Detailed AFM Force Spectroscopy of the Interaction Between CD44–IgG Fusion Protein and Hyaluronan Aernout A. Martens & Marcel Bus & Peter C. Thüne & Tjerk H. Oosterkamp & Louis C. P. M. de Smet Published online: 24 July 2014 # Springer Science+Business Media New York 2014 Abstract Atomic force microscopy (AFM) force spectrosco- py was used to study the single-molecule rupture events of the interaction between hyaluronan (HA) and the binding domain of its cell surface receptor CD44. AFM probes were amino terminated with 3-aminopropyl triethoxy silane (APTES) followed by covalent coupling of protein A, enabling the binding of the CD44–HA-binding domain, as part of a CD44–Fc fusion protein. HA was covalently bound to APTES-coated silicon surfaces. Single-rupture events were recorded at various loading rates revealing an energy barrier: E b =24±1 kT and characteristic distance: x β =1.3±0.1 nm for this interaction. This quantification will be of interest in ap- plications and research involving the use of the CD44–Fc fusion protein since we observe a weaker interaction between HA and CD44–Fc than what has been reported for the entire native CD44 molecule. Keywords CD44 link domain . Fusion protein . Hyaluronan . Force microscopy 1 Introduction Fusion proteins consisting of a receptor and the Fc region of IgG are used for various applications, both clinical and non- clinical [1–3]. Companies producing Fc–receptor fusions claim that these constructs are useful in elucidating the inter- actions between a receptor and its ligands. Some also claim that dimerization via the disulfide bonds of the Fc regions enhances the receptor’ s affinity to its ligand, especially when dimerization is needed for activation of the receptor. With CD44–Fc chimera and its ligand hyaluronan (HA) as an example, we demonstrate that the binding properties of an Fc–receptor fusion protein as obtained by an atomic force microscopy (AFM) force spectroscopy may not accurately represent those of native receptors and that they may even be weaker instead of being enhanced. The CD44–HA inter- action [4] is of special interest as it is involved in many processes, including wound healing, fetal development, tissue remodeling, lymphocyte rolling and extravasation, and cancer metastases [5–7]. Research involving CD44–Fc includes its use as fluores- cent labels in vitro on living cells [8], binding studies of short and long HA chains [9], and different (proteo)glycan ligands [10, 11]. Recent AFM-based studies report on the affinity and specificity of CD44 to HA and other potential ligands [12–14]. In these studies, the glycan ligand was always at- tached to the AFM tip, while CD44 was present on the probed (cell) surface. However, to probe for HA present on (cell) surfaces by AFM, one would need an AFM tip with a HA receptor. In this study, we use CD44–Fc to prepare such a tip. The interaction of the modified tip with HA-covered surfaces was subsequently measured by AFM force spectroscopy [15]. Electronic supplementary material The online version of this article (doi:10.1007/s12668-014-0143-8) contains supplementary material, which is available to authorized users. A. A. Martens : M. Bus : L. C. P. M. de Smet (*) Department of Chemical Engineering, Delft University of Technology, Julianalaan 136, Delft 2628 BL, The Netherlands e-mail: l.c.p.m.desmet@tudelft.nl P. C. Thüne Department of Chemical Engineering and Chemistry, Catalysis & Energy, Eindhoven University of Technology, Eindhoven 5600 MB, The Netherlands T. H. Oosterkamp Kamerling Onnes Laboratory, Leiden University, Niels Bohrweg 2, Leiden 2333 CA, The Netherlands Present Address: P. C. Thüne Fontys Hogescholen, Rachelsmolen 1, Eindhoven 5612 MA, The Netherlands BioNanoSci. (2014) 4:232–239 DOI 10.1007/s12668-014-0143-8