Peptidoglycan Isolation and Binding Studies with LysM-Type Pattern Recognition Receptors
In the last decade, more and more plant receptors for complex carbohydrate structures have been described. However, studies on receptor binding to glycan ligands are often hampered due to the technical challenge to obtain pure preparations of homogeneous carbohydrate ligands such as bacterial peptidoglycan (PGN) in amounts suitable for studying protein–glycan interactions. Also, most approaches rely on the availability of defined soluble ligands, which in the case of glycans can rarely be synthesized but have to be purified from the respective microorganism. In this chapter, we describe the purification of complex PGN from sources such as gram-positive bacteria, from which PGN isolation is facilitated due to its larger content in their cell wall. Insoluble PGN can subsequently be used in simple carbohydrate pull-down assays to test for interaction with plant proteins. In this respect, lysin motif (LysM)-domain containing proteins are of particular interest. All plant receptors described to date to be involved in the perception of N-Acetylglucosamine-containing ligands (such as PGN or chitin) have been shown to belong to this protein class. Thus, this chapter will also include the production of recombinant LysM proteins to analyze their PGN interaction.
Key wordsPeptidoglycan Chitin LysM Carbohydrate affinity assay Protein–glycan interaction
We thank the Deutsche Forschungsgemeinschaft (SFB 766) for support to U.B. and A.A.G. Roland Willmann is acknowledged for preparing Fig. 1 and for helpful discussions on the manuscript.
- 11.Erbs G, Silipo A, Aslam S, De Castro C, Liparoti V, Flagiello A, Pucci P, Lanzetta R, Parrilli M, Molinaro A, Newman MA, Cooper RM (2008) Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity. Chem Biol 15:438–448CrossRefPubMedGoogle Scholar
- 17.Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D, Tsuda K, Katagiri F, Fliegmann J, Bono JJ, Cullimore JV, Jehle AK, Gotz F, Kulik A, Molinaro A, Lipka V, Gust AA, Nürnberger T (2011) Arabidopsis lysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proc Natl Acad Sci U S A 108:19824–19829CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Wong JE, Midtgaard SR, Gysel K, Thygesen MB, Sorensen KK, Jensen KJ, Stougaard J, Thirup S, Blaise M (2015) An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase. Acta Crystallogr D Biol Crystallogr 71:592–605CrossRefPubMedPubMedCentralGoogle Scholar
- 25.Broghammer A, Krusell L, Blaise M, Sauer J, Sullivan JT, Maolanon N, Vinther M, Lorentzen A, Madsen EB, Jensen KJ, Roepstorff P, Thirup S, Ronson CW, Thygesen MB, Stougaard J (2012) Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding. Proc Natl Acad Sci U S A 109:13859–13864CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Hayafune M, Berisio R, Marchetti R, Silipo A, Kayama M, Desaki Y, Arima S, Squeglia F, Ruggiero A, Tokuyasu K, Molinaro A, Kaku H, Shibuya N (2014) Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization. Proc Natl Acad Sci U S A 111:E404–E413CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Maolanon NN, Blaise M, Sorensen KK, Thygesen MB, Clo E, Sullivan JT, Ronson CW, Stougaard J, Blixt O, Jensen KJ (2014) Lipochitin oligosaccharides immobilized through oximes in glycan microarrays bind LysM proteins. Chembiochem (A European journal of chemical biology) 15:425–434CrossRefGoogle Scholar