Lectins as probes for assessing the accessibility of N‐linked glycans in relation to the conformational changes of fibronectin

Abstract

Fibronectin, a ≈ 450-kDa protein with 4-9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal-induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose-binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal-induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research.

Vitronectin (Vn) glycosylation patterned by lectin affinity assays—A potent glycoproteomic tool to discriminate plasma Vn from cancer ascites Vn

“…The binding activity of human Vn has been found to increase under heating. Our group has recently described the effect of thermal treatment on the glycan accessibility of human plasma Fn, another adhesive glycoprotein, using the mannose‐binding ConA lectin . ConA is widely used as molecular probe in glyco‐biotechnology, and our present study demonstrated distinct ConA binding patterns according to Vn origin.…”

“…It belongs to the calcium‐dependent (C‐type) lectin family, which shares structural and functional homologies with animal C‐type lectins as blood plasma mannan binding proteins . ConA is one of the most widely studied lectins due to its wide range biological functions, such as immune‐stimulating activity, and to its biotechnological applications to characterize glycoproteins from clinical specimens, as shown by our group …”

“…Attached glycans are known to be involved in a diverse array of cellular functions, including cell‐cell interactions and protein folding, stability, and activity . As compared with other ECM proteins like Fn (5%–8% glycosylated), human Vn is more glycosylated, with ~30% of its molecular mass being attributed to glycosylation. Changes in glycosylation of plasma Vn have been associated to significant alterations in its binding activities and cellular functions, related to different physiological states .…”

Controlling Cell Behavior through the Design of Biomaterial Surfaces: A Focus on Surface Modification Techniques

“…For example, Pro–His–Ser–Arg–Asn is an amino acid sequence, found in fibronectin, whose activation results in the enhancement of signaling responses that initiate stem cell recruitment . Despite these merits, ECM proteins have several major shortcomings that limit their clinical use, such as potential risk of pathogen transmission, unsolicited adverse immune response (against cadaveric or xenogenic proteins), and bioactivity loss owing to conformational changes during conjugation to scaffolds …”

Biosensing with optical fiber gratings

“…An important role in understanding biomolecule interaction and in the study of extracellular membranes is played by glycoprotein association with lectins and integrins [82]. In this area, two groups have developed FBG-based biosensors exploiting the interaction between concanavalin A (ConA) and mannose-functionalized dendrimers [32] and between fibronectin and fibroblast cells to study the cell response to stimuli [36].…”