{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T12:07:35Z","timestamp":1762603655231},"reference-count":35,"publisher":"Portland Press Ltd.","issue":"3","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2010,6,1]]},"abstract":"<jats:p>Legumain has been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates. The present study reports the first characterization of legumain cDNA from the protochordate Branchiostoma belcheri. The deduced 435-amino-acid-long protein is structurally characterized by the presence of a putative N-terminal signal peptide, a peptidase_C13 superfamily domain with the conserved Lys123-Gly124-Asp125 motif and catalytic dyad His153 and Cys195 and two potential Asn-glycosylation sites at Asn85 and Asn270. Phylogenetic analysis demonstrates that B. belcheri legumain forms an independent cluster together with ascidian legumain, and is positioned at the base of vertebrate legumains, suggesting that B. belcheri legumain gene may represent the archetype of vertebrate legumain genes. Both recombinant legumain expressed in yeast and endogenous legumain are able to be converted into active protein of ~37\u00a0kDa via a C-terminal autocleavage at acid pH values. The recombinant legumain efficiently degrades the legumain-specific substrate Z-Ala-Ala-Asn-MCA (benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine-4-methylcoumaryl-7-amide) at optimum pH\u00a05.5; and the enzymatic activity is inhibited potently by iodoacetamide and N-ethylmaleimide, partially by hen's-egg white cystatin, but not by E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane], PMSF and pepstatin A. In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. Altogether, these results suggest that B. belcheri legumain plays a role in the degradation of macromolecules in food.<\/jats:p>","DOI":"10.1042\/bsr20090049","type":"journal-article","created":{"date-parts":[[2009,6,30]],"date-time":"2009-06-30T10:31:46Z","timestamp":1246357906000},"page":"177-186","update-policy":"http:\/\/dx.doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Identification and functional characterization of legumain in amphioxus <i>Branchiostoma belcheri<\/i>"],"prefix":"10.1042","volume":"30","author":[{"given":"Lei","family":"Teng","sequence":"first","affiliation":[{"name":"Department of Marine Biology, Ocean University of China, Qingdao 266003, People's Republic of China"},{"name":"Department of Biology, Medical College of Qingdao University, Qingdao 266071, People's Republic of China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hiroshi","family":"Wada","sequence":"additional","affiliation":[{"name":"Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shicui","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Marine Biology, Ocean University of China, Qingdao 266003, People's Republic of China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","published-online":{"date-parts":[[2009,12,15]]},"reference":[{"key":"2021111618081131400_B1","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/BF01673722","article-title":"A novel protease from jack bean seeds: asparaginyl endopeptidase","volume":"11","author":"Ishii","year":"1992","journal-title":"J. Protein Chem."},{"key":"2021111618081131400_B2","doi-asserted-by":"crossref","first-page":"575","DOI":"10.1017\/S0031182000077052","article-title":"Asparaginyl endopeptidase activity in adult Schistosoma mansoni","volume":"111","author":"Dalton","year":"1995","journal-title":"Parasitology"},{"key":"2021111618081131400_B3","doi-asserted-by":"crossref","first-page":"8090","DOI":"10.1074\/jbc.272.12.8090","article-title":"Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase","volume":"272","author":"Chen","year":"1997","journal-title":"J. Biol. Chem."},{"key":"2021111618081131400_B4","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1042\/bj3390743","article-title":"Pig kidney legumain: an asparaginyl endopeptidase with restricted specificity","volume":"339","author":"Dando","year":"1999","journal-title":"Biochem. J."},{"key":"2021111618081131400_B5","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1016\/j.gene.2004.03.002","article-title":"Two novel asparaginyl endopeptidase-like cysteine proteinases from the protist Trichomonas vaginalis: their evolutionary relationship within the clan CD cysteine proteinases","volume":"335","author":"Le\u00f3n-F\u00e9lix","year":"2004","journal-title":"Gene"},{"key":"2021111618081131400_B6","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1016\/S0020-7519(00)00022-9","article-title":"Toxocara canis: genes expressed by the arrested infective larval stage of a parasitic nematode","volume":"30","author":"Maizels","year":"2000","journal-title":"Int. J. Parasitol."