Membrane-associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG)
A Widespread Protein Superfamily

PER-JOHAN JAKOBSSON, RALF MORGENSTERN, JOSEPH MANCINI, ANTHONY FORD-HUTCHINSON, and BENGT PERSSON

Department of Medical Biochemistry and Biophysics and Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden; and Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada

	THE MEMBERS AND THEIR RELATIONSHIP

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A widespread superfamily designated MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) has been defined according to enzymatic activities, sequence motifs, and structural properties (1). The family consists of six human proteins including 5-lipoxygenase-activating protein (FLAP), leukotriene C₄ (LTC₄) synthase, microsomal glutathione S-transferase 1 (MGST1), MGST2, MGST3, and MGST1-like 1 (MGST1-L1). In addition, several nonmammalian members have been identified, including those from plants (Arabidopsis thaliana, Oryza sativa, and Ricinus communis), fungi (Aspergillus nidulans), and bacteria (Synechocystis sp. [SynMGST], Escherichia coli, and Vibrio cholerae).

On the basis of the multiple sequence alignments of these 13 proteins (1) the evolutionary tree (Figure 1) (2, 3) clearly shows that the MAPEG family can be subdivided into four subgroups. The first subfamily consists of the members FLAP, LTC₄ synthase, and MGST2 and the second subfamily consists of MGST3 together with the members found in plants and fungi. The third and fourth subfamilies are composed of the proteins identified in bacteria (E. coli and V. cholerae) and MGST1 and MGST1-L1, respectively. SynMGST is presently positioned between the top two groups and cannot be significantly grouped to any of these two subfamilies. This is in line with the hypothesis that SynMGST might constitute an ancestral form to the group I and II proteins. However, the four distinct subfamilies make functional interpretations and speculations possible, where the proteins involved in leukotriene biosynthesis (FLAP and LTC₄ synthase) form a subfamily with MGST2 (which also catalyzes the biosynthesis of LTC₄; see below). The plant and fungus proteins form a subgroup with human MGST3, the bacterial proteins form a group on their own, and the human MGST1 and MGST1-L1 proteins constitute a fourth subgroup possibly involved in cytoprotection (see below).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Evolutionary tree of the MAPEG family. The tree is calculated with CLUSTAL W (2), on the basis of the multiple sequence alignments reported in Reference 1, and drawn with PHYLO_WIN (3). Branch lengths are proportional to the sequence dissimilarity, with the scale bar giving the distance corresponding to 8%. Numbers at branching points represent bootstrap values (2) of 1,000 trials. The brackets show the four subfamilies, denoted I-IV. These four groups are clearly separated with bootstrap values above 933/1,000. The exact branching order of the proteins of the first subfamily cannot be judged at the present stage (bootstrap value, 624/1,000). It is also too early to assign the exact branching order of the four subfamilies.

	MAPEG MEMBERS AND LEUKOTRIENE BIOSYNTHESIS

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5-Lipoxygenase-activating Protein

Leukotrienes are important mediators of inflammation (4). 5-Lipoxygenase catalyzes the formation of leukotriene A₄ (LTA₄) from arachidonic acid. In this reaction, 5-hydroperoxyeicosatetraenoic acid (5-HPETE) forms an intermediate metabolite and is also important for the regulation of 5-lipoxygenase activity (5). Calcium induces membrane association of soluble 5-lipoxygenase (6) and incubation of intact blood leukocytes with the calcium ionophore A23187 results in a calcium-dependent translocation of 5-lipoxygenase (7, 8). In 1989, MK-886 was reported to be an efficient leukotriene biosynthesis inhibitor (9). It was demonstrated that MK-886 inhibited leukotriene biosynthesis in intact human polymorphonuclear leukocytes stimulated with the calcium ionophore A23187 but not in corresponding homogenates or purified 5-lipoxygenase. The mechanism of action was first believed to involve inhibition of the 5-lipoxygenase translocation from the cytosol to the membrane (10, 11) and as a consequence, the hypothesis at that time was that FLAP constituted a membrane-anchoring protein for 5-lipoxygenase, a function that could be blocked by MK-886. However, several studies demonstrated lack of correlation between leukotriene biosynthesis inhibition by MK-886 and 5-lipoxygenase translocation, which is why this hypothesis has been questioned (12, 13). For the more recent results and ideas regarding the process of translocation and intracellular localization of 5-lipoxygenase, the reader is referred to articles in this journal supplement that deal with 5-lipoxygenase (e.g., see References 14 and 15).

The cellular target for MK-886 was identified with a radioactive photoaffinity probe that specifically detected an 18-kD protein only in cells capable of producing leukotrienes. The protein was then purified by affinity chromatography and sequence data were obtained (16). This allowed for the cloning and expression of this protein in osteosarcoma cells (17). Only cells expressing both FLAP and 5-lipoxygenase were found to synthesize leukotrienes after stimulation with the calcium ionophore A23187, and MK-886 was able to block the cellular leukotriene biosynthesis (17). The MK-886-binding protein was thus termed 5-lipoxygenase-activating protein (FLAP) (16, 17). The subcellular location of FLAP was investigated by immunogold labeling of cryosections from resting and ionophore A23187-activated human leukocytes and the protein was found to reside on the nuclear envelope in both resting and activated cells (18).

