Live Cells versus Fixated Cells: Kinetic Measurements of Biomolecular Interactions with the LigandTracer Method and Surface Plasmon Resonance Microscopy

Abstract

Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.

Surface plasmon resonance microscopy identifies glycan heterogeneity in pancreatic cancer cells that influences mucin-4 binding interactions

“…The SPRm 200 (surface plasmon resonance microscope model 200 from Biosensing Instrument Inc., Tempe, Arizona), which has an inverted microscope for simultaneous bright field imaging and SPR microscopy with CCD camera (600 μm × 450 μm area) [4,[50][51][52] was used in this study with BxPC3 adherent pancreatic cancer cells (Fig 1). The running buffer was 1X PBS with a flow rate of 100 μL/min.…”

“…The SPRM kinetic analysis results of all ROIs were aggregated for statistical analysis. Histograms of the kinetic parameters were fitted with Gaussian distributions to extract the mean and 95% confidence interval of the cell population [4,[50][51][52]. ImageSPR software (Biosensing Instrument Inc., Tempe Az) was used to generate the sensorgrams, fitting, and statistical analysis of binding interactions and heterogeneity.…”

“…In vitro whole-cell binding kinetics without disrupting the native environment of membrane glycoproteins is critical to revealing more biologically relevant binding interaction information. Although there are various technologies, such as Quartz Crystal Microbalance (QCM) [45,[76][77][78][79] and Ligand Tracer (LT) [80,81] that can also reveal glycan binding kinetics in whole cells, such technologies do not have the sub-cellular resolution of SPRM that enables detailed statistical studies of binding heterogeneity [4,[50][51][52][53]. Aberrant glycosylation is a well-established hallmark of cancer [10][11][12][82][83][84][85][86][87][88][89][90][91], and aberrant N-glycosylation has been studied extensively and shown to play a major role in binding interactions of critical therapeutic targets [7,8,35,45].…”

“…Membrane proteins play critical functions in cell biology, ranging from cell signaling, protein transport, and translocation [1]. Roughly 60% of all membrane proteins are the targets of therapeutic drugs [2][3][4]. However, to effectively understand the binding kinetics of new or potential therapeutic drugs, it is crucial to study binding in the context of the cell surface that contains a complex array of carbohydrate structures (glycans) associated with the membrane proteome, which are often overlooked.…”

Cell-Surface Glycan Labeling and Sensing

“…This is the first study reporting on the binding and kinetic parameters of WGA interactions with two different glycosylated sites. They also studied the interaction of lectins with glycans in living and fixed cells [113]. Luo et al [114] developed a localized surface plasmon resonance (LSPR) method using U-shaped gold nanoparticle (AuNP)-modified optical fibers.…”