U.S. patent application number 10/677865 was filed with the patent office on 2005-05-26 for polypeptide expressed in the horny layer of the epidermis and its use.
This patent application is currently assigned to SOCIETE L'OREAL S.A.. Invention is credited to Serre, Guy Bruno Rene, Simon, Michel, Weber-Vivat, Marina.
Application Number | 20050112150 10/677865 |
Document ID | / |
Family ID | 26233428 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112150 |
Kind Code |
A1 |
Serre, Guy Bruno Rene ; et
al. |
May 26, 2005 |
Polypeptide expressed in the horny layer of the epidermis and its
use
Abstract
The present invention relates to a cosmetic or pharmaceutical
composition comprising, in a physiologically acceptable medium, a
purified natural or synthetic epidermis-specific polypeptide which
is involved in horny layer cell cohesion. The invention also
relates to a cosmetic or pharmaceutical composition comprising a
mixture of polypeptides derived from the proteolysis of the
purified polypeptide of the present invention. The present
invention further relates to a method for strengthening horny layer
cell cohesion which comprises applying the cosmetic or
pharmaceutical composition of the present invention to the skin.
Finally, the present invention relates to a method for reducing
horny layer cell cohesion, and thereby promoting exfoliation,
wherein said method comprises applying to the skin a cosmetic or
pharmaceutical composition comprising an effective amount of at
least one protease active on the polypeptide of the present
invention.
Inventors: |
Serre, Guy Bruno Rene;
(Toulouse, FR) ; Simon, Michel; (Belberaud,
FR) ; Weber-Vivat, Marina; (Villefranche de
Lauragais, FR) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
SOCIETE L'OREAL S.A.
Paris
FR
|
Family ID: |
26233428 |
Appl. No.: |
10/677865 |
Filed: |
October 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10677865 |
Oct 3, 2003 |
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09381656 |
Dec 29, 1999 |
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6645509 |
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09381656 |
Dec 29, 1999 |
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PCT/FR98/00636 |
Mar 27, 1998 |
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Current U.S.
Class: |
424/401 ;
424/70.14; 514/18.8 |
Current CPC
Class: |
A61P 17/00 20180101;
A61K 8/606 20130101; A61P 43/00 20180101; C07K 14/47 20130101; A61K
38/47 20130101; A61Q 19/00 20130101; A61K 38/47 20130101; Y10S
514/844 20130101; Y10S 514/943 20130101; A61K 8/64 20130101; A61K
8/66 20130101; C07K 16/18 20130101; A61K 2300/00 20130101; A61K
38/00 20130101; A61K 2800/86 20130101 |
Class at
Publication: |
424/401 ;
514/012; 424/070.14 |
International
Class: |
A61K 007/06; A61K
007/11; A61K 038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 28, 1997 |
FR |
97/03899 |
Sep 11, 1997 |
FR |
97/11317 |
Claims
1-17. (canceled)
18. A cosmetic or pharmaceutical composition comprising, in a
physiologically acceptable medium, an effective quantity of at
least one protease active on a polypeptide comprising
Endoproteinase LysC, Endoproteinase GluC, Proline-Endopeptidase,
Thrombin, Pepsin, Myxobacter AL117 and Elastase; wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO:1.
19. The composition according to claim 18, further comprising, a
glycosidase.
20. A method for preventing or treating hyperkeratosis, comprising
administering an effective amount of the composition according to
claim 18 to a patient in need of such prevention or treatment.
21. A method for preventing or treating xerosis, ichthyoses,
psoriasis, benign or malignant hyperkeratotic tumour lesions or
reactive keratoses, comprising administering an effective amount of
the composition according to claim 18 to a patient in need of such
prevention or treatment.
22. A method for preventing or treating leukokeratosis of the
uterine neck during prolapsus, buccal leukokeratoses, or benign or
malignant hyperkeratotic tumour lesions of the Malpighian mucosae,
comprising administering an effective amount of the composition
according to claim 18 to a patient in need of such prevention or
treatment.
23. A method of cosmetic treatment for reducing intercorneocyte
cohesion or for promoting desquamation, comprising administering an
effective amount of the composition as described in claim 18,
wherein the composition is applied to the skin of the subject in
need of such reduction or promotion.
24. Cosmetic or pharmaceutical composition comprising, in a
physiologically acceptable medium, at least the following coding
nucleotide sequence SEQ ID NO: 2:
3 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 TTT AAG CCA GGA ACG GGG TAT
TCC CAG GTC AGC TAC TCC TCC GGA 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 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 TCT 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 GAC AAA TCC TAT GGT GGC TAC GAG GTG GTG GGT 901 GGC TCC
TCT GAG 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 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 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 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 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
25. The composition according to claim 24, characterized in that
the nucleotide sequence comprises all or part of the nucleotide
sequence as described in claim 24.
27. (canceled)
28. A method for the preparation of a ribonucleic acid, comprising
using the nucleic acid sequence according to claim 24.
29. A method for preparing or purifying any molecule capable of
binding specifically to a purified polypeptide or to purified
proteolysis fragments or to a synthetic peptide; wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO:1.
30. A method for preparing or purifying structural proteins
specific to corneodesmosomes or enzymes of the horny layer of the
"protease" or "glycosidase" type, comprising using a composition
comprising one or more purified natural or synthetic polypeptides,
wherein each of the polypeptides comprises the amino acid sequence
of SEQ ID NO:1.
31. (canceled)
Description
[0001] The subject of the invention is a cosmetic or pharmaceutical
composition comprising, in a physiologically acceptable medium, at
least one purified natural or synthetic polypeptide, specific to
the epidermis, having a role in intercorneocyte cohesion. The
subject of the invention is also a cosmetic or pharmaceutical
composition comprising a mixture of polypeptides derived from the
proteolysis of the purified polypeptide, a method of cosmetic
treatment for strengthening intercorneocyte cohesion and a method
of cosmetic treatment for reducing intercorneocyte cohesion, and
therefore for promoting desquamation.
[0002] Human skin consists of two compartments, namely a deep
compartment, the dermis, and a superficial compartment, the
epidermis.
[0003] The dermis provides the epidermis with a solid support. It
is also its feeder component. It consists mainly of fibroblasts and
an extracellular matrix itself mainly composed of collagen, elastin
and a substance called ground substance, these components being
synthesized by the fibroblast. Leukocytes, mastocytes or tissue
macrophages are also found therein. It also consists of blood
vessels and of nerve fibres.
[0004] Natural human epidermis is composed mainly of three types of
cells which are the keratinocytes, which are highly predominant,
the melanocytes and the Langerhans' cells. Each of these cell types
contributes, through its specific functions, to the essential role
played by the skin in the organism.
[0005] The epidermis is conventionally divided into a basal layer
of keratinocytes which constitutes the germinative layer of the
epidermis, a so-called prickle cell layer consisting of several
layers of polyhedral cells arranged on the germinative cells, a
so-called granular layer consisting of flattened cells containing
distinct cytoplasmic inclusions, the keratohyalin granules, and
finally a top layer called horny layer (or stratum corneum),
consisting of keratinocytes at the final stage of their
differentiation, called corneocytes. These are anucleated,
mummified cells which are derived from the keratinocytes.
[0006] The corneocytes are mainly composed of a fibrous matrix
containing cytokeratins, surrounded by a very resistant structure
15 nm thick, called horny or hornified envelope. The stacking of
these corneocytes constitutes the horny-layer which is responsible
for the barrier function of the epidermis. During the normal
desquamation process, the most superficial corneocytes become
detached from the surface of the epidermis.
[0007] Intercellular structures derived from the desmosomes, called
corneosomes or corneodesmosomes, have been described in the horny
layer. Recent studies have shown their key importance in
intercorneocyte cohesion as well as in the desquamation process. In
particular, a close correlation exists between cell dissociation
and proteolysis of certain corneodesmosomal components such as
desmoglein I.
[0008] Several serine proteases of the trypsin or chymotrypsin type
appear to be involved in the proteolysis of the corneodesmosomes,
in particular the chymotryptic enzyme of the horny layer (stratum
corneum chymotryptic enzyme).
[0009] Numerous pathological conditions of the skin are
characterized by the production of a thick horny layer and by an
abnormal desquamation, that is to say by hyperkeratosis. The latter
may occur on any anatomical skin area and in a wide variety of
clinical contexts. Its physiopathological substratum and its cause
are varied. By way of example, there may be mentioned:
[0010] xerosis (or dryness of the skin),
[0011] ichthyoses,
[0012] psoriasis,
[0013] certain benign or malignant tumour lesions,
[0014] reactive hyperkeratoses.
