Investigations of prebiotics and of inter- and intra-molecular glycan-protein interactions

Abstract

Carbohydrate chains play key roles in living organisms. Novel techniques have been developed in recent years to investigate the structure/function of carbohydrates. 2D nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulation in conjunction with NMR refinement, as well as surface plasmon resonance (SPR), have been employed in this thesis to map structures and biological functions of glycans. Chapter 1 gives an overview of the concept of prebiotics, probiotics and synbiotics, with particular emphasis on carbohydrates as possible prebiotics. Significant results obtained in recent clinical studies are also reported. In Chapter 2 the studies carried out in the framework of the European Union project “Novel Food Additives and bio-active components from milk for innovative engineering (NOFA)” are reported. To overcome the major bottleneck of obtaining naturally occurring oligosaccharides in sufficient amounts to perform structural characterization and biological testing, an improved protocol to isolate large quantities of lactose-free oligosaccharide fractions from non-human milk was developed. We demonstrated that naturally occurring galacto-oligosaccharides could be isolated in large amounts from skimmed goat milk (or colostrum) oligosaccharide pools, when incorporating a beta-galactosidase (E. coli) digestion in the protocol. Major components from goat colostrum were isolated and structurally characterized by high-pH anion-exchange chromatography, MALDI-TOF mass spectrometry, and NMR spectroscopy. Bacterial adhesion studies with isolated acidic oligosaccharide fractions showed a significant inhibition of the adherence of faecal Salmonella fyris B8132 to cultured intestinal epithelial cells. In Chapter 3 the isolation of tri- and tetrasaccharides from goat milk after enzymatic lactose hydrolysis with E. coli beta-galactosidase, and their structural analysis by methylation analysis, mass spectrometry, and NMR spectroscopy, is described. In Chapter 4, in an attempt to understand the biological significance of a recently discovered form of glycosylation, i.e. C-mannosylation, the conformation of Man1alpha-Trp in human RNase 2 was investigated. NMR experiments and molecular modeling calculations for (C2-alpha-D-Man-)Trp demonstrated that the C-linked mannopyranosyl residue exists in an ensemble of conformations, among which 1C4 is the most represented. For isolated glycopeptides, NMR showed no evidence for long-range connectivities and secondary structure, arguing against a stabilization of the analyzed glycopeptides, due to the C-linked mannopyranosyl residue. For native RNase 2, molecular modeling studies and NMR data revealed that the mannopyranosyl residue interacts with the loop residues Asp115-123 of RNase 2, the end of the beta strands Met105-Arg114 and the beginning of the beta strands Pro124-Ile134. These interactions stabilize not only to the mannose residue and Trp7 in a specific orientation, but also the N-terminal loop of the protein. Chapter 5 focuses on the feasibility of surface plasmon resonance (SPR) to detect oligosaccharide-protein interactions. A serious constraint imposed by this technique concerns the dimension of the molecules to be employed as analytes. We developed an organoplatinum(II) label to increase the SPR response of low-molecular-mass analytes. Hence, we demonstrated to be able to detect binding events between monosaccharides and lectins even at very low analyte concentrations. The organoplatinum(II) complex did not influence the interaction, and platinum(II) was shown to be essential for the SPR signal enhancement.