Balabanova L et al. (2014), A novel bifunctional hybrid with marine bacteri...

ECB-ART-43691

PLoS One 2014 Jan 01;911:e112729. doi: 10.1371/journal.pone.0112729.

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A novel bifunctional hybrid with marine bacterium alkaline phosphatase and Far Eastern holothurian mannan-binding lectin activities.

Balabanova L , Golotin V , Kovalchuk S , Bulgakov A , Likhatskaya G , Son O , Rasskazov V .

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A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%. The bifunctional hybrid holothurian''s lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

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Genes referenced: LOC583464 LOC594261 mgat4c

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References [+] :

Arai, Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593. 2014, Pubmed

	Figure 1. Scheme of expression plasmid pET40CmAP/MBL-AJ.
	Figure 2. Optimization of pET40CmAP/MBL-AJ expression: black squares - specific AP activity; red circles - total activity; blue triangles - total protein.
	Figure 3. SDS-PAGE of CmAP/MBL-AJ eluted from Superdex 200: 1 –molecular weight marker (Bio-rad); 2 –CmAP/MBL-AJ.
	Figure 4. Stability of CmAP in different buffers.The enzyme activity was measured using the standard enzyme assay (0.1 M Tris-HCl buffer, pH 9.0) after 5 h incubation at different pH values. Squares – 0.03 M Na COOH3 (4.0–6.0); circles - 0.05 M Tris-HCl (7.0–9.0); diamonds – 1 M DEA (8.0–10.3); down triangle - 0.1 M NaHCO3 (9.0–11.0); up triangle - 0.1 M glycine (9.0–11.0) (n = 9, p≤0.05).
	Figure 5. Determination of MBL-AJ-binding embryonic alpha-1-acid glycoproteins by the two methods: with the use of hybrid CmAP/MBL-AJ (black) and MBL-AJ-antibody-horse peroxidase conjugates (red).
	Figure 6. The homology model of CmAP monomer with binding sites for two Zn2+ and one Mg2+ ions.
	Figure 7. The homology model of MBL-AJ monomer (A), the superimposition of MBL-AJ model with template CEL-IV (B) and the structural model of MBL-AJ dimer (C).(a) - Graphic representation of MBL-AJ monomer with disulfide bonds (Cys24-Cys35, Cys52-Cys168) and free Cys60 and Cys172 group at C-terminus of lectin displayed as sticks. (b) - Superimposition of the MBL-AJ structural model (orange) and template CEL-IV (purple). Loop L of MBL-AJ (orange) with significant difference is indicated. (c) – The structural model of MBL-AJ dimer build from monomer M1 (orange) and M2 (purple) with two intermonomer disulfide bonds Cys′60-Cys172 and Cys60-Cys′172.
	Figure 8. The structural model of the hybrid CmAP/MBL-AJ monomer.The C-terminal region of CmAP is linked via a linker (G4S)3 with the N-terminal region of MBL-AJ. Blue spheres indicate two ions Zn2+, red sphere indicates ion Mg2+ in CmAP; green sphere indicates ion Ca2+ in MBL-AJ.
	Figure 9. The putative structural model of hybrid CmAP/MBL-AJ dimer built from monomers M1 (orange) and M2 (purple) on the base of the model MBL-AJ dimer.
	Figure 10. In silico docking the model of oligosaccharide α-D-Manp-(1→6)-[α-D-Manp-(1→2)-α-D-Manp-(1→2)]-α-D-Manp(1→6)-D-Manp inside of the MBL-AJ carbohydrate-recognition domain.Surface representation of the MBL-AJ binding site hydrophobic regions are in green, mildly polar regions are in blue and hydrogen bonding are in purple. Oligosaccharide structure is shown in the stick form, Ca2+ ion is shown as space filling.
	Figure 11. 2D-Diagram of the oligosaccharide binding with MBL-AJ and contacts of the oligosaccharide with MBL-AJ.
	Figure 12. 2D-Diagram of the oligosaccharide molecular contacts with A155N/F159K MBL-AJ mutant and contacts of model oligosaccharide with A155N/F159K MBL-AJ mutant.
	Figure 13. Lectin-binding activity of the bifunctional hybrid CmAP/MBL-AJ mutants. The lectin-bound complexes with mucin (axis X) were monitored by measuring the phosphatase activity of CmAP/MBL-AJ (axis Y).