FR2761362A1 - New polypeptide, corneodesmosin, and related nucleic acid - Google Patents

Abstract

Natural or synthetic, purified polypeptide (I), designate corneodesmosin, having 529 amino acids, reproduced, and its fragments are new. Also claimed are: (i) mixture of polypeptides (Ia) produced by proteolysis of (I) or its fragments; (ii) nucleic acid (II) encoding (I), specifically a 1590 bp sequence reproduced; and (iii) cosmetic or pharmaceutical composition containing a protease able to breakdown (I).

Description

The subject of the invention is a purified polypeptide, a mixture of polypeptides derived from the proteolysis of the purified polypeptide, compositions containing them, a cosmetic treatment method intended to reinforce intercorneocytic cohesion and a cosmetic treatment method intended to decrease intercorneocytic cohesion , therefore to favor the desquamation.

Human skin is made up of two compartments, namely a deep compartment, the dermis, and a superficial compartment, the epidermis.

The dermis provides the epidermis with solid support. It is also its nourishing element. It is mainly composed of fibroblasts and an extracellular matrix itself composed mainly of collagen, elastin and a substance, called fundamental substance, components synthesized by the fibroblast. There are also leukocytes, mast cells or tissue macrophages. It also consists of blood vessels and nerve fibers.

The natural human epidermis is mainly composed of three types of cells which are the keratinocytes, very majority, the melanocytes and the cells of
Langerhans. Each of these cell types contributes by its own functions to the essential role played in the body by the skin.

The epidermis is conventionally divided into a basal layer of keratinocytes which constitutes the germinal layer of the epidermis, a so-called thorny layer consisting of several layers of polyhedral cells arranged on the germ cells, a so-called granular layer consisting of flattened cells containing inclusions distinct cytoplasmic, keratohyaline grains, and finally an upper layer called the stratum corneum (or stratum corneum), consisting of keratinocytes at the terminal stage of their differentiation called corneocytes. These are mummified, anucleated cells that are derived from keratinocytes.

The corneocytes are mainly composed of a fibrous matrix containing cytokeratins, surrounded by a very resistant structure 15 nm thick, called the corneal or cornified envelope. The stack of these corneocytes constitutes the stratum corneum which is responsible for the barrier function of the epidermis. During the normal scaling process, the most superficial corneocytes detach from the surface of the epidermis.

Intercellular structures derived from desmosomes, called corneosomes or corneodesmosomes, have been described in the stratum corneum. Recent studies have shown their major importance in intercorneocyte cohesion as well as in the scaling process. In particular, a close correlation exists between cell dissociation and the proteolysis of certain corneodesmosomal components such as desmogleine I.

Several serine proteases of the trypsin or chymotrypsin type seem to be involved in the proteolysis of corneodesmosomes, in particular the chymotrypsic enzyme of the stratum corneum (stratum corneum chymotryptic enzyme).

Many skin pathologies are characterized by the production of a thickened stratum corneum and by abnormal scaling, that is to say by hyperkeratosis. This can occur on any cutaneous anatomical territory and in very varied clinical contexts. Its pathophysiological substratum and its cause are varied.

Examples include: - xerosis (or dry skin), - ichthyoses, - psoriasis, - certain benign or malignant tumor lesions, - reactive hyperkeratoses.

Other pathologies are characterized by transdifferentiation or metaplasia, at the level of mucous membranes, squamous or not, but normally not cornified, which become cornified, that is to say, are coated with an abnormal epithelium, producing on its surface d 'a stratum corneum. Although the genital mucosa and those of the upper aerodigestive tract are most often affected, these metaplasias can occur in various anatomical territories.

Examples include: - leukkeratosis of the cervix during prolapse, - oral leukkeratosis, - benign keratotic tumor lesions of the squamous mucosa.

Conversely, certain pathological manifestations lead to a thinning of the epidermis and in particular of the stratum corneum, resulting in an excessive fragility of the skin covering. It can sit in various anatomical territories, its cause is variable and it can be constitutional or acquired.

Examples include: - trophic skin disorders of the lower limbs in patients with vascular varicose pathologies, arteriopathies (diabetes, arteriosclerosis ...), - trophic skin disorders in the context of an algo syndrome- dystrophic, - trophic disorders following an abnormal scarring.

The purification and the knowledge of the polypeptides implied in intercorneocyte cohesion is one of the ways which could allow the development of products intended to fight against the effects of an excess or a defect in polypeptides of this type in particular on the surface of the skin.

One of the objects of the invention is to provide a polypeptide involved in intercorneocyte cohesion in purified form.

After long and laborious work due to its low representation among the proteins of the epidermis, its great instability and its high sensitivity to proteases, the Applicant has highlighted, isolated and purified by biochemical techniques from human epidermis, a polypeptide specific for cornified epithelia. This polypeptide, which will also be called elsewhere in the text "corneodesmosine" is expressed in the stratum corneum of the epidermis and is involved in intercorneocytic cohesion. The Applicant has determined the primary amino acid sequence.

The subject of the invention is therefore a purified polypeptide, characterized in that it
responds to the amino acid sequence: 1MGSSRAPWMGRVGGHGMM ALLLAGL
26 LLPGTLAKSIGTFSDPCKDPT RITS
51 PNDPCLTGKGDSSGFSSYSGS SSSG
76 SSISSARSSGGGSSGSSSGSS IA HQ
101 GSAGSF k PGTGYSQVSYSSGSGSSL
126 QGASGSSQLGSSSSHSGSSGS HSGS
151 SSSHSSSSSSFQFSSSSFQVG NGSA
176 LPTNDNSYRGILNPSQPGQSS SSSQ
201 TS GV SSSG QSV SSNQRPCSSDIPDS
226 PCSGGPIVSHSGPYIPSSHSV SGGQ 251 RPVVVVVDQHGS GA P GV VQGPPCSN 276 GGLPGKPCPPITSVDKSYGGY EVVG 301 GSSDSYLVPGMTYS k G k IYPVGYFT 326 KENPVKGSPGVPSFAAGPPIS EGKY 351 FSSNPIIPS QS AASSAIAFQPV GT G 376 GVQLCGGGSTGSKGPCSPSSS RVPS 401 SSSISSSAGSPYHPCGSASQS PSAP 426 PGTGSFSSSSSSQSSGKIILQ PCGS 451 KSSSSGHPC MS VSSLTLTGGPDGSP 476 HPDPSAGAKPCGSSSAGKIPC RSIR 501 DILAQV k PLGPQLADPEVFLPQGEL 526 LDSP
The primary amino acid sequence of the polypeptide is presented here in a form which uses the letter code commonly used in the prior art and which can be explained in "Principles of Biochemistry" Lehninger A.

L., Flammarion éditeur, 1985. The numbering begins on the aminoterminal side of the polypeptide and continues until the carboxyterminal end thereof.

The polypeptide of the invention can be of natural or synthetic origin. By synthetic is meant here any polypeptide obtained chemically or by production in an organism after introduction into this organism of the elements necessary for this production.

The polypeptide of the invention can come from any possible origin, namely either animal, in particular mammal and even more particularly human, or plant, or from microorganisms (viruses, phages, bacteria among others) or also from fungi, without prejudging whether it is present naturally or not in the said organism of origin.

Preferably, the polypeptide of the invention is of natural origin, purified from mammalian tissues, particularly from mammalian skin.

Preferably, the polypeptide of the invention is purified from human skin and even more preferably from human epidermis.

As previously indicated, intercorneocyte cohesion is apparently due, among other things, to the existence in the horny layer of specific polypeptides of the structures involved in the intercorneocyte junction.

Thus, the polypeptide of the invention is specific for the stratum corneum and the granular layer and, preferably, the polypeptide according to the invention is specific for the structures involved in the intercorneocytic junction, particularly corneodesmosomes.

It is known that in general the mature polypeptides which are found in cells originate from the maturation of precursors which contain in their sequence the sequence of the mature polypeptide.

Thus, the invention also relates to any polypeptide whose sequence is partly constituted by the sequence of the polypeptide of the invention.

 It is also known that the polypeptides can undergo post-translational modifications such as the formation of disulfide bonds, specific proteolytic cleavages, the addition of carbohydrates (glycosylation), phosphorylation in particular at the level of serines and / or threonines and / or tyrosines, and / or the association with lipids.

The invention therefore relates to the polypeptide of the invention having undergone or not post-translational modifications.

