DE19742725A1 - DNA encoding UV-repressible serin protease inhibitor (hurpin - Google Patents

Abstract

Three cDNA clones of a gene for a UV-repressible serine protease inhibitor expressed in HaCaT epidermoid cells, called hurpin ("HaCaT UV repressible serpin"), are new. The clones are referred to as R7-1.1, R7-16.1 and R7-12.2 and have defined sequences of 3142, 2834 and 2654 bp, respectively, given in the specification. The following are claimed: (1) a nucleic acid comprising the following nucleotide sequence: R7-1.1 R7-16.1 R7-12.2 no punctuation; (2) the nucleotide sequence of a gene or gene fragment contained in the following vectors: pBS-HP1.1 pBS-HP16.1 pBS-HP12.2 no punctuation; (3) a nucleic acid sequence that hybridises to the nucleotide sequences of (1) and (2) or the complementary sequences under stringent conditions; (4) an amino acid sequence encoded by the sequences of (1); (5) a vector containing the sequences of (1), (2) or (3); (6) an expression vector containing the nucleotide sequence of (1), (2) or (3) linked to regulatory elements for controlling expression of the nucleotide sequence in a host cell; (7) a recombinantly produced host cell containing the nucleotide sequence of (1), (2), (3) or it is stated (4); (8) a recombinantly produced host cell containing the nucleotide sequence of (1), (2), (3) or it is stated (4) linked to regulatory elements for controlling expression of the nucleotide sequence in the host cell; (9) a purified gene product encoded by the nucleic acids of (1), (2), (3) or it is stated (4); (10) an antibody that immunospecifically binds the gene product of (9); (11) a transgenic animal in which the nucleic acids of (1), (2), (3) or it is stated (4) forms sic, as an expressed transgene, a component of the animal's genome; (12) a transgenic animal in which the expression of a genomic sequence encoding the gene product of is stated (8) is inhibited or suppressed.

Description

Skin homeostasis disorders are found in malignant skin tumors such as basalioma or squamous cell carcinoma, but also in benign proliferative Skin diseases such as psoriasis. At the expression level This abnormal phenotype of the keratinocytes involved is documented by a changed gene expression compared to keratinocytes in healthy skin.

Ultraviolet (UV) also has a direct influence on gene expression in the skin Component of sunlight. Especially the middle spectrum of UV radiation (UVB, 280-210 nm) is responsible for this effect in keratinocytes. In addition to acute Effects like sunburn and immunosuppression can be long-term effects Skin tumors develop. The connection between UV radiation and deregulated Gene expression in skin diseases suggests that genes that are involved in the physiological UV response are also involved in pathological processes in the Skin are important.

The molecular processes in the physiological UV response and the UV car zinogenesis are only incompletely elucidated and only a few genes were able to do so Context to be identified.

Due to the methodological approaches, the repressing effect on the Gene expression of UV radiation not systematically recorded (1-3).

By using the differential mRNA display polymerase chain reaction (DD-PCR) (4) offered new possibilities for identification and cloning UV-re gulated genes. Similar to subtractive cDNA hybridization, no knowledge is available about the sequence of the genes to be isolated is necessary. Just a quantitative difference relevant sequences in the cell populations to be compared is for identification required. This enables the simultaneous recording of known and unknown Genes. In contrast to subtractive cDNA hybridization, this is the case with DD-PCR comparative analysis is not limited to 2 cell populations, but can as many populations are analyzed side by side as are handled simultaneously  can be. Since quantitative differences can also be shown, This enables the expression kinetics of UV-regulated genes to be recorded. You can unlike other methods at the same time both UV-induced as well UV-repressed genes can be detected.

The family of serine proteinase inhibitors (serpins) includes high molecular weight (40-60 kDa) Proteins with a single amino acid chain, which is characterized by a highly ordered Tertiary structure is characterized. This tertiary structure is defined by α1-antitrypsin (α1-PI) (5), the prototype of all serpins. That from 9 α-helices and 3 β-sheets existing tertiary structure with specific folding properties is elementary for the Function of the protease inhibitors. The inhibitory specificity is primarily by the amino acid residues in P1-P1 'position in the reactive center near the C-terminus of the protein. This metastable part of the protein makes Interaction with the reactive center of a target protease involves a conformational change and finally forms a covalent serpin-proteinase complex (6).

