Elucidation of Carbohydrate Molecular Interaction Mechanism of Recombinant and Native ArtinM

Abstract

The quartz crystal microbalance (QCM) technique has been applied for monitoring the biorecognition of ArtinM lectins at low horseradish peroxidase glycoprotein (HRP) concentrations, using a simple kinetic model based on Langmuir isotherm in previous work.18 The latter approach was consistent with the data at dilute conditions but it fails to explain the small differences existing in the jArtinM and rArtinM due to ligand binding concentration limit. Here we extend this analysis to differentiate sugar-binding event of recombinant (rArtinM) and native (jArtinM) ArtinM lectins beyond dilute conditions. Equivalently, functionalized quartz crystal microbalance with dissipation monitoring (QCM-D) was used as real-time label-free technique but structural-dependent kinetic features of the interaction were detailed by using combined analysis of mass and dissipation factor variation. The stated kinetic model not only was able to predict the diluted conditions but also allowed to differentiate ArtinM avidities. For instance, it was found that rArtinM avidity is higher than jArtinM avidity whereas their conformational flexibility is lower. Additionally, it was possible to monitor the hydration shell of the binding complex with ArtinM lectins under dynamic conditions. Such information is key in understanding and differentiating protein binding avidity, biological functionality, and kinetics.

ArtinM Binding Effinities and Kinetic Interaction with Leukemia Cells: A Quartz Crystal Microbalance Bioelectroanalysis on the Cytotoxic Effect

“…This model allows to calculate the equilibrium binding affinity, , from a mathematical fit of real time binding kinetic pattern obtained directly from QCM measurements . Beyond the diluents limit conditions of the kinetic model based on Langmuir isotherms applied to evaluate ArtinM‐HRP binding, the QCM−D additionally allows to investigate viscoelastic properties . …”

“…The applicability of the QCM technology for monitoring real‐time binding of lectins with carbohydrates or glycoproteins has previously been evaluated with immobilized lectins ,, and carbohydrates . Indeed, lectin‐cell interaction has been evaluated with immobilized lectins or cells on chip surface and opens a new possibility of characterization of tumor cells glycan.…”

“…The rArtinM used herein was obtained as described in reference . The procedure leads to a system that was not only able to produce a good protein yield, but also a functional lectin, since rArtinM was able to bind to horseradish peroxidase (HRP) in a similar way to that of native ArtinM ,.…”

Glycoprotein assay based on the optimized immittance signal of a redox tagged and lectin-based receptive interface

“…The advantage of these techniques is the capability to investigate interaction kinetics in real-time (Lebed et al, 2006;Pedroso et al, 2008;Pesquero et al, 2010). This technique can also incorporate analysis of the energy dissipation factor (QCM-D), additionally allowing investigation of the viscoelastic properties of proteinprotein binding/interactions (Höök and Kasemo, 2007), eventually associated with different binding processes or recognition events (Giménez-Romero et al, 2013;Santos et al, 2015a). Similar to QCM, Surface Plasmon Resonance (SPR) is a technique in which the transducing signal is intrinsically and related to changes in the optical properties of the investigated receptive surface.…”

Evaluating the Equilibrium Association Constant between ArtinM Lectin and Myeloid Leukemia Cells by Impedimetric and Piezoelectric Label Free Approaches

“…The affinity parameters of the lectin–carbohydrate interaction have been proposed using label-free techniques, including surface plasmon resonance [ 10 , 11 , 12 ], piezoelectric [ 13 , 14 , 15 , 16 , 17 , 18 , 19 ] and impedimetric approaches [ 20 , 21 ]. Formally, the affinity interactions between a receptor and multiple sites in a ligand is an avidity interaction [ 12 , 22 ] and the observed K a is an averaged “equilibrium association constant”.…”