Four Distinct Membrane-bound Dipeptidase RNAs Are Differentially Expressed and Show Discordant Regulation with γ-Glutamyl Transpeptidase
1996; Elsevier BV; Volume: 271; Issue: 27 Linguagem: Inglês
10.1074/jbc.271.27.16273
ISSN1083-351X
AutoresGeetha M. Habib, Roberto Barrios, Zheng-Zheng Shi, Michael W. Lieberman,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoMembrane-bound dipeptidase (MBD) participates in the degradation of glutathione by cleaving the cysteinyl-glycine bond of cystinyl bisglycine (oxidized cysteinyl-glycine) following removal of a γ-glutamyl group by γ-glutamyl transpeptidase (GGT). In the mouse, MBD RNA is most abundant in small intestine, kidney, and lung and is represented by four distinct RNA species. These are generated by transcription from two promoters located 6 kilobases apart in the 5′ flanking region of the gene and by the use of two different poly(A) addition sites. Promoter I is used primarily in small intestine and kidney, whereas promoter II is most active in lung and kidney. We found a discordance in the expected co-expression of MBD and GGT; as expected, MBD and GGT are both expressed at high levels in the kidney and small intestine. However, in the lung, MBD is expressed at high levels, whereas GGT is almost undetectable. The reverse is true in the seminal vesicles and fetal liver. Thus, although both enzymes may function in concert to metabolize glutathione in kidney and small intestine, in other tissues they appear to act independently, suggesting that they have independent roles in other biological processes. Membrane-bound dipeptidase (MBD) participates in the degradation of glutathione by cleaving the cysteinyl-glycine bond of cystinyl bisglycine (oxidized cysteinyl-glycine) following removal of a γ-glutamyl group by γ-glutamyl transpeptidase (GGT). In the mouse, MBD RNA is most abundant in small intestine, kidney, and lung and is represented by four distinct RNA species. These are generated by transcription from two promoters located 6 kilobases apart in the 5′ flanking region of the gene and by the use of two different poly(A) addition sites. Promoter I is used primarily in small intestine and kidney, whereas promoter II is most active in lung and kidney. We found a discordance in the expected co-expression of MBD and GGT; as expected, MBD and GGT are both expressed at high levels in the kidney and small intestine. However, in the lung, MBD is expressed at high levels, whereas GGT is almost undetectable. The reverse is true in the seminal vesicles and fetal liver. Thus, although both enzymes may function in concert to metabolize glutathione in kidney and small intestine, in other tissues they appear to act independently, suggesting that they have independent roles in other biological processes. INTRODUCTIONMembrane-bound dipeptidase (MBD 1The abbreviations used are: MBDmembrane-bound dipeptidaseGGTγ-glutamyl transpeptidaseRT-PCRreverse transcriptionpolymerase chain reactionRACErapid amplification of cDNA endskbkilobase(s)bpbase pair(s). ; dehydropeptidase-I; renal dipeptidase; microsomal dipeptidase; EC) was initially identified for its β-lactamase activity and has been shown to catalyze the hydrolysis of a number of dipeptides, including antibiotics such as penem and carbapenem derivatives (1Kozak E.M. Tate S.S. J. Biol. Chem. 1982; 257: 6322-6327Abstract Full Text PDF PubMed Google Scholar, 2Kropp H. Sundelof J.G. Hajdu R. Kahan F.M. Antimicrob. Agents Chemother. 