},{"key":"2021111618081131400_B7","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1017\/S0031182006000229","article-title":"Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus","volume":"133","author":"Oliver","year":"2006","journal-title":"Parasitology"},{"key":"2021111618081131400_B8","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1016\/j.ijpara.2006.12.020","article-title":"IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus","volume":"37","author":"Sojka","year":"2007","journal-title":"Int. J. Parasitol."},{"key":"2021111618081131400_B9","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/j.molbiopara.2007.07.006","article-title":"Comparative molecular analysis of two asparaginyl endopeptidases and encoding genes from Fasciola gigantica","volume":"156","author":"Adisakwattana","year":"2007","journal-title":"Mol. Biochem. Parasitol."},{"key":"2021111618081131400_B10","doi-asserted-by":"crossref","first-page":"911","DOI":"10.1016\/j.ibmb.2007.04.010","article-title":"Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis","volume":"37","author":"Alim","year":"2007","journal-title":"Insect. Biochem. Mol. Biol."},{"key":"2021111618081131400_B11","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1042\/bj3350111","article-title":"Cloning and expression of mouse legumain, a lysosomal endopeptidase","volume":"335","author":"Chen","year":"1998","journal-title":"Biochem. J."},{"key":"2021111618081131400_B12","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1042\/bj3520327","article-title":"Activation of human prolegumain by cleavage at a C-terminal asparagine residue","volume":"352","author":"Chen","year":"2000","journal-title":"Biochem. J."},{"key":"2021111618081131400_B13","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1006\/abbi.1993.1274","article-title":"The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays","volume":"303","author":"Kembhavi","year":"1993","journal-title":"Arch. Biochem. Biophys."},{"key":"2021111618081131400_B14","first-page":"1651","article-title":"Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni","volume":"5","author":"Hara-Nishimura","year":"1993","journal-title":"Plant Cell"},{"key":"2021111618081131400_B15","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/S1074-7613(00)80191-0","article-title":"Control of antigen presentation by a single protease cleavage site","volume":"12","author":"Antoniou","year":"2000","journal-title":"Immunity"},{"key":"2021111618081131400_B16","doi-asserted-by":"crossref","first-page":"27747","DOI":"10.1074\/jbc.274.39.27747","article-title":"Identification of human asparaginyl endopeptidase (Legumain) as an inhibitor of osteoclast formation and bone resorption","volume":"274","author":"Choi","year":"1999","journal-title":"J. Biol. Chem."},{"key":"2021111618081131400_B17","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1038\/25379","article-title":"An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation","volume":"396","author":"Manoury","year":"1998","journal-title":"Nature"},{"key":"2021111618081131400_B18","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.1016\/j.febslet.2007.02.064","article-title":"Legumain\/asparaginyl endopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells","volume":"581","author":"Morita","year":"2007","journal-title":"FEBS Lett."},{"key":"2021111618081131400_B19","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/j.pt.2004.03.004","article-title":"Blood \u2018n\u2019guts: an update on schistosome digestive peptidases","volume":"20","author":"Caffrey","year":"2004","journal-title":"Trends Parasitol."},{"key":"2021111618081131400_B20","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/S0166-6851(03)00194-4","article-title":"Functional expression and characterization of Schistosoma mansoni cathepsin B and its transactivation by an endogenous asparaginyl endopeptidase","volume":"131","author":"Sajid","year":"2003","journal-title":"Mol. Biochem. Parasitol."},{"key":"2021111618081131400_B21","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1111\/j.1469-7998.2008.00497.x","article-title":"Chordate phylogeny and evolution: a not so simple three taxon problem","volume":"276","author":"Stach","year":"2008","journal-title":"J. Zool."