文昌鱼Legumain基因的克隆、表达和功能研究
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摘要
半胱氨酸蛋白酶(EC3.4.22)是一类催化活性位点含有亲核的半胱氨酸残基的肽链内切酶。Legumain(C13)(EC3.4.22.34),又称天冬酰胺內肽酶(asparaginyl endopeptidase,AEP),是半胱氨酸蛋白酶家族新成员。近年来,在植物、寄生虫及哺乳动物中对于Legumain基因、蛋白的结构和功能研究都取得了一定程度的进展。但是,却未见文昌鱼的Legumain基因或蛋白的相关报道。文昌鱼是现存的与脊椎动物原始祖先最接近的头索动物,一直被认为是研究脊椎动物起源和进化的模式动物。本文克隆了青岛文昌鱼(Branchiostoma belcheri tsingtaunese)的具有完整开放阅读框的Legumain基因(BbLGMN),并对其结构、进化、表达和功能进行了研究。
首先,克隆得到一个全长1 637 bp的cDNA序列,其开放阅读框为1 308 bp,编码一个435个氨基酸残基的多肽链,理论分子量约48.9 kDa。氨基末端有一个由15个氨基酸残基组成的信号肽。内部有一个肽酶C13超家族结构域。结构域内有一个His153和Cys195催化二联体和一个保守的KGD (Lys123-Gly124-Asp125)基序。多个高度保守的半胱氨酸残基分别位于第226、386、398、418及435位。与曼氏血吸虫、长角血蜱、海鞘、斑马鱼、非洲爪蟾,牛,人的Legumain的同源性分别为36.6%、45.9%、46.1%、49.8%、49.4%、47.1%和48.5%。系统进化分析显示BbLGMN和海鞘的Legumain独聚一支,位于脊椎动物的基部,提示文昌鱼Legumain基因可能是脊椎动物Legumain基因的原祖型。
其次,采用毕赤酵母X-33真核表达系统得到50 kDa的rBbLGMN,进而对蛋白的生物学特征进行研究。发现rBbLGMN及内源性的BbLGMN都能在酸性条件下,在Asn347位点自切去除C末端肽段,形成分子量大小为37 kDa的有活性的成熟酶,能特异性切割多肽链中天冬酰胺残基羧基端的肽键。而在中性及碱性环境中就会失去自切及对特异性底物(Z-Ala-Ala-Asn-MCA)的酶切活性;成体文昌鱼肝盲囊中内源性BbLGMN在pH5.5的缓冲环境中活性最高。rBbLGMN的活性,能被碘乙酰胺及N-乙基顺丁烯二酰亚胺抑制;被蛋清半胱氨酸蛋白酶抑制剂部分抑制,却不被苯甲基磺酰氟化物,胃酶抑素A和E-64抑制。
再者,我们研究了BbLGMN在成体文昌鱼各组织中的表达情况。内源性酶活性检测及Western blot分析结果都表明BbLGMN蛋白主要在肝盲囊及后肠表达,Northern blot和qRT-PCR分析也进一步证明了这一点。文昌鱼BbLGMN呈现出组织特异性表达模式。文昌鱼的肝盲囊被认为是哺乳动物的肝脏和胰脏的起源。Legumain在文昌鱼消化道内呈现的组织特异性分布提示这种酶可能和食物大分子的降解有关。
最后,鉴于哺乳动物Legumain参与机体免疫过程中抗原的加工与递呈,我们对BbLGMN在免疫方面的功能进行了初步探讨。在LPS处理文昌鱼后,BbLGMN的表达量增加,提示BbLGMN可能参与了文昌鱼的免疫防御,但是BbLGMN却没有抑菌作用。
所以,BbLGMN在消化道内可能主司食物大分子的降解消化,关于它是否参与文昌鱼体内的免疫过程,尚不能确定。
Legumains(C13) (EC3.4.22.34) , or asparaginyl endopeptidases, are a recently identified family of cyteine-class endopeptides. They have been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates, the amphioxus Branchiostoma belcheir. Amphioxus or lancelet, a cephalochordate, has long been regarded as the closest relative of vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus legumain gene (BbLGMN).
The full-length cDNA of BbLGMN was 1 637 bp long, containing an open reading frame (ORF) of 1 308 bp which encoded a protein of 435 amino acids with a predicted molecular mass of approximately 48.9 kDa . The deduced protein had a signal peptide (15 amino acids) and had a single peptidase_C13 superfamily domain with the conserved KGD (Lys123-Gly124-Asp125) motif and the catalytic dyad His153 and Cys195, which are both characteristic of legumains. It also possessed all the highly conserved cysteines at the residual positions 226, 386, 398, 418 and 435. Alignment analysis showed that B. belcheri legumain shared 36.6%, 45.9%, 46.1%, 49.8%, 49.4%, 47.1% and 48.5% identity to the legumains of S. mansoni, H. longicornis, C. intestinalis, D. rerio, X. laevis, B. taurus and H. sapiens, respectively. The phylogenetic tree constructed using the sequences of representative legumains including that of B. belcheri demonstrated that BbLGMN formed an independent group together with C. intestinalis legumain, which is positioned at the base of vertebrate cluster, suggesting that BbLGMN gene may be the archetype of vertebrate legumain genes.
Both rBbLGMN expressed in yeast and endogenous BbLGMN are able to be converted into active protein of ~37 kDa via a C-terminal autocleavage at acid pH values to efficiently degrade the legumain-specific substrate Z-Ala-Ala-Asn-MCA. The endogenous BbLGMN efficiently hydrolyses the legumain-specific substrate at optimum pH5.5. Both autocatalytic activity and the enzymatic activity are inhibited potently by idoacetamide and N-ethylmaleimide, partially by egg white cystatin, but not by E-64, PMSF and pepstain 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. The hepatic caecum has been thought to be the precursor of liver/pancreas in vertebrates The tissue-specific distribution of BbLGMN therefore suggests that this enzyme may play a role in the degradation of macromolecules in food.
Considering that mammalian legumain is involved in processing of the microbial tetanus toxin C fragment for MHCⅡantigen presentation in the lysosomal system, we initially approached immune function of BbLGMN. Expression of BbLGMN is up-regulated in the digestive system of B. belcheri challenged with LPS, indicating BbLGMN may be involved in immune defense in B. belcheri, But rBbLGMN expressed in P. pastoris dose not inhibit the growth of E. coli and S. aureus.
In summary, this study reports the first characterization of legumain in lower chordates, and demonstration in the hepatic caecum and hind-gut of B. belcheri suggests a function for this enzyme associated with the degradation of food.
