Cell Binding

Cells, the fundamental functional units in organisms, don’t do their job alone. They need to communicate with other cells and extra-cellular matrices (ECM) to retrieve information for proliferation, differentiation and immune responses, etc. Cells have many recognition molecules expressed on their surfaces such as carbohydrate epitopes and protein receptors. These interact with other cells and the ECM. Our group is developing cell-binding assay methods to screen the various interactions that occur between cells and biomolecules. The foundation of these assays are array technologies and surface chemistry techniques that were developed in our laboratories.



Figure 1: Glycocalyx, a dense layer of carbohydrates, on a mamalian cell surface. The depth of the glycocalyx can range up to 500nm.

Cell surface carbohydrates (Figure 1) play important roles in many fundamental biological processes, such as proliferation, migration and differentiation. Diseased cells frequently have altered carbohydrate expression patterns when compared with healthy cells. In order to study and understand the implications of this altered carbohydrate expression it is essential to have effective testing methods. Lectins are the natural binding partners of carbohydrates. Our group hypothesized that lectins, if immobilized on a surface, would be capable of capturing cells through their unique lectin:carbohydrate binding interaction. Taken to the next step, an array of different lectins could then be used to profile the presence or absence of each corresponding surface-expressed carbohydrate. Ideally, one array would be capable of profiling cell surface carbohydrate expression patterns.



Figure 2: Binding patterns of different cells on lectin arrays.

Lectins are a group of proteins of non-immune origin that can recognize and bind to specific carbohydrate epitopes. Hundreds of lectins have been isolated from plants and animals and their specificities are studied and described in the literature. Various lectins were covalently attached to self-assembled monolayer modified gold substrates using chemistry developed by our research group. Cell suspensions were then incubated on the arrays to facilitate occurrence of the binding interaction. After incubation and washing, the binding between cells and the array was studied with a standard optical microscope. Different cell lines showed distinct lectin binding patterns, which correspond to the expression of carbohydrates on the cell surface (Figure 2). To validate the specificity of the binding, a number of different sugars were added to the incubation buffer. These sugars were provided in excess and intended to block binding. Indeed, binding between the cells and certain lectins were significantly reduced when blocking sugars were added (Figure 3). In addition, only the targeted binding event was affected. Other specific interactions occurred to the expected level.



Figure 3: Binding of BHK-21 cells to lectins in the presence of various blocking sugars.

Similarly, we are also fabricating growth factor arrays to interrogate the binding between cells and growth factors. The growth factors can be immobilized onto chip surfaces using chemistry analogous to the chemistry used for the fabrication of lectin arrays. The goal is to create arrays with several different immobilized growth factors. Cell binding patterns on the chip would then be indicative of the expression of receptors on the cell surface. As proof-of-concept, we immobilized basic fibroblast growth factor 2 (BFGF2) on a self-assembled monolayer modified gold chip. Baby hamster kidney cells (BHK21), which expressed receptors to BFGF2 bound specifically to the place on the chip that contained the immobilized BFGF2 (Figure 4A). Heparin sulfate on the cell-surface is known to facilitate the binding. To validate that the binding between immobilized BFGF2 and BHK21 cells was specific, we treated the cells prior to incubation with an enzyme that destroys heparin sulfate (heparitinase). Cells treated with this enzyme remained alive but did not engage in binding (Figure 4B).



Figure 4: binding of BHK-21 cells with FGF-2 . A: BHK-21 cells; BHK-21 cells treated with heparitinas

It is also interesting to note that the cells that are bound to the growth factors on the gold substrates can be directly analyzed by MALDI (matrix assisted laser desorption ionization) mass spectrometry (Figure 5). The spectra obtained thusly add a significant amount of information to the assay. Specifically, the spectra provide a profile of the highly expressed proteins in the cell samples. We are beginning to look for correlations between the binding patters and the protein expression.




Figure 5: MALDI spectrum from captured BHK-21 cells with FGF 2