1982; 22: 62-70Crossref PubMed Scopus (335) Google Scholar); it is an ectoenzyme anchored to the plasma membrane by glycosylphosphatidylinositol (3Kenny A.J. Turner A.J. Kenny A.J. Turner A.J. Mammalian Ectoenzymes. Elsevier Science Publishing Co. Inc., New York1987: 329Google Scholar). MBD has also been implicated in the degradation of glutathione. Following removal of a γ-glutamyl group by γ-glutamyl transpeptidase (GGT; EC), MBD is thought to cleave cystinyl bisglycine to its component amino acids. Thus, the sequential action of these two enzymes is believed to be essential in the recycling of GSH via the γ-glutamyl cycle (4Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York1995: 1461Google Scholar, 5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar) and the formation of the mercapaturic acid derivatives of toxins and xenobiotics (1Kozak E.M. Tate S.S. J. Biol. Chem. 1982; 257: 6322-6327Abstract Full Text PDF PubMed Google Scholar, 4Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York1995: 1461Google Scholar, 5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar). The metabolism of peptidyl leukotrienes, prostaglandins, and the transport of cysteine and perhaps other amino acids are other biological processes in which MBD participates (5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar, 6Hannigan M.H. Ricketts W.A. Biochemistry. 1993; 32: 6302-6306Crossref PubMed Scopus (236) Google Scholar, 7Keppler D. Rev. Physiol. Biochem. Pharmacol. 1992; 121: 1-30Crossref PubMed Google Scholar, 8Gonzalez J. Esteller A. Vina J. Glutathione Metabolism and Physiological Functions. CRC Press Inc., Boca Raton, FL1990: 295Google Scholar). Inactivation of these pathways by homologous recombination or inhibitors of enzyme function results in massive thiolurea and reduced levels of plasma cysteine and intracellular GSH (4Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York1995: 1461Google Scholar, 9Griffith O.W. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 268-272Crossref PubMed Scopus (204) Google Scholar, 10Anderson M.E. Bridges R.J. Meister A. Biochem. Biophys. Res. Commun. 1980; 96: 848-853Crossref PubMed Scopus (129) Google Scholar, 11Huber M. Keppler D. Sies H. Ketterer B. Glutathione Conjugation. Academic Press, New York1988: 449Google Scholar). 2M. W. Lieberman, A. L. Wiseman, Z.-Z. Shi, B. Z. Carter, C-N. Ou, R. Barrios, P. Chevez-Barrios, G. M. Habib, J. C. Goodman, S. L. Huang, R. M. Lebovitz, and M. M. Matzuk, submitted for publication. The complete primary sequence of MBD has been elucidated for a variety of mammalian species by cDNA cloning (12Satoh S. Keida Y. Konta Y. Maeda M. Matsumoto Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1163: 234-242Crossref PubMed Scopus (22) Google Scholar, 13Rached E. Hooper N.M. James P. Semenza G. Turner A.J. Biochem. J. 1990; 271: 755-760Crossref PubMed Scopus (38) Google Scholar, 14An S. Schmidt F.J. Campbell B.J. Biochim. Biophys. Acta. 1994; 1226: 337-340Crossref PubMed Scopus (10) Google Scholar). The human MBD gene has been cloned and found to span about 6 kb and consists of nine coding exons (15Satoh S. Kusunoki C. Konta Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1172: 181-183Crossref PubMed Scopus (12) Google Scholar). Recently, Adachi et al. (16Adachi H. Katayama T. Nakazato H. Tsujimoto M. Biochim. Biophys. Acta. 1993; 1163: 42-48Crossref PubMed Scopus (20) Google Scholar) demonstrated the importance of Glu-125 in the catalytic activity of human MBD, and this residue is also conserved in mouse MBD (12Satoh S. Keida Y. Konta Y. Maeda M. Matsumoto Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1163: 234-242Crossref PubMed Scopus (22) Google Scholar) and is present in coding exon 5 (this paper). Because of the potential therapeutic importance of β-lactam antibiotics, most of these efforts appear to have been directed toward understanding the structure-function relationship and the development of better inhibitors of MBD (16Adachi H. Katayama T. Nakazato H. Tsujimoto M. Biochim. Biophys. Acta. 1993; 1163: 42-48Crossref PubMed Scopus (20) Google Scholar, 17Hikida M. Kawashima K. Nishiki K. Furukawa Y. Nishizawa K. Saito I. Kuwao S. Antimicrob. Agents Chemother. 