Binding studies of adhesion/growth-regulatory galectins with glycoconjugates monitored by surface plasmon resonance and NMR spectroscopy.
Functionalized fluorescent glycans have the potential to act as tools to detect and analyze protein-carbohydrate interactions. We present here a facile strategy for immobilization of functionalized lactose as a model disaccharide. Bioactivity was tested with three members of the adhesion/growth-regulatory galectins family in different types of assay, i.e. matrix in surface plasmon resonance (SPR), free ligand in solution by STD/trNOESY and docking measurements. In all cases, the activity of the disaccharide was maintained. The attachment of this new fluorescent glycoconjugate to the surface results in a well-defined interface, enabling desired orientational flexibility and enhanced access of binding partners. The results indicate that this new glycoconjugate exhibits binding affinity to galectin-1, 3 and CG-16. Kinetic analysis of the interaction between these galectins and immobilized glycoconjugate by SPR yielded a K(D) of 1.01 mM for galectin-1, 83.5 microM for galectin-3 and 0.28 mM for CG-16. No major contacts to the aglyconic part were detected, which might compromise the specificity of the binding process with other headgroups. Thus, testing these proteins offers the potential for medical applications to detect these endogenous effectors or further derivatives and characterize their carbohydrate specificity.
Muñoz
F Javier
FJ
Departamento de Química Orgánica y Farmacéutica, Universidad Complutense de Madrid, Pz/Ramón y Cajal s/n, 28040, Madrid, Spain.
Santos
J Ignacio
JI
Ardá
Ana
A
André
Sabine
S
Gabius
Hans-Joachim
HJ
Sinisterra
José V
JV
Jiménez-Barbero
Jesús
J
Hernáiz
María J
MJ
D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes / Q000378:metabolism; D037161:Galectins / Q000737:chemistry / Q000378:metabolism*; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
D005456:Fluorescent Dyes; D037161:Galectins; D006001:Glycoconjugates