Abstract
Asparaginyl endopeptidase is a cysteine endopeptidase that has strict substrate specificity toward the carboxy side of asparagine residues. Vigna mungo processing enzyme 1, termed VmPE-1, occurs in the cotyledons of germinated seeds of V. mungo, and is possibly involved in the post-translational processing of a vacuolar cysteine endopeptidase, designated SH-EP, which degrades seed storage protein. VmPE-1 also showed a substrate specificity to asparagine residues, and its enzymatic activity was inhibited by NEM but not E-64. In addition, purified VmPE-1 had a potential to process the recombinant SH-EP precursor to its intermediate in vitro. cDNA clones for VmPE-1 and its homologue, named VmPE-1A, were identified and sequenced, and their expressions in the cotyledons of V. mungo seedlings and other organs were investigated. VmPE-1 mRNA and SH-EP mRNA were expressed in germinated seeds at the same stage of germination although the enzymatic activity of VmPE-1 rose prior to that of SH-EP. The level of VmPE-1A mRNA continued increasing as germination proceeded. In roots, stems and leaves of fully grown plants, and in hypocotyls, VmPE-1 and VmPE-1A were little expressed. We discuss possible functions of VmPE-1 and VmPE-1A in the cotyledons of germinated seeds.
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Abe Y, Shirane K, Yokosawa H, Matsushita H, Mitta M, Kato I, Ishii S: Asparaginyl endopeptidase of jack bean seeds. J Biol Chem 268: 3525–3529 (1993).
Akasofu H, Yamauchi D, Mitsuhashi W, Minamikawa T: Nucleotide sequence of cDNA for sulfhydryl-endopeptidase (SH-EP) from cotyledons of germinating Vigna mungo seeds. Nucl Acids Res 17: 6733.
Alonso JM, Granell A: A putative vacuolar processing enzyme is regulated by ethylene and also during fruit riping in Citrus fruit. Plant Physiol 109: 541–547 (1995).
Becker C, Shutov AD, Nong VH, Senyuk VI, Jung R, Horstmann C, Fischer J, Nielsen NC, Müntz K: Purification, cDNA cloning and characterization of proteianse B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds. Eur J Biochem 228: 456–462 (1995).
Chen J-M, Dando PM, Rawlings ND, Brown MA, Young NE, Stevens RA, Hewitt E, Watts C, Barret AJ: Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase. J Biol Chem 272: 8090–8098 (1997).
Chrispeels MJ: Sorting of proteins in the secretory system. Annu Rev Plant Physiol Plant Mol Biol 42: 21–53 (1991).
Chrispeels MJ, Raikhel NV: Short peptide domains target proteins to plant vacuoles. Cell 68: 613–616 (1992).
Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791 (1985).
Hara-Nishimura I, Inoue K, Nishimura M: A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett 294: 89–93 (1991).
Hara-Nishimura I, Takeuchi Y, Nishimura M: Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni. Plant Cell 5: 1651–1659 (1993).
Haraguchi H, Yamauchi D, Minamikawa T: Multiple forms of acid phosphatase in cotyledons of Vigna mungo seedlings: immunological detection and quantitation. Plant Cell Physiol 31: 917–923 (1990).
Holwerda BC, Galvin NJ, Baranski TJ, Rogers JC: In vitro processing of aleurain, a barley vacuolar thiol protease. Plant Cell 2: 1091–1106 (1990).
Ishii S: Legumain: asparaginyl endopeptidase. Meth Enzymol 244: 604–615.
James MNG, Sielecki AR: Molecular structure of an aspartic proteinase zymogen, porcine pepsinogen, at 1.8 Å resolution. Nature 319: 33–38 (1986).
Kassell B, Kay J: Zymogens of proteolytic enzymes. Science 180: 1022–1027 (1973).
Kembhavi AA, Buttle DJ, Knight CG, Barrett AJ: The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays. Arch Biochem Biophys 303: 208–213 (1993).
