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-Glutamyl Leukotrienase, a Novel Endothelial Membrane Protein, Is Specifically Responsible for Leukotriene D4 Formation in Vivo
From the Departments of Pathology,*Medicine,and Molecular and Cellular Biology,Baylor College of Medicine, Houston, Texas
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Abstract |
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-glutamyl transpeptidase (GGT) family, -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 C4 to leukotriene D4, the most potent of the cysteinyl leukotrienes and the immediate precursor of leukotriene E4. 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 D4 in vivo either in blood when exogenous leukotriene C4 is administered intravenously or in bronchoalveolar lavage fluid of Aspergillus fumigatus extract-induced experimental asthma. Further, GGL-deficient mice show leukotriene C4 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 D4 synthesis in vivo and in inflammatory processes.
-glutamyl residue. Although -glutamyl transpeptidase (GGT) is known to catalyze LTD4 formation in the test tube,8,9
the mechanism of LTD4 formation in vivo is unknown. Because LTD4 is more potent than its precursor and binds with much higher affinity to the cysteinyl LT1 receptor than LTC4,10
understanding LTD4 metabolism is important in clarifying its role in disease. In addition, clearance of Cyst LTs in the urine as leukotriene E4 (LTE4) requires LTD4 formation because it is the immediate precursor of LTE4.11-13
It is generally accepted that GGT is responsible for LTC4/LTD4 conversion in vivo.13,14
However, we recently identified and cloned a mouse GGT family member designated -glutamyl leukotrienase (GGL) because its primary natural substrate appears to be LTC4.15,16
The enzyme also cleaves several other S-substituted glutathiones, but not GSH itself. The GGL gene is located 
3-kb upstream of GGT, and the two cDNAs share a 57% nucleotide identity.16
Because all known substrates for GGL are also substrates for GGT,16,17
it is not possible to assay for GGL in tissues from wild-type (WT) mice without also detecting GGT. To circumvent this problem we have used GGT-deficient mice for assays of GGL activity and developed antibodies specific for GGL.4,15-18
GGL is expressed at highest levels in the spleen and uterus.4,16
This observation is puzzling because it is not readily apparent what physiological function an enzyme such as GGL would have in tissues as different as spleen and uterus. Although the relative contributions of GGL and GGT to Cyst LT metabolism are unknown, our previous data indicate that no other enzymes in the mouse cleave LTC4.4
Thus it is possible to take advantage of mice deficient in GGL, GGT, or both enzymes to evaluate their roles in Cyst LT metabolism in vivo and in pathophysiology.4,17
Differences in organ distribution of the two enzymes suggest both different functions and different substrate specificities in vivo.4,15-18 For the most part, GGT is expressed on absorptive and secretory epithelium (the luminal surface of the proximal tubular cells of the kidney, the villous epithelium of the small intestine, the ductular epithelium of the pancreas, and the secretory epithelial cells of the seminal vesicle).9,19 These locations are consistent with the generally held idea that the major function of GGT is GSH cleavage.9 Although LTC4 is a substrate for GGT in the test tube,8 the distribution of the enzyme contrasts with the known distribution and function of Cyst LTs.20 Using GGL-deficient mice, we have recently found that in Zymosan A-induced peritonitis most of the initial neutrophil accumulation is dependent on GGL activity and that GGL is responsible for most or all of the cleavage of endogenous LTC4 that accumulates in the peritoneal cavity.4 These findings underscore the potential role of GGL in inflammation and provide evidence that it functions separately from GGT. Further studies of the characteristics of GGL and its roles in different pathophysiological conditions are essential for the understanding of its biological function.
Here we demonstrate that GGL is a glycoprotein composed of two polypeptide chains and is located on the surface of endothelial cells. GGL converts exogenously administered LTC4 in the circulation, as well as formed in A. fumigatus extract (CF)-induced experimental asthma, to LTD4 to limit potentially life-threatening airway hyperreponsiveness (AHR). These findings suggest the importance of GGL in the pathophysiology of disease.