[0015] Other pathological conditions are characterized by
transdifferentiation or metaplasia, at the level of the mucosae,
Malpighian or otherwise, but normally nonhornified, which become
hornified, that is to say which become covered with an abnormal
epithelium, producing a horny layer at its surface. Although the
genital mucosae and those of the upper aerodigestive tracts are
most often involved, these metaplasias may be seated in various
anatomical areas. By way of examples, there may be mentioned:
[0016] leukokeratosis of the uterine neck during prolapsus,
[0017] buccal leukokeratoses,
[0018] keratotic benign tumour lesions of the Malpighian
mucosae.
[0019] By contrast, some pathological manifestations cause thinning
of the epidermis and in particular of the horny layer, resulting in
excessive fragility of the skin covering. It may be seated in
various anatomical areas, its cause is variable and it may be
constitutional or acquired.
[0020] By way of examples, there may be mentioned:
[0021] trophic skin disorders of the lower limbs in patients
carrying vascular pathological conditions: varicose veins,
arteriopathies (diabetes, arteriosclerosis and the like),
[0022] trophic skin disorders in the context of an algodystrophic
syndrome,
[0023] trophic disorders following abnormal cicatrization.
[0024] The purification and knowledge of the polypeptides involved
in intercorneocyte cohesion is one of the routes which could allow
the production of products for combating the effects of an excess
or a deficiency of polypeptides of this type, in particular at the
surface of the skin.
[0025] One of the objects of the invention is to provide a
composition comprising a polypeptide involved in intercorneocyte
cohesion in purified form.
[0026] After long and laborious studies because of its low
representation among the proteins of the epidermis, and its high
instability and its high sensitivity to proteases, the applicant
has identified, isolated and purified by biochemical techniques,
from a human epidermis, a polypeptide specific to the hornified
epithelia. This polypeptide, which will also be called elsewhere in
the text "corneodesmosine" is expressed in the horny layer of the
epidermis and is involved in intercorneocyte cohesion. The
applicant has determined the primary amino acid sequence
thereof.
[0027] The subject of the invention is therefore a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, at least one purified natural or synthetic
polypeptide, the said polypeptide being characterized in that it
corresponds to the following amino acid sequence SEQ ID NO: 1:
1 Met Gly Ser Ser Arg Ala Pro Trp Met Gly Arg Val Gly Gly His Gly 1
5 10 15 Met Met Ala Leu Leu Leu Ala Gly Leu Leu Leu Pro Gly Thr Leu
Ala 20 25 30 Lys Ser Ile Gly Thr Phe Ser Asp Pro Cys Lys Asp Pro
Thr Arg Ile 35 40 45 Thr Ser Pro Asn Asp Pro Cys Leu Thr Gly Lys
Gly Asp Ser Ser Gly 50 55 60 Phe Ser Ser Tyr Ser Gly Ser Ser Ser
Ser Gly Ser Ser Ile Ser Ser 65 70 75 80 Ala Arg Ser Ser Gly Gly Gly
Ser Ser Gly Ser Ser Ser Gly Ser Ser 85 90 95 Ile Ala Gln Gly Gly
Ser Ala Gly Ser Phe Lys Pro Gly Thr Gly Tyr 100 105 110 Ser Gln Val
Ser Tyr Ser Ser Gly Ser Gly Ser Ser Leu Gln Gly Ala 115 120 125 Ser
Gly Ser Ser Gln Leu Gly Ser Ser Ser Ser His Ser Gly Ser Ser 130 135
140 Gly Ser His Ser Gly Ser Ser Ser Ser His Ser Ser Ser Ser Ser Ser
145 150 155 160 Phe Gln Phe Ser Ser Ser Ser Phe Gln Val Gly Asn Gly
Ser Ala Leu 165 170 175 Pro Thr Asn Asp Asn Ser Tyr Arg Gly Ile Leu
Asn Pro Ser Gln Pro 180 185 190 Gly Gln Ser Ser Ser Ser Ser Gln Thr
Ser Gly Val Ser Ser Ser Gly 195 200 205 Gln Ser Val Ser Ser Asn Gln
Arg Pro Cys Ser Ser Asp Ile Pro Asp 210 215 220 Ser Pro Cys Ser Gly
Gly Pro Ile Val Ser His Ser Gly Pro Tyr Ile 225 230 235 240 Pro Ser
Ser His Ser Val Ser Gly Gly Gln Arg Pro Val Val Val Val 245 250 255
Val Asp Gln His Gly Ser Gly Ala Pro Gly Val Val Gln Gly Pro Pro 260
265 270 Cys Ser Asn Gly Gly Leu Pro Gly Lys Pro Cys Pro Pro Ile Thr
Ser 275 280 285 Val Asp Lys Ser Tyr Gly Gly Tyr Glu Val Val Gly Gly
Ser Ser Asp 290 295 300 Ser Tyr Leu Val Pro Gly Met Thr Tyr Ser Lys
Gly Lys Ile Tyr Pro 305 310 315 320 Val Gly Tyr Phe Thr Lys Glu Asn
Pro Val Lys Gly Ser Pro Gly Val 325 330 335 Pro Ser Phe Ala Ala Gly
Pro Pro Ile Ser Glu Gly Lys Tyr Phe Ser 340 345 350 Ser Asn Pro Ile
Ile Pro Ser Gln Ser Ala Ala Ser Ser Ala Ile Ala 355 360 365 Phe Gln
Pro Val Gly Thr Gly Gly Val Gln Leu Cys Gly Gly Gly Ser 370 375 380
Thr Gly Ser Lys Gly Pro Cys Ser Pro Ser Ser Ser Arg Val Pro Ser 385
390 395 400 Ser Ser Ser Ile Ser Ser Ser Ala Gly Ser Pro Tyr His Pro
Cys Gly 405 410 415 Ser Ala Ser Gln Ser Pro Cys Ser Pro Pro Gly Thr
Gly Ser Phe Ser 420 425 430 Ser Ser Ser Ser Ser Gln Ser Ser Gly Lys
Ile Ile Leu Gln Pro Cys 435 440 445 Gly Ser Lys Ser Ser Ser Ser Gly
His Pro Cys Met Ser Val Ser Ser 450 455 460 Leu Thr Leu Thr Gly Gly
Pro Asp Gly Ser Pro His Pro Asp Pro Ser 465 470 475 480 Ala Gly Ala
Lys Pro Cys Gly Ser Ser Ser Ala Gly Lys Ile Pro Cys 485 490 495 Arg
Ser Ile Arg Asp Ile Leu Ala Gln Val Lys Pro Leu Gly Pro Gln 500 505
510 Leu Ala Asp Pro Glu Val Phe Leu Pro Gln Gly Glu Leu Leu Asp Ser
515 520 525 Pro
[0028] The polypeptide of the invention may be of natural or
synthetic origin. Synthetic is understood here to mean any
polypeptide obtained chemically or by production in an organism
after introducing into this organism the components necessary for
this production.
[0029] The polypeptide of the invention may be derived from any
possible origin, namely either animal, in particular mammalian and
still more particularly human, origin or plant origin or from
microorganisms (inter alia viruses, phages or bacteria) or from
fungi, without prejudging the fact that it is present naturally or
otherwise in the said organism of origin.
[0030] Preferably, the polypeptide of the invention is of natural
origin, purified from mammalian tissues, particularly from
mammalian skin.
[0031] Preferably, the polypeptide of the invention is purified
from human skin and still more preferably from human epidermis.
[0032] As indicated above, intercorneocyte cohesion is apparently
due, inter alia, to the existence, in the horny layer, of
polypeptides specific to the structures involved in the
intercorneocyte junction.
[0033] Accordingly, the polypeptide of the invention is specific to
the horny layer and to the granular layer and, preferably, the
polypeptide according to the invention is specific to the
structures involved in the intercorneocyte junction, particularly
of the corneodesmosomes.
[0034] It is known, in general, that the mature polypeptides which
are found in cells are derived from the maturation of precursors
which contain, in their sequence, the sequence of the mature
polypeptide. Accordingly, the invention also relates to a cosmetic
or pharmaceutical composition comprising, in a physiologically
acceptable medium, any polypeptide whose sequence partly consists
of the sequence of the polypeptide of the invention.
[0035] It is also known that the polypeptides may undergo
post-translational modifications such as the formation of
disulphide bonds, specific proteolytic cleavages, the addition of
carbohydrates (glycosylation), phosphorylation, in particular at
the level of the serines and/or of the threonines and/or of the
tyrosines, and/or combination with lipids.
[0036] The invention therefore relates to a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, at least one polypeptide of the invention which
has undergone post-translational modifications or not.
[0037] The polypeptide of the invention may have undergone one or
more post-translational modifications.
[0038] Preferably, the polypeptide according to the invention is
glycosylated and/or phosphorylated.
[0039] It is known to classify polypeptides according to their
isoelectric point. Preferably, the polypeptide of the invention is
basic.
