Expression cloning of a cDNA for human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathione to leukotriene A4.
Leukotriene (LT) C4 synthase, an integral microsomal membrane protein, conjugates LTA4, an epoxide intermediate, to reduced glutathione (GSH) to form a proinflammatory mediator, LTC4. A sensitive fluorescence-linked immunoassay for LTC4 was used to screen a KG-1 cDNA expression library for LTC4 synthase activity after transfection of COS cells and addition of substrate LTA4. Stepwise resolution of 240,000 colonies in 96 pools led to the identification of individual clones with maximal LTC4 synthase activity that contained a 694-bp cDNA insert. This insert was composed of a 54-bp 5' nontranslated region, an ATTAAA polyadenylylation signal, and a poly(A)+ tail. The open reading frame encodes a 16.5-kDa protein with a pI of 11.05. Hybridization with a cDNA probe demonstrated a mRNA transcript of 0.7 kbp in RNAs from human eosinophils and KG-1 cells, which contain LTC4 synthase. The nucleotide and deduced amino acid sequences of the LTC4 synthase cDNA show no significant homology to GSH S-transferases but share 31% overall amino acid identity with 5-lipoxygenase activating protein (FLAP). The identity at the N-terminal two-thirds of these two proteins is 44%, with some regions of near identity. Peptide structural analysis of the deduced LTC4 synthase predicts the presence of three transmembrane domains nearly superimposable on those of FLAP. Moreover, LTC4 synthase is inhibitable by a FLAP inhibitor, MK-886. Therefore, LTC4 synthase is distinct from the known GSH S-transferases by nucleotide and consensus amino acid sequences, and its GSH-conjugating function represents a distinct integral membrane protein belonging to a distinct gene family.
Lam
B K
BK
Department of Medicine, Harvard Medical School, Boston, MA 02115.
Penrose
J F
JF
Freeman
G J
GJ
Austen
K F
KF
D058927:5-Lipoxygenase-Activating Proteins; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002451:Cell Compartmentation; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D017572:Leukotriene A4 / Q000378:metabolism; D017997:Leukotriene C4 / Q000096:biosynthesis; D008565:Membrane Proteins / Q000235:genetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D018076:DNA, Complementary; D017572:Leukotriene A4; D008565:Membrane Proteins; D011994:Recombinant Proteins; D017997:Leukotriene C4; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase; D005978:Glutathione