Sponges (Porifera), the simplest and earliest multicellular organisms, are thought to have evolved from their unicellular ancestors about 1 billion years ago by developing cell-recognition and adhesion mechanisms to discriminate against ''non-self.'' Consequently, they are used as models for investigating recognition phenomena. Cellular adhesion of marine sponges is an event involving adherence of extracellular proteoglycan-like molecules, otherwise known as aggregation factors (AFs). In a calciumindependent process the AFs adhere to the cell surface, and in a calcium-dependent process they exhibit AF self-association. A mechanism which has been implied but not definitely proven to play a role in the calcium-dependent event is self-recognition of defined carbohydrate epitopes. For the red beard sponge, Microciona prolifera, two carbohydrate epitopes, a sulfated disaccharide and a pyruvylated trisaccharide, have been implicated in cellular adhesion. To investigate this phenomenon a system has been designed, by using surface plasmon resonance detection, to mimic the role of carbohydrates in cellular adhesion of M. prolifera. The results show self-recognition of the sulfated disaccharide to be a major force behind the calcium-dependent event. The interaction is not simply based on electrostatic interactions, as other sulfated carbohydrates analyzed by using this procedure did not selfassociate. Furthermore, the interaction is completely eradicated on substitution of Ca 2؉ ions by either Mg 2؉ or Mn 2؉ ions. This physiologically relevant recognition mechanism confirms the existence of true carbohydrate self-recognition, and may have significant implications for the role of carbohydrates in cellular recognition of higher organisms. W eak polyvalent interactions play an important role in biological processes. There is growing evidence that carbohydrates, found on the surfaces of all living cells, are functional constituents in cell-cell interactions. At present, only a few examples of low-affinity carbohydrate-carbohydrate interactions are known (1-5). For example, pioneering work by Hakomori and his colleagues (1-3) has shown glycosphingolipid self-interaction to occur by way of multivalent interaction of Lewis X epitopes.Since 1900, marine sponges have been used as primitive models for studying the phenomenon of cell recognition. Knowledge of the recognition mechanisms of these simple organisms, hypothetically situated at the foot of the metazoan kingdom, may contribute to the understanding of cell-cell adhesion events within higher organisms. Adhesion of marine sponges is an event that involves both calcium-independent adherence of proteoglycan-like molecules, named aggregation factors (AFs), to the cell surface, and calcium-dependent AF self-association (6-8). The calcium-dependent event is species-specific, as illustrated by the rapid self-association and sorting, on the addition of calcium ions, of a mixture of colored (pink, yellow, and white) proteoglycan-coated beads, each color corresponding to a different s...

An accurate, rapid, and sensitive method for characterizing the carbohydrate binding properties of lectins using a BIAcore apparatus and the detection method of surface plasmon resonance is described. As a model study, the sialic acid binding lectins from Sambucus nigra and Maackia amurensis, which are specific for the epitopes Neu5Ac(␣2-6)Gal and Neu5Ac(␣2-3)Gal, respectively, were chosen as suitable candidates. Two systems, one for the analysis of oligosaccharides and the other for glycoproteins, were developed after a rigorous analysis and evaluation of such parameters as binding conditions, buffers, and regeneration conditions. The systems take into account nonspecific binding, using the respective denatured lectin as negative blank, and avoid loss of activity: regeneration of the surface using either 10 mM NaOAc (pH 4.3) buffer (oligosaccharide system) or 20 mM HCl (glycoprotein system). The specificity of the lectins is well illustrated, while the kinetics parameters are shown to be sensitive to subtle changes in the recognized epitopes, and to be affected by steric hindrance. Surface plasmon resonance is a suitable technique for the analysis and characterization of lectins.

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed b-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.

Long-chain polysialic acid (PSA) is expressed on the vertebrate neural cell adhesion molecule (NCAM) during neuronal plasticity. Its structural similarity to the capsular PSAs of some pathogenic bacteria has hampered the development of polysaccharide vaccines against meningitis. The antibodies formed during immunization require a long epitope for binding, and cross-react with host tissue PSA. The nature of the epitope and possible external effectors involved are still unclear. We have evaluated the interaction of PSA with its antibody mAb735 by surface plasmon resonance. The influences of PSA chain length, pH, temperature, ionic environment, and polyamines were also determined.The antibody binding affinity was found to dramatically increase with PSA chain length. A sub-nanomolar dissociation constant (K D = 8.5 × 10 −10 M) was obtained for the binding of very long chain native MenB polysaccharides (∼200 Neu5Ac-residues). Colominic acid from Escherichia coli K1 (∼100 residues) and shorter polymers exhibited progressively weaker affinities. The antibody also bound tightly (K D ∼ 5 × 10 −9 M) to polysialylated glycopeptides from human embryonal brain. The effects of pH and ionic strength suggested that the interaction is largely electrostatic. Ca 2+ and Mn 2+ ions promoted the observed surface plasmon resonance response in a concentration dependent fashion. Spermine increased the response in a similar way. Our results suggest that divalent cations and polyamines may play significant role in the regulation of the PSA epitope presentation in vivo.

A polysaccharide containing 2-acetamido-2-deoxy-~-galacturonic acid (GalNAcA), 2,4-diacetamido-2,4,6-trideoxy-~-glucose (QuiNAc4NAc), and D-ahnine (Ala) was isolated from the water-soluble lipopolysaccharide (LPS) originating from the reference strain for Acinetobacter huemolyticus (DNA group 4) strain ATCC 17906. The polysaccharide, characterised by means of monosaccharide analyses and NMR studies, was shown to be based on a linear trisaccharide repeating unit, as shown below, with the alanine group amide-bound to position 6 of one GalNAcA residue. It was specifically recognised in western blots by polyclonal rabbit antisera. D-Ala