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VOL. 72 (4), 629-642, 2006 PROTEIN PROCESSING IN PLASMODIUM FALCIPARUM?

DISCUSSION

The data presented above indicate that this unique bifunctional
Plasmodium G6PD-6PGL protein varies in size depending on the
parasite developmental stage. Thus, the 69 KDa band detected by
the anti-G6PD domain antibody coincides with the expected size of
the G6PD domain at the C-terminal, and the 73 kDa band identified
by the anti-6PGL domain antibody could have a size that including
the essential insertion (24) corresponds to the N-terminal of the
protein with the 6PGL activity. Thus, it seems that the bifunctional
protein could mature to render two different polypeptides with sepa-
rate enzyme activities but sharing the essential insertion.

To assess specific biological gene function in the parasite, several
systems for the functional analysis of P. falciparum genes have been
developed, including gene silencing by antisense RNA (14, 18) or
more recently, by RNA interference (16-18). Antisense RNA has
been found in humans, mice, plants and protozoan parasites such as
P. falciparum. The fact that endogenous antisense RNAs are
widespread in P. falciparum, suggests that they could be a natural
gene expression regulatory mechanism (28, 29). In our model,
P. falciparum G6PD-6PGL was silenced in vivo through a dsRNA.
Although mechanisms of RNAi silencing in many organisms are not
well known, this technique has been used to study gene function in
a great variety of organisms including other parasites (30). Despite
the fact that, so far, the genes encoding the required RNAi machinary
have not been detected in any of the currently available Plasmodium
databases, RNAi silencing has been achieved in Plasmodium (16-18).
Thus, it could be that the data reported for Plasmodium, as well as
our results using dsRNA-G6PD, are the consequence of an antisense
RNA rather than a direct RNAi effect. However, it is also true that,
to date, 60% of the genes predicted for P. falciparum have no known
homologs, and we have no clues as to their function (4).

The protein expression patterns examined by gene silencing
showed that after 3 h of transfection, when most of the parasites are
at the ring stage, the complete 107 kDa band predominates in both
transfected and not transfected parasites. However, after 24 h of
electroporation when the parasites were mainly at the trophozoite
stage, the main band was the 73 kDa band. Again, this data suggest

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