Metabolism of Leukotriene C4 in γ-Glutamyl Transpeptidase-deficient Mice

Abstract

LTC 4 -converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC 4 , S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (␥-glutamyl-p-nitroanilide and ␥-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC 4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named ␥-glutamyl leukotrienase.Peptidyl leukotrienes, cysteine-containing derivatives of arachidonic acid, are potent inducers of airway constriction, vasoconstriction, smooth muscle contraction, edema, and inflammation (1-4). LTC 4 1 is formed by conjugation of leukotriene A 4 with GSH (5) and is known to be cleaved by GGT, which removes the glutamyl moiety to form LTD 4 (6). LTC 4 conversion to LTD 4 has long been thought to be mediated solely by GGT (7,8). Recently, however, the existence of an activity termed GGT-rel has been identified in humans (9). GGT-rel shares an overall 40% amino acid sequence identity with human GGT and is capable of cleaving the ␥-glutamyl linkage of LTC 4 , but it is unable to hydrolyze synthetic substrates that are commonly used for assaying GGT. This activity has been reported to be absent in mice (9).The role of GGT in leukotriene metabolism is of interest because of the great potency of these compounds in responses to injury. However, the distribution of GGT activity and the sites of peptidyl leukotriene actions are not concordant. In the mouse, GGT tends to be expressed at very high levels in epithelia concerned with catabolism of GSH and reabsorption of its constituent amino acids (kidney and small intestine) and other ductular and secretory epithelia (pancreas and seminal vesicles) (7, 10 -12). It is characteristically low in organs in which leukotrienes may play a role in responses to injury (e.g. lung, heart, and lymphoid tissue (spleen)). In addition, the types of responses mediated by the peptidyl leukotrienes (vasoconstriction, bronchoconstriction, increases in vascular permeability, and mucus formation) do not occur in close proximity to sites where GGT is most abundant (13). Although there are reports of GGT activity in endothelium (14 -16), the discordance between peptidyl leukotriene targets and GGT activity raises the possibility that there are other LTC 4 -cleaving enzymes in the mouse.We have recently used homologous recombination to inactivate GGT in the mouse (12). Mice homozygous for th...

Leukotriene D 4 and cystinyl-bis-glycine metabolism in membrane-bound dipeptidase-deficient mice

“…The remaining cystinyl-bis-glycine and its conversion products were then incubated with 5 l of 10 mM DTT to convert them to cysteinyl glycine and cysteine. The samples were derivatized with 2,4-dinitroflurobenzene and analyzed by reversed-phase ion exchange HPLC as described previously (8,36).…”

“…They are generated by the conjugation of the epoxide intermediate LTA 4 with glutathione (GSH) to form LTC 4 (6). This compound is degraded by the action of ␥-glutamyl transpeptidase (GGT, EC 2.3.2.2) and the more recently characterized enzyme ␥-glutamyl leukotrienase; these enzymes cleave the ␥-glutamyl moiety from LTC 4 to form the cysteinyl glycine conjugate of LTA 4 (7,8). LTD 4 is then converted to LTE 4 by a dipeptidase.…”

Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

“…GGT also cleaves exogenous glutathione for use as a cysteine and nitrogen source in Escherichia coli, yeast, and mammalian cells (3)(4)(5). In mammals, GGT is involved in the conversion of leukotriene C 4 to D 4 , although GGT is not the only enzyme that catalyzes this reaction (6,7). GGT is a key enzyme in glutathione metabolism, and genetic diseases of GGT deficiency, including glutathionemia and glutathionuria, are associated with mental retardation (8,9).…”

Donor substrate specificity of bovine kidney gamma-glutamyltransferase

“…HPLC was used to monitor the reactions with LTC 4 , GSH and GSSG; spectrophotometric analysis was used for GpNA andglutamyl-4-methoxy-2-naphthylamide. It was concluded that there was enzyme activity present that was able to cleave LTC 4 , and was named γ-glutamylleukotrienase (GGL) (Carter et al, 1997). Other physiological donor substrates such as GSH, GSSG, or chromogenic donor substrates such as GpNA and -glutamyl-4-methoxy-2-naphthylamide were not hydrolyzed by GGL (Carter et al, 1997).…”

“…Carter and colleagues studied GGT-knock-out mice that were generated by embryonic stem cell technology (Lieberman et al, 1996;Carter et al, 1997). A recombinant plasmid carrying the mouse GGT-knock-out gene was transfected into embryonic stem cells; transformed embryonic stem cells were inserted into mouse blastocysts; blastocysts were implanted into a female mouse (Lieberman et al, 1996).…”