Biomedical Engineering Reference
In-Depth Information
into peptides and amino acids that can be transported across the intestinal
lumen.
11
This process has been best described in the hookworms, N. amer-
icanus
12
and A. caninum,
13
in which Hb is digested by a cooperative cascade of
mechanistically distinct proteases that are expressed in and released from the
intestinal microvilli. Remarkably similar Hb digestion cascades have been
reported from phylogenetically distant blood-feeding parasites including the
platyhelminth blood fluke, Schistosoma mansoni
7,14,15
and the protozoan
malaria parasite, Plasmodium falciparum,
16,17
providing an excellent example of
convergent evolution.
8.2.1 Aspartic Proteases
Aspartic proteases belong to clan AA (http://merops.sanger.ac.uk/) and are
optimally active at acidic pH. Most molecules consist of two structurally
identical lobes, each containing a single aspartic acid residue partially
responsible for peptide hydrolysis. It is only upon proper folding, however, that
the lobes form an active site cleft containing both aspartic acid residues that act
cooperatively to cleave substrate.
18
The enzymes responsible for initiating the
digestion of Hb in human and dog hookworms are the cathepsin-D-like
aspartic proteases, Na-APR-1 and Ac-APR-1 respectively.
19
Both enzymes
cleave the intact Hb tetramer at numerous sites, revealing promiscuous subsite
specificities, but a preference for hydrophobic P1 and P1
0
residues is evi-
dent.
12,19
Hb hydrolysis by APR-1 allows subsequent proteolysis of globin
fragments by other enzymes including cysteine and metallo-proteases.
12,13,19
Moreover, each hookworm's protease will preferentially cleave Hb from its
respective mammalian host despite high levels of sequence identity
19
, a phe-
nomenon which illustrates the finely tuned co-evolution of the host-parasite
relationship, and further demonstrates the critical roles of these enzymes in the
feeding process and the pursuit of developing vaccines that target them.
Dogs vaccinated with recombinant Ac-APR-1 and challenged with L3 of A.
caninum had significant reductions in adult hookworm burdens and faecal egg
counts compared to control dogs that received adjuvant alone.
2
Importantly,
vaccinated dogs had reduced blood loss, and most did not develop anemia (the
main pathology associated with hookworm disease). Furthermore, IgG from
vaccinated animals decreased the catalytic activity of the recombinant enzyme
in vitro, and the antibody bound in situ to the intestines of worms recovered
from vaccinated dogs, implying that vaccination induced antibodies which were
ingested by feeding worms, and that these antibodies interfered with the
parasite's ability to digest its bloodmeal.
2
Further evidence of the ecacy of
this molecule was demonstrated in a vaccine trial conducted with Ac-APR-1 in
hamsters (given a heterologous challenge with N. americanus), which resulted in
a 44% reduction in worm burden in vaccinated compared to control animals.
20
While such reductions in worm burdens might appear modest, it is important to
note that most parasitic helminths do not reproduce asexually within the host,
so even partial reductions in worm numbers can equate to reduced pathology
