DE19515514C1 - DNA encoding nucleolar-endosomal auto-antigen - Google Patents

Abstract

Nucleolar/endosomal auto-antigen (I) with the 1411 amino acid sequence given in the specification, and its functional derivs. and fragments, against which an auto-antibody (Ab) is directed, is new. Also new are: (1) DNA which encodes (I), having the 4709 bp nucleic acid sequence given in the specification, and its fragments, hybrids and degenerates; (2) expression plasmid contg. the DNA of (1); and (3) transformed cell contg. the plasmid of (2).

Description

The present invention relates to a nucleolar / endosomal autoantigen, a such a coding DNA and a method for producing such like its use.

Autoimmune diseases are characterized by the appearance of autoanti body, d. H. Antibodies that are against components of your own organism judge. Autoantibodies can damage the organism, an organ or induce an organ component and thereby sometimes serious trigger life-threatening diseases. The emergence of such a he disease occurs through various pathomechanisms, such as through neutra lization of antigens, e.g. B. hormones, by blocking or stimulation a receptor for biologically active substances, e.g. B. by autoantibodies against the receptor of the thyroid stimulating hormone in hyperthyroidism, by binding to certain cell or tissue structures with induction Complement-mediated inflammation, e.g. B. Autoanti glomerulonephritis body against glomerulus basement membranes. Tissue damage can also occur through cell-mediated mechanisms (autoantibody-dependent cellular cytoto xicity) or by localized and systemic immune complex deposits after binding of the autoantibodies to soluble antigens.

In addition to such obviously directly damaging autoantibodies occur in the People have a variety of autoantibodies to blood, cell, or tissue components that have so far not been clearly identified as a tissue-damaging role can be awarded.  

The range of forms of rheumatic diseases, especially inflammatory ones rheumatic diseases to which collagen diseases are attributed is characterized by the appearance of numerous autoantibodies. These react e.g. B. with antigens of the cell nucleus, such as with double-stranded DNA, Single-stranded DNA, RNA, histones, non-histone proteins, ribonucleoproteins, Chromosome-associated antigens, e.g. B. centromeres or spindle apparatus, or with antigens that are only in certain phases of the cell cycle, e.g. B. cyclin, be expressed.

For diseases such as lupus erythematosus, Sjögren's syndrome, mixed collagen se, polymyositis, scleroderma, CREST syndrome, Wegener's granulomatosis and dermatomyositis, the above autoantibodies are found. Your after usually takes place through the implementation of kernel extracts from veal or rabbit fur chen thymocytes. However, this is a differential diagnosis of this crane not possible. However, such is a prerequisite for the choice of Therapy.

The present invention is therefore based on the object of providing a means with which rheumatic diseases are diagnosed more precisely can.

According to the invention, this is achieved by providing the objects in the Claims achieved.

The invention thus relates to a nucleolar / endosomal autoantigen which comprises the amino acid sequence of FIG. 2 or a functional derivative or fragment thereof.

The expression "nucleolar / endosomal autoantigen" indicates that the Autoantigen can be present in both nucleoli and endosomes.

The term "functional derivative or fragment" includes any derivative or fragment of the amino acid sequence of Fig. 2 against which an autoantibody can be raised. In particular, the term refers to epitopes in the amino acid sequence that can act as antigens. It also goes without saying that the amino acid sequence of FIG. 2 can have additions, substitutions and / or deletions of one or more amino acids, which also applies to the functional derivatives or fragments thereof.

Another object of the invention is one for an autoantigen above coding nucleic acid. This can be an RNA or a DNA. The latter can e.g. B. be a genomic DNA or a cDNA. Preferred is a DNA that includes the following:

• (a) the DNA of Figure 2 or a portion thereof,
• (b) a DNA hybridizing with the DNA of (a), or
• (c) one with the DNA from (a) or (b) above the degenerate genetic Code related DNA.

The term "hybridizing DNA" indicates a DNA that is commonly used Conditions, especially at 20 ° C below the melting point of the DNA with a DNA from (a) hybridizes.

The DNA of FIG. 2 was deposited with the DSM (German Collection of Microorganisms and Cell Cultures) as E. coli P162 under DSM 9915 on April 12, 1995.

A DNA according to the invention or one encoded by it are described below Autoantigen with "P162 DNA" ("P162 cDNA) or" P162 Antigen "designated net. This is due to the fact that the molecular weight of the Autoantigen according to the invention is 162 465 Daltons.

To produce a P162 cDNA, it is favorable to screen a lambda phage expression library with sera from patients with rheumatic diseases. Positive clones can then be sequenced and used for another screening if necessary. The preparation of a P162 cDNA according to the invention is described in Examples 1-2. Their structure elucidation as well as those of P162 antigen encoded by it are described in Example 3 and Fig. 5 beschrie ben.

According to the invention, a P162 cDNA in a vector or expression vector present. Examples of such are known to the person skilled in the art. in case of an Expression vectors for E. coli are z. B. pGEMEX, pUC derivatives, pGEX-2T and pET3b, the latter being preferred. For expression in yeast z. B. To name pY100 and Ycpad1, while for expression in animal cells e.g. B. pKCR, pEFBOS, cDM8 and pCEV4 are to be specified.

The person skilled in the art knows suitable cells, around one, in an expression vector to express the present P162 cDNA. Examples of such cells include the E. coli strains HB101, DH1, x1776, JM101, JM 109, and BL21, the latter rer is preferred, the yeast strain Saccharomyces cerevisiae and the animal Cells L, 3T3, FM3A, CHO, COS, Vero and HeLa.

The person skilled in the art knows how a P162 cDNA is expressed vector must be inserted. He also knows that this DNA is in Verbin dung with a DNA coding for another protein or peptide can be so that the P162 expresses cDNA in the form of a fusion protein can be.

Furthermore, the person skilled in the art knows conditions, transformed or trans cultivate infected cells. He is also familiar with methods that expressed P162 Isolate and purify antigen. A foregoing, recombinant prepared P162 antigen (hereinafter referred to as rP162 antigen), whereby this can also be a fusion protein is therefore also an object the invention.  

Autoantigens according to the invention are distinguished in that they have rheumatism recognize table-specific autoantibodies. They are therefore suitable for differential diagnosis of rheumatic diseases. Such a diagnosis can be found in usual detection methods, especially in a Western blot, an ELISA, immunoprecipitation or immunofluorescence. You can do this the autoantigens according to the invention, if appropriate, be marked or used in combination with labeled antibodies directed against them will.

Furthermore, autoantigens according to the invention can be present in a kit. This can combine the autoantigens as stated in the above form with usual additives, such as buffers and carrier material. Such a Kit is also the subject of the invention.

In addition, autoantigens according to the invention are also suitable for therapy table purposes. For example, they can have autoantibodies affinity chro be removed matographically from the circulation of rheumatic patients. There is also a need to remove rheumatism-specific autoantibody products lymphocytes by coupling the autoantigens according to the invention Thinking about cell toxins.

Also suitable are nucleic acids according to the invention, in particular DNAs, for diagnostic purposes of all kinds. Such nucleic acids can also be used for therapeutic measures are used. For example, the Nucleic acids are inserted into conventional expression vectors and these in Patients are introduced. By expression of the nucleic acids Receive autoantigens that can then intercept the autoantibodies.

Brief description of the drawings

Figure 1 shows the cloning and composition of the P162 cDNA. The upper part of the figures shows a scale in kb. The thick line corresponds to the open reading frame with restriction interfaces indicated by arrows (E = Eam 1105l; N = Nhel; S = Spel; Sa = Sacl; H = Hpal; A = Accl; I = Esp3l). The four thin lines represent isolated clones; the clones sequenced in both directions are marked with dots. The small clone shown at the bottom left was obtained according to the RACE protocol (cf. Frohmann, MA et al., Proc. Natl. Acad. Sci. USA 85 (1988), 8998-9002). 1: nt 314-4780; 2: nt 278-2912; 3: nt 2514-4372; 4: nt 1-325,

Fig. 2 shows the nucleotide sequence and the deduced amino acid sequence of P162 cDNA

Figure 3 shows a Northern blot analysis of P162 gene expression in HeLa and HEp-2 cells. RNA samples were separated with the aid of formaldehyde agarose gel electrophoresis according to their size and hybridized with 32 P-labeled P162 cDNA with high stringency. Lane 1: 2 µg poly (A) ⁺ RNA from HeLa cells; Lane 2: 2 µg poly (A) ⁺ RNA from HEp-2 cells. The size of the bands in the Autora diogram corresponds to approximately 7.5 kb,

