Phage display mapping for peptide 11 sensitive sequences binding to laminin-1.
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.
Kazmin
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DA
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
Hoyt
T R
TR
Taubner
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L
Teintze
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M
Starkey
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D007797:Laminin / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D018716:Molecular Mimicry; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D019151:Peptide Library; D011485:Protein Binding; D011498:Protein Precursors; D017433:Protein Structure, Secondary; D017474:Receptors, Laminin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
D003432:Cross-Linking Reagents; D000939:Epitopes; D006493:Heparin; D006497:Heparan Sulfate; D007797:Laminin; D010446:Peptide Fragments; D019151:Peptide Library; D011498:Protein Precursors; D017474:Receptors, Laminin; C121346:laminin 1