bp of flanking sequence analysed, two C CAAT boxes and a presumed TATA box sequence (TCATAAA) w ere identified at position of 27 bp upstream of the transcription initiation site. Im m ediately upstream of the CAAT boxes is a potential ras response elem ent (A G A C TC T) at nucleotide position -126 to -120. A glucocorticoid response elem ent (G R E ) was also localized in the S'- flanking region between nucleotides -3S3 and -2 3 9 although no classic G R E sequence (TG TTC T) was identified within this region (Fan e ta !., 1992).

1 .4 .3 Pi class GST gene

Unlike the alpha and mu classes of G S T subunits that are encoded by gene families, human pi class G S T subunit is encoded by a single gene (Kano e t a/., 1987) (Cowell et a/., 1988) (Morrow e t a/., 1 9 89). The human G S T P subunit contains 209 amino acids with a My of 2 3 ,0 0 0 . The gene spans about 2.8 kb and has seven exons. O ver 2200 bp of the S'-flanking region of the G S T P gene has been characterised, it contains four putative transcription regulatory motifs. A TATA box is located at 29 bp upstream from the start site of transcription. Two S p l recognition sequences (G G G C G G ) are located between nucleotides -46 and -4 1 , -56 and -51. An activator protein-1 (A P -1 ) site (T G A C T C A ), originally identified responsive to the 12 -0 -te tra d e c a n o y l-p h o rb o l-1 3 -a c e ta te (TP A ), is located at nucleotides between -69 and -6 3 (Morrow e t a/., 1989) (Morrow e t a /., 1990b ). Of interest is the finding that the rat genom e contains several

processed pi class GST pseudogenes. These pseudogenes have no introns and are thought to arise from reverse transcription of m R N A and insertion of the resultant cD N A into the genome. The 5'- and 3'-flanking sequences of these pseudogenes are completely different from the active rat g ene, and these pseudogenes are not transcriptionally active (Okuda et al., 1987).

1 .4 .4 Theta class GST gene

Two theta class GSTs have been found in human liver which probably represent only about 0.01% of the soluble liver protein (M a y e r a t al., 1991) (Hussey and Hayes, 1992). Partial analysis of the primary structures of these two G STs show they are related to three rat theta class GSTs, and a dog theta class G S T described to date (Hiratsuka at al., 1990) (M eyer a t al., 1991) (Igarashi a t al., 1991). Neither of the human theta class G S T have been cloned. However, two cDNAs encoding rat Theta class G ST subunits have been cloned and sequenced. One has an open reading frame of 732 bp encoding a polypeptide of 244 amino acids residues with a of 27,311 (Ogura at al., 1991). Another has an open reading fram e of 240 amino acids with a M f of 2 7 ,0 0 0 (R am ble and Taylor, 1992). They share about 58% nucleotide sequence identity.

1 .4 .5 Microsomal GST gene

The cDNA clone of human microsomal G S T encodes a 154 amino acid polypeptide with a of 17,450. It shares 7 7 % nucleotide similarity and 83% amino acids similarity with that of rat (DeJong a t al., 1988b ). H ow ever, the microsomal G S T possesses only limited sequence homology with the cytosolic G ST