Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

Molecular cloning, occurrence, and expression of a novel partially secreted protein "psoriasin" that is highly up-regulated in psoriatic skin.

Analysis of the protein patterns of normal and psoriatic noncultured unfractionated keratinocytes has revealed several low-molecular-weight proteins that are highly up-regulated in psoriatic epidermis. Here, we have cloned and sequenced the cDNA (clone 1085) for one of these proteins that we have termed psoriasin. The deduced sequence predicted a protein of molecular weight of 11,457 daltons and a pI of 6.77. The protein co-migrated with psoriasin as determined by two-dimensional (2D) gel analysis of [35S]-methionine-labeled proteins expressed by RK13 cells transfected with clone 1085 using the vaccinia virus expression system. Analysis of the predicted sequence revealed a potential calcium-binding sequence of the EF-hand type, as well as the absence of a signal sequence at its amino terminal. Psoriasin is not related to other proteins that migrate closely in 2D gels (MRP 14, also known as calgranulin B, L1 and calprotectin; MRP 8, or calgranulin A and cystatin A or stefin A), and bears no significant sequence homology with any other protein of known primary structure. Increased expression of psoriasin mRNA in psoriatic keratinocytes was confirmed by Northern blotting and in situ hybridization. Psoriasin showed a restricted occurrence in fetal human tissues as determined by 2D gel electrophoresis. Of 21 tissues analyzed, only ear, skin, and tongue showed significant levels of this protein. Psoriasin was not detected in normal human fibroblasts, lymphocytes, endothelial cells and transformed epithelial cells of keratinocyte origin. Granulocyte extracts contained this protein suggesting that its overexpression by psoriatic keratinocytes may be linked to the inflammatory stimuli.