Molecular basis of the interaction between glycosaminoglycans, mimetics and proteins

Heparin (H) and heparan sulfate (HS) are polysaccharides belonging to the glycosaminoglycan (GAG) family. As a proteoglycan, HS is present either in the outer face of the cell membrane or in the extra cellular matrix (ECM). HS is involved in the modulation of the communication between cells and cell-ECM. Like polyanionic molecules, H/HS are involved in a great number of GAG-protein interactions and biological activities such as blood coagulation, lipid metabolism, cell adhesion and growth factor regulation (Ori, A., 2008; Capila, I., 2002). Recently, it has been shown an increasing in H/HS-protein complexes structural studies. This lead to an improved understanding of H/HS-protein interactions. Does H/HS interaction network represent the crossroad of the cell-ECM molecular net? The aim of the present study is oriented to clarify some H/HS network aspects, for instance the H/HS-fibroblast growth factor (FGF) family interactions, the H/HS-\u3b2amyloid (\u3b2A) and the H/HS-antithrombin III (AT) interactions. Different methods were applied to elucidate biochemical and structural data: Nuclear Magnetic Resonance (NMR), Dynamic Light Scattering (DLS) and Surface Plasmon Resonance (SPR). Literature results showed as oligosaccharides derived from H (H oligosaccharides) are able to mimic H/HS effects in H/HS-protein binding and biological activity (Linhardt, R. J., 1999; Yates, E. A., 2004; Guerrini M., 2002). According to H oligosaccharides properties, we decide to use H oligosaccharides having different sulfation pattern and derived by partial depolimerization of beef lung and pig mucosal heparin. NMR and DLS data confirm the great importance of \u3b2A folding for the chemo-physical environment as previously reported (Zagorski M. G., 1992; Mandal P. K., 2004; Valerio M., 2008). Preliminary NMR results of \u3b2As (\u3b2A (1-40), \u3b2A (1-28) and \u3b2A (25-35))/H oligosaccharides in solution suggest a critical role in \u3b2As stability. 1H-15N heteronuclear correlation spectroscopy (HSQC) spectra acquired before and after H tetrasaccharide and AGA*IA (see methods) addition show some interesting differences, showing the influence of sulfation and length of the chains in the aggregation phenomena. SPR results confirm the importance of sulfation pattern and monosaccharides sequence in FGF/H oligosaccharides interactions. Changing in sulfation pattern (6-O desulfation or 2-O desulfation) strongly affects FGF-1/H oligosaccharides-binding mechanisms

ADDRESSING THE MOLECULAR BASIS OF THE INTERACTION BETWEEN GLYCOSAMYNOGLYCAN, MIMETICS AND PROTEINS

Glycosaminoglycans (GAGs) are some of the most relevant biological molecules, involved in important biological, physiological activities and also used as pharmaceutical agents. GAG research activities oriented to understand their nature, molecular population composition, characterisation, protein-GAG interaction network and putative pharmaceutical properties are essential to develop new drug with a more controlled and described activity. Recently experimental and clinical informations had increased the attention over heparan sulfate (HS)/ heparin (Hep) coagulant and not coagulant activity opening new fields of research oriented to develop new therapeutical strategy involving heparin usage. Hep mimetics/dericvatives can be extremely helpful from this perspective, due to their controlled nature, molecular features and structure, thus they can be employed as performance drugs. My Ph.D. thesis deals with statistical characterisation of commercial and not commercial pharmaceutical heparin, composition and structural characterisation of heparin mimetics and characterisation of protein-heparin interaction for drug design strategy. Thesis focus was oriented on three different subject: 1) build a library of accepted and well charaterised heparins, set up a statistical analysis based on a principal component analysis (PCA) approach to statistically define heparin molecular nature and to develop a new heparin quality control procedure. 2) 1D/2D NMR interaction studies (1H, TOCSY, NOESY, ROESY, trNOE and STD) to understand the importance of heparin antithrombin (AT) binding domain (AT-bd, the pentasaccharide \u3b2-D-N-acetylated,6-O-sulfated glucosamine -\u3b1(1->4)-D-glucuronic acid -\u3b2(1->4)-D-N-sulfated,3,6-O-sulfated glucosamine -\u3b1(1->4)-D-2-O-sulfated iduronic acid -\u3b1(1->4)-D-N-sulfated,6-O-sulfated glucosamine, also abbreviated as AGA*IA) flanking residues over AT activation on octasaccharides bearing the pentasaccharide sequence with normal or modified reducent residue in complex with AT. 3) dynamic light scattering (DLS) studies on amyloid beta 1-40 (A\u3b21-40)-heparin or heparin mimetics/derivatives as a new strategy to counter act A\u3b2 aggregation progression involved in Alzheimer disease (AD) and better understand the deep relationship between A\u3b2 peptide and GAGs

Backbone and side-chain (1)H, (15)N, (13)C assignment and secondary structure of BPSL1445 from Burkholderia pseudomallei

BPSL1445 is a lipoprotein produced by the Gram-negative bacterium Burkholderia pseudomallei (B.pseudomallei), the etiological agent of melioidosis. Immunodetection assays against sera patients using protein microarray suggest BPSL1445 involvement in melioidosis. Herein we report backbone, side chain NMR assignment and secondary structure for the recombinant protein

Cyclic steps at the head of channelized features along the calabrian margin (Southern Tyrrhenian Sea, Italy)

High-resolution multibeam bathymetry enabled to identify coaxial trains of crescent-shaped bedforms within the heads of channelized features lying in shallow-water sectors along the tectonically-controlled Calabrian Margin. These bedforms have wavelengths of tens or few hundreds of meters and wave heights of some meters, and their crest-lines trend perpendicular to the maximum slope gradients. Repeated multibeam surveys realized in 2007, 2008, 2012 and 2013 showed a rapid and significant morphological evolution of the channel’s floors, with the generation or upslope migration of the bedforms. Based on their size, upslope migration and similarities with crescent-shaped bedforms recognized in other active canyon's heads, these features can be interpreted as cyclic steps. The bedforms are, in fact, formed or modified by frequent slope failures and related sedimentary flows, whose occurrence is favored by the concurrent presence of several predisposing and triggering mechanisms, such as high sedimentary rate due to steep coastal creek, severe storms and seismic events

J Biol Chem

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (ANAc,6S-GlcUA-ANS,3,6S-I2S-ANS,6S). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin

Structure-based design of a B cell antigen from B. pseudomallei

Burkholderia pseudomallei is the etiological agent of melioidosis, a severe endemic disease in South-East Asia, causing septicemia and organ failure with high mortality rates. Current treatments and diagnostic approaches are largely ineffective. The development of new diagnostic tools and vaccines toward effective therapeutic opportunities against B. pseudomallei is therefore an urgent priority. In the framework of a multidisciplinary project tackling melioidosis through reverse and structural vaccinology, BPSL1050 was identified as a candidate for immunodiagnostic and vaccine development based on its reactivity against the sera of melioidosis patients. We determined its NMR solution structure and dynamics, and by novel computational methods we predicted immunogenic epitopes that once synthesized were able to elicit the production of antibodies inducing the agglutination of the bacterium and recognizing both BPSL1050 and B. pseudomallei crude extracts. Overall, these results hold promise for novel chemical biology approaches in the discovery of new diagnostic and prophylactic tools against melioidosis