首先,克隆得到一个全长1 637 bp的cDNA序列,其开放阅读框为1 308 bp,编码一个435个氨基酸残基的多肽链,理论分子量约48.9 kDa。氨基末端有一个由15个氨基酸残基组成的信号肽。内部有一个肽酶C13超家族结构域。结构域内有一个His153和Cys195催化二联体和一个保守的KGD (Lys123-Gly124-Asp125)基序。多个高度保守的半胱氨酸残基分别位于第226、386、398、418及435位。与曼氏血吸虫、长角血蜱、海鞘、斑马鱼、非洲爪蟾,牛,人的Legumain的同源性分别为36.6%、45.9%、46.1%、49.8%、49.4%、47.1%和48.5%。系统进化分析显示BbLGMN和海鞘的Legumain独聚一支,位于脊椎动物的基部,提示文昌鱼Legumain基因可能是脊椎动物Legumain基因的原祖型。
其次,采用毕赤酵母X-33真核表达系统得到50 kDa的rBbLGMN,进而对蛋白的生物学特征进行研究。发现rBbLGMN及内源性的BbLGMN都能在酸性条件下,在Asn347位点自切去除C末端肽段,形成分子量大小为37 kDa的有活性的成熟酶,能特异性切割多肽链中天冬酰胺残基羧基端的肽键。而在中性及碱性环境中就会失去自切及对特异性底物(Z-Ala-Ala-Asn-MCA)的酶切活性;成体文昌鱼肝盲囊中内源性BbLGMN在pH5.5的缓冲环境中活性最高。rBbLGMN的活性,能被碘乙酰胺及N-乙基顺丁烯二酰亚胺抑制;被蛋清半胱氨酸蛋白酶抑制剂部分抑制,却不被苯甲基磺酰氟化物,胃酶抑素A和E-64抑制。
再者,我们研究了BbLGMN在成体文昌鱼各组织中的表达情况。内源性酶活性检测及Western blot分析结果都表明BbLGMN蛋白主要在肝盲囊及后肠表达,Northern blot和qRT-PCR分析也进一步证明了这一点。文昌鱼BbLGMN呈现出组织特异性表达模式。文昌鱼的肝盲囊被认为是哺乳动物的肝脏和胰脏的起源。Legumain在文昌鱼消化道内呈现的组织特异性分布提示这种酶可能和食物大分子的降解有关。
最后,鉴于哺乳动物Legumain参与机体免疫过程中抗原的加工与递呈,我们对BbLGMN在免疫方面的功能进行了初步探讨。在LPS处理文昌鱼后,BbLGMN的表达量增加,提示BbLGMN可能参与了文昌鱼的免疫防御,但是BbLGMN却没有抑菌作用。
所以,BbLGMN在消化道内可能主司食物大分子的降解消化,关于它是否参与文昌鱼体内的免疫过程,尚不能确定。
Legumains(C13) (EC3.4.22.34) , or asparaginyl endopeptidases, are a recently identified family of cyteine-class endopeptides. They have been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates, the amphioxus Branchiostoma belcheir. Amphioxus or lancelet, a cephalochordate, has long been regarded as the closest relative of vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus legumain gene (BbLGMN).
The full-length cDNA of BbLGMN was 1 637 bp long, containing an open reading frame (ORF) of 1 308 bp which encoded a protein of 435 amino acids with a predicted molecular mass of approximately 48.9 kDa . The deduced protein had a signal peptide (15 amino acids) and had a single peptidase_C13 superfamily domain with the conserved KGD (Lys123-Gly124-Asp125) motif and the catalytic dyad His153 and Cys195, which are both characteristic of legumains. It also possessed all the highly conserved cysteines at the residual positions 226, 386, 398, 418 and 435. Alignment analysis showed that B. belcheri legumain shared 36.6%, 45.9%, 46.1%, 49.8%, 49.4%, 47.1% and 48.5% identity to the legumains of S. mansoni, H. longicornis, C. intestinalis, D. rerio, X. laevis, B. taurus and H. sapiens, respectively. The phylogenetic tree constructed using the sequences of representative legumains including that of B. belcheri demonstrated that BbLGMN formed an independent group together with C. intestinalis legumain, which is positioned at the base of vertebrate cluster, suggesting that BbLGMN gene may be the archetype of vertebrate legumain genes.
Both rBbLGMN expressed in yeast and endogenous BbLGMN are able to be converted into active protein of ~37 kDa via a C-terminal autocleavage at acid pH values to efficiently degrade the legumain-specific substrate Z-Ala-Ala-Asn-MCA. The endogenous BbLGMN efficiently hydrolyses the legumain-specific substrate at optimum pH5.5. Both autocatalytic activity and the enzymatic activity are inhibited potently by idoacetamide and N-ethylmaleimide, partially by egg white cystatin, but not by E-64, PMSF and pepstain 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. The hepatic caecum has been thought to be the precursor of liver/pancreas in vertebrates The tissue-specific distribution of BbLGMN therefore suggests that this enzyme may play a role in the degradation of macromolecules in food.
Considering that mammalian legumain is involved in processing of the microbial tetanus toxin C fragment for MHCⅡantigen presentation in the lysosomal system, we initially approached immune function of BbLGMN. Expression of BbLGMN is up-regulated in the digestive system of B. belcheri challenged with LPS, indicating BbLGMN may be involved in immune defense in B. belcheri, But rBbLGMN expressed in P. pastoris dose not inhibit the growth of E. coli and S. aureus.
In summary, this study reports the first characterization of legumain in lower chordates, and demonstration in the hepatic caecum and hind-gut of B. belcheri suggests a function for this enzyme associated with the degradation of food.
引文
方永强,翁幼竹,胡晓霞。文昌鱼内柱结构与功能及其神经内分泌调节。自然科学进展,2002,12(10):1090-1094
方永强,周寿康,翁幼竹等。LHβmRNA在文昌鱼脑和哈氏窝的定位及其表达。自然科学进展,2001,11:992-994
方永强,王龙。文昌鱼轮器哈氏窝匀浆对幼体蟾蛛睾丸发育的初步探讨。实验生物学报,1984,17(1):115-117
方永强。文昌鱼生态习性及其资源的保护。动物学杂志,1987,22:41-45
金德祥等.厦门文昌鱼.动物学报,1953, 5(1):65-78
王玢,左明雪。人体及动物生理学。高等教育出版社(第二版)北京。2001。300-301
张士璀,郭斌,梁宇君。我国文昌鱼研究50年。生命科学,2008,20(1):64-68
张致一,朱益陶,陈大元。促黄体素(LH)在文昌鱼哈氏窝中的免疫细胞化学定位。1982,(15):946-947
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, 268(5):3525-9.