The polypeptide of the invention may have undergone one or more post-translational modifications.

Preferably, the polypeptide according to the invention is glycosylated and / or phosphorylated.

We know how to classify polypeptides according to their isoelectric point.

Preferably, the polypeptide of the invention is basic.

Of course, the primary amino acid sequence, as well as the various post-translational modifications undergone by the polypeptide, mean that said polypeptide can be characterized by its molecular weight, expressed in kilodaltons.

The polypeptide of the invention has an apparent molecular weight determined by SDS polyacrylamide gel electrophoresis (PAGE-SDS) of between 50 and 60 kilodaltons, preferably between 52 and 56 kilodaltons.

Very preferably, the polypeptide of the invention is a glycosylated, phosphorylated, basic polypeptide with an apparent molecular weight of between 50 and 60, preferably between 52 and 56, kilodaltons.

 It is also known that the primary amino acid sequence of a polypeptide determines sites specifically recognized by proteases which, once recognition of these sites is effective, will, with or without attachment to said polypeptide, induce its cleavage by proteolysis.

Thus, the invention also relates to a mixture of polypeptides derived from the proteolysis of the polypeptide of the invention.

As previously described, certain pathologies may be due to an excess of desquamation which it can be assumed to be due to a defect in polypeptides involved in intercorneocyte cohesion.

Another object of the invention is therefore to provide cosmetic or pharmaceutical compositions comprising, in a physiologically acceptable medium, at least one polypeptide or a mixture of polypeptides as described above.

Preferably, the composition according to the invention is intended to be applied to the surface of the skin.

As seen previously, certain pathological manifestations lead to a thinning of the epidermis and in particular of the stratum corneum, resulting in an excessive fragility of the cutaneous covering, which can sit in any cutaneous anatomical territory, be of various constitutional or acquired cause.

The invention also relates to the use of a polypeptide or of a mixture as described above for reinforcing the intercorneocyte cohesion and / or inducing the thickening of the stratum corneum. As such, the polypeptide of the invention or the mixture of polypeptides can be used to treat thinning of the epidermis and in particular of the stratum corneum or even excessive fragility of the skin covering.

Particularly the polypeptide of the invention or the mixture of polypeptides can be used to treat skin trophic disorders, for example patients carrying vascular pathologies such as varicose veins or arteriopathies (diabetes, arteriosclerosis ...), trophic skin disorders patients with algo-dystrophic syndromes or those consecutive to scarring disorders.

In the same way, the polypeptide of the invention or the mixture of polypeptides can be used to cause a localized thickening of the stratum corneum in the skin areas which are to undergo repetitive strain injuries.

By way of example, mention may be made of the preventive treatment of subepidermal bubbles ("blisters") in athletes.

The invention also relates to the use of a polypeptide or of a mixture as described above in a cosmetic composition or for the preparation of a pharmaceutical composition intended to reinforce intercorneocytic cohesion and / or induce the thickening of the layer. cornea. It also relates to a cosmetic treatment process intended to reinforce intercorneocyte cohesion and / or induce the thickening of the stratum corneum, characterized in that a composition as described above is applied to the skin of the subject to be treated .

Analysis of the primary amino acid sequence of the protein according to the invention shows that it has recognition and binding sites for known proteases or specific sites for cleavage by chemical agents. We can cite as an example the sites for Chymotrypsin,
Cathépsine, proteinase K, Subtilisine, protease V8, Thermolysine,
Thrombin, Trypsin, Papain, Pepsin, Proline-Endopeptidase,
GluC Endoproteinase, LysC Endoproteinase, AspN Endoproteinase,
Endoproteinase ArgC (Clostripaine), Myxobacter ALI 17, Elastase, chymotrypsic enzyme of the stratum corneum, cyanogen bromide, Nchlorosuccinimide or specific sites of acid hydrolysis (70% formic acid , 7M Guanidine-HCI, 40 "C, 24h).

Intercorneocyte cohesion is apparently due to the existence, in the stratum corneum, of polypeptides specific for the structures involved in the intercorneocyte junction, such as in particular the polypeptide of the invention. It has been seen that certain hyperkeratotic pathologies could be linked to an excess of intercorneocytic cohesion, due in particular to the polypeptide of the invention.

Another object of the invention is to provide a cosmetic treatment method for combating excess intercoreocytic cohesion and therefore for increasing flaking and in particular the excess due to the polypeptide of the invention, method which consists in applying to the skin a cosmetic composition comprising at least one protease having a specific site for recognition and / or fixation and cleavage within the primary amino acid sequence of the polypeptide of the invention.

Thus, the subject of the invention is a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, an effective amount of at least one protease active on the polypeptide of the invention, chosen from Endoproteinase LysC, Endoproteinase GluC , Proline-Endopeptidase,
Thrombin, Pepsin, Myxobacter AL117 and Elastase.

These proteases can be isolated from plants, animals, bacteria, viruses or fungi.

The polypeptide of the invention can be glycolysed. Thus, the composition as defined above can also contain a glycosidase which can be chosen from those isolated from plants, animals, fungus or even microorganisms, in particular bacteria. By way of example, mention may be made of neuraminidases, mannosidases, galactosidases, glucosidases,
N-acetylglucosaminidases and N-acetylgalactosaminidases.

As seen previously, certain pathologies are characterized by the production of a thickened epidermal stratum corneum and by abnormal scaling, that is to say by hyperkeratosis which can occur in any cutaneous anatomical territory, in various clinical contexts and including the substratum pathophysiology and cause can be varied.

The subject of the invention is also a cosmetic treatment method for combating excess intercoreocytic cohesion consisting in the application to the skin of a cosmetic composition comprising a protease as defined above.

The invention also relates to a pharmaceutical composition comprising a protease, as defined above, intended to treat for example xerosis (or dry skin), ichthyoses, psoriasis, hyperkeratosis of certain benign or malignant tumor lesions, reactive keratoses .

The invention also relates to a pharmaceutical composition comprising a protease, as defined above, intended for treating pathologies characterized by transdifferentiation or metaplasia, in the mucous membranes, squamous or not, but normally not cornified, which become cornified, for example leukkeratosis of the cervix during prolapse, oral leukkeratosis, or benign or malignant hyperkeratotic tumor lesions of the squamous mucosa.

It is known that a protein is synthesized in cells from a matrix of deoxyribonucleic acids coding for said protein. We also know that the genetic code is degenerate. Thus, the amino acid sequence of the polypeptide of the invention can be derived from different sequences of deoxyribonucleic acids, natural or synthetic. By synthetic deoxyribonucleic acid sequence is meant here any sequence obtained chemically or by genetic manipulation.

Said deoxyribonucleic acid sequences can come from all possible origins, namely either animal, in particular mammals and even more particularly human, or plant, or from microorganisms (viruses, phages, bacteria among others) or also from fungi, without prejudging whether they are present naturally or not in the said organism of origin.

The Applicant has isolated, purified and sequenced by molecular biology techniques, in particular the screening of expression libraries of complementary deoxyribonucleic acids prepared from human epidermis, a fragment of deoxyribonucleic acids coding for the polypeptide of the invention. .

The subject of the invention is therefore also all sequences of deoxyribonucleic acids, natural or synthetic, all or part of which codes for the primary amino acid sequence of the polypeptide of the invention.

During this work, the applicant was able to isolate and purify a sequence of deoxyribonucleic acids coding for the primary amino acid sequence of the polypeptide of the invention from human skin.

Thus, the subject of the invention is also an isolated nucleotide sequence coding for the polypeptide of the invention or any natural or synthetic nucleotide sequences containing said sequence.

