Dataset Information

Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus.

ABSTRACT: IRS-2 from the hard tick Ixodes ricinus belongs to the serpin family of protease inhibitors. It is produced in the salivary glands of the tick and its anti-inflammatory activity suggests that it plays a role in parasite-host interaction. Recombinant IRS-2 prepared by heterologous expression in a bacterial system was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the primitive tetragonal space group P4(3) and diffracted to 1.8?Å resolution. Mass-spectrometric and electrophoretic analyses revealed that IRS-2 was cleaved by contaminating proteases during crystallization. This processing of IRS-2 mimicked the specific cleavage of the serpin by its target protease and resulted in a more stable form (the so-called relaxed conformation), which produced well diffracting crystals. Activity profiling with specific substrates and inhibitors demonstrated traces of serine and cysteine proteases in the protein stock solution.

SUBMITTER: Kovarova Z

PROVIDER: S-EPMC3001646 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus.

Kovářová Zuzana Z Chmelař Jindřich J Sanda Miloslav M Brynda Jiří J Mareš Michael M Rezáčová Pavlína P

Acta crystallographica. Section F, Structural biology and crystallization communications 20101028 Pt 11

IRS-2 from the hard tick Ixodes ricinus belongs to the serpin family of protease inhibitors. It is produced in the salivary glands of the tick and its anti-inflammatory activity suggests that it plays a role in parasite-host interaction. Recombinant IRS-2 prepared by heterologous expression in a bacterial system was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the primitive tetragonal space group P4(3) and diffracted to 1.8 Å resolution. Mass-spectrometri ...[more]

PMID: 21045293

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Project description:BackgroundIxodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed.MethodsRNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV).ResultsDifferential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway.ConclusionsThis systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.

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Dataset Information

Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus.

Publications

Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus.

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