Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

Abstract

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan-protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan-protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan-protein-protein interactions, as well as a molecular-level understanding of those complexes. G lycosaminoglycans (GAGs) regulate a wide range of physiological processes, including viral invasion, blood coagulation, cell growth, and spinal cord injury (1-4). Assembled from repeating disaccharide units, GAGs display diverse patterns of sulfation (SI Appendix, Fig. S1). These sulfation patterns are believed to have important functional consequences, enabling the polysaccharides to interact with a wide variety of proteins (1, 2). However, the precise sulfation motifs involved in protein recognition are understood in only a few cases (1,4,5). Moreover, studies of heparan sulfate (HS) interactions with fibroblast growth factors suggest that GAGs can assist in the assembly of multimeric protein complexes, thereby modulating signal transduction pathways (6-10). Yet, only a few such examples have been elucidated, and the extent to which other GAGs such as chondroitin sulfate (CS) engage in the formation of multimeric protein complexes remains unknown. Elucidating the interactions of specific GAG substructures with proteins and large protein-protein complexes will be critical for understanding the structure-activity relationships of GAGs and the mechanisms underlying important biological processes.Several methods have been developed to study GAG-protein interac...

Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E Motifs in Rodent Brains

“…Carbohydrate Microarray Assays-Microarrays containing natural GAGs enriched in the CS-A, CS-C, CS-D, and CS-E motifs (Seikagaku Corp.), dermatan sulfate (also called CS-B; Sigma), hyaluronic acid (HA; Sigma), heparin (Hep; Neoparin, Alameda, CA), HS (Sigma), or chondroitin sulfate (CS; Sigma) were printed on poly-D-lysine-coated glass surfaces as described previously (31,32). Arrays were blocked with 10% FBS in 1ϫ PBS with gentle rocking at room temperature for 1 h, followed by a brief rinse with 1ϫ PBS.…”

A sulfated carbohydrate epitope inhibits axon regeneration after injury

“…Microarrays were incubated with PTPσ-Fc, followed by a Cy3-conjugated antihuman IgG secondary antibody, and analyzed using a GenePix 5000a scanner. In light of our results showing that CS-E is a major inhibitory motif on CSPGs, we examined the potential interaction between CS-E and PTPσ using carbohydrate microarrays (26). A soluble PTPσ-Fc fusion protein, but not other receptors such as EphA2-Fc or Fc alone, bound efficiently to CS-E polysaccharides arrayed on poly-lysine-coated glass slides (Fig.…”

Functional Proteomics: Mapping Lipid-Protein Interactomes

“…To assess metabolites-protein interactions, protein arrays are exposed to fluorescently labelled metabolites (Zhu et al 2001). Small molecules arrays are exposed to individual proteins (Rogers et al 2011) or cellular lysates containing tagged proteins and interactions are detected using antibody recognising the specific tag (Gallego et al 2010).…”