Abi-Rached L, Gilles A, Shiina T, et al. Evidence of en bloc duplication in vertebrate genomes. Nat Genet, 2002, 31(1): 100-105
Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Motobu M et al. Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis. Insect Biochem Mol Biol 2007, 37(9):911–22.
Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Hatta T, Fujisaki K.HlLgm2, a member of asparaginyl endopeptidases/legumains in the midgut of the ixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion. J Insect Physiol, 2008, 54(3):573-85
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSL-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997, 25: 3389~3402
Antoniou AN, Blackwood SL, Mazzeo D, Watts C. Control of antigen presentation by a single protease cleavage site. Immunity, 2000,12:391–98
Barrett, AJ, Rawlings, ND. Families and clans of cysteine peptidases. Perspect Drug DiscovDesign, 1996, 6:1-11.
Barrington EJW. The localization of organically bound io dine in the endostyle of Amphioxus. Mar. Biol. Ass. U.K. 1958, 37: 117-126
Bernd W. Structure-function relationship in class CA1 cysteine peptidase propeptides. Acta Biochemical Polonica, 2003, 50(3:6912713.
Brindley PJ, Kalinna BH, Dalton JP, Day SR, Wong JY, Smythe ML, McManus DP.Proteolytic degradation of host hemoglobin by schistosomes. Mol Biochem Parasitol, 1997, 89(1):1-9
Burland TG. DNASTAR's Lasergene sequence analysis software. Methods Mol Biol, 2000, 132: 71-91.
Caffrey CR, Mathieu MA, Gaffney AM, Salter JP, Sajid M, Lucas KD, Franklin C, Bogyo M, McKerrow JH. Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris. FEBS Lett, 2000,466(2-3):244-8
Cannon JP, Haire RN, Litman GW. Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate. Nat Immunol, 2002, 3(12): 1200-1207
Castro L F C, Furlong R F and Holland P W H. An antecedent of the MHC-linked genomic region in amphioxus. Immunogenetics, 2004, 55: 782–784
Chen JM, Dando PM, Rawlings ND, Brown MA, Young NE, Stevens RA, Hewitt E, Watts C, Barrett AJ. Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase.J Biol Chem, 1997, 272(12):8090-8
Chen JM, Dando PM, Stevens RA, Fortunato M, Barrett AJ. Cloning and expression of mouse legumain, a lysosomal endopeptidase. Biochem J, 1998a, 335 ( Pt 1):111-7
Chen, J.-M., Rawlings, N. D., Stevens, R. A. E. and Barrett, A. J. Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases. FEBS Lett. 1998b, 441:361-365
Chen JM, Fortunato M, Barrett AJ.Activation of human prolegumain by cleavage at a C-terminal asparagine residue.Biochem J, 2000, 352 Pt 2:327-34
Choi SJ, Reddy SV, Devlin RD, Menaa C, Chung H, Boyce BF, Roodman GD. Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption. J Biol Chem, 1999, 274(39):27747-53
Choi SJ, Kurihara N, Oba Y, Roodman GD.Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment.J Bone Miner Res, 2001, 16(10):1804-11
Dalton JP, Hola Jamriska L, Brindley PJ. Asparaginyl endopeptidase actiyity in adult Schistosoma mansoni . Parasitology, 1995, 111 (5):575-80
Dando PM, Fortunato M, Smith L, Knight CG, McKendrick JE, Barrett AJ. Pig kidney legumain: an asparaginyl endopeptidase with restricted specificity. Biochem J, 1999, 339 ( Pt 3):743-9
Dehal P, Satou Y, Campbell R K, Chapman J, Degnan B, De Tomaso A, et al. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science, 2002, 298: 2157-2167
Dickinson DP. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease.Crit Rev Oral Biol Med, 2002, 13(3):238-75.
Dong M, Fu Y, Yu C, et al. Identification and characterization of a homolog of an activation gene for the recombination activating gene 1 (RAG 1) in amphioxus. Fish Shellfish Immunol, 2005, 19(2): 165-174.
Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G.Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver.Int J Biochem Cell Biol. 2007, 39(2):450-61.
Fan C, Zhang S, Li L, Chao Y. Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.Mol Immunol. 2008, 45(12):3338-46.
Ferrier DE, Minguillón C, Holland PW, Garcia-Fernàndez J. The amphioxus Hox cluster: deuterostome posterior flexibility and Hox14. Evol Dev. 2000, 2(5):284-93
Fresriksson G, Ericaon LE, Olsson R. Iodine binding in the endostyle of larval Branchiostoma Lanceolatum. Gen. Comp. Endocrinol, 1984, 56:177-84
Gao YY, Zhang DF, Li H, et al.Proteomic approach for caudal trauma-induced acute phage proteins reveals that creatine kinase is a key acute phage protein in amphioxus humoral fluid. Journal of Proteome Research , 2007, 6(11): 4321-4329
Grudkowska M ,Zagdanska B. Multifunctional role of plant cysteine proteinases . Acta Biochemical Polonica, 2004, 51 (3) :6092624.
Hammar JA. Zur Kenntnis der Leberentwicklung bei Amphioxus. Anat Anz, 1898, 14: 602-606
Han L, Zhang S, Wang Y Sun X. Immunohistochemical localization of vitellogenin in the hepatic diverticulum of the amphioxus Branchiostoma belcheri tsingtauense, with implications for the origin of the liver. Invertebr Bio, 2006, 125: 172-176
Hara-Nishimura I, Nishimura M. Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons. Plant Physiol, 1987, 85:440-445
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, 1991, 294:89-93
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, 5:1651-1659
Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S, Kondo M, Nishimura M, Hara-Nishimura I. A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death. Science, 2004, 305, 855-858
He Y, Tang B, Zhang S, Liu Z, Zhao B, Chen L. Molecular and immunochemical demonstration of a novel member of Bf/C2 homolog in amphioxus Branchiostoma belcheri: implications for involvement of hepatic cecum in acute phase response. Fish Shellfish Immunol. 2008, 24(6):768-78
Holland LZ, Gibson-Brown J J. The Ciona intestinalis genome: when the constraints are off. Bioessays, 2003, 25: 529–532
Holland LZ, Laudet V, Schubert M. The chordate amphioxus: an emerging model organism for developmental biology. Cell Mol Life Sci, 2004, 61: 2290-2308
Holland LZ, Schubert M, Kozmik Z Holland ND. AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest. Evol. Dev, 1999, 1: 153-165
Holland L Z., Schubert M, Holland N D and Neuman T. Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Dev. Biol, 2000, 226: 18-33
Holland ND, Panganiban G, Henyey EL, Holland LZ. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous118system: insights into evolution of craniate forebrain and neural crest. Development, 1996, 122(9): 2911-2920
Holland PWH, Garcia-Fernandez J, Williams NA, Sidow A. Gene duplications and the origins of vertebrate development. Dev Suppl, 1994, 125-133
Holland PW, Koschorz B, Holland LZ, Herrmann BG. Conservation of Brachyury (T) genes in amphioxus and vertebrates: developmental and evolutionary implications. Development. 1995, 121(12): 4283-4291
Ishii S, Abe Y, Mitta M, Matsushita H, Kato I. A novel protease from jack bean seeds: Asparaginyl endopeptidase. J Protein Chem 1992, 11:367–8.