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Materials and Methods |
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LTC4, LTD4, and LTE4 were from Cayman Chemical Company (Ann Arbor, MI). Papain and N-glycosidase F were purchased from Sigma Chemical Co. (St. Louis, MO). Endoglycosidase H was from Boehringer Mannheim Co. (Indianapolis, IN). Anti-mouse CD31 monoclonal antibody was from Pharmingen (San Diego, CA). Texas Red-conjugated anti-rat antibody and Oregon Green conjugated anti-rabbit antibody were from Molecular Probes (Eugene, OR). The immunohistochemistry kit was from BioGenes (San Ramon, CA).
Animals
All GGT-, GGL-, and GGT/GGL-deficient mice were developed in our laboratory.4,17 All mice including WT controls were on a C57BL/129SvEv background and were used between 6 to 8 weeks of age. They were supplied with N-acetyl cysteine in the drinking water.17
Generation of Anti-GGL Polyclonal Antibody
Rabbit anti-mouse GGL was generated by using an N-terminus truncated recombinant GGL protein. GGL cDNA fragment (nucleotides 426 to 1721) was expressed in-frame in an expression vector, pET-30a (+) (Novagen, Madison, WI) in Escherichia coli. The truncated protein with 100 amino acids at the N-terminus removed was then purified by using the Xpress System Protein Purification kit (Invitrogen, Carlsbad, CA). New Zealand White rabbits were injected with 200 µg of the protein in complete Freund’s adjuvant and boosted with 100 µg of protein in incomplete Freund’s adjuvant every 2 weeks. Rabbits made high-titer-specific anti-GGL antibodies as judged by Western blot analysis.
Induction of Experimental Asthma
A. fumigatus culture filtrate allergen (CF) (lot no. DC980809) was prepared and used as previously described.21 Mice were challenged as previously described;21 briefly, 50 µl of CF or saline control was administered intranasally to mice anesthetized with Metofane (Janssen, Toronto, Canada). Mice were challenged five times with 4 days between each challenge. Fifteen hours after the final CF challenge, airway resistance was measured and AHR determined by C200, and bronchoalveolar lavage fluid (BALF), serum, and lung tissue were collected. BALF total and differential cell counts, BALF mucine, and lung histology were evaluated as described.21 Data are representative of two independent experiments with seven to eight mice in each groups.
Western Blot Analysis
Tissue homogenates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence or absence of 0.1 mol/L of dithiothreitol and electrophoretically transferred to nitrocellulose membrane. The anti-serum was used at 1:20,000 dilution. The detecting system was a Phototope-HRP Detection Kit (New England BioLabs, Beverly MA).
For deglycosylation of GGL with endoglycosidases, tissue homogenates from spleen and uterus were subjected to endoglycosidase H (Boehringer Mannheim Co.) or N-glycosidase F (Sigma Chemical Co.) treatment as previously described.22-24 The reactions were performed at 37°C for 18 hours followed by Western blot analysis.
For dissociation of GGL from cell membranes, homogenates of WT uterus were centrifuged at 43,000 x g for 30 minutes at 4°C, and the membrane fraction was subjected either to dithiothreitol reduction or to papain digestion. The samples were reduced with 0.1 mol/L of dithiothreitol in the presence of 0.02% SDS at 60°C for 1 hour or incubated with papain at a final concentration of 1 mg of papain/1.5 g membrane protein at 25°C for 30 minutes. The reactants were centrifuged at 43,000 x g at 4°C for 10 minutes, and both supernatant and pellet were analyzed by Western blot.
For assaying GGL protein level in the lungs after asthma induction, lung homogenates were directly analyzed by Western blot and differences were quantified by scanning densitometry.
LTC4/LTD4 Conversion Assay
LTC4 conversion activity was assayed by high performance liquid chromatography (HPLC) as described previously.15 Specific activity was expressed as nmol of LTC4 converted/mg protein/hour by measuring the formation of LTD4 and LTE4; the latter is formed by the action of membrane-bound dipeptidase on LTD4.12,15,16
To assay LTC4/LTD4 conversion activity in phosphate-buffered saline (PBS)-treated and A. fumigatus-treated lungs, organs were homogenized in 0.1 mol/L of Tris-HCl, pH 8.0, and the homogenates were directly incubated with LTC4 for activity assay.
