Adriana Carvalho de Souza

Identification of the Smallest Structure Capable of Evoking Opsonophagocytic Antibodies against Streptococcus pneumoniae Type 14

ABSTRACT Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), {6)-[β-d-Galp-(1→4)-]β-d-GlcpNAc-(1→3)-β-d-Galp-(1→4)-β-d-Glcp-(1→}n, were conjugated to CRM197 protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria. Earlier studies have reported that the oligosaccharide corresponding to one structural repeating unit of Pn14PS, i.e., Gal-Glc-(Gal-)GlcNAc, induces a specific antibody response to Pn14PS. The broader study described here, which evaluated 16 oligosaccharides, showed that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the nonreducing end contributes clearly to the immunogenicity of the epitope. Only the oligosaccharide conjugates that produce antibodies recognizing Pn14PS were capable of promoting the phagocytosis of S. pneumoniae type 14. In conclusion, the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc may be a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14.

Gold Glyconanoparticles as Probes to Explore the Carbohydrate- Mediated Self-Recognition of Marine Sponge Cells

Cell aggregation in the red-beard marine sponge Microciona prolifera is mediated by a 2 10 kDa proteoglycan-like macromolecular aggregation factor (MAF), and is based on two highly polyvalent functional properties; a Ca-dependent proteoglycan self-interaction and a Ca-independent cell-binding activity. MAF, the first circular proteoglycan described, is composed of two N-glycosylated proteins, MAFp3 and MAFp4, with twenty units of each glycoprotein forming the central ring and the radiating arms, respectively. Each MAFp3 carries one or two copies of a 200 kDa acidic glycan, g-200, whereas each MAFp4 carries about 50 copies of a 6 kDa glycan, g-6. The MAFp4 arms of the sunburst-like proteoglycan are linked to cell-surface binding receptors, while the MAFp3 ring exposes the g-200 glycans so that they can engage in the Ca-dependent self-association (for a detailed review, see ref. [4]). By making use of MAF-specific monoclonal antibodies, it could be demonstrated that the self-association of MAF occurs through highly repetitive epitopes on the g-200 glycan. One of these epitopes was shown to be the sulfated disaccharide GlcpNAc3S(b1–3)Fucp. To gain insight into the role of carbohydrate interactions in MAF self-aggregation, we designed a challenging system for mimicking the g-200 self-association. By using the synthetic sulfated disaccharide, multivalently presented as a bovine serum albumin conjugate, and surface plasmon resonance spectroscopy, it was shown that Ca-dependent carbohydrate self-recognition is a major force in the g-200 association phenomenon. Gold glyconanoparticles have been successfully used as inert multivalent systems to explore either carbohydrate self-interactions or carbohydrate binding to proteins. In the present study, water-soluble gold glyconanoparticles coated with synthetic carbohydrates related to the sulfated disaccharide fragment (Scheme 1) were used as multivalent systems to investigate the g-200 glycan–glycan interaction by transmission electron microscopy (TEM). Very recently, an NMR study of intact MAF glycans suggested the presence of a-Fuc residues. However, earlier structural analysis of oligosaccharide fragments obtained from a partial acid hydrolysate of the g-200 glycan could not identify the anomeric configuration of the fucose residue in these fragments. 16] Therefore, gold glyconanoparticles coated with the aor the b-anomer (Au-1a and Au-1b) of the native sulfated disaccharide epitope were used in the aggregation experiments. The importance of each of the two monosaccharide units for the self-recognition process of the disaccharide epitope was determined by studying the gold glyconanoparticles Au-2 and Au-3. The three gold glyconanoparticle systems Au-4 (a-l-Fucp replaced by a-l-Galp), Au-5 (bd-GlcpNAc3S replaced by b-d-GlcpNAc), and Au-6 (b-dGlcpNAc3S replaced by b-d-Glcp3S) were used to evaluate the relevance of the modified sites in the self-recognition process. Scheme 1. Gold glyconanoparticles Au-1a/b to Au-6, related to the MAF sulfated disaccharide epitope.

