Skip to main content

Advertisement

SpringerLink
Log in

Identification and characterization of an asparaginyl endopeptidase from Angiostrongylus cantonensis

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

Asparaginyl endopeptidase, also known as legumain, is a family of cysteine proteases in many organisms. In this study, an asparaginyl endopeptidase (Ac-AEP) was identified from the cDNA library of Angiostrongylus cantonensis. The full-length of Ac-AEP was determined to be 1,472 bp with an open reading frame of 1,341 bp encoding a putative protein with 446 amino acids. This putative protein was determined to have 37–65 % identity in the amino acid sequences of the asparaginyl endopeptidases of other parasitic helminths. By real-time quantitative PCR analysis, Ac-AEP was revealed to be more abundantly expressed in the female adult worms than in other development stages. A recombinant asparaginyl endopeptidase (rAc-AEP) was then produced by a Pichia pastoris expression system. Posttranslational modification was shown to occur via N-linked glycosylation in this recombinant enzyme. The proteolytic activity of rAc-AEP was inhibited by iodoacetamide but not affected by E64, pepatain A, AEBSF, and EDTA. Moreover, the purified rAc-AEP was recognized by IgG in serum samples from BALB/c or ICR mice with A. cantonensis infection and patients with eosinophilic meningitis. These findings indicate that the rAc-AEP may have the potential for detecting A. cantonensis infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Abe Y, Shirane K, Yokosawa H, Matsushita H, Mitta M, Kato I, Ishii S (1993) Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis. J Biol Chem 268:525–3529

    Google Scholar 

  • Adisakwattana P, Viyanant V, Chaicumpa W, Vichasri-Grams S, Hofmann A, Korge G, Sobhon P, Grams R (2007) Comparative molecular analysis of two asparaginyl endopeptidases and encoding genes from Fasciola gigantica. Mol Biochem Parasitol 156:102–116

    Article  CAS  PubMed  Google Scholar 

  • Baheti NN, Sreedharan M, Krishnamoorthy T, Nair MD, Radhakrishnan K (2008) Neurological picture. Eosinophilic meningitis and an ocular worm in a patient from Kerala, south India. J Neurol Neurosurg Psychiatry 79:271

    Article  CAS  PubMed  Google Scholar 

  • Brockelman CR, Chusatayanond W, Baidikul V (1976) Growth and localization of Angiostrongylus cantonensis in the molluscan host, Achatina fulica. Southeast Asian J Trop Med Public Health 7:30–37

    Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Lett 24:45–66

    Article  CAS  Google Scholar 

  • Chang SH, Tang P, Wang LC (2011) A transcriptomic study on the pepsin-activated infective larvae of Angiostrongylus cantonensis. Mol Biochem Parasitol 179:47–50

    Article  CAS  PubMed  Google Scholar 

  • Chang SH, Tang P, Yen CM, Chow KP, Wang LC (2014) A transcriptomic analysis on gene expressions in the infective third and pathogenic fifth larval stages of Angiostrongylus cantonensis. Parasitol Int 63:42–48

    Article  CAS  PubMed  Google Scholar 

  • Chen JM, Fortunato M, Barrett AJ (2000) Activation of human prolegumain by cleavage at a C-terminal asparagine residue. Biochem J 352:327–334

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen D, Zhang Y, Shen H, Wei Y, Huang D, Tan Q, Lan X, Li Q, Chen Z, Li Z, Ou L, Suen H, Ding X, Luo X, Li X, Zhan X (2011) Epidemiological survey of Angiostrongylus cantonensis in the west-central region of Guangdong Province, China. Parasitol Res 109:305–314

    Article  PubMed  Google Scholar 

  • Chen JX, Chen MX, Ai L, Chen JH, Chen SH, Zhang YN, Cai YC, Zhu XQ, Zhou XN (2012) A protein microarray for the rapid screening of patients suspected of infection with various food-borne helminthiases. PLoS Negl Trop Dis 6:e1899

