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Laminin-1, a major component of basement membranes, consists of three different chains designated ␣1, ␤1, and ␥1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin ␥1 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 ␥1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-␣ 2 ␤ 1 integrin 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 ␥2 chain. These results suggest that the specific sequences on the laminin ␥1 chain are biologically active and interact with distinct cell surface receptors.Laminin-1, a major component of the basement membrane matrix, has multiple biological activities including promotion of cell adhesion, spreading, growth, neurite outgrowth, tumor metastasis, and collagenase IV secretion (1-4). There are at least 11 isoforms of laminin, each consisting of three different chains (5). The most extensively characterized laminin, laminin-1 (M r ϭ 900,000) from the mouse Engelbreth-Holm-Swarm tumor consists of ␣1, ␤1, and ␥1 chains, which assemble into a triple-stranded cross-like structure with a coiled-coil domain at the long arm (6).Several active sequences on laminin-1 have been identified using proteolytic fragments, recombinant proteins and synthetic peptides (7,8). The YIGSR sequence located on the ␤1 chain promotes cell adhesion and migration, and inhibits angiogenesis and tumor metastasis (9 -11). The PDSGR and F-9 (RYVVLPR) sequences located on the ␤1 chain also promote cell adhesion (12, 13). An IKVAV sequence located on the C-terminal end of the long arm of the ␣1 chain promotes cell adhesion, neurite outgrowth, experimental metastasis, collagenase IV activity, angiogenesis, plasminogen activator activation, cell growth, and tumor growth (14 -18). Recently, we identified five cell binding sequences in the mouse laminin ␣1 chain C-terminal globular domain, G domain (positions 2111-3060), by systematic peptide screening using overlapping peptide-polystyrene beads and free peptides.Although the laminin ␥1 chain is present in most laminin isoform...

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A protocol has been developed for the synthesis of peptides containing 0-phosphorylated tyrosines, serines, and/or threonines, The procedure involves incorporation of dimethyl-protected 0-phosphorylated amino acid derivatives (1-3) into peptides using standard Boc chemistry and subsequent removal of Me groups using a two-step deprotection method consisting of high-acidic and low-acidic treatments. Optimized deprotection conditions for the protected resins (4-6) were established, which consist of a combination of the first-step reagent (1 M TMSOW-thioanisole in TFA (loo), m-cresol (5), EDT (5), (v/v)) and the second-step reagent (first-step reagent (110) + DMS-TMSOTf(30:20 to 40:10), (v/v)). The two-step deprotection protocol can be conducted in one pot by appropriate modification of the first-step reagent. The second deprotection step proceeds by an S N~ mechanism with little tendency to induce side reactions resulting from harsh acid treatment. A 19-residue MAP-kinase peptide 10 possessing not only two phosphoamino acids but also Met and Trp was subjected to this synthetic procedure and was obtained in 24% yield based on the protected resin. The present synthetic method afforded phosphoamino acid-containing peptides in high yield without significant accompanying side reactions (e.g., loss of phosphate groups, migration of phosphate groups, or alkylation of Met and Trp residues).

A practical synthesis of phosphopeptides was achieved using a combination of dimethyl-protected phosphoamino acids and two-step deprotection protocols consisting of high acidic (SNI/SN2) and low acidic ( S N ~) reagent systems.

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A new practical strategy has been developed for the synthesis of long-chain phosphopeptide. Both the 2-chlorobenzyloxycarbonyl (CIZ) group for Lys and methyl (Me) for phosphoamino acids remained intact, while other commonly used side-chain protecting groups were cleaved quantitatively, during the reaction using a highly acidic trifluoromethanesulfonic acid (TFMSA)-based reagent system (High TFMSA: TFMSA-TFA-m-cresol=1:9:1, v/v). Selective deprotection of the CIZ and Me group-containing protected phosphopeptide resin with the High TFMSA gave a partially protected phosphopeptide fragment suitable for thioester-mediated fragment condensation. A deprotection protocol of the 9-fluorenylmethyloxycarbonyl (Fmoc) group, which evades significant side reaction toward the protected phosphoamino acid, was also developed. These two new findings enabled us to synthesize long-chain phosphopeptide via thioester-mediated fragment condensation.