Carbohydrate-lectin interactions assayed by SPR.
Surface plasmon resonance is a valuable tool to determine the affinity between glycoconjugates and sugar-binding proteins such as plant and animal lectins. The main interest of using such an approach is that neither the lectins - which are proteins - nor their ligands - natural compounds such as glycoproteins, oligosaccharides, polysaccharides, or synthetic glycoconjugates such as glycoclusters or neoglycoproteins - require any tag. Because lectins bear several binding sites, they behave like immunoglobulin eliciting avidity phenomena. This peculiarity may lead to erroneous results if special conditions are not applied. We obtained best and reproducible results when the lectin was immobilized and its ligands were used as soluble analytes. With heterogeneous glycoconjugates such as neoglycoproteins (which are heterogeneous in terms of nature, number, and position of sugar residues) or a mixture of oligosaccharides, the data may be more accurately gathered by using the Sips approach, which has been used to determine mean binding constants of polyclonal antibodies. With small analytes such as oligosaccharides, we found it convenient to determine binding constants by using an inhibitory approach: a neoglycoprotein (M (r) = approximately 80,000) was allowed to bind to the immobilized lectin and small oligosaccharides were used as inhibitors. With larger glycoconjugates such as peptides substituted with glycoclusters, direct binding measurements gave accurate results. Because of the availability of low-cost simple sugars (mono- or disaccharides) it is very convenient to use large concentrations of such carbohydrates to clean the sensor chips instead of more drastic cleaning solutions such as acids or alkali, in such a way that the immobilized lectin is stable for many experiments.
Duverger
Eric
E
Glycobiologie, Centre de Biophysique Moléculaire, CNRS et Université d'Orléans, Orléans, France.
Lamerant-Fayel
Nathalie
N
Frison
Natacha
N
Monsigny
Michel
M
D053000:Analytic Sample Preparation Methods; D000818:Animals; D050260:Carbohydrate Metabolism; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D037181:Lectins, C-Type / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
D015815:Cell Adhesion Molecules; D055767:Immobilized Proteins; D037102:Lectins; D037181:Lectins, C-Type; D008358:Mannose; D011956:Receptors, Cell Surface; D012710:Serum Albumin, Bovine; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin