Akira Otaka

Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening

Laminin-1, a major component of basement membranes, consists of three different chains designated α1, β1, and γ1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin γ1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the γ1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-α2β1integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin γ2 chain. These results suggest that the specific sequences on the laminin γ1 chain are biologically active and interact with distinct cell surface receptors.

Synthesis of 4-phosphono(difluoromethyl)-D,L-phenylalanine and N-boc and N-Fmoc derivatives suitably protected for solid-phase synthesis of nonhydrolyzable phosphotyrosyl peptide analogues

Abstract Synthesis of 4-phosphono(difluoromethyl)-D,L-phenylalanine as well as its diethyl phosphonate analogues bearing either Boc or Fmoc-amino protection are reported. The latter two derivatives were utilized for the solid-phase synthesis of SH2-related peptides containing nonhydrolyzable phosphotyrosyl mimetics.

Deprotection and cleavage methods for protected peptide resins containing 4-[(diethylphosphono)difluoromethyl]-D,L-phenylalanine residues

Abstract Ethyl groups on 4-[(diethylphosphono)difluoromethyl]-D,L-phenylalanine are efficiently removed using S N 2-type acidic reagents. These methods were utilized for the solid-phase synthesis of SH2-binding peptides containing nonhydrolyzable phosphotyrosyl mimetics.

Synthesis of phosphonomethyl-phenylalanine and phosphotyrosine containing cyclic peptides as inhibitors of protein tyrosine kinase/SH2 interactions.

Abstract Linear and cyclic hexameric peptides were synthesized with the amino acid sequence Gly-Tyr-Val-Pro-Met-Leu, which corresponds to the autophosphorylation segment around Y751 of PDGF receptor β subunit. The natural L -tyrosine (position 2) was also substituted in peptide analogs with D -Tyr, L -tyrosine phosphate, and L - and D -4-phosphonomethyl-phenylalanine (Pmp). Fmoc chemistry-based SPPS methodology, and Rink resin support were used with diphenylphosphoryl azide as the cyclizing agent. The linear and cyclic peptides were characterized by circular dichroism spectroscopy. The peptides described were designed as inhibitors of receptor tyrosine kinase / src homology region 2 interactions that mediate mitogenic signal transduction pathways.

Norleucine as a replacement for methionine in phosphatase-resistant linear and cyclic peptides which bind to p85 SH2 domains

Abstract A Met residue in the pTyr+3 position has previously been shown to be an important determinant for high affinity binding of peptides to PI 3-kinase p85 SH2 domains. In the present work, a series of linear and cyclic peptides based on the sequence “Gly-pTyr-Val-Pro-Met-Leu” as well as analogues having pTyr replaced by the phosphatase-resistant pTyr mimetics, phosphonomethyl phenylalanine (Pmp) or difluorophosphonomethyl phenylalanine (F 2 Pmp), were synthesized and their binding potency in p85 SH2 domain preparations compared with corresponding peptides in which the Met has been substituted by Nle. Nle is a chemically more stable, isosteric Met homologue in which the sulfur has been replaced by a methylene. Significant binding potency was retained by the Nle-containing peptides, indicating that Met is not absolutely essential for high affinity binding to this SH2 domain.

Glycosylation of the active sequence ser-ile-lys-val-ala-val from the α1 chain of laminin reduces tumor cell attachment activity

Abstract N-terminal serine O-glycopeptides of SIKVAV-, a sequence located on the long arm of the α1 chain of laminin, were synthesized to study the effect of glycosylation on the adhesive properties of this biologically relevant peptide. The data show that covalently linked carbohydrates decrease the cell-attachment activity in a structurally dependent manner.