Gamma-glutamyl leukotrienase, a novel endothelial membrane protein, is specifically responsible for leukotriene D(4) formation in vivo

Abstract

The metabolism of cysteinyl leukotrienes in vivo and the pathophysiological effects of individual cysteinyl leukotrienes are primarily unknown. Recently we identified an additional member of the gamma-glutamyl transpeptidase (GGT) family, gamma-glutamyl leukotrienase (GGL), and developed mice deficient in this enzyme. Here we show that in vivo GGL, and not GGT as previously believed, is primarily responsible for conversion of leukotriene C(4) to leukotriene D(4), the most potent of the cysteinyl leukotrienes and the immediate precursor of leukotriene E(4). GGL is a glycoprotein consisting of two polypeptide chains encoded by one gene and is attached at the amino terminus of the heavy chain to endothelial cell membranes. In mice it localizes to capillaries and sinusoids in most organs and in lung to larger vessels as well. In contrast to wild-type and GGT-deficient mice, GGL-deficient mice do not form leukotriene D(4) in vivo either in blood when exogenous leukotriene C(4) is administered intravenously or in bronchoalveolar lavage fluid of Aspergillus fumigatus extract-induced experimental asthma. Further, GGL-deficient mice show leukotriene C(4) accumulation and significantly more airway hyperreponsiveness than wild-type mice in the experimental asthma, and induction of asthma results in increased GGL protein levels and enzymatic activity. Thus GGL plays an important role in leukotriene D(4) synthesis in vivo and in inflammatory processes.

Figure 1.

Characterization on GGL protein by SDS-PAGE and Western blotting. A: Structural analysis on GGL. Lane 1 shows the truncated recombinant GGL protein used for immunization (reduced). Samples in lanes 2 to 5 were run under reducing conditions and those in lanes 6 to 9 were run under nonreducing conditions. Lanes 2 and 6 were samples from WT spleen, lanes 3 and 7 from GGL^−/− spleen. Lanes 4 and 8 were samples from WT uterus, lanes 5 and 9 from GGL^−/− uterus. B: Deglycosylation of GGL with endoglycosidase H and N-glycosidase F. Samples in lanes 1 to 4 were WT spleen homogenates and those in lanes 5 to 8 were WT uterus homogenates. Lanes 1 and 5 show untreated samples. Lanes 2 and 6 show samples incubated with the buffer in the absence of enzymes. Lanes 3 and 7 show samples treated with endoglycosidase H, and lanes 4 and 8 show samples treated with N-glycosidase F. C: Dissociation of GGL from the cell membrane. The left panel shows the result of releasing GGL protein from a uterus membrane preparation with dithiothreitol. Lane 1 is untreated sample, lane 2 is the supernatant after dithiothreitol treatment, and lane 3 is the pellet after dithiothreitol treatment. The right panel shows the result of limited papain digestion on a uterus membrane preparation. Lane 1 is the untreated sample, lane 2 is the supernatant from a papain-digested sample, lane 3 is the pellet from a papain-digested sample, lane 4 is the supernatant of the sample incubated with buffer in the absence of papain, and lane 5 is the pellet. The numbers at the left side of the figures indicate the molecular weights in kd. Twenty μg of total protein from spleen homogenates and 10 μg of total protein from uterus homogenates were analyzed in all of the experiments.

Figure 2.

LTC₄/LTD₄ conversion activity in different fractions of spleen from WT (□), GGT^−/− (▪), and GGL^−/− (▨) mice. The cleavage of LTC₄ was analyzed by HPLC as described in Materials and Methods. The specific activity is expressed as nmol/mg protein/hour (n = 3). Values are mean ± SEM. *, Significant difference from the other two groups (P < 0.01) by Student’s t-test.

Figure 3.

Localization of GGL by immunohistochemistry and immunofluorescence. A: Section of GGL-deficient spleen. B: Section of WT spleen. The black arrow points to positive staining on endothelium of a sinusoid. C: Section of WT liver. The black arrow points to positive staining on endothelium of a sinusoid; the white arrow points to lack of staining on endothelium of a central vein. D: Section of WT kidney. The black arrow points to positive staining on endothelium of capillaries in a glomerulus; the white arrow points to lack of staining on endothelium of a small artery. E: Section of WT brain. The black arrow points to positive staining on endothelium of a capillary. F: Section of WT heart. The black arrow points to positive staining on endothelium of a capillary in myocardium. G to L: Results of immunofluorescence co-localization of GGL and CD31. G to I: Sections of WT liver. J to L: Sections of WT brain. G and J: GGL expression detected with Oregon Green. H and K: CD31 expression detected with Texas Red. I and L: GGL and CD31 co-localization with Oregon Green and Texas Red merged. Original magnifications, ×400.

Figure 4.

Analysis of plasma leukotrienes after exogenous administration of LTC₄. HPLC was applied as described in Materials and Methods. A, WT; B, GGT-deficient; C, GGL-deficient; D, GGT/GGL-deficient; E, WT pretreated with d-penicillamine; F, GGT-deficient pretreated with d-penicillamine. LTC₄, LTD₄, and LTE₄ peaks were authenticated by comparing them with retention times of external standards. Peak UI is an unidentified peak that is also observed in the control plasmas (no exogenous LTC₄ administration) (data not shown). Three mice were used for each group, and all mice in the same group showed similar results.

Figure 5.

Analysis on BALF Cyst LTs levels in mice with experimental asthma. A: LTC₄ levels in BALF. B: LTE₄ levels in BALF. C: AHR after acetylcholine challenge. AHR was measured as the amount of acetylcholine that induced a 200% rise in pulmonary resistance from the baseline during challenge (PC₂₀₀). WT, GGL^−/−, GGT^−/−, and GGL^−/−/GGT^−/− mice were treated with PBS (□) or CF (▪). Data are representative of two separate experiments with seven to eight mice per group and are plotted as means ± SEM *, Significant differences (P < 0.01) between PBS treatment and CF treatment; #, significant differences (P < 0.01) from the WT group, by Student’s t-test.

Figure 6.

Response of GGL to asthma induction. A: Western blot analysis of GGL protein in lungs from mice treated with PBS or CF. The figure shows the light chain of GGL protein with a molecular weight of 20 kd (see Materials and Methods). Lane 1 is from the lung of a PBS-treated GGL-deficient mouse, used as a negative control for GGL; lanes 2 and 3, samples from PBS-treated WT mice; lanes 4 and 5, samples from CF-treated WT mice; lanes 6 and 7, samples from PBS-treated GGT-deficient mice; and lanes 8 and 9, samples from CF-treated GGT-deficient mice. B: LTC₄/LTD₄ conversion activity in lungs. WT, GGT^−/−, and GGL^−/− mice were treated with either PBS (□) or CF (▪). Conversion of LTC₄ to LTD₄ and to LTE₄ was analyzed by HPLC as described in Materials and Methods. The specific activity is expressed as nmol/mg protein/hour (n = 3 to 6). Values are mean ± SEM. For GGT (assayed in GGL-deficient mice) the difference between PBS treatment and CF treatment, P < 0.05; for GGL (assayed in GGT-deficient mice) the difference between PBS treatment and CF treatment, P < 0.001; both tested by Student’s t-test.