[0040] Of course the primary amino acid sequence as well as the
various post-translational modifications undergone by the
polypeptide are responsible for the fact that the said polypeptide
may be characterized by its molecular weight, expressed in
kilodaltons.
[0041] The polypeptide of the invention has an apparent molecular
weight, determined by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE), of between 50 and 60 kilodaltons, preferably between 52
and 56 kilodaltons.
[0042] Most preferably, the polypeptide of the invention is a
basic, phosphorylated, glycosylated polypeptide having an apparent
molecular weight of between 50 and 60, preferably between 52 and 56
kilodaltons.
[0043] Thus preferably, the subject of the invention is a cosmetic
or pharmaceutical composition comprising, in a physiologically
acceptable medium, at least one polypeptide of the invention, the
said polypeptide being phosphorylated, basic and glycosylated and
having an apparent molecular weight of between 50 and 60
kilodaltons, preferably between 52 and 56 kilodaltons.
[0044] It is also known that the primary amino acid sequence of a
polypeptide determines sites specifically recognized by proteases
which, once the recognition of these sites has been achieved, will,
with or without attachment to the said polypeptide, induce its
cleavage by proteolysis.
[0045] Accordingly, the invention also relates to a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, at least one mixture of polypeptides derived
from the proteolysis of the polypeptide of the invention.
[0046] As described above, some pathological conditions may be due
to excessive desquamation which may be assumed to be due to a
deficiency of polypeptides involved in intercorneocyte
cohesion.
[0047] Some pathological manifestations cause thinning of the
epidermis and, in particular, of the horny layer, resulting in
excessive fragility of the skin covering, which may be seated in
any anatomical skin area and may have various causes,
constitutional or acquired.
[0048] Accordingly, the composition according to the invention is
intended for treating the thinning of the epidermis, and in
particular the horny layer, and/or for treating excessive fragility
of the skin covering and/or for strengthening intercorneocyte
cohesion and/or inducing the thickening of the horny layer.
[0049] Likewise, the composition according to the invention is
intended for treating the thinning of the epidermis, and in
particular of the horny layer and/or for treating excessive
fragility of the skin covering and/or for strengthening
intercorneocyte cohesion and/or inducing the thickening of the
horny layer.
[0050] Likewise, the composition of the invention may be used for
causing localized thickening of the horny layer at the level of the
skin regions which have to be subjected to repeated
microtraumas.
[0051] By way of example, there may-be mentioned the preventive
treatment of subepidermal blisters ("ampullae") in
sportspeople.
[0052] The subject of the invention is also a method of cosmetic
treatment for treating the thinning of the epidermis, and in
particular of the horny layer, and/or for treating excessive
fragility of the skin covering and/or for strengthening
intercorneocyte cohesion and/or inducing the thickening of the
horny layer, characterized in that a cosmetic composition according
to the invention is applied to the skin of the subject to be
treated.
[0053] The subject of the invention is also a method of cosmetic
treatment for treating trophic skin disorders, for example of
patients carrying vascular pathological conditions such as varicose
veins or arteriopathies (diabetes, arteriosclerosis and the like),
trophic skin disorders of patients carrying algodystrophic
syndromes or those following cicatrization disorders.
[0054] 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. There may be mentioned, as example, the sites for
Chymotrypsin, Cathepsin, proteinase K, Subtilisin, protease V8,
Thermolysin, Thrombin, Trypsin, Papain, Pepsin,
Proline-Endopeptidase, Endoproteinase GluC, Endoproteinase LysC,
Endoproteinase AspN, Endoproteinase ArgC (Clostripain), Myxobacter
AL117, Elastase, chymotryptic enzyme of the horny layer, cyanogen
bromide, N-chlorosuccinimide or specific sites for acid hydrolysis
(70% formic acid, 7 M Guanidine-HCl, 40.degree. C., 24 h).
[0055] Intercorneocyte cohesion is apparently due to the existence,
in the horny layer, of polypeptides specific to the structures
involved in the intercorneocyte junction such as, in particular,
the polypeptide of the invention. It has been seen that certain
hyperkeratotic pathological conditions could be linked to an
excessive intercorneocyte cohesion, due in particular to the
polypeptide of the invention.
[0056] Accordingly, the subject of the invention is a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, an effective quantity 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.
[0057] These proteases may be isolated from plants, animals,
bacteria, viruses or fungi.
[0058] The polypeptide of the invention may be glycolysed.
Accordingly, the composition as defined above may contain, in
addition, a glycosidase which may be chosen from those isolated
from plants, animals, fungi or microorganisms, in particular
bacteria. There may be mentioned, by way of example,
neuraminidases, mannosidases, galactosidases, glucosidases,
N-acetylglucosaminidases and N-acetylgalactos-aminidases.
[0059] As seen above, some pathological conditions are
characterized by the production of a thickened epidermal horny
layer and by abnormal desquamation, that is to say by
hyperkeratosis which may occur in any anatomical skin area, in
various clinical contexts and whose physiopathological substratum
and cause may be varied.
[0060] The invention also relates to a cosmetic or pharmaceutical
composition comprising a protease, as defined above, for treating,
for example, hyperkeratosis, xerosis (or dryness of the skin),
ichthyoses, psoriasis, certain benign or malignant hyperkeratotic
tumour lesions, and reactive keratoses.
[0061] The invention also relates to a cosmetic or pharmaceutical
composition comprising a protease, as defined above, for treating
pathological conditions which are characterized by
transdifferentiation or metaplasia, at the level of mucosae,
Malpighian or otherwise, but normally nonhornified, which become
hornified such as, for example, leukokeratosis of the uterine neck
during prolapsus, buccal leukokeratoses, or benign or malignant
hyperkeratotic tumour lesions of the Malpighian mucosae.
[0062] Another object of the invention is to provide a method of
cosmetic treatment for combating excessive intercorneocyte cohesion
and therefore for increasing desquamation and in particular the
excesses due to the polypeptide of the invention, which method
consists in applying to the skin a cosmetic composition comprising
at least one protease having a specific recognition and/or binding
and cleavage site within the primary amino acid sequence of the
polypeptide of the invention.
[0063] It is known that a protein is synthesized in the cells from
a template of deoxyribonucleic acids encoding the said protein. It
is also known that the genetic code is degenerate. Accordingly, the
amino acid sequence of the polypeptide of the invention may be
derived from various natural or synthetic deoxyribonucleic acid
sequences. Synthetic deoxyribonucleic acid sequence is understood
here to mean any sequence obtained chemically or by genetic
engineering.
[0064] The said deoxyribonucleic acid sequences may be derived from
any possible origin, namely either animal, in particular mammalian
and still more particularly human origin or plant origin or from
microorganisms (inter alia viruses, phages, or bacteria) or from
fungi, without prejudging the fact that they are present naturally
or otherwise in the said organism of origin.
[0065] The applicant has isolated, purified and sequenced, using
molecular biology techniques, in particular the screening of
libraries for expression of complementary deoxyribonucleic acids
prepared from human epidermis, a deoxyribonucleic acid fragment
encoding the polypeptide of the invention.
[0066] The subject of the invention is also therefore a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, any deoxyribonucleic acid sequences, natural or
synthetic, all or part of which encodes the primary amino acid
sequence of the polypeptide of the invention.
[0067] During these studies, the applicant has been able to isolate
and purify a deoxyribonucleic acid sequence encoding the primary
amino acid sequence of the polypeptide of the invention from human
skin.
[0068] Particularly, the subject of the invention is a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, an isolated deoxyribonucleic acid fragment
comprising at least the following coding nucleotide sequence SEQ ID
NO: 2:
2 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 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 ACC AGC
ACC TTT CAG TTC AGC AGC 496 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 TCT TCC CAA ACC TCT GGG GTA TCC ACC ACT
CCC CAA AGC 631 CTC ACC TCC AAC CAC CCT CCC TGT AGT TCG GAC ATC CCC
CAC 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 GAC AAA 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
GGG GTC CCT TCC 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 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 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 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
[0069] The subject of the invention is also a cosmetic or
pharmaceutical composition comprising, in a physiologically
acceptable medium, a sense or antisense ribonucleic acid sequence
corresponding to the said sequence SEQ ID NO: 2.
[0070] Its subject is also the use of the said deoxyribonucleic
acid sequences 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 synthesis by organisms.
[0071] The 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 the
said purified polypeptide or to the said purified proteolysis
fragments or to the said synthetic peptide. This molecule may in
particular correspond to other structural proteins specific to the
corneodesmosomes and various enzymes of the horny layer, of the
"protease", "glycosidase" or "phosphatase" type.
[0072] The subject of the invention is also the use of the
polypeptide of the invention or of its proteolytic fragments and of
any synthetic peptide deduced from its sequence, for preparing
specific monoclonal antibodies and antisera, intended in particular
for purifying this protein and its fragments. By extension, the
subject of the invention is also any use of the said sequence for
producing recombinant antibodies or antibody fragments, regardless
of the biological system used for producing the latter.
[0073] Whatever their nature, the compositions of the invention may
be ingested, injected or applied to the skin (over any skin area of
the body) or the mucosae (buccal, jugal, gingival, genital,
conjunctival and the like).
[0074] Preferably, the compositions of the invention are applied to
the skin or the mucosae.
[0075] Depending on the mode of administration, the compositions
according to the invention may be provided in any of the galenical
forms normally used.
[0076] For a 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 fatty phase in an aqueous phase (O/W) or conversely (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 and/or nonionic type or of
foams or alternatively in the form of aerosol compositions also
comprising a pressurized propellant. These compositions are
prepared according to the customary methods.
[0077] For injection, the composition may be provided in the form
of an aqueous or oily lotion or in the form of a serum. For the
eyes, it may be provided in the form of drops, and for ingestion,
it may be provided in the form of capsules, granules, syrups or
tablets.
[0078] The quantities of the various constituents of the
compositions according to the invention are those conventionally
used in the fields considered.
[0079] These compositions constitute in particular cleansing,
protective, treatment or care creams for the face, for the hands,
for the feet, for the large anatomical folds or for the body, (for
example day creams, night creams, make-up removing creams,
foundation creams, antisun creams), fluid foundations, make-up
removing milks, protective or care body milks, antisun milks,
skincare lotions, gels or foams, such as cleansing lotions, antisun
lotions, artificial tanning lotions, bath compositions, deodorant
compositions containing a bactericidal agent, aftershave gels or
lotions, depilatory creams, compositions against insect bites,
antipain compositions or compositions for treating certain skin
diseases such as eczema, rosacea, psoriasis, lichens and severe
pruritus.
[0080] The compositions according to the invention may also consist
of solid preparations constituting cleansing soaps or cakes.
[0081] The compositions may also be packaged in the form of an
aerosol composition also containing a pressurized propellant.
[0082] The composition according to the invention may also be a
composition for the care of the scalp, especially a shampoo, a hair
setting lotion, a treatment lotion, a hair styling cream or gel, a
dyeing (especially oxidation dyeing) composition optionally in the
form of dyeing shampoos, restructuring lotions for the hair, a
permanent-waving composition (especially a composition for the
first stage of a permanent waving), a lotion or gel against hair
loss, an antiparasitic shampoo and the like.
[0083] The composition may also be for dentibuccal use, for example
a toothpaste. In this case, the composition may contain customary
adjuvants and additives for compositions for buccal use and
especially surfactants, thickening agents, humectants, polishing
agents such as silica, various active ingredients such as
fluorides, in particular sodium fluoride, and optionally sweetening
agents such as sodium saccharinate.
[0084] When the composition is an emulsion, the proportion of fatty
phase may 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.
[0085] When the composition is an oily gel or a solution, the fatty
phase may represent more than 90% of the total weight of the
composition.
[0086] In a known manner, the cosmetic composition may also contain
adjuvants common in the cosmetic field, such as hydrophilic or
lipophilic gelling agents, hydrophilic or lipophilic additives,
preservatives, antioxidants, solvents, perfumes, fillers, screening
agents, odour absorbers and colouring materials. The quantities of
these various adjuvants are those conventionally used in the
cosmetic field, and for example from 0.01% to 10% of the total
weight of the composition. These adjuvants, depending on their
nature, may be introduced into the fatty phase, into the aqueous
phase and/or into the lipid spherules.
[0087] As oils or waxes which can be used in the invention, there
may be mentioned mineral oils (petroleum jelly), vegetable oils
(liquid fraction of shea butter, sunflower oil), animal oils
(perhydrosqualene), synthetic oils (Purcellin oil), silicone oils
or waxes (cyclomethicone) and fluorinated oils
(perfluoropolyethers), beeswaxes, carnauba or paraffin waxes. Fatty
alcohols and fatty acids (stearic acid) may be added to these
oils.
[0088] As emulsifiers which can be used in the invention, there may
be mentioned for example glycerol stearate, polysorbate 60 and the
PEG-6/PEG-32/Glycol Stearate mixture sold under the name TefoseR 63
by the company Gattefosse.
[0089] As solvents which can be used in the invention, there may be
mentioned the lower alcohols, especially ethanol and isopropanol,
and propylene glycol.
[0090] As hydrophilic gelling agents which can be used in the
invention, there may be mentioned the carboxyvinyl polymers
(carbomer), acrylic copolymers such as acrylate/alkylacrylate
copolymers, polyacrylamides, polysaccharides such as
hydroxypropylcellulose, natural gums and clays, and, as lipophilic
gelling agents, there may be mentioned modified clays such as
bentones, metal salts of fatty acids such as aluminium stearates
and hydrophobic silica, ethylcellulose and polyethylene.
[0091] 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.
[0092] As lipophilic active agents, there may be used retinol.
(vitamin A) and its derivatives, tocopherol (vitamin E) and its
derivatives, essential fatty acids, ceramides, essential oils,
salicylic acid and its derivatives.
[0093] According to the invention, the composition may combine at
least one extract of at least one Iridaceae with other active
agents intended especially for the prevention and/or treatment of
skin conditions. Among these active agents, there may be mentioned,
by way of example:
[0094] agents. reducing skin differentiation and/or proliferation
and/or pigmentation such as retinoic acid and its isomers, retinol
and its esters, vitamin D and its derivatives, oestrogens such as
oestradiol, kojic acid or hydroquinone;
[0095] antibacterials such as clindamycin phosphate, erythromycin
or antibiotics of the tetracycline class;
[0096] antiparasitic agents, in particular metronidazole,
crotamiton or pyrethroids;
[0097] antifungal agents, in particular the compounds belonging to
the imidazole class such as econazole, ketoconazole or miconazole
or their salts, the polyene compounds, such as amphotericin B, the
compounds of the allylamine family, such as terbinafine, or
octopirox;
[0098] antiviral agents such as acyclovir;
[0099] steroidal anti-inflammatory agents such as hydrocortisone,
betamethasone valerate or clobetasol propionate, or nonsteroidal
anti-inflammatory agents such as, for example, ibuprofen and its
salts, diclofenac and its salts, acetylsalicylic acid,
acetaminophen or glycyrrhizic acid;
[0100] anaesthetic agents such as lidocaine hydrochloride and its
derivatives;
[0101] antipruritic agents such as thenaldine, trimeprazine or
cyproheptadine;
[0102] keratolytic agents such as .alpha.- and
.beta.-hydroxycarboxylic or .beta.-ketocarboxylic acids, their
salts, amides or esters and more particularly hydroxy acids such as
glycolic acid, lactic acid, salicylic acid, citric acid and, in
general, fruit acids and 5-n-octanoylsalicylic acid;
[0103] anti-free-radical agents, such as a-tocopherol or its
esters, superoxide dismutases, certain metal chelators or ascorbic
acid and its esters;
[0104] antiseborrhoeic agents such as progesterone;
[0105] antidandruff agents such as octopirox or zinc
pyrithione;
[0106] anti-acne agents such as retinoic acid or benzoyl
peroxide.
[0107] Accordingly, according to a specific embodiment, the
composition according to the invention also comprises at least one
agent chosen from antibacterial, antiparasitic, antifungal,
antiviral, anti-inflammatory, antipruritic, anaesthetic,
keratolytic, anti-free-radical, antiseborrhoeic, antidandruff and
anti-acne agents, and/or agents reducing skin differentiation
and/or proliferation and/or pigmentation.
EXAMPLES
[0108] Monoclonal Antibodies:
[0109] The murine monoclonal antibodies G36-19, F28-27 and B17-21
(IgG1) which are specific for the polypeptide of the invention,
corneodesmosine, form part of a series of antibodies directed
against epidermal differentiation antigens, produced after
immunization of a mouse with a human plantar horny layer
homogenate, and then characterized. The ascites of the monoclonal
antibody MOPC-21 (IgG1) (Sigma Chemical Co., St Louis, Mo.) was
used as 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.
[0110] Isolation and Characterization of the Polypeptide:
[0111] Sequential Protein Extraction:
[0112] Starting with mammary skin (obtained from reductive
mammaplasty), the epidermis was mechanically separated from the
dermis after heat treatment of the skin for 5 minutes at 56.degree.
