Characterization of the wheat germ agglutinin binding to self-assembled monolayers of neoglycoconjugates by AFM and SPR.
Carbohydrate-protein interactions govern many crucial life processes involved in cell recognition events, but are often difficult to study because the interactions are weak, and multivalent exposure appears to be crucial for their biological function. We have used self-assembled monolayers (SAMs) of neoglycoconjugates as a model system to probe the specific interactions between the lectin wheat germ agglutinin (WGA) and monosaccharides by surface plasmon resonance (SPR) and atomic force microscopy (AFM) force measurements. SAMs presenting N-acetyl-D-glucosamine (GlcNAc) as a neoglycoconjugate were produced on gold surfaces, where the SAM formation was monitored using a quartz crystal microbalance (QCM) and shown to be a very rapid process. In the AFM force measurements WGA was covalently coupled to flexible polyethylene glycol (PEG) molecules at a probe surface using amine coupling. GlcNAc-specific binding events were detected with a WGA-modified probe on the GlcNAc-neoglycoconjugate SAM at bond rupture forces of 47 +/- 15 pN. Additionally, less frequent GlcNAc-specific unbinding events were detected at higher forces (120 +/- 20 pN) which are believed to originate from simultaneous detachment of multiple binding sites from the SAM surface. SPR measurements confirmed that WGA has higher affinity toward the immobilized GlcNAc-SAM than toward the soluble free monosaccharide. The binding constants obtained for soluble chitinoligosaccharides suggested up to three subsites within one carbohydrate-binding site of the WGA molecule and also provided further evidence of the multivalent binding character of the WGA dimer.
Lienemann
Michael
M
VTT Technical Research Centre of Finland, VTT, Finland.
Paananen
Arja
A
Boer
Harry
H
de la Fuente
Jesús M
JM
García
Isabel
I
Penadés
Soledad
S
Koivula
Anu
A
D000117:Acetylglucosamine / Q000737:chemistry*; D001665:Binding Sites; D006001:Glycoconjugates / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D014909:Wheat Germ Agglutinins / Q000737:chemistry*
D006001:Glycoconjugates; D014909:Wheat Germ Agglutinins; D000117:Acetylglucosamine