Limits...
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 - PLoS ONE (2014)

Bottom Line: 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 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 double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Marine Biochemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232515&req=5

pone-0112729-g010: 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.

Mentions: Since the lectin MBL-AJ is a functionally active dimer, the hybrid CmAP/MBL-AJ model was constructed based on the MBL-AJ dimer. The CmAP/MBL-AJ dimer model has the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other (Fig. 9). The MBL-AJ lectin exhibits high specificity for branched mannan [5]. Molecular docking predicted the structural model of MBL-AJ lectin binding with a model oligosaccharide (Fig. 10). The mannose residues of the main chain Man2 and from branching chain Man4 that are not the terminal mannose residues form contacts with the MBL-AJ carbohydrate-recognition domain. Analysis of the contacts in MBL-AJ carbohydrate-recognition domain showed that the model oligosaccharide forms hydrogen bonds with the lectin and Ca2+ ion (Fig. 11). According to the lectin-oligosaccharide complex model, the residue Glu134 forms hydrogen bonds with O3 and O4 of the mannose residue Man2 from the main chain; Ca2+ ion forms ionic and metal bonds with O3 of the mannose residue Man4; residue Asn137 forms hydrogen bond with O2 of the mannose residue Man5.


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 - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232515&req=5

pone-0112729-g010: 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.
Mentions: Since the lectin MBL-AJ is a functionally active dimer, the hybrid CmAP/MBL-AJ model was constructed based on the MBL-AJ dimer. The CmAP/MBL-AJ dimer model has the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other (Fig. 9). The MBL-AJ lectin exhibits high specificity for branched mannan [5]. Molecular docking predicted the structural model of MBL-AJ lectin binding with a model oligosaccharide (Fig. 10). The mannose residues of the main chain Man2 and from branching chain Man4 that are not the terminal mannose residues form contacts with the MBL-AJ carbohydrate-recognition domain. Analysis of the contacts in MBL-AJ carbohydrate-recognition domain showed that the model oligosaccharide forms hydrogen bonds with the lectin and Ca2+ ion (Fig. 11). According to the lectin-oligosaccharide complex model, the residue Glu134 forms hydrogen bonds with O3 and O4 of the mannose residue Man2 from the main chain; Ca2+ ion forms ionic and metal bonds with O3 of the mannose residue Man4; residue Asn137 forms hydrogen bond with O2 of the mannose residue Man5.

Bottom Line: 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 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 double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25 ± 5%.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Marine Biochemistry, G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russian Federation.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus