Search

Type 4 filaments (T4F)--of which type 4 pili (T4P) are the archetype--are a superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of one major pilin, which also contain minor pilins whose roles are often poorly understood. Here, we complete the structure/function analysis of the full set of T4P pilins in the opportunistic bacterial pathogen Streptococcus sanguinis. We determined the structure of the minor pilin PilA, which is unexpectedly similar to one of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F. We found that PilA interacts and dramatically stabilizes the minor pilin PilC. We determined the structure of PilC, showing that it is a modular pilin with a lectin module binding a subset of glycans prevalent in the human glycome, the host of S. sanguinis. Altogether, our findings support a model whereby the minor pilins in S. sanguinis T4P form a tip-located complex promoting adhesion to various host receptors. This has general implications for T4F. [ABSTRACT FROM AUTHOR]

The β1,2-glucans produced by bacteria are important in invasion, survival and immunomodulation in infected hosts be they mammals or plants. However, there has been a lack of information on proteins which recognize these molecules. This is partly due to the extremely limited availability of the sequence-defined oligosaccharides and derived probes for use in the study of their interactions. Here we have used the cyclic β1,2-glucan (CβG) of the bacterial pathogen Brucella abortus, after removal of succinyl side chains, to prepare linearized oligosaccharides which were used to generate microarrays. We describe optimized conditions for partial depolymerization of the cyclic glucan by acid hydrolysis and conversion of the β1,2-gluco-oligosaccharides, with degrees of polymerization 2-13, to neoglycolipids for the purpose of generating microarrays. By microarray analyses, we show that the C-type lectin receptor DC-SIGNR, like the closely related DC-SIGN we investigated earlier, binds to the β1,2-gluco-oligosaccharides, as does the soluble immune effector serum mannosebinding protein. Exploratory studies with DC-SIGN are suggestive of the recognition also of the intact CβG by this receptor. These findings open the way to unravelling mechanisms of immunomodulation mediated by β1,2-glucans in mammalian systems. [ABSTRACT FROM AUTHOR]

The oligosaccharide moieties of glycoproteins, glycolipids, proteoglycans and polysaccharides are highly diverse, the reason for this diversity is not yet understood. Neoglycolipid technology allows the generation of oligosaccharide probes with lipid tags from desired sources and is showing promise as a basis for oligosaccharide microarrays. Such microarrays would allow surveys of glycomes and proteomes to be carried out, which would enable the molecular definition of carbohydrate-recognition systems in whole organisms. [ABSTRACT FROM AUTHOR]

The number of receptors of the ‘C‐type’ lectin family is greater than previously thought with a considerable proportion on cells (dendritic cells and macrophages) critical for innate immunity. Establishing that they bind carbohydrates, unravelling and comparing details of their ligands is crucial for understanding the molecular basis of the cell–cell and cell–pathogen interactions that they mediate. Here we use carbohydrate arrays as a new approach to discovering the ligands of three recently described C‐type lectin‐type receptors on antigen‐presenting cells: murine SIGN‐R1, SIGN‐R3 and langerin. The arrays encompass an extensive panel including polysaccharides, glycoproteins, oligosaccharides and monosaccharides. These are probed with soluble forms of the receptors (IgG–Fc chimeras). The dominant specificities found for SIGN‐R1 and SIGN‐R3 are mannose‐ and fucose‐related, as expressed on high mannose type N‐glycans and Lewisa/b/Lewisx/y‐type sequences, respectively, with subtle differences between the receptors. The dominant specificity for langerin is unique so far: a Lewisx‐related sequence with sulfate at position 6 of the terminal galactose. The polysaccharide dextran, known from classical studies to elicit a T‐independent response, and whose cellular uptake has been shown recently to be mediated by membrane‐associated SIGN‐R1, gave no binding signals with the soluble form of the protein. We highlight here the additional need for cell‐based assays for detecting biologically relevant low affinity ligands, for we show with SIGN‐R1‐transfected cells that dextran is such a low affinity ligand for SIGN‐R1 that binding is detectable only with the cell membrane‐associated receptor. But there is a close relationship between dextran recognition and mannose/fucose recognition, with dextran‐ and mannose‐conjugates co‐localizing in intracellular compartments. [ABSTRACT FROM AUTHOR]

Structures of the N-linked oligosaccharides of a recombinant soluble form of human CD4 glycoprotein (sCD4) have been investigated by enzymic microsequencing. The glycoprotein has two N-glycosylation sites, Asn271 and Ash300, at both of which evidence for the presence of complex type biantennary sialo-oligosaccharides has been obtained previously by mass spectrometric analyses [Carr, S.A., Hemling, M.E., Folena-Wasserman, G., Sweet, R. W., Anumula, K., Barr, J. R., Huddleston, M.J. & Taylor, P. (1989) J. Biol. Chem. 264, 21286–21295]. Among oligosaccharides released from sCD4 by hydrazinolysis and labelled with NaB3H4, neutral (12.8%) and acidic (87.2%) oligosaccharides were detected by paper electrophoresis. The latter were rendered neutral following sialidase treatment indicating that acidity was due exclusively to the presence of sialic acid residues. By enzymic microsequencing of the sialidase-treated oligosaccharides (fractionated on affinity columns of Ricinis communis agglutinin 120 and concanavalin A) in conjunction with methylation data from the earlier study, 14 sequences were identified. These accounted for over 80% of the sialidase-treated oligosaccharides of sCD4 as follows: [This symbol cannot be presented in ASCII format] where ± indicates residues present on only a proportion of chains. The spectrum of oligosaccharide structures released from each glycosylation site was assessed as being similar to that of total oligosaccharides on the basis of their chromatographic profiles on the lectin columns and on Bio-Gel P-4. [ABSTRACT FROM AUTHOR]

The reactions of two monoclonal IgM lambda cold agglutinins, Sch and Sher, have been studied in detail with human and animal erythrocyte antigens. Although they were unusual in having lambda and not kappa polypeptide chains, they could be assigned to the anti I and anti-Pr1 groups of cold agglutinins. The findings with serum Sher indicated that the Pr1 antigen may be more complex than previously thought. The occurrence of unexpectedly large numbers of specificities among monoclonal anti-I, anti-i and anti-Pr antibodies is discussed and it is suggested that each monoclonal antibody may recognize only a limited portion of a complex red cell antigen. [ABSTRACT FROM AUTHOR]

Desialylated human blood group O erythrocyte glycopeptides were digested with the endo-β-galactosidase of Bacteroides fragilis and the enzyme-released products reduced with NaBH4 and purified by Bio-Gel P-4 Chromatography. Three linear and six branched oligosaccharidcs of poly(N- acetyllactosamine) type, which together accounted for 90% of the oligosaecharide alditols, were characterised by fast-atom-bombardment mass spectrometry and gas-liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence: GlcNAcβ1 6 Galβ1-4GlcNAcβ1-3Gal.ol 3- GlcNAcβ1 and (b) there was no evidence for the terminal branch-point sequence: - GlcNAcβ1 6 Gal.ol 3- GlcNAcβ1 Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Haut'land, P., Uemura, K. & Feizi, T. (1984) J. Biol. Chem. 259, 6586-6592], the endo-β-galactosidase of Bacteroides fragilis cannot hydrolyse branch-point β-galactosidic linkages on erythrocyte membrane glycopeptides. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "Engineering Sweet Targets for Magic Bullets," that was previously published in the 2004 issue of the journal "Nature Biotechnology."