Fig. 4 shows a Kyte-Doolittle hydrophobicity plot in which is hydrophilic nature can be seen from P162,

Fig. 5 shows the profile of the coiled-coil segments (heptad repeats) of P is 162. values <0.5 indicate potential coiled-coil structures. Almost all of the protein consists of heptad repeats. Only the ends of the molecule and five other regions (for AS 110, 220, 280, 350 and 1050) have different structures,

Figure 6 shows the synthesis and purification of a recombinant P162 fusion polypeptide. A: Analysis of the individual protein fractions using SDS-PAGE. 1 : supernatant of the cell lysate with which the Ni ²⁺ affinity column was loaded; 2: passage through the column; 3 and 4: elution fractions. B: Immunoblot analysis of the protein fractions shown. A rabbit anti-P162 serum was used as an immunological sample. The molecular weights are given in kD on the left,

Fig. 7 shows immunoprecipitations ³⁵S-methionine-labeled proteins from HEp-2 cells with human or rabbit serum. 1: Protein A-Sepharose alone; 2: serum of a healthy person; 3: serum of a patient D; 4: Affinity-purified antibodies from patient D; 5: rabbit pre-immune serum; 6: rabbit immune serum; 7: Rabbit affinity-purified antibodies. The molecular weights are given in kD on the left,

Figure 8 shows the isolation of early endosomes from BHK cells. To present a cytoplasmic fraction, the cells were broken up by careful shearing (homogenization). The various cytoplasmic components were obtained by sucrose gradient centrifugation. The distribution of P162 in the different fractions was determined in the immunoblot, which was sampled with affinity-purified rabbit anti-P162. 1: late endosomes; 2: early endosomes; 3: remaining cytoplasmic fraction,

Fig. 9 shows the staining of the endosomes in the form of cytoplasmic granules of HEp-2 cells with a natural antibodies from a patient serum (A) and with a rabbit antibody (B), which is directed against recombinant P162. HEp-2 cells were incubated simultaneously with antibody-containing human serum or rabbit serum. Goat anti-human IgG, which was FITC-labeled, was used to display the bound human antibodies. Goat anti-rabbit IgG labeled with rhodamine was used to display the bound rabbit antibodies. The figure shows that both antibodies stain the same structures within the cytoplasm of the HEp-2 cells (see arrows), in which case the human antibody has a stronger fluorescence than the rabbit antibody, and

Figure 10 shows K562 cells incubated with antibodies raised against recombinant P162 in rabbits. In addition to granular fluorescence of the endosomes in the cytoplasm (arrow), there is also a clear fluorescence of the nucleoli in the cell nucleus. Depending on the cell type and probably also on the functional state of the cells, the antibody also reacts with an antigen that is located in the cell nucleus.

The following examples illustrate the invention.

example 1 Screening and isolation of recombinant lambda phages

The human anti-P162 positive serum used as an immunological sample also contained anti-Ro 52 kD antibodies. The latter were removed by binding to membrane-immobilized Ro 52 kD protein. The treated serum was used to screen approximately 500,000 plaques from an oligo (dT) -primed HeLa cDNA expression library (λ UNIZAPXR, Stratagene, Heidelberg). After repeated purification, three positive clones were obtained, one of which was sequenced (see FIG. 1). In addition, a shorter 5'-clone was obtained with an Amplifinder RACE kit (Clontech, Palo Alto, CA, USA) and cloning into the plasmid Bluescript KS (Stratagene). A total of 4 clones were isolated.

Example 2 Sequencing of the clones and construction of the entire P162 cDNA

Bluescript KS plasmids were cut out of the λ clones with helper phages according to the manufacturer's instructions (Stratagene) and the DNAs were characterized by hybridization and purification with Quiagen columns (Quiagen GmbH, Düsseldorf) using a sequencing machine (ALF, Pharmacia , Freiburg) sequenced using the dideoxy method with fluorescence ATP (Boehringer GER Mannheim). All sequences of the clones were created according to a wal king primer strategy, the clones No. 1 and No. 4 (see FIG. 1) were sequenced in both directions. The sequence data were combined using the Assemgel software from PC-Gene (IntelliGenetics Inc. Brussels, Belgium).