By comparing amino acids, characteristic properties of proteins and Genome organization was able to manage the ovalbumin family within the large serpine family (Ov-Serpine) can be identified from intracellular Serpine (7).

Serpins are linked to a variety of important proteinase-mediated physiological Functions such as blood clotting, fibrinolysis, inflammation, cell migration, remodeling extracellular matrix (8) and activation of interleukin precursors (9).

A growing number of new data shows that serpins are involved in regulation of proteinases that are key to apoptotic processes (10-13). In human keratinocytes are serpins, as shown for PAI-1 (14), in migration and Differentiation of cells and in the context of wound healing is important. For PAI-2 was able to participate in intracellular processes in keratinocytes associated with the terminal differentiation and formation of the cornified cell wall ("cornified envelope") in Related, demonstrated (15).

The present application describes the identification, clarification and analysis of the complete cDNA sequences of a new serine protease inhibitor and its Assignment to the ovalbumin serpine family. The new Serpin is in the Keratinozy ten-cell line HaCaT was isolated as a UVB-repressible sequence and was therefore called "hurpin" for HaCaT UV repressible serpin. The genome data base  nomenclature committee has the systematic name proteinase for hurpin inhibitor 13 (PI13) reserved (confidential). Expression studies show that hurpin is specifically expressed in keratinocytes. In addition, the results indicate on a link between hurpin and the proliferation and activation of human Keratinocytes. Furthermore, psoriasis shows a clear overexpression of hurpin in infested skin compared to unaffected skin.

A possible function of hurpin in this context could be prevention of apoptotic processes in keratinocytes.

The identification of a new UV-repressible serine protease inhibitor and the association with a benign, proliferative skin disease (psoriasis) offers starting points for:

• - the elucidation of the molecular mechanisms of the disease.
• - the establishment of new clinical parameters for follow-up.
• - the development of new treatment strategies.

Below are those in the course of the analysis of differential gene expression, Cloning, and sequencing methods listed.  

Methods Chemicals, radioisotopes and enzymes

Unless otherwise stated, the chemicals used were p.A. quality purchased from Flow Laboratories, Gibco BRL, Merck, Roth, Serva or Sigma. The radiochemicals used were obtained from ICN. Restriction endonucleases and other enzymes were, unless otherwise noted, by Boehringer (Mannheim), Gibco BRL, New England BioLabs or Pharmacia.

Cell culture

The epidermoid celline HaCaT (16) was provided by Prof. Fusenig. Primary keratinocytes are removed from pieces of skin that are removed during plastic surgery and are usually discarded. For this, the one stored in 1 × PBS Skin is freed of fatty tissue under sterile conditions and cut into small pieces (approx. 5 mm × 5 mm) divided. The pieces were washed in 1 × PBS and then in Petri dishes for 12 to 16 hours at 4 ° C with the epidermis upwards in dispase (Grade II, 2.4 U / ml, Boehringer Mannheim). Then the epidermis of the Leather skin removed and washed in 1 × PBS. The pieces are for 5 to max. 15 min in a trypsin / EDTA solution (0.2% trypsin and 0.2% EDTA dissolved in 1 × PBS) Incubated at 37 ° C. The keratinocytes were made from the enzymatically decomposed keratin scaffold mechanically released. By adding at least three times the volume Trypsinization was stopped in 10% FCS. The pelleted primary Keratinocytes are resuspended in medium (see below) and used for further cultivation Petri dishes distributed.

The cell cultures are cultivated in an incubator at 37 ° C. in a water-saturated atmosphere with 5% CO ₂ .

HaCaT medium Dulbecco's Mod. Eagle Medium w. Sodium pyruvate (GibcoBRL)

5 ml of antibiotic (penicillin / streptomycin, 10000 µg / ml, Flow-La boratories), 5 ml L-glutamine (200 mM), 50 ml FCS (100%).

Keratinocyte medium KBM (Keratinocytes Basal Medium, Clonetics)

500 µl gentamicin sulfate (50 mg / ml), 500 µl am-photericin (50 µg / ml) are added, 500 µl insulin bovine (5 mg / ml), 300 µl epidermal growth factor (0.1 µg / ml), 500 µl Hydrocortisone (0.5 mg / ml).  

