Analysis of mutually exclusive alternatively spliced serpin-1 isoforms and identification of serpin-1 proteinase complexes in Manduca sexta hemolymph.
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.
Ragan
Emily J
EJ
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
An
Chunju
C
Yang
Celeste T
CT
Kanost
Michael R
MR
D017398:Alternative Splicing / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D006458:Hemolymph / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018402:Manduca / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019476:Insect Proteins; D020033:Protein Isoforms