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J. Biol. Chem., Vol. 280, Issue 14, 14341-14348, April 8, 2005

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Manduca sexta Serpin-6 Regulates Immune Serine Proteinases PAP-3 and HP8

cDNA CLONING, PROTEIN EXPRESSION, INHIBITION KINETICS, AND FUNCTION ELUCIDATION^*

Zhen Zou and Haobo Jiang

From the Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, Oklahoma 74078

Analogous to blood coagulation and complement activation in mammals, some insect defense responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in hemolymph. We recently isolated Manduca sexta serpin-6 from hemolymph of the bacteria-challenged larvae, which selectively inhibited proPO-activating proteinase-3 (PAP-3) (Wang, Y., and Jiang, H. (2004) Insect Biochem. Mol. Biol. 34, 387–395). To further characterize its structure and function, we cloned serpin-6 from an induced fat body cDNA library using a PCR-derived probe. M. sexta serpin-6 is 55% similar in amino acid sequence to Drosophila melanogaster serpin-5, an immune-responsive protein. We produced serpin-6 in an Escherichia coli expression system and purified the soluble protein by nickel affinity and hydrophobic interaction chromatography. The recombinant protein specifically inhibited PAP-3 and blocked proPO activation in vitro in a concentration-dependent manner. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that the cleavage site of serpin-6 is between Arg³⁷³ and Ser³⁷⁴. Serpin-6 is constitutively present in hemolymph of naïve larvae, and its mRNA and protein levels significantly increase after a bacterial injection. The association rate constant of serpin-6 and PAP-3 is 2.6 x 10⁴ M^-1 s^-1, indicating that serpin-6 may contribute to the inhibitory regulation of PAP-3 in the hemolymph. We also identified the covalent complex of serpin-6 and PAP-3 in induced hemolymph by immunoaffinity chromatography and mass spectrometry. Furthermore, immulectin-2, serine proteinase homologs, proPO, PO, attacin-2, and a complex of serpin-6 and hemolymph proteinase-8 were also detected in the proteins eluted from the immunoaffinity column using serpin-6 antibody. These results suggest that serpin-6 plays important roles in the regulation of immune proteinases in the hemolymph.

Received for publication, January 18, 2005 , and in revised form, February 3, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY672799.

^* This work was supported by National Institutes of Health Grant GM58634. This article was approved for publication by the Director of Oklahoma Agricultural Experimental Station and supported in part under Project OKLO2450. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Ctr., Stillwater, OK 74078. Tel.: 405-744-9400; Fax: 405-744-6039; E-mail: haobo{at}okstate.edu.

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