The invention relates in particular to an isolated fragment of deoxyribonucleic acid comprising at least the following coding nucleotide sequence: 1 ATG GGC TCG TCT CGG GCA CCC TGG ATG GGG CGT GTG GGT GGG CAC 46 GGG ATG ATG GCA CTG CTG CTG GCT GGT CTC CTC CTG CCA GGG ACC 91 TTG GCT AAG AGC ATT GGC ACC TTC TCA GAC CCC TGT AAG GAC CCC 136 ACG CGT ATC ACC TCC CCT AAC GAC CCC TGC CTC ACT GGG AAG GGT 181 GAC TCC AGC GGC TTC AGT AGC TAC AGT GGC TCC AGC AGT TCT GGC 226 AGC TCC ATT TCC AGT GCC AGA AGC TCT GGT GGT GGC TCC AGT GGT 271 AGC TCC AGC GGA TCC AGC ATT GCC CAG GGT GGT TCT GCA GGA TCT 316 TTT AAG CCA GGA ACG GGG TAT TCC TAC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC TCC GGA 361 TCT GGC TCT AGT CTA CAA GGT GCA TCC GGT TCC TCC CAG CTG GGG 406 AGC AGC AGC TCT CAC TCG GGA AGC AGC GGC TCT CAC TCG GGA AGC 451 AGC AGC TCT CAT TCG AGC AGC AGC AGC AGC TTT CAG TTC AGC AGC 49 AGC AGC TTC CAA GTA GGG AAT GGC TCT GCT CTG CCA ACC AAT GAC 541 AAC TCT TAC CGC GGA ATA CTA AAC CCT TCC CAG CCT GGA CAA AGC 586 TCT TCC TC T TCC CAA ACC TCT GGG GTA TCC AGC AGT GGC CAA AGC 631 GTC AGC TCC AAC CAG CGT CCC TGT AGT TCG GAC ATC CCC GAC TCT 676 CCC TGC AGT GGA GGG CCC ATC GTC TCG CAC TCT GGC CCC TAC ATC 721 CCC AGC TCC CAC TCT GTG TCA GGG GGT CAG AGG CCT GTG GTG GTG 766 GTG GTG GAC CAG CAC GGT TCT GGT GCC CCT GGA GTG GTT CAA GGT 811 CCC CCC TGT AGC AAT GGT GGC CTT CCA GGC AAG CCC TGT CCC CCA 856 ATC ACC TCT GTA GTA TCC TAT GGT GGC TAC GAG GTG GTG GGT 901 GGC TCC TCT GAC AGT TAT CTG GTT CCA GGC ATG ACC TAC AGT AAG 946 GGT AAA ATC TAT CCT GTG GGC TAC TTC ACC AAA GAG AAC CCT GTG 991 AAA GGC TCT CCA GCC GTC CTC TTT GCA GCT GGG CCC CCC ATC 1036 TCT GAG GGC AAA TAC TTC TCC AGC AAC CCC ATC ATC CCC AGC CAG 1081 TCG GCA GCT TCC TCG GCC ATT GCG TTC CAG CCA GTG GGG ACT GGT 1126 GGG GTC CAG CTC TGT GGA GGC GGC TCC AC GGC TCC AAG GGA CCC 1171 TGC TCT CCC TCC AGT TCT CGA GTC CCC AGC AGT TCT AGC ATT TCC 1216 AGC AGC GCC GGT TCA CCC TAC CAT CCC TGC GGC AGT GCT TCC CAG 1261 AGC CCC TGC TCC CCA CCA GGC ACC GGC TCC TTC AGC AGC AGC TCC 1306 AGT TCC CAA TCG AGT GGC AAA ATC ATC CTT CAG CCT TGT GGC AGC 1351 AAG TCC AGC TCT TCT GGT CAC CCT TGC ATG TCT GTC TCC TCC TTG 1396 ACA CTG ACT GGG GGC CCC GAT GGC TCT CCC CAT CCT CCC TCC 1441 GCT GGT GCC AAG CCC TGT GGC TCC AGC AGT GCT GGA AAG ATC CCC 1486 TGC CGC TCC ATC CGG GAT ATC CTA GCC CAA GTG AAG CCT CTG GGG 1531 CCC CAG CTA GCT GAC CCT GAA GTT TTC CTA CCC CAA GGA GAG TTA 1576 CTC GAC AGT CCA TAA
The subject of the invention is also a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least said sequence of deoxyribonucleic acids, or a sequence of corresponding sense or antisense ribonucleic acids. More particularly, said composition is intended to strengthen or reduce intercorneocyte cohesion.

It also relates to the use of said sequences of deoxyribonucleic acids for the production of the polypeptide of the invention or of a corresponding ribonucleic acid by any known technique such as for example synthesis in vitro, from reconstituted media or the synthesis by organizations.

A subject of the invention is also the use of the polypeptide of the invention or of its proteolysis fragments, and of any synthetic peptide deduced from its sequence, for preparing or purifying, optionally from the epidermis, any molecule, structural or functional, capable of binding specifically to said purified polypeptide or to said purified proteolysis fragments or to said synthetic peptide. This molecule can in particular correspond to other specific structural proteins of the corneodesmosomes and various enzymes of the stratum corneum, of the "protease", "glycosidase" or "phosphatase" type.

A subject of the invention is also the use of the polypeptide of the invention or of its proteolysis fragments and of any synthetic peptide deduced from its sequence, for preparing specific antisera and monoclonal antibodies, aiming in particular at purifying this protein and its fragments . By extension,
A subject of the invention is also any use of said sequence to produce antibodies or fragments of recombinant antibodies, whatever the biological system used to produce the latter.

Whatever their nature, the compositions of the invention can be ingested, injected or applied to the skin (on any cutaneous zone of the body) or the mucous membranes (buccal, jugale, gingival, genital, conjunctival, etc.).

Preferably, the compositions of the invention are applied to the skin or the mucous membranes.

According to the mode of administration, the compositions according to the invention can be presented in all the galenical forms normally used.

For topical application to the skin, the composition may take the form in particular of an aqueous or oily solution or of a dispersion of the lotion or serum type, of emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a phase oily in an aqueous phase (O / W) or vice versa (W / O), or of suspensions or emulsions of soft consistency of the aqueous or anhydrous cream or gel type, or of microcapsules or microparticles, or of vesicular dispersions of the ionic type and / or nonionic or foam or also in the form of aerosol compositions also comprising a propellant under pressure. These compositions are prepared according to the usual methods.

For injection, the composition may be in the form of an aqueous, oily lotion or in the form of a serum. For the eyes, it can be in the form of drops and for ingestion, it can be in the form of capsules, granules, syrups or tablets.

The amounts of the various constituents of the compositions according to the invention are those conventionally used in the fields considered.

These compositions constitute in particular cleansing, protective, treatment or care creams for the face, for the hands, for the feet, for large anatomical folds or for the body (for example day creams, night creams, creams make-up removers, foundation creams, sunscreen creams), fluid foundations, make-up removal milks, body protection or care milks, anti-sun milks, lotions, gels or foams for the care of the skin, such as cleansing lotions, sunscreen lotions, artificial tanning lotions, bath compositions, deodorant compositions comprising a bactericidal agent, after-shaving gels or lotions, depilatory creams, compositions against stings of insects, pain-relieving compositions, compositions for treating certain skin diseases such as eczema, rosacea, psoriasis, lichens, severe pruritus.

The compositions according to the invention may also consist of solid preparations constituting soaps or cleaning bars.

The compositions can also be packaged in the form of an aerosol composition also comprising a propellant under pressure.

The composition according to the invention can also be a composition for caring for the scalp, and in particular a shampoo, a styling lotion, a treating lotion, a styling cream or gel, a composition of dyes (in particular dyes of oxidation) possibly in the form of coloring shampoos, restructuring hair lotions, a perm composition (in particular a composition for the first time of a perm), a fall prevention lotion or gel, an antiparasitic shampoo, etc.

The composition can also be for oral use, for example a toothpaste. In this case, the composition may contain adjuvants and additives customary for compositions for oral use and in particular surfactants, thickening agents, humectants, polishing agents such as silica, various active ingredients such as fluorides, in particular particularly sodium fluoride, and optionally sweetening agents such as sodium saccharinate.

When the composition is an emulsion, the proportion of the fatty phase can range from 5% to 80% by weight, and preferably from 5% to 50% by weight relative to the total weight of the composition. The oils, waxes, emulsifiers and coemulsifiers used in the composition in the form of an emulsion are chosen from those conventionally used in the cosmetic field.

The emulsifier and the coemulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5 to 20% by weight relative to the total weight of the composition. The emulsion may, in addition, contain lipid vesicles.

When the composition is a shea butter, sunflower oil), animal oils (perhydrosqualene), synthetic oils (Purcellin oil), silicone oils or waxes (cyclomethicone) and fluorinated oils (perfluoropolyethers), beeswaxes , carnauba or paraffin.

Fatty alcohols and fatty acids (stearic acid) can be added to these oils.

