heparitin-sulfate and lipoteichoic-acid

In addition to its role in the nucleoid, the histone-like protein (HlpA) of Streptococcus pyogenes is believed to act as a fortuitous virulence factor in delayed sequelae by binding to heparan sulfate-proteoglycans in the extracellular matrix of target organs and acting as a nidus for in situ immune complex formation. To further characterize this protein, the hlpA genes were cloned from S. pyogenes, S. gordonii, S. mutans, and S. sobrinus, using PCR amplification, and sequenced. The encoded HlpA protein of S. pyogenes has 91 amino acids, a predicted molecular mass of 9,647 Da, an isoelectric point of 9.81, and 90% to 95% sequence identity with HlpA of several oral streptococci. The consensus sequence of streptococcal HlpA has 69% identity with the consensus sequence of the histone-like HB protein of Bacillus species. Oral viridans group streptococci, growing in chemically defined medium at pH 6.8, released HlpA into the milieu during stationary phase as a result of limited cell lysis. HlpA was not released by these bacteria when grown at pH 6.0 or below. S. pyogenes did not release HlpA during growth in vitro; however, analyses of sera from 155 pharyngitis patients revealed a strong correlation (P < 0.0017) between the production of antibodies to HlpA and antibodies to streptolysin O, indicating that the histone-like protein is released by group A streptococci growing in vivo. Extracellular HlpA formed soluble complexes with lipoteichoic acid in vitro and bound readily to heparan sulfate on HEp-2 cell surfaces. These results support a potential role for HlpA in the pathogenesis of streptococcus-induced tissue inflammation.

Topics: Amino Acid Sequence; Antibodies, Bacterial; Bacillus; Bacterial Proteins; Base Sequence; Cells, Cultured; Cloning, Molecular; Culture Media; DNA-Binding Proteins; DNA, Bacterial; Epithelial Cells; Gene Expression; Heparitin Sulfate; Lipopolysaccharides; Molecular Sequence Data; Plant Proteins; Polymerase Chain Reaction; Protein Binding; S Phase; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Streptococcus; Streptolysins; Teichoic Acids

The same 70-kDa protein, present on the surface of mouse lymphocytes, served as the predominant binding site for heparin, heparinoids, and bacterial lipoteichoic acids, as well as peptidoglycan and lipopolysaccharides. This conclusion was supported by the following results: (a) all of these compounds photoaffinity cross-linked to one major 70-kDa 6.5-7.0 pI protein that co-migrated on two-dimensional polyacrylamide gel electrophoresis; (b) peptide maps of the 70-kDa proteins digested with chymotrypsin, subtilisin, protease V, or papain yielded the same peptides for heparin-, lipoteichoic acid-, peptidoglycan-, and lipopolysaccharide-binding proteins; (c) cross-linking of peptidoglycan, lipopolysaccharide, lipoteichoic acid, and heparin was competitively inhibited by the same compounds with the same order of potency, i.e. carboxyl-reduced sulfated heparin > peptidoglycan > pentosan polysulfate > heparin > chitin > dextran sulfate > trestatin sulfate > polyanetholesulfonate > fucoidan > beta-cyclodextrin tetradecasulfate > heparan sulfate > carrageenan lambda > lipoteichoic acids > Re-lipopolysaccharide > lipopolysaccharide > lipid A > polygalacturonic acid; and (d) cross-linking of each of these ligands was not inhibited by carboxyl-reduced heparin, dextran, beta-cyclodextrin, trestatin, carrageenan kappa, chondroitin 4-sulfate, chondroitin 6-sulfate, beta-D-glucan, carboxy-methylcellulose, levan, alpha-D-mannan, and glycogen. The minimum size of the molecule that bound was 7-9 glycan residues, whereas, di- and trisaccharides did not bind. There was a logarithmic linear relationship between the strength of the binding and the length of the polymer (up to > 1500 glycan residues), which indicates an avidity effect of the cooperative binding of one polymeric molecule to several receptor molecules on the cell surface. The 70-kDa receptor, therefore, has a broad, but limited specificity of binding for non-charged (peptidoglycan and chitin), highly negatively charged (heparin and heparinoids), and weakly negatively charged (lipoteichoic acids, lipopolysaccharides, and lipid A) ligands.

Topics: Acute-Phase Proteins; Amino Acid Sequence; Animals; Carbohydrate Conformation; Carbohydrate Sequence; Carrier Proteins; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Heparin; Heparitin Sulfate; Lipopolysaccharides; Lymphocytes; Membrane Glycoproteins; Mice; Molecular Sequence Data; Molecular Structure; Molecular Weight; Polysaccharides; Receptors, Cell Surface; Structure-Activity Relationship; Teichoic Acids