The hydrophobicity profile of FLAP indicated three hydrophobic regions, separated by two hydrophilic parts (17). Mutagenesis studies have shown that there are several amino acid residues essential for the binding of MK-886 analogs within the first hydrophilic region (19), but also that Tyr-101 in the second hydrophilic region is critical for inhibitor binding (20). Important for the present view on the function of FLAP was the demonstration that FLAP binds a photoaffinity analog of arachidonic acid. This binding was inhibited by both arachidonic acid and MK-886 (21). Furthermore, in Spodoptera frugiperda (Sf9) cells expressing both FLAP and 5-lipoxygenase, FLAP stimulated the use of arachidonic acid by 5-lipoxygenase and increased the efficiency with which 5-lipoxygenase catalyzed the conversion of 5-HPETE to LTA₄ (22). These studies were in agreement with results obtained with human polymorphonuclear leukocytes stimulated with calcium ionophore in the presence of zileuton, a direct 5-lipoxygenase inhibitor (23), which protected 5-lipoxygenase from self-inactivation (7, 8). In these cells, 5-lipoxygenase that accumulated in the membrane fraction was more efficient in converting 5-HPETE into LTA₄ compared with the cytosolic enzyme (23). In addition, the membrane-associated 5-lipoxygenase could metabolize alternative substrates such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 15(S)-HETE to 5,12-DHETE and 5,15-DHETE, respectively. These findings have also been confirmed and extended with Sf9 cells after dual infection with recombinant baculovirus producing FLAP or various FLAP mutants and 5-lipoxygenase (20). It was shown that FLAP causes a 190-fold increase in the conversion of 12(S)- HETE to 5,12-DHETE when coexpressed with 5-lipoxygenase. In conclusion, the nuclear envelope integral membrane protein FLAP causes increased conversion of arachidonic acid to LTA₄ by 5-lipoxygenase. FLAP also changes the substrate specificity of 5-lipoxygenase. Although the exact mechanism of action remains unclear, it is presently hypothesized that FLAP acts as a substrate provider for 5-lipoxygenase.

LTC₄ Synthase

The cysteinyl-leukotrienes (LTC₄, LTD₄, and LTE₄) are important mediators of airway obstruction. LTC₄ synthase specifically catalyzes the conjugation of leukotriene A₄ with glutathione. LTC₄ synthase was successfully purified from THP-1 cells as an 18-kD membrane-associated protein, active as a homodimer (24). Thereafter, two groups independently cloned and characterized the gene product (25, 26). The deduced amino acid sequence demonstrated that FLAP and LTC₄ synthase are homologs with 31% amino acid sequence identity. Also, the LTC₄ synthase polypeptide displayed a similar hydropathy pattern compared with FLAP. The genes encoding FLAP and LTC₄ synthase are different in size but have the same intron/exon organization consisting of five small exons and four large introns (27, 28). In a study in which several site-directed mutants of LTC₄ synthase were investigated, it was concluded that Arg-51 functions as a proton donor for the opening of the LTA₄ epoxide and Tyr-93 as a base for the formation of the thiolate anion of glutathione (29). For more information on LTC₄ synthase the reader is referred to the review article in this supplement that describes this enzyme (30).

MGST2 and MGST3

To investigate whether FLAP and LTC₄ synthase were members of a larger protein family, the GenBank database was searched for sequences homologous to FLAP and LTC₄ synthase. These computer-assisted searches of the expressed sequence tag (EST) databases first uncovered an EST clone that, after cDNA sequencing, protein expression, and enzyme characterizations, clearly represented a novel protein homologous to both FLAP and LTC₄ synthase. The protein was termed MGST2. Soon after, another EST clone was identified after database searches using the polypeptide sequence of MGST2. After characterization this enzyme was named MGST3.

MGST2 and MGST3 were produced in a baculovirus/insect cell expression system (31, 32). The microsomal fractions from insect cells infected with virus encoding either MGST2 or MGST3 were both found to catalyze the conjugation of LTA₄ with reduced glutathione, thus leading to the formation of LTC₄. The apparent K_m for LTA₄ was higher for MGST2 (41 µM) compared with that of LTC₄ synthase (7 µM). The K_m for LTA₄ was not determined for MGST3. Human MGST2 protein expression was analyzed by a combination of Western blot and LTC₄ synthase activity assays, and found predominantly in liver microsomes, endothelial cells, and more sparsely in lung membranes (33). On the other hand, LTC₄ synthase was the predominant enzyme responsible for LTC₄ production in human lung membranes, platelet homogenates, and eosinophilic HL-60 cells. The role of MGST2 and MGST3 in LTC₄ production must be further investigated. In particular, their role in LTC₄ production in endothelial cells (34) and hepatocytes (35) is interesting and should be further examined. In contrast to LTC₄ synthase, both MGST2 and MGST3 possess a wider substrate specificity and also catalyze other type of reactions (see below).

