Characterization of the wheat germ agglutinin binding to self-assembled monolayers of neoglycoconjugates by AFM and SPR

Glycobiology vol. 19 no. 6 pp. 633–643, 2009 doi:10.1093/glycob/cwp030 Advance Access publication on February 24, 2009 Characterization of the wheat germ agglutinin binding to self-assembled monolayers of neoglycoconjugates by AFM and SPR Michael Lienemann3 , Arja Paananen3 , Harry Boer3 , Jesús M de la Fuente2,4 , Isabel Garcı́a4,5 , Soledad Penadés4,5 , and Anu Koivula1,3 3 VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland; 4 Grupo de Carbohidratos, IIQ-CSIC, Americo Vespucio s/n, 41092 Sevilla, Spain; and 5 Laboratory of GlycoNanotechnology, CIC biomaGUNE and CIBER-BBN, Parque Tecnológico, P◦ de Miramón 182, 20009 San Sebastian, Spain 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. GlcNAcspecific 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. Keywords: AFM force spectroscopy/protein–carbohydrate interaction/self-assembled monolayer/surface plasmon resonance/wheat germ agglutinin 1 To whom correspondence should be addressed: Tel: +358-20-7225110; Fax: +358-20-7227071; e-mail: 2 Present address: Instituto de Nanociencia de Aragón (INA), University of Zaragoza, 50009 Zaragoza, Spain. Carbohydrate sequences on glycoproteins, glycolipids, and proteoglycans are key ligands in different molecular recognition systems involved in many normal and pathogenic processes ranging from fertilization to viral/bacterial infections and metastasis formation. In particular, cell-adhesion and cell-activation events triggered by the carbohydrate–protein interaction are among the current topics of active research (McEver et al. 1995; Walsh and Jefferis 2006). Carbohydrate–protein interactions are usually difficult to study because the interactions are subtle. The weak affinity is overcome in nature by several simultaneous contacts between carbohydrates that are clustered on cell surfaces, and protein receptors that contain multiple carbohydratebinding sites (Mammen et al. 1998). The polyvalent display of carbohydrates can lead to remarkably high binding avidities. Understanding the molecular mechanisms of carbohydrate recognition would be of importance for resolving their biological role and combating disease. Specific protein–carbohydrate interactions can also be exploited in bioanalytical applications in detection of, e.g., certain carbohydrate epitopes on cell surfaces. Direct measurements of protein–carbohydrate interactions between single molecules are therefore of high interest. Due to the complexity of cell surfaces, simplified model systems are needed to study polyvalent carbohydrate–protein interactions using either two- or three-dimensional surfaces. A considerable number of immobilization chemistries have been reported in the recent years using either carbohydrates isolated in scarce amounts from natural sources or synthesized from commercially available monosaccharides (Park and Shin 2002; Seeberger and Werz 2007). Carbohydrate immobilization has been achieved by conjugation of (methyl-)amino-modified carbohydrates to commercially available carboxylated surfaces using EDC/NHS chemistry (Nahálková et al. 2002) and by noncovalently binding biotinylated oligosaccharides to a streptavidincontaining dextran matrix (Shinohara et al. 1997; for a recent review see also Paulson et al. 2006). Self-assembled monolayers (SAMs) of alkanethiolates on gold provide another convenient way to display carbohydrates on surfaces with control over the average in-plane density of the carbohydrate ligand (Poirier and Tarlov 1994; De La Fuente and Penadés 2004; Love et al. 2005). The use of an aliphatic carbon chain as a linker leads to a wellpacked SAM on a gold surface and the carbohydrates presented in these set-ups resemble biological membranes on cell surfaces where multivalent interactions with, e.g., lectins are possible (Love et al. 2005; Paulson et al. 2006). Atomic force microscopy (AFM) force spectroscopy has emerged as a powerful tool for measuring binding properties of biological interactions at the single molecule level (for a review see Willemsen et al. (2000); Chen and Moy (2002); and Dufrêne and Hinterdorfer (2008)). In these measurements, c The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:  633 Received on October 31, 2008; revised on February 17, 2009; accepted on February 18, 2009 Introduction M Lienemann et al. Fig. 1. Preparation of N-acetyl-β-D-glucosamine neoglycoconjugate. Results QCM analysis of GlcNAc-neoglycoconjugate adsorption onto gold surfaces QCM measurements were carried out to follow the adsorption of the GlcNAc-neoglycoconjugate onto a gold-coated quartz crystal and to verify the SAM formation. The process was compared to the adsorption of a reference sample of the unconjugated alkanethiol 11-mercapto-1-undecanol linker, which is part of the 634 Fig. 2. Adsorption of the GlcNAc-neoglycoconjugate onto a gold surface monitored by QCM. The plot shows the change of frequency and dissipation (lower and upper pair of graphs, respectively) of a QCM sensor during injection of 400 μL of 1 mM GlcNAc-neoglycoconjugate (black) and 11-mercapto-1-undecanol (gray) dissolved in ethanol, as a function of time. Solutions were injected at a flow rate of 0.1 mL min−1 and the start of the thiol injection and the ethanol wash are marked with arrows (left and right, respectively). The flow through the sample cell was interrupted after injection of the SAM-forming agent for 3 min f (...truncated)