Microbead analysis of cell binding to immobilized lectin: an alternative to microarrays in the development of carbohydrate drugs and diagnostic tests

Abstract

SummaryMicroarray technology is currently used in the development of carbohydrate drugs and diagnostic tests. Here we model an inexpensive alternative to microarrays using derivatized microbeads. In this model we examine the binding of mannose-rich yeast to microbeads derivatized with concanavalin A (Con A), a mannose-binding lectin, in the presence of 30 different sugars and 9 different pH conditions. We developed a listing of effective saccharide inhibitors of immobilized Con A based on 3901 replicates. We suggest that this is the most extensive saccharide inhibitor list ever developed for this lectin and it may be useful to use this listing to replace the less extensive lists that have been in the literature for decades. Information is also provided on pH effects on immobilized Con A binding based on 918 trials. Two assays to study binding, one which qualitatively scores more or less binding than control in thousands of replicate samples, and another that quantitatively evaluates binding by counting the number of cells bound to each bead, are also modeled here. We know of no previous studies that provide such as extensive information on saccharide inhibition and pH effects on the binding of immobilized Con A. We suggest that this microbead approach, using beads derivatized with lectins or sugars, and the two simple assays presented here, can in some cases, substitute for more expensive microarray technology in the development of carbohydrate drugs and diagnostic tests. If, for example, our model Saccharomyces cerevisiae was a pathogen, these studies show that it binds via cell surface mannose residues and drugs to prevent binding could be developed using the inhibitors of binding identified here. The beads could be also used in the development of diagnostic tests that identify the presence of the organism in blood samples, etc. in much the same way as microarray technology is being used today.

A glycobiology review: Carbohydrates, lectins and implications in cancer therapeutics

“…Many human pathogens utilize cell surface glycans as either receptors or ligands to initiate adhesion and infection (Sharon and Lis, 1989, 2003; Zem et al, 2006; Hyun et al, 2007; Oppenheimer et al, 2008; Magalhaes et al, 2009; Mukhopadhyay et al, 2009). Escherichia coli ( E. coli ), for example, binds to host mannosides, while influenza virus binds to host sialic acids (Mukhopadhyay et al, 2009).…”

“…However, complex carbohydrates found in honey may also contribute to the antimicrobial activity of honey. Complex carbohydrates such as the trisaccharide D(+)melezitose (α-D-Glucopyranosyl-β-1,3-fructofuranosyl-α-glucopyranoside), found in honey in concentrations of up to 3.4 mg/g, were shown to be potent inhibitors of yeast-Con A bead binding system at low concentrations (Aso et al, 1960; Zem et al, 2006; de la Fuente et al, 2007). …”

“…As discussed in the section on Carbohydrates in host-pathogen interactions , many human pathogens utilize cell surface glycans as either receptors or ligands to initiate adhesion and infection (Kyogashima et al, 1989; Sharon and Lis, 1989, 2003; Thankavel et al, 1999; Zem et al, 2006; Hyun et al, 2007; Oppenheimer et al, 2008; Mukhopadhyay et al, 2009; Rek et al, 2009). Therefore, using specific carbohydrates or their analogs to interfere with the pathogen lectin-host carbohydrate interactions may prevent and treat microbial infections or diseases (Fig.…”

Carbohydrate-based experimental therapeutics for cancer, HIV/AIDS and other diseases

“…Serological markers have been identified for Crohn's disease using microarrays (Dotan et al, 2006). In addition to microarrays, carbohydrate and lectin derivatized microbeads have been used as model systems for development of carbohydrate-based drugs and diagnostic tests (Zem et al, 2006).…”

Activation of G-Protein-Coupled Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads

“…Considering the fact that the function of GPCRs critically depends on the composition of their lipid environment, GPCR signaling studies in native supported membranes are particularly interesting. Different methods have been developed to transfer native cell plasma membranes to planar − and spherical − solid supports. They are principally based on unspecific interactions of the surface of the plasma membrane with the surface of the solid support by either (i) binding living cells to the supporting material and subsequently disrupting the cell by applying mechanical shear forces ,,,,,− ,, or (ii) first preparing plasma membrane vesicles from cells which are subsequently bound to solid surfaces under conditions in which they open to planar membrane sheets. ,,,,,,,− …”