Leukotriene D4 and cystinyl-bis-glycine metabolism in membrane-bound dipeptidase-deficient mice

Abstract

We have developed mice deficient in membrane-bound dipeptidase (MBD, EC 3.4.13.19), the enzyme believed to be responsible for the conversion of leukotriene D4 (LTD4) to leukotriene E4 (LTE4). The MBD mutation generated by us was demonstrated to be a null mutation by Northern blot analysis and the absence of beta-lactamase activity in lung, kidney, small intestine, and heart. MBD gene deletion had no effect on viability or fertility. The mutant mice retain partial ability to convert LTD4 to LTE4, ranging from 80-90% of the wild-type values in small intestine and liver to 16% in kidney and 40% in lung, heart, and pancreas. MBD is also believed to function consecutively after gamma-glutamyl transpeptidase to cleave cystinyl-bis-glycine (cys-bis-gly) generated from glutathione cleavage. Our data indicate that kidney homogenates from MBD-deficient mice retain approximately 40% of their ability to cleave cys-bis-gly, consistent with only modest elevations (3-5-fold) of cys-bis-gly in urine from MBD-deficient mice. These observations demonstrate that the conversion of LTD4 to LTE4 and the degradation of cys-bis-gly are catalyzed by at least two alternative pathways (one of which is MBD) that complement each other to varying extents in different tissues.

Figure 1

Targeting strategy for homologous recombination at the MBD locus. (A) Partial restriction maps of the endogenous MBD gene, deletion targeting vector, and the recombinant mutant allele are shown. Coding sequences and introns that are replaced by homologous recombination are indicated by the following: vertical open box, noncoding exon of type I MBD RNA; vertical hatched box, common splice site necessary for all MBD RNAs; vertical solid box, part of the coding exon 1 containing the initiation site ATG and the introns (thin lines). Solid horizontal boxes represent the 5′ and 3′ arms. A phosphoglycerate kinase-hypoxanthine phosphoribosyltransferase expression cassette was used as the positive selectable marker, and an MC1tk expression cassette was used as a negative selectable marker as shown (31). B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; Sp, SphI. (B) Southern blot analysis of BamHI-digested tail DNA; hybridization was performed using a 3′ external probe. wt, wild-type; +/−, heterozygote, −/−, homozygote. (C) Northern blot analysis of total RNA from various tissues of wild-type and MBD-deficient mice. The blots were hybridized using a 620-bp ³²P-labeled mouse MBD cDNA probe corresponding to bases +610 to +1230 of the mouse MBD cDNA (28). The blot was stripped and reprobed with glyceraldehyde-3-phosphate dehydrogenase cDNA to verify equal loading in all lanes.

Figure 2

Analysis of LTD₄ metabolism by tissue homogenates of kidney, lung, and heart of wild-type (Left) and MBD-deficient (Right) mice by HPLC. The reaction conditions are as described under Materials and Methods. The peaks labeled as 1 and 2 refer to LTD₄ and LTE₄, respectively. For kidney (Top), 100 μg of protein and an incubation time of 30 min was used. Lung extracts were assayed using 50 μg of protein for 30 min (Middle). Heart extracts were incubated for 75 min and contained 100 μg of protein (Bottom).

Figure 3

Time course of LTD₄ metabolism by small intestine, liver, and pancreas of wild-type and MBD-deficient mice. Total homogenates from these tissues were assayed for LTD₄ to LTE₄ conversion as described under Materials and Methods. For all assays, 100 μg of protein was used. Squares indicate wild-type and diamonds denote MBD-deficient assays. All assays were carried out at 37°C.

Figure 4

Analysis of cys-bis-gly metabolism in kidney homogenates from wild-type and MBD-deficient mice. For details on assay conditions and sample preparation, see Materials and Methods. (A) HPLC profile of cys-bis-gly metabolism by homogenates of wild-type kidney. The peaks labeled 1, 2, and 3 represent cys-gly, cysteine, and the internal standard γ-glu-glu, respectively. (B) HPLC profile of cys-bis-gly metabolism in kidney homogenates from MBD-deficient mice.