Gene Targeting of Envoplakin, a Cytoskeletal Linker Protein and Precursor of the Epidermal Cornified Envelope

Abstract

Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function.The functional endpoint of epidermal differentiation is assembly of the cornified envelope (CE), a covalently crosslinked protein layer that is deposited at the cytoplasmic face of the plasma membrane and forms a barrier between the living cell layers of the skin and the outside environment (16,26,27,35). The importance of the epidermal barrier is highlighted by the phenotype of mice which lack the gene for transglutaminase 1 (25), the key enzyme responsible for the cross-linking of envelope precursor proteins, in which a failure in cornified envelope assembly leads to excessive transepidermal water loss and neonatal lethality. Inactivating mutations in the human gene also result in severe perturbation of epidermal differentiation and function (15). Although there are a multitude of proteins that become incorporated into the cornified envelope, recent biochemical, cell biological, and immunoelectron microscopical data suggest that cornified envelope biogenesis is an ordered process, in which cross-linking of one specific group of proteins at the membrane precedes incorporation of abundant late precursors such as loricrin and the small proline-rich proteins (4,24,32,33,34).Two of the early envelope precursors are envoplakin and periplakin (1,28,29). They belong to the plakin family of cytolinker proteins, which includes desmoplakin, plectin, and BPAG1 (6,12,14,20,30,37). Prior to cornified envelope assembly, envoplakin and periplakin localize to desmosomes and form an interconnecting subplasmalemmal network between the desmosomes (28, 29, 31). Envoplakin and periplakin can heterodimerize via their rod domains, and the periplakin N-terminal globular domain appears to target heterodimers to...

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