Purification and primary structural characterization of prophenoloxidases from Aedes aegypti larvae

There are more prophenoloxidase (proPO) genes in mosquitoes than other model insect species studied to date. The high sequence similarity among mosquito proPOs makes it extremely difficult to use histochemical methods to determine the presence of individual proPOs in different stages of mosquito development or their tissue locations. As a consequence, there always are questions when attempting to assign any observed functions to a particular proPO. By following the PO fractions of Aedes aegypti larval proteins during chromatographic separations, we were able to isolate two proPO fractions. Each displayed a single protein band on SDS-PAGE gel. The two fractions showed relative molecular weights of 75 and 60k Da. In-gel trypsin-digestion of the two protein bands and subsequent mass spectrometry of their tryptic peptides confirmed their proPO identities. The 75 kDa protein was a new Aedes aegypti proPO that has not been described in databases, whereas the 60 kDa band contained three previously described Aedes aegypti proPO sequences, with the absence of approximately 125-128 residues at their carboxyl end as compared with their deduced sequences, which suggests that some proPOs might undergo specific proteolytic processing after synthesis. Comparison between the transcriptional profiles of different proPOs and the number of isolated proPO proteins in late-stage larvae indicates that individual proPOs might be transcribed during the earlier stages of larval development, and that resulting proPO proteins persist through all larval stages. Results of this study provide a basis for developing a comprehensive understanding of structure/function relationships of individual proPOs in mosquitoes.