Molecular cloning and expression of human leukotriene-C4 synthase.
AUTOR(ES)
Welsch, D J
RESUMO
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., Ali, A., Vaillancourt, J.P., Calaycay, J.R., Mumford, R.A., Zamboni, R.J. & Ford-Hutchinson, A. W. (1993) Proc. Natl. Acad. Sci. USA 90, 2015-2019] and specifically bound detergent-solubilized LTC4S 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 LTC4S from THP-1 cells. The cDNA contains an open reading frame that encodes a 150-amino acid protein (M(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 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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=44893Documentos Relacionados
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