EP1114157A2 - Proteine regulatrice issue de keratinocytes humains - Google Patents

Proteine regulatrice issue de keratinocytes humains

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Publication number
EP1114157A2
EP1114157A2 EP99969420A EP99969420A EP1114157A2 EP 1114157 A2 EP1114157 A2 EP 1114157A2 EP 99969420 A EP99969420 A EP 99969420A EP 99969420 A EP99969420 A EP 99969420A EP 1114157 A2 EP1114157 A2 EP 1114157A2
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Prior art keywords
protein
nucleic acid
pke
sequence
keratinocytes
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EP99969420A
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German (de)
English (en)
Inventor
Michael Kramer
Michael Bechtel
Jeanette Reinartz
Birgit Schäfer
Reinhard Wallich
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Individual
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Individual
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Publication of EP1114157A2 publication Critical patent/EP1114157A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • the invention relates to an isolated polypeptide which is similar to or similar to a protein which occurs naturally in human keratinocytes and which is expressed more strongly in the activated state of the keratinocytes.
  • the protein also relates to an isolated nucleic acid which is such a polypeptide which is typical of human keratinocytes or protein encoded, and the use of this polypeptide and this nucleic acid for detecting, in particular diagnostic, and / or for therapeutic purposes or the use of reagents, in particular recombinant vector molecules and antibodies, against such molecules
  • drugs with a broad spectrum of activity such as locally or systemically administered glucocorticoids, vitamin A
  • glucocorticoids such as glucocorticoids, vitamin A
  • epidermal disorders such as the autoimmune dermatoses “Pemphigus vulgaris” and “Bulloses Pemphigoid” Acid derivatives, antimetabolites and cytostatics, or it is treated with more or less unspecific measures such as “dye therapy” or "light therapy”.
  • the known active substances or measures all have the disadvantage that they are not very specific and therefore naturally cause numerous side effects
  • the provision of more specific active ingredients has so far failed due to the fundamental problem that has existed for a long time in dermatology, that the number of cellular target molecules (target structures, targets), which act as a point of attack for a (specific) influence on the cellular metabolism - especially under medical or also cosmetic Viewpoints - could serve in epidermal keratinocytes is narrowly limited
  • the object of the present invention is therefore to provide new target structures in epidermal keratinocytes which can serve as a point of attack for diagnostics, therapeutics, cosmetics or in general for influencing the cellular metabolism
  • One solution to this problem is to provide a protein of the type mentioned at the outset, which upregulates activated keratinocytes, ie is expressed or produced more and is kept at a higher concentration level, and that either in the sequence listing SEQ ID NO: 2 or in the sequence listing SEQ ED NO: 3 amino acid sequence shown or an allele or derivative formed by amino acid substitution, deletion, insertion, or inversion from one of these two amino acid sequences.
  • the polypeptide with the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 is shown in hereinafter also referred to as protein pKe # 122
  • a further solution to this problem consists in the provision of an isolated nucleic acid which encodes a protein which is similar to or similar to a protein which occurs naturally in human keratinocytes and which is expressed more intensely in the activated state of the keratinocytes, and which is either that described in the sequence listing SEQ ID NO: 1 or the nucleotide sequence shown in the sequence listing SEQ ID NO: 4 or a nucleotide sequence complementary to one of these two or a partial sequence of one of these two shown or complementary nucleotide sequences or a nucleotide sequence hybridizing wholly or partially with one of the aforementioned nucleotide sequences, with these two sequence listing instead of "T” can also stand for "TJ".
