Juan F. Medina

MOLECULAR CLONING AND EXPRESSION OF MOUSE LEUKOTRIENE A4 HYDROLASE CDNA

A cDNA clone for mouse leukotriene A4 hydrolase encoding the full sequence of the enzyme was isolated from a mouse spleen lambda ZAP-II library. The identification was ascertained by expression of enzyme activity in Escherichia coli. The encoded protein has 610 amino acids and exhibits an extensive identity (93%) with the human leukotriene A4 hydrolase. A region spanning between residues 233 and 340, where the zinc binding site is located, was found to be perfectly conserved between the two species. We found six sites of polymorphism in the cDNA sequence of mouse LTA4 hydrolase, one of which leads to a difference in the encoded amino acid. The polymorphism of cDNA was confirmed by reverse transcription-PCR sequencing of mouse spleen total RNA, prepared as a mixture from ten different strains.

Recombinant mouse leukotriene A4 hydrolase : a zinc metalloenzyme with dual enzymatic activities

Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of zinc/mol of enzyme. Apparent kinetic constants (Km and Vmax) for the conversion of leukotriene A4 to leukotriene B4 (at 0 degree C, pH 8) were 5 microM and 900 nmol/mg per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine-4-nitroanilide. Km and Vmax for this reaction (at 37 degrees C, pH 8) were 680 microM and 365 nmol/mg per min, respectively. Apo-leukotriene A4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A4 resulted in a dose-dependent inactivation of both enzyme activities.

Leukotriene A4: metabolism in different rat tissues

The transformation of leukotriene A4 into dihydroxyeicosatetraenoic acids and sulfidopeptide leukotrienes was determined in homogenates of rat tissues supplied with glutathione and albumin. The highest production of leukotriene B4 was found in spleen, lung and small intestine, while leukotriene C4 dominated in liver and lung. 5(S),6(R)-Dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5,6-DHETE) was formed in all tissues, most prominently in kidney, heart and brain. We also found another isomer of 5,6-dihydroxyeicosatetraenoic acid produced in the kidney. This compound was derived from 5,6-DHETE by isomerization, probably of the 11-cis double bond to 11-trans, and the process appeared to be catalyzed by a membrane-bound factor.

Leukotriene A4 hydrolase: a critical role of glutamic acid-296 for the binding of bestatin.

Leukotriene A(4) hydrolase is a bifunctional Zn(2+)-containing enzyme catalysing the formation of the potent chemotaxin leukotriene B(4). From an analysis of three mutants of Glu-296 we have found that this catalytic residue is critical for the binding of bestatin, a classical aminopeptidase inhibitor. For bestatin, but not for three other tight-binding inhibitors, the IC(50) values for inhibition of the epoxide hydrolase activity decreased in the mutants to 0.7-0.003% of the control. Hence Glu-296 is an important structural determinant for binding of bestatin to leukotriene A(4) hydrolase; this conclusion might also apply to other members of the M1 family of metallopeptidases.

B-lymphocytic cell line Raji expresses the leukotriene A4 hydrolase gene but not the 5-lipoxygenase gene

The expression of LTA hydrolase and 5-lipoxygenase genes was studied in Raji cells, a Burkitt lymphoma derived B-cell line. Northern and Western blot analyses clearly showed the expression, both at the transcriptional and translational level, of the LTA4 hydrolase gene in these cells. However, expression of the 5-lipoxygenase gene was undetectable. Thus, the genes coding for the two enzymes required for biosynthesis of leukotriene B4 from arachidonic acid, 5-lipoxygenase and LTA4 hydrolase, were differentially expressed in the Raji cells.

The Role of Leukotriene A4 Hydrolase in Cells and Tissues Lacking 5-Lipoxygenase

Leukotriene (LT) A4 hydrolase converts the unstable epoxide intermediate LTA4 into the potent proinflammatory compound LTB4. The formation of LTA4 is catalyzed by the enzyme 5-lipoxygenase and involves the dioxygenation of arachidonic acid with subsequent epoxide formation (1).

Mutations of the Three Zinc-Ligands of Leukotriene A4 Hydrolase: Effects on Zinc Content and Enzyme Activities

X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus sequences for catalytic and structural zinc binding sites (1). Such reference structures have served to predict zinc binding ligands among members of related zinc enzyme families and also, more noticeable, in apparently unrelated proteins. Thus, comparison of the amino acid sequence of LTA4 hydrolase with the corresponding primary structures of certain aminopeptidases and neutral proteases, typified by thermolysin, revealed the presence of a putative catalytic zinc site (1, 2). Consequently, LTA4 hydrolase was found, not only to contain one zinc atom per molecule, essential for the catalytic activity, but also to possess a zinc-dependent peptidase activity towards synthetic amides (3–5).