Molecular cloning and characterization of Ac-mep-1, a developmentally regulated gut luminal metalloendopeptidase from adult Ancylostoma caninum hookworms.
A zinc metalloendopeptidase cDNA (Ac-mep-1) was cloned from Ancylostoma caninum adult hookworms. Ac-mep-1 is encoded by a 2.8 kb mRNA with a predicted open reading frame (ORF) of 870 amino acids (predicted pI=5.5, m.w.=98.7 kDa) that contains four potential N-linked glycosylation sites and predicted zinc-binding domains (HExxH and ENxADxGG). These domains represent signature sequences of the Neutral Endopeptidase 24.11 (neprilysin) family of enzymes. The ORF corresponding to Ac-MEP-1 exhibited strong similarity to metalloproteases from the trichostrongyle Haemonchus contortus as well as Caenorhabditis elegans. RT-PCR analysis of A. caninum eggs, L1, non-activated and activated L3 and adult cDNA identify transcription of Ac-MEP-1 only in the adult stage of the parasite. Mouse antibody raised to the expressed protein recognized proteins of approximately 90 and 100 kDa in adult hookworm extracts. Adult worm sections probed with these antisera localized Ac-mep-1 to the microvilli of the worm gastrointestinal tract suggesting a possible role for this enzyme in digestion of the parasite blood meal.
Jones
Brian F
BF
Medical Helminthology Laboratory, Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, CT 06510, USA.
Hotez
Peter J
PJ
D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D018507:Gene Expression Regulation, Developmental; D015801:Helminth Proteins; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism
D015801:Helminth Proteins; D012333:RNA, Messenger; C449009:MEP-1 protein, Ancylostoma caninum; D008666:Metalloendopeptidases; D015032:Zinc