title: Identification of active sequences in the L4a domain of laminin α5 promoting neurite elongation.
abstract: Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1β1. However, the antibodies to integrin α1 and β1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1β1 seems to modulate neurite elongation on the short arm of α5.
authors.affiliation: Laboratory of Clinical Biochemistry, Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan.
authors.forename: Fumihiko
authors.initials: F
authors.lastname: Katagiri
authors.forename: Misuzu
authors.initials: M
authors.lastname: Sudo
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hozumi
authors.forename: Motoyoshi
authors.initials: M
authors.lastname: Nomizu
authors.forename: Yamato
authors.initials: Y
authors.lastname: Kikkawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002448:Cell Adhesion; D057809:HEK293 Cells; D006801:Humans; D016023:Integrins / Q000276:immunology / Q000378:metabolism; D007797:Laminin / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000906:Antibodies; D016023:Integrins; D007797:Laminin; D011994:Recombinant Proteins; C097043:laminin alpha5