As emulsifiers which can be used in the invention, there may be mentioned for example glycerol stearate, polysorbate 60 and the mixture of PEG-6 / PEG-32 / Glycol
Stearate sold under the name of TefoseR 63 by the company Gattefosse.

As solvents which can be used in the invention, mention may be made of lower alcohols, in particular ethanol and isopropanol, propylene glycol.

As hydrophilic gelling agents which can be used in the invention, mention may be made of carboxyvinyl polymers (carbomer), acrylic copolymers such as acrylatealkylacrylate copolymers, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural gums and clays, and, as lipophilic gelling agents, mention may be made of modified clays such as bentones, metal salts of fatty acids such as aluminum stearates and hydrophobic silica, ethylcellulose, polyethylene.

The composition may contain other hydrophilic active agents such as proteins or protein hydrolysates, amino acids, polyols, urea, allantoin, sugars and sugar derivatives, water-soluble vitamins, plant extracts and hydroxy acids.

As lipophilic active agents, retinol (vitamin A) and its derivatives, tocopherol (vitamin E) and its derivatives, essential fatty acids, ceramides, essential oils, salicylic acid and its derivatives can be used.

According to the invention, the composition can combine at least one extract of at least one
Iridacea to other active agents intended in particular for the prevention and / or treatment of skin conditions. Among these active agents, there may be mentioned by way of example:
agents reducing the differentiation and / or proliferation and / or skin pigmentation such as retinoic acid and its isomers, retinol and its esters, vitamin D and its derivatives, estrogens such as estradiol, kojic acid or hydroquinone;
- antibacterials such as clindamycin phosphate, erythromycin or antibiotics of the tetracycline class;
- antiparasitics, in particular metronidazole, crotamiton or pyrethroids;
- antifungals, in particular compounds belonging to the class of imidazoles such as econazole, ketoconazole or miconazole or their salts, polyene compounds, such as amphotericin B, compounds of the allylamine family, such as terbinafine, or octopirox;
- antiviral agents such as acyclovir;
- steroidal anti-inflammatory agents, such as hydrocortisone, betamethasone valerate or clobetasol propionate, or non-steroidal anti-inflammatory agents such as, for example, ibuprofen and its salts, diclofenac and its salts, acid acetylsalicylic acid, acetaminophen or glycyrrhizic acid;
- anesthetic agents such as lidocaine hydrochloride and its derivatives;
- antipruritic agents such as thenaldine, trimeprazine or cyproheptadine;
keratolytic agents such as a- and p-hydoxycarboxylic or ss-ketocarboxylic acids, their salts, amides or esters and more particularly hydroxy acids such as glycolic acid, lactic acid, salicylic acid,
Citric acid and generally fruit acids, and n-octanoyl-5salicylic acid;
- anti-free radical agents, such as α-tocopherol or its esters, su peroxide dismutases, certain metal chelators or ascorbic acid and its esters;
- antiseborrheics such as progesterone;
- anti-dandruff agents such as octopirox or zinc pyrithione;
- anti-acne drugs such as retinoic acid or benzoyl peroxide.

Thus, according to a particular embodiment, the composition according to the invention also comprises at least one agent chosen from antibacterial, antiparasitic, antifungal, antiviral, anti-inflammatory, antipruritic, anesthetic, keratolytic, anti-free radicals, anti-seborrheic agents, anti-dandruff, anti-acne and / or agents that decrease differentiation and / or proliferation and / or skin pigmentation.

EXAMPLES:
Monoclonal antibodies:
The murine monoclonal antibodies G36-19, F28-27 and B17-21 (IgG1) specific for the polypeptide of the invention, corneodesmosine, are part of a series of antibodies directed against epidermal differentiation antigens, produced after immunization of 'a mouse with a homogenous stratum of the human plantar, then characterized. The ascites of the monoclonal antibody MOPC-21 (IgG1) (Sigma Chemical Co., St Louis, MO) was used as a negative control. The anti-phosphoserine antibody kit from Biomol Feinchenukalien GmbH (monoclonal antibody 1C8, 4A3, 4A9 and 4H4) and the monoclonal antibody PSR45 from Sigma were also used.

Isolation and characterization of the polypeptide:
Sequential protein extraction.

From breast skin (from reduction breast plasties),
The epidermis was mechanically separated from the dermis after heat treatment of the skin for 5 minutes at 56 "C in a phosphate buffer, then sequentially homogenized, at 4" C, in equal volumes of the following buffers (three times in each buffer: i) TE buffer: 40 mM Tris-HCl pH 7.5; 10 mM ethylenediaminetetraacetate (EDTA); 0.25 mM phenylmethylsulfonyl fluoride and 2 Cig / ml of each of the following inhibitors: aprotinin, pepstatin A and leupeptin; ii) TENP40: TE buffer containing a detergent,
Nonidet P40 at 0.5%; iii) TEU buffers: TE containing different concentrations of urea (from 2 to 8 M). After each extraction, the homogenates were centrifuged for 15 minutes at 15,000 g and the supernatants removed. The first supernatant of each extraction was kept at -30 ° C until use.

Finally, the pellet corresponding to the last extraction in the buffer containing 8M urea was homogenized in the TUDTT buffer: Tris cul 35 mM pH 6.8; 8 M urea; 50 mM dithiothreitol; glycerol 5%; 0.25 mM phenylmethylsulfonyl fluoride and 2 μgiml of each of the same inhibitors, incubated for 30 minutes at 95 ° C. and centrifuged as above. The protein concentrations were measured using the Pierce system (Coomassie Plus protein assay,
Pierce Chemical Co., Rockford, IL).

Protein electrophoresis and Immunodetectlon
The proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) in gels of 7.5 or 10% acrylamide, or by two-dimensional electrophoresis in the presence of urea, using the system of
Pharmacia (PhastSystem; Pharmacia LKB). Isoelectric focusing (IEF) or electrophoresis interrupted before pH equilibrium (NEpHGE: No
Equilibrium pH Gel Electrophoresis) were performed in the first dimension, then an SDS-PAGE was performed in the second dimension with 12.5% polyacrylamide gels. For two-dimensional electrophoresis, the proteins of the extracts in TENP40 buffer were precipitated with ethanol, collected by centrifugation and dissolved in 50 mM Tris-HCl pH 7.4; 8 M urea and 0.5% ss-mercaptoethanol. The marker proteins as well as the two-dimensional electrophoresis standards (2-D SDS-PAGE standardsTM) from
Bio-Rad have been used as molecular weight markers or as references for isoelectric points.

After electrophoresis, the proteins were stained with Coomassie blue or using silver nitrate (silver stain plus Bio-Rad Lab kit), or transferred to a reinforced nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) .

The membranes were then stained with Ponceau red or
Protogold (British BioCell International, Cardiff, UK) and immunodetected with monoclonal antibodies, as previously described. The reactivities were revealed with the ECLTM kit from Amersham (ECL Western blotting kit, Amersham
International, Aylesbury, UK), according to the manufacturer's protocol.

Results.

The 52-56 kDa polypeptide was detected in the TE buffer extracts and
TEU (from a urea concentration equal to 6 M), but not in the sequential extracts in TENP40 and TUDTT buffers. In addition, if the TE buffer extract was centrifuged for 30 minutes at 100,000 xg, the polypeptide was completely found in the supernatant. The monoclonal antibody G36-19 also recognized several polypeptides of lower apparent molecular weight, which were only partially extracted in the presence of urea (even at the concentration of 8 M), and were partially extracted in the presence of a reducing agent. Whether in TE buffer or in the presence of urea, no polypeptide was immunodetectable in the last of the three extracts produced. This indicates that at each step,
The extraction was complete. Control experiments carried out in the absence of primary antibody or with the antibody MOPC-21 have always been negative. It is likely that the forms of immunodetected low molecular weight polypeptides were not degradation products generated during the extraction steps, since their proportion did not vary from one preparation to another and since the extractions have always been performed in the presence of protease inhibitors.

Human corneodesmosin is a component of cornified husks:
In order to confirm at the biochemical level that corneodesmosine is covalently linked to the cornified envelopes, fragments, generated by proteolysis of highly purified envelopes from human plantar epidermis, were analyzed with the monoclonal antibody G36-19.

Preparation and analysis of cornified envelopes:
Human cornified envelopes were purified from the plantar stratum corneum and mammary epidermis, as described previously.

