Fabrication of Carbohydrate Surfaces by Using Nonderivatised Oligosaccharides, and their Application to Measuring the Assembly of Sugar–Protein Complexes

Abstract

This way up. Dual polarisation interferometry was used to design and characterise a surface on which the orientation and density of immobilised carbohydrates was suitable for studying their interactions with proteins. Lactoferrin was shown to adopt two orientations: "end-on" or "side-on", while for FGF-2 a single monolayer of protein was observed. The new surface can be used to elucidate the binding of proteins to carbohydrates and the geometry of the complexes, a frequently controversial area. Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index and mass of adsorbates 100 nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates is suitable for studying their interactions with proteins and where nonspecific binding is reduced to less than 5 % of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional crosslinker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulfate (KS) chondroitin sulfate (CS) and heparin, that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG. The heparin DP-10 surfaces were then used to determine the performance of the immobilised DP-10 with respect to binding of two well-characterised proteins, lactoferrin (Lf) and fibroblast growth factor-2. The results show that Lf could adopt two different orientations, at high protein loadings the protein layer thickness corresponded to an "end-on" orientation of Lf, whilst rinsing with buffer saw the Lf molecules adopt a "side-on" configuration. In the case of FGF-2, a single monolayer of protein bound to DP-10 was observed. These results demonstrate that the new surface can be used to resolve key questions relating to the binding of proteins to carbohydrates, including, when used in DPI, the resolution of the geometry of complexes, an area that is frequently controversial.

Characterisation of the interaction of neuropilin-1 with heparin and a heparan sulfate mimetic library of heparin-derived sugars

“…The same heparin (17 kDa average molecular weight) (Celsus Lab, Cincinnati, OH, USA) was used throughout, including for the production of the chemically modified polysaccharides (Table 1) and oligosaccharides of defined size. Heparin derivatives were as characterised previously (Yates et al, 1996) and heparin derived oligosaccharides were obtained from partial heparinase I (IBEX Technologies, Montreal, Canada) digestion of porcine heparin performed, as described previously (Popplewell et al, 2009; Powell et al, 2010). …”

“…The experiment was performed as described previously (Popplewell et al, 2009). Briefly, a thiol chip was derivatized at 30 °C with 5 mg/mL N-( β -maleimidopropionic acid) hydrazide (BMPH) (Thermo) applied at 8 µL/ min to each flow cell to produce a surface with hydrazide groups.…”

Comment on “Tuning the bioactivity of bone morphogenetic protein-2 with surface immobilization strategies” by Chen et al.

“…88,89 DPI has been used to study the binding of protein to glycosaminoglycans (GAGs) as chondroitin sulfate, keratin sulfate, and heparin. 71 The reducing end of GAGs has been grafted onto a hydrazide-functionalized surface and their surface coverage, the thickness of the layer, and its density were followed with DPI [ Fig. 2(c 0 )].…”

Immobilization of chondroitin sulfate to lipid membranes and its interactions with ECM proteins

“…Several immobilization techniques have been presented in the literature, e.g. immobilizing biotinylated GAGs to (strept)avidin modified surfaces [16,17], coupling of thiolated carbohydrates to a maleimide presenting surface or vice versa [18,19] or direct coupling of the GAG via the reducing end to a hydrazide presenting surface [20]. When the GAG is chemically modified, changes are induced in the primary structure and care must be taken as to not influence the binding characteristics to other biomolecules.…”