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.
Ranjit
Najju
N
Helminth Biology Laboratory, Division of Infectious Diseases, Queensland Institute of Medical Research, Australia.
Zhan
Bin
B
Hamilton
Brett
B
Stenzel
Deborah
D
Lowther
Jonathan
J
Pearson
Mark
M
Gorman
Jeffrey
J
Hotez
Peter
P
Loukas
Alex
A
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