1992; 36: 481-483Crossref PubMed Scopus (111) Google Scholar, 18Keynan S. Hooper N.M. Turner A.J. FEBS Lett. 1994; 349: 50-54Crossref PubMed Scopus (24) Google Scholar).In contrast, other functions of MBD have been less extensively studied (19Curthoys N.P. Vina J. Glutathione Metabolism and Physiological Functions. CRC Press Inc., Boca Raton, FL1990: 217Google Scholar, 20Turner A.J. Turner A.J. Molecular and Cell Biology of Membrane Proteins. Ellis Harwood, New York1990: 129Google Scholar). In view of the importance of MBD in the γ-glutamyl cycle and in the metabolism of leukotrienes and xenobiotics, more information about non-antibiotic aspects of MBD would be welcome. As a first step in the analysis of the regulation of MBD expression and its role in mammalian physiology, we have studied MBD expression in the mouse. We have examined tissue-specific steady-state MBD RNA levels on a comparative basis with GGT, identified four distinct MBD RNA species, and demonstrated that they are encoded by a single MBD gene.DISCUSSIONWe have investigated the expression of MBD in the mouse and found that steady state RNA levels are highest in the small intestine, kidney, and lung. At least four different types of RNA are expressed, and these are generated by a combination of the use of two different promoters and two different poly(A) addition sites. Because we have found that a significant portion (∼40-50%) of lung MBD RNA is not protected in nuclease protection assays by probes specific for type I or type II MBD RNA (Fig. 4C), additional MBD RNAs must exist. It is not clear at present whether these are generated by the use of other promoters or alternative splicing or both. All tissues examined use both poly(A) addition sites but with different frequencies (Fig. 1A), and in kidney at least, all four types of MBD RNA have been identified (Fig. 7). It seems likely that the MBD like GGT and other genes may utilize transcription from different promoters as a strategy to achieve unique patterns of tissue-specific expression (5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar, 27Sepulveda A.R. Carter B.Z. Habib G.M. Lebovitz R.M. Lieberman M.W. J. Biol. Chem. 1994; 269: 10699-10705Abstract Full Text PDF PubMed Google Scholar, 28Toda K. Simpson E.R. Mendelson C.R. Shizula Y. Kilgore M.W. Mol. Endocrinol. 1994; 8: 210-217PubMed Google Scholar, 29Kengaku M. Misawa H. Deguchi T. Mol. Brain Res. 1993; 18: 71-76Crossref PubMed Scopus (78) Google Scholar). It appears that the MBD gene uses these different 5′- and 3′-ends to achieve fine adjustments in mRNA levels, depending on the physiological requirements of each tissue. Whether this heterogeneity of expression has biological importance is not clear.Mouse MBD is a single copy gene consisting of 11 exons and is similar in structure to the human gene (15Satoh S. Kusunoki C. Konta Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1172: 181-183Crossref PubMed Scopus (12) Google Scholar). Using the nomenclature established for human MBD RNA (15Satoh S. Kusunoki C. Konta Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1172: 181-183Crossref PubMed Scopus (12) Google Scholar), type I RNA is derived from exon 1. Type II RNA has its origin ∼6 kb 5′ of this region in an exon that we have designated −1 (Fig. 6). Type II transcripts are spliced at a site 3′ of the origin of type I RNA and common to both types I and II RNA (Fig. 6).Because MBD acts in concert with GGT to cleave GSH into its component amino acids, we evaluated the coexpression of these two genes. In tissues like kidney and small intestine, where significant extracellular GSH degradation occurs, not surprisingly, we have found high expression of both genes (Fig. 1). However, the cellular distribution of MBD and GGT are somewhat different in the kidney (Fig. 2, A and B); while both are expressed in the proximal convoluted tubules, MBD is also expressed in some cortical collecting duct epithelial cells. Although the reason for expression there is unclear, it implies that MBD and GGT function are not always tightly coupled. Additional evidence for this formulation comes from evaluation of expression in lung and seminal vesicles. Small intestine and kidney have high steady state levels of both MBD and GGT RNAs; in contrast, we could not detect GGT expression in mouse lung by Northern analysis or in histochemistry. We know that lung expresses low levels of GGT (∼0.3% of kidney levels) from other studies. 