Open AccessEpitope mapping of the O‐chain polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide
by saturation‐transfer‐difference NMR spectroscopy
Author(s) -
Kooistra Oliver,
Herfurth Lars,
Lüneberg Edeltraud,
Frosch Matthias,
Peters Thomas,
Zähringer Ulrich
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.0014-2956.2001.02684.x
Subject(s) - epitope , chemistry , legionella pneumophila , residue (chemistry) , biochemistry , stereochemistry , nuclear magnetic resonance spectroscopy , methylation , polysaccharide , epitope mapping , amino acid , peptide sequence , bacteria , biology , antibody , genetics , gene , immunology
Two modifications of 5‐acetimidoylamino‐7‐acetamido‐3,5,7,9‐tetradeoxy‐ d ‐ glycero ‐ d ‐ galacto ‐non‐2‐ulosonic acid (5‐ N ‐acetimidoyl‐7‐ N ‐acetyllegionaminic acid) in the O‐chain polysaccharide (OPS) of the Legionella pneumophila serogroup 1 lipopolysaccharide (LPS) concern N‐methylation of the 5‐N‐acetimidoyl group in legionaminic acid. Both N‐methylated substituents, the ( N , N ‐dimethylacetimidoyl) amino and acetimidoyl( N ‐methyl)amino group, could be allocated to one single legionaminic acid residue in the long‐ and middle‐chain OPS, respectively. Using mutants devoid of N‐methylated legionaminic acid derivatives, it could be shown that N‐methylation of legionaminic acid correlated with the expression of the mAb 2625 epitope. In the present study we investigated the binding of the LPS‐specific monoclonal antibody mAb 2625 to isolated OPS with surface‐plasmon‐resonance biomolecular interaction analysis and saturation‐transfer‐difference (STD) NMR spectroscopy in order to map the mAb 2625 epitope on a molecular level. It could be demonstrated that the binding affinity of the N‐methylated legionaminic acid derivatives was independent from the size of the isolated OPS molecular species. In addition, STD NMR spectroscopic studies with polysaccharide ligands with an average molecular mass of up to 14 kDa revealed that binding was mainly mediated via the N‐methylated acetimidoylamino group and via the closely located 7‐ N ‐acetyl group of the respective legionaminic acid residue, thus indicating these derivatives to represent the major epitope of mAb 2625.