Groot, K.R.; (2005) Functional analysis of the cornified envelope precursors, periplakin and envoplakin. Doctoral thesis , University of London.

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Abstract

Periplakin and envoplakin are components of the epithelial cornified cell envelope (CE). The CE is a crosslinked protein structure that forms beneath the plasma membrane of differentiating keratinocytes and is essential for epidermal barrier function. Periplakin and envoplakin belong to the plakin family of cytolinker proteins. Their N termini are thought to mediate their interaction with the plasma membrane. I found that the N terminus of envoplakin does not efficiently localise to desmosomes or microvilli, suggesting a dependence on periplakin for membrane localisation. I found that the first 133 amino acids of periplakin targets the protein to the apical plasma membrane. In a yeast two-hybrid screen, using this region as 'bait', a novel protein was identified and named kazrin. There are four alternatively spliced transcripts, encoding three proteins with different N termini. The interaction between periplakin and all three kazrin isoforms was confirmed by pull-down reactions. Kazrin was expressed in all layers of stratified squamous epithelia and was incorporated into the CEs of cultured keratinocytes. Kazrin localised to the cell periphery of differentiated cells, however it was more diffusely localised in cells of the basal layer. In stratified keratinocyte cultures kazrin partially colocalised with desmoplakin and periplakin at the desmosomes and periplakin at the interdesmosomal plasma membrane. In transient transfections, kazrin proteins localised predominantly to the apical plasma membrane of primary keratinocytes. The exogenous expression of kazrin proteins caused changes in keratinocyte cell shape and the actin cytoskeleton. I carried out in vivo analysis of the function of periplakin and envoplakin using mice lacking periplakin, envoplakin and involucrin expression. Triple knockout mice produced slightly larger CEs and had a slight delay in epidermal barrier formation relative to control mice. In future it would be interesting to establish the consequence of deleting the kazrin gene in mice.

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