3B. Z. Carter, A. L. Wiseman, R. Orkiszewski, K. Ballard, J. E. Shields, Y. Will, D. J. Reed, C.-N. Ou, and M. W. Lieberman, submitted for publication. 4M. W. Lieberman and A. L. Wiseman, unpublished results. Thus, in lung, MBD appears to function independently of GGT, but its role in lung physiology is at present unclear. Its expression in bronchiolar epithelial cells and Clara cells may indicate its involvement in the detoxification of xenobiotics or leukotriene metabolism in the lung.The reverse situation occurs in seminal vesicle, where GGT is clearly expressed in luminal epithelial cells2 (Fig. 2, E and F), but MBD expression is not detectable. This observation raises the question of whether seminal fluid has high levels of cysteinyl glycine (generated by the action of GGT on GSH) or some other dipeptidase cleaves this dipeptide there. Similarly, fetal liver expresses GGT (Fig. 1; 24Habib G.M. Carter B.Z. Sepulveda A.R. Shi Z.-Z. Wan D.-F. Lebovitz R.M. Lieberman M.W. J. Biol. Chem. 1995; 270: 13711-13715Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar); however, we were unable to detect MBD in this tissue. These observations underscore the discordance of MBD and GGT expression.It is likely that MBD plays multiple roles in biological processes. Its main function in kidney may be the recycling of GSH in conjunction with GGT, while in the intestine it may participate in similar recycling and also play a role in the catabolism of dietary proteins. It is likely that MBD may be involved in the metabolism of eicosanoids, including leukotriene D4. Thus, relative tissue levels of leukotrienes C4, D4, and E4, and the renal and gastrointestinal clearance of these compounds, may be regulated by the relative activities of MBD, GGT, and a leukotrienase that we have recently described.3 In this regard, the absence of MBD in tissues in which GSH metabolism and/or eicosanoid activity is high may tell us as much about the function of this enzyme as its presence. INTRODUCTIONMembrane-bound dipeptidase (MBD 1The abbreviations used are: MBDmembrane-bound dipeptidaseGGTγ-glutamyl transpeptidaseRT-PCRreverse transcriptionpolymerase chain reactionRACErapid amplification of cDNA endskbkilobase(s)bpbase pair(s). ; dehydropeptidase-I; renal dipeptidase; microsomal dipeptidase; EC) was initially identified for its β-lactamase activity and has been shown to catalyze the hydrolysis of a number of dipeptides, including antibiotics such as penem and carbapenem derivatives (1Kozak E.M. Tate S.S. J. Biol. Chem. 1982; 257: 6322-6327Abstract Full Text PDF PubMed Google Scholar, 2Kropp H. Sundelof J.G. Hajdu R. Kahan F.M. Antimicrob. Agents Chemother. 1982; 22: 62-70Crossref PubMed Scopus (335) Google Scholar); it is an ectoenzyme anchored to the plasma membrane by glycosylphosphatidylinositol (3Kenny A.J. Turner A.J. Kenny A.J. Turner A.J. Mammalian Ectoenzymes. Elsevier Science Publishing Co. Inc., New York1987: 329Google Scholar). MBD has also been implicated in the degradation of glutathione. Following removal of a γ-glutamyl group by γ-glutamyl transpeptidase (GGT; EC), MBD is thought to cleave cystinyl bisglycine to its component amino acids. Thus, the sequential action of these two enzymes is believed to be essential in the recycling of GSH via the γ-glutamyl cycle (4Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York1995: 1461Google Scholar, 5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar) and the formation of the mercapaturic acid derivatives of toxins and xenobiotics (1Kozak E.M. Tate S.S. J. Biol. Chem. 1982; 257: 6322-6327Abstract Full Text PDF PubMed Google Scholar, 4Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York1995: 1461Google Scholar, 5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar). The metabolism of peptidyl leukotrienes, prostaglandins, and the transport of cysteine and perhaps other amino acids are other biological processes in which MBD participates (5Lieberman M.W. Barrios R. Carter B.Z. Habib G.M. Lebovitz R.M. Rajagopalan S. Sepulveda A.R. Shi Z.-Z. Wan D.F. Am. J. Pathol. 