ENZYME KINETICS AND BIOCHEMICAL PROPERTIES OF SERINE PROTEASES FROM THE MIDGUT OF THE DENGUE MOSQUITO, AEDES AEGYPTI

Abstract

Dengue fever has re-emerged as a global health risk over the past 10 years. The Dengue virus is transmitted by the Aedes aegypti mosquito, which becomes infected with the virus upon blood feeding on an infected human host. Blood feeding, and hence blood meal digestion, is required to obtain the proper nutrients for completion of the gonotrophic cycle. Serine proteolytic enzymes digest the blood meal proteins, and although there are functional studies that have demonstrated the important roles these serine proteases play in blood meal metabolism, no one has biochemically characterized these proteases in vitro. I have engineered recombinant protease constructs that enable us to express, purify, and activate mosquito proteases in vitro using a bacterial expression system and a denaturation/refolding strategy. The four major midgut proteases studied (AaET, AaLT, AaSPVI, and AaSPVII), were purified to near homogeneity and were shown to be active in in vitro enzyme assays. Kinetic parameters, using the artificial trypsin substrate BApNA, were determined for three of the four mosquito proteases. AaLT, which was originally believed to be the major trypsin involved in blood meal digestion in the later phase portion of digestion, was shown not to be a classic trypsin based on in vitro BApNA assays. However, this same AaLT enzyme preparation was shown to cleave natural blood meal protein substrates (serum albumin and hemoglobin), confirming its proteolytic activity. Determination of mosquito protease activities and partial proteolysis of bovine serum albumin (BSA) provide evidence that the four proteases have selective cleavage sites. We also present initial evidence that mosquito proteases may be autocatalytic, based on in vitro auto-cleavage assays with AaET and AaLT.