Briefly, the samples were extracted by repeated boiling with vigorous stirring in a solution containing 2% SDS (w / v) and dithiothreitol at a concentration of 25 mM, then at 37 "C for 72 hours in a solution containing 1 urea at a concentration of 8 M and dithiothreitol at a concentration of 25 mM The envelopes extracted with urea were sedimented, then suspended in a solution containing 0.1% of SDS, glycine at a concentration of 192 mM and Tris at a concentration of 125 mM Finally, they were electrodialysed against this same buffer for 72 hours. The purified cornified envelopes were collected by centrifugation, washed with distilled water and counted. They were analyzed morphologically, then by indirect immunofluorescence, as described in the prior art. V8 protease digestion and immunodetection of the proteolysis products were carried out as described in the prior art.

Results.

The envelopes have been incubated for increasing times with the protease
V8, and the fragments produced were separated by SDS-PAGE and immunodetected.

The monoclonal antibody G36-19 strongly marked a large number of bands of apparent molecular weight greater than 50 kDa, some of which were located at the upper limit of the gel. This indicates that corneodesmosine is incorporated inside large heteropolymeric structures resulting from the proteolysis of the envelope. Several bands stained by Protogold were not immunodetected, which confirms the specificity of the reaction.

No band was clearly immunodetected with the monoclonal antibody
G36-19 in the absence of proteolysis, which proves the existence of covalent links between the corneodesmosine and other constituents of the cornified envelopes.

None of the fragments produced by digestion of cornified envelopes purified from mammary epidermis were immunodetected by the monoclonal antibody.
G36-19. In agreement with this result, these envelopes were much less marked, and only at their periphery, when they were analyzed with the monoclonal antibody G36-19 in indirect immunofluorescence.

52-56 kDa human corneodesmosine is a basic phosphoprotein
To determine the isoelectric point of corneodesmosine, human epidermis was extracted directly into a buffer containing a detergent (TENP40 buffer), and the extracted proteins were separated by two-dimensional electrophoresis (NEpHGE / SDS-PAGE) and immunodetected with the monoclonal antibody G36-19. The 52-56 kDa corneodesmosine showed a basic isoelectric point greater than 8. This result was confirmed by immunodetection after an IEF / SDS-PAGE separation. The protein immunodetected by the monoclonal antibody G36-19 was not visible after staining with Coomassie blue, even when 100 μg of proteins of the extract in TENP40 buffer were loaded on the gel. This therefore suggests that the 52-56 kDa corneodesmosine is a quantitatively minor protein, representing less than 0.1% of the proteins extracted. In addition, the immunoreactive protein migrated in the form of several juxtaposed spots, suggesting the presence of post-translational modifications. To analyze the possible phosphorylation of human corneodesmosine, it was purified by affinity on a sepharose matrix (HiTrap-NHS, Pharmacia) activated by N-hydroxysuccinimide groups and then coupled with the monoclonal antibody.
G36-19. Then, it was analyzed by immunoblotting with antibodies specific for phosphoserine or phosphotyrosine. One of the five monoclonal anti-phosphoserine antibodies used has specifically immunodetected corneodesmosine. On the contrary, neither the monoclonal antibody
PY20 and an antiserum, directed against phosphotyrosine, did not recognize corneodesmosine.

Human corneodesmosine 52-56 kDa is a glycoprotein: Affinodetection with lectins:
The 52-56 kDa corneodesmosine was partially purified from an extract in TENP40 buffer by affinity chromatography. The fixed corneodesmosine was eluted with 3% SDS, deposited on electrophoresis gel and transferred to nitrocellulose membranes. The membranes were incubated in blocking buffer, then with biotinylated lectins sold by Pierce.
Chemical Co., diluted to 1 pglml: agglutinin from Pisum sativum (PSA), wheat germ (WGA), Ricinus communis (RCA), Dolichos bifluorus (DBA) and
Arachis hypogaea (PNA). After rinsing, the lectins were detected with streptavidin labeled with peroxidase (diluted 1: 400,000), and the kit
ECLTM from Amersham, as described above.

Deglycosylation experiments
The TENP40 buffer extract (10 µg) was kept boiling for 3 minutes in 20 µl of pH 7.2 sodium phosphate buffer containing 1% (w / v) SDS. Of
Nonidet P40 and EDTA were added to reach a final concentration of 1% and 20 mM, respectively. 2.4 units of N-glycosidase F (EC 3.2.2.18,
Boehringer Mannheim) were added and the reaction mixture was incubated at 37 "C for 6 hours. Proteins (34 pg) from a TENP40 buffer extract were also incubated with 5 mU endo-aN-acetylgalactosaminidase (EC 3.2.1.97) at 37 "C for 6 hours, with or without N-glycosidase F or neuraminidase (EC 3.2.1.18), under the conditions described by the manufacturer (Oxford
GlycoSystems Ltd, Abingdon, UK). The reactions were stopped by 2 minutes of boiling in sample buffer. The deglycosylation of fetuin, used as a positive control, was tested by SDS-PAGE and with biotinylated lectins.

The treated and untreated samples were separated by electrophoresis and analyzed by immunodetection and affinodetection, as described above.

Chromatography on Concanavalin A-Sepharose.

A TENP40 buffer extract was directly injected, with a flow rate of 0.5 ml / minute, onto a column of Concanavalin A sepharose 4B (ConA Sepharose,
Sigma) which had been previously equilibrated with washing buffer: Tris cul 20 mM pH 7.4 containing 0.2 M NaCl. After washing, with a flow rate of 1 ml / minute, with 15 ml of this buffer, the adsorbed proteins were eluted with a flow rate of 0.5 ml / minute with methyl-aD mannopyranoside (Sigma) diluted to 0.5 M in the wash buffer. The proteins were then separated by
PAGE-SDS and analyzed as described above.

Results
The proteins of a TENP40 buffer extract, containing 52-56 kD corneodesmosine, were treated with various glycosidases and analyzed in immunoblot with two monoclonal antibodies directed against the polypeptide.

The staining of the total proteins of the extract did not show any apparent degradation during the incubation. Treatment with N-glycosidase F induced a decrease of approximately 5 kDa in the apparent molecular weight of corneodesmosine. This result strongly suggests an N-glycosylation. On the contrary, treatments with endo-a-N-acetylgalactosaminidase and / or neuraminidase did not modify the migration of corneodesmosine. To confirm this result, human corneodesmosine was affinity purified and analyzed using biotinylated lectins. Wheat germ agglutinin (WGA) strongly bound the purified protein, unlike the other lectins tested which did not or very little recognition. In addition, corneodesmosine is linked to concanavalin A coupled to a sepharose matrix. It could be eluted with methyl a-D-mannopyranoside, specific sugar of this lectin, at a concentration of 0.5 M, which proves the specificity of the bond.

Purification of corneodesmosine:
After dermo-epidermal cleavage, the epidermis was homogenized in a TEA buffer: 40 mM Tris-HCl pH 7.5; 10 mM EDTA; aprotinin 10 ugiml and 4- (2 aminoethyl) -benzenesulfonyl fluoride (Interchim, Paris, France) used at 0.8 mM.

The homogenate was centrifuged for 15 minutes at 15,000 g. The supernatant was clarified by filtration on filters with a pore diameter of 0.45 μm (Puradisc TM 25 AS; Whatman, Clifton, NJ), and injected with a flow rate of 0.3 ml / minute on an exchange column. anion (Hi Trap Q, Pharmacia LKB) which had been equilibrated in the washing buffer: Tris-HCl 20 mM pH 7.5. The proteins not retained (ie approximately 5% of the total proteins of the extract) were directly injected at the same rate on an affinity column prepared as follows: approximately 2 mg of the monoclonal antibody F28-27 were coupled to 1 ml of Sepharose 4B matrix activated by N-hydroxysuccinimide groups (HiTrap-NHS), as recommended by the manufacturer Pharmacia LKB. The column was washed thoroughly with a flow rate of 1 ml / minute with the washing buffer in the presence then in the absence of 1 M NaCl. The immunoadsorbed proteins were eluted at a flow rate of 0.3 ml / minute with 0.2 M glycine at pH 2.5; the pH of the eluted fractions was immediately neutralized with Tris base at a concentration of 2 M. The proteins of the different fractions were analyzed by immunoblotting with the monoclonal antibody G36-19, or with the control monoclonal antibody, MOPC- 21. The fractions containing the proteins thus eluted were mixed, then lyophilized. Proteins
The lyophilisates were analyzed as described above, by mono or two-dimensional electrophoresis. The corneodesmosine was then cut from the gel in order to be sequenced.