C. in a phosphate buffer, and then sequentially homogenized, at
4.degree. C., in equal volumes of the following buffers (three
times in each buffer: i) TE buffer: 40 mM Tri.sctn.-HCl, pH 7.5; 10
mM ethylenediaminetetraacetate (EDTA); 0.25 mM
phenylmethylsulphonyl fluoride and 2 pg/ml of each of the following
inhibitors: aprotinin, pepstatin A and leupeptin; ii) TENP40
buffer: TE containing a detergent, Nonidet P-40 at 0.5%; iii) TEU
buffers: TE containing various 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 collected. The first
supernatant from each extraction was kept at -30.degree. C. until
the time of use. Finally, the pellet corresponding to the last
extraction in the buffer containing 8 M urea was homogenized in
TUDTT buffer: 35 mM Tris-HCl, pH 6.8; 8 M urea; 50 mM
dithiothreitol; 5% glycerol; 0.25 mM phenylmethylsulphonyl fluoride
and 2 .mu.g/ml of each of the same inhibitors, incubated for 30
minutes at 95.degree. C. and centrifuged as above. The protein
concentrations were measured using the Pierce system (Coomassie
Plus protein assay, Pierce Chemical Co., Rockford, Ill.).
[0113] Electrophoresis of the Proteins and Immunodetection:
[0114] The proteins were separated by polyacrylamide gel
electrophoresis in the presence of SDS (SDS-PAGE) in 7.5 or 10%
acrylamide gels or by two-dimensional electrophoresis in the
presence of urea, using the Pharmacia system (PhastSystem.TM.;
Pharmacia LKB). An isoelectric focusing (IEF) or a non-equilibrium
pH gel electrophoresis (NEpHGE) was carried out in the first
dimension, and then an SDS-PAGE was carried out in the second
dimension with 12.5% polyacrylamide gels. For the two-dimensional
electrophoreses, the proteins in 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%
.beta.-mercaptoethanol. The marker proteins as well as the
two-dimensional electrophoresis standards (2-D SDS-PAGE
standards.TM.) from Bio-Rad were used as molecular weight markers
or as isoelectric point references.
[0115] After electrophoresis, the proteins were stained with
Coomassie blue or with the aid of silver nitrate (silver stain plus
kit Bio-Rad Lab.), or transferred to a reinforced nitrocellulose
membrane (Schleicher & Schuell, Dassel, Germany). The membranes
were then stained with Ponceau red or with Protogold (British
BioCell International, Cardiff, UK) and immunodetected with
monoclonal antibodies, as described above. The reactivities were
revealed with the ECL.TM. kit from Amersham (ECL.TM. Western
blotting kit, Amersham International, Aylesbury, UK), according to
the manufacturer's protocol.
[0116] Results:
[0117] The polypeptide of 52-56 kDa was detected in the extracts in
TE and TEU buffers (starting from a urea concentration equal to 6
M), but not in the sequential extracts in TENP40 and TUDTT buffers.
Furthermore, if the extract in TE buffer was centrifuged for 30
minutes at 100,000.times.g, the polypeptide was completely present
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 the extraction was complete
in each of the steps. The control experiments carried out in the
absence of primary antibody or with the antibody MOPC-21 always
proved to be negative. It is probable that the immunodetected forms
of polypeptides of low molecular weight were not products of
degradation generated during the extraction steps, since their
proportion did not vary from one preparation to another and since
the extractions were always carried out in the presence of protease
inhibitors.
[0118] Human Corneodesmosine is a Component of the Horny
Envelopes:
[0119] In order to confirm, at the biochemical level, that the
corneodesmosine is covalently linked to the horny envelopes,
fragments, generated by proteolysis of envelopes highly purified
from human plantar epidermis, were analysed with the monoclonal
antibody G36-19.
[0120] Preparation and Analysis of the Horny Envelopes:
[0121] Human horny envelopes were purified from the plantar horny
layer and mammary epidermis, as described above. Briefly, the
samples were extracted by repeated boiling, with vigorous stirring,
in a solution containing 2% SDS (w/v) and dithiothreitol at the
concentration of 25 mM, and than at 37.degree. C. for 72 hours in a
solution containing urea at the concentration of 8 M and
dithiothreitol at the concentration of 25 mM. The envelopes
extracted in urea were sedimented and then suspended in a solution
containing 0.1% SDS, glycine at the concentration of 192 mM and
Tris at the concentration of 125 mM. Finally, they were
electrodialysed against this same buffer for 72 hours. The purified
horny envelopes were then collected by centrifugation, washed with
distilled water and counted. They were analysed morphologically,
and then by indirect immunofluorescence, as described in the prior
art. Digestion with protease V8 and immunodetection of the
proteolysis products were carried out as described in the prior
art.
[0122] Results:
[0123] The envelopes were incubated for increasing periods with
protease V8, and the fragments produced were separated by SDS-PAGE
and immunodetected. The monoclonal antibody G36-19 strongly
labelled a large number of bands having an apparent molecular
weight greater than 50 kDa, some of which were located at the upper
limit of the gel. This indicates that the corneodesmosine is
incorporated inside the large-sized heteropolymeric structures
resulting from the proteolysis of the envelope. Several bands
stained with 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 bonds between
corneodesmosine and other constituents of the horny envelopes.
[0124] None of the fragments produced by dig estion of horny
envelopes purified from mammary epidermis was immunodetected by the
monoclonal antibody G36-19. In agreement with this result, these
envelopes were a lot less labelled, and only at their periphery,
when they were analysed with the monoclonal antibody G36-19 in
indirect immunofluorescence.
[0125] Human Corneodesmosine of 52-56 kDa is a Basic
Phosphoprotein:
[0126] To determine the isoelectric point of corneodesmosine, human
epidermis was extracted directly in a buffer containing a detergent
(TENP40 buffer), and the proteins extracted were separated by
two-dimensional electrophoresis (NEpHGE/SDS-PAGE) and
immunodetected with the monoclonal antibody G36-19. The
corneodesmosine of 52-56 kDa showed a basic isoelectric point
greater than 8. This result was confirmed by immunodetection after
IEF/SDS-PAGE separation. The protein immunodetected by the
monoclonal antibody G36-19 was not visible after Coomassie blue
staining, even when 100 .mu.g of proteins in the extract in TENP40
buffer were loaded on the gel. This therefore suggests that the
corneodesmosine of 52-56 kDa is a quantitatively minor protein,
representing less than 0.1% of the proteins extracted. Furthermore,
the immunoreactive protein migrated in the form of several
juxtaposed spots, suggesting the presence of post-translational
modifications. To analyse the possible phosphorylation of human
corneodesmosine, the latter was affinity-purified on a Sepharose
matrix (HiTrap-NHS, Pharmacia) activated with N-hydroxysuccinimide
groups and then coupled with the monoclonal antibody G36-19. Next,
it was analysed by immunotransfer with antibodies specific for
phosphoserine or for phosphotyrosine. One of the five
anti-phosphoserine monoclonal antibodies used immunodetected
corneodesmosine in a specific manner. In contrast, neither the
monoclonal antibody PY20 nor an antiserum, directed against
phosphotyrosine, recognized corneodesmosine.
[0127] Human Corneodesmosine of 52-56 kDa is a Glycoprotein:
[0128] Affinodetection with Lectins:
[0129] The corneodesmosine of 52-56 kDa was partially purified from
an extract in TENP40 buffer by affinity chromatography. The bound
corneodesmosine was eluted with 3% SDS, deposited on an
electrophoresis gel and transferred onto nitrocellulose membranes.
The membranes were incubated in a blocking buffer; and then with
biotinylated lectins sold by Pierce Chemical Co., diluted to 1
.mu.g/ml: agglutinin from Pisum sativum (PSA), from wheat germ
(WGA), from Ricinus communis (RCA), from Dolichos bifluorus (DBA)
and from Arachis hypogaea (PNA). After rinsing, the lectins were
detected with streptavidin labelled with peroxidase (diluted
1/400,000), and the ECL.TM. kit from Amersham, as described
above.
[0130] Deglycosylation Experiments:
[0131] The extract in TENP40 buffer (10 .mu.g) was kept boiling for
3 minutes in 20 .mu.l of a sodium phosphate buffer at pH 7.2
containing 1% (w/v) of SDS. Nonidet P40 and EDTA were added in
order to obtain a final concentration of, respectively, 1% and 20
mM. 2.4 units of N-glycosidase F (EC 3.2.2.18, Boehringer Mannheim)
were added and the reaction mixture was incubated at 37.degree. C.
for 6 hours. The proteins (34 .mu.g) of an extract in TENP40 buffer
were also incubated with 5 mU of
endo-.alpha.-N-acetylgalactosaminidase (EC 3.2.1.97) at 37.degree.
C. for 6 hours, in the presence or otherwise of 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 positive control, was tested
by SDS-PAGE and with the biotinylated lectins.
[0132] The treated and untreated samples were separated by
electrophoresis and analysed by immunodetection and
affinodetection, as described above.
