earticle

영어

Living organisms use simple or complex types of diverse biomolecule interactions (i.e. protein-protein, proteincompound, protein-carbohydrate, and carbohydrate-carbohydrate). Such interactions initiate biologically meaningful events and consequently, cell or organism can maintain its metabolism. Carbohydrate-protein interaction plays critical roles in living organisms; cell-cell communication, signaling, cell adhesion, fertilization, and immunological process.
These interactions also initiate infection of host cells by bacterial toxin protein and viruses. Therefore, understanding of the molecular relation for carbohydrate-protein interaction not only provides useful information on biological processes in living organisms but also is helpful for development of potent biomedical agents. Interaction between ganglioside GM1 and Vibrio cholera toxin proteins has been generally regarded as a good model for carbohydrateprotein
interaction. Pentameric B subunits of Vibrio cholera toxin are well known to interact individually with pentasaccharide of ganglioside GM1. Monomeric A subunit interacts with the membrane surface through pentameric B subunit and is known as enhancing avidity of whole interaction.
In the present work, we employed three nanobio-analytical techniques, surface plasmon resonance (SPR),
electrochemical detection, and atomic force microscopy (AFM), to detect and analyze more accurate interactions among three biomolecule components, ctxA, ctxB, and GM1 pentasaccharide.
To analyze interaction, biomolecules should be immobilized on solid surface. Since ganglioside GM1 that includes ceramide portion having highly hydrophobic carbon chain was selected as a target biomolecule, it was difficult to immobilize this molecule covalently onto solid surface. Therefore, we designed novel carbohydrate modification method to introduce thiol-group to reducing sugar and successfully modified mono- and pentasaccharide using this method. The proposed modification method has several advantages over previously reported methods,
including direct and rapid one-step immobilization onto a gold surface without surface pretreatment(s) by thiol group coupling in mild reaction environment, and exposure of functional carbohydrate moieties through oriented immobilization of the terminal reducing sugar.
We found that kinetic result of equilibrium constant (2.71 x 10-10 M) for ctxB-GM1 from SPR analysis was in good agreement with other reports even though we used novel immobilization method for GM1 pentasaccharide on gold surface which is more facile method. This result indicated that ctxB-GM1 has the highest affinity among the carbohydrate-lectin interaction. From the comparison analyses to reveal the role of ctxA, ctxAB-GM1 is expected to have high interaction compared with other receptor-ligand interactions. However, in the relative interaction analyses including SPR and electrochemical method, ctxAB-GM1 showed lower degree of binding than due to steric hindrance from different protein size than can affect the exact interaction values. However, cholera toxin ctxA increased the binding affinity through the complexation with ctxB from the unbinding force measurement using AFM.
From structural analyses using the GM1 analogues, we conclude that sialic acid and terminal galactose residues are mostly important in cholera toxin-GM1 pentasacchariede interaction and reorganizing in the binding sites occurred in the absence of its complementary fragments. Finally, interaction strength of several carbohydrates with complex cholera toxin showed following order: GM1 >> LST-b > LST-a > asialo GM1 > GM3.
Collectively, we obtained interaction information about kinetics and affinity from SPR, highly sensitive specificity and affinity from electrochemical detection, and direct interaction unbinding force from AFM. These analytical systems have individual advantages and disadvantages for interaction analysis. We noted that specificity can not simply decide affinity because other binding factors can affect binding affinity. In addition, it is hard to say that high affinity interaction decides high specificity. Therefore, the data from all three analytical methods should be
integrated and collectively analyzed to obtain more accurate information about the interaction.