Cloning and characterization of four Anopheles gambiae serpin isoforms, differentially induced in the midgut by Plasmodium berghei invasion

The genomic locus SRPN10 of the malaria vector Anopheles gambiae codes for four alternatively spliced serine protease inhibitors of the serpin superfamily. The four 40- to 42-kDa isoforms differ only at their C terminus, which bears the reactive site loop, and exhibit protein sequence similarity with other insect serpins and mammalian serpins of the ovalbumin family. Inhibition experiments with recombinant purified SRPN10 serpins reveal distinct and specific inhibitory activity of three isoforms toward different proteases. All isoforms are mainly expressed in the midgut but also in pericardial cells and hemocytes of the mosquito. The cellular localization of SRPN10 serpins is nucleocytoplasmic in pericardial cells, in hemocytes and in a hemocyte-like mosquito cell line, but in the gut the proteins are mostly localized in the nucleus. Although the transcript levels of all SRPN10 isoforms are marginally affected by bacterial challenge, the transcripts of two isoforms (KRAL and RCM) are induced in female mosquitoes in response to midgut invasion by Plasmodium berghei ookinetes. The KRAL and RCM SRPN10 isoforms represent new potential markers to study the ookinete midgut invasion process in anopheline mosquitoes.