Results.

The 52-56 kDa corneodesmosin was purified by anion exchange chromatography followed by affinity chromatography. All of the extracted corneodesmosine bound specifically to the affinity column, from which it could be eluted in the presence of glycine. Protogold staining of the transferred proteins shows the high degree of purification obtained, since corneodesmosine is in the majority in these fractions. Similar results were obtained when the affinity chromatography was carried out on a matrix coupled with the monoclonal antibody F28-27 or with the monoclonal antibody G36-19. Two-dimensional gel analysis confirmed the basic isoelectric point of purified corneodesmosine and showed that it is the only eluted protein with an apparent molecular weight of 52-56 kDa. The corneodesmosine thus purified was separated by electrophoresis and the strip of the gel containing this protein was cut, then the protein was sequenced.

Instability of the purified polypeptide:
During its numerous attempts to purify corneodesmosine, the Applicant has encountered two major problems: (1) the low proportion of this protein among the epidermal proteins and (2) its instability, probably due to its great sensitivity to action of proteases.

Various tests have been implemented in an attempt to stabilize the corneodesmosine: addition of zinc (inhibitor of the chymotrypsic enzyme of the horny layer), reducing agents (ss-mercaptoethanol or dithiothreitol), denaturing agents (SDS), glycerol, specific serine protease inhibitors (PMSF, aprotinin) or a cocktail of various protease inhibitors (aprotinin, leupeptin, pepstatin, benzamidine, phenanthroline, PMSF) or removal of the freeze / thaw steps. Finally, the absence of the freezing / thawing steps, the presence of 2% of SDS or the presence of the cocktail of inhibitors, and preferably a combination of these conditions, have proved to be the most effective in stabilizing the corneodesmosine.

Construction of a human epidermis expression library:
The cloning of the cDNA of human corneodesmosine could not be carried out from preexisting expression libraries. In fact, these were produced either from the epidermis of mice or from human keratinocytes cultivated in a monolayer, conditions which do not allow the expression of genes characteristic of epidermal terminal differentiation. The Applicant has therefore built a human epidermal expression bank.

Extraction of poly (A) t ribonucleic acids (RNA) from the human epidermis
It was carried out from breast skin samples, surgical waste obtained after plastic surgery. The subcutaneous adipose tissue was dissected, then skin flaps were cut using a dermatome and incubated for 2 hours at 37 "C, in a solution of trypsin (solution
A, Gibco BRL), epidermal side up. The dermo-epidermal cleavage was then carried out with forceps. The epidermal sheets obtained were rinsed in a phosphate buffer (PBS, pH 7.4). The extraction of poly (A) + RNA was carried out according to the protocol proposed in the "mRNA purification kit" kit marketed by Dynal (Oslo), after homogenization of the sheets using a "Turax", directly in the supplied buffer (Lysis / Binding buffer). The principle of this kit uses the affinity of messenger RNA (mRNA) comprising a poly end (A) for magnetic beads covered with oligo (dT) 25. After 5 minutes of incubation at room temperature in the lysate, the beads were isolated with a magnet and subjected to 3 washes. The poly (A) + RNA eluted by incubating the beads at 65 "C for 2 minutes in a 2 mM EDTA solution, pH 8, were assayed using the" DNA DipStick "colorimetric kit marketed by
Invitrogen (San Diego, CA).

Construction of the bank.

The bank was built with the "ZAP Express cDNA Gigapack II Gold cloning kit" box sold by Stratagene (La Jolla, CA), following the protocol proposed by the supplier. The complementary DNAs (cDNAs) were synthesized from 2 μg of poly (A) + RNA, with the reverse transcriptase of the Moloney murine leukemia virus (MMLV-RT), using oligo (dT) primers. comprising a restriction site for the enzyme Xhol and in the presence of 5-methyl cytidine triphosphate. The synthesis of the second strand was carried out with DNA polymerase I from E. coli, and the ends of the cDNAs were made blunt with the recombinant pfu polymerase. After addition of EcoR I adapters (T4 DNA ligase), and phosphorylation of the 5 'ends (T4 polynucleotide kinase), the cDNAs were subjected to digestion with the enzyme Xho I. The selection of
CDNA larger than 500 base pairs was produced with a column of Sephacryl S-500. Ligation of cDNAs with both arms of phage ZAP
Express was performed with phage T4 ligase. The encapsulation of the recombinant phage was carried out with the extracts "Gigapack II Gold Packaging Extract" supplied by Stratagene. The E. coli XL-1 blue MRF 'strain was used for the titration and spreading of the library.

Results.

This library was produced from 2 μg of mRNA, extracted from a fragment of human epidermis within hours of its collection, by unidirectional cloning in the phage ZAP Express. It consists of approximately 2x105 independent clones.

Cloning of complementary deoxyribonucleic acid for corneodesmosine: Immunological screening.

The immunological screening, carried out without a prior amplification step, was done on nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), incubated in an isopropyl-1-thio-BD-galactopyranoside solution (I PTG,
Stratagene) at 10 mM for 10 minutes, dried, and incubated for 4 h at 37 "C on the spread bank. The immunological screening procedure was carried out as described in the prior art with a cocktail of the three monoclonal antibodies G3619, F28-27 and B17-21, used at the respective concentrations of 0.2, 0.2 and 2 μg. The positive clones were isolated by conventional techniques and tested with each of the three monoclonal antibodies in isolation.

 Sequencing.

The ZAP Express phages corresponding to the purified clones were subjected to excision in vivo, with the aid of an ExAssist phage, as indicated by Stratagene.

The plasmids obtained were amplified with the Qiagen kit (Hilden,
Germany), then sequenced at the ends of the inserts, with primers T3 and M13 (Stratagene), using a Perkin-Elmer box (ABI PRISM Dye
Terminator cycle sequencing kit, Perkin Elmer, Foster city, CA). The comparison of the sequences obtained with international databases was carried out with the Blast program.

Results.

The library not having been amplified beforehand, the screening made it possible to isolate 5 independent clones. The human cDNA fragments contained in the clones were sequenced pa earlier. These results also make it possible to order on the molecule the epitopes recognized by each of the monoclonal antibodies. Indeed, the clones having been constructed by reverse transcription from the 3 ′ end of the mRNAs, which corresponds to the COOH end of the protein, it is possible to conclude that
The epitope recognized by F28-27 is the closest to this end, followed by
The epitope of G36-19 and finally that of B17-21. Analysis by enzymatic restriction of the size of these different cDNA fragments, confirmed that the 3 clones recognized by the 3 monoclonal antibodies have the longest sequences (from 2 to 1.5 kb), while that which only recognizes F28-27 is the shortest (1 kb), the last, recognized by 2 monoclonal antibodies, having an intermediate size of 1.3 kb.

Thus, all or part of the cDNA encoding human corneodesmosin, a protein characterized by the 3 monoclonal antibodies B17-21, G36-19 and F28-27 which recognize different epitopes, ordered on the protein, has been cloned. indicated, from the NH2 end to the COOH end.

The partial sequences of each clone, compared to the sequences included in the international databases, revealed an identity of 99% with the sequence of the S gene, registered in the GenBank database under the number L20815. This analysis also showed that among the 5 clones obtained, 4 correspond to the short form of the mRNA, only one, clone 5.1, corresponding to the long form. These forms come from an alternative choice of the polyadenylation site at the level of the 3 ′ non-coding part of the primary transcript.

This analysis made it possible to specify the location of the epitopes recognized by our 3 monoclonal antibodies: the epitope recognized by B17-21 is located in the part of the protein corresponding to nucleotides 594 to 762, G36-19 and F28-27 being respectively located in area 762-1044 and 1044-stop codon. Finally, clone 1.1, which has the longest coding sequence, begins at nucleotide 348.

Cloning of the complete cDNA coding for corneodesmosine:
The preceding analyzes showed that the clones obtained did not cover the part of the cDNA going from nucleotide 1 to nucleotide 347. The Applicant therefore had to clone this missing part.

