Background The mosquito invasive form of the malaria parasite, the ookinete, secretes chitinases critical for mosquito invasion and continuation of the parasite life cycle. This class of enzyme is validated as a target for interrupting malaria transmission. A recombinant zymogen form of the P. gallinaceum chitinase rPgCHT1 was previously shown to be activated in vitro by the serine protease Endoproteinase Lys-C.
Objective To test the hypothesis that an ookinete-produced serine protease activates pro-PgCHT1, we sought to characterize this enzyme toward the goal of validating this protease as a target of blocking malaria transmission.
Methods Conditioned ookinete culture medium was subjected to a benzamidine (serine protease inhibitor) affinity column. The ability of the eluate to activate pro-rPgCHT1 in the presence and absence of the serine protease inhibitor AEBSF and the aspartic acid protease inhibitor pepstatin A was assessed using a microfluorimetry assay, which measured chitinase activity expressed as relative fluorescence units (RFU)/minute.
Results Eluate containing 11.1 μg of protein increased the activity of 11.1 μg of rPgCHT1 from 30 RFU/minute to 488 RFU/minute (p = .002). This effect was diminished by 21% following the addition of the serine protease inhibitor AEBSF (p = .046) but not by the aspartic protease inhibitor pepstatin A. Eluate without rPgCHT1 had 477 RFU/minute of chitinase activity, significantly higher than rPgCHT1 by itself (p = .003) but not significantly different from eluate and rPgCHT1 together (p = .64). The addition of AEBSF to eluate decreased activity by 23% (p = .017), with no effect seen following the addition of pepstatin A.
Discussion Benzamidine-column eluate of P. gallinaceum ookinete culture supernatants proteolytically activated pro-rPgCHT1. This eluate also possessed intrinsic chitinase activity, suggesting the cosecretion of PgCHT1 and serine protease activity in a complex. These findings are novel for apicomplexan parasites and have practical application toward developing novel approaches to blocking malaria transmission.
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