Identification and characterization of an asparaginyl endopeptidase from Angiostrongylus cantonensis.
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.
Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-yuan, 333, Taiwan.
Shih-Hsin
SH
Chang
Kuang-Yao
KY
Chen
Lian-Chen
LC
Wang
D000595:Amino Acid Sequence; D017185:Angiostrongylus cantonensis / Q000201:enzymology* / Q000235:genetics; D000818:Animals; D000909:Antibodies, Helminth; D000918:Antibody Specificity; D001482:Base Composition; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D008807:Mice, Inbred BALB C; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017206:Strongylida Infections; D000099240:Asparaginyl Endopeptidase
D000909:Antibodies, Helminth; D003546:Cysteine Endopeptidases; D007074:Immunoglobulin G; D000099240:Asparaginyl Endopeptidase