Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph[S]

• Published in: The Journal of biological chemistry 2010-09, Vol.285 (38), p.29642-29650
• Main Authors: Ragan, Emily J., An, Chunju, Yang, Celeste T., Kanost, Michael R.
• Format: Article
• Language: English
• Subjects: Alternative Splicing - genetics; Alternative Splicing - physiology; Amino Acid Sequence; Animals; Electrophoresis, Gel, Two-Dimensional; Genomics and Proteomics; Hemolymph; Hemolymph - metabolism; Immunology; Innate Immunity; Insect; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Manduca - genetics; Manduca - metabolism; Manduca sexta; Mass Spectrometry (MS); Molecular Sequence Data; Polymerase Chain Reaction; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Structure, Secondary; Sequence Homology, Amino Acid; Serine Protease; Serpin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.125419

Mutually exclusive alternative splicing produces transcripts for 12 serpin-1 isoforms in Manduca sexta that differ only in the region encoding the carboxyl-terminal 36–40-amino acid residues. This variable region includes the reactive center loop, which determines the inhibitory selectivity of the serpin. We investigated mRNA levels of individual serpin-1 isoforms by quantitative PCR. The 12 isoforms were expressed at similar levels in hemocytes, but in fat body isoform B mRNA was present at significantly higher levels than isoforms C, D, E, F, G, J, K, and Z. To investigate the presence of individual serpin-1 isoforms in plasma we used immunoaffinity purification of serpin-1 isoforms from M. sexta plasma, followed by two-dimensional PAGE and identification of protein spots by digestion with a series of proteinases and analysis of the resulting peptides by MALDI-TOF/TOF. We identified nine of the 12 serpin-1 isoforms and, through analysis of putative serpin-1-proteinase complexes, identified three endogenous M. sexta proteinase targets of serpin-1. Our results suggest that M. sexta serpin-1 isoforms A, E, and J can inhibit hemolymph proteinase 8, which activates the cytokine spätzle. At least one isoform of serpin-1 can inhibit hemocyte proteinase 1, another M. sexta blood proteinase. In addition, a complex of serpin-1K in a complex with M. sexta midgut chymotrypsin was identified, suggesting serpin-1 isoforms may also function to protect insect tissues from digestive proteinases that may leak into the hemocoel.