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.
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
Kevin J
KJ
Hamill
Lutz
L
Langbein
Jonathan C R
JC
Jones
W H Irwin
WH
McLean
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