Biomedical Engineering Reference
In-Depth Information
and the sexual stages of development take place in the mosquito. Through all
the stages of parasite development, proteolytic enzymes may be involved in the
invasion of cells and passage through tissue barriers in addition to their roles in
hemoglobin degradation.
11.2.1 Plasmepsins as Targets for Drug Discovery Against
Malaria
Our detailed understanding of the role of proteolytic enzymes in the degra-
dation of hemoglobin in the food vacuole of the malaria parasite P. falciparum
was initiated by the work of the Goldberg laboratory with the discovery of two
enzymes, called hemoglobinases 1 and 2.
6
These were isolated by column
chromatography from a sonicated extract of frozen trophozoites. The sequence
of the first peak of activity (hemoglobinase I) was identified by cloning and
sequencing. Subsequently, Dame and colleagues
7
cloned the DNA encoding the
second activity, determined the sequence, expressed the protein, characterized
its activity, and modeled the structure by homology with the aspartic proteinase
class of enzymes, based on the 33% identity with human cathepsin D
(HuCatD). Hemoglobinases 1 and 2 were unusual only in that their proseg-
ments (the N-terminal self-inhibitory extensions common to this class of pep-
tidase) were approximately 124 amino acids long, over two times as large as
those of the classic zymogen forms of pepsin and gastricsin, which are about 50
amino acids in length. The enzymes were named plasmepsin 1 and plasmepsin
2, in order of their discovery, where the names are derived from Plasmodium
pepsin. More detail on these two enzymes and others in the family will be
provided in later sections of this chapter.
Other proteases may play critical roles, so other families of proteases could
provide additional targets for drug discovery.
8-13
However, the plasmepsin
family has elicited great interest and attention.
14
11.2.2 Plasmepsin Gene Family in the Malaria Parasite
With the completion of the sequencing of the genome of the malaria parasite,
15
it was found that there are in fact 10 genes for aspartic peptidases in all.
16
In the
paper by Coombs et al., it is stated that 'The use of diagnostic inhibitors that
block heme release and kill the parasite in culture has implicated two aspartic
proteases, plasmepsins I and II, in the early stages of this degradative pro-
cess'.
16
However, now that we know that 10 different enzymes of this class exist
in the parasite, it is not clear that blocking hemoglobin degradation and killing
of the parasite are related. First, the assumption that the compounds used were
in fact 'diagnostic' inhibitors presumes that the effect is only upon the two
known (at that time) plasmepsins. Second, we now know that other enzymes
within the food vacuole contribute to hemoglobin digestion. As described
below, we now know that both plasmepsin I and II can be knocked out, and the
parasite remains viable.
