chapter 2 - Bentham Science

Synthesis and Biological Applications of Glycoconjugates, 2011, 255-266 255
Analytical Tools for Protein-Carbohydrate Interaction Studies
Cédric Goyer and Pierre Labbé *
Olivier Renaudet and Nicolas Spinelli (Eds)
All rights reserved - © 2011 Bentham Science Publishers
CHAPTER 14
Département de Chimie Moléculaire, UMR-CNRS 5250 & ICMG FR 2607, Université Joseph Fourier, 38041
Grenoble Cedex 9, France
Abstract: A wide range of assays has been used to characterize protein-carbohydrates interactions allowing access to
various quantitative information including thermodynamic ones such as stoichiometry of binding, binding constant,
enthalpy of binding as well as kinetics and mechanistic ones. An understanding of each technique is essential in order
to propose and validate appropriate interaction models that rationalize experimental data. Due to the complexity of
carbohydrate-lectins interactions as an inherent consequence of multivalent interactions, a careful control of
experimental conditions is necessary in order to avoid misinterpretation. This article will briefly present the various
techniques that are commonly used to characterize sugar-lectin interaction: inhibition of hemagglutination assay
(HIA), enzyme-linked lectin assays (ELLA), isothermal titration calorimetry (ITC) and surface plasmon resonance
(SPR). Recently introduced, force spectroscopy by atomic force microscopy (AFM) is a new approach for measuring
carbohydrate-protein interaction at the level of a single protein.
Keywords: Biomolecular interaction, biosensors, surface plasmon resonance, isothermal calorimetric titration,
atomic force spectroscopy.
INTRODUCTION
Lectins are carbohydrate binding proteins other than enzymes and antibodies [1] and exist in most living organisms
ranging from viruses and bacteria to plants and animals [2]. They constitute cellular receptors that play a fundamental
role in a wide range of biological processes including physiological and pathological events [3] such as adhesion of
infectious agents to host cells, inflammatory processes, cell interactions in the immune system, growth and metastasis
of tumour cells. Interactions between lectins and carbohydrates occur in the highly hydrated biological environment
where hydration and solvation effects play a determinant role [4]. Carbohydrates primarily bind to polar groups of the
lectin active site through hydrogen-bonding of their hydroxyl groups. Complementary hydrophobic interactions also
occur between hydrophobic patches of the carbohydrate structure and the aromatic moieties that are present in the lectin
constituents. It has been early recognized that an individual carbohydrate ligand binds to a lectin site weakly, with
affinity constant generally in the range 1µm-1mM. The reason for this weakness lies in the solvent-exposed nature of
the lectin binding-site, which are shallow pockets making few direct contacts with the ligands [5]. Considering the high
specificity of biorecognition processes, such a weak binding is unlikely to be of any biological significance. However it
is now well established that the weak binding affinities are efficiently overcome through so-called multivalent
interactions, also known as “cluster glycoside effect”, that allow increased binding affinities and higher specificity
required for a biologically relevant recognition process. Polyvalent associations that occur throughout biology present a
number of characteristics that monovalent interactions do not exhibit. In most cases, polyvalent interactions rather than
a strong single one are used in biological systems. Thus, the ability of lectins to distinguish between subtle variations of
sugar structure makes them particularly efficient for decoding such carbohydrate-encoded information known as
“glycocode”. Since lectins and carbohydrates are both commonly present at the cell surface and because sugars posses
tremendous coding capacity, they constitute excellent cell recognition markers [6] that can be consequently exploited
for therapeutic and diagnostic applications.
A complete comprehension of the mechanism of protein-carbohydrate interaction at the molecular scale requires the
elucidation of both structural and energetic aspects of the process. A large amount of structural data is now
available, essentially from X-ray crystallography and NMR spectroscopy that have been used to elucidate the
*Address correspondence to Pierre Labbé: Département de Chimie Moléculaire, UMR-CNRS 5250 & ICMG FR 2607, Université Joseph
Fourier, 38041 Grenoble Cedex 9, France; E-mail: pierre.labbé@ujf-grenoble.fr