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

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    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

    Cite this

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    title = "Identification of a novel family of laminin N-terminal alternate splice isoforms structural and functional characterization",
    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.",
    keywords = "EXTRACELLULAR-MATRIX PROTEINS, MONOCLONAL-ANTIBODY GB3, GAMMA-2 CHAIN, DIFFERENTIAL EXPRESSION, BASEMENT-MEMBRANES, CELL-ADHESION, ALPHA-CHAINS, EXON USAGE, GENES, MOUSE",
    author = "Hamill, {Kevin J.} and Lutz Langbein and Jones, {Jonathan C. R.} and McLean, {W. H. Irwin}",
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    doi = "10.1074/jbc.M109.052811",
    language = "English",
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    pages = "35588--35596",
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    Identification of a novel family of laminin N-terminal alternate splice isoforms structural and functional characterization. / Hamill, Kevin J. (Lead / Corresponding author); Langbein, Lutz; Jones, Jonathan C. R.; McLean, W. H. Irwin.

    In: Journal of Biological Chemistry, Vol. 284, No. 51, 18.12.2009, p. 35588-35596.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Identification of a novel family of laminin N-terminal alternate splice isoforms structural and functional characterization

    AU - Hamill, Kevin J.

    AU - Langbein, Lutz

    AU - Jones, Jonathan C. R.

    AU - McLean, W. H. Irwin

    PY - 2009/12/18

    Y1 - 2009/12/18

    N2 - 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.

    AB - 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.

    KW - EXTRACELLULAR-MATRIX PROTEINS

    KW - MONOCLONAL-ANTIBODY GB3

    KW - GAMMA-2 CHAIN

    KW - DIFFERENTIAL EXPRESSION

    KW - BASEMENT-MEMBRANES

    KW - CELL-ADHESION

    KW - ALPHA-CHAINS

    KW - EXON USAGE

    KW - GENES

    KW - MOUSE

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    U2 - 10.1074/jbc.M109.052811

    DO - 10.1074/jbc.M109.052811

    M3 - Article

    VL - 284

    SP - 35588

    EP - 35596

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 51

    ER -