Assessing carbohydrate-carbohydrate interactions by NMR spectroscopy: the trisaccharide epitope from the marine sponge Microciona prolifera

9 páginas, 7 figuras, 2 tablas, 1 esquema -- PAGS nros. 511-519Weak recognition processes: Weak calcium-mediated carbohydrate–carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide-CaII-trisaccharide complexes based on results from molecular dynamics simulations are proposed. Diffusion-ordered NMR spectroscopy (DOSY-NMR) and TR-NOESY-NMR experiments are used to detect ligand binding to macromolecular receptors. These techniques have been applied to detect weak carbohydrate–carbohydrate self-recognition in solution, making use of sugar-decorated gold nanoparticles as the “macromolecule” and the same carbohydrate as the ligand. Changes in the diffusion coefficient of the free carbohydrate in the presence of the glyconanoparticle (only with CaII ions in the sample solution), as well as changes in the sign of the sugar NOE peaks—positive for the free sugar (in the presence or absence of CaII) and negative for the sugar only in the simultaneous presence of the glyconanoparticle and CaII ions—have been taken as proof of weak CaII-mediated carbohydrate–carbohydrate interactions in solution. Although different methods such as SPR, TEM, and AFM have been used in the past to detect carbohydrate–carbohydrate interactions with the aid of gold nanoparticles and gold selfassembled monolayers, they are restricted to high-affinity ranges. The methods used in this study allow expansion of the number of techniques to tackle this relevant biological problem, also for approaching ligand–receptor interactions below the high-affinity range. Additionally, 3D models of trisaccharide-CaII-trisaccharide complexes based on results from molecular dynamics simulations are proposedThe group at CIB-CSIC thanks MEC/MCINN for financial support (Grant CTQ2006–10874-C02–01). S.M.-S. also thanks MEC for her Ramón y Cajal contract. The group at Utrecht University thanks the European Commission for financial support (Glycogold project; Contract No: MRTN-CT-2004–005645)Peer reviewe

Cartilage Aggrecan Can Undergo Self-Adhesion

Here it is reported that aggrecan, the highly negatively charged macromolecule in the cartilage extracellular matrix, undergoes Ca2+-mediated self-adhesion after static compression even in the presence of strong electrostatic repulsion in physiological-like solution conditions. Aggrecan was chemically end-attached onto gold-coated planar silicon substrates and gold-coated microspherical atomic force microscope probe tips (end radius R ≈ 2.5 μm) at a density (∼40 mg/mL) that simulates physiological conditions in the tissue (∼20–80 mg/mL). Colloidal force spectroscopy was employed to measure the adhesion between opposing aggrecan monolayers in NaCl (0.001–1.0 M) and NaCl + CaCl2 ([Cl−] = 0.15 M, [Ca2+] = 0 – 75 mM) aqueous electrolyte solutions. Aggrecan self-adhesion was found to increase with increasing surface equilibration time upon compression (0–30 s). Hydrogen bonding and physical entanglements between the chondroitin sulfate-glycosaminoglycan side chains are proposed as important factors contributing to aggrecan self-adhesion. Self-adhesion was found to significantly increase with decreasing bath ionic strength (and hence, electrostatic double-layer repulsion), as well as increasing Ca2+ concentration due to the additional ion-bridging effects. It is hypothesized that aggrecan self-adhesion, and the macromolecular energy dissipation that results from this self-adhesion, could be important factors contributing to the self-assembled architecture and integrity of the cartilage extracellular matrix in vivo