Ligand specificity of CS-35, a monoclonal antibody that recognizes mycobacterial lipoarabinomannan: a model system for oligofuranoside-protein recognition.
The CS-35 antibody is widely used in the characterization of glycans containing D-arabinofuranose residues, in particular polysaccharides present in the mycobacterial cell wall. A detailed understanding of the combining site of this antibody and the measurement of its binding to different ligands is of interest as this knowledge will have implications in the characterization of arabinofuranose-containing glycoconjugates that are increasingly recognized as important biological molecules. Of even greater significance is that an in-depth study of this carbohydrate-protein interaction will provide insights into the mechanisms by which oligosaccharides containing furanose rings are bound by proteins, an area that has, to date, received little attention. This system has been refractory to X-ray crystallography, and thus we report here a study of the interaction of CS-35 with its ligands using a combination of chemical synthesis, mass spectrometry, titration microcalorimetry, and NMR spectroscopy. Through these investigations we have established that the binding pocket recognizes, as a minimum epitope, a linear tetrasaccharide motif and that the residues at the reducing and non-reducing end of the oligosaccharide are essential for tight binding. The residue at the non-reducing end appears to be bound in an aliphatic pocket, whereas the rest of the tetrasaccharide interacts more strongly with aromatic amino acids.
Rademacher
Christoph
C
Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
Shoemaker
Glen K
GK
Kim
Hyo-Sun
HS
Zheng
Ruixiang Blake
RB
Taha
Hashem
H
Liu
Chunjuan
C
Nacario
Ruel C
RC
Schriemer
David C
DC
Klassen
John S
JS
Peters
Thomas
T
Lowary
Todd L
TL
D000911:Antibodies, Monoclonal / Q000276:immunology*; D002151:Calorimetry; D002241:Carbohydrates / Q000737:chemistry; D002460:Cell Line; D000939:Epitopes / Q000276:immunology; D006027:Glycosides / Q000276:immunology*; D008024:Ligands; D008070:Lipopolysaccharides / Q000276:immunology*; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008954:Models, Biological; D015394:Molecular Structure; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D000939:Epitopes; D006027:Glycosides; D008024:Ligands; D008070:Lipopolysaccharides; C050016:lipoarabinomannan