Determination of the primary structure and carboxyl pKAs of heparin-derived oligosaccharides by band-selective homonuclear-decoupled two-dimensional 1H NMR

Determination of the structure of heparin-derived oligosaccharides by 1H NMR is challenging because resonances for all but the anomeric protons cover less than 2 ppm. By taking advantage of increased dispersion of resonances for the anomeric H1 protons at low pD and the superior resolution of band-selective, homonuclear-decoupled (BASHD) two-dimensional 1H NMR, the primary structure of the heparin-derived octasaccharide ∆UA(2S)-[(1 → 4)-GlcNS(6S)-(1 → 4)-IdoA(2S)-]3-(1 → 4)-GlcNS(6S) has been determined, where ∆UA(2S) is 2-O-sulfated ∆4,5-unsaturated uronic acid, GlcNS(6S) is 6-O-sulfated, N-sulfated β-d-glucosamine and IdoA(2S) is 2-O-sulfated α-l-iduronic acid. The spectrum was assigned, and the sites of N- and O-sulfation and the conformation of each uronic acid residue were established, with chemical shift data obtained from BASHD-TOCSY spectra, while the sequence of the monosaccharide residues in the octasaccharide was determined from inter-residue NOEs in BASHD-NOESY spectra. Acid dissociation constants were determined for each carboxylic acid group of the octasaccharide, as well as for related tetra- and hexasaccharides, from chemical shift–pD titration curves. Chemical shift–pD titration curves were obtained for each carboxylic acid group from sub-spectra taken from BASHD-TOCSY spectra that were measured as a function of pD. The pKAs of the carboxylic acid groups of the ∆UA(2S) residues are less than those of the IdoA(2S) residues, and the pKAs of the carboxylic acid groups of the IdoA(2S) residues for a given oligosaccharide are similar in magnitude. Relative acidities of the carboxylic acid groups of each oligosaccharide were calculated from chemical shift data by a pH-independent method

Human Apolipoprotein A-I-Derived Amyloid: Its Association with Atherosclerosis

Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.3131556

Platelet endothelial cell adhesion molecule (PECAM-1) and its interactions with glycosaminoglycans 2. Biochemical analyses

Platelet endothelial cell adhesion molecule 1 (PECAM-1) (CD31), a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules with six Ig-like domains, has a range of functions, notably its contributions to leukocyte extravasation during inflammation and in maintaining vascular endothelial integrity. Although PECAM-1 is known to mediate cell adhesion by homophilic binding via domain 1, a number of PECAM-1 heterophilic ligands have been proposed. Here, the possibility that heparin and heparan sulfate (HS) are ligands for PECAM-1 was reinvestigated. The extracellular domain of PECAM-1 was expressed first as a fusion protein with the Fc region of human IgG1 fused to domain 6 and second with an N-terminal Flag tag on domain 1 (Flag-PECAM-1). Both proteins bound heparin immobilized on a biosensor chip in surface plasmon resonance (SPR) binding experiments. Binding was pH-sensitive but is easily measured at slightly acidic pH. A series of PECAM-1 domain deletions, prepared in both expression systems, were tested for heparin binding. This revealed that the main heparin-binding site required both domains 2 and 3. Flag-PECAM-1 and a Flag protein containing domains 1-3 bound HS on melanoma cell surfaces, but a Flag protein containing domains 1-2 did not. Heparin oligosaccharides inhibited Flag-PECAM-1 from binding immobilized heparin, with certain structures having greater inhibitory activity than others. Molecular modeling similarly identified the junction of domains 2 and 3 as the heparin-binding site and further revealed the importance of the iduronic acid conformation for binding. PECAM-1 does bind heparin/HS but by a site that is distinct from that required for homophilic binding