In about 70% confluent grown cells, the cultures were irradiated with UVB and for incubated for different lengths of time before going for isolation of the RNA were harvested.

UVB radiation

An irradiation bench made of four FS20 sun lamps (Westinghouse Electric Corp., Pittsburgh) was used for the UVB irradiation. The medium was exchanged for PBS for the irradiation and the cells were exposed to UV light (100 J / m ² ) without a lid.

Molecular biological methods RNA isolation

The Trizol reagent (Gibco-BRL) is used to isolate the total RNA Manufacturer information used. For the RNA extraction from cultured cells Petri dish 1.6 ml trizole inserted. Deviating from the manufacturer's protocol, the Avoid DNA contamination the aqueous phase of the first extraction extracted a second time with trizole.

For the RNA preparation from skin biopsies (0.3 mm dermatome sections), the Tissue pieces immediately frozen in liquid nitrogen after removal. The Tissue pieces were stored at -70 ° C until preparation. With help of a Dismembrators (Braun) became the frozen tissue under liquid nitrogen powdered. The chamber was opened on ice and the tissue powder with 1.5 ml trizole overlaid. The sample was shaken again in the dismembrator for 30 seconds. After this Opening the chamber, the frozen trizole was thawed at RT and then in a Transfer 2 ml Eppendorf tube. The rest of the work was done as for the RNA isolation from culture cells.

DNase treatment of RNA

10 µg RNA was filled up to a volume of 40 µl with DEPC water. For the corresponding number of samples, a master mix is prepared, which contains 60 µl DEPC water, 1 µl 0.1 M DTT, 1 µl 1 M MgCl ₂ , 1 µl DNase I (RNase free, 10 U / µl , Boehringer Mannheim) and 0.4 µl RNase inhibitor (40 U / µl, Boehringer Mannheim). The mixture was incubated for 30 min at 37 ° C. in a water bath.

Trizol was used to extract the RNA (1 ml trizole + 200 µl chloroform). After the alcoholic precipitation, the pellet is taken up in 10 µl DEPC water and dissolved in a water bath at 42 ° C for a few minutes. The purity control was carried out photometric and with the help of an analytical agarose gel.

cDNA synthesis

333 ng of DNase-treated RNA in 11 μl of DEPC-treated water were used for cDNA synthesis. After adding 1 .mu.l of the respective 3 'dT primer (TA, TG and TC; 2 .mu.M each), the samples were denatured for 10 min at 70.degree. C. and then incubated on ice for a further 10 min. From a master mix, 8 µl was distributed to the RNA. For each batch, this contained 2 ul 0.1 M DTT (Gibco BRL), 1 ul RNAse Inhibitor (40 U / ul, Boehringer Mannheim), 4 ul 5 × reaction buffer (50 mM Tris.CL (pH 7.6), 60 mM KCl and 10 mM MgCl ₂ , Gibco BRL) and 1 µl dNTP mix (400 µM).

After the batches had been preheated to 37 ° C., the reaction was carried out by adding 200 U Superscript II reverse transcriptase (Gibco BRL) started. The samples were for 40 min at 37 ° C in a water bath and then a heating block at 40 ° C for a further 20 min incubated. The approaches were then used to inactivate the reverse transcriptase Incubated for 5 min at 70 ° C. The cDNA preparations were then at -20 ° C stored.

Differential mRNA display (DD-PCR)

For each of the samples examined, 2 μl of the corresponding cDNA were used in a 0.5 ml Eppendorf tube. The remaining components were prepared in the form of a master mix. The following components were used for each approach:

2 µl primer 1 (2 µM)
2 µl primer 2 (2 µM)
1.8 µl Taq polymerase buffer (10 ×)
2 µl gelatin (0.1%)
1.6 µl dNTP mix (25 µM)
6.2 µl ddH ₂ O
0.4 ul α- ³² P-dCTP.

16 µl each of the master mix was added to the cDNA and with 80 µl mineral oil as Evaporation protection layered.

The Taq polymerase is also prepared in the form of a master mix and after first denaturation and binding phase (annealing) added to the approach ("hot start").

Enzyme master mix

0.4 µl Taq enzyme (5 U / µl)
0.2 µl Taq polymerase buffer (10 ×)
1.4 µl ddH ₂

O.