To assay the activity in different compartments of the spleen, we prepared spleen fractions. The organs were teased apart with fine forceps in PBS to release free cells. The cell suspension and the initial residues were separated by allowing the preparation to settle on ice for 5 minutes. The cells were collected by centrifugation at 1000 x g for 5 minutes. The supernatant was combined with the initial residues and this was designated the residual stroma. Both the cell suspension and the residual stroma were homogenized for the assay.
To examine the intravascular metabolism of exogenous LTC4, LTC4 at 100 µg/ml in ethanol was diluted in normal saline to 40 µg/ml, and 50 µl was injected intravenously through the tail veil (2 µg/mouse). Three mice from each of the WT, GGT-, GGL-, and GGT/GGL-deficient group were treated. Ten minutes after the injection, blood samples were harvested by heart puncture and plasma was collected using Brand Serum Separators (Becton Dickinson, Franklin Lakes, NJ). Plasma from control mice (no LTC4 administration) was used as a negative control. For inhibition of LTD4/LTE4 conversion, D-penicillamine (Sigma Chemical Co.) was injected intraperitoneally 5 minutes before the administration of LTC4 at 5 mmol/kg body weight. Cyst LTs in plasma were analyzed by HPLC as described above.
LTC4 and LTE4 Measurement in BALF
BALF was collected in PBS and centrifuged. Supernatants were collected and stored at -80°C until assayed. BALF was first extracted and Cyst LTs concentrated using Sep-Park cartridges (Waters Corp., Milford, MA). They were assayed for LTC4 and LTE4 using leukotriene C4 and leukotriene E4 EIA kits (Cayman Chemical Company). To rule out the cross-reactivity among Cyst LTs, in some experiments samples were further fractionated by HPLC based on the retention times established with standards after extraction by Sep-Park cartridges4 and assayed for LTC4 and LTE4 by enzyme-linked immunosorbent assay. Data are representative of two independent experiments with seven to eight mice in each group.
Immunohistochemistry and Immunofluorescence Analysis
Formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated using a standard protocol. The endogenous peroxidase was quenched by incubating the slides in 3% H2O2 in methanol for 5 minutes followed by two 5-minute PBS washes. The slides were incubated with trypsin (Trypsin 3-Pack; BioGenex, San Ramon, CA) for 20 minutes at 37°C, blocked with 1.5% normal goat serum in PBS at room temperature for 30 minutes, and incubated with protein A-purified anti-GGL polyclonal antibody at 10 µg/ml at 25°C for 1 hour. Peroxidase staining was achieved using BioGenex’s Supersensitive detection kit and liquid diaminobenzidine. The sections were counterstained with hematoxylin. For immunofluorescence co-localization of mouse CD31 and GGL, we used zinc-fixed sections because the rat anti-mouse CD31 does not react well with the formalin-fixed antigen. The zinc fixative solution was 0.1 mol/L Tris buffer at pH 7.4, containing 0.05% calcium acetate, 0.5% zinc acetate, and 0.5% zinc chloride. Both rabbit anti-GGL and rat anti-CD31 (Pharmingen, San Diego, CA) antibodies were used at a concentration of 10 µg/ml. The fluorescence-labeled secondary antibodies were Texas Red-conjugated goat anti-rat IgG and Oregon Green conjugated goat anti-rabbit IgG (Molecular Probes). The fluorescence was detected with a fluorescence microscope (Nikon Optiphot-2; Nikon Inc., Nelville, NY) with a 568-nM filter for Texas Red and a 488 nM filter for Oregon Green.
Statistical Analysis
Statistical analyses were performed using Student’s t-test. Data are presented as mean ± SEM.
