Identification of a novel family of laminin N-terminal alternate splice isoforms: structural and functional characterization.
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) alpha3 and LaNt alpha5, 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 alpha3 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 alpha3 and LaNt alpha3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt alpha3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt alpha3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.
Hamill
Kevin J
KJ
Epithelial Genetics Group, Human Genetics Unit, Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom. k-hamill@northwestern.edu
Langbein
Lutz
L
Jones
Jonathan C R
JC
McLean
W H Irwin
WH
D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002448:Cell Adhesion / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D007797:Laminin / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D009928:Organ Specificity / Q000502:physiology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014945:Wound Healing / Q000502:physiology
D000911:Antibodies, Monoclonal; D007797:Laminin; D020033:Protein Isoforms; D012333:RNA, Messenger; C097043:laminin alpha5; C095567:laminin alpha 3