80 ng of poly (A) RNA + extract of human epidermis as previously described, were subjected to reverse transcription from random primers, using the "SuperScript" kit sold by Gibco BRL. Amplification of the 5 ′ end of the cDNA of corneodesmosine was carried out from 1/20 of the first strand synthesis reaction, with an oligonucleotide corresponding to the sequence 2-20 (GenBank L20815) upstream and also comprising a Spe I adapter, and, downstream an oligonucleotide complementary to the sequence
1017-1035. The conditions for the PCR polymerase chain reaction were as follows: 3 minutes at 94 "C followed by 35 cycles with 80 sec at 94" C, 80 sec at 57 "C and 2 minutes at 72" C . The single fragment obtained, purified after electrophoresis in TBE agarose gel, digested with
Spe I and EcoN I (nucleotides 2 to 1003), was cloned into the plasmid pBK-CMV1.1 (isolated by screening the library) digested with the same enzymes, which made it possible to obtain the plasmid pS11. The complete cDNA contained in the plasmid pS11 has been sequenced at least 3 times up to nucleotide 1700. Comparison with the previously published sequence revealed four point differences which were analyzed at the genomic level.

Genomic analysis of corneodesmosine:
Four point differences have been noted between the sequence of the complete cDNA isolated by the applicant and that of the S gene published in the prior art (Zhou Y. and Chaplin DD, PNAS usa, Vol. 90, pp. 9470-9474, October 1993).

These four point differences were analyzed by PCR, at the genomic level from ten control samples of DNA extracted from human blood according to conventional methods.

Analysis of the possible insertion of a guanine at position 1514 was carried out after PCR amplification of the genomic region between nucleotides 1446 and 1786 (GenBank, L20815), carried out under standard conditions. The DNA fragment obtained for each sample was subjected to digestion with Bsi MI on the one hand and Nci I on the other hand. NuSieve GTG 3% gel electrophoresis (FMC, Rockland, ME) was performed in TBE buffer.

The study of the sequence at position 66 was carried out according to the ASA method (allele specific amplification). Two PCR reactions were carried out in parallel with downstream, an oligonucleotide complementary to the sequence 572-91 and, upstream, an oligonucleotide corresponding to the sequence 52-65 and ending in position 66 either with A or with T. PCR reactions were carried out with 0.5 μg of genomic DNA, 2.5 pmol of each oligonucleotide, and 0.5 U of Taq polymerase (Appligene). The amplification conditions are 94 "C, 2 minutes then 30 cycles with 50 seconds at 94" C, 1 minute at 56 "C and 2.5 minutes at 72" C.

The analysis of the bases in position 1118 and 1236 was carried out after amplification with a pair of single oligonucleotides: sense oligonucleotide 908-27, antisense 1573-93. The PCR conditions are the same as above, with a polymerization time limited to 1 minute. The fragments were subjected to digestion with Bsm I or Hin6 I for the respective study of positions 1118 and 1236 and then separated by electrophoresis on 3% NuSieve gel.

Results
The insertion of a guanine at position 1514 causes a shift in the reading frame and moves the stop codon from position 1523 to position 1603. The protein produced by this sequence differs in the last two amino acids (I and S). which are replaced by the sequence: DILAQVKPLGPQLADPEVFLPQGELLDSP which corresponds to an addition of 27 amino acids compared to the published sequence. The 5 previously isolated cDNAs all presenting this insertion, the study was extended to other individuals. Ten control samples of human genomic DNA from ten individuals were amplified in this region and analyzed by enzyme restriction. Indeed, the published sequence corresponds to the presence of a Bsi MI restriction site in position 1510 which disappears in the sequence comprising an additional guanine, while an Nci I site appears in 1512. The results are the same for the 10 individuals , namely that only the Nci I site is present. This means that in these ten individuals, corneodesmosine is produced in a longer form than the published form of the S gene.

Three other point differences, of substitution type, were found between the clones and the published sequence. Analysis of the ten control genomic samples by PCR and enzymatic restriction or by ASA, showed that it is a polymorphism of the gene. The Applicant has demonstrated in position 66 a conservative mutation T / A (Leu-Met), in position 1118, an A / G polymorphism, corresponding to a silent Ala-Ala mutation and, finally, in 1236 an nG polymorphism corresponding to a non-conservative mutation
Will be there.

Production of recombinant corneodesmosin:
The complete cDNA coding for corneodesmosine was isolated, in the form of an Ec1136 I / Not I fragment of 2106 base pairs from the plasmid pS11. The subcloning was carried out in the vector pCDNA amp (InVitrogen) previously digested with EcoR V and Not I, resulting in the plasmid p14-9.

The in vitro translation of corneodesmosine was carried out with the "TNT" kit
T7 Quick coupled transcriptionltranslation system "marketed by Promega, starting from plasmid p14-9. This kit combines in one step, transcription by phage T7 RNA polymerase, with translation by a lysate of rabbit reticulocytes. These reactions have were carried out with 0.6 μg of plasmid in a volume of 25 μl, according to the supplier's instructions.

Corneodesmosine was expressed in COS-7 cells after transfection with the plasmid p14-9. The cells were transfected according to the conventional protocol with DEAE-dextran, supplemented by the addition of chloroquine and shock to
DMSO, using 1 µg of plasmid / 3.10 cells. After 48 h of expression, the cells are washed twice with PBS pH 7.4 and lysed in a 40 mM Tris-HCl buffer pH 7.5, 10 mM EDTA, 0.5% NP40, containing a cocktail of inhibitors proteases (Pharmingen, Inc). After separation on SDS-PAGE, and electrotransfer, the samples are immunodetected, as previously described.

Results
The new complete sequence of the protein predicts a molecular weight of 51.45 kDa, in the absence of any post-translational modification. Corneodesmosine synthesized in glass, in the absence of microsomal membranes, migrates with an apparent molecular weight of approximately 60 kDa. This shows that this protein has an aberrant migration in SDS-PAGE gel. Co-migration with the majority epidermal form extracted in a low ionic strength buffer, demonstrates that the latter corresponds to a fairly widely truncated form of the protein, all the more so since the Applicant has shown that this epidermal form is glycosylated.

Transfection of COS-7 cells with plasmid 14-9 reveals an immunoreactive protein which also migrates around 60 kDa. The same results were obtained with the human neuroepithelioma line CHP 126, transfected by electroporation. All of these results strongly suggest that in the epidermis, corneodesmosine undergoes, early in its maturation, a specific proteolysis.

Claims (31)