[0133] Chromatography on Concanavalin A-Sepharose
[0134] An extract in TENP40 buffer was directly injected, with a
flow rate of 0.5 ml/minute, into a Concanavalin A Sepharose 4B
column (ConA Sepharose, Sigma) which had been previously
equilibrated with washing buffer: 20 mM Tris-HCl, 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-.alpha.-D-mannopyranoside (Sigma) diluted to 0.5 M in
washing buffer. The proteins were then separated by SDS-PAGE and
analysed as described above.
[0135] Results:
[0136] The proteins of an extract in TENP40 buffer, containing the
52-56 kDa corneodesmosine, were treated with various glycosidases
and analysed in immunotransfer with two monoclonal antibodies
directed against the polypeptide. The staining of the total
proteins of the extract did not show apparent degradation during
the incubation. The treatment with N-glycosidase F induced a
reduction of about 5 kDa in the apparent molecular weight of the
corneodesmosine. This result very strongly suggests an
N-glycosylation. In contrast, the treatments with
endo-.alpha.-N-acetyl-galactosaminidase and/or neuraminidase did
not modify the migration of corneodesmosine. To confirm this
result, human corneodesmosine was affinity-purified and analysed
with the aid of biotinylated lectins. Wheat germ agglutinin (WGA)
was strongly bound to the purified protein, unlike the other
lectins tested which did not or very weakly recognized it.
Furthermore, corneodesmosine bound to Concanavalin A coupled to a
Sepharose matrix. It was possible to elute it with methyl
.alpha.-D-mannopyranoside, a sugar which is specific for this
lectin, at the concentration of 0.5 M, which proves the specificity
of the binding.
[0137] Purification of Corneodesmosine:
[0138] After dermoepidermal cleavage, the epidermis was homogenized
in TEA buffer: 40 mM Tris-HCl, pH 7.5; 10 mM EDTA; 10 .mu.g/ml
aprotinin and 4-(2-aminoethyl)benzenesulphonyl 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 whose pore diameter is 0.45 .mu.m (Puradisc.TM. 25 AS;
Whatman, Clifton, N.J.), and injected with a flow rate of 0.3
ml/minute on an anion-exchange column (Hi Trap Q, Pharmacia LKB)
which had been equilibrated in the washing buffer: 20 mM Tris-HCl,
pH 7.5. The proteins not retained (that is to say about 5% of the
total proteins of the extract) were directly injected at the same
flow rate into an affinity column prepared as follows: about 2 mg
of monoclonal antibody F28-27 were coupled to 1 ml of Sepharose 4B
matrix activated by N-hydroxysuccinimide groups (HiTrap-NHS), as is
recommended by the manufacturer Pharmacia LKB. The column was
washed exhaustively with a flow rate of 1 ml/minute with the
washing buffer in the presence and then in the absence of 1 M NaCl.
The immunoadsorbed proteins were eluted with 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
analysed in immunotransfer with the monoclonal antibody G36-19, or
with the control monoclonal antibody MOPC-21. The fractions
containing the proteins thus eluted were mixed, and then
lyophilized. The proteins of the lyophilizate were analysed as
described above, by one- or two-dimensional electrophoresis. The
corneodesmosine was then cut out of the gel so as to be
sequenced.
[0139] Results:
[0140] The corneodesmosine of 52-56 kDa was purified by an
anion-exchange chromatography followed by affinity chromatography.
The entire corneodesmosine extracted is specifically bound to the
affinity column, from which it was possible for it to be eluted in
the presence of glycine. The staining with Protogold of the
transferred proteins shows the high degree of purification
obtained, since the corneodesmosine is highly predominant 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.
A two-dimensional gel analysis confirmed the basic isoelectric
point of the purified corneodesmosine and showed that it is the
only protein eluted having an apparent molecular weight of 52-56
kDa. The corneodesmosine thus purified was separated by
electrophoresis and the gel band containing this protein was cut
out and then the protein was sequenced.
[0141] Instability of the Purified Polypeptide:
[0142] During its numerous trials to purify corneodesmosine, the
applicant encountered two major problems: (1) the low proportion of
this protein among the epidermal proteins and (2) its instability,
possibly due to its high sensitivity to the action of
proteases.
[0143] Different tests were used to try to stabilize the
corneodesmosine: addition of zinc (inhibitor of the chymotryptic
enzyme of the horny layer), reducing agents (.beta.-mercaptoethanol
or dithiothreitol), denaturing agents (SDS), glycerol, inhibitors
specific for serin proteases (PMSF, aprotinin) or a cocktail of
inhibitors of various proteases (aprotinin, leupeptin, pepstatin,
benzamidine, phenanthroline, PMSF) or suppression of the
freezing/thawing steps. Finally, the absence of freezing/thawing
steps, the presence of 2% SDS or the presence of the cocktail of
inhibitors, and preferably a combination of these conditions proved
to be the most effective for stabilizing corneodesmosine.
[0144] Sequencing of the Purified Polypeptide:
[0145] For the sequencing, the bands which correspond to
corneodesmosine or to fragments of corneodesmosine (52-56 kDa, 45
kDa), identified on the electrophoresis gels were cut out. The
proteins were then directly digested in the gel with the
endopeptidase lys-C (EC 3.4.99.30) and the peptides generated were
purified by HPLC using a DEAE-C18 column. The peptides thus
selected were sequenced by the method of Edman degradation cycles
on a Procise Sequencer apparatus from the company Applied
Biosystems, according to the supplier's instructions.
[0146] Results:
[0147] No sequencing from the NH.sub.2-terminal end was possible,
suggesting the protection of the protein at this end.
[0148] Internal sequencings made it possible to establish the
sequence of two fragments, namely:
[0149] A: Lys Ser Tyr Gly Gly Tyr Glu Val Val Gly Gly Ser Ser Asp
Ser Gly
[0150] B: Lys Ile Tyr Pro Val Gly Tyr Phe Thr Lys
[0151] This result confirms that obtained by the molecular biology
techniques which allowed the deduction of the amino acid sequence
of the polypeptide of the invention from the nucleotide
sequence.
[0152] Construction of a Human Epidermis Expression Library:
[0153] The cloning of the cDNA for human corneodesmosine could not
be carried out using preexisting expression libraries. Indeed, the
latter were produced either from mouse epidermis, or from human
keratinocytes cultured in a monolayer, conditions which do not
allow the expression of the genes characteristic of epidermal
terminal differentiation. The applicant therefore constructed a
human epidermal expression library.
[0154] Extraction of poly(A).sup.+Ribonucleic Acids (RNA) from
Human Epidermis:
[0155] It was carried out using samples of mammary skin, surgical
waste obtained after plastic surgery. The subcutaneous adipose
tissue was dissected and then the skin pieces were cut up with the
aid of a dermatome and incubated for 2 hours at 37.degree. C., in a
trypsin solution (solution A, Gibco BRL), epidermal face facing
upwards. The dermo-epidermal cleavage was then carried out with
tweezers. The epidermal sheets obtained were rinsed in phosphate
buffer (PBS, pH 7.4). The extraction of the poly(A).sup.+ RNAs was
carried out according to the protocol proposed in the kit "mRNA
purification kit" marketed by Dynal (Oslo), after homogenization of
the sheets with the aid of a "Turax", directly in the buffer
provided (lysis/binding buffer). The principle of this kit uses the
affinity of the messenger RNA (mRNA) containing a poly(A) end for
the magnetic beads coated with oligo(dT).sub.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).sup.+
RNAs eluted by incubating the beads at 65.degree. 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,
Calif.).
[0156] Construction of the Library:
[0157] The library was constructed with the kit "ZAP Express cDNA
Gigapack II Gold cloning kit" marketed by Stratagene (La Jolla,
Calif.), following the protocol proposed by the supplier. The
complementary DNAs (cDNA) were synthesized from 2 .mu.g of poly(A)+
RNA, with the Moloney murine leukaemia virus reverse transcriptase
(MMLV-RT), using oligo(dT)18 primers comprising a restriction site
for the enzyme XhoI and in the presence of 5-methylcytidine
triphosphate. The synthesis of the second strand was carried out by
E. coli DNA polymerase I, and the ends of the cDNAs ere made blunt
with recombinant pfu polymerase. After addition of EcoRI adaptors
(T4 DNA ligase), and phosphorylation of the 51 ends (T4
polynucleotide kinase), the cDNAs were subjected to digestion with
the enzyme XhoI. The selection of the cDNAs having a size greater
than 500 base pairs was carried out with a Sephacryl S-500 column.
The ligation of the cDNAs with the two arms of the ZAP Express
phage was carried out with T4 phage ligase. The encapsulation of
the recombinant phage was carried out with the extracts "Gigapack
II Gold Packaging Extract" provided by Stratagene. The strain XL-1
blue MRF' of E. coli was used for the titration and the spreading
of the library.
[0158] Results:
[0159] This library was prepared from 2 .mu.g of mRNA, extracted
from a fragment of human epidermis within the hours following its
collection, by unidirectional cloning into the ZAP Express phage.
It consists of about 2.times.10.sup.5 independent clones.
[0160] Cloning of the Complementary Deoxyribonucleic Acid (cDNA)
Encoding Corneodesmosine:
[0161] Immunological Screening:
[0162] The immunological screening, carried out without a prior
amplification step, was performed on nitrocellulose membranes
(Schleicher & Schuell, Dassel, Germany), incubated in an
isopropyl-1-thio-.beta.-D-ga- lacto-pyranoside (IPTG, Stratagene)
solution at 10 mm for 10 minutes, dried and incubated for 4 h at
37.degree. C. on the spread library. The immunological screening
procedure was carried out as described in the prior art with a
cocktail of the three monoclonal antibodies G36-19, F28-27 and
B17-21, used at the respective concentrations of 0.2, 0.2 and 2
.mu.g/ml. The positive clones were isolated by conventional
techniques and tested with each of the three monoclonal antibodies
separately.
[0163] Sequencing:
[0164] 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), and then sequenced
at the ends of the inserts, with primers T3 and M13 (Stratagene),
using a Perkin-Elmer kit (ABI PRISM Dye Terminator cycle sequencing
kit, Perkin Elmer, Foster city, CA). Comparison of the sequences
obtained with international databases was carried out with the
Blast program.
[0165] Results:
[0166] Given that the library was not amplified beforehand, the
screening made it possible to isolate 5 independent clones. The
human cDNA fragments contained in the clones were partially
sequenced from the ends, which made it possible to show that they
were all overlapping and therefore all encoded fragments of the
same protein. The 5 clones were also tested with each of the three
monoclonal antibodies separately. Three clones (1.1, 4.4 and 5.1)
proved to be reactive with the three monoclonal antibodies, the
clone 5.5 with two monoclonal antibodies (F28-27 and G36-19) and
finally the clone 1.2 was-recognized only by the monoclonal
antibody F28-27. These results conclusively demonstrate that the
three monoclonal antibodies recognize the same protein, thus
confirming what had been strongly suggested by previous biochemical
studies. These results also make it possible to order on the
molecule the epitopes recognized by each of the monoclonal
antibodies. Indeed, given that the clones were constructed by
reverse transcription starting 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 closest to this
end, followed by the epitope for G36-19 and finally that for
B17-21. Enzymatic restriction analysis of the size of these
different cDNA fragments confirmed that the 3 clones recognized by
the 3 monoclonal antibodies have the Iongest sequences (from 2 to
1.5 kb), whereas that which only F28-27 recognizes is the shortest
(1 kb), the last, recognized by 2 monoclonal antibodies, having an
intermediate size of 1.3 kb.
[0167] Accordingly, all or part of the cDNA encoding human
corneodesmosine was cloned, a: protein characterized by the 3
monoclonal antibodies B17-21, G36-19 and F28-27 which recognize
different epitopes, ordered on the protein in the order indicated,
from the NH.sub.2 end to the COOH end.
[0168] The partial sequences of each clone, compared with the
sequences included in the international databanks, revealed a 99%
identity with the sequence of the S gene, registered in the GenBank
databank 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 are derived from an alternative choice of the polyadenylation
site at the level of the noncoding 3' 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 the 762-1044 and 1044-stop codon zone.
Finally, the clone 1.1, which contains the longest coding sequence,
starts at nucleotide 348.
[0169] Cloning of the Complete cDNA Encoding Corneodesmosine:
[0170] The preceding analyses showed that the clones obtained did
not cover the cDNA part extending from nucleotide 1 to nucleotide
347. The applicant therefore had to clone this missing part.
[0171] 80 ng of poly(A).sup.+ RNA extracted from human epidermis as
described above were subjected to reverse transcription starting
with random primers, using the kit "SuperScript" marketed by Gibco
BRL. The amplification of the 5' end of the cDNA for
corneodesmosine was carried out starting with 1/20 of the first
strand synthesis reaction, with, upstream, an oligonucleotide
corresponding to the sequence 2-20(GenBank L20815) and also
comprising an SpeI adapter, and, downstream, an oligonucleotide 25
complementary to the sequence 1017-1035. The polymerase chain
reaction (PCR) conditions were the following: 3 minutes at
94.degree. C. followed by 35 cycles with 80 sec at 94.degree. C.,
80 sec at 57.degree. C. and 2 minutes at 72.degree. C. The single
fragment obtained, purified after agarose-TBE gel electrophoresis,
digested with SpeI and EcoNI (nucleotides 2 to 1003), was cloned
into the plasmid pBK-CMV-1.1 (isolated by screening of the library)
digested with the same enzymes, which made it possible to arrive at
the plasmid pS11. The complete cDNA contained in the plasmid pS11
was sequenced at least 3 times up to nucleotide 1700. Comparison
with the sequence previously published revealed four localized
differences which were analysed at the genomic level.
[0172] Genomic Analysis of Corneodesmosine:
[0173] Four localized differences were observed 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 D. D.,
P.N.A.S. usa, Vol. 90, pp. 9470-9474, October 1993). These four
localized differences were analysed by PCR, at the genomic level
starting with ten control samples of DNA extracted from human blood
according to conventional methods.
[0174] 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 conventional conditions. The DNA fragment obtained for each
sample was subjected to digestion with BsiMI on the one hand and
NciI on the other. Electrophoresis on a 3% NuSieve GTG gel (FMC,
Rockland, Me.) was carried out in TBE buffer.
[0175] The study of the sequence at position 66 was carried out
according to the ASA (allele specific amplification) method. 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
at position 66 either with A or with T. The PCR reactions were
carried out with 0.5 .mu.g of genomic DNA, 2.5 pmol of each
oligonucleotide, and 0.5 U of Taq polymerase (Appligene). The
amplification conditions are 94.degree. C., 2 minutes and then 30
cycles with 50 seconds at 94.degree. C., 1 minute at 56.degree. C.
and 2.5 minutes at 72.degree. C.
[0176] Analysis of the bases at position 1118 and 1236 was carried
out after amplification with a single oligonucleotide pair: 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 BsmI or Hin6 I for
the respective study of positions 1118 and 1236 and then separated
by electrophoresis on a 3% NuSieve gel.
[0177] Results:
[0178] The insertion of a guanine at position 1514 causes a reading
frame shift and moves the stop codon from position 1523 to position
1603. The protein produced by this sequence differs at the level of
the last two amino acids (I and S) which are replaced by the
sequence:
[0179] Asp Ile Leu Ala Gln Val Lys Pro Leu Gly Pro Gln Leu Ala Asp
Pro Glu Val Phe Leu Pro Gln Gly Glu Leu Leu Asp Ser Pro
[0180] which corresponds to an addition of 27 amino acids relative
to the published sequence. The 5 cDNAs previously isolated all
exhibited this insertion; the study was extended to other
individuals. Ten control samples of human genomic DNA, obtained
from ten individuals, were amplified in this region and analysed by
enzymatic restriction. Indeed, the published sequence corresponds
to the presence of a BsiMI restriction site at position 1510 which
disappears in the sequence comprising an additional guanine,
whereas an NciI site appears at 1512. The results are the same for
the 10 individuals, namely that only the NciI 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.
[0181] Three other localized differences, of the substitution type,
were found between the clones and the published sequence. The
analysis of the ten control genomic samples by PCR and enzymatic
restriction or by ASA, showed that polymorphism of the gene is
involved. The applicant has identified at position 66 a
conservative mutation T/A (Leu-Met), at position 1118 a
polymorphism A/G, corresponding to a silent mutation Ala-Ala and,
finally, at 1236 a polymorphism T/G corresponding to a
nonconservative mutation Ser-Ala.
[0182] Production of Recombinant Corneodesmosine:
[0183] The complete cDNA encoding corneodesmosine was isolated, in
the form of an Ec1136 I/NotI fragment of 2106 base pairs, from the
plasmid pS11. The subcloning was carried out into the vector pCDNA
amp. (In Vitrogen) previously digested with EcoRV and NotI, leading
to the plasmid p14-9.
[0184] The in vitro translation of corneodesmosine was carried out
with the kit "TNT T7 Quick coupled transcription/translation
system" marketed by Promega starting with the plasmid p14-9. This
kit combines in one step the transcription with T7 phage RNA
polymerase with translation with a rabbit reticulocyte lysate.
These reactions were carried out with 0.6 .mu.g of plasmid in a
volume of 25 .mu.l, according to the manufacturer's
instructions.
[0185] Corneodesmosine was expressed in COS-7 cells after
transfection with the plasmid p14-9. The cells were transfected
according to the conventional DEAE-dextran protocol, supplemented
by the addition of chloroquine and a DMSO shock, using 1 .mu.g of
plasmid/3.times.10.sup.5 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, containing 10 mM EDTA and 0.5% NP40, and containing
a cocktail of protease inhibitors (Pharmingen, Inc.). After
SDS-PAGE separation and electrotransfer, the samples are
immunodetected, as described above.
[0186] Results:
[0187] The new complete sequence of the protein predicts a
molecular weight of 51.45 kDa, in the absence of any
post-translational modification. The corneodesmosine synthesized in
vitro, in the absence of microsomal membranes, migrates with an
apparent molecular weight of about 60 kDa. This shows that this
protein has an aberrant migration in SDS-PAGE gel. Comigration with
the predominant epidermal form extracted in a low-ionic strength
buffer demonstrates that the latter corresponds to a fairly
extensively truncated form of the protein, all the more so since
the applicant has shown that this epidermal form is
glycosylated.
[0188] The transfection of COS-7 cells with the plasmid 14-9
reveals an immunoreactive protein which also migrates in the region
of 60 kDa. The same results were obtained with the human
neuroepithelioma line CHP 126, transfected by electroporation. All
these results strongly suggest that in the epidermis,
corneodesmosine undergoes, prematurely and during its maturation, a
specific proteolysis.
Sequence CWU 1
1
2 1 529 PRT Human 1 Met Gly Ser Ser Arg Ala Pro Trp Met Gly Arg Val
Gly Gly His Gly 1 5 10 15 Met Met Ala Leu Leu Leu Ala Gly Leu Leu
Leu Pro Gly Thr Leu Ala 20 25 30 Lys Ser Ile Gly Thr Phe Ser Asp
Pro Cys Lys Asp Pro Thr Arg Ile 35 40 45 Thr Ser Pro Asn Asp Pro
Cys Leu Thr Gly Lys Gly Asp Ser Ser Gly 50 55 60 Phe Ser Ser Tyr
Ser Gly Ser Ser Ser Ser Gly Ser Ser Ile Ser Ser 65 70 75 80 Ala Arg
Ser Ser Gly Gly Gly Ser Ser Gly Ser Ser Ser Gly Ser Ser 85 90 95
Ile Ala Gln Gly Gly Ser Ala Gly Ser Phe Lys Pro Gly Thr Gly Tyr 100
105 110 Ser Gln Val Ser Tyr Ser Ser Gly Ser Gly Ser Ser Leu Gln Gly
Ala 115 120 125 Ser Gly Ser Ser Gln Leu Gly Ser Ser Ser Ser His Ser
Gly Ser Ser 130 135 140 Gly Ser His Ser Gly Ser Ser Ser Ser His Ser
Ser Ser Ser Ser Ser 145 150 155 160 Phe Gln Phe Ser Ser Ser Ser Phe
Gln Val Gly Asn Gly Ser Ala Leu 165 170 175 Pro Thr Asn Asp Asn Ser
Tyr Arg Gly Ile Leu Asn Pro Ser Gln Pro 180 185 190 Gly Gln Ser Ser
Ser Ser Ser Gln Thr Ser Gly Val Ser Ser Ser Gly 195 200 205 Gln Ser
Val Ser Ser Asn Gln Arg Pro Cys Ser Ser Asp Ile Pro Asp 210 215 220
Ser Pro Cys Ser Gly Gly Pro Ile Val Ser His Ser Gly Pro Tyr Ile 225
230 235 240 Pro Ser Ser His Ser Val Ser Gly Gly Gln Arg Pro Val Val
Val Val 245 250 255 Val Asp Gln His Gly Ser Gly Ala Pro Gly Val Val
Gln Gly Pro Pro 260 265 270 Cys Ser Asn Gly Gly Leu Pro Gly Lys Pro
Cys Pro Pro Ile Thr Ser 275 280 285 Val Asp Lys Ser Tyr Gly Gly Tyr
Glu Val Val Gly Gly Ser Ser Asp 290 295 300 Ser Tyr Leu Val Pro Gly
Met Thr Tyr Ser Lys Gly Lys Ile Tyr Pro 305 310 315 320 Val Gly Tyr
Phe Thr Lys Glu Asn Pro Val Lys Gly Ser Pro Gly Val 325 330 335 Pro
Ser Phe Ala Ala Gly Pro Pro Ile Ser Glu Gly Lys Tyr Phe Ser 340 345
350 Ser Asn Pro Ile Ile Pro Ser Gln Ser Ala Ala Ser Ser Ala Ile Ala
355 360 365 Phe Gln Pro Val Gly Thr Gly Gly Val Gln Leu Cys Gly Gly
Gly Ser 370 375 380 Thr Gly Ser Lys Gly Pro Cys Ser Pro Ser Ser Ser
Arg Val Pro Ser 385 390 395 400 Ser Ser Ser Ile Ser Ser Ser Ala Gly
Ser Pro Tyr His Pro Cys Gly 405 410 415 Ser Ala Ser Gln Ser Pro Cys
Ser Pro Pro Gly Thr Gly Ser Phe Ser 420 425 430 Ser Ser Ser Ser Ser
Gln Ser Ser Gly Lys Ile Ile Leu Gln Pro Cys 435 440 445 Gly Ser Lys
Ser Ser Ser Ser Gly His Pro Cys Met Ser Val Ser Ser 450 455 460 Leu
Thr Leu Thr Gly Gly Pro Asp Gly Ser Pro His Pro Asp Pro Ser 465 470
475 480 Ala Gly Ala Lys Pro Cys Gly Ser Ser Ser Ala Gly Lys Ile Pro
Cys 485 490 495 Arg Ser Ile Arg Asp Ile Leu Ala Gln Val Lys Pro Leu
Gly Pro Gln 500 505 510 Leu Ala Asp Pro Glu Val Phe Leu Pro Gln Gly
Glu Leu Leu Asp Ser 515 520 525 Pro 2 1590 DNA Human 2 atgggctcgt
ctcgggcacc ctggatgggg cgtgtgggtg ggcacgggat gatggcactg 60
ctgctggctg gtctcctcct gccagggacc ttggctaaga gcattggcac cttctcagac
120 ccctgtaagg accccacgcg tatcacctcc cctaacgacc cctgcctcac
tgggaagggt 180 gactccagcg gcttcagtag ctacagtggc tccagcagtt
ctggcagctc catttccagt 240 gccagaagct ctggtggtgg ctccagtggt
agctccagcg gatccagcat tgcccagggt 300 ggttctgcag gatcttttaa
gccaggaacg gggtattccc aggtcagcta ctcctccgga 360 tctggctcta
gtctacaagg tgcatccggt tcctcccagc tggggagcag cagctctcac 420
tcgggaagca gcggctctca ctcgggaagc agcagctctc attcgagcag cagcagcagc
480 tttcagttca gcagcagcag cttccaagta gggaatggct ctgctctgcc
aaccaatgac 540 aactcttagc gcggaatact aaacccttcc cagcctggac
aaagctcttc ctcttcccaa 600 acctctgggg tatccagcag tggccaaagc
gtcagctcca accagcgtcc ctgtagttcg 660 gacatccccg actctccctg
cagtggaggg cccatcgtct cgcactctgg cccctacatc 720 cccagctccc
actctgtgtc agggggtcag aggcctgtgg tggtggtggt ggaccagcac 780
ggttctggtg cccctggagt ggttcaaggt cccccctgta gcaatggtgg ccttccaggc
840 aagccctgtc ccccaatcac ctctgtagac aaatcctatg gtggctacga
ggtggtgggt 900 ggctcctctg acagttatct ggttccaggc atgacctaca
gtaagggtaa aatctatcct 960 gtgggctact tcaccaaaga gaaccctgtg
aaaggctctc caggggtccc ttcctttgca 1020 gctgggcccc ccatctctga
gggcaaatac ttctccagca accccatcat ccccagccag 1080 tcggcagctt
cctcggccat tgcgttccag ccagtgggga ctggtggggt ccagctctgt 1140
ggaggcggct ccacgggctc caagggaccc tgctctccct ccagttctcg agtccccagc
1200 agttctagca tttccagcag cgccggttca ccctaccatc cctgcggcag
tgcttcccag 1260 agcccctgct ccccaccagg caccggctcc ttcagcagca
gctccagttc ccaatcgagt 1320 ggcaaaatca tccttcagcc ttgtggcagc
aagtccagct cttctggtca cccttgcatg 1380 tctgtctcct ccttgacact
gactgggggc cccgatggct ctccccatcc tgatccctcc 1440 gctggtgcca
agccctgtgg ctccagcagt gctggaaaga tcccctgccg ctccatccgg 1500
gatatcctag cccaagtgaa gcctctgggg ccccagctag ctgaccctga agttttccta
1560 ccccaaggag agttactcga cagtccataa 1590
* * * * *