title: Co-assembly of envoplakin and periplakin into oligomers and Ca(2+)-dependent vesicle binding: implications for cornified cell envelope formation in stratified squamous epithelia.
abstract: Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, although co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca(2+). These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca(2+)] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly.
authors.affiliation: Laboratory of Skin Biology and Laboratory of Structural Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892, USA. kalinina@mail.nih.gov
authors.forename: Andrey E
authors.initials: AE
authors.lastname: Kalinin
authors.forename: William W
authors.initials: WW
authors.lastname: Idler
authors.forename: Lyuben N
authors.initials: LN
authors.lastname: Marekov
authors.forename: Peter
authors.initials: P
authors.lastname: McPhie
authors.forename: Blair
authors.initials: B
authors.lastname: Bowers
authors.forename: Peter M
authors.initials: PM
authors.lastname: Steinert
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D000838:Anions / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D010743:Phospholipids / Q000378:metabolism; D051179:Plakins; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000838:Anions; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D008055:Lipids; D008565:Membrane Proteins; C491674:PPL protein, human; D010743:Phospholipids; D051179:Plakins; D011498:Protein Precursors; D011994:Recombinant Proteins; C100754:envoplakin; D002118:Calcium