Molecular cloning and expression of human leukotriene-C sub(4) synthase

Leukotriene-C sub(4) synthase (LTC sub(4)S; 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 h...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1994-01, Vol.91 (21), p.9745-9749
Main Authors: Welsch, D J, Creely, D P, Hauser, S D, Mathis, K J, Krivi, G G, Isakson, P C
Format: Article
Language:English
Subjects:
amino acid sequence
cDNA
gene expression
leukotriene-C4 synthase
LTC subS gene
man
nucleotide sequence
THP-1 cells
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Summary:Leukotriene-C sub(4) synthase (LTC sub(4)S; 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 LTC sub(4)S and specifically bound detergent-solubilized LTC sub(4)S obtained from THP-1 cells, confirming that the published sequence is associated with enzyme activity. Inosine-containing oligonucleotides based on the partial protein sequence were used to isolate a 679-bp cDNA for LTC sub(4)S from THP-1 cells. The cDNA contains an open reading frame that encodes a 150-amino acid protein (M sub(r) = 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 LTC sub(4)S is unique and shows no homology to other glutathione S-transferases. LTC sub(4)S 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 LTC sub(4) in incubation mixtures containing LTA sub(4) and reduced glutathione. The cloning and expression of human LTC sub(4)S provide the basis for a better understanding of this key enzyme in peptidoleukotriene biosynthesis.
ISSN:0027-8424