Ju L, Zhang S, Liang Y, Sun X. Identification, expression and antibacterial activity of a tachylectin-related homolog in amphioxus Branchiostoma belcheri with implications for involvement of the digestive system in acute phase response. Fish Shellfish Immunol. FishShellfish Immunol. 2009, 26(2):235-42
Kapitonov VV, Jurka J. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PloS Biol, 2005, 3(6): 998~1011
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, 1993, 303(2):208-13
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. 1995a, 29(1):81-9
Kinoshita T, Nishimura M, Hara-Nishimura I.The sequence and expression of the gamma-VPE gene, one member of a family of three genes for vacuolar processing enzymes in Arabidopsis thaliana. Plant Cell Physiol. 1995b, 36(8):1555-62
Kinoshita T, Yamada K, Hiraiwa N, Nishimura M, Hara-Nishimura I). Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions. Plant J, 1999,19:43-53
Kortschak RD, Samuel G, Saint R, Miller DJ. EST analysis of the cnidarian Acropora millepora reveals extensivegene loss and rapid sequence divergence in the model invertebrates. Curr Biol. 2003, 13: 2190-2195.
Kozmik Z, Holland ND, et al. Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structure,and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development, 1999, 126: 1295-1304
Kubota K, Wakabayashi K, Matsuoka T.Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography. Proteomics, 2003, 3(5):616-26
Langeland JA, Tomsa JM, Jackman WR Jr and Kimmel C. B. An amphioxus snail gene: expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo. Dev. Genes Evol, 1998, 208: 569-577.
Lazzaro D, Price M et al. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development, 1991, 113:1093-1104
Lewēn S, Zhou H, Hu HD, Cheng T, Markowitz D, Reisfeld RA, Xiang R, Luo Y. A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis. Cancer Immunol Immunother, 2008, 57(4):507-15
Li X, Zhang SC, Liu ZH, Li HY. Ribosomal protein genes S23 and L35 from amphioxus Branchiostoma belcheri tsingtauense: identification and copy number. Acta Biochim Biophys Sin (Shanghai). 2005, 37(8):573-9
Li ZM, Zhang SC, Wang CF, et al. Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: Implications for a dual role of O-antigens in the resistance to bactericidal activity. Fish Shellfish Immunology, 2008, 24: 215-222
Liang Y, Zhang S, Lun L, Han L. Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver. Cell and Tissue Research, 2006a, 323: 537-541
Liang Y, Zhang S. Demonstration of plasminogen-like protein in amphioxus with implications for the origin of vertebrate liver. Acta Zoologica, 2006b, 87(2): 141-145.
Liqht S F. Amphioxus fishers near the university of Amoy, China. Science, 1923,Ⅷ: 57-60
Liu C, Sun C, Huang H, Janda K, Edgington T. Overexpression of legumain in tumors is significant for invasion/metastasis and a candidate enzymatic target for prodrug therapy. Cancer Res, 2003, 63(11):2957-64
Manoury B, Hewitt EW, Morrice N, Dando PM, Barrett AJ, Watts C. An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation. Nature, 1998, 396(6712):695-9
Manoury B, Mazzeo D, Li DN, Billson J, Loak K, Benaroch P, Watts C. Asparagine endopeptidase can initiate the removal of the MHC class II invariant chain chaperone. Immunity. 2003, 18(4):489-98
Meulemans D. and Bronner-Fraser M. Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns. Development , 2002, 129: 4953-4962.
Minguillón C, Gardenyes J, Serra E, Castro LFC, Hill-Force A, Holland PWH, Amemiya CT, Garcia-Fernàndez J. No more than 14: the end of the amphioxus Hox cluster. Int J Biol Sci, 2005, 1: 19-23
Moller PC, Philpott CW. The circulatory system of amphioxus (Branchiostoma floridae). I. Morphology of the major vessels of the pharyngeal area. J Morphol, 1973, 139: 389-406
Monaco F, Dominici R, Andreoli M, De Pirro R, Roche J. Thyroid homone fomation in thyroglobulin synthesized in the amphioxus (Branchiostoma lanceolatum Pallas). Comp Biochem Physiol, 1981, 70B: 341
Morita Y, Araki H, Sugimoto T, Takeuchi K, Yamane T, Maeda T et al. Legumain/asparaginylendopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells. FEBS Lett 2007, 581(7):1417–24.
Müller J. Ueber den Bau und die Lebenserscheinungen des Branchiostoma lubricum Costa, Amphioxus Lanceolatus. Yarrell Abh K Preuss Akad Wiss Berl, 1884, 1844: 79-116
Murthy RV, Arbman G, Gao J, Roodman GD, Sun XF. Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer. Clin Cancer Res, 2005, 11(6):2293-9
Nakaune S, Yamada K, Kondo M, Kato T, Tabata S, Nishimura M, Hara-Nishimura I.A vacuolar processing enzyme, deltaVPE, is involved in seed coat formation at the early stage of seed development.Plant Cell. 2005, 17(3):876-87
Oliver EM, Skuce PJ, McNair CM, Knox DP. Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus. Parasitology, 2006, 133(Pt 2):237-44
Pang QX, Zhang SC, Liu XM, et al. Humoral immune responses of amphioxus Branchiostoma belcheri to challenge with Escherichia coli. Fish Shellfish Immunol, 2006, 2:139-145
Pang QX, Zhang SC, Shi XD, et al. Purication and characterization of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense. Fish Shellfish Immunol, 2005, 19:139-148
Panopoulou GD, Clark MD, Holland LZ, Lehrach H, Holland ND. AmphiBMP2/4, an amphioxus bone morphogenetic protein closely related to Drosophila decapentaplegic and vertebrate BMP2 and BMP4: insights into evolution of dorsoventral axis specification. Dev Dyn, 1998, 213(1): 130-139
Rawlings ND, Barrett Al. Families of cysteine peptidases. Methods Enzymol, 1994, 244:461-86.
