A bacteria-induced, intracellular serpin in granular hemocytes of Manduca sexta.
Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.
Gan
H
H
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Wang
Y
Y
Jiang
H
H
Mita
K
K
Kanost
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006434:Hemocytes / Q000201:enzymology*; D007424:Intracellular Fluid; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008837:Micrococcus / Q000276:immunology; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000378:metabolism; D015843:Serpins / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C434683:serpin-2