	MAPEG MEMBERS AND DETOXIFICATION

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MGST1

Microsomal glutathione S-transferase 1 (MGST1) was isolated in 1982 (36). This 17-kD enzyme has a wide substrate specificity, and is widely expressed. Liver microsomes are an exceptionally rich source (3% of protein being MGST1) (36). Among the many substrates for this enzyme are halogenated arenes such as 1-chloro-2,4-dinitrobenzene (CDNB) as well as various polyhalogenated unsaturated hydrocarbons (37). In contrast to certain cytosolic glutathione S-transferases, LTA₄ and other epoxides are poor substrates for MGST1 (38, 39). This enzyme should therefore not contribute to LTC₄ biosynthesis. In addition to the glutathione S-transferase activity, MGST1 also catalyzes a glutathione-dependent reduction of certain lipid hydroperoxides such as organic hydroperoxides, fatty acid hydroperoxides, and phospholipid hydroperoxides (40, 41). These reactions may be of importance for protection against lipid peroxidation under conditions of oxidative stress (42). An interesting property of MGST1 is the effective binding of LTC₄ (43). The role of the LTC₄ binding is not known and requires further studies but may suggest a storage function of the enzyme. This latter topic is further discussed in the article dealing with cysteinyl-leukotriene (cys-LT) receptors (44).

MGST2 and MGST3

Both MGST2 and MGST3 were also found to catalyze the glutathione-dependent reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (32). The apparent K_m of 5-HPETE was determined to be approximately 7 µM for MGST2 and 21 µM for MGST3. Neither FLAP nor LTC₄ synthase, when produced in the same expression system (Sf9 cells), possessed peroxidase activity. Interestingly, as opposed to MGST2, MGST3 was not found to use 1-chloro-2,4-dinitrobenzene (a general GST substrate). In conclusion, these enzymes clearly catalyze both glutathione transferase as well as glutathione peroxidase reactions. Therefore, both MGST2 and MGST3 may function in detoxification of xenobiotics and/or protection against harmful metabolites generated during oxidative stress. Their precise role in physiology remains to be elucidated.

MGST1-L1

The protein MGST1-L1, as well as other proteins and enzymes involved in redox regulation, have been reported to be under the control of p53 (45). The protein was also independently identified as an EST clone, and the full-length sequence was deposited in the GenBank database (P. J. Jakobsson, J. A. Mancini, and A. W. Ford-Hutchinson, unpublished results, 1997). This novel homolog of MGST1 is referred to as MGST1-L1 (microsomal glutathione S-transferase 1-like 1). The finding that MGST1-L1 was upregulated after p53 expression in a colorectal cancer cell line (45) suggests an important biological function possibly associated with cancer or apoptosis.

	SUMMARY

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The members of the MAPEG superfamily have been aligned and found to be distantly related (1), with a common pattern of hydropathy (1). Figure 2A shows the multiple sequence alignments of the human members and Figure 2B the corresponding superimposed hydropathy profiles. The alignment in Figure 2A demonstrates a total of six strictly conserved residues. The Arg-51 in LTC₄ synthase has been suggested to function as a proton donor for the opening of the LTA₄ epoxide (29). This arginine is found in all but the FLAP sequences, in accordance with the observation that FLAP has no known enzyme activity. Also, the Tyr-93 in LTC₄ synthase has been suggested to function as a base for the formation of the thiolate anion of glutathione. This tyrosine is not conserved in MGST1 or MGST1-L1. Table 1 summarizes some other properties of the individual human proteins. They are all of the same size, ranging from 147 to 161 amino acids. Only FLAP differs, in that its isoelectric point is more neutral than that of the other, more basic proteins. The genes encoding these proteins all reside on different chromosomes (when known) (Table 1).

View larger version (33K): [in this window] [in a new window]   View larger version (36K): [in this window] [in a new window]   Figure 2.   (A) Multiple sequence alignment of the human members within the MAPEG family. Alignment is based on comparisons of all MAPEG members (1). Positions conserved in all six proteins are drawn against a black background whereas those conserved in the top two subfamilies are drawn against a gray background. (B) Hydropathy curves corresponding to the alignments in (A). For further details, see Reference 1.

In addition to the human proteins, MAPEG members have been identified in plants, fungi, and bacteria. It is clearly a challenge to elucidate their role in these different phyla in relation to their defined physiological functions in humans.

	Footnotes

Correspondence and requests for reprints should be addressed to Per-Johan Jakobsson, Ph.D., Department of Biochemistry and Biophysics (MBB), Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail: per-johan.jakobsson{at}mbb.ki.se

	References

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