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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2003 as doi:10.1096/fj.02-0899fje. |
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,2
Departments of
* Pathology and
Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
2Correspondence: Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: mikel{at}bcm.tmc.edu
SPECIFIC AIMS
Membrane-bound dipeptidase-1 (MBD-1) is known to catalyze the conversion of leukotriene D4 (LTD4) to leukotriene E4 (LTE4) and the cleavage of cystinyl-bis-glycine (cys-bis-gly); however, MBD-1-deficient mice retain the ability to catalyze both reactions, suggesting that additional dipeptidases can perform these functions. The aim of the present experiments was to identify and characterize additional members of the MBD family.
PRINCIPAL FINDINGS
1. Identification of two additional MBDs (MBD-2 and MBD-3) and determination of their DNA sequences and deduced amino acid sequences
Mouse spleen cDNA from an MBD-1-deficient mouse was screened by PCR using primers based on sequence information from EST H10870 that had sequence similarity to the known MBD-1. The resultant cDNA (MBD-2) comprises a single open reading frame of 1734 bp, a 5' untranslated region of 56 bp, a 9 bp 3' untranslated region, and a poly(A) signal (GenBank acc. no. AF488552). The predicted open reading frame encodes a protein of 578 amino acids with two potential N-glycosylation sites (Fig. 1
). A similar approach was used to obtain additional family members from an MBD-1-deficient mouse testis cDNA that resulted in the cloning of a full-length cDNA (MBD-3). This cDNA has a single open reading frame of 1479 bp, a 128 bp 5' untranslated region, a 3' untranslated region of 72 bp, and a poly(A) tail (GenBank acc. no. AF488553). The predicted protein sequence consists of 493 amino acids with six putative N-glycosylation sites. Overall, MBD-2 and MBD-3 show 33% and 39% identity, respectively, with MBD-1 at the amino acid level. MBD-2 and MBD-3 show an overall amino acid identity of 55%. There are three putative N-glycosylation sites in MBD-1, 2 in MBD-2, and 6 in MBD-3. An alignment of amino acid sequences of all three MBDs reveals that two potential N-glycosylation sites are conserved between MBD-2 and MBD-3 and six cysteine residues are conserved among all three MBDs.
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2. MBD-1, MBD-2, and MBD-3 genes are tightly linked on the same chromosome
DNA gel blot analysis of mouse kidney DNA digested with various restriction enzymes, and MBD-1, MBD-2, and MBD-3 cDNA probes indicated that all three are single copy genes. Analysis of MBD-1 genomic phages using an MBD-2 cDNA probe revealed that MBD-2 is located 
5 kb upstream of MBD-1. MBD-2 was cloned from a 
Fix II genomic library using MBD-2 cDNA probes. Screening of MBD-2 genomic phages with an MBD-3 cDNA probe demonstrated that MBD-3 is located 12 kb upstream of MBD-2.
3. MBD-1 and MBD-2 are widely expressed whereas MBD-3 has a more restricted pattern of expression
Comparison of steady mRNA levels of MBD-1, MBD-2, and MBD-3 by Northern analysis using type-specific cDNA probes indicated that MBD-1 was expressed in heart, lung, skeletal muscle, and kidney (1.6 kb and 2.2 kb) (Fig. 2
). Liver expressed only the 2.2 kb mRNA whereas 2.2 and 1.3 kb mRNAs were both detectable in the testis. We did not detect MBD-1 expression in spleen and brain. Heart and lung expressed two species of MBD-2 RNA (2.0 and 1.6 kb) whereas testis expressed only the 2.0 kb mRNA. Liver, spleen, and skeletal muscle expressed very low levels of MBD-2 RNA (visible upon longer exposure) whereas expression was undetectable in kidney and brain. MBD-3 was detectable only in testis (1.7 kb). These differences could arise from differential utilization of poly(A) sites and variations in the 5' untranslated region. The blot was stripped and reprobed with ß-actin cDNA to correct for differences in loading. The expression levels of specific RNAs were quantified by PhosphorImaging analysis and normalized to the expression of ß-actin RNA.
