Comp. Biochem. Physiol. Vol. 98B, No. 4, pp. 511-515, 1991 0305-0491/91 $3.00 + 0.00 Printed in Great Britain © 1991 Pergamon Press pie

THE ISOLATION AND CHARACTERIZATION OF THE MAJOR GLUTATHIONE S-TRANSFERASE FROM THE

SQUID LOLIGO VULGARIS

JONATHAN HARRIS, BRIAN COLES, DAVID J. MEYER and BRIAN KETTERER Cancer Research Campaign Molecular Toxicology Research Group, Department of Biochemistry,

University College and Middlesex School of Medicine, London WlP 6DB, UK (Tel: 071-380 9309)

(Received 17 August 1990)

Abstract--1. The major glutathione S-transferase ((3ST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8.

2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane.

3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs.

4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.

INTRODUCTION MATERIALS AND METHODS

In mammals glutathione S-transferases (GSTs) (EC 2.5.1.18) are a supergene family of multifunctional enzymes, which play an important part in dctoxifica- tion by catalysing conjugation of glutathione (GSH) with clectrophili c metabolites of natural or man- made hydrophobic chemicals, some of which are cytotoxins and carcinogens. GSTs may also be im- portant in preventing oxygen toxicity both by their peroxidase activity towards lipid hydroperoxides and catalysis of the GSH conjugation of hydroxy- alkenals, both of which are products of lipid peroxi- dation. They also bind a number of non-substrate ligands including haem, bilirubin and steroid hor- mones. In addition to detoxification, GSTs have a synthetic function, catalysing the formation of pros- taglandins and leukotrien¢ C (Ketterer et al., 1988).

Interspecies relationships in gene family structure and function have been studied in some detail in rat, mouse and man and a high degree of conservation observed, enabling sub-classification of the GSTs into g, # and ~ classes. A similar comparison has been carried out on two schistosome species, but otherwise detailed information is limited. There are complete sequences from the prokaryote Methylobacterium sp; the plant Zea mays and the insect Drosophila melanogaster and partial (N-terminal) sequences for GSTs from the cestodes Moniezia expansa and Sehistocephalus solidus. In the present paper the principal GST from the common squid, Loligo vulgaris, a cephalopod mollusc, has been isolated. L. vulgaris is of interest because its phylogenetic relationship to other organisms, so far studied, is distant and also because it has a very active aerobic existence, it may need powerful protection against oxygen toxicity. Its N-terminal sequence and enzy- mological properties compared with data available from other species is discussed (Ketterer et al., 1988; Pickett et al., 1984).

Chemicals All chemicals, which were commercially available, were

used without further purification, with the exception of 1-chloro-2,4-dinitrobenzene (CDNB), which was recrystal- lized from ethanol. Ghitathione and ethacrynic acid were obtained from Sigma Chemical Co. (St Louis, MO, USA), p-nitrobenzyl chloride (PNBC), trans-4-phenyl-3-buten-2- one and 1,2-dichloro-4-nitrobenzene (DCNB) were ob- tained from Aldrich Chemical Co. (Milwaukee, WI, USA); 1,2-epoxy-3-(p-hitrophenoxy)propane (EPNP) was ob- tained from Eastman Kodak Company (Rochester, NY, USA). 4-Hydroxynon-2-enal was the kind gift of Professor Bengt Mannervik, University of Uppsala, Sweden and linoleate hydroperoxide was prepared according to O'Brien (1969). Immunochemicals were supplied by Bio-Rad (Rich- mond, CA 94801, USA) and (3ST antibodies were supplied by Medlabs (Dublin, Eire).

Enzyme assays GST enzyme activities were determined using the sub-

strates listed below according to Habig et al. (1974) and glutathione peroxidase activity was determined according to Prohaska and Ganther (1976).

Purification and characterization of the principal glutathione transferase

L. vulgaris was obtained frozen from Wainwright and Daughter, London. After thawing the digestive gland was dissected out and homogenized in 10 mM Na phosphate buffer pH 7.0 containing 0.15 M KC1, 2 mM dithiothreitol, 25#M phenyl methyl suiphonyl fluoride, I mM EDTA and I #g/ml leupeptin. The soluble supcmatant was pre- pared by centrifugation for I hr at 80,000 g and the GSH transferase fraction was obtained there from by GSH-agarose affinity chromatography (Simons and Van der Jagt, 1977).

