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. 1998 Oct 1;335(Pt 1):111–117. doi: 10.1042/bj3350111

Cloning and expression of mouse legumain, a lysosomal endopeptidase.

J M Chen 1, P M Dando 1, R A Stevens 1, M Fortunato 1, A J Barrett 1
PMCID: PMC1219758  PMID: 9742219

Abstract

Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain.

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Selected References

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  1. Abe Y., Shirane K., Yokosawa H., Matsushita H., Mitta M., Kato I., Ishii S. Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis. J Biol Chem. 1993 Feb 15;268(5):3525–3529. [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Anastasi A., Brown M. A., Kembhavi A. A., Nicklin M. J., Sayers C. A., Sunter D. C., Barrett A. J. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem J. 1983 Apr 1;211(1):129–138. doi: 10.1042/bj2110129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barrett A. J. Cellular proteolysis. An overview. Ann N Y Acad Sci. 1992 Dec 31;674:1–15. doi: 10.1111/j.1749-6632.1992.tb27472.x. [DOI] [PubMed] [Google Scholar]
  5. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  6. Berg T., Gjøen T., Bakke O. Physiological functions of endosomal proteolysis. Biochem J. 1995 Apr 15;307(Pt 2):313–326. doi: 10.1042/bj3070313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  8. Bohley P., Seglen P. O. Proteases and proteolysis in the lysosome. Experientia. 1992 Feb 15;48(2):151–157. doi: 10.1007/BF01923508. [DOI] [PubMed] [Google Scholar]
  9. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  10. Chen J. M., Changco A., Brown M. A., Barrett A. J. Immunolocalization of thimet oligopeptidase in chicken embryonic fibroblasts. Exp Cell Res. 1995 Jan;216(1):80–85. doi: 10.1006/excr.1995.1010. [DOI] [PubMed] [Google Scholar]
  11. Chen J. M., Dando P. M., Rawlings N. D., Brown M. A., Young N. E., Stevens R. A., Hewitt E., Watts C., Barrett A. J. Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase. J Biol Chem. 1997 Mar 21;272(12):8090–8098. doi: 10.1074/jbc.272.12.8090. [DOI] [PubMed] [Google Scholar]
  12. Chen J. M., Little C. D. Cells that emerge from embryonic explants produce fibers of type IV collagen. J Cell Biol. 1985 Oct;101(4):1175–1181. doi: 10.1083/jcb.101.4.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. D'Souza S. E., Ginsberg M. H., Plow E. F. Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci. 1991 Jul;16(7):246–250. doi: 10.1016/0968-0004(91)90096-e. [DOI] [PubMed] [Google Scholar]
  14. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dalton J. P., Hola-Jamriska L., Brindley P. J. Asparaginyl endopeptidase activity in adult Schistosoma mansoni. Parasitology. 1995 Dec;111(Pt 5):575–580. doi: 10.1017/s0031182000077052. [DOI] [PubMed] [Google Scholar]
  16. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  17. 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. 1993 Nov;5(11):1651–1659. doi: 10.1105/tpc.5.11.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kembhavi A. A., Buttle D. J., Knight C. G., Barrett A. J. The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays. Arch Biochem Biophys. 1993 Jun;303(2):208–213. doi: 10.1006/abbi.1993.1274. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lusson J., Benjannet S., Hamelin J., Savaria D., Chrétien M., Seidah N. G. The integrity of the RRGDL sequence of the proprotein convertase PC1 is critical for its zymogen and C-terminal processing and for its cellular trafficking. Biochem J. 1997 Sep 15;326(Pt 3):737–744. doi: 10.1042/bj3260737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maunsbach A. B. Isolation of kidney lysosomes. Methods Enzymol. 1974;31:330–339. doi: 10.1016/0076-6879(74)31035-x. [DOI] [PubMed] [Google Scholar]
  22. Nakayama K. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J. 1997 Nov 1;327(Pt 3):625–635. doi: 10.1042/bj3270625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rawlings N. D., Barrett A. J. FLUSYS: a software package for the collection and analysis of kinetic and scanning data from Perkin-Elmer fluorimeters. Comput Appl Biosci. 1990 Apr;6(2):118–119. doi: 10.1093/bioinformatics/6.2.118. [DOI] [PubMed] [Google Scholar]
  24. Rawlings N. D., Barrett A. J. Families of cysteine peptidases. Methods Enzymol. 1994;244:461–486. doi: 10.1016/0076-6879(94)44034-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ruoslahti E., Pierschbacher M. D. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
  26. 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. 1994 Sep;116(3):541–546. doi: 10.1093/oxfordjournals.jbchem.a124559. [DOI] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. el Meanawy M. A., Aji T., Phillips N. F., Davis R. E., Salata R. A., Malhotra I., McClain D., Aikawa M., Davis A. H. Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites. Am J Trop Med Hyg. 1990 Jul;43(1):67–78. doi: 10.4269/ajtmh.1990.43.67. [DOI] [PubMed] [Google Scholar]

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