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Results |
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Because the mature GGT protein consists two disulfide-linked peptide chains generated from a single precursor polypeptide and GGL is a membrane protein with similarities to GGT,16,25
we examined the structure of GGL protein in tissue homogenates from spleen and uterus. Using SDS-PAGE and Western blotting with anti-GGL antibodies, we found that GGL protein consists of two polypeptides with molecular weights of 57 kd and 20 kd under reducing conditions and runs as a single band of 75 kd under nonreducing conditions (Figure 1A)
. These bands were not detected in homogenates of spleen and uterus from GGL-deficient mice, confirming the specificity of our antibodies. These results indicate that, like GGT, GGL consists of two polypeptides chains bonded through one or more S-S linkages
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is greater than the predicted molecular weight of 63 kd (573 amino acids) for the heavy chain, we examined protein glycosylation. For this purpose, tissue homogenates were incubated with either of two endoglycosidases, N-glycosidase F and endoglycosidase H. N-glycosidase F hydrolyzes all N-linked carbohydrate chains from glycoproteins whereas hydrolysis by endoglycosidase H is restricted to high mannose and hybrid types.22-24
The deduced amino acid sequence of GGL heavy chain has six potential N-linked glycosylation sites.16
If each site is glycosylated, the predicted molecular weight is 60 kd (assuming an average of 3 kd per complex type of N-linked sugar), whereas the predicted molecular weight of the unglycosylated heavy chain is 42.8 kd.16
After N-glycosidase F treatment, the apparent molecular weight of the heavy chain decreased from 57 kd to 42.5 kd, indicating the removal of most or all carbohydrate (Figure 1B
, lane 4). After treatment with endoglycosidase H, the heavy chain decreased from 57 kd to 54 kd (Figure 1B
, lane 3), indicating that most carbohydrate chains of the protein are the complex type, which is a feature of plasma membrane glycoproteins.26
Neither of the enzymes changed the migration of the light chain (Figure 1B
, lanes 3 and 4), indicating the absence of N-linked glycosylation.
To characterize the physical anchoring of GGL to cells, we reduced a membrane preparation with dithiothreitol; this treatment released only the light chain, indicating that GGL is anchored to the membrane through the heavy chain (Figure 1C
, left). As a second approach, we released GGL from the membrane with limited papain digestion.27
Papain treatment released the intact GGL light chain and a protein of 42 kd representing a shortened heavy chain. An 15-kd band cleaved from the heavy chain was also detected in the supernatant (Figure 1C
, right). These results provide additional evidence that GGL is anchored in the membrane through the heavy chain. An analysis of the amino acid hydropathy revealed a hydrophobic region (amino acids 11 to 29) located at the N-terminus of GGL,13
suggesting that like GGT, membrane association of GGL involves the N-terminus of the heavy chain.
Tissue Localization of GGL
We examined the distribution of GGL in spleen by separating it into a cell suspension and residual stroma. By light microscopy, the suspension contained red blood cells, lymphocytes, neutrophils, a small number of macrophages and fibroblasts, and a few endothelial cells, whereas the residual stroma contained predominantly sinusoidal and reticular material with attached endothelial cells, collagen fibers (with fibroblasts), macrophages, and a few lymphocytes. In both WT and GGT-deficient spleen, the residual stroma was 10-fold enriched in LTC4 cleavage activity (Figure 2)
. In GGL-deficient spleen, LTC4 cleavage is approximately eightfold lower than in WT spleen and is associated with the suspended cells; this cleavage represents residual GGT activity.15,16
Thus in contrast to GGT, which is known to be expressed on lymphocytes in spleen (at relatively low levels),28
GGL activity is associated with nonlymphoid cells in stroma.
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. As expected, GGL-deficient spleen did not stain (Figure 3A)
. Sinusoid endothelial cells in WT liver were also stained (Figure 3C)
. Capillary endothelial cells in brain (Figure 3D)
, glomerulus (Figure 3E)
, and myocardium (Figure 3F)
also expressed GGL, as did endothelial cells in endometrium and the lamina propria of the small intestine (data not shown). Larger blood vessels in most organs, as exemplified by the central veins in liver (Figure 3C)
, small arteries in kidney (Figure 3E)
, and aorta and vena cava (data not shown), were negative. The lungs were an exception, and we found that in addition to capillary endothelium, endothelium in larger vessels was also positive (data not shown). In the organs examined, we did not detect staining in endothelium of lymphatics. To confirm the endothelial localization of GGL we performed fluorescent microscopy and found that CD31 (PECAM-1), an integral membrane protein constitutively expressed on the surface of endothelial cells,29,30
and GGL co-localized (Figure 3
; G to L).