  • This group of nucleic acids or nucleotide sequences according to the invention also includes, in particular, splice variants and sense or antisense oligonucleotides which correspond to those in the sequence listing SEQ ID NO: 1 or with hybridize the nucleotide sequence shown in the sequence listing SEQ ID NO: 4, preferably identical to or complementary to at least one of these two
  • the invention consequently also includes proteins or polypeptides of the type mentioned at the outset, which have an amino acid sequence which results from such a splice variant, in particular from the splice variant of an mRNA which corresponds to that in the sequence listing SEQ ID NO: 1 or in the sequence listing SEQ ID NO: 4 indicated nucleotide sequence is identical or complementary to it
  • the sense or antisense oligonucleotides according to the invention comprise at least 6, preferably 8 to 25 nucleotides
  • hybridized refers to the hybridization methods known in the prior art under customary, in particular under highly stringent hybridization conditions, the person skilled in the art chooses the specific hybridization parameters based on the nucleotide sequence used and his general specialist knowledge (see Current Protocols in Molecular Biology, Vol 1, 1997, John Wiley & Sons Ine, Suppl 37, Cha ⁇ ter 4 9 14)
  • nucleic acid (s) according to the invention can be obtained both from a natural source and also synthetically or semi-synthetically. In practice, their implementation as cDNA has proven particularly effective
  • the polypeptide which has the amino acid sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 and which is encoded by the nucleic acid shown in the sequence listing SEQ ID NO: 1 or in the sequence listing SEQ ID NO: 4, and which is hereinafter referred to as protein pKe # 122, is upregulated in human epidermal keratinocytes, namely expressed more (produced) and kept at a concentration level that is significantly higher than in the initial state when these cells are in the "activated” state, ie in the state of proliferation and / or among other things Migration, eg after an accident-related skin injury or in the autoimmunologically triggered bullose dermatoses "Pemphigus vulgaris" (triggered by autoantibodies against desmosomes) and "Bulloses pemphigoid” (triggered by autoantibodies against hemidesmosomes)
  • the activated state of Human epidermal keratinocytes are also expressed in an increased expression of the known activation markers
  • the pKe # 122 protein has a serine / threonine kinase motif, several (four) tyrosine kinase phosphorylation motifs and a kinase domain with an ATP binding site. It is obviously involved in signaling processes, is very likely to have a serine / threonine kinase function and is active a presumed role in the formation of cell-cell and / or cell-matrix connections and / or of desmosomes and / or of hemidesmosomes
  • Serine / threonine kinases also play an important role in the formation of hemidesmosomes (cf. Mainiero F, Pepe A, Wary KK, Spinardi L, Mohammadi M, Schlessinger J Giancotti FG, 1995 Signal transduction by the alpha6beta4 integrm: distinct beta4 subunit sites mediale recruitment of Shc / Grb2 and association with the cytoskeleton of hemidesmosomes.EMBO J 14 4470-4481)
  • the invention further relates to recombinant DNA vector molecules which comprise a nucleic acid according to the invention and which have the ability to express in a prokaryotic a protein which occurs in human keratinocytes and is expressed in the activated state of the keratinocytes, in particular the pKe # 122 protein or eukaryotic cell.
  • the DNA vector molecules are preferably the plasmid pUEX-1 and / or the plasmid pGEX-2T and / or the plasmid pBK-CMV and / or the plasmid pHR 2 (a derivative of Bluescript KS [Stratagene, Heidelberg], contains the human keratin 14 promoter), since these vectors have proven to be very suitable in practice
  • Particularly suitable eukaryotic cells are cells from cell cultures, for example COS cells, but the cell in question can equally well be part of a living organism, for example a transgenic mouse
  • the invention therefore also encompasses transformed host cells which contain a nucleic acid according to the invention which is coupled to an activatable promoter which is contained in these cells naturally or as a result of recombination and which (as a result) has the ability to express a keratinocyte in human protein that occurs and is increasingly expressed in the activated state of the keratinocytes, in particular the protein pKe # 122
  • the invention further relates to the use of a nucleic acid according to the invention or a vector molecule according to the invention for the production of transgenic mammals, in particular mice or rats
  • the transfectants according to the invention open up the possibility for research and development work with regard to a further elucidation of the changes in cell morphology induced by the protein pKe # 122 and cellular basic functions such as proliferation, adhesion, migration and differentiation, in particular with a view to answering the questions Question whether the protein pKe # 122 itself has a "pathogenic" activity
  • the present invention also relates to a reagent for the indirect detection of a protein which occurs in human keratinocytes and which is expressed more strongly in the activated state of the keratinocytes, in particular the protein pKe # 122, this reagent being characterized in that it comprises at least one nucleic acid according to the invention.