1995; 147: 1175-1185PubMed Google Scholar, 6Hannigan M.H. Ricketts W.A. Biochemistry. 1993; 32: 6302-6306Crossref PubMed Scopus (236) Google Scholar, 7Keppler D. Rev. Physiol. Biochem. Pharmacol. 1992; 121: 1-30Crossref PubMed Google Scholar, 8Gonzalez J. Esteller A. Vina J. Glutathione Metabolism and Physiological Functions. CRC Press Inc., Boca Raton, FL1990: 295Google Scholar). Inactivation of these pathways by homologous recombination or inhibitors of enzyme function results in massive thiolurea and reduced levels of plasma cysteine and intracellular GSH (4Meister A. Larsson A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Diseases. McGraw-Hill Inc., New York1995: 1461Google Scholar, 9Griffith O.W. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 268-272Crossref PubMed Scopus (204) Google Scholar, 10Anderson M.E. Bridges R.J. Meister A. Biochem. Biophys. Res. Commun. 1980; 96: 848-853Crossref PubMed Scopus (129) Google Scholar, 11Huber M. Keppler D. Sies H. Ketterer B. Glutathione Conjugation. Academic Press, New York1988: 449Google Scholar). 2M. W. Lieberman, A. L. Wiseman, Z.-Z. Shi, B. Z. Carter, C-N. Ou, R. Barrios, P. Chevez-Barrios, G. M. Habib, J. C. Goodman, S. L. Huang, R. M. Lebovitz, and M. M. Matzuk, submitted for publication. The complete primary sequence of MBD has been elucidated for a variety of mammalian species by cDNA cloning (12Satoh S. Keida Y. Konta Y. Maeda M. Matsumoto Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1163: 234-242Crossref PubMed Scopus (22) Google Scholar, 13Rached E. Hooper N.M. James P. Semenza G. Turner A.J. Biochem. J. 1990; 271: 755-760Crossref PubMed Scopus (38) Google Scholar, 14An S. Schmidt F.J. Campbell B.J. Biochim. Biophys. Acta. 1994; 1226: 337-340Crossref PubMed Scopus (10) Google Scholar). The human MBD gene has been cloned and found to span about 6 kb and consists of nine coding exons (15Satoh S. Kusunoki C. Konta Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1172: 181-183Crossref PubMed Scopus (12) Google Scholar). Recently, Adachi et al. (16Adachi H. Katayama T. Nakazato H. Tsujimoto M. Biochim. Biophys. Acta. 1993; 1163: 42-48Crossref PubMed Scopus (20) Google Scholar) demonstrated the importance of Glu-125 in the catalytic activity of human MBD, and this residue is also conserved in mouse MBD (12Satoh S. Keida Y. Konta Y. Maeda M. Matsumoto Y. Niwa M. Kohsaka M. Biochim. Biophys. Acta. 1993; 1163: 234-242Crossref PubMed Scopus (22) Google Scholar) and is present in coding exon 5 (this paper). Because of the potential therapeutic importance of β-lactam antibiotics, most of these efforts appear to have been directed toward understanding the structure-function relationship and the development of better inhibitors of MBD (16Adachi H. Katayama T. Nakazato H. Tsujimoto M. Biochim. Biophys. Acta. 1993; 1163: 42-48Crossref PubMed Scopus (20) Google Scholar, 17Hikida M. Kawashima K. Nishiki K. Furukawa Y. Nishizawa K. Saito I. Kuwao S. Antimicrob. Agents Chemother. 1992; 36: 481-483Crossref PubMed Scopus (111) Google Scholar, 18Keynan S. Hooper N.M. Turner A.J. FEBS Lett. 1994; 349: 50-54Crossref PubMed Scopus (24) Google Scholar).In contrast, other functions of MBD have been less extensively studied (19Curthoys N.P. Vina J. Glutathione Metabolism and Physiological Functions. CRC Press Inc., Boca Raton, FL1990: 217Google Scholar, 20Turner A.J. Turner A.J. Molecular and Cell Biology of Membrane Proteins. Ellis Harwood, New York1990: 129Google Scholar). In view of the importance of MBD in the γ-glutamyl cycle and in the metabolism of leukotrienes and xenobiotics, more information about non-antibiotic aspects of MBD would be welcome. As a first step in the analysis of the regulation of MBD expression and its role in mammalian physiology, we have studied MBD expression in the mouse. We have examined tissue-specific steady-state MBD RNA levels on a comparative basis with GGT, identified four distinct MBD RNA species, and demonstrated that they are encoded by a single MBD gene.
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