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In mice, GGT and GGL are the only enzymes known to generate LTD4 from LTC4 in the test tube.4,15,16
The endothelial location of GGL suggests that GGL may perform this function in vivo. To test this hypothesis, we administered exogenous LTC4 intravenously to WT, GGT-, GGL-, and GGT/GGL-deficient mice and analyzed the Cyst LT profile in blood by HPLC.4,12
Ten minutes after injection, 25 to 30% of the LTC4 had been converted to LTE4 in both WT (Figure 4A)
and GGT-deficient mice (Figure 4B)
, whereas little, if any, conversion was detected in blood from GGL-deficient mice (Figure 4C)
. Similarly, LTE4 was also absent from the blood of GGT/GGL-deficient mice (Figure 4D)
. When mice were pretreated with D-penicillamine, a specific inhibitor of membrane-bound dipeptidase,31
LTD4 was also detected in the blood from WT and GGT-deficient mice (Figure 4, E and F)
, indicating that the LTD4 is quickly converted to LTE4 in blood. These findings demonstrate that GGL is responsible for LTD4 synthesis in blood.
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Because asthma is one of the most pressing medical problems associated with Cyst LT production, we examined the role of GGL in CF-induced experimental asthma in mice. Although it is generally believed that GGT is responsible for LTC4 to LTD4 conversion in asthma,3
our results on LTC4 metabolism in blood (Figure 4)
and in Zymosan A-induced peritonitis4
suggest that GGL might catalyze LTC4 to LTD4 conversion in asthma. To resolve this discrepancy and to assess the relative roles of GGL and GGT, we induced asthma in mice using CF and studied mice deficient in GGL, GGT, or both GGL and GGT. We found that the level of LTC4 in BALF increases sixfold to eightfold in CF-treated GGL- and GGL/GGT-deficient mice compared to saline-treated controls whereas little change is seen in WT mice and GGT-deficient mice (Figure 5A)
. These data indicate that during the asthmatic response GGL is the principal enzyme that converts LTC4 to LTD4. Because the antibody used to measure LTC4 has some cross reactivity with LTD4, we separated Cyst LTs in BALF by HPLC and verified that in GGL- and GGL/GGT-deficient mice only LTC4 is present (data not shown). In parallel with this finding, we found that there was a threefold to fivefold increase in LTE4 levels in CF-treated WT mice and GGT-deficient mice (Figure 5B)
. We also detected what appear to be very low levels of LTE4 in all PBS-treated mice, including mice deficient in GGL and both GGL and GGT. It is likely that this result stems from cross-reactivity with other arachidonic derivatives because LTE4 was not detectable in these samples after HPLC fractionation (data not shown). Our data indicate that GGL deficiency prevents the generation of LTD4 and LTE4 in experimental asthma and are further confirmation that GGL is the principle enzyme for LTC4 to LTD4 conversion in vivo.
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and increased eosinophil infiltration and goblet cell metaplasia with mucus overproduction (data not shown). All mice exhibited similar total and differential cell counts and mucin levels in BALF at the time of BALF collection (data not shown). These results indicate that CF can effectively induce asthma in mice whether or not LTD4 is present. We found that in all groups of mice CF treatment resulted in a greater than twofold decrease in PC200 compared to saline treatment (Figure 5C)
. This result is not surprising because both LTC4 and LTD4 are known to be involved in asthma.2,3
It is interesting that the PC200 values in CFtreated GGL- and GGL/GGT-deficient mice are significantly lower (P < 0.01) than that for CF-treated WT mice; this finding indicates that deficiency in GGL further exacerbates AHR. Although the difference between the PC200 for GGT-deficient mice and that for WT mice after CF treatment is not statistically significant (Figure 5C)
, it shows a trend in that direction. Whether this result indicates involvement of GGT functions unrelated to Cyst LT metabolism in asthma or is not a real difference remains to be determined. Response of GGL Enzymatic Activity and Protein to Asthma Induction
The observation that LTC4 does not accumulate in CF-treated WT and GGT-deficient mice led us to examine GGL protein levels and LTC4/LTD4 conversion activity in lungs after CF-treatment. We used Western blot to determine GGL protein levels in lungs in WT and GGT-deficient mice, and found that after CF treatment these mice exhibit a twofold increase in GGL protein as quantified by scanning densitometry (Figure 6A)
. This result indicates that expression of GGL protein is increased in response to asthma development.