    Article  PubMed Central  PubMed  Google Scholar 

  • Dalton JP, Brindley PJ (1996) Schistosome asparaginyl endopeptidase SM32 in hemoglobin digestion. Parasitol Today 12:125

    Article  CAS  PubMed  Google Scholar 

  • Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorak J, Hsieh I, Bahgat M, Dissous C, McKerrow JH (2006) A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem 281:39316–39329

    Article  CAS  PubMed  Google Scholar 

  • Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27:297–306

    CAS  PubMed  Google Scholar 

  • Dissanaike AS, Cross JH (2004) Ocular parastrongyliasis (=angiostrongyliasis): probable first report of human infection from a patient in Ceylon (Sri Lanka). Parassitologia 46:315–316

    CAS  PubMed  Google Scholar 

  • Dorta-Contreras AJ, Padilla-Docal B, Moreira JM, Robles LM, Aroca JM, Alarcon F, Bu-Coifiu-Fanego R (2011) Neuroimmunological findings of Angiostrongylus cantonensis meningitis in Ecuadorian patients. Arq Neuropsiquiatr 69:466–469

    Article  PubMed  Google Scholar 

  • Dvorak J, Mashiyama ST, Sajid M, Braschi S, Delcroix M, Schneider EL, McKerrow WH, Bahgat M, Hansell E, Babbitt PC, Craik CS, McKerrow JH, Caffrey CR (2009) SmCL3, a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni. PLoS Negl Trop Dis 3:e449

    Article  PubMed Central  PubMed  Google Scholar 

  • Eamsobhana P, Yong HS (2009) Immunological diagnosis of human angiostrongyliasis due to Angiostrongylus cantonensis (Nematoda: Angiostrongylidae). Int J Infect Dis 13:425–431

    Article  PubMed  Google Scholar 

  • El-Sayed LH, Ghoneim H, Demian SR, El-Sayed MH, Tawfik NM, Sakr I, Abou-Basha LM, Renganathan E, Klinkert MQ, Abou-Rawash N (1998) Diagnostic significance of Schistosoma mansoni proteins Sm31 and Sm32 in human schistosomiasis in an endemic area in Egypt. Trop Med Int Health 3:721–727

    Article  CAS  PubMed  Google Scholar 

  • Han YP, Li ZY, Li BC, Sun X, Zhu CC, Ling XT, Zheng HQ, Wu ZD, Lv ZY (2011) Molecular cloning and characterization of a cathepsin B from Angiostrongylus cantonensis. Parasitol Res 109:369–378

    Article  PubMed  Google Scholar 

  • Hewitson JP, Grainger JR, Maizels RM (2009) Helminth immunoregulation: the role of parasite secreted proteins in modulating host immunity. Mol Biochem Parasitol 167:1–11

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Huang CJ, Damasceno LM, Anderson KA, Zhang S, Old LJ, Batt CA (2011) A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol 90:235–247

    Article  CAS  PubMed  Google Scholar 

  • Intapan PM, Maleewong W, Sawanyawisuth K, Chotmongkol V (2003) Evaluation of human IgG subclass antibodies in the serodiagnosis of angiostrongyliasis. Parasitol Res 89:425–429

    PubMed  Google Scholar 

  • Ju JW, Joo HN, Lee MR, Cho SH, Cheun HI, Kim JY, Lee YH, Lee KJ, Sohn WM, Kim DM, Kim IC, Park BC, Kim TS (2009) Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms. Proteomics 9:3066–3078

    Article  CAS  PubMed  Google Scholar 

  • Klinkert MQ, Felleisen R, Link G, Ruppel A, Beck E (1989) Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases. Mol Biochem Parasitol 33:113–122

