Identification of a novel family of laminin N-terminal alternate splice isoforms structural and functional characterization

Kevin J. Hamill (Lead / Corresponding author), Lutz Langbein, Jonathan C. R. Jones, W. H. Irwin McLean

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    Abstract

    The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5'-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) alpha 3 and LaNt alpha 5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt alpha 3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt alpha 3 and LaNt alpha 3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha 3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha 3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

    Original languageEnglish
    Pages (from-to)35588-35596
    Number of pages9
    JournalJournal of Biological Chemistry
    Volume284
    Issue number51
    DOIs
    Publication statusPublished - 18 Dec 2009

    Keywords

    • EXTRACELLULAR-MATRIX PROTEINS
    • MONOCLONAL-ANTIBODY GB3
    • GAMMA-2 CHAIN
    • DIFFERENTIAL EXPRESSION
    • BASEMENT-MEMBRANES
    • CELL-ADHESION
    • ALPHA-CHAINS
    • EXON USAGE
    • GENES
    • MOUSE

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