Profilaggrin, a high-molecular-weight precursor of filaggrin in human epidermis and cultured keratinocytes

Abstract

Filaggrin is a histidine-rich matrix protein of keratinized epidermis. Filaggrin is synthesized as a high-M(r), phosphorylated precursor, profilaggrin, that is processed to form the lower-M(r) product present in cornified cells. This study reports the identification of profilaggrin in human epidermis and in usually well-differentiated cultured human keratinocytes with the use of polyclonal antihuman filaggrin antiserum. Polyclonal antiserum raised against human filaggrin stained keratohyaline granules and stratum corneum in tissue sections of human skin. Analysis of epidermal extracts showed an immonoreactive low-M(r) band (37,000), previously identified as filaggrin, and an immunoreactive high-M(r) band (> 220,000). Both [32P]phosphate and [3H]histidine were incorporated into the high-M(r) band after pulse labeling for 3 h. After a 15-h chase, [3H]histidine, but not [32P]phosphate appeared in filaggrin. Human foreskin keratinocytes cultured on a 3T3 feeder layer were unusually well differentiated. Numerous well-formed keratohyaline granules, complete desmosomes, lamellar granules, and cornified cell envelopes were observed. Immunofluorescence with antihuman filaggrin antiserum showed a granular staining pattern in the more stratified cells. Extracts of cultured cells contained a diffuse high-M(r) immunoreactive band but no immunoreactive equivalent of filaggrin. These studies suggest that human skin filaggrin, like rodent filaggrin, is synthesized as a high-M(r), phosphorylated, histidine-rich precursor (profilaggrin) that is processed via posttranslational modification to filaggrin. In human keratinocyte cultures a similar high-M(r) precursor is present, but evidence of processing to the lower-M(r) product, filaggrin, is lacking.