Proteolytic degradation of hemoglobin in the intestine of the human hookworm Necator americanus.
Blood-feeding parasites use mechanistically distinct proteases to digest hemoglobin (Hb), often as multienzyme cooperative cascades. We investigated the roles played by 3 distinct proteases from adults of the human hookworm Necator americanus. The aspartic protease Na-APR-1 and the cysteine protease Na-CP-3 were expressed in catalytically active form in yeast, and the metalloprotease Na-MEP-1 was expressed in catalytically active form in baculovirus. Antibodies to all 3 proteases were used to immunolocalize each native enzyme to the intestine of adult N. americanus. Recombinant Na-APR-1 cleaved intact human Hb. In contrast, Na-CP-3 and Na-MEP-1 could not cleave Hb but instead cleaved globin fragments that had been hydrolyzed by Na-APR-1, implying an ordered process of hemoglobinolysis. Seventy-four cleavage sites within Hb alpha- and beta-chains were characterized after digestion with all 3 proteases. All of the proteases demonstrated promiscuous subsite specificities within Hb; noteworthy preferences included aromatic and hydrophobic P1 residues and hydrophobic P1' residues for Na-APR-1 and hydrophobic P1 residues for Na-MEP-1. We conclude that Hb digestion in N. americanus involves a network of distinct proteases, some of which act in an ordered fashion, providing a potential mechanism by which some of these hemoglobinases exert their efficacy as recombinant vaccines against hookworm infection.
Helminth Biology Laboratory, Division of Infectious Diseases, Queensland Institute of Medical Research, Australia.
Najju
N
Ranjit
Bin
B
Zhan
Brett
B
Hamilton
Deborah
D
Stenzel
Jonathan
J
Lowther
Mark
M
Pearson
Jeffrey
J
Gorman
Peter
P
Hotez
Alex
A
Loukas
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D017221:Necator americanus / Q000201:enzymology / Q000378:metabolism*; D009332:Necatoriasis / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010963:Plasmodium falciparum / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D006454:Hemoglobins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C070077:alpha(A) globin; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases