Molecular cloning and expression of human leukotriene-C4 synthase.

Abstract

Leukotriene-C4 synthase (LTC4S; EC 2.5.1.37) catalyzes the committed step in the biosynthesis of the peptidoleukotrienes, which are important in the pathogenesis of asthma. Antibodies were generated to a synthetic peptide based on the partial amino acid sequence previously reported for human LTC4S [Nicholson, D. W (M, = 16,568) that has a calculated pI value of 11.1. The deduced protein sequence is composed predominantly of hydrophobic amino acids; hydropathy analysis predicts three transmembrane domains connected by two hydrophilic loops. Analysis of the deduced sequence identified two potential protein kinase C phosphorylation sites and a potential N-linked glycosylation site. The amino acid sequence for human LTC4S is unique and shows no homology to other glutathione S-transferases. LTC4S was found to be most similar to 5-lipoxygenase activating protein (31% identity, 53% similarity), another protein involved in leukotriene biosynthesis. Active enzyme was expressed in bacterial, insect, and mammalian cells as shown by the biosynthesis of LTC4 in incubation mixtures containing LTA4 and reduced glutathione. The cloning and expression of human LTC4S provide the basis for a better understanding of this key enzyme in peptidoleukotriene biosynthesis.Leukotrienes (LTs) are potent biological mediators derived from arachidonic acid and are formed in response to a variety ofimmunologic and inflammatory stimuli (1-3). It is now well established that LTs affect leukocyte chemotaxis, pulmonary smooth muscle contraction, vascular tone and permeability, and mucous secretion (4-8). As such, the LTs have been implicated as potential mediators of immediate hypersensitivity and inflammatory conditions (9, 10).Initially, the production of LTs results from the oxygenation and subsequent dehydration of arachidonic acid to yield the unstable epoxide intermediate LTA4; both of these enzymatic reactions are catalyzed by 5-lipoxygenase (5-LO) (11). Cellular 5-LO activity requires an additional cofactor, 5-LO activating protein (FLAP) (12). It has been suggested that FLAP activates 5-LO by specifically binding arachidonic acid and transferring this substrate to the 5-LO enzyme (13). The LTA4 formed by 5-LO-FLAP can then be stereoselectively hydrolyzed to biologically active LTB4 by the cytosolic enzyme LTA4 hydrolase or conjugated with reduced glutathione by the membrane-bound enzyme LTC4 synthase (LTC4S; EC 2.5.1.37) to form the peptidoleukotriene LTC4.LTC4 is then converted to LTD4 by y-glutamyltranspeptidase and subsequently metabolized to LTE4 by cysteinylglycine dipeptidase (14).Collectively, the peptidoleukotrienes (LTC4, LTD4, and LTE4) have been demonstrated to be involved in inflammatory and anaphylactic reactions. They were first isolated and identified as the active mediators of the slow-reacting substance of anaphylaxis and are released from the lung tissue of asthmatics upon exposure to specific allergens (15). Additionally, exogenous application of peptidoleukotrienes results in many phenomena characteristic o...

Targeted lipidomics reveals mPGES-1-PGE2 as a therapeutic target for multiple sclerosis

“…2). The discrepancy be- tween transcripts and metabolites is explained by posttranscriptional/translational (such as phosphorylation) regulation of enzymes (42)(43)(44) and/or functional coupling with cPLA 2 ␣. Furthermore, COX enzymes were up-regulated in the SC around the same time (Fig.…”

Phorbol ester‐induced suppression of leukotriene C4 synthase activity in human granulocytes

“…The enzyme was recently cloned and the deduced amino acid sequence demonstrated to be similar to FLAP, but not to any known cytosolic or microsomal glutathione S-transferase [6,20]. The presence of LTC 4 synthase in human lung tissue, alveolar macrophages, eosinophils and platelets was recently demonstrated, using a polyclonal antibody against the enzyme [8].…”

The membrane organization of leukotriene synthesis

“…FLAP may serve as an arachidonic acidbinding protein, allowing arachidonic acid to be presented to 5-LO for conversion to both 5-HPETE and LTA 4 (14)(15)(16)(17)(18). To form LTC 4 , LTA 4 is conjugated with reduced glutathione by LTC 4 synthase, a 17-kDa integral membrane protein that shares a high identity with FLAP (19)(20)(21)(22)(23). Two untested models for the membrane organization of LTC 4 synthesis can be proposed.…”