Characterization of the binding of wisteria floribunda agglutinin to chondroitin sulfate

Abstract

Chondroitin sulfate proteoglycans (CSPGs) are found in specialized brain extracellular matrix structures termed perineuronal nets (PNNs). The chondroitin sulfate chains of these CSPGs are thought to have a strong effect on neuroplasticity, along with development, injury, and diseased states of the brain. Wisteria floribunda agglutinin (WFA) is a plant lectin used to identify PNN via staining; the pattern of this staining is changed upon schizophrenia. As such, one powerful method of probing the identity of the CS chains of PNNs and addressing what changes in CS identity occur during schizophrenia is to characterize the features of the CS which bind to the lectin. Methods for characterization of WFA-CS binding and their biological relevance were developed and evaluated. Commercially available CS was used to probe the binding affinity of the agglutinin to various regions of CS via hemagglutination inhibition assays and affinity gradient elution of CS bound to WFA. The size, sulfation extent, and fragment location in the CS chain from these eluates were determined using HILIC-LC-MS. As commercial sources can be used to elucidate the binding specificity of WFA, but not the actual relevant binding partner of WFA within the brain, PNN CS extractions were performed with a modified method aimed at reducing the timescale at which PNN CS can be obtained so as to allow similar experimentation on CS directly from PNN. The results pave the way for further determination of WFA-CS binding.