Following the first annealing phase at 41 ° C, the samples remained at 42 ° C. The The oil phase of each batch was pierced and 2 ul of Taq polymerase Mas ter mixes added. As soon as all samples are provided with enzyme, the first Elongation phase started.

DD-PCR program

After all PCR cycles had ended, a 5 'elongation phase was carried out at 72 ° C attached.

An aliquot (5-7 µl) of each batch was mixed with 1/5 5 × TBE loading buffer. The samples were denatured for 3 min at 75 ° C and stored on ice until applied.

Equal sample volumes were then denatured in a 5% PAA gel Conditions separated at a constant 40-45 W until the xylene-xylanol color marker of Loading buffer had reached the lower edge of the gel.

By treating one of the two glass plates surrounding the gel with binding silane, the gel adhered to the treated plate after disassembly. That at the Glass plate adhesive gel was covered with household film. To the location of the for There were several to exactly record autoradiography of the X-ray film  Markings using "tracker tape" (RPN 2050, Amersham) on the edge of the gel appropriate. The X-ray film was exposed at -70 ° C without an intensifying screen.

Elution of the cDNAs from polyacrylamide gels

Differential bands are marked with lateral dots on the X-ray film and the film is brought into its original position on the gel with the aid of the “tracker tape” markings and fixed there. The film was pierced at the marking points with the aid of a needle, thus marking the position of the band on the PAA gel. After removing the film, a thin strip of the gel was cut out between the puncture sites with a clean scalpel. This piece was transferred to a 1.5 ml Eppendorf tube with 100 ul ddH ₂ O. To elute the cDNA from the gel fragment, the samples were heated to 80 ° C. for 20 min and then centrifuged for 2 min at 13,000 rpm (15,000 g). The gel-free supernatant is transferred to a new vessel. By adding 10 µl 3 M NaAc (pH 5.2), 2.5 µl glycogen (20 mg / ml) and 450 µl ice-cold ethanol (100%), the eluted cDNA is precipitated at -20 ° C overnight (optional for at least 2 hours in ethanol on dry ice). The cDNAs are pelleted at 4 ° C for 30 min at 13,000 rpm (15,000 g), the supernatant is removed and the pellet is washed with 75% EtOH. The pellet is dissolved in TE * (10 mM Tris pH 7.5, 1 mM EDTA).

Reamplification of differential cDNAs

The reamplification of the eluted cDNA fragments was carried out in a 40 µl PCR approach with the same primer combinations that were used for their original generation DD-PCR were used.

4 µl each of the eluted cDNA in TE * are mixed with:

4 µl primer 1 (2 µM)
4 µl primer 2 (2 µM)
3.8 µl Taq polymerase buffer (10 ×)
4 µl gelatin (0.1%)
3.2 µl dNTP mix (250 µM)
15 µl DEPC water

combined and covered with mineral oil. To reduce pipetting inaccuracies the components for several approaches were combined in the form of a master mix.

The Taq enzyme was, as in the DD-PCR, in 2 ul 1 × Taq polymerase buffer in "hot-start" process added.

In contrast to the DD-PCR program, a "touch down" protocol was used to increase the specificity of the amplification. Here, an increased anealing temperature was used in the first 5 cycles of the PCR, which is reduced per cycle so that the optimal temperature for the primer combination is reached from the 6th cycle. Due to the initially more stringent conditions, the amplification effectiveness is lower, but it is ensured that the desired product is preferentially amplified. After sufficient quantities of the desired template have been obtained in this way, amplification can be carried out with high efficiency at the analysis temperature matched to the primers. The success of this procedure was shown in the following preparative agarose gel by hardly any unspecific PCR products.

Cloning of PCR products

For the ligation, the reamplified cDNA fragments after size fractionation eluted from the gel in agarose gel with JetSorb (Genomed) according to the manufacturer's instructions. For TA cloning of PCR products became the Invitrogen TA cloning kit Manufacturer information used. The ligation was carried out in the vector: cDNA ratio of 1: 3.