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. (Recall that GGL enzyme activity can only be measured directly in GGT-deficient mice.) This increase is similar to the increase in GGL protein levels (Figure 6A)
. As expected, there was no detectable LTC4 to LTD4 conversion activity in GGL/GGT-deficient mice (data not shown).16
With respect to GGT, we found a decrease in enzyme activity in CF-treated GGL-deficient mice (Figure 6B)
. Why GGT enzyme activity is reduced after CF treatment in our experiments remains to be determined. The total LTC4/LTD4 conversion activity in CF-treated mice was the same as that for saline-treated mice, reflecting the increase in GGL and decrease in GGT activity. These data provide further evidence that GGL is the enzyme responsible for the initiation of Cyst LT metabolism in asthmatic inflammation. They also raise the questions of the physiological role of GGT in lung. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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. In contrast to GGT, which is located primarily on the luminal surface of epithelial cells,9,19
GGL is expressed on the surface of endothelial cells of capillaries and sinusoids in most organs and larger vessels in the lung (Figure 3
and data not shown). Our results do not rule out low-level GGL expression on other endothelial cells or even other cell types at levels below the sensitivity of our techniques, but it is likely that any such expression is quantitatively minor.15,16
Our data also indicate that GGL plays a key role in regulating conversion of intravascular LTC4 as well as those formed in CF-induced asthma, to LTD4 in mice (Figures 4 and 5)
.
Previously we found that spleen and uterus expressed the highest levels of GGL.4,15
Our current study provides an explanation for this phenomenon: the abundant sinusoidal endothelium in spleen and the rich capillary network in endometrium contribute to the high levels of GGL in these organs (Figure 3B
and data not shown). The fact that endothelium of large vessels in most organs expresses little, if any, of GGL protein highlights variations in its expression among different types of endothelial cells and the physiological importance of GGL. Using in situ hybridization we have previously reported tissue distribution of GGL;16
these results show some differences with our current results. The reason for this discrepancy is not readily apparent, but because our chemical and immunohistochemistry results agree, we believe that the results reported here accurately reflect the distribution of GGL.
Our data also clarify the relation between GGL and GGT function. GGT has long been thought to be the enzyme that catalyzes LTC4/LTD4 conversion in vivo.13
However, our results on LTC4 metabolism in the blood and in experimental asthma indicate that GGL performs much, if not all this function in vivo (Figures 4 and 5)
. In addition to its expression on secretory and absorptive epithelium, GGT has been reported to be expressed on capillary endothelial cells in the brain.32
However, it is absent from capillaries in other organs and from brain capillaries that lack a blood-brain barrier.33
Functionally, GGT in brain capillaries is apparently involved in the transport of neutral amino acids across blood-brain barrier.34
Lymphocytes also express low levels of GGT.28
Thus it is theoretically possible that GGT could function to a limited extent in some brain capillaries or lymphocytes to convert circulating LTC4 to LTD4; however, our data argue against any significant participation of GGT in the intravascular metabolism of Cyst LTs in that we could not detect metabolism of LTC4 in GGL-deficient mice (which express GGT) (Figure 4)
. Because GGL has a more limited substrate spectrum than GGT and does not cleave GSH,15
its presence on endothelium allows synthesis of LTD4 without interfering with circulating GSH. In addition, the Km of GGL for LTC4 is 10-fold lower than that of GGT,15
further implicating the role of GGL in LTD4 synthesis in vivo. All of these observations argue for a central role for GGL in LTD4 synthesis and in the regulation of responses to injury.
In humans Cyst LTs are important agonists of AHR in asthma, and blocking the action of Cyst LTs by Cyst LT receptor antagonists is an important part of asthma therapy.35-37
Our findings elucidate the mechanism by which Cyst LTs are metabolized during the development of experimental asthma and demonstrate that GGL plays a key role in the conversion of LTC4 to LTD4 during asthmatic inflammation. This finding is significant because it clarifies the mechanism of how LTD4 is synthesized in vivo. In our study, AHR shows significant increase in GGL-deficient mice (Figure 5C)
. Because LTC4 formed is quickly converted to LTD4 by GGL and further to LTE4 by membrane-bound dipeptidase in WT and GGT-deficient mice (Figure 5, A and B)
,12
we reason that the increase in AHR in GGL- and GGL/GGT-deficient mice results from LTC4 accumulation. This result indicates that GGL not only converts LTC4 to more potent LTD4, but also serves as a critical enzyme to promote the chemical clearance of Cyst LTs, thus to limit potentially deleterious airway obstruction. This finding is pathophysiologically important because defect and insufficiency on GGL function caused by any pathological factors may lead to the development and/or exacerbation of asthma. The fact that GGL expression is increased during asthma underscores the importance of this enzyme in regulating the metabolism of Cyst LTs (Figure 6)
.
