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First published online June 29, 2006
Journal of Experimental Biology 209, 2651-2659 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02267

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A novel secreted endonuclease from Culex quinquefasciatus salivary glands

Eric Calvo and José M. C. Ribeiro^*

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Twinbrook III (12735 Twinbrook Parkway), Room 2E-32D, Rockville, MD 20852, USA

View larger version (39K): [in a new window]   Fig. 1. (A) Amino acid (aa) sequence alignment of conserved elements among different endonuclease active sites. Conserved aa residues are highlighted in red. Most known endonucleases have the conserved R(K)GH triad. CuquEndo also contains other aa residues implicated in the nucleophilic attack of DNA substrate and stabilization of the active site. (B) Molecular modeling of CuquEndo and active site comparison with Smarcens endonuclease (PDB id 1G8T). The similarity of the active site geometry suggests that both enzymes might have a similar mechanism of action on DNA substrates. (C) Phylogenetic analysis of the endonuclease family. The unrooted neighbor-joining tree (10,000 bootstraps) was generated by MEGA 3.1 software. CuquEndo, Culex quinquefasciatus; Smarcens, Serratia marcescens; Mjaponic, Marsupenaeus japonicus; Pcamtsch, Paralithodes camtschaticus; Hsapiens, Homo sapiens; Gmorsitans, Glossina morsitans; Longipalpis, Lutzomyia longipalpis; Pariasi, Phlebotomus ariasi; Agambiae, Anopheles gambiae. NCBI accession numbers in parentheses.

View larger version (34K): [in a new window]   Fig. 2. Endonuclease activity in salivary gland extract of female Culex quinquefasciatus. Double-stranded plasmid DNA (400 ng) was incubated in a final volume of 20 µl with different salivary gland dilutions (serial dilutions of one salivary gland) and incubated for 10 min at 37°C. (A) 10 µl from each sample was electrophoresed in a 1.2% agarose gel and visualized under UV light. NSG, no salivary gland, negative control. (B) 5 µl from each reaction were diluted in 100 µl of Hoechst dye mix and the fluorescence measured using a fluorimeter with 365/460-nm (excitation/emission, 6.1 V) filters.

View larger version (83K): [in a new window]   Fig. 3. Secretion of CuquEndo in Cx. quinquefasciatus saliva. Starved mosquitoes were allowed to probe in an agarose gel containing 10 mmol l-1 NaHCO3, 100 mmol l-1 NaCl, 1 mmol l-1 MgCl2, 50 mmol l-1 Tris, pH 7.5, and 200 µg ml-1 of double-stranded plasmid DNA. After 30 min incubation at 37°C, the gel was stained with Ethidium Bromide and visualized under UV light. Arrows indicate the hydrolysis of plasmid DNA at the biting sites within the gel.

View larger version (41K): [in a new window]   Fig. 4. Expression of recombinant CuquEndo in 293 cells and SDS-PAGE agarose gel assay. (A) In vitro assay showing the presence of endonuclease activity in supernatant of cells transfected with the CuquEndo-VR2001 construct. No plasmid degradation was observed in the supernatant of 293 cells transfected with an empty VR2001 plasmid (mock control) P, 500 ng of doubled stranded plasmid DN, Ladder, 1 kb Plus DNA ladder. (B) Concentrated 293 cell supernatant (crude) containing the recombinant CuqueEndo was resolved in a NuPAGE gel. After in gel renaturation (see Materials and methods) the NuPAGE gel was exposed to a 1% agarose gel containing 0.54 ng ml-1 of double stranded plasmid DNA. Degradation of plasmid DNA occurred at the position of the calculated molecular mass of recombinant CuquEndo, as visualized with Ethidium Bromide under UV light. (C) The Coomassie Blue-stained NuPAGE gel (arrow indicates position of CuquEndo). Blue, prestained standard proteins.

View larger version (26K): [in a new window]   Fig. 5. Effect of divalent cations on recombinant CuquEndo activity. Reaction mixtures containing 1 µl of recombinant CuquEndo and 500 ng of plasmid DNA in a final volume of 20 µl were incubated for 10 min at 37°C in buffers with cation concentration ranging from 0 to 10 mmol l-1. The digested DNA plasmids were resolved in 1.2% agarose gels and visualized under UV light. (A) MgCl2. (B) CaCl2.

View larger version (19K): [in a new window]   Fig. 6. Chromatography of CuquEndo hydrolysis of different nucleic acid substrates on an IECDEAENPR column. (A) PolyT standard used to calibrate the column. Peaks represent polymers of 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 and 2 nucleotides. (B,C) The effect of incubation of double-stranded plasmid DNA with (B) salivary gland extract or (C) recombinant enzyme-containing supernatant. Both enzyme sources hydrolyzed the plasmid substrate with no sequence specificity. (D,E) Negative controls are (D) undigested double-stranded plasmid DNA and (E) recombinant enzyme-containing supernatant alone.

View larger version (21K): [in a new window]   Fig. 7. Substrate specificity of recombinant CuquEndo-containing supernatant on different single- or double-stranded polynucleotides. Hydrolysis products were resolved using an IEC-DEAE-NPR column. Recombinant CuquEndo hydrolyzed only double-stranded DNA with no sequence specificity. No endonuclease activity was detected on single-stranded substrates.