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4. Recombinant MBD-2 and MBD-3 share functional characteristics with MBD-1
MBD-1 along with MBD-2 cloned from MBD-1-deficient spleen and MBD-3 cloned from MBD-1-deficient testis were subcloned into pCMV vectors under the control of the cytomegalovirus (CMV) promoter and transfected into COS-7 cells. We chose COS-7 cells for transfection because we could not detect any endogenous dipeptidase activity in the untransfected cells as measured by the cleavage of LTD4 to LTE4. Recombinant MBD-1 converted LTD4 to LTE4 in a time-dependent fashion (specific activity, 89.6±15.4 nmol·mg protein–1·h–1, mean±SD, n=3). MBD-2 also hydrolyzed LTD4 to LTE4 in a time-dependent fashion (specific activity, 72.6±19.1 nmol·mg protein–1·h–1, mean±SD, n=3). We did not detect any LTD4-to-LTE4 cleavage by MBD-3.
The only known physiological substrate for MBD-1 other than LTD4 is cys-bis-gly. We assayed recombinant MBD-1, MBD-2, and MBD-3 for their ability to cleave cys-bis-gly. MBD-1 cleaved cys-bis-gly (specific activity of cleavage, 908 µmol·mg protein–1·h–1) in a time-dependent manner. MBD-2 had no activity against cys-bis-gly whereas MBD-3 can cleave cys-bis-gly (specific activity of conversion 30.9 µmol·mg protein–1·h–1). Of the three enzymes we tested, only MBD-1 was capable of hydrolyzing ß-lactam substrates.
Similar to MBD-1, MBD-2- and MBD-3-specific activities can be enriched in the supernatant 10- to 12-fold by treatment with phosphatidylinositol-specific phospholipase C, indicating they are anchored on the plasma membrane by a glycosylphosphatidylinositol linkage. The activities of the three enzymes are inhibitable by penicillamine (90% inhibition at 1 mM), suggesting that, like MBD-1, MBD-2 and MBD-3 are metalloproteases.
The apparent Kms of MBD-1 and MBD-2 for LTD4 were determined to be 10 and 5 µM, respectively. MBD-1 and MBD-3 had apparent Kms of 2.5 and 0.45 mM, respectively, with cys-bis-gly as the substrate.
CONCLUSIONS
The present study describes the characterization of two additional mouse cDNAs that are not only structurally similar to MBD-1, but also code for MBD-1-like activities. MBD-2 and MBD-3 map immediately upstream of the mouse MBD-1 gene, indicating that all three genes are tightly linked and belong to the same gene family. Attempts to find additional family members through a GenBank search and by RT-PCR using primers specific for mouse MBD-1, 2, and 3 failed to reveal any other related genes.
Comparison of mouse MBD sequences against the GenBank revealed that mouse MBD-2 has a high sequence identity to the human cDNA (predicted identity 80% at the amino acid level, GenBank acc. no. AJ295149). Mouse MBD-3 is highly similar to a human cDNA (predicted identity 68% at the amino acid level, GenBank acc. no. AJ291679), suggesting they are homologues.
Because MBD-1 is undetectable in spleen, it appears that only MBD-2 cleaves LTD4 and other GSH conjugates in spleen, where it may also participate in immune/inflammatory processes involving leukotrienes. These MBDs are both expressed in lung and are likely to be involved in the clearance of LTD4, the most potent of the leukotrienes in asthma. This concept is supported by the observation that the apparent Km values of MBD-1 and MBD-2 for LTD4 are similar (10 µM vs. 5 µM). All three MBDs are expressed at relatively high levels in the testis, but it is unclear what role, if any, they play in the testicular function.
Studies of the properties of MBD-3 indicate it can hydrolyze cys-bis-gly but not LTD4. Determination of Km values suggests that MBD-1 and MBD-3 have similar capacities to cleave cys-bis-gly. Although MBD-3 can cleave cys-bis-gly, it is not clear whether this is the preferred endogenous substrate for MBD-3 in the testis.
MBD-1 is the only enzyme known to date that is capable of hydrolyzing ß-lactam substrates. It is unclear whether any of these MBDs have additional physiological substrates.
In conclusion, we have identified and characterized two additional members of the MBD family that differ in their tissue distribution and substrate preference. It should now be possible to delineate their roles in pathophysiological processes. It is possible that MBD-1 and MBD-2 may participate in immune/inflammatory processes like asthma, and it is known from our previous work that MBD-1 is involved in cys-bis-gly cleavage in the kidney. It is interesting to imagine why all three MBDs are expressed in testis, since the need for cleavage of LTD4 and cys-bis-gly in testis remains unclear.
A schematic representation summarizing the reactions catalyzed by the MBDs and other members of the 
-glutamyl cycle is shown in Fig. 3
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0899fje; doi: 10.1096/fj.02-0899fje 
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