Rhodes CP, Ratcliffe NA, Rowley AF. Presence of coelomocytes in the primitive chordate amphioxus (Branchiostoma lanceolatum). Science, 1982, 217: 263-265
Ridder R, et al. Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris. Biotechnology (N Y), 1995, 13 (3): 255-260
Ruppert EE. 1997. Cephalochordata (Acrania). In Harrison, F. W. & Ruppert, E. E. (Eds.), Microscopic anatomy of invertebrates. (Vol. 15. pp. 349–504). Wiley, New York.
Sembrat K.Effect of the endostyle of the lancelet (Branchiostoma lanceolatum) on the metamorphosis of axolotl. Zool Polon, 1953, 6: 3
Shimada T, Yamada K, Kataoka M, Nakaune S, Koumoto Y, Kuroyanagi M, Tabata S, Kato T,Shinozaki K, Seki M, Kobayashi M, Kondo M, Nishimura M, Hara-Nishimura I.Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana.J Biol Chem, 2003, 278(34):32292-9.
Shirahama-Noda K, Yamamoto A, Sugihara K, Hashimoto N, Asano M, Nishimura M, Hara-Nishimura I. Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice. J Biol Chem, 2003, 278:33194-33199
Silva JRMC, Mendes EG, Mariano M. Wound repair in the amphioxus (Branchiostoma platae), an animal deprived of inflammatory phagocytes. J Invertebr Pathol, 1995, 65: 147-151
Skelly PJ ,Shoemaker CB. Schistosoma mansoni proteases Sm31 ( cathepsin B) and Sm32 (legumain) are expressed in the cecuma and protonephridia of cercariae. JParasitol, 2001, 87 (5) :1218-1221
Sojka D, Hajdusek O, Dvorák J, Sajid M, Franta Z, Schneider EL, Craik CS, VancováM, BuresováV, Bogyo M, Sexton KB, McKerrow JH, Caffrey CR, Kopácek P. IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int J Parasitol, 2007, 37(7):713-24.
Stach T. Chordate phylogeny and evolution: a not so simple threetaxon problem. J Zool. 2008, 1–25.
Stern L, Perry R, Ofek P, Many A, Shabat D, Satchi-Fainaro R. A novel antitumor prodrug platform designed to be cleaved by the endoprotease legumain.Bioconjug Chem, 2009, [Epub ahead of print]
Stokes MD, Holland ND. The lancelet: also known as "amphioxus," this curious creature has returned to the limelight as a player in the phylogenetic history of the vertebrates. American Scientist, 1998, 86: 552-560
Suzuki MM, Satoh N. Genes expressed in the amphioxus notochord revealed by EST analysis.Dev Biol. 2000, 224(2):168-77.
Tchang S, KooKC. Description of a new variety of Branchiostoma belcheri Gray from Kiaochow Bay, Shantung, China. Contr Inst Zool Nat Acad Peiping, 1936, 3 (4):77-114.
Thomas I M. The accumulation of radioactive iodine by amphioxus. J Mar Biol Ass UK,1956, 35:203.
Urano A, Suzuki MM, Zhang P, Satoh N, Satoh G. Expression of muscle-related genes and two MyoD genes during amphioxus notochord development. Evol Dev. 2003, 5(5):447-58.
Venkatesh TV, Holland ND, Holland LZ, Su MT, Bodmer R. Sequence and developmental expression of AmphiNk2-1: Insights into the evolution origin of the vertebrate thyroid gland and forebrain. Dev. Genes Evol. 1999, 209: 254-259
Venkatesh TV, Holland ND, Holland LZ, Su MT, Bodmer R. Sequence and developmental expression of AmphiNk2-1: Insights into the evolution origin of the vertebrate thyroid glandand forebrain. Dev. Genes Evol. 1999, 209: 254-259
Wang CF, Zhang SC, Luo Y, et al. Presence and induction by bacteria of D-galactoside-specific lectins in the humoral fluids of amphioxus Branchiostoma belcheri tsingtauense. Inflammopharmacol, 2002, 9(3): 241-248
Wang G, Zhang S, Wang Z.Responses of alternative complement expression to challenge with different combinations of Vibrio anguillarum, Escherichia coli and Staphylococcus aureus: evidence for specific immune priming in amphioxus Branchiostoma belcheri. Fish Shellfish Immunol. 2009, 26(1):33-9
Welsch U. The fine structure of the pharynx, cyrtopodocytes and digestive caecum of Amphioxus (Branchiostoma lanceolatum). Symposium of the Zoological Society, London, 1975, 36:17-41
Yamane T, Takeuchi K, Yamamoto , Li YH, Fujiwara M, Nishi K, Takahashi S, Ohkubo I. Legumain from bovine kidney: its purification, molecular cloning, immunohisto- chemical localization and degradation of annexin II and vitamin D-binding protein .Biochim Biophys Acta, 2002, 1596(1):108-20.
Yuan J, Zhang S, Liu Z, Luan Z, Hu G. Cloning and phylogenetic analysis of an amphioxus myogenic bHLH gene AmphiMDF. Biochemical and Biophysical Research Communications, 2003, 301: 960-967
Zhang HW. Granulocytes and macrophages in amphioxus. Zool Sci, 1992, 9(1): 113-118
方永强,周寿康,翁幼竹等。LHβmRNA在文昌鱼脑和哈氏窝的定位及其表达。自然科学进展,2001,11:992-994
方永强,王龙。文昌鱼轮器哈氏窝匀浆对幼体蟾蛛睾丸发育的初步探讨。实验生物学报,1984,17(1):115-117
方永强。文昌鱼生态习性及其资源的保护。动物学杂志,1987,22:41-45
金德祥等.厦门文昌鱼.动物学报,1953, 5(1):65-78
王玢,左明雪。人体及动物生理学。高等教育出版社(第二版)北京。2001。300-301
张士璀,郭斌,梁宇君。我国文昌鱼研究50年。生命科学,2008,20(1):64-68
张致一,朱益陶,陈大元。促黄体素(LH)在文昌鱼哈氏窝中的免疫细胞化学定位。1982,(15):946-947
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, 268(5):3525-9.