  • the protein pKe # 122 and the polypeptides related to it that is to say with the amino acid sequence shown in the sequence listing SEQ ID NO: 2 or in the sequence listing SEQ ID NO: 3, namely the polypeptides which are obtained by substitution, deletion, insertion and / or inversion of a these amino acid sequences can be derived according to SEQ ID NO: 2 or SEQ ID NO: 3, or which have an amino acid sequence which results from a splice variant of an mRNA which corresponds to the nucleotide sequence according to the sequence listing SEQ ID NO: 1 or to the nucleotide sequence according to the sequence listing SEQ ID NO: 4 or with a partial sequence of these nucleotide sequences is identical or complementary to it, or at least hybridized, offer diverse application possibilities in the field of dermatological research and development.
  • antibodies against these polypeptides that is to say with the amino acid sequence shown in the sequence listing SEQ ID NO: 2 or in the sequence listing SEQ ID NO: 3, namely the polypeptides which are
  • the invention consequently also includes the use of such a protein or polypeptide for the production of an (monoclonal, polyclonal or recombinant) antibody against this polypeptide, said antibody itself and also its use for the diagnostic and / or therapeutic treatment of dermatological diseases, for cosmetic purposes Treatment of the epidermis and for the diagnostic, therapeutic and / or cosmetic treatment of other tissues or organs expressing the protein pKe # 122
  • sense and / or antisense oligonucleotides are also suitable as active ingredients for pharmacotherapy (cf. G Hartmann et al 1998 antisense
  • the present invention therefore also relates to the use of sense or antisense oligonucleotides according to the invention for diagnostic and / or therapeutic purposes Treatment, in particular of dermatological diseases, or for cosmetic treatment, in particular of the epidermis
  • a technically and economically important possibility of using a polypeptide according to the invention or a nucleic acid according to the invention is that with the aid of such a molecule, a very large number of available substances can be selected from a very large number of available substances in a screening process, which are specific to the nucleic acid in question or that Binding the relevant polypeptide These substances can then serve as the starting material (lead structure) for the development of pharmacologically usable substances and thus offer the prerequisites for the development of alternative pharmaceuticals for diagnosis and therapy, in particular the dermatological diseases mentioned at the beginning
  • the invention also relates to the use of a polypeptide according to the invention or a nucleic acid according to the invention for the identification of pharmacologically usable substances which bind to the polypeptide or the nucleic acid and thereby influence its or its function and / or expression, in particular have an inhibiting or activating effect
  • A) Obtaining or producing a polynucleotide. encoding the protein pKe # 122 Human epidermal keratinocytes of a cell culture or a cell culture model served as the polynucleotide source, which is described in the publication by Schafer BM, Reinartz J, Bechtel MJ, Inndorf S, Lang E, and Kramer MD, 1996 dispase mediated basal detachment of cultured keratinocytes induces urokinase-type plasminogen activator (uPA) and its receptor (uPA-R, CD87), Exp. Cell Res 228, S 246-253, is described in detail.
  • uPA urokinase-type plasminogen activator
  • uPA-R urokinase-type plasminogen activator
  • this cell culture model is characterized in that it allows keratinocytes to be destroyed by enzymatic destruction of the cell / matrix contacts, ie by means of a dispase-induced detachment of the keratinocytes from the culture matrix, from the resting [uPA ⁇ 7uPA-R ⁇ ] to the activated [uPA + / uPA-R + ] state.
  • the induction of the activated state is reversible the (renewed)
  • NHEK normal human epidermal keratinocytes
  • the uPA / uPA-R upregulation was detectable from the incubated using known techniques such as enzyme-linked immunosorbent assay (ELISA), in-situ hybridization and immunofluorescence Cells were extracted using the guanidinium-thiocyanate-phenol-chloroform extraction method known in the prior art (cf. Chromczynski P and Sacchi N, 1986 single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal Biochem 162 156-159) the entire RNA obtained ("RNA-Clean" kit from AGS from Heidelberg) From the total -RNA, the mRNA was isolated by binding to poly-T-coated spheres. This mRNA served as the starting material for the next process section of subtraction cloning
  • ELISA enzyme-linked immunosorbent assay
  • mRNA was isolated from adherent keratinocyte sheets according to the same procedure as described above, except for the difference that for the duration of the dispase treatment, a dispase inhibitor, for example phosphoramidon (100 ⁇ g / ml), was used in addition to the dispase. was applied
  • a gene bank which preferably contained cDNA of the dyshasions-induced genes, ie those genes which were increasingly expressed in these (or their cells) after detachment of the keratinocyte sheets.
  • the mRNA obtained from the cells of the adherent keratinocyte sheets was generated again bound to poly-T-coated spheres, rewritten thereon into single-stranded cDNA and then hybridized against the mRNA of detached, ie non-adherent, keratinocyte sheets.
  • Those mRNA molecules which only express in the non-adherent state, ie after dyshasion were and as a result found no hybridization partner remained in the supernatant. They were rewritten into cDNA and chlorinated into the cloning vector pUEX-1
  • the resulting gene library was then subjected to a Southern blot method with [ 32 P] -labeled cDNA-adherent and non-adherent keratinocyte sheets for the purpose of checking. That cDNA or rather the host cell clones containing it - here the E coli strain MC 1061 - which after dyshasion one showed clear upregulation, were then cultivated overnight at 30 ° C. under normal culture conditions or increased.
  • the plasmid DNA (pUEXl cDNA) was excised from these E coli clones, and the cDNA fragments cut out from the pUEXl vector were analyzed by random -priming [ 32 P] -labeled
  • the labeled cDNA was used as a probe in Northern blots with RNA from adherent and non-adherent keratinocyte sheets.
  • the clones which contained cDNA when used as a probe in the Northern blot method, had little or no signal with the RNA adherent keratinocytes, on the other hand a clear signal with RNA non-adherent kerat inocyte sheets were selected for the subsequent process step of the sequencing
  • the nucleotide sequence of the gene pKe # 122 contains a stop codon at the 3 'end at position 2373-2375 according to SEQ ID NO: 1 and accordingly at position 2472-2474 according to SEQ ID NO: 4, which specifies the putative location of the transcription end and the like exactly 28 nucleic acids before the poly-A site follows a sequence very similar to the so-called "polyadenylation site" (AATAAA), namely AATAA
  • this gene or polynucleotide has the nucleotide sequence shown in the sequence listing SEQ IN NO: 1 or the sequence listing SEQ IN NO: 4 or has a partial sequence of one of these two nucleotide sequences or comprises or consists of a nucleotide sequence that is complementary to one of the nucleotide sequences shown or one of its partial sequences, or that this gene or polynucleotide is wholly or partly identical to that described in the sequence listing SEQ ID NO: 1 or im Sequence listing SEQ ID NO: 4 hybridized with a partial sequence of one of these two nucleotide sequences or with a sequence complementary to these shown nucleotide sequences or their partial sequences, wherein in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 4 instead of "T" can also be a "U", and that from this gene or polynucleotide has the nucleotide sequence shown in the sequence listing SEQ IN NO: 1 or the sequence listing SEQ IN NO: 4 or has
  • amino acid sequence from positions 40 to 63 corresponds to known protein kinase motifs with an ATP binding site
  • amino acid sequence from positions 152 to 164 corresponds to known serine / threonine protein kinase motifs
  • TLR amino acid sequences from positions 238 to 240 (TLR), from positions 475 to 477 (TGR), from positions 485 to 487 (STR) and from positions 600 to 603 (TTR) according to SEQ D3 NO: 2 and from positions 271 to 273 (TLR), from positions 508 to 510 (TGR), from positions 518 to 520 (STR) and from positions 633 to 635 (TTR) according to SEQ D3 NO: 3 correspond to known phosphorylation sites for protein kinase C,
  • the amino acid sequence from positions 138 to 156 (WQILSAVEYCHDHHIVHRD) according to SEQ ID NO: 2 and accordingly from positions 171 to 189 according to SEQ ID NO: 3 represents the pKe # 122-l peptide against which the anti-peptide antibody " Anti- pKe # 122-1 "was produced in the rabbit (see Example 2),
  • amino acid sequence from positions 481 to 499 (LAEVSTRLSPLTAPCINNS) according to SEQ ID NO: 2 and accordingly from positions 514 to 532 according to SEQ ID NO: 3 represents the pKe # 122-2 peptide against which the anti-peptide antibody " Anti-pKe # 122-2 "was produced in the rabbit (see Example 2),
  • amino acid sequence from position 339-352 (NHFAAIYYLLLERL) according to SEQ ID NO: 2 and accordingly from position 372-385 according to SEQ ID NO: 3 represents the pKe # 122-3 peptide against which the anti-peptide antibody " Anti-pKe # 122-3 "was produced in the rabbit (see Example 2),
  • FIGS. 2A and 14A show the protein kinase motif with ATP binding sites, the serine / threonine protein kinase motif and the four phosphorylation sites for protein kinase C
  • Figures 2B and 14B show the sequence segments against which anti-peptide antibodies were raised in rabbits
  • Example 2 Use of the amino acid sequence of the protein pKe # 122 for the production of polyclonal anti-peptide antibodies
  • the postimmune sera were first subjected to an ammonium sulfate precipitation and thereby the IgG fraction Enriched with this enriched IgG fraction Immunoaffinity chromatography performed
  • the four peptides pKe # 122-1 to -4 used for immunization were immobilized on Sepharose 4B and these peptide-Sepharose-4B conjugates were used in immunoaffinity chromatography.
  • the polyclonal anti-peptide antibody pKe # 122-1 was also tested with the recombinant ca 100 kD GST-pKe # 122 fusion protein (fraction 85 in FIG. 10 B) described here in Example 5 using the immunoblot method.
  • the polyclonal anti-peptide - Antibody pKe # 122-1 reacted with the fusion protein This positive reaction was confirmed by comparison with control experiments in which an anti-GST antibody or normal rabbit IgG was used instead of the anti-peptide antibody pKe # 122-1
  • the results are shown in Table 1 and in FIG. 4. Lane “a” in FIG. 4 shows the control batch with goat normal IgG, lane "b" in FIG.
  • FIG. 4 shows the batch with goat anti-GST IgG, lane " c "in FIG. 4 shows the approach with rabbit normal IgG and lane” d "in FIG. 4 shows the approach with rabbit anti-pKe # 122-1
  • Example 3 Use of the protein pKe # 122 or against the protein or against the mRNA of the pKe # 122-directed reagents for the detection of the activated state of human epidermal keratinocytes
  • PBS normal serum of the species from which the second antibody originates, here goat normal serum
  • the sections are incubated in PBS containing 5 ⁇ g / ml anti-peptide pKe # 122-1 for 1 hour at room temperature.
  • the sections are then incubated in PBS containing 0.2 % (Weight / volume) of bovine serum albumin. This is followed by incubation with an antibody from goat, for example biotin-labeled and directed against rabbit IgG (1,500 diluted in PBS / 0.2% BSA, 30 minutes at room temperature) a further washing step and the application of a streptavidin labeled with the fluorescent dye Cy3 (1 1 000 diluted in PB S / 0.2% BSA).
  • FIG. 5 A The anti-pKe # 122-1 IgG antibody stains keratinocytes on normal skin sections in the area of the epidermal basement membrane zone (FIG. 5 A).
  • FIG. 5 B When staining biopsies of lasional skin due to the diseases Pemphigus vulgaris (FIG. 5 B), bullous pemphigoid (FIG. 5 C) or psoriasis vulgaris (FIG. 5 D), there is a strikingly strong staining in epidermal keratinocytes, particularly in the area of epidermal lesions. Accordingly, there has been increased expression and an obvious upregulation of the protein pKe # 122
  • FIG. 3 shows the detection of the pKe # 122 protein by means of the Westemblot method using anti-pKe # 122-1.
  • the proteins were analyzed using a standard method Blotted nitrocellulose membrane To block unspecific binding sites, an incubation with 5% by weight milk powder / TBS buffer was carried out.
  • the (protein) strips with the designation "anti- 122-1" were then added in 3% milk powder / TBS buffer with the addition of anti-pKe # 122-1 antibodies (1 ⁇ g / ml) and the (protein) strips labeled “rblgG” in 3% milk powder / TBS buffer with the addition of rabbit normal IgG (1 ⁇ g / ml ) incubated at 4 ° C for approx. 18 hours (overnight).
  • the nitrocellulose membrane was then washed with TBS / Tween and TBS buffer and with an enzyme-labeled anti-rabbit IgG antibody in 3% milk powder / TBS - Buffer incubated After washing again with TBS / Tween and TBS, the bound antibodies were made visible with a peroxidase-specific luminescent substrate (here, for example, the ECL system from Amersham-Buchler) and also represented by autoradiography. An alternative label with chromogenic substrate was also shown aten is well possible The cell lysates can also be blotted directly onto a nitrocellulose membrane without prior electrophoretic separation and further treated as described above
  • Microtiter plates are coated with recombinant pKe # 122 / GST fusion protein in different concentrations (10-0 ng / ml). Unspecific binding sites are blocked by treatment with 0.1% by weight gelatin in PBS (PB S / gelatin). Then the coated wells are blocked incubated with anti-pKe # 122-1 IgG (1 ⁇ g / ml) for 1 hour at room temperature (see FIG. 6 A, closed circles) The control mixture is carried out with rabbit normal IgG in the same concentrations (see FIG.
  • FIG. 6.A shows that the color concentration of the amount of the pKe # 122 fusion protein bound to the plate It is proportional from this that when samples of known antigen concentrations, so-called standards, are used, it is possible to quantify an unknown amount of antigen by comparison
  • a sandwich ELISA For the quantification of the pKe # 122 protein in complex solutions, the implementation of a sandwich ELISA (FIG. 6 B) is preferred.
  • a microtiter plate with an antibody directed against pKe # 122 eg rabbit anti-pKe # 122 / GST fusion protein, 1 ⁇ g / Nertie Stahl
  • the remaining unspecific binding sites of the microtiter plate are blocked with PB S / gelatin.
  • the microtiter plate is mixed with different concentrations of the pKe # 122 / GST protein (10 - 0 ng / ml).
  • peroxidase-labeled anti-pKe # 122 antibody eg peroxidase-labeled rabbit anti-pKe # 122-1 (peptide) antibody
  • Peroxidase here stands for practically any labeling of the antibody, for example with enzymes, fluorescent molecules or luminescent molecules
  • Example 4 Detection of pKe # 122-specific mRNA in cells by means of a reverse polymerase chain reaction
  • PCR polymerase chain reaction
  • This cDNA was subjected to a PCR in which a partial fragment of ⁇ 350 kb was amplified from the pKe # 122-specific cDNA
  • 10 ng of cDNA with 10 ⁇ M primer each were used together with a mixture of heat-stable DNA polymerase, ATP, TTP, GTP, CTP and polymerase buffer (cf. e.g. Current protocols in Molecular Biology, Vol.
  • Lane 1 molecular weight marker
  • lane 2 NHEK T0
  • lane 3 NHEK T2
  • Lane 4 NHEK T4
  • Lane 5 NHEK T8
  • Lane 6 HaCaT
  • Lane 7 free
  • Lane 8 positive control (pUEX-1; with pKe # 122 as insert)
  • Lane 9 negative control (approach with cDNA without primer)
  • Lane 10 kit-specific positive control for checking the functionality of the PCR
  • lane 11 negative control (approach with primer without cDNA)
  • lane 12 molecular weight marker.
  • a PCR product of the expected size of 350 kb was detected in lanes 3, 4, 5, 6 and 8, that is to say: pKe # 122-specific mRNA was found in cells of keratinocyte sheets at time 2 (T2), 4 (T4 ) and 8 (T8) hours after dispase-induced detachment and also in HaCaT cells.
  • This technique enables the detection of pKe # 122 expression even in cases in which the detection of pKe # 122 protein is not possible due to the expression level being too low using immunohistological methods, the ELISA, dot blot or western emblot method.
  • Example 5 Production of vector molecules with the ability to express the protein pKe # 122 in prokaryotic or eukaryotic cells
  • pKe # 122-gluthathione-S-transferase (GST) fusion proteins ⁇ Ke # 122 / GST-I and pKe # 122 / GST-II, (vector pGEX; see FIG. 8) were used for the purpose of expression in Bacteria (E. coli DH5 ⁇ ) are produced.
  • GST pKe # 122-gluthathione-S-transferase
  • the pKe # 122-gluthathione-S-transferase (GST) fusion proteins were expressed in E. coli (DH5 ⁇ ) by LPTG induction. After the induction, the bacterial lysate was examined in a western emblot with anti-GST antibodies, in comparison to pKe # 122-gluthathione-S-transferase (GST) vector-bearing, non-induced bacterial lysate and to lysate of bacteria that exclusively GST expressed. The product of this western emblot is shown in Fig.
  • Lane (a) shows the control transfectant (GST without insert) before LPTG induction
  • lane (b) shows the control transfectant (GST without insert) after LPTG induction
  • Lane (c) shows pKe # 122 / GST-I before LPTG induction
  • lane (d) shows pKe # 122 / GST-I after LPTG induction
  • lane (e) shows ⁇ Ke # 122 / GST-II before LPTG- Induction
  • lane (f) shows pKe # 122 / GST-II after LPTG induction.
  • the highest molecular band had an apparent molecular weight of approx. 100 kD (see Fig. 10 B, fraction 85). This allows the conclusion that the 100 kD pKe # 122-GST fusion protein consists of the GST protein and an approximately 70-75 kD fragment of the Protein pKe # 122 exists (see also FIG. 4 and the associated description in Example 2)
  • the pBK-CMV-pKe # 122 vector (FIG. 9) was transformed into so-called Cos cells, ie into cells of the Cos cell line which is generally known in the prior art.
  • the Cos cells were removed by treatment with DEAE by standard methods -Dextran / chloroquine brought to take up the plasmid DNA.
  • the transformed cells were then incubated for two days under standard conditions (37 ° C. and 7% CO 2 ).
  • the Cos cells were lysed and analyzed in the immuno-blot method using an antibody against the FLAG epitope or the anti-peptide antibody anti-pKe # 122-1 IgG.
  • Lane 11 shows the product of the immunoblot
  • Lane a shows the non-transfected Cos cells
  • lane b shows a FLAG control protein
  • lane c shows pKe # 122-FLAG vector construct transfected Cos cells.
  • Lane c has a band with an approximate molecular weight of 80 kD, which was stained by the anti-FLAG antibody.
  • Lane b shows a FLAG-labeled control protein which is functional itat the anti-FLAG antibody demonstrated
  • Antisense nucleotides are taken up by cells, including keratinocytes (cf. G Hartmann et al 1998 antisense oligonucleotides, Inc. Cardiol actin, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12, IL-12 (cf. G Hartmann et al 1998 antisense oligonucleotides, Inc 24, C1 115-C1119) and bind to the mRNA present in the cell and inhibit its translation and thus its translation Expression (cf.
  • Suitable antisense oligonucleotides were identified the pKe # 122-specific nucleotide sequence (SEQ ID NO: 1 or SEQ ID NO: 4) was prepared. They were adjusted to a concentration of 100 ⁇ M with a suitable buffer medium (so-called “oligo buffer”). HaCaT cells were at 37 ° C.
  • the cells were trypsinized (10 minutes 0.2% EDTA, 5-10 minutes 0.1% trypsin) and adjusted to a concentration of 25,000 cells / ml. 100 ⁇ l of cell suspension (corresponding to 2,500 cells) were pipetted into each well of a 96-well plate The cells were incubated for 1 hour, followed by the addition of the antisense oligonucleotide (2 ⁇ l of a 100 ⁇ M solution) and a further incubation of 24-48 hours. Cell batches were used as a negative control, to which oligonucleotide with the same base distribution but a randomly selected sequence were added
  • FIG. 12 a shows subconfluent HaCaT cultures which contain pKe # 122-specific antisense oligonucleotides
  • 12 b shows subconfluent HaCaT cultures which have been treated with control oligonucleotides
  • FIG. 13 a shows confluent HaCaT cultures which have been treated with pKe # 122-specific antisense oligonucleotides
  • FIG. 13 b shows confluent HaCaT cultures which have been treated with control oligonucleotides
  • FIG. 13 c shows a detail from FIG. 13 a
  • the microscopic examination results demonstrate that, in comparison to control oligonucleotides, the number of cells in the cultures treated with the specific antisense oligonucleotide is significantly reduced. This suggests a decrease in cellular proliferation caused by the antisense oligonucleotide in the HaCaT cultures treated with antisense oligonucleotides, greatly enlarged cells which were not found in the cultures treated with control oligonucleotides. These large cells correspond in their morphology to differentiated keratinocytes. The finding suggests that pKe # 122- specific antisense oligonucleotides treated cells have an increased tendency to differentiate

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un polypeptide isolé qui est équivalent ou similaire (en termes de fonction et d'effet) à une protéine intervenant de manière naturelle dans des kératinocytes humains et qui est amplifiée à l'état actif des kératinocytes. L'invention concerne en outre un acide nucléique isolé qui code un polypeptide ou une protéine typique des kératinocytes humains. L'invention concerne par ailleurs l'utilisation de ce polypeptide et de cet acide nucléique à des fins d'analyse, notamment à des fins de diagnostic, et/ou à des fins thérapeutiques. L'invention concerne également l'utilisation de réactifs, notamment de molécules vecteurs et d'anticorps, à l'encontre de molécules de ce type. L'invention obtenue selon l'invention présente soit un protocole séquentiel SEQ ID NO:2, soit la séquence d'aminoacide présentée dans le protocole séquentiel SEQ ID NO:3, soit un allèle ou un dérivé de cette séquence d'aminoacide, apparu par substitution, suppression, insertion ou inversion d'aminoacide. L'acide nucléique de l'invention présente soit la séquence nucléotide figurant dans le protocole séquentiel SEQ ID NO:1, soit la séquence nucléotide figurant dans le protocole séquentiel SEQ ID NO:4, soit une séquence nucléotide qui en est complémentaire, soit une séquence nucléotide hybridisée en totalité ou en partie avec une des séquences nucléotides précitées.
EP99969420A 1998-09-19 1999-09-06 Proteine regulatrice issue de keratinocytes humains Withdrawn EP1114157A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19842863 1998-09-19
DE19842863A DE19842863A1 (de) 1998-09-19 1998-09-19 Regulatorisches Protein aus humanen Keratinozyten
PCT/DE1999/002865 WO2000017232A2 (fr) 1998-09-19 1999-09-06 Proteine regulatrice issue de keratinocytes humains

Publications (1)

Publication Number Publication Date
EP1114157A2 true EP1114157A2 (fr) 2001-07-11

Family

ID=7881448

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99969420A Withdrawn EP1114157A2 (fr) 1998-09-19 1999-09-06 Proteine regulatrice issue de keratinocytes humains

Country Status (6)

Country Link
EP (1) EP1114157A2 (fr)
JP (1) JP2002526060A (fr)
AU (1) AU1149100A (fr)
CA (1) CA2342957A1 (fr)
DE (2) DE19842863A1 (fr)
WO (1) WO2000017232A2 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1240194A2 (fr) * 1999-11-24 2002-09-18 Sugen, Inc. Nouvelles proteines kinases humaines et enzymes analogues
AU2001245974A1 (en) * 2000-03-24 2001-10-08 Millennium Pharmaceuticals, Inc. 3714, 16742, 23546, and 13887 novel protein kinase molecules and uses therefor
GB0400122D0 (en) * 2004-01-06 2004-02-11 Badrilla Ltd Method of quantifying binding
US6946028B1 (en) * 2004-09-07 2005-09-20 Kerr-Mcgee Chemical Llc Surface-treated pigments
JP2015072226A (ja) * 2013-10-03 2015-04-16 住友ベークライト株式会社 検査方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0017232A2 *

Also Published As

Publication number Publication date
DE19842863A1 (de) 2000-04-27
JP2002526060A (ja) 2002-08-20
AU1149100A (en) 2000-04-10
DE19981873D2 (de) 2002-01-24
WO2000017232A2 (fr) 2000-03-30
CA2342957A1 (fr) 2000-03-30
WO2000017232A3 (fr) 2000-10-12

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