It seems unlikely that GGT functions in Cyst LT metabolism during asthma. GGT enzyme activity falls during asthma (Figure 5B)
, and there is no evidence of conversion of LTC4 to LTD4 (as measured by LTE4 accumulation, Figure 4B
; in Figure 4B
, GGT activity is assessed in GGL-deficient mice). We have previously reported that lung homogenates from untreated mice contain more GGT activity than GGL activity (see also Figure 6B
).4,16
Although this finding may seem counterintuitive in terms of asthma, GGT probably functions primarily in GSH homeostasis, which is known to be critical for protection against oxidative damage in lung.9,37
Previous studies have shown that LTD4 can mediate eosinophil infiltration, mucus production, and bronchial contraction.3
Because GGL- and GGL/GGT-deficient mice do not synthesize LTD4 after CF challenge (Figure 5)
and develop eosinophil infiltration and mucus production similar to those in CF-treated WT mice and GGT-deficient mice (data not show), it follows that LTC4 plays a role similar to that of LTD4 in asthma. We previously observed a delayed neutrophil infiltration in Zymosan A-induced peritonitis in the GGL-deficient mice.4
In the current study, we found similar total and differential cell counts and mucin levels in BALF from WT, GGL-, GGT-, and GGL/GGT-deficient mice. The difference between these two studies may reflect differences in inducing agent, physiological location, or the inflammatory process.
In summary, GGL appears to have evolved from GGT to cleave a substrate related to inflammatory processes. Most of the structural features of GGT have been preserved in the structure of GGL, but its expression appears to be restricted to some endothelial cells. The importance of GGL in pathophysiological events isunderscored by its role in regulating metabolism of Cyst LTs in vivo.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant ES 07827 and a grant from the Sandler Program for Asthma Research.
Accepted for publication April 29, 2002.
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-glutamyl leukotrienase results in disruption of leukotriene D4 synthesis in vivo and attenuation of the acute inflammatory response. Mol Cell Biol 2001, 21:5389-5395-glutamyl transpeptidase; concomitant formation of leukotriene D4 and -glutamyl amino acids. Proc Natl Acad Sci USA 1982, 79:1088-1091-Glutamyl transpeptidase: what does the organization and expression of a multipromoter gene tell us about its functions? Am J Pathol 1995, 147:1175-1185[Abstract]
-glutamyl transpeptidase-deficient mice. J Biol Chem 1997, 272:12305-12310-Glutamyl leukotrienase, a -glutamyl transpeptidase gene family member, is expressed primarily in spleen. J Biol Chem 1998, 273:28277-28285-Glutamyl leukotrienase cleavage of leukotriene C4. Honn KV Marrett LJ Nigam S Dennis EA eds. Eicosanoid and Other Bioactive Lipids in Cancer, Inflammation, and Injury, ed 4 1999:pp 301-306 Kluwer Academic/Plenum Publisher, New York
-glutamyl transpeptidase-deficient mice. Proc Natl Acad Sci USA 1996, 93:7923-7926-Glutamyl transpeptidase. Methods Enzymol 1981, 77:237-253[Medline]
-Glutamyl transpeptidase, a lymphoid cell-surface marker: relationship to blastogenesis, differentiation, and neoplasia. Proc Natl Acad Sci USA 1976, 73:2414-2418-Glutamyl transpeptidase: molecular cloning and structural and functional features of a blood-brain barrier marker protein. Pardiridge WM eds. The Blood-Brain Barrier. 1993:pp 339-369 Raven, New York
-glutamyl transpeptidase and alkaline phosphatase. Anat Embryol 1999, 199:29-34[Medline]
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astn F: Effect of -glutamyl transpeptidase inhibitors on the transport of glutamate into neuronal and glial primary cultures. Int J Dev Neurochem 1992, 10:225-230
-glutamyl transpeptidase-deficient mice. Lung 2002, 179:1-12This article has been cited by other articles:
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), 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.