Abi-Rached L, Gilles A, Shiina T, et al. Evidence of en bloc duplication in vertebrate genomes. Nat Genet, 2002, 31(1): 100-105
Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Motobu M et al. Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis. Insect Biochem Mol Biol 2007, 37(9):911–22.
Alim MA, Tsuji N, Miyoshi T, Islam MK, Huang X, Hatta T, Fujisaki K.HlLgm2, a member of asparaginyl endopeptidases/legumains in the midgut of the ixodid tick Haemaphysalis longicornis, is involved in blood-meal digestion. J Insect Physiol, 2008, 54(3):573-85
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and PSL-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997, 25: 3389~3402
Antoniou AN, Blackwood SL, Mazzeo D, Watts C. Control of antigen presentation by a single protease cleavage site. Immunity, 2000,12:391–98
Barrett, AJ, Rawlings, ND. Families and clans of cysteine peptidases. Perspect Drug DiscovDesign, 1996, 6:1-11.
Barrington EJW. The localization of organically bound io dine in the endostyle of Amphioxus. Mar. Biol. Ass. U.K. 1958, 37: 117-126
Bernd W. Structure-function relationship in class CA1 cysteine peptidase propeptides. Acta Biochemical Polonica, 2003, 50(3:6912713.
Brindley PJ, Kalinna BH, Dalton JP, Day SR, Wong JY, Smythe ML, McManus DP.Proteolytic degradation of host hemoglobin by schistosomes. Mol Biochem Parasitol, 1997, 89(1):1-9
Burland TG. DNASTAR's Lasergene sequence analysis software. Methods Mol Biol, 2000, 132: 71-91.
Caffrey CR, Mathieu MA, Gaffney AM, Salter JP, Sajid M, Lucas KD, Franklin C, Bogyo M, McKerrow JH. Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris. FEBS Lett, 2000,466(2-3):244-8
Cannon JP, Haire RN, Litman GW. Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate. Nat Immunol, 2002, 3(12): 1200-1207
Castro L F C, Furlong R F and Holland P W H. An antecedent of the MHC-linked genomic region in amphioxus. Immunogenetics, 2004, 55: 782–784
Chen JM, Dando PM, Rawlings ND, Brown MA, Young NE, Stevens RA, Hewitt E, Watts C, Barrett AJ. Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase.J Biol Chem, 1997, 272(12):8090-8
Chen JM, Dando PM, Stevens RA, Fortunato M, Barrett AJ. Cloning and expression of mouse legumain, a lysosomal endopeptidase. Biochem J, 1998a, 335 ( Pt 1):111-7
Chen, J.-M., Rawlings, N. D., Stevens, R. A. E. and Barrett, A. J. Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases. FEBS Lett. 1998b, 441:361-365
Chen JM, Fortunato M, Barrett AJ.Activation of human prolegumain by cleavage at a C-terminal asparagine residue.Biochem J, 2000, 352 Pt 2:327-34
Choi SJ, Reddy SV, Devlin RD, Menaa C, Chung H, Boyce BF, Roodman GD. Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption. J Biol Chem, 1999, 274(39):27747-53
Choi SJ, Kurihara N, Oba Y, Roodman GD.Osteoclast inhibitory peptide 2 inhibits osteoclast formation via its C-terminal fragment.J Bone Miner Res, 2001, 16(10):1804-11
Dalton JP, Hola Jamriska L, Brindley PJ. Asparaginyl endopeptidase actiyity in adult Schistosoma mansoni . Parasitology, 1995, 111 (5):575-80
Dando PM, Fortunato M, Smith L, Knight CG, McKendrick JE, Barrett AJ. Pig kidney legumain: an asparaginyl endopeptidase with restricted specificity. Biochem J, 1999, 339 ( Pt 3):743-9
Dehal P, Satou Y, Campbell R K, Chapman J, Degnan B, De Tomaso A, et al. The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science, 2002, 298: 2157-2167
Dickinson DP. Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease.Crit Rev Oral Biol Med, 2002, 13(3):238-75.
Dong M, Fu Y, Yu C, et al. Identification and characterization of a homolog of an activation gene for the recombination activating gene 1 (RAG 1) in amphioxus. Fish Shellfish Immunol, 2005, 19(2): 165-174.
Fan C, Zhang S, Liu Z, Li L, Luan J, Saren G.Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver.Int J Biochem Cell Biol. 2007, 39(2):450-61.
Fan C, Zhang S, Li L, Chao Y. Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.Mol Immunol. 2008, 45(12):3338-46.
Ferrier DE, Minguillón C, Holland PW, Garcia-Fernàndez J. The amphioxus Hox cluster: deuterostome posterior flexibility and Hox14. Evol Dev. 2000, 2(5):284-93
Fresriksson G, Ericaon LE, Olsson R. Iodine binding in the endostyle of larval Branchiostoma Lanceolatum. Gen. Comp. Endocrinol, 1984, 56:177-84
Gao YY, Zhang DF, Li H, et al.Proteomic approach for caudal trauma-induced acute phage proteins reveals that creatine kinase is a key acute phage protein in amphioxus humoral fluid. Journal of Proteome Research , 2007, 6(11): 4321-4329
Grudkowska M ,Zagdanska B. Multifunctional role of plant cysteine proteinases . Acta Biochemical Polonica, 2004, 51 (3) :6092624.