},{"key":"2021111618081131400_B22","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"2021111618081131400_B23","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein\u2013dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"2021111618081131400_B24","doi-asserted-by":"crossref","first-page":"4350","DOI":"10.1073\/pnas.76.9.4350","article-title":"Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications","volume":"76","author":"Towbin","year":"1979","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021111618081131400_B25","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1016\/j.biocel.2006.09.013","article-title":"Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver","volume":"39","author":"Fan","year":"2007","journal-title":"Int. J. Biochem. Cell Biol."},{"key":"2021111618081131400_B26","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1016\/j.jinsphys.2007.12.006","article-title":"HlLgm2, a member of asparaginyl endopeptidases\/legumains in the midgut of the ixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion","volume":"54","author":"Alim","year":"2008","journal-title":"J. Insect Physiol."},{"key":"2021111618081131400_B27","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/0014-5793(91)81349-D","article-title":"A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms","volume":"294","author":"Hara-Nishimura","year":"1991","journal-title":"FEBS Lett."},{"key":"2021111618081131400_B28","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1016\/S0014-5793(99)01798-6","article-title":"Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris","volume":"466","author":"Caffrey","year":"2000","journal-title":"FEBS Lett."},{"key":"2021111618081131400_B29","doi-asserted-by":"crossref","first-page":"38980","DOI":"10.1074\/jbc.M305930200","article-title":"Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo","volume":"278","author":"Li","year":"2003","journal-title":"J. Biol. Chem."},{"key":"2021111618081131400_B30","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1016\/0076-6879(94)44044-1","article-title":"Legumain: asparaginyl endopeptidase","volume":"244","author":"Ishii","year":"1994","journal-title":"Methods Enzymol."},{"key":"2021111618081131400_B31","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1017\/S0025315400014879","article-title":"The localization of organically bound iodine in the endostyle of Amphioxus","volume":"37","author":"Barrington","year":"1958","journal-title":"Mar. Biol. Ass."},{"key":"2021111618081131400_B32","first-page":"602","article-title":"Zur Kenntnis der Leberentwicklung bei Amphioxus","volume":"14","author":"Hammar","year":"1898","journal-title":"Anat. Anz."},{"key":"2021111618081131400_B33","first-page":"17","article-title":"The fine structure of the pharynx, cyrtopodocytes and digestive caecum of Amphioxus (Branchiostoma lanceolatum)","volume":"36","author":"Welsch","year":"1975","journal-title":"Symp. Zool. Soc."},{"key":"2021111618081131400_B34","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1007\/s10126-004-4084-9","article-title":"Characterization and expression of AmphiCL encoding cathepsin L proteinase from amphioxus Branchiostoma belcheri tsingtauense","volume":"27","author":"Wang","year":"2005","journal-title":"Mar. Biotechnol. (NY)"},{"key":"2021111618081131400_B35","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/j.ejcb.2007.10.002","article-title":"Gut-specific expression of cathepsin L and B in amphioxus Branchiostoma belcheri tsingtauense larvae","volume":"87","author":"Wang","year":"2008","journal-title":"Eur. J. Cell Biol."}],"container-title":["Bioscience Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/bioscirep\/article-pdf\/30\/3\/177\/474025\/bsr0300177.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/bioscirep\/article-pdf\/30\/3\/177\/474025\/bsr0300177.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,16]],"date-time":"2021-11-16T18:21:46Z","timestamp":1637086906000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/bioscirep\/article\/30\/3\/177\/55878\/Identification-and-functional-characterization-of"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2009,12,15]]},"references-count":35,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2010,6,1]]}},"URL":"https:\/\/doi.org\/10.1042\/bsr20090049","relation":{},"ISSN":["0144-8463","1573-4935"],"issn-type":[{"value":"0144-8463","type":"print"},{"value":"1573-4935","type":"electronic"}],"subject":[],"published":{"date-parts":[[2009,12,15]]}}}