Biomedical Engineering Reference
In-Depth Information
Attempts to determine whether PfA-M1 and PfA-M17 are genetically
essential have so far proven inconclusive.
22
Generation of transgenic Pf
parasites that over-expressed the full-length, functionally active neutral ami-
nopeptidases were less susceptible to killing by bestatin when compared to wild-
type parasites.
29,30
These data support the hypothesis that both the PfA-M1
and PfA-M17 neutral aminopeptidases are main targets for the anti-malarial
activity of bestatin. These data show the need to investigate the similarities and
differences in the structure of these enzymes to enhance our prospect of
developing potent anti-malarials, either as a single drug that blocks the two
enzymes or as a combination with overlapping inhibitory properties.
7.2 Pf Neutral Aminopeptidases
7.2.1 PfA-M1 Alanyl Aminopeptidase
The M1 alanyl aminopeptidase gene is present in a single copy and located on
chromosome 13 of Pf. The gene is 3257 bp in length and encodes a protein of
1085 amino acids and a predicted full length of 122 kDa. The enzyme is a
member of the Clan MA, Family M1 and hence is termed PfA-M1 (http://
merops.sanger.ac.uk). The full-length amino acid sequence of PfA-M1 exhibits
B
70% sequence similarity with M1 aminopeptidase orthologues of the various
rodent malaria sp. (P. berghei, P. chabaudi chabaudi,andP. yoelii); the
sequences are most divergent at the large non-conserved N-terminal extension
(
194 amino acids) which contains three asparagine-rich low-complexity
regions and a putative transmembrane domain. PfA-M1 is expressed at all
developmental stages of the intra-erythrocytic parasite. In soluble parasite
extracts the enzyme has been identified as having three forms—each designated
by their molecular weight (p120, p96 and p68).
26,34
B
7.2.1.1 Proteolytic Maturation and Localization
The maturation of PfA-M1 in Pf parasites begins with the inactive precursor
p120 being targeted to the parasitophorous vacuole via the parasite endo-
plasmic reticulum/Golgi, where it is converted into the transient p96 form.
26,35
The conversion from p120 to p96 involves the removal of the N-terminal signal
sequence of the protein.
26
This p96 form is eventually redirected into the
parasite, and further processing of the protein results in the proteolytic removal
of the entire C-terminal domain to yield p68.
35
The p68 protein is only mar-
ginally (approx. 16%) delivered to the parasite food vacuole with the majority
of the protein remaining in the parasite cytosol.
35
To date, enzymatic analysis
has been restricted to the combined protease activity of both the p96 and p68
forms isolated from soluble extracts of malaria parasites.
26,34
Functional ana-
lysis of the recombinant p96 protein shows that it is functional only at neutral
pH in vitro, thus arguing for a cytosolic location in the parasite.
30
However,
studies by Dalal and Klemba that tracked a C-terminally tagged version of the
protein, clearly showed that PfA-M1 was found in the DV lumen and the