    Article  CAS  PubMed  Google Scholar 

  • Laha T, Sripa J, Sripa B, Pearson M, Tribolet L, Kaewkes S, Sithithaworn P, Brindley PJ, Loukas A (2008) Asparaginyl endopeptidase from the carcinogenic liver fluke, Opisthorchis viverrini, and its potential for serodiagnosis. Int J Infect Dis 12:e49–e59

    Article  PubMed Central  PubMed  Google Scholar 

  • Leone S, De Marco M, Ghirga P, Nicastri E, Esposito M, Narciso P (2007) Eosinophilic meningitis in a returned traveler from Santo Domingo: case report and review. J Travel Med 14:407–410

    Article  PubMed  Google Scholar 

  • Lima AR, Mesquita SD, Santos SS, Aquino ER, Rosa Lda R, Duarte FS, Teixeira AO, Costa ZR, Ferreira ML (2009) Alicata disease: neuroinfestation by Angiostrongylus cantonensis in Recife, Pernambuco, Brazil. Arq Neuropsiquiatr 67:1093–1096

    Article  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Maley F, Trimble RB, Tarentino AL, Plummer TH Jr (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180:195–204

    Article  CAS  PubMed  Google Scholar 

  • Mattis A, Mowatt L, Lue A, Lindo J, Vaughan H (2009) Ocular angiostrongyliasis—first case report from Jamaica. West Indian Med J 58:383–385

    CAS  PubMed  Google Scholar 

  • Morassutti AL, Levert K, Pinto PM, da Silva AJ, Wilkins P, Graeff-Teixeira C (2012) Characterization of Angiostrongylus cantonensis excretory-secretory proteins as potential diagnostic targets. Exp Parasitol 130:26–31

    Article  CAS  PubMed  Google Scholar 

  • Ni F, Wang Y, Zhang J, Yu L, Fang W, Luo D (2012) Cathepsin B-like and hemoglobin-type cysteine proteases: stage-specific gene expression in Angiostrongylus cantonensis. Exp Parasitol 131:433–441

    Article  CAS  PubMed  Google Scholar 

  • Oliver EM, Skuce PJ, McNair CM, Knox DP (2006) Identification and characterization of an asparaginyl proteinase (legumain) from the parasitic nematode, Haemonchus contortus. Parasitology 133:237–244

    Article  CAS  PubMed  Google Scholar 

  • Ranjit N, Zhan B, Hamilton B, Stenzel D, Lowther J, Pearson M, Gorman J, Hotez P, Loukas A (2009) Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator americanus. J Infect Dis 199:904–912

    Article  CAS  PubMed  Google Scholar 

  • Rawlings ND, Barrett AJ (1994) Families of cysteine peptidases. Methods Enzymol 244:461–486

    Article  CAS  PubMed  Google Scholar 

  • Rawlings ND, Barrett AJ, Bateman A (2012) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 40:D343–D350

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rosen L, Loison G, Laigret J, Wallace GD (1967) Studies on eosinophilic meningitis. 3. Epidemiologic and clinical observations on Pacific islands and the possible etiologic role of Angiostrongylus cantonensis. Am J Epidemiol 85:17–44

    CAS  PubMed  Google Scholar 

  • Rotari VI, Dando PM, Barrett AJ (2001) Legumain forms from plants and animals differ in their specificity. Biol Chem 382:953–959

    Article  CAS  PubMed  Google Scholar 

  • Sajid M, McKerrow JH (2002) Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 120:1–21

    Article  CAS  PubMed  Google Scholar 

  • Sajid M, McKerrow JH, Hansell E, Mathieu MA, Lucas KD, Hsieh I, Greenbaum D, Bogyo M, Salter JP, Lim KC, Franklin C, Kim JH, Caffrey CR (2003) Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase. Mol Biochem Parasitol 131:65–75

    Article  CAS  PubMed  Google Scholar 

  • Santos-Silva LK, Soares-Costa A, Gerald LT, Meneghin SP, Henrique-Silva F (2012) Recombinant expression and biochemical characterization of sugarcane legumain. Plant Physiol Biochem 57:181–192