Hammar JA. Zur Kenntnis der Leberentwicklung bei Amphioxus. Anat Anz, 1898, 14: 602-606
Han L, Zhang S, Wang Y Sun X. Immunohistochemical localization of vitellogenin in the hepatic diverticulum of the amphioxus Branchiostoma belcheri tsingtauense, with implications for the origin of the liver. Invertebr Bio, 2006, 125: 172-176
Hara-Nishimura I, Nishimura M. Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons. Plant Physiol, 1987, 85:440-445
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, 1991, 294:89-93
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, 5:1651-1659
Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S, Kondo M, Nishimura M, Hara-Nishimura I. A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death. Science, 2004, 305, 855-858
He Y, Tang B, Zhang S, Liu Z, Zhao B, Chen L. Molecular and immunochemical demonstration of a novel member of Bf/C2 homolog in amphioxus Branchiostoma belcheri: implications for involvement of hepatic cecum in acute phase response. Fish Shellfish Immunol. 2008, 24(6):768-78
Holland LZ, Gibson-Brown J J. The Ciona intestinalis genome: when the constraints are off. Bioessays, 2003, 25: 529–532
Holland LZ, Laudet V, Schubert M. The chordate amphioxus: an emerging model organism for developmental biology. Cell Mol Life Sci, 2004, 61: 2290-2308
Holland LZ, Schubert M, Kozmik Z Holland ND. AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest. Evol. Dev, 1999, 1: 153-165
Holland L Z., Schubert M, Holland N D and Neuman T. Evolutionary conservation of the presumptive neural plate markers AmphiSox1/2/3 and AmphiNeurogenin in the invertebrate chordate amphioxus. Dev. Biol, 2000, 226: 18-33
Holland ND, Panganiban G, Henyey EL, Holland LZ. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis and nervous118system: insights into evolution of craniate forebrain and neural crest. Development, 1996, 122(9): 2911-2920
Holland PWH, Garcia-Fernandez J, Williams NA, Sidow A. Gene duplications and the origins of vertebrate development. Dev Suppl, 1994, 125-133
Holland PW, Koschorz B, Holland LZ, Herrmann BG. Conservation of Brachyury (T) genes in amphioxus and vertebrates: developmental and evolutionary implications. Development. 1995, 121(12): 4283-4291
Ishii S, Abe Y, Mitta M, Matsushita H, Kato I. A novel protease from jack bean seeds: Asparaginyl endopeptidase. J Protein Chem 1992, 11:367–8.
Ju L, Zhang S, Liang Y, Sun X. Identification, expression and antibacterial activity of a tachylectin-related homolog in amphioxus Branchiostoma belcheri with implications for involvement of the digestive system in acute phase response. Fish Shellfish Immunol. FishShellfish Immunol. 2009, 26(2):235-42
Kapitonov VV, Jurka J. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PloS Biol, 2005, 3(6): 998~1011
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, 1993, 303(2):208-13
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. 1995a, 29(1):81-9
Kinoshita T, Nishimura M, Hara-Nishimura I.The sequence and expression of the gamma-VPE gene, one member of a family of three genes for vacuolar processing enzymes in Arabidopsis thaliana. Plant Cell Physiol. 1995b, 36(8):1555-62
Kinoshita T, Yamada K, Hiraiwa N, Nishimura M, Hara-Nishimura I). Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions. Plant J, 1999,19:43-53
Kortschak RD, Samuel G, Saint R, Miller DJ. EST analysis of the cnidarian Acropora millepora reveals extensivegene loss and rapid sequence divergence in the model invertebrates. Curr Biol. 2003, 13: 2190-2195.
Kozmik Z, Holland ND, et al. Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structure,and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development, 1999, 126: 1295-1304
Kubota K, Wakabayashi K, Matsuoka T.Proteome analysis of secreted proteins during osteoclast differentiation using two different methods: two-dimensional electrophoresis and isotope-coded affinity tags analysis with two-dimensional chromatography. Proteomics, 2003, 3(5):616-26
Langeland JA, Tomsa JM, Jackman WR Jr and Kimmel C. B. An amphioxus snail gene: expression in paraxial mesoderm and neural plate suggests a conserved role in patterning the chordate embryo. Dev. Genes Evol, 1998, 208: 569-577.
Lazzaro D, Price M et al. The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development, 1991, 113:1093-1104
Lewēn S, Zhou H, Hu HD, Cheng T, Markowitz D, Reisfeld RA, Xiang R, Luo Y. A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis. Cancer Immunol Immunother, 2008, 57(4):507-15
Li X, Zhang SC, Liu ZH, Li HY. Ribosomal protein genes S23 and L35 from amphioxus Branchiostoma belcheri tsingtauense: identification and copy number. Acta Biochim Biophys Sin (Shanghai). 2005, 37(8):573-9
Li ZM, Zhang SC, Wang CF, et al. Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: Implications for a dual role of O-antigens in the resistance to bactericidal activity. Fish Shellfish Immunology, 2008, 24: 215-222
Liang Y, Zhang S, Lun L, Han L. Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver. Cell and Tissue Research, 2006a, 323: 537-541
Liang Y, Zhang S. Demonstration of plasminogen-like protein in amphioxus with implications for the origin of vertebrate liver. Acta Zoologica, 2006b, 87(2): 141-145.
Liqht S F. Amphioxus fishers near the university of Amoy, China. Science, 1923,Ⅷ: 57-60
Liu C, Sun C, Huang H, Janda K, Edgington T. Overexpression of legumain in tumors is significant for invasion/metastasis and a candidate enzymatic target for prodrug therapy. Cancer Res, 2003, 63(11):2957-64
Manoury B, Hewitt EW, Morrice N, Dando PM, Barrett AJ, Watts C. An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation. Nature, 1998, 396(6712):695-9
Manoury B, Mazzeo D, Li DN, Billson J, Loak K, Benaroch P, Watts C. Asparagine endopeptidase can initiate the removal of the MHC class II invariant chain chaperone. Immunity. 2003, 18(4):489-98
Meulemans D. and Bronner-Fraser M. Amphioxus and lamprey AP-2 genes: implications for neural crest evolution and migration patterns. Development , 2002, 129: 4953-4962.
Minguillón C, Gardenyes J, Serra E, Castro LFC, Hill-Force A, Holland PWH, Amemiya CT, Garcia-Fernàndez J. No more than 14: the end of the amphioxus Hox cluster. Int J Biol Sci, 2005, 1: 19-23
Moller PC, Philpott CW. The circulatory system of amphioxus (Branchiostoma floridae). I. Morphology of the major vessels of the pharyngeal area. J Morphol, 1973, 139: 389-406
Monaco F, Dominici R, Andreoli M, De Pirro R, Roche J. Thyroid homone fomation in thyroglobulin synthesized in the amphioxus (Branchiostoma lanceolatum Pallas). Comp Biochem Physiol, 1981, 70B: 341
Morita Y, Araki H, Sugimoto T, Takeuchi K, Yamane T, Maeda T et al. Legumain/asparaginylendopeptidase controls extracellular matrix remodeling through the degradation of fibronectin in mouse renal proximal tubular cells. FEBS Lett 2007, 581(7):1417–24.
