A novel synthetic peptide from the B1 chain of laminin with heparin-binding and cell adhesion-promoting activities.

Abstract

Abstract. Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]hepafin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin

“…It has previously been shown that not only do integrins, such as «,0,, a2ß,, a3ß,, a6ß,, «,0,, and aßß3 bind laminin, but also that other nonintegrin molecules bind laminin as well. There are reports suggesting that cell surface heparin-like molecules or heparan sulfate proteoglycans can interact with specific regions on laminin (Charonis et al ., 1988 ;Skubitz et al ., 1991) . Other cell surface molecules also have been reported to bind laminin including a 67-kD protein (Terranova et al, 1983) and 01,4 galactosyltransferase (Begovac et al, 1991) .…”

“…The IgG was incubated in the wells for 1 h at 37°C. 100 Al of radiolabeled cells at lWlml were added to each well and incubated for 30 min at 37°C, at which time the wells were washed and adherent cells were solubilized and quantitated in a scintillation counter as described previously (Charonis et al ., 1988) .…”

“…The criteria for peptide selection was based on the net positive charge and hydrophilicity of specific regions of murine EHS-laminin such that the laminin-derived peptides would have an increased potential for cell surface interactions . Peptide sequences were derived from the published sequences of the EHS-laminin A and BI chains (Sasaki et al ., 1988) and synthesized by the Merrifield solid-phase method as previously described (Charonis et al ., 1988) . Peptides were purified by HPLC and verified by amino acid analysis using an amino acid analyzer (Beckman Instrs ., Inc., Fullerton, CA) and sequence determination done by sequential Edman degradation on a gas phase sequenator.…”

“…Recently, several synthetic peptides derived from the amino acid sequence of the murine laminin A, Bl, and B2 chains have been described that mimic certain functional activities of intact laminin (Graf et al, 1987;Charonis et al, 1988;Tashiro et al, 1989;Skubitz et al, 1990Skubitz et al, , 1991 . Interestingly, only a peptide containing an RGD sequence from the A chain near the cross region has been shown to interact with an integrin receptor, a.ß3 (Tashiro et al, 1991;Kramer et al, 1990) .…”

Active peptides from the carboxyl‐terminal globular domain of laminin α2 and Drosophila α chains

“…[27], PDSGR from Kleinman et al [28], IKVAV from Tashiro et al [29], F-9 (RYVVLPR) from Charonis et al [30] and Skubitz et al [31], RGD from Tashiro et al [32], AG-10, AG-22, AG-32, AG-56, AG-73 from Nomizu et al [14]. E8 and E3 designate previously described proteolytic fragments active in cell adhesion [33].…”