Production of a lambda cDNA bank

In order to isolate full-length cDNA clones, a cDNA library was produced in Uni-ZAP-XR (Stratagene, Heidelberg) using poly (A) ⁺-RNA from HaCaT cells. The enrichment of poly (A) ⁺ mRNA from total RNA was carried out using the Oligotex mRNA Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions. The ZAP-cDNA cloning kit (Stratagene, Heidelberg, Germany) was used to produce the cDNA bank from 4 µg poly (A) ⁺-RNA according to the manufacturer's suggestions with the exception of the following modifications:

• - Superscript II reverse transcriptase was used for the production of the 1st strand cDNA (Gibco, BRL, Eggenstein) used.
• an S-400 was used for the size fractionation of the Xho I-cleaved cDNA Centrifugation column used.

After ligation of the cDNA fraction with molecules <500 bp in Uni-ZAP-XR vector arms the bank became using the Gigapack III Gold packaging extract (Stratagene, Heidelberg, Germany) packed in λ phage particles.

The cDNA library obtained after a single amplification had a titer of 1.4 × 10 ¹⁰ pfu / ml.

Cloning of full-length cDNA clones

For the identification of hurpin cDNA clones, 10 ⁶ pfu were transferred to Hybond-N⁺ nylon membranes (Amersham, Braunschweig) using standard methods (17). The 412 bp HUR7 cDNA was used as the probe, after radioactive labeling with [α- ³² P] dCTP using the Megaprime DNA labeling kit (Amersham, Braunschweig, Germany). The nylon membranes were prehybridized in QuickHyb (Stratagene, Heidelberg, Germany) for 2 h at 68 ° C. Hybridization with the radiolabelled probe was then carried out in the same solution overnight at 68 ° C. The membranes were then washed under stringent conditions (final at 55 ° C. in 0.1 × SSC, 0.1% SDS) and positive plaques were made visible by autoradiography with hyper film (Amersham). Individual phages that hybridized with the HUR 7 cDNA were identified by a second filter hybridization with immobilized phages, each with a positive plaque. The cDNA inserts of these phages were prepared by in vivo excision of the pBlueScript SK⁻ phagemid from the phage genome according to the manufacturer's instructions.

Restriction analysis of the cDNA clones

The cDNA inserts from the pBS vectors were analyzed with Eco RI (3 U / µg) and Xho I cut out. Because the pBS vector is only a slightly different size than that cDNA fragments had been used for better identification and separation of the fragments additionally cut the vector with Pvu I (1.7 U / ug). In all restriction approaches worked with Boehringer "high salt" buffer. For further restriction analyzes the Plasmids cut with Notl, Pst I, Xba I, Sac I and Bam Hl according to standard instructions (17) and analyzed in a 1% agarose gel.

Northern blot analysis

10 µg total RNA from HaCaT cells were in a 1% formaldehyde agaro Size gel fractionated and by capillary blot on Hybond N⁺-Ny ion membranes (Amersham) transferred. Northern blots with poly (A) ⁺ mRNA Different human tissues (MTN: multiple tissue Northern blots) were from Clontech related.

For the hybridization of filters, the DNA fragments which were used as hybridization probes were labeled with [ ³² P] -dCTP (3000 Ci / mmol; ICN) using the "Megaprime labeling kit" from Amersham. Free nucleotides were separated by chromatography on an S300 centrifugation column (Pharmacia). The incorporation rate of [ ³² P] -dCTP in the DNA was determined by measuring an aliquot of the labeling reaction in a liquid scintillation counter. The specific activity was 1-2 × 10 ⁹ cpm / µg DNA.

The hybridization was done in DIG-Easy-Hyb solution (Boehringer Mannheim) overnight carried out. After stringent washing of the membranes (final 55-60 ° C, 0.1% SDS, 0.1 × SSC) were signals by exposure to an X-ray film (Hyperfilm, Amersham) Amplifier film made visible at -70 ° C.

RT-PCR analyzes

For the reverse transcription of RNA in cDNA, a recombinant Mo-MLV reverse transcriptase without RNase-H activity (Superscript II, Gibco BRL) was used with the 5 × buffer system [250 mM Tris-HCl (pH 8.3), 375 mM, KCl provided , 15 mM MgCl ₂ ] used. 1 µg total RNA in ddH ₂ O and 10 pmol dT _12-18 primer (Pharmacia) was used per batch (total volume: 11 µl). After heat denaturation (10 'at 70 ° C) of the RNA and cooling on ice, the following were added:

4 µl 5 × buffer
2 µl 0.1 M DTT
1 µl dNTP mix (10 mM each dATP, dGTP, dCTP, dTTP)
1 µl RNasin (40 U, Promega).