Müller J. Ueber den Bau und die Lebenserscheinungen des Branchiostoma lubricum Costa, Amphioxus Lanceolatus. Yarrell Abh K Preuss Akad Wiss Berl, 1884, 1844: 79-116
Murthy RV, Arbman G, Gao J, Roodman GD, Sun XF. Legumain expression in relation to clinicopathologic and biological variables in colorectal cancer. Clin Cancer Res, 2005, 11(6):2293-9
Nakaune S, Yamada K, Kondo M, Kato T, Tabata S, Nishimura M, Hara-Nishimura I.A vacuolar processing enzyme, deltaVPE, is involved in seed coat formation at the early stage of seed development.Plant Cell. 2005, 17(3):876-87
Oliver EM, Skuce PJ, McNair CM, Knox DP. Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus. Parasitology, 2006, 133(Pt 2):237-44
Pang QX, Zhang SC, Liu XM, et al. Humoral immune responses of amphioxus Branchiostoma belcheri to challenge with Escherichia coli. Fish Shellfish Immunol, 2006, 2:139-145
Pang QX, Zhang SC, Shi XD, et al. Purication and characterization of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense. Fish Shellfish Immunol, 2005, 19:139-148
Panopoulou GD, Clark MD, Holland LZ, Lehrach H, Holland ND. AmphiBMP2/4, an amphioxus bone morphogenetic protein closely related to Drosophila decapentaplegic and vertebrate BMP2 and BMP4: insights into evolution of dorsoventral axis specification. Dev Dyn, 1998, 213(1): 130-139
Rawlings ND, Barrett Al. Families of cysteine peptidases. Methods Enzymol, 1994, 244:461-86.
Rhodes CP, Ratcliffe NA, Rowley AF. Presence of coelomocytes in the primitive chordate amphioxus (Branchiostoma lanceolatum). Science, 1982, 217: 263-265
Ridder R, et al. Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris. Biotechnology (N Y), 1995, 13 (3): 255-260
Ruppert EE. 1997. Cephalochordata (Acrania). In Harrison, F. W. & Ruppert, E. E. (Eds.), Microscopic anatomy of invertebrates. (Vol. 15. pp. 349–504). Wiley, New York.
Sembrat K.Effect of the endostyle of the lancelet (Branchiostoma lanceolatum) on the metamorphosis of axolotl. Zool Polon, 1953, 6: 3
Shimada T, Yamada K, Kataoka M, Nakaune S, Koumoto Y, Kuroyanagi M, Tabata S, Kato T,Shinozaki K, Seki M, Kobayashi M, Kondo M, Nishimura M, Hara-Nishimura I.Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana.J Biol Chem, 2003, 278(34):32292-9.
Shirahama-Noda K, Yamamoto A, Sugihara K, Hashimoto N, Asano M, Nishimura M, Hara-Nishimura I. Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice. J Biol Chem, 2003, 278:33194-33199
Silva JRMC, Mendes EG, Mariano M. Wound repair in the amphioxus (Branchiostoma platae), an animal deprived of inflammatory phagocytes. J Invertebr Pathol, 1995, 65: 147-151
Skelly PJ ,Shoemaker CB. Schistosoma mansoni proteases Sm31 ( cathepsin B) and Sm32 (legumain) are expressed in the cecuma and protonephridia of cercariae. JParasitol, 2001, 87 (5) :1218-1221
Sojka D, Hajdusek O, Dvorák J, Sajid M, Franta Z, Schneider EL, Craik CS, VancováM, BuresováV, Bogyo M, Sexton KB, McKerrow JH, Caffrey CR, Kopácek P. IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int J Parasitol, 2007, 37(7):713-24.
Stach T. Chordate phylogeny and evolution: a not so simple threetaxon problem. J Zool. 2008, 1–25.
Stern L, Perry R, Ofek P, Many A, Shabat D, Satchi-Fainaro R. A novel antitumor prodrug platform designed to be cleaved by the endoprotease legumain.Bioconjug Chem, 2009, [Epub ahead of print]
Stokes MD, Holland ND. The lancelet: also known as "amphioxus," this curious creature has returned to the limelight as a player in the phylogenetic history of the vertebrates. American Scientist, 1998, 86: 552-560
Suzuki MM, Satoh N. Genes expressed in the amphioxus notochord revealed by EST analysis.Dev Biol. 2000, 224(2):168-77.
Tchang S, KooKC. Description of a new variety of Branchiostoma belcheri Gray from Kiaochow Bay, Shantung, China. Contr Inst Zool Nat Acad Peiping, 1936, 3 (4):77-114.
Thomas I M. The accumulation of radioactive iodine by amphioxus. J Mar Biol Ass UK,1956, 35:203.
Urano A, Suzuki MM, Zhang P, Satoh N, Satoh G. Expression of muscle-related genes and two MyoD genes during amphioxus notochord development. Evol Dev. 2003, 5(5):447-58.
Venkatesh TV, Holland ND, Holland LZ, Su MT, Bodmer R. Sequence and developmental expression of AmphiNk2-1: Insights into the evolution origin of the vertebrate thyroid gland and forebrain. Dev. Genes Evol. 1999, 209: 254-259
Venkatesh TV, Holland ND, Holland LZ, Su MT, Bodmer R. Sequence and developmental expression of AmphiNk2-1: Insights into the evolution origin of the vertebrate thyroid glandand forebrain. Dev. Genes Evol. 1999, 209: 254-259
Wang CF, Zhang SC, Luo Y, et al. Presence and induction by bacteria of D-galactoside-specific lectins in the humoral fluids of amphioxus Branchiostoma belcheri tsingtauense. Inflammopharmacol, 2002, 9(3): 241-248
Wang G, Zhang S, Wang Z.Responses of alternative complement expression to challenge with different combinations of Vibrio anguillarum, Escherichia coli and Staphylococcus aureus: evidence for specific immune priming in amphioxus Branchiostoma belcheri. Fish Shellfish Immunol. 2009, 26(1):33-9
Welsch U. The fine structure of the pharynx, cyrtopodocytes and digestive caecum of Amphioxus (Branchiostoma lanceolatum). Symposium of the Zoological Society, London, 1975, 36:17-41
Yamane T, Takeuchi K, Yamamoto , Li YH, Fujiwara M, Nishi K, Takahashi S, Ohkubo I. Legumain from bovine kidney: its purification, molecular cloning, immunohisto- chemical localization and degradation of annexin II and vitamin D-binding protein .Biochim Biophys Acta, 2002, 1596(1):108-20.
Yuan J, Zhang S, Liu Z, Luan Z, Hu G. Cloning and phylogenetic analysis of an amphioxus myogenic bHLH gene AmphiMDF. Biochemical and Biophysical Research Communications, 2003, 301: 960-967
Zhang HW. Granulocytes and macrophages in amphioxus. Zool Sci, 1992, 9(1): 113-118