title: Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy.
abstract: The laminin alpha5 chain is a component of laminin-10 (alpha5beta1gamma1) and -11 (alpha5beta2gamma1). In this study, we have screened 113 overlapping synthetic peptides from the laminin alpha5 globular domain (G-domain) for cell attachment activity with B16-F10 cells using peptide-coated dishes. Eleven attachment-active peptides were identified. In vivo experimental B16-F10 pulmonary metastasis and primary tumor growth assays found that 4 of the 11 peptides inhibited tumor metastasis and growth and increased apoptosis. These four peptides also blocked tumor cell migration, invasion, and angiogenesis. Two of the peptides were highly homologous and showed significant similarity to sequences in collagens. We sought to identify the B16-F10 cell surface receptors for each of the four active peptides using peptide affinity chromatography. Only one peptide recognized a cell surface protein. Peptide A5G27 (RLVSYNGIIFFLK, residues 2892-2904) bound a diffuse M(r) approximately 120,000-180,000 band that eluted with 2 m NaCl. Glycosidase digestion of the 2 m eluate yielded protein bands of M(r) 90,000 and 60,000 that reacted in Western blot analysis with antibodies to CD44. Immunoprecipitation of the A5G27-bound membrane proteins with various cell surface proteoglycan antibodies confirmed CD44 as the surface receptor for A5G27. Finally, attachment assays to A5G27 in the presence of soluble glycosaminoglycans (GAGs) identified the GAGs of CD44 as the binding sites for A5G27. Our results suggest that A5G27 binds to the CD44 receptor of B16-F10 melanoma cells via the GAGs on CD44 and, thus, inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner.
authors.affiliation: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research/NIH, 30 Convent Drive, Bethesda, MD 20892, USA.
authors.forename: Suguru
authors.initials: S
authors.lastname: Hibino
authors.forename: Masahiko
authors.initials: M
authors.lastname: Shibuya
authors.forename: Jean A
authors.initials: JA
authors.lastname: Engbring
authors.forename: Mayumi
authors.initials: M
authors.lastname: Mochizuki
authors.forename: Motoyoshi
authors.initials: M
authors.lastname: Nomizu
authors.forename: Hynda K
authors.initials: HK
authors.lastname: Kleinman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002642:Chick Embryo; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism* / Q000494:pharmacology; D008175:Lung Neoplasms / Q000517:prevention & control / Q000556:secondary; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000378:metabolism* / Q000556:secondary; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D006025:Glycosaminoglycans; D018960:Hyaluronan Receptors; D007797:Laminin; D009842:Oligopeptides; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C097043:laminin alpha5