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: 3.34). 02/2011; 79(6):1999-2003. DOI: 10.1002/prot.23002
Source: PubMed
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    ABSTRACT: In vector-borne diseases, the complex interplay between pathogen and its vector's immune system determines the outcome of infection and therefore disease transmission. Serpins have been shown in many animals to be key regulators of innate immune reactions. Their control over regulatory proteolytic cascades ultimately decides whether the recognition of a pathogen will lead to an appropriate immune response. In mosquitoes, serpins (SRPNs) regulate the activation of prophenoloxidase and thus melanization, contribute to malaria parasite lysis, and likely Toll pathway activation. Additionally, in culicine mosquitoes, SRPNs are able to regulate hemostasis in the vertebrate host, suggesting a crucial role during bloodfeeding. This review summarizes the annotation, transcriptional regulation, and current knowledge of SRPN function in the three mosquito species for which the complete genome sequence is available. Additionally, we give a brief overview of how SRPNs may be used to prevent transmission of vector-borne diseases.
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    ABSTRACT: Serine proteinase inhibitors of the serpin family are well known as negative regulators of hemostasis, thrombolysis and innate immune responses. Additionally, non-inhibitory serpins serve functions as chaperones, hormone transporters, or anti-angiogenic factors. In the African malaria mosquito, Anopheles gambiae s.s., at least three serpins (SRPNs) are implicated in the innate immune response against malaria parasites. Based on reverse genetic and cell biological analyses, AgSRPN6 limits parasite numbers and transmission and has been postulated to control melanization and complement function in mosquitoes. This study aimed to characterize AgSRPN6 biophysically and determine its biochemical mode of action. The structure model of AgSRPN6, as predicted by I-Tasser showed the protein in the native serpin fold, with three central β-sheets, nine surrounding α-helices, and a protruding reactive center loop. This structure is in agreement with biophysical and functional data obtained from recombinant (r) AgSRPN6, produced in Escherichia coli. The physical properties of purified rAgSRPN6 were investigated by means of analytical ultracentrifugation, circular dichroism, and differential scanning calorimetry tools. The recombinant protein exists predominantly as a monomer in solution, is composed of a mixture of α-helices and β-sheets, and has a mid-point unfolding temperature of 56°C. Recombinant AgSRPN6 strongly inhibited porcine pancreatic kallikrein and to a lesser extent bovine pancreatic trypsin in vitro. Furthermore, rAgSRPN6 formed inhibitory, SDS-stable, higher molecular weight complexes with prophenoloxidase-activating proteinase (PAP)1, PAP3, and Hemolymph protein (HP)6, which are required for melanization in the lepidopteran model organism, Manduca sexta. Taken together, our results strongly suggest that AgSRPN6 takes on a native serpin fold and is an inhibitor of trypsin-like serine proteinases.
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