Effect of Amino Acid Substitution in the Penaeus monodon LGBP and Specificity Through Mutational Analysis
- 35 Downloads
Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) is a pattern recognition protein (PRP) purified from the Penaeus monodon by Blue-Sepharose, Phenyl-Sepharose followed by Sephadex G-100 chromatography. P. monodon LGBP consist of 36 and 48 kDa subunits on 10% SDS-PAGE under reducing and non-reducing conditions respectively. Purified P. monodon LGBP agglutinates the fungal pathogen Candida glabrata, due to the presence of β-glucan (βG) on its surface. This agglutination was cross checked with the in silico docking analysis of LGBP-βG and LGBP-laminarin (isomeric form βG) interaction. As part of a strategy, to determine the precise role of P. monodon LGBP (Pm-LGBP) in pattern recognition mechanism mutations were introduced by in silico approach. In crustacean LGBP, RGD motif (Arg, Gly, Asp) plays the vital role in the cell adhesion and pattern recognition mechanism. Role of Asp in RGD motif was determined through amino acid substitution, introduction of a specific mutation D134K into a central area of the sugar-binding (βG) site resulted in complete loss of pathogen recognition and binding of Pm-LGBP to βG. These results demonstrate that, the RGD motif of Asp134 is essential for sugar binding in P. monodon. To our knowledge, P. monodon D134K is the first mutant shrimp LGBP which is unable to bind with the sugar residues. This mutant could be useful in the discovery of actual function of Pm-LGBP in the recognition of homologous symbionts.
KeywordsDocking Homology modelling LGBP Molecular simulation Mutation RMSD SDS-PAGE
The study was supported by the National Research Foundation of Korea, which is funded by the Korean Government [NRF-2018-R1A6A1A-03024314]. The authors Chandrabose Selvaraj and Sanjeev Kumar Singh thankfully acknowledge financial support of RUSA-Phase 2.0 grant sanctioned vide Letter No: F.24-51/2014-U, Policy (TNmulti-Gen), Dept. of Edn, Govt. of India, Dt.09.10.2018.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Amparyup P, Sutthangkul J, Charoensapsri W, Tassanakajon A (2012) Pattern recognition protein binds to lipopolysaccharide and β-1,3-glucan and activates shrimp prophenoloxidase system. J BiolChem 13:10060–10069Google Scholar
- Jiang H, Ma C, Lu ZQ, Kanost MR (2004) Beta-1,3-glucan recognition protein-2 (betaGRP-2)from Manduca sexta; an acute-phase protein that binds beta-1,3-glucan and lipoteichoic acid to aggregate fungi and bacteria and stimulate prophenoloxidase activation. Insect Biochem Mol Biol 34:89–100CrossRefGoogle Scholar
- Romo-Figueroa MG, Vargas-Requena C, Sotelo-Mundo RR, Vargas-Albores F, Higuera-Ciapara I, Soderhall K, Yepiz-Plascencia G (2004) Molecular cloning of a beta-glucan pattern-recognition lipoprotein from the white shrimp Penaeus (Litopenaeus) vannamei: correlations between the deduced amino acid sequence and the native protein structure. Dev Comp Immunol 28:713–726CrossRefGoogle Scholar
- Sali A, Blundell TL (1994) Comparative protein modelling by satisfaction of spatial restraints. Protein structure by distance analysis. Sci Res 64:C86Google Scholar
- Sivakamavalli J, Selvaraj C, Singh SK, Vaseeharan B (2013) Exploration of protein–protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies. J Biomol Struct Dyn. https://doi.org/10.1080/07391102.2013.838909 CrossRefPubMedGoogle Scholar
- Vargas-Albores F, Yepiz-Plascencia G, Galvan TG, Garcia-Banuelos M (2000) Synthesis of hemolymph high-density lipoprotein β-glucan binding protein by Penaeus vannamei shrimp hepatopancreas. Mar Biotechnol (NY) 2:485–492Google Scholar