Sciellin and uses thereof

Details Sciellin and uses thereof Sciellin and uses thereof

1999-03-26

2003-12-02

Bugaisky, Gabriele E. (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S252300, C435S325000, C435S254110, C536S023500

Reexamination Certificate

active

06656705

ABSTRACT:

BACKGROUND OF THE INVENTION
The cornified envelope is a fifteen (15) nm thick insoluble protein layer that is formed under the plasma membrane in the upper layers of epidermis and keratinizing stratified epithelium (Reichert, U. et al. (1993)
Molecular Biology of the Skin
, 107-150). It appears to play a major role in the physical barrier properties of the stratum comeum (Elias, P. M. and D. S. Friend (1975)
J. Cell. Biol
. 65:180-191). The envelope is formed from several precursor proteins by the calcium dependent enzyme transglutaminase, which catalyzes formation of &egr;-(&ggr;-glutamyl) lysine crosslinks (Polakowska, R. R. and L. A. Goldsmith (1991)
Physiology, Biochemistry and Molecular Biology of the Skin
, 168-201) that are resistant to proteolytic digestion. It has been postulated that crosslinking of an envelope related protein, involucrin, to the plasma membrane is a first step in envelope assembly (Ishida-Yamamoto, A. et al. (1997)
J. Invest. Dermatol
. 108:12-16). This is followed by crosslinking of the less abundant precursors such as SPRR proteins, elafin, envoplakin, filaggrin, keratin filaments and cystatin a (Steinert, P. M. and L. N. Marekov (1995)
J. Biol. Chem
. 270:17702-17711; Takahashi, H. et al. (1997)
J. Invest. Dermatol
. 108:843-847; Ruhrberg, C. et al. (1996)
J. Cell. Biol
. 134:715-729; Takahashi, M. et al. (1996)
Arch. Biochem. and Biophys
. 329:123-126). Finally loricrin covers the cytoplasmic side of the envelope (Candi, E. et al. (1995)
J. Biol. Chem.
270: 26382-26390).
Several reports have suggested that multiple components are necessary for envelope structure and function. Involucrin for example acts as the framework for the attachment of other envelope components and is covalently linked to the lipids which are important components of the barrier of stratum comeum cells (Downing, D. T. (1992)
J. Lipid Res
. 33:301-313). The pancornulin proteins have been shown to act as molecular bridges and are able to cross-link with two different proteins (Li, V. W. et al. (1996)
Dermatology Clinics
745-751). Loricrin can impart flexibility as a result of its high glycine content and insolubility from disulfide bonds.
Gene mutation and knockout studies have been used to gather information on the function of epidermal proteins, with the keratins being the most well known (Fuchs, E. et al. (1992)
PNAS
89:6906-6910; Vassar, R. et al. (1991)
Cell
64:365-380). Only one study of envelope related proteins has been reported, a loricrin knockout mouse (deviragh, P. A. et al. (1996)
J. Invest. Dermatol
. 106:844; deviragh, P. A. et al. (1997)
J. Invest. Dermatol
. 108:555). Heterozygous mice are normal, while homozygotes have abnormal skin during the first few days, but the animals appear normal as adults However, the mice have a defect in barrier function and respond abnormally to the application of some chemicals. A mutation of the loricrin gene has been observed in patients with a rare autosomal dominant palmoplantar keratoderma, Vohwinkel's Keratoderma, as well as in Progressive, Symmetric Erythrokeratoderma (Ishida-Yamamoto, A. et al. (1997)
J. Invest. Dermatol
. 108:12-16). Loss of epidermnal transglutaminase activity from mutations in the gene results in the human disease, lamellar ichthyosis, which is characterized by ia thickened stratum corneum, disturbed epidermal keratinization and inflammatory changes (Huber, M. et al. (1995)
Science
267:525-528).
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of the gene which encodes the epidermal protein, sciellin. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of sciellin, or a sciellin polypeptide.
In a preferred embodiment, sciellin has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human sciellin, e.g., the human sciellin of SEQ ID NO:2. Sciellin can be identical to a human sciellin sequence, e.g., that of SEQ ID NO:2. In another embodiment, sciellin is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:1. In addition, sciellin can have substantially the same electrophoretic mobility as human sciellin, e.g., it appears as an electrophoretic band of about 75.3 kDa on reducing gels. Yet another preferred embodiment of the invention features a sciellin which is reactive with a sciellin-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 34D11, or a polyclonal antibody SC4. Antibodies against sciellin can be made by methods exemplified herein.
In another preferred embodiment, sciellin is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic animal. Cultured cells can include CHO cells or SF8 cells. Expression of sciellin in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode sciellin or a fragment thereof are expressed in a preferred cell-type by a tissue specific prtomoter, e.g., a K14 promoter. Exemplary sequences encoding fragments of sciellin include, e.g., a sequence encoding the central domain of sciellin, e.g., one or more of repeats 1-16, or a sequence encoding a LIM domain.
In a preferred embodiment, the recombinant sciellin differs from sciellen isolated from tissue in one or more of the following: its pattern of glycosylation, myristilation, phosphorylation, or other posttranslational modifications.
In a preferred embodiment, the recombinant sciellin preparation is free of other keratinocyte proteins, placental proteins, or other human proteins.
In a preferred embodiment, the recombinant sciellin preparation contains at least 1, 10, or 100 &mgr;g of sciellin, or a sciellin polypeptide.
In a preferred embodiment, the recombinant sciellin preparation contains at least 1, 10, or 100 mg of sciellin, or a sciellin polypeptide.
In a preferred embodiment, the sciellin polypeptide has the following biological acitivities: 1) it is a precursor of the cornified envelopelof keratinizing tissues; 2) it provides structural support to the comified envelopes of stratum corneum cells; 3) it promotes adhesion between tissue elements; 4) it promotes intracIellular signalling; 5) it defines cell shape; 6) it can act as an adaptor element to promote the assembly and targeting of multiprotein complexes; 7) it forms homotrimeric beta helices; (8) it is involved in the terminal differentiation of keratinocytes; and (9) it plays a role in development. In other preferred embodiments: the sciellin polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:2; the sciellin polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:2; the sciellin polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the sciellin polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:2; the sciellin polypeptide is either, an agonist or an antagonist, of a biological activity of naturally occurring sciellin; the sciellin polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, sciellin polypeptide.
In preferred embodiments: the sciellin polypeptide is encoded by the nucleic acid in SEQ ID NO:1, or by a nucleic acid having at least aboutl 85%, more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with the nucleic acid from SEQ ID NO:1.
In preferred embodiments, the sciellin polypeptide includes an amino terminal domain, a central domain containing comprised of sixteen repeats, and/or a carboxy terminal domain containing a LIM domain.
In preferred embodiments, th

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