Identification and biochemical characterization of a Kunitz‐type serine protease inhibitor from an insect, Manduca sexta (772.1)

Abstract

Hemolymph (blood) of insects contains serine proteases and serine protease inhibitors that function in innate immune responses. The objective for this research was to investigate low molecular weight protease inhibitors from hemolymph of a lepidopteran insect, Manduca sexta. We identified serine protease inhibitor activity against trypsin, chymotrypsin, and elastase in plasma from naïve larvae and in plasma from larvae that were previously injected with Micrococcus luteus. We separated hemolymph plasma proteins using size exclusion chromatography, and the fractions with protease inhibitor activity were analyzed by SDS‐PAGE and liquid chromatography‐electrospray ionization tandem mass spectrometry. A serine protease inhibitor belonging to the Kunitz family was identified, and using primers based on the sequence encoding this protein found in the M. sexta genome sequence, we cloned its cDNA from larval fat body RNA. The cDNA encodes a protein with a secretion signal sequence followed by the mature secreted inhibitor protein of 56 amino acid residues. This sequence is highly similar to a Kunitz inhibitor previously purified from M. sexta (Ramesh, N., Sugumaran, M., Mole, J.E. 1988. J Biol Chem. 263:11523‐7). Using a synthesized and codon‐optimized DNA encoding the protease inhibitor, we are currently producing the recombinant inhibitor in E. coli for further biochemical characterization. We will study its inhibitory activity as a potential regulator of proteases in hemolymph that function in the innate immune system of M. sexta. The genome of M. sexta contains nine genes for single‐domain Kunitz proteins closely spaced on a single scaffold. The ultimate goal of this project is to understand the biological functions of these Kunitz protease inhibitors in innate immunity.Grant Funding Source: Supported by NIH grant GM41247.