Example 3 Nucleotide sequence and deduced amino acid sequence of the P162 cDNA

Computer-aided sequence analyzes were carried out using the GCG software (Genetics Computer Group Madison, Wisconsin, USA). The EMBL nucleic acid and the SWISS-PROT protein sequence databases were analyzed with the BLASTN and BLASTP algorithms (cf. Altschul, SF et al., J. Mol. Biol. 215 (1990), 403-410) for the presence of local similarities with individual sequences scanned in the P162 nucleic acid / amino acid sequence. The search for functional motifs was carried out using the PROSITE algorithm (cf. Bairoch, A., Nucleic Acids Res. 19 (1991), 2241-2245, supplement). The cloning and sequencing strategy gave the nucleic acid sequence shown in FIG. 2 and the amino acid sequence derived therefrom. The first ATG (nt 71) lies in an environment favored for translocation initiation and starts an open reading frame of 4233 bp, which codes for a protein with 1411 amino acids (162 465 daltons).

The P162 antigen is a hydrophilic, acidic protein (pl = 5.49) with one uniform distribution of charged or polar and hydrophobic amino acids found in N-glycosylation and potential phosphorylation motifs  are embedded.

The search for sequence similarities between the P162 antigen and others Proteins showed no pronounced homology to those in the databases deposited sequences. However, there were two functional domains in the P162 Protein can be clearly identified. The most outstanding feature are the almost the entire protein spanning coiled-coil structures. The second functional region is one in the N-terminus (A.S. 45-65) local based zinc finger motif characteristic of nucleic acid binding proteins.

Example 4 Size of the P162 mRNA

Total RNA and poly (A) ⁺ RNA were isolated from a human cell line, separated by size electrophoretically in a formaldehyde agarose gel and after transfer to a membrane hybridized with radioactively labeled P162 cDNA. The autoradiogram ( Fig. 3) shows a signal at approximately 7.5 kb. This experiment demonstrates that the P162 mRNA is large enough to contain the 4.78 kb P162 cDNA.

Example 5 Synthesis of part of the coding region of the P162 cDNA in a bacterial len expression system

A segment in the N-terminal part of the P162 cDNA (nt 2238-4307) ( Fig. 2) was provided with a BamHl site at the 5'-end and after ligation into the bacterial fusion expression vector pEx34b in E. coli 537 (cf. Remaut , E. et al., Gene 22, 1983, 103-113; Seelig, HP et al., J. Immunol. Methods 143, 1991, 11-24. After culturing an 80 ml culture overnight at 30 This culture was diluted and induced with 320 ml of LB medium at 47 ° C. After three hours of growth (42 ° C.), the bacteria were harvested, lysed with lysozyme and suspended in 20 ml of 8 M urea Centrifugation removed (16,000 x g, 10 min., 20 ° C.) and the soluble proteins were obtained using Ni ²⁺ chelate affinity chromatography (Quiagen). The yield was approximately 50 mg of recombinant P162. The size of the P162 antigen according to the estimate in SDS-PAGE, the derived molecular weight of approx. 80 kD (70.5 kD P162 portion and 10.3 kD M S2 polymerase fusion fraction).

Example 6 Localization of P162 in early endosomes

The early endosomes of BHK cells were marked by internalization of horseradish peroxidase (5 min at 37 ° C). The cytoplasmic fraction was separated after homogenization of the cells using a sucrose-D₂O gradient. The interphase between 16 and 10% sucrose in D₂O contained the early endosomes (see Gorvel, J.-P. et al., Cell 64, 1991, 915-925). The proteins of the early and late endosomes as well as the remaining cytoplasmic fraction were analyzed in a Western blot. The vast majority of P162 was found in the early endosome fraction (see Figures 8 and 9).

Example 7 Localization of P162 in Nucleoli

K562 cells were incubated with rabbit anti-rP162 serum and the bound antibodies were prepared with goat anti-rabbit IgG labeled with rhodamine. In contrast to the exclusive endosomal localization of the P162 antigen shown in FIG. 9, the K562 cells additionally show a clear fluorescence of the nucleoli in the cell nucleus.

Claims (6)

1. A nucleolar / endosomal autoantigen comprising the amino acid sequence of Fig. 2, wherein Fig. 2 is part of this claim, or a functional derivative or fragment thereof against which an autoantibody can be directed. 2. DNA according to claim 1, wherein the DNA comprises:

• . (a) the DNA of Figure 2, Fig. 2 part of this