title: A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus.
abstract: mRNAs encoding cathepsin B-like cysteine proteases (CatBs) are abundantly expressed in the genomes of blood-feeding nematodes. Recombinant CatBs have been partially efficacious in vaccine trials in animal models of hookworm infection, supporting further investigation of these enzymes as new control tools. We recently described a family of four distinct CatBs (Na-CP-2, -3, -4, -5) from the human hookworm, Necator americanus. Here we show that these N. americanus CatBs form a robust clade with other hookworm CatBs and are most similar to intestinal CatBs from Haemonchus contortus. All four mRNAs (Na-cp-2, -3, -4 and -5) are up-regulated during the transition from a free-living larva to a blood-feeding adult worm and are also expressed in gut tissue of adult N. americanus that was dissected using laser microdissection microscopy. Recombinant Na-CP-3 was expressed in soluble, secreted form in the yeast Pichia pastoris, while Na-CP-2, -4 and -5 were expressed in insoluble inclusion bodies in Escherichia coli. Recombinant Na-CP-3 was not catalytically active when secreted by yeast but underwent auto-activation to an active enzyme at low pH in the presence of dextran sulphate. Activated Na-CP-3 digested gelatin and cleaved the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin (AMC) but not Z-Arg-Arg-AMC. Recombinant Na-CP-3 did not digest intact hemoglobin but digested globin fragments generated by prior hydrolysis with N. americanus aspartic hemoglobinases. Antibodies raised in mice to all four recombinant proteins showed minimal cross-reactivity with each other, and each antiserum bound to the intestine of adult N. americanus, supporting the intestinal expression of their mRNAs. These data show that N. americanus expresses a family of intestinal CatBs, many of which are likely to be involved in nutrient acquisition and therefore are potential targets for chemotherapies and vaccines.
authors.affiliation: School of Life Sciences, Queensland University of Technology, Brisbane, QLD, Australia.
authors.forename: Najju
authors.initials: N
authors.lastname: Ranjit
authors.forename: Bin
authors.initials: B
authors.lastname: Zhan
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Stenzel
authors.forename: Jason
authors.initials: J
authors.lastname: Mulvenna
authors.forename: Ricardo
authors.initials: R
authors.lastname: Fujiwara
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hotez
authors.forename: Alex
authors.initials: A
authors.lastname: Loukas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000096:biosynthesis* / Q000378:metabolism; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D006224:Cricetinae; D004151:Dipeptides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005780:Gelatin / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006189:Haemonchus / Q000201:enzymology; D006454:Hemoglobins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017221:Necator americanus / Q000201:enzymology*; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015854:Up-Regulation
chemical_list: D003374:Coumarins; D004151:Dipeptides; D006454:Hemoglobins; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D005780:Gelatin; D002401:Cathepsin B