Kinoshita T, Nishimura M, Hara-Nishimura I: The sequence and expression of theγ-VPE gene, one member of a family of three genes for vacuolar processing enzymes in Arabidopsis thaliana. Plant Cell Physiol 36: 1555–1562 (1995).
Kinoshita T, Nishimura M, Hara-Nishimura I: Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana. Plant Mol Biol 29: 81–89 (1995).
Koehler SM, Ho T-HD: Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layars. Plant Cell 2: 769–783 (1990).
Kuheji R, Dolinar M, Pungercar J., Turk V: The preparation of catalytically active human cathepsin B from its precursor expressed in Eschericia coli in the form of inclusion bodies. Eur J Biochem 229: 533–539 (1995).
Mach L, Mort JS, Glössl J: Maturation of human procathepsin B. J Biol Chem 269: 13030–13035 (1994).
Mitsuhashi W, Koshiba T, Minamikawa T: Separation and characterization of two endopeptidases from cotyledons of germinating Vigna mungo seeds. Plant Physiol 80: 628–634 (1986).
Mitsuhashi W, Minamikawa T: Synthesis and posttranslational activation of sulfhydryl-endopeptidase in cotyledons of germinating Vigna mungo seeds. Plant Physiol 89: 274–279 (1989).
Müntz K: Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds. J Exp Bot 298: 605–622 (1996).
Okamoto T, Minamikawa T: Purification of a processing enzyme (VmPE-1) that is involved in post-translational processing of a plant cysteine endopeptidase (SH-EP). Eur J Biochem 231: 300–305 (1995).
Okamoto T, Miura-Izu Y, Ishii S, Minamikawa T: Asparaginyl endopeptidase in developing and germinating legume seeds: immunological detection and quantitation. Plant Sci 115: 49–57 (1996).
Okamoto T, Nakayama H, Seta K, Isobe T, Minamikawa T: Posttranslational processing of a carboxy-terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase (SH-EP). FEBS Lett 351: 31–34 (1994).
Rowan AD, Mason P, Mach L, Mort JS: Rat procathepsin B. J Biol Chem 267: 15993–15999 (1992).
Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenic trees. Mol Biol Evol 4: 406–425 (1987).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
Shimada T, Hiraiwa N, Nishimura M, Hara-Nishimura I: Vacuolar processing enzyme of soybean that converts proproteins to the corresponding mature forms. Plant Cell Physiol 35: 713–718 (1994).
Smith SM, Gottesman MM: Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli. J Biol Chem 264: 20487–20495 (1989).
Suzuki Y, Minamikawa T: On the role of stored mRNA in protein synthesis in embryonic axes of germinating Vigna unguiculata seeds. Plant Physiol 79: 327–331 (1985).
Takeda O, Miura Y, Mitta M, Matsushita H, Kato I, Abe Y, Yokosawa H, Ishii S: Isolation and analysis of cDNA encoding a precursor of Canavalia ensiformis asparaginyl endopeptidase (legumain). J Biochem 116: 541–546 (1994).
Vernet T, Khouri HE, Laflamme P, Tessier DC, Musil R, Gour-Salin BJ, Storer AC, Thomas DY: Processing of the papain precursor. J Biol Chem 266: 21451–21457 (1991).
Yamauchi D, Akasofu H, Minamikawa T: Cysteine endopeptidase from Vigna mungo: gene structure and expression. Plant Cell Physiol 33: 789–797 (1992).
Yamauchi D, Takeuchi H, Minamikawa T: Structure and expression of_-amylase gene from Vigna mungo. Plant Cell Physiol 35: 705–711 (1994).
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Okamoto, T., Minamikawa, T. Molecular cloning and characterization of Vigna mungo processing enzyme 1 (VmPE-1), an asparaginyl endopeptidase possibly involved in post-translational processing of a vacuolar cysteine endopeptidase (SH-EP). Plant Mol Biol 39, 63–73 (1999). https://doi.org/10.1023/A:1006170518002
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DOI: https://doi.org/10.1023/A:1006170518002