Analysis of serpin‐1 isoforms in hemolymph of the insect Manduca sexta

Hemolymph of the caterpillar Manduca sexta contains no less than 25 serine proteases. At least some of these proteases are involved aspects of innate immunity, such as activation of prophenoloxidase. Also in hemolymph are serine protease inhibitors, including those from seven different serpin genes. Serpin‐1 has an alternatively spliced ninth exon that codes for production of 12 serpin‐1 isoforms that differ in sequences of their reactive center loop and in inhibitory selectivity. Serpin‐1 is expressed in both fat body and hemocytes and secreted into hemolymph. Little is known about the regulation of serpin‐1 isoform expression or which proteases are inhibited by these serpin‐1 isoforms. Our goals are to understand which serpin‐1 isoforms are present in plasma and to identify some protease targets of these isoforms. We used two dimensional polyacrylamide gel electrophoresis followed by matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) to separate and quantify serpin‐1 isoforms in plasma. The serpin‐1 isoforms differ in their concentration in plasma, suggesting that regulation of splicing occurs. We also used antibodies to serpin‐1 to construct an affinity column for serpin‐1 to isolate serpin‐1 protease complexes from Manduca plasma. Identification by MALDI‐TOF of proteases that form complexes with individual serpin‐1 isoforms will provide information about in vivo protease targets of the serpin‐1 isoforms. Knowing the serpin‐1 isoforms that inhibit specific proteases enhances our understanding of proteolytic cascades in Manduca and provides inhibitors for specific steps in those cascades. Research supported by NIH R01‐GM41247