The reaction was started by adding 1 μl (200 U) of reverse transcriptase (RT) and was carried out at 42 ° C. for 1 h. The RT was inactivated by incubating the reaction mixture at 70 ° C. for 5 minutes. The cDNA thus prepared was stored at -20 ° C. For the detection of specific transcripts, the corresponding cDNA fragment was amplified in the PCR using two specific primers. The sequence of the primers used was optimized with the help of the computer program "Oligo", version 3.3 (control of duplex formation, homology of the 3 'and 5' ends to one another, self-complementarity, melting temperature of the primer). In order to optimize the reaction conditions in the PCR, several test PCR analyzes were carried out with each primer pair and a positive control, in which the MgCl ₂ concentration (1.5-2.5 mM) and the temperature (variation of the "Oligo" determined temperature) were varied during the primer binding step ("annealing").

Composition of a PCR approach sscDNA (RT-PCR) or dsDNA

4.7 µl 10x Taq buffer [100m Tris-HCl (pH 8.3 at 25 ° C), 500mM KCl, 15mM, MgCl ₂

]
1 µl dNTP mix (10 mM each dATP, dGTP, dCTP, dTTP)
0.5 µl Primer 1 (10 µm / ul)
0.5 µl Primer 2 (10 µm / µl)
x µl 60 mM MgCl ₂

(depending on the primer to reach a final concentration of 2.0 mM or 2.5 mM)
ad 47 µl ddH ₂

O.

List of primers used in RT-PCR analyzes

Evaporation during incubation was achieved by overlaying the Prevention of reaction with mineral oil prevented. About the creation of non-specific products restrict, a "hot start" was carried out, in which 2 U Taq polymerase in 3 µl 1 × Taq buffer for the reaction mixture after the first denaturation step of the PCR was added.

The batches were incubated in a programmable thermal cycler from the company Biometra (Trioblock).

Standard PCR program

94 ° C: 1 min
50 ° -68 ° C: 1 min (primer specific)
72 ° C: 2 min
Repeat for 25-30 cycles
72 ° C: 5 min.

The PCR products were analyzed in 1.5% agarose gels. The DNA fragments were stained with ethidium bromide and used under long-wave UV light for the Documentation made visible.

The invention is illustrated in detail by the following examples, illustrations and sequences explains:  

Fig. 1 shows the autoradiogram of the differential display analysis to identify the first partial hurpin cDNA clone (HUR 7) as a UV-repressed sequence. The corresponding gang is marked by an arrow.

Fig. 2 shows the schematic representation of the organization of the full-length hurpin cDNA clones according to the underlying sequence analysis.

Tab. 1 shows the results of the homology comparison of the amino acid sequence of hurpin with various well-known members of the ovalbumin branch of the Serpine family together.

Fig. 3 shows the homology comparison of the amino acid sequence of hurpin and the "squamous cell carcinoma antigen" (SCCA) 1 and SCCA 2. Identical amino acids in the same position are highlighted accordingly. The position of the amino acids preserved in serpins is marked by bars above the amino acids. Black bars indicate conserved amino acids in hurpin, while unfilled bars indicate a deviation from the conserved amino acid. Hatched bars show amino acids that typically differ from the conserved amino acid in ovalbumin serpins. The amino acids of the reactive center in position P ₁₇ -P ₈ conserved in inhibitory serpins are framed and the consensus sequence for inhibitory serpins is marked by a dashed line below the hurpin sequence. The amino acids of the reactive center in the P ₁ -P ₁ position are also marked.

Fig. 4 shows the autoradiogram of a hybridization of the radioactively labeled cDNA insert of the hurpin clone 1.1 against total RNA from irradiated and unirradiated HaCaT cells.

Fig. 5 RT-PCR analysis for the detection of hurpin-specific transcripts in human, cultured retinal pigment epithelial cells (RPE), iris epithelial cells (IPE), normal skin, primary, cultured keratinocytes (NHK), the epidermal cell line HaCaT, and infected (a) and unaffected (u) skin from three patients with psoriasis.

Fig. 6-8 Restriction maps of the plasmid vectors. The plasmid vectors are pBlueScript SK⁻ in which the full length hurpin / PI13 cDNAs are cloned via Eco RI and Xho I.

