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The protein encoded by the C1orf10 gene was described to be esophageal-specific and a marker for cancer development. This protein, however, has the previously unreported structural features of the "fused gene" family combining sequences and structural similarities of both the S100 proteins and precursor proteins of the cornified cell envelope as in profilaggrin, trichohyalin, and repetin. Since all members of this family are expressed in keratinocytes, we suspected a role in epidermal differentiation and named the protein cornulin. Here, we report that human cornulin mRNA is expressed primarily in the upper layers of differentiated squamous tissues including the epidermis. Using polyclonal peptide antibodies, we show that cornulin is expressed in the granular and lower cornified cell layers of scalp epidermis and foreskin, as well as in calcium-induced differentiated cultured keratinocytes. Ca(2+)-overlay assay indicated that EF-hand domains of cornulin are functional and bind calcium. In HeLa cells, cornulin, co-transfected with transglutaminase 1, was diffusely distributed throughout the cytoplasm in contrast to small proline-rich 4, which localized to the cell periphery. We conclude that cornulin is a new member of the "fused gene" family, does not appear to be a precursor of the cornified cell envelope by itself, and is a marker of late epidermal differentiation.

The human repetin gene is a member of the "fused" gene family and localized in the epidermal differentiation complex on chromosome 1q21. The "fused" gene family comprises profilaggrin, trichohyalin, repetin, hornerin, the profilaggrin-related protein and a protein encoded by c1orf10. Functionally, these proteins are associated with keratin intermediate filaments and partially crosslinked to the cell envelope (CE). Here, we report the isolation and characterization of the human repetin gene and of its protein product. The repetin protein of 784 amino acids contains EF (a structure resembling the E helix-calcium-binding loop-F helix domain of parvalbumin) hands of the S100 type and internal tandem repeats typical for CE precursor proteins, a combination which is characteristic for "fused" proteins. Repetin expression is scattered in the normal epidermis but strong in the acrosyringium, the inner hair root sheat and in the filiform papilli of the tongue. Ultrastructurally, repetin is a component of cytoplasmic non-membrane "keratohyalin" F-granules in the stratum granulosum of normal epidermis, similar to profilaggrin. Finally, we show that EF hands are functional and reversibly bind Ca(2+). Our results indicate that repetin is indeed a member of the fused gene family similar to the prototypical members profilaggrin and trichohyalin.

A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P′3 (Met8→ Leu CMTI I), i.e. at the third position downstream of the reactive site bond (Arg5-Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system. The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies. After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse-phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture. Association constants of recombinant Leu-8-CMTI I with bovine β-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.