Identification and Characterization of a Novel Human Microsomal Glutathione S-Transferase with Leukotriene C4 Synthase Activity and Significant Sequence Identity to 5-Lipoxygenase-activating Protein and Leukotriene C4 Synthase

Abstract

5-Lipoxygenase-activating protein (FLAP) and leukotriene C4 (LTC4) synthase, two proteins involved in leukotriene biosynthesis, have been demonstrated to be 31% identical at the amino acid level. We have recently identified and characterized a novel member of the FLAP/LTC4 synthase gene family termed microsomal glutathione S-transferase II (microsomal GST-II). The open reading frame encodes a 16.6-kDa protein with a calculated pI of 10.4. Microsomal GST-II has 33% amino acid identity to FLAP, 44% amino acid identity to LTC4 synthase, and 11% amino acid identity to the previously characterized human microsomal GST (microsomal GST-I). Microsomal GST-II also has a similar hydrophobicity pattern to FLAP, LTC4 synthase, and microsomal GST-I. Fluorescent in situ hybridization mapped microsomal GST-II to chromosomal localization 4q28-31. Microsomal GST-II has a wide tissue distribution (at the mRNA level) and was specifically expressed in human liver, spleen, skeletal muscle, heart, adrenals, pancreas, prostate, testis, fetal liver, and fetal spleen. In contrast, microsomal GST-II mRNA expression was very low (when present) in lung, brain, placenta, and bone marrow. This differs from FLAP mRNA, which was detected in lung, various organs of the immune system, and peripheral blood leukocytes, and LTC4 synthase mRNA, which could not be detected in any tissues by Northern blot analysis. Microsomal GST-II and LTC4 synthase were expressed in a baculovirus insect cell system, and microsomes from Sf9 cells containing microsomal GST-II or LTC4 synthase were both found to catalyze the production of LTC4 from LTA4 and reduced glutathione. Microsomal GST-II also catalyzed the formation of another product, displaying a conjugated triene UV absorption spectra with a maximum at 283 nm, suggesting less catalytic stereospecificity compared with LTC4 synthase. Also, the apparent Km for LTA4 was higher for microsomal GST-II (41 microM) than LTC4 synthase (7 microM). In addition, unlike LTC4 synthase, microsomal GST-II was able to catalyze the conjugation of 1-chloro-2, 4-dinitrobenzene with reduced glutathione. Therefore, it is proposed that this novel membrane protein is a member of the microsomal glutathione S-transferase family, also including LTC4 synthase, with significant sequence identities to both LTC4 synthase and FLAP.