ABSTRACT
We utilized a 9-mer random phage display library to identify sequences which bind to laminin-1 and elute with heparan sulfate or peptide 11 (CDPGYIGSR). Laminin-1 derivatized plates were used for biopanning. Three consecutive rounds of low pH elutions were carried out, followed by three rounds of specific elutions, each consisting of a heparan sulfate elution followed by a peptide 11 elution. The random sequence inserts were sequenced for phage populations eluted at low pH, by heparan sulfate and by peptide 11. Specifically eluted phage populations exhibited three classes of mimotopes for different regions in the cDNA derived amino acid sequence of the 67 kDa laminin binding protein (LBP). These regions were (1) a palindromic sequence known as peptide G, (2) a predicted helical domain corresponding to LBP residues 205-229, and (3) TEDWS-containing C-terminal repeats. All elution conditions also yielded phage with putative heparin binding sequences. We modeled the LBP(205-229) domain, which is strongly predicted to have a helical secondary structure, and determined that this region likely possesses heparin-binding characteristics located to one side of the helix, while the opposite side appears to contain a hydrophobic patch where peptide 11 could bind. Using ELISA plate assays, we demonstrated that peptide 11 and heparan sulfate individually bound to synthetic LBP(205-229) peptide. We also demonstrated that the QPATEDWSA peptide could inhibit tumor cell adhesion to laminin-1. These data support the proposal that the 67 kDa LBP can bind the beta-1 laminin chain at the peptide 11 region, and suggest that heparan sulfate is a likely alternate ligand for the binding interactions. Our results also confirm previous data suggesting that the most C-terminal region of the LBP, which contains the TEDWS repeats, is involved in cell adhesion to laminin-1, and we specifically implicate the repeat sequence in that activity.
Subject(s)
Laminin/chemistry , Laminin/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Library , Protein Precursors , Receptors, Laminin/chemistry , Amino Acid Sequence , Animals , Binding, Competitive , Cell Adhesion/drug effects , Cell Line , Cricetinae , Cross-Linking Reagents , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , Epitopes/pharmacology , Heparin/metabolism , Heparitin Sulfate/metabolism , Mice , Models, Molecular , Molecular Mimicry , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Protein Binding , Protein Structure, Secondary , Receptors, Laminin/immunology , Receptors, Laminin/metabolism , Repetitive Sequences, Amino AcidABSTRACT
The synthesis of a series of novel analogues of lipid A, the active principle of lipopolysaccharide, is reported. In these compounds, the 1-O-phosphono and (R)-3-hydroxytetradecanoyl moieties of native Salmonella minnesota R595 lipid A have been replaced with hydrogen and the length of the normal fatty acyl residues has been systematically varied. Normal fatty acid chain length in the 3-O-desacyl monophosphoryl lipid A (MLA) series is shown to be a critical determinant of iNOS gene expression in activated mouse macrophages and the induction of proinflammatory cytokines in human peripheral monocytes. Examination of pyrogenicity in rabbits and lethal toxicity in D-galactosamine-treated mice shows that toxic effects in the MLA series can be ameliorated by modifying fatty acid chain length. When used as an adjuvant for tetanus toxoid vaccines, certain MLA derivatives enhance the production of tetanus toxoid-specific antibodies in mice.