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Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis.

Panwar D, Rawal L, Sehgal N, Ali S - PLoS ONE (2015)

Bottom Line: The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface.Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex.This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.

ABSTRACT
Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

No MeSH data available.


Bubalus bubalis KGF and KITLG genes amplification.Representative gel picture showing the amplicons corresponding to KGF and KITLG genes. For size marker, 100 bp ladder was used.
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pone.0127993.g001: Bubalus bubalis KGF and KITLG genes amplification.Representative gel picture showing the amplicons corresponding to KGF and KITLG genes. For size marker, 100 bp ladder was used.

Mentions: The amplification of the PCR products corresponding to Bubalus bubalis KGF and KITLG are shown in Fig 1. The full length coding sequence of KGF and KITLG were deduced to be of 585 bp and 825 bp, respectively. Accordingly, in silico analysis showed the presence of 194 and 274 amino acids corresponding to KGF and KITLG proteins, respectively. The recombinant His-KGF and GST-KITLG proteins were purified using immobilized metal and glutathione affinity chromatography techniques, respectively. The SDS-PAGE and western blot analysis showed purified KGF (His-tagged) and KITLG (GST-tagged) proteins with bands corresponding to 23 and 57 kDa, respectively, thus, in accordance with their theoretical molecular weights (Fig 2A and 2B).


Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis.

Panwar D, Rawal L, Sehgal N, Ali S - PLoS ONE (2015)

Bubalus bubalis KGF and KITLG genes amplification.Representative gel picture showing the amplicons corresponding to KGF and KITLG genes. For size marker, 100 bp ladder was used.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4470682&req=5

pone.0127993.g001: Bubalus bubalis KGF and KITLG genes amplification.Representative gel picture showing the amplicons corresponding to KGF and KITLG genes. For size marker, 100 bp ladder was used.
Mentions: The amplification of the PCR products corresponding to Bubalus bubalis KGF and KITLG are shown in Fig 1. The full length coding sequence of KGF and KITLG were deduced to be of 585 bp and 825 bp, respectively. Accordingly, in silico analysis showed the presence of 194 and 274 amino acids corresponding to KGF and KITLG proteins, respectively. The recombinant His-KGF and GST-KITLG proteins were purified using immobilized metal and glutathione affinity chromatography techniques, respectively. The SDS-PAGE and western blot analysis showed purified KGF (His-tagged) and KITLG (GST-tagged) proteins with bands corresponding to 23 and 57 kDa, respectively, thus, in accordance with their theoretical molecular weights (Fig 2A and 2B).

Bottom Line: The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface.Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex.This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Genetics Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India.

ABSTRACT
Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

No MeSH data available.