Appendix: Sequences of the hurpin / PI13 cDNA clones.  

Examples example 1 1. Differential Display (DD-PCR) analyzes

HaCaT cells were irradiated with a sublethal UVB dose (100 J / m ² ) and harvested 0.5.1 h, 2 h, 4 h, 6 h, 8 h and 24 h later. For the DD-PCR analyzes, the RNA from cells was used 8 h and 24 h after UVB irradiation and from unirradiated cells at the beginning of the experiment and after 24 h in culture. The parallel analysis of these 4 cell populations makes it possible to exclude bands that randomly vary between the different tracks on the display and thus reduces the probability of selecting false positive bands for the subsequent analyzes. Furthermore, this parallel analysis allows the potential of DD-PCR to be used to detect quantitative differences as an additional criterion in the identification of sequences regulated by UVB.

Fig. 1 shows part of such a "differential display" (DD) analysis. Independent DD-PCR analyzes showed that a specific band pattern could be produced reproducibly with the protocol developed for a specific primer combination. The complex band pattern shows that UVB radiation leads to both induction and repression of different genes within 24 hours after the radiation.

For hurpin, this analysis was able to regulate differentially after UVB radiation cDNA band of 412 bp can be shown. Since UVB radiation is a had a repressing effect on the transcript amount of the corresponding gene, was chosen as the name for this cDNA HUR 7, for HaCaT UV repressed.

Example 2 2. Isolation of full-length hurpin cDNA clones

Using the HUR7 cDNA, full-length hurpin cDNA clones were subsequently isolated from a HaCaT cDNA library. After hybridization of the radiolabelled HUR 7 cDNA against 10 ⁶ λ cDNA clones, 67 clones showed a positive signal. Of these, 20 were selected for a second hybridization. Of the 15 positive clones from this second screening, 12 were selected for the in vivo excision of the insert-bearing pBlueScript SK p phagemid from the phage genome. A restriction analysis of the phagemids obtained showed that 3 clones did not correspond to HUR 7 and the other phagemids. The 9 remaining clones could be divided into 3 groups based on their size and their restriction pattern.

Example 3 3. Sequence analysis of the hurpin cDNA clones

A representative clone of the 3 phagemid groups was completely sequenced. A summary of the properties of these clones is shown in Fig. 2. Clone 1.1 with a 3130 bp cDNA insert represents the 3.4 kb transcript, while clone 12.2 with a 2637 bp cDNA insert represents the smaller 3.0 kb transcript. Clone 16.1 also corresponds to the 3.0 kb transcript, but contains a 188 bp insertion that is missing in the other clones. Since this insertion interrupts the open reading frame, it is not an alternative spliced RNA variant. Although a perfect splice consensus sequence could not be found, the insertion probably represents an unspliced intron. Comparative analysis of the sequence of the different clones showed that the distinct two transcripts observed in Northern blot are due to the use of different polyadenylation signals.

The 3.0 kb transcript is generated using a Consensus polyadenylation signal AAUAAA 13 nt above the poly (A) tail generated. The 3 'untranslated region (3' UTR) of the 3.4 kb transcript is found two functional variants of the consensus signal with the sequence AAUAAU and AAUAUA, which are 56 nt and 22 nt above the poly (A) tail.

All cDNA clones contain a single open reading frame (ORF) with one length from 1176 bp starting from position 103 of the sequence of clone 1.1 with an ATG in a Kozak consensus sequence. Clone 1.1 contains the longest 5 'with 102 bp untranslated area (5 'UTR).

The putative protein consists of 391 amino acids and has a calculated molecular Mass of about 44 kDa and a pI of 5.47.  

Database searches against the EMBL sequence database showed that the protein encoded by the cDNA clones is new, but has clear homologies to members of the ovalbu min branch of the serpin superfamily of protease inhibitors (Table 1). The greatest agreement in the amino acid sequence was found between hurpin and the "squamous cell carcinoma antigen" (SCCA) 1 (59.1%), SCCA 2 (58.5%) and bomapin / PI10 (43.5%). An analysis of the amino acid sequence showed that hurpin is a typical serpin, in which 46 of the 51 amino acids (5) described as characteristic for serpins are conserved ( Fig. 3).