The affinity column eluate was transferred into 10 mM ethanolamine-HCl pH9.1 by gel filtration (PD-10, Pharmacia-LKB, Uppsala, Sweden) and loaded onto a mono Q HR 5/5 anion exchange column (Pharmacia-LKB) pre-equilibrated with ethanolamine buffer. This was eluted

511

512 JONATHAN HARRIS et al.

T 0.2

l

0 I I I 5 0 z 0 310 410 5 0 Time {rain)

Fig. 1. Reverse phase HPLC analysis o f L. vulgaris GSTs. GSH-alfmity column purified GST fraction of L. vulgaris digestive gland was analysed by RP-HPLC using a Dynamax C18 300A column

(300 x4 .6 ram) and a 35-55% acetonitrile gradient containing 0.1% trifluoroacetic acid.

with a linear gradient of NaC1 (0-0.5 M) at 0.35 ml/min, protein being monitored by absorbance at 280 nm and enzymic activity determined with 1-chloro-2,4-dinitro-

benzene (CDNB) as substrate. The principal protein com- ponents and the major peak of enzymic activity coincided and were judged to be pure by SDS-polyacrylamide gel

A280 i 1

f

J J

J J

f

J J

J I oJ

J f

f f

f i f

f 2.0

I I l I I 0 +0 Vol. of el2°uent (+mlJ--+ 3 0 40

Fig. 2. Separation of GSTs from L. vulgaris digestive gland by anion exchange F'PLC. The GST fraction obtained by GSH--agrarose affinity chromatography was subject to anion exchange FPLC as described in the text. Protein elution was monitored from A280 (solid line); the dashed line denotes salt concentration.

Squid GST 513

electrophoresis (PAGE) according to Laemmli (1970), gel isoelectric focusing carried out on preeast PAG plates (Pharmacia-LKB) in the pH range 3-10 as described by Kispert et al. (1989), analytical reverse phase (RP) HPLC by the method of Ostlund Farrants et al. (1987) using a Dynamax 300A, 300 x 4.6mm octadecasilane column (Rainin Instrument Co., Woburn, MA, USA) and amino acid sequence determined using an Applied Biosystems 470A gas phase sequencer.

The use of the above techniques also enabled the determi- nation of mol. wt, isoelectric point, retention time of RP-HPLC and the sequence of the first 40 amino acids of the N-terminus, respectively.

The immuno-cross-reactivity of the major L. vulgaris GST was tested by the immunoblot technique of Towbin et al. (1979). Twenty #1 of a 0.1 mg/ml solution were immobi- lized by dot-blotting onto nitrocellulose and probed with polyclonal antibodies raised to rat GSTs 1-1, 3-4, 7-7 and 8-8 and human GSTs a,/~ and n, all at a dilution of 1 : 500. A second anti-rabbit antibody/alkaline phosphatase conju- gate was used to detect bound anti-GST antibodies.

RESULTS

The CDNB-GST activity in the cytosol of L. vulgaris digestive gland was four times higher (5.5 pmol/min/mg total protein) than a soluble super- natant of similar tissue content from the rat liver (1.4/~moi/min/mg total protein). Approximately 96% of this CDNB-GST activity was recovered from the affinity column in the GSH elutate. On analysis by RP-HPLC, this fraction yielded one major (90%) and one minor (10%) protein peak (Fig. 1). Anion-exchange FPLC yielded a major GST fraction separated from some minor components (Fig. 2). The major isoenzyme migrated as a single band with an apparent pI of 6.8 on gel isoelectric focussing and as a single band of apparent M~ 24,000 on SDS-PAGE (see Fig. 3a and b). It was assayed with a number of substrates and the activities obtained are recorded in Table 1.

Twenty # g of the major isoenzyme from the anion- exchange step were desalted and concentrated by RP-HPLC. A single peak was obtained which co- eluted with the major peak from the GSH-agarose affinity eluate. N-terminal amino acid sequencing identified the first 40 residues of the protein, with the exception of residue 35. The sequence is compared with that of rat GST subunit 1 (Pickett et al., 1984) and the N-terminal sequence of a GST-like protein which is a lens crystallin of the squid Ommastrephes sloanei pacificus (Tomarev and Zinovieva, 1988) as shown in Fig. 4.

DISCUSSION

L. vulgaris is a cephalopod, and as such occupies a position of singular isolation in evolution, having anatomy and behaviour that is more complex than those of other non-cephalopod molluscs. It has a diet of fish and is highly adapted for raptorial feeding, swimming powerfully and forming great shoals in open water (Worms, 1983). The present study describes the major GST from L. vulgaris' digestive gland, a digestive/storage organ, functionally analagous to the mammalian liver (Boucand-Camou and Rodoni, 1983), showing it to be a homodimeric enzyme of sub-unit M r 24,000 and pI 6.8. Its enzymic activities

are compared with representatives of the u, # and gene families from the rat and also with the major GST of the parasitic helminths Schistosoma mansoni

(~)

(b)

,°41 5.92~

Fig. 3. (a) Analysis of major L. oulgaris GST by SDS-polyaerylamide-gel eleetrophoresis (SDS-PAGE). SDS-PAGE was performed according to Laemmli (1970). Lane 1, mol. wt markers; lane 2, GST markers from rat liver; lane 3, purified GST from L. vulgaris digestive gland. (b) Determination of the pI of the major L. vulgaris GST. Isoelectric focusing was performed at 10°C on pre-east gels as described in the text. Lane l, standard proteins of known pl; lane 2, FPLC purified major GST from L. vulgaris

digestive gland.