1. Purified natural or synthetic polypeptide, characterized in that it responds to the amino acid sequence: 1 MGS S RAPWMGRVGGHGMMA L L L AGL  26 L L P G T L A K S I G T F S D P C K D P T R I T S  51 P N D P C L T G k G D S S G F S S Y S G S S S S G  76 S S I S S A R S S G G G S S G S S S G S S I A Q G  101 G S A G S F k P G T G Y S Q V S Y S S G S G S S L  126 Q G A S G S S Q L G S S S S H S G S S G S H S G S  151 S S S H S S S S S S F Q F S S S S F Q V G N G S A  176 L P T N D N S Y R G I L N P S Q P G Q S S S S S Q  201 T S GV S S S G Q S V S S N Q R P C S S D I P D S  226 P C S G G P I V S H S G P Y I P S S H S V S G G Q  251 R P V V V V V D Q H G S GA P GV V Q G P P C S N  276 G G L P G K P C P P I T S V D K S Y G G Y E VV G  301 GS S D S Y L V P GMT Y S K GK I Y P V G Y F T  326 K E N P V K G S P G V P S F A A G P P I S E G k Y  351 F S S N P I I P S QS A A S S A I A F Q P V GT G  376 G V Q L C G G G S T G S K G P C S P S S S R V P S  401 S S S I S S S A G S P Y H P C G S A S Q S P C S P  426 P G T G S F S S S S S S Q S S G K I I L Q P C G S  451 K S S S S GH P C MS V S S L T L T GG P D GS P  476 H P D P S A G A K P C G S S S A G K I P C R S I R  501 D I L A Q V k P L G P Q L A D P E V F L P Q G E L  526 L D S P 2. Polypeptide according to claim 1, characterized in that it is purified from mammals. 3. Polypeptide according to one of the preceding claims, characterized in that it is purified from the skin of mammals. 4. Polypeptide according to one of the preceding claims, characterized in that it is purified from human skin. 5. Polypeptide according to one of the preceding claims, characterized in that it is purified from human epidermis. 6. Polypeptide according to one of the preceding claims, characterized in that it is involved in intercorneocyte cohesion. 7. A polypeptide whose sequence is partly constituted by the sequence of the polypeptide as described in claim 1. 8. Polypeptide according to one of the preceding claims, characterized in that it has an apparent molecular weight of between 50 and 60 kilodaltons, preferably between 52 and 56 kilodaltons. 9. Polypeptide according to one of the preceding claims, characterized in that it is basic. 10. Polypeptide according to one of the preceding claims, characterized in that it is glycosylated. 11. Polypeptide according to one of the preceding claims, characterized in that it is phosphorylated. 12. Polypeptide according to one of the preceding claims, characterized in that it is glycosylated, basic, phosphorylated and that it has an apparent molecular weight of between 50 and 60 kilodaltons, preferably between 52 and 56 kilodaltons. 13. Mixture of polypeptides derived from the proteolysis of the polypeptide as described in claims 1 to 12. 14. Cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, at least one polypeptide or a mixture of polypeptides as described in one of claims 1 to 13. 15. Cosmetic treatment method intended to treat skin trophic disorders or those consecutive to scarring disorders, characterized in that a cosmetic composition as described in claim 14 is applied to the skin of the subject to be treated. 16. Cosmetic treatment method intended to treat the thinning of the epidermis, and in particular of the stratum corneum, and to treat an excessive fragility of the cutaneous coating and / or to reinforce the intercorneocytic cohesion and / or to induce the thickening of the stratum corneum, characterized in that a composition as described in claim 14 is applied to the skin of the subject to be treated. 17. Pharmaceutical composition according to claim 14 intended to treat skin trophic disorders or those consecutive to scarring disorders. 18. Pharmaceutical composition according to claim 14 intended to treat the thinning of the epidermis and in particular of the stratum corneum and / or to treat an excessive brittleness of the cutaneous coating and / or to reinforce the inter-corneocytic cohesion and / or to induce thickening of the stratum corneum. 19. Cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, an effective amount of at least one protease active on the polypeptide according to one of claims 1 to 12, chosen from 'Endoproteinase LysC, Endoproteinase GluC, Proline -Endopeptidase, the Thrombin, Pepsin, Myxobacter ALI 17 and Elastase. 20. Composition according to claim 19, characterized in that it further comprises a glycosidase. 21. Cosmetic treatment method for decreasing intercorneocyte cohesion or for promoting flaking, characterized in that a composition as described in claims 19 or 20 is applied to the skin of the subject to be treated. 22. Pharmaceutical composition according to any one of claims 19 or 20 intended to treat hyperkeratosis. 23. Pharmaceutical composition according to any one of claims 19 or 20 intended to treat xerosis, ichthyoses, psoriasis, benign or malignant hyperkeratotic tumor lesions or reactive keratoses. 24. Pharmaceutical composition according to any one of claims 19 or 20 intended to treat leukoeratosis of the cervix during prolapse, oral leukoeratosis, or even benign or malignant hyperkeratotic tumor lesions of the squamous mucosa. 26. Nucleotide sequence coding for the polypeptide as defined in one of claims 1 to 12. 27. Isolated fragment of deoxyribonucleic acid comprising at least the following coding nucleotide sequence 1 ATG GGC TCG TCT CGG GCA CCC TGG ATG GGG CGT GTG GGT GGG CAC 46 GGG ATG ATG GCA CTG CTG CTG GCT GGT CTC CTC CTG CCA GGG ACC 91 TTG GCT AAG AGC ATT GGC ACC TTC TCA GAC CCC TGT AAG GAC CCC 136 ACG CGT ATC ACC TCC CCT AAC GAC CCC TGC CTC ACT GGG AAG GGT 181 GAC TCC AGC GGC TTC AGT AGC TAC AGT GGC TCC AGC AGT TCT GGC 226 AGC TCC ATT TCC AGT GCC AGA AGC TCT GGT GGT GGC TCC AGT GGT 271 AGC TCC AGC GGA TCC AGC ATT GCC CAG GGT GGT TCT GCA GGA TCT 316 TTT AAG CCA GGA ACG GGG TAT TCC CAG GTC AGC TAC TCC TCC GGA 361 TCT GGA CAA GGT GCA TCC GGT TCC TCC CAG CTG GGG 406 AGC AGC AGC TCT CAC TCG GGA AGC AGC GGC TCT CAC TCG GGA AGC 451 AGC AGC TCT CAT TCG AGC AGC AGC AGC AGC TTT CAG TTC AGC AGC 496 AGC AGC TTC CAA GTA G GGC TCT GCT CTG CCA ACC AAT GAC 541 AAC TCT TAC CGC GGA ATA CTA AAC CCT TCC CAG CCT GGA CAA AGC 586 TCT TCC TCT TCC CAA ACC TCT GGG GTA TCC AGC AGT GGC C AA AGC 631 GTC AGC TCC AAC CAG CGT CCC TGT AGT TCG GAC ATC CCC GAC TCT 676 CCC TGC AGT GGA GGG CCC ATC GTC TCG CAC TCT GGC CCC TAC ATC 721 CCC AGC TCC CAC TCT GTG TCA GGG GGT CAG AGG CCT GTG GTG GTG GTG GTG GTG 766 GTG GTG GAC CAG CAC GGT TCT GGT GCC CCT GGA GTG GTT CAA GGT 811 CCC CCC TGT AGC AAT GGT GGC CTT CCA GGC AAG CCC TGT CCC CCA 856 ATC ACC TCT GTA GAC AAA TCC TAT GGT GGC TAC GAG GTG GTG GGT 90 TCC TCT GAC AGT TAT CTG GTT CCA GGC ATG ACC TAC AGT AAG 946 GGT AAA ATC TAT CCT GTG GGC TAC TTC ACC AAA GAG AAC CCT GTG 991 AAA GGC TCT CCA GGG GTC CCT TCC TTT GCA GCT GGG CCC CCC ATC 1036 TCT GAG GCC AAA TAC TTC TCC AGC AAC CCC ATC ATC CCC AGC CAG 1081 TCG GCA GCT TCC TCG GCC ATT GCG TTC CAG CCA GTG GGG ACT GGT 1126 GGG GTC CAG CTC TGT GGA GGC GGC TCC ACG GGC TCC AAG GGA CCC 1171 TGC TCT CCC TCC AGT TCT CGA GTC CCC AGC AGT TCT AGC ATT TCC 1216 AGC AGC GCC GGT TCA CCC TAC CAT CCC TGC GGC AGT GCT TCC CAG 1261 AGC CCC TGC TCC CCA CCA GGC ACC GGC TCC TTC AGC AGC AGC TCC 1306 AGT TCC CAA TCG AGT GG C AAA ATC ATC CTT CAG CCT TGT GGC AGC 1351 AAG TCC AGC TCT TCT GGT CAC CCT TGC ATG TCT GTC TCC TCC TTG 1396 ACA CTG ACT GGG GGC CCC GAT GGC TCT CCC CAT CCT GAT CCC TCC 1441 GCT GGT GCC AAG CCC TGT GGC TCC AGC AGT GCT GGA AAG ATC CCC 1486 TGC CGC TCC ATC CGG GAT ATC CTA GCC CAA GTG AAG CCT CTG GGG 1531 CCC CAG CTA GCT GAC CCT GAA GTT TTC CTA CCC CAA GGA GAG TTA 1576 CTC GAC AGT CCA TAA 28. Nucleotide sequence comprising all or part of the nucleotide sequence as described in either of claims 26 or 27. 29. Cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, a sequence as described in any one of claims 26 to 28. 30. Use of the acid sequence as described in any one of claims 26 to 28 to prepare a polypeptide or a mixture of polypeptides as described in claims 1 to 13. 31. Use of the acid sequence as described in any one of claims 26 to 28 to prepare a ribonucleic acid. 32. Use of the purified polypeptide or of its purified proteolysis fragments or of any synthetic peptide as described in claims 1 to 13, for preparing or purifying any molecule capable of specifically binding to said purified polypeptide or to said purified proteolysis fragments or to said synthetic peptide. 33. Use according to the preceding claim for preparing or purifying specific structural proteins of corneodesmosomes or enzymes of the stratum corneum of the "protease" or "glycosidase" type. 34. Use of the purified polypeptide or of its purified proteolysis fragments or of any synthetic peptide as described in claims I to 13, for preparing or purifying specific antisera or monoclonal antibodies.