Effect of Amino Acid Substitution in the Penaeus monodon LGBP and Specificity Through Mutational Analysis

  • Jeyachandran SivakamavalliEmail author
  • Chandrabose Selvaraj
  • Sanjeev Kumar Singh
  • Kiyun Park
  • Ihn-Sil Kwak
  • Baskaralingam VaseeharanEmail author


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.

Graphic Abstract

Docking analysis of LGBP binding towards β-glucan (i) LGBP without mutationv (ii) LGBP with mutation.


Docking 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.

Supplementary material

10989_2019_9960_MOESM1_ESM.doc (184 kb)
Supplementary file1 (DOC 184 kb)


  1. 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
  2. Aspán A, Söderhäll K (1991) Purification of prophenoloxidase from crayfish blood cells, and its activation by an endogenous serine proteinase. Insect Biochem 21:363–373CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Chen C, Rowley AF, Newton RP, Ratcliffe NA (1999) Identification, purification and properties of a beta-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions. Comp Biochem Physiol B 122:309–319CrossRefGoogle Scholar
  5. Cheng W, Liu CH, Tsai CH, Chen JC (2005) Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 18:297–310CrossRefGoogle Scholar
  6. Christophides GK, Vlachou D, Kafatos FC (2004) Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae. Immunol Rev 198:127–148CrossRefGoogle Scholar
  7. Du XJ, Zhao XF, Wang JX (2007) Molecular cloning and characterization of a lipopolysaccharide and beta-1,3-glucan binding protein from fleshy prawn (Fenneropenaeus chinensis). Mol Immunol 44:1085–1094CrossRefGoogle Scholar
  8. Duvic B, Soderhall K (1990) Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus. J Biol Chem 265:9327–9332PubMedGoogle Scholar
  9. Dziarski R (2004) Peptidoglycan recognition proteins (PGRPs). Mol Immunol 40:877–886CrossRefGoogle Scholar
  10. Fazil MH, Kumar S, Rao NS, Selvaraj C, Singh SK, Pandey HP, Singh DV (2012) Comparative structural analysis of two proteins belonging to quorum sensing system in Vibrio cholerae. J Biomol Struct Dyn 30:574–584CrossRefGoogle Scholar
  11. Flegel TW (2012) Historic emergence, impact and current status of shrimp pathogens in Asia. J Invertebr Pathol 110:166–173CrossRefGoogle Scholar
  12. Goncalves P, Vernal J, Rosa RD, Yepiz-Plascencia G, Batista de Souza CR, Barracco MA et al (2013) Evidence for a novel biological role for the multifunctional β-1,3-glucan binding protein in shrimp. Mol Immunol 51:363–367CrossRefGoogle Scholar
  13. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Ann Rev Immunol 20:197–216CrossRefGoogle Scholar
  14. 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
  15. Kanagawa M, Satoh T, Ikeda A, Adachi Y, Ohno N, Yamaguchi Y (2011) Structural insights into recognition of triple-helical beta-glucans by an insect fungal receptor. J Biol Chem 286:29158–29165CrossRefGoogle Scholar
  16. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  17. Lee SY, Soderhall K (2002) Early events in crustacean innate immunity. Fish Shellfish Immunol 12:421–437CrossRefGoogle Scholar
  18. Lee SY, Wang R, Soderhall K (2000) A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning. J Biol Chem 275:1337–1343CrossRefGoogle Scholar
  19. Lin YC, Vaseeharan B, Chen JC (2008) Identification and phylogenetic analysis on lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) of kuruma shrimp Marsupenaeus japonicus. Dev Comp Immunol 32:1260–1269CrossRefGoogle Scholar
  20. Liu F, Li F, Dong B, Wang X, Xiang J (2009a) Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis. Mol Biol Rep 36:471–477CrossRefGoogle Scholar
  21. Liu Y, Li F, Wang B, Dong B, Zhang X, Xiang J (2009b) A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge. Fish Shellfish Immunol 26:345–351CrossRefGoogle Scholar
  22. Lüthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with threedimensional profiles. Nature 356:83–85CrossRefGoogle Scholar
  23. Martin GG, Graves BL (1985) Fine structure and classification of shrimp hemocytes. J morphol 185:339–348CrossRefGoogle Scholar
  24. Medzhitov R (2007) Recognition of microorganisms and activation of the immune response. Nature 449:819–826CrossRefGoogle Scholar
  25. Medzhitov R, Janeway CA Jr (1997) Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 9:4–9CrossRefGoogle Scholar
  26. Medzhitov R, Janeway C Jr (2000) Innate immune recognition: mechanisms and pathways. Immunol Rev 173:89–97CrossRefGoogle Scholar
  27. Nagaraju GPC, Siva Kumari N, Prasad GLV, Rajitha B, Madan M, Sreenivasa RM, Reddya Naik B (2009) Computational analysis and structural prediction of VIH related peptides from selected crustacean species. Bioinformation 4:6–11CrossRefGoogle Scholar
  28. 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
  29. Roux MM, Pain A, Klimpel KR, Dhar AK (2002) The lipopolysaccharide and beta-1,3-glucan binding protein gene is upregulated in white spot virus-infected shrimp (Penaeus stylirostris). J Virol 76:7140–7149CrossRefGoogle Scholar
  30. Sali A, Blundell TL (1994) Comparative protein modelling by satisfaction of spatial restraints. Protein structure by distance analysis. Sci Res 64:C86Google Scholar
  31. Sivakamavalli J, Vaseeharan B (2013) Purification, characterization and functional analysis of a novel beta-1, 3-glucan binding protein from green tiger shrimp Penaeus semisulcatus. Fish Shellfish Immunol 35:689–696CrossRefGoogle Scholar
  32. 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. CrossRefPubMedGoogle Scholar
  33. Soderhall K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28CrossRefGoogle Scholar
  34. Sritunyalucksana K, Lee SY, Soderhall K (2002) A beta-1,3-glucan binding protein from the black tiger shrimp, Penaeus monodon. Dev Comp Immunol 26:237–245CrossRefGoogle Scholar
  35. Valli JS, Vaseeharan B (2012) cDNA cloning, characterization and expression of lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) gene from the Indian white shrimp Fenneropenaeus indicus. Comp Biochem Physiol A 163:74–81CrossRefGoogle Scholar
  36. Vargas-Albores F, Guzmán MA, Ochoa JL (1993) An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp Penaeus californiensis. Comp Biochem Physiol A 106:299–303CrossRefGoogle Scholar
  37. Vargas-Albores F, Jimenez-Vega F, Soderhall K (1996) A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by β-1,3-glucan. Dev Comp Immunol 20:299–306CrossRefGoogle Scholar
  38. 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

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Jeyachandran Sivakamavalli
    • 1
    • 2
    Email author
  • Chandrabose Selvaraj
    • 3
  • Sanjeev Kumar Singh
    • 3
  • Kiyun Park
    • 1
  • Ihn-Sil Kwak
    • 1
    • 4
  • Baskaralingam Vaseeharan
    • 2
    Email author
  1. 1.Department of Fisheries Research InstituteChonnam National UniversityYeosuSouth Korea
  2. 2.Crustacean Molecular Biology and Genomics Lab, Department of Animal Health and ManagementAlagappa UniversityKaraikudiIndia
  3. 3.Computer Aided Drug Designing and Molecular Modeling Lab, Department of BioinformaticsAlagappa UniversityKaraikudiIndia
  4. 4.Faculty of Marine TechnologyChonnam National UniversityChonnamRepublic of Korea

Personalised recommendations