514 JONATHAN HARRIS et al.

Table 1. GST activities of L. vulgaris digestive gland major GST in comparison to rat isoenzyrnes from % # and n families; S. mansoni major GST, M. expansa GST EII and S. solidus GST SII

Activity (pmol/min/mg protein)

Source of enzymes Rat isoenzymes ~

L. vulgaris 1-1 3-3 7-7 S. mansoni 2 M. expansa 3 S. solidus 4 Substrate Major GST (~) (#) (n) GST GST Eli GST SII l-Chloro-2,4-dinitrobenzene 237 40 50 20 124 25.8 6.7 1,2-Dichlor o-4-nitrobenzene 0.6 0.15 8.4 < 0.05 nd nd nd p-Nitrobenzyl chloride 2.6 0.1 11.4 nd nd nd nd Ethaerynic acid nd 0.3 0.4 4.0 0.8 0.29 0.52 1,2-Epoxy-3-(p-nitrophenoxy)propane nd 0.7 0.2 1.0 <0.5 tad nd trans-4-Phenyl-3-bumn.2-one tad 0.1 0.1 0.02 0.4 0.13 0.44 4-Hydroxynon-2-enal 17.7 2.6 2.7 nd nd 2.07 nd Cumene hydroperoxide 1.2 1.4 0.1 <0.01 0.4 0.10 nd Linoleate hydroperoxide 3.1 3.0 0.2 1.5 5.5 nd nd nd, not detectable. lData from Ketterer et al. (1988). 2Data from Taylor et al. (1987). 3Data from Brophy et al. (1989a). 4Data from Brophy et aL (1989b).

N-terminal sequence • ee • • •

Ommastrephes pacif icus MPNYTLYYF NGRGR~EMIC~GVQYTDARF_EFNEWDKY

Lo!i~o vulKaris HI(YTLHYFTLKARAE~LPAFLVANGEEFI(DRIEFSEXDNIKA ee • @e@

rat GST l HSGKPVLHYFNARGRME_CIRWI/AAAGVEFDEKFIQSPF~_LEKL

Fig. 4. A comparison of the N-terminal amino acid sequence of the major GST of L. vulgaris digestive gland with that of the rat subunit 1 (Pickett et al., 1984) and the lens erystallin of the squid O. pacificus (Tomarev and Zinovieva, t988). Identical amino acids are underlined and similar amino acids are dotted.

Indicates a space inserted to maximize similarity and X indicates an unknown amino acid.

(Taylor et al., 1988) and Moniez ia expansa (Brophy et al., 1989a) and Schistocephalus solidus (Brophy et aL, 1989b). Its activity with CDNB is one of the highest yet recorded, and although it has a wide range of activities for a single enzyme, it does not express activity towards all the model substrates used to characterize the multiple isoenzymes of the mammals GSTs. The xenobiotic substrates which Loligo might normally encounter are un- known. Its activity with substrates which might arise endogenously from oxygen toxicity, namely linoleic acid hydroperoxide and 4-hydroxynon-2-enal, is noteworthy, particularly in view of the Lol igo 's aerobic existence with its a t tendant exposure to oxy- gen toxicity (Wells, 1983) and the presence of high levels of polyunsaturated fatty acids in its fins (Shchepkin et aL, 1976). GSTs from the cestodes M. expansa and S. solidus which live a much less active life are notable for their low activity with these substrates.

Structural data for the major L. vulgaris GST are limited to the first 40amino acids from the N-terminus. This sequence has a degree of identity with that of rat subunit 1 (~ family), but much less with that of subunit 4 0z family) and subunit 7 (~r family). It may be significant that high activities towards hydroperoxides and alkenals is a property of the c¢ family in mammals. This cephalopod GST has no epitopes in common with known mammal ian GSTs. On the other hand the lens crystallin from another squid, O. pacificus (Tomarev and Zinovieva, 1988) shows 55% identity to rat subunit 1 over its entire length but has negligible CDNB-GST activity. The lens is believed to recruit

its crystallins from proteins with high solubility and thermodynamic stability and these are character- istics of many of the GSTs. Apparently a gene similar to a mammal ian GST transferase gene exists in this squid, but the product does not function as a GST. The lens crystallin of L. vulgaris has yet to be determined.

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