In addition to the amino acid level matches, hurpin has all the structural features that separate the ovalbumin serpine from the other members of the serpin superfamily:

• - hurpin has no N-terminal extension and the ORF starts with the amino acid 23 of α-1 antitrypsin (α-1-PI).
• - hurpin has no c-terminal extension and ends with Pro ³⁹¹ , corresponding to Pro ³⁹¹ in α-1-PI.
• - The penultimate amino acid is Ser and not Asp, as with the other members of the Super family.
• - hurpin has a variable amino acid instead of valine at position 388 regarding α-1-PI.
• - hurpin has no typical N-terminal hydrophobic signal peptide.

By comparison with other ovalbumin serpins, the specificity-determining positions P ₁ -P _{1 'of} the potential reactive center could be identified as Thr ^356- Ser ³⁵⁷ . The large identity of the "hinge" region of hurpin with the postulated consensus sequence of the inhibitory serpins (18, 19) suggests a protease inhibitory activity for hurpin ( Fig. 3).

Example 4 4. Northern blot analyzes

When using the full-length cDNA inserts for HUR 7 as radioactive probe in Northern blot with HaCaT cells total RNA, the same hybridization pattern as with the original "differential display" clone (HUR 7) could be generated. The detected two transcripts of approx. 3.0 and 3.4 kb showed the same decrease in the steady-state mRNA amount after UVB irradiation with 100 J / m ² as after hybridization with the original HUR 7 fragment. As with the original HUR 7 cDNA clone, no signal could be detected with the full-length cDNA clones in other human tissues (MTN Northern blots: heart, brain, placenta, lung, liver, skeletal muscle, pancreas).

Example 5 5. RT-PCR analyzes

In RT-PCR analyzes with specific primers for hurpin, expression could be detected not only in HaCaT cells, but also in cultivated primary keratinocytes and in biopsy material from healthy skin. In contrast, cultured human retinal pigment epithelial cells and iris epithelial cells showed no hurpin expression. The lower expression of hurpin in normal skin compared to the expression in HaCaT cells or cultured primary generation cells suggested an association of hurpin with the proliferation state of keratinocytes. A characteristic feature of psoriatic skin is the hyperproliferation of keratinocytes. In order to test the association of hurpin with the proliferation of keratinocytes, the expression of hurpin in infected and unaffected skin of 2 psoriatic patients was examined by RT-PCR. The affected skin showed an expression of hurpin which was up to 10-14 times stronger in comparison with the unaffected skin of the same patient. Hurpin / PI 13 sequence: clone R7-1.1
Total number of nucleotides: 3143

Hurpin / PI 13 sequence: clone R7-16.1
Total number of nucleotides: 2832

Hurpin / PI 13 sequence: clone R7-12.2
Total number of nucleotides: 2654

Table 1

Amino acid homology between hurpin and known ovalbumin serpins

Claims (12)

1. A nucleic acid that contains the following nucleotide sequence:
R7-1.1
R7-16.1
R7-12.2. 2. Nucleotide sequence of a gene or gene fragment which is contained in the following vectors:
pBS-HP1.1
pBS-HP16.1
pBS-HP12.2. 3. A nucleic acid which, under stringent conditions, contains nucleotide sequences from claim 1 and 2 or the complementary sequence hybridizes. 4. An amino acid sequence for which the sequences from claim 1 code. 5. A vector containing the sequences from claim 1, 2 or 3. 6. An expression vector which is the nucleotide sequence from claim 1, 2 or 3 in functional Association with regulatory elements to control the expression of the Contains nucleotide sequence in a host cell. 7. A genetically engineered host cell that contains the nucleotide sequence from claim 1, 2, 3 or 4 contains. 8. A genetically engineered host cell that contains the nucleotide sequence from claim 1, 2, 3 or 4 in functional association with regulatory elements to control the expression of the Contains nucleotide sequence in the host cell. 9. A purified gene product encoded by the nucleic acids in claim 1, 2, 3 or 4. 10. An antibody that binds the gene product from claim 8 in an immunospecific manner. 11. A transgenic animal in which the nucleic acids from claim 1, 2, 3 or 4 are expressed as Transgene forms part of the animal's genome. 12. A transgenic animal in which the expression of a genomic sequence that the Gene product of claim 8 is encoded, prevented or suppressed.