Immune-related protein complexes and serpin-1 isoforms in Manduca sexta plasma



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Kansas State University


Manduca sexta is a large insect species well-suited for biochemical analysis of proteins in the hemolymph (blood) that respond to infection. Insects lack adaptive immunity and rely entirely on innate immunity to prevent and manage infection. Immune response proteins include proteins that bind pathogens and activate serine proteases, which function in proteolytic cascades that trigger effector responses, such as antimicrobial peptide production and prophenoloxidase activation. Phenoloxidase catalyzes melanin synthesis, which leads to microbial killing.
I used MALDI-TOF/TOF mass spectrometry and immunoblotting to identify M. sexta proteins present in putative immune complexes. From analyses of high molecular weight gel filtration fractions of plasma activated by microbial polysaccharides, I detected hemocytin, prophenoloxidase, and cleaved serine protease homologs, suggesting prophenoloxidase and serine protease homologs form large complexes in plasma. I used in vitro bacterial binding assays to identify hemolymph proteins that bind either directly or indirectly to the surface of bacteria or curdlan. Prophenoloxidase, annexin IX, and hemocyte aggregation inhibitor protein were found bound to all the samples tested, indicating they play a role in the early stage of immune response.
Serpins regulate specific active proteases by covalently binding and forming serpin-protease complexes. Serpin-1, an abundant plasma protein, has an alternatively spliced ninth exon encoding 12 serpin-1 isoforms that differ in inhibitory selectivity. RT-PCR showed that all 12 isoforms are expressed in hemocytes, fat body, and midgut. Comparisons of naïve and immune-challenged hemocytes and fat body indicated the immune-related upregulation of serpin-1A but not the other isoforms. Using immunoaffinity chromatography I isolated two serpin-1-protease complexes from plasma after activation with bacterial lipopolysaccharide. MALDI-TOF/TOF analysis of these serpin-1-protease complexes identified the digestive enzyme chymotrypsin as a specific target of serpin-1K. Nine out of the twelve serpin-1 isoforms were identified from control plasma at the protein level using 2D-PAGE. Serpin-1 protease complexes were identified by 2D-PAGE analysis: serpin-1A, E and J were found to be complexed with hemolymph proteinase-8 and an unidentified isoform of serpin-1 was complexed with hemolymph proteinase-1. Discovering the serpin-1 isoforms that inhibit specific proteases enhances our understanding of the regulation of proteolytic cascades in M. sexta.



Manduca sexta, hemolymph, serpin, innate immunity, insect, proteomics

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Doctor of Philosophy


Department of Biochemistry

Major Professor

Michael R. Kanost