    Article  CAS  PubMed  Google Scholar 

  • Sojka D, Hajdusek O, Dvorak J, Sajid M, Franta Z, Schneider EL, Craik CS, Vancova M, Buresova V, Bogyo M, Sexton KB, McKerrow JH, Caffrey CR, Kopacek P (2007) IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus. Int J Parasitol 37:713–724

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Takeda O, Miura Y, Mitta M, Matsushita H, Kato I, Abe Y, Yokosawa H, Ishii S (1994) Isolation and analysis of cDNA encoding a precursor of Canavalia ensiformis asparaginyl endopeptidase (legumain). J Biochem 116:541–546

    CAS  PubMed  Google Scholar 

  • Tkalcevic J, Ashman K, Meeusen E (1995) Fasciola hepatica: rapid identification of newly excysted juvenile proteins. Biochem Biophys Res Commun 213:169–174

    Article  CAS  PubMed  Google Scholar 

  • Tseng YT, Tsai HC, Sy CL, Lee SS, Wann SR, Wang YH, Chen JK, Wu KS, Chen YS (2011) Clinical manifestations of eosinophilic meningitis caused by Angiostrongylus cantonensis: 18 years’ experience in a medical center in southern Taiwan. J Microbiol Immunol Infect 44:382–389

    Article  CAS  PubMed  Google Scholar 

  • Wang LC, Chao D, Chen ER (1989) Acquired immunity in rats against Angiostrongylus cantonensis infection. Int J Parasitol 19:617–620

    Article  CAS  PubMed  Google Scholar 

  • Wang QP, Lai DH, Zhu XQ, Chen XG, Lun ZR (2008) Human angiostrongyliasis. Lancet Infect Dis 8:621–630

    Article  PubMed  Google Scholar 

  • Wang QP, Wu ZD, Wei J, Owen RL, Lun ZR (2012) Human Angiostrongylus cantonensis: an update. Eur J Clin Microbiol Infect Dis 31:389–395

    Article  PubMed  Google Scholar 

  • Wang LC, Chen KY, Chang SH, Chung LY, Gan RC, Cheng CJ, Tang P (2013) Transcriptome profiling of the fifth-stage larvae of Angiostrongylus cantonensis by next-generation sequencing. Parasitol Res 112:3193–3202

    Article  PubMed Central  PubMed  Google Scholar 

  • Wawrzyniak I, Texier C, Poirier P, Viscogliosi E, Tan KS, Delbac F, El Alaoui H (2012) Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite. Parasitol Int 61:437–442

    Article  CAS  PubMed  Google Scholar 

  • Yamane T, Takeuchi K, Yamamoto Y, Li YH, Fujiwara M, Nishi K, Takahashi S, Ohkubo I (2002) Legumain from bovine kidney: its purification, molecular cloning, immunohistochemical localization and degradation of annexin II and vitamin D-binding protein. Biochim Biophys Acta 1596:108–120

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported in part by a grant from the Chang Gung Memorial Hospital Research Grant CMRPD1D0352, CMRPD1A0113 and National Science Council, Executive Yuan, ROC (NSC102-2320-B-182-028-MY3).

Author information

Authors and Affiliations

  1. Department of Parasitology, College of Medicine, Chang Gung University, Tao-Yuan, 333, Taiwan

    Lian-Chen Wang

  2. Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-yuan, 333, Taiwan

    Shih-Hsin Chang, Kuang-Yao Chen & Lian-Chen Wang

Authors
  1. Shih-Hsin Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kuang-Yao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lian-Chen Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lian-Chen Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chang, SH., Chen, KY. & Wang, LC. Identification and characterization of an asparaginyl endopeptidase from Angiostrongylus cantonensis . Parasitol Res 113, 2143–2152 (2014). https://doi.org/10.1007/s00436-014-3866-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-014-3866-0

Keywords

Search

Navigation