Genome-wide transcriptional changes in Streptococcus gordonii in response to competence signaling peptide.

Streptococcus gordonii is a primary colonizer of the multispecies biofilm on tooth surfaces forming dental plaque and a potential agent of endocarditis. The recent completion of the genome sequence of the naturally competent strain Challis allowed the design of a spotted oligonucleotide microarray to examine a genome-wide response of this organism to environmental stimuli such as signal peptides. Based on temporal responses to synthetic competence signaling peptide (CSP) as indicated by transformation frequencies, the S. gordonii transcriptome was analyzed at various time points after CSP exposure. Microarray analysis identified 35 candidate early genes and 127 candidate late genes that were up-regulated at 5 and 15 min, respectively; these genes were often grouped in clusters. Results supported published findings on S. gordonii competence, showing up-regulation of 12 of 16 genes that have been reported to affect transformation frequencies in this species. Comparison of CSP-induced S. gordonii transcriptomes to results published for Streptococcus pneumoniae strains identified both conserved and species-specific genes. Putative intergenic regulatory sites, such as the conserved combox sequence thought to be a binding site for competence sigma factor, were found preceding S. gordonii late responsive genes. In contrast, S. gordonii early CSP-responsive genes were not preceded by the direct repeats found in S. pneumoniae. These studies provide the first insights into a genome-wide transcriptional response of an oral commensal organism. They offer an extensive analysis of transcriptional changes that accompany competence in S. gordonii and form a basis for future intra- and interspecies comparative analyses of this ecologically important phenotype.

Selective binding of N-acetylglucosamine to the chicken hepatic lectin.

Among Ca2+-dependent (C-type) animal lectins, the chicken hepatic lectin (CHL) is unique in displaying almost complete selectivity for N-acetylglucosamine over other monosaccharide ligands. The crystal structures of the carbohydrate-recognition domain (CRD) from serum mannose-binding protein (MBP) and of a complex between the CRD from liver MBP and the methyl glycoside of N-acetylglucosamine were used to model the binding site in CHL. Substitution of portions of CHL into the MBP framework did not substantially increase selectivity. A bacterial expression system for the CRD of CHL was developed so that specific residues predicted to be near the 2-acetamido substituent of N-acetylglucosamine could be altered by site-directed mutagenesis. The results indicate that the ligand is bound to CHL in the same orientation as it binds to liver MBP. A tyrosine and a valine residue that probably contact the the N-acetyl group have been identified. These results, together with studies of ligand-binding selectivity, suggest that these residues form part of a binding pocket for the N-acetyl group, which confers selective binding of N-acetylglucosamine.

Selective sugar binding to the carbohydrate recognition domains of the rat hepatic and macrophage asialoglycoprotein receptors.

Asialoglycoprotein receptors on the surfaces of both hepatocytes and peritoneal macrophages bind terminal galactose residues of desialylated glycoproteins and mediate endocytosis and eventual degradation of these ligands. The hepatic receptor binds oligosaccharides with terminal N-acetylgalactosamine residues more tightly than ligands with terminal galactose residues, but the macrophage receptor shows no such differential binding affinity. Carbohydrate recognition domains from the macrophage receptor and the major subunit of the hepatic receptor have been expressed in a bacterial system and have been shown to retain the distinct binding selectivities of the receptors from which they derive. Binding of a series of N-acyl derivatives of galactosamine suggests that the 2-substituent of these sugars interacts with the surface of the hepatic receptor with highest affinity binding observed for the N-propionyl derivative. Chimeric sugar-binding domains have been used to identify three regions of the hepatic receptor that are essential for establishing selectivity for N-acetylgalactosamine over galactose. Based on these results and the orientation of N-acetylgalactosamine when bound to an homologous galactose-binding mutant of rat serum mannose-binding protein, a fourth region likely to interact with N-acetylgalactosamine has been identified and probed by site-directed mutagenesis. The results of these studies define a binding pocket for the 2-substituent of N-acetylgalactosamine in the hepatic asialoglycoprotein receptor.

Introduction of selectin-like binding specificity into a homologous mannose-binding protein.

The structures of the ligand-binding C-type carbohydrate-recognition domains of selectin cell adhesion molecules and of mannose-binding proteins (MBPs) are similar to each other even though these proteins bind very different carbohydrate ligands. Our current understanding of ligand binding by E-selectin is based on structural studies of unliganded E-selectin and of MBP-carbohydrate complexes, combined with results from mutagenesis of E-selectin. Five regions of E-selectin that differ in sequence from the corresponding regions of MBP have been introduced into the carbohydrate-recognition domain of MBP. Four of the changes have little effect on ligand binding. Insertion of one stretch of positively charged amino acids alters the sugar binding selectivity of the domain so that it now binds HL-60 cells and serum albumin derivatized with sialyl-Lewis X tetrasaccharide, thus mimicking the properties of E-selectin.

Binding of sugar ligands to Ca(2+)-dependent animal lectins. I. Analysis of mannose binding by site-directed mutagenesis and NMR.

The Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesis to determine the importance of individual residues in ligation of mannose and related sugars. The effects of the mutations were assessed by direct binding assays, competition binding studies, partial proteolysis, and NMR analysis of sugar-CRD titrations. As suggested by the crystal structure of the mannose-binding CRD complexed with oligosaccharide ligand, asparagine and glutamic acid residues that interact with hydroxyl groups 3 and 4 of the sugar, as well as with one of the two bound Ca2+, are critical for ligand binding. In addition, the beta-carbon of His189 contributes substantially to the binding affinity, apparently through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His189 and the 2 hydroxyl group of mannose, and between Ile207 and C-6 of mannose, observed in the crystal structure, contribute less to stability of the ligand complex. The effects of changes at positions 189 and 207 on the ability of the CRD to distinguish between alpha-and beta-methyl L-fucosides suggest that fucose may bind in an alternative orientation compared to the arrangement originally proposed based on the mannose-CRD complex.

Binding of sugar ligands to Ca(2+)-dependent animal lectins. II. Generation of high-affinity galactose binding by site-directed mutagenesis.

Changes have been introduced into the Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein by site-directed mutagenesis to model the binding sites of homologous galactose-binding CRDs. Binding assays reveal that galactose-binding activity nearly identical to that of the CRD from the asialoglycoprotein receptor can be introduced into the mannose-binding site by 3 single amino acid changes and insertion of a segment of 5 amino acids. Separate changes are required to establish high-affinity binding to galactose and create high selectivity by exclusion of mannose from the binding site. The mutagenesis studies and NMR analysis of sugar-CRD titrations demonstrate that an important component of high-affinity galactose binding is interaction between the B face of the sugar and tryptophan. The binding properties of the C-type CRD from the cartilage proteoglycan, aggrecan, can also be modeled based on the mannose-binding CRD frame-work. This lower affinity binding site involves stacking of a phenylalanine residue against the sugar ligand.