SPR Studies of Carbohydrate–Protein Interactions: Signal Enhancement of Low-Molecular-Mass Analytes by Organoplatinum(II)-Labeling

The relatively insensitive surface plasmon resonance (SPR) signal detection of low‐molecular‐mass analytes that bind with weak affinity to a protein—for example, carbohydrate–lectin binding—is hampering the use of biosensors in interaction studies. In this investigation, low‐molecular‐mass carbohydrates have been labeled with an organoplatinum(II) complex of the type [PtCl(NCNR)]. The attachment of this complex increased the SPR response tremendously and allowed the detection of binding events between monosaccharides and lectins at very low analyte concentrations. The platinum atom inside the organoplatinum(II) complex was shown to be essential for the SPR‐signal enhancement. The organoplatinum(II) complex did not influence the specificity of the biological interaction, but both the signal enhancement and the different binding character of labeled compounds when compared with unlabeled ones makes the method unsuitable for the direct calculation of biologically relevant kinetic parameters. However, the labeling procedure is expected to be of high relevance for qualitative binding studies and relative affinity ranking of small molecules (not restricted only to carbohydrates) to receptors, a process of immense interest in pharmaceutical research.

Analysis of Carbohydrate–Carbohydrate Interactions Using Gold Glyconanoparticles and Oligosaccharide Self‐Assembling Monolayers

Carbohydrates are the most extended structures exposed at the surface of most cells. These carbohydrate chains, when arranged in polyvalent clusters, offer a rich supply of low-affinity binding sites, making them a reliable and flexible system to regulate cell adhesion and recognition. The very first model system for cell-cell recognition by means of carbohydrate-carbohydrate interactions in the animal kingdom came from a primitive invertebrate animal: the marine sponge. During the past 50 years, studies have shown that highly repetitive carbohydrate motives on extracellular proteoglycan supramolecular complexes of marine sponge cells are involved in the species-specific adhesion. In this chapter, some glyconanotechnology procedures are described for the detailed investigation of the role of a carbohydrate epitope in the marine sponge cell recognition. The various protocols are generally applicable in other areas of glycoscience.

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

Weak 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.

The application of neoglycopeptides in the development of sensitive surface plasmon resonance-based biosensors

The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide libraries with Ricinus communis agglutinin (RCA(120)) and human adhesion/growth-regulatory galectin-1 (h-Gal-1). Glycopeptides were immobilized on Au sensor chips functionalized with mixed self-assembled monolayers of different ratios of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid, and of 3-mercapto-1-propanol and 11-mercaptoundecanoic acid. The biosensors were optimized for the detection of RCA(120), and a detection limit of 0.13 nM was obtained. Subsequent experiments with h-Gal-1 indicated a detection limit of at least 0.9 nM for this lectin. Additionally, the effect of interfering proteins on the sensitivity of the optimized biosensor was investigated.

Synthesis and conjugation of oligosaccharide analogues of fragments of the immunoreactive glycan part of the circulating anodic antigen of the parasite Schistosoma mansoni

The gut-associated circulating anodic antigen (CAA) is one of the major excretory antigens produced by the parasite Schistosoma mansoni. The immunoreactive part of CAA is a threonine-linked polysaccharide composed of long stretches of the unique repeating disaccharide-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1-->. Previously, using surface plasmon resonance and ELISA techniques, it has been shown that some anti-CAA IgM monoclonal antibodies (MAbs) also recognize members of a series of bovine serum albumin (BSA)-coupled synthetic di- to penta-saccharide fragments of the CAA glycan. To generate information on the molecular level about the glycan specificity of the relevant IgM MAbs, two series of oligosaccharides related to the CAA disaccharide epitope were synthesized, and coupled to BSA. The first three analogues, beta-D-GlcpA-(1-->3)-[small beta]-D-GlcpNAc-(1-->O), beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O), and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O), wherein the native beta-D-GalpNAc moiety was replaced by beta-D-GlcpNAc, were synthesized to investigate the specificity of the selected MAbs to the carbohydrate backbone of CAA. The second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), wherein the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S, was synthesized to evaluate the importance of the type/nature of the charge of CAA for the MAb recognition.

Studying Carbohydrate Self-Recognition in Marine Sponges Using Synthetic Aggregation Factor Epitopes

Sponges (Porifera) are the simplest and earliest multicellular organisms. They do not produce complex patterned structures (i.e., organ systems). Epithelial cells (pinacocytes) line the outer surface and the internal system of openings, channels, and chambers, through which water is continuously pumped by flagellated cells called choanocytes. The generated water current supplies food particles and oxygen and removes metabolic waste products. The major part of the sponge biomass consists of a gelatinous extracellular matrix (ECM) containing a large number of highly motile cells. This part of the sponge body is called the mesohyl. The mesohyl also contains the skeletal elements of the sponge body: spicules (needle-like structures made of either silicon or calcium carbonate) and spongin (collagenous fibers).