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

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

UR - https://www.scopus.com/pages/publications/71449095202

U2 - 10.1074/jbc.M109.052811

DO - 10.1074/jbc.M109.052811

M3 - Article

C2 - 19773554

SN - 0021-9258

VL - 284

SP - 35588

EP - 35596

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 51

ER -