A family of cathepsin B cysteine proteases expressed in the gut of the human hookworm, Necator americanus

• Published in: Molecular and biochemical parasitology 2008-08, Vol.160 (2), p.90-99
• Main Authors: Ranjit, Najju, Zhan, Bin, Stenzel, Deborah J., Mulvenna, Jason, Fujiwara, Ricardo, Hotez, Peter J., Loukas, Alex
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cathepsin B; Cathepsin B - biosynthesis; Cathepsin B - metabolism; Cloning, Molecular; Coumarins - metabolism; Cricetinae; Cysteine protease; Dipeptides - metabolism; Escherichia coli - genetics; Gelatin - metabolism; Gene Expression; Gene Expression Profiling; Haemonchus - enzymology; Hemoglobin; Hemoglobins - metabolism; Hookworm; Mice; Molecular Sequence Data; Necator americanus; Necator americanus - enzymology; Phylogeny; Pichia - genetics; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Up-Regulation
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/j.molbiopara.2008.04.008

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.