Article

Crystal structure of native Anopheles gambiae serpin-2, a negative regulator of melanization in mosquitoes

Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.92). 06/2011; 79(6):1999-2003. DOI: 10.1002/prot.23002
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Available from: Kristin Michel, Jun 12, 2015
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    ABSTRACT: Serine protease cascade-mediated prophenolxidase activation is a prominent innate immune response in insect defense against the invading pathogens. Serpins regulate this reaction to avoid excessive activation. However, the function of serpins in most insect species, especially in some non-model agriculture insect pests, is largely unknown. We here cloned a full-length cDNA for a serpin, named as serpin-3, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of serpin-3 encodes 462-amino acid residue protein with a 19-redidue signal peptide. It contains a reactive center loop strikingly similar to the proteolytic activation site in prophenoloxidase. Sequence comparison indicates that O. furnacalis serpin-3 is an apparent ortholog of Manduca sexta serpin-3, a defined negative regulator of melanization reaction. Serpin-3 mRNA and protein levels significantly increase after a bacterial or fungal injection. Recombinant serpin-3 efficiently blocks prophenoloxidase activation in larval plasma in a concentration-dependent manner. It forms SDS-stable complexes with serine protease 13 (SP13), and prevents SP13 from cleaving prophenoloxidase. Injection of recombinant serpin-3 into larvae results in decreased fungi-induced melanin synthesis and reduced the expression of attacin, cecropin, gloverin, and peptidoglycan recognition protein-1 genes in the fat body. Altogether, serpin-3 plays important roles in the regulation of prophenoloxidase activation and antimicrobial peptide production in O. furnacalis larvae. Copyright © 2015. Published by Elsevier Ltd.
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    ABSTRACT: Serpin-2 (SRPN2) is a key negative regulator of the melanization response in the malaria vector Anopheles gambiae. SRPN2 irreversibly inhibits CLIPB9, which functions in a serine proteinase cascade culminating in the activation of pro-phenoloxidase (proPO) and melanization. Silencing of SRPN2 in An. gambiae results in spontaneous melanization and decreased lifespan and is therefore a promising target for vector control. The previously determined structure of SRPN2 revealed a partial insertion of the hinge region of the reactive center loop (RCL) into β sheet A. This partial hinge insertion participates in heparin-linked activation in other serpins, notably antithrombin III. SRPN2 does not contain a heparin binding site and any possible mechanistic function of the hinge insertion was previously unknown. To investigate the function of the SRPN2 hinge insertion, we developed three SRPN2 variants in which the hinge regions are either constitutively expelled or inserted and analyzed their structure, thermostability, and inhibitory activity. We determined that constitutive hinge expulsion resulted in a 2.7-fold increase in the rate of CLIPB9Xa inhibition, which is significantly lower than previous observations of allosteric serpin activation. Furthermore, we determined that stable insertion of the hinge region did not appreciably decrease the accessibility of the RCL to CLIPB9. Together, these results indicate the partial hinge insertion in SRPN2 does not participate in the allosteric activation observed in other serpins and instead represents a molecular trade-off between RCL accessibility and efficient formation of an inhibitory complex with the cognate proteinase.
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    ABSTRACT: Serpin 2 (SRPN2) is a key negative regulator of the serine proteinase cascade that controls activation of prophenoloxidase (proPO) and thus melanization by inhibiting CLIP domain serine proteinase B9 (CLIPB9) in malaria mosquito Anopheles gambiae. An unusual feature of native SRPN2 is the partial insertion of hinge region of the reactive center loop (RCL) into β sheet A, which is only seen in three other mammalian and one beetle serpin. To determine whether this partial insertion of the hinge region contributes to target proteinase inhibition, two mutated forms of SRPN2 were produced in which the serine residue at residue 358 was mutated to a glutamate (S358E) or a tryptophan (S358W). In one of the variants, S358E, but not S358W, the hinge region was expelled out from β sheet A as revealed by x-ray crystallography. However, in vitro inhibition assays with CLIPB9Xa show that the stoichiometry of inhibition (SI) and second order rate constant (ka) of the variant S358E are comparable to that of the wild-type SRPN2. These data suggest that in contrast to mammalian serpins such as anti-thrombin III, complete hinge expulsion may not be required for optimal proteinase inhibition. An alternative explanation is that hinge expulsion does not require co-factors, such as sulfated glycosaminoglycans, and is instead mediated by exosite interaction with CLIPB9 prior to cleavage of the scissile bond. The latter proposes a previously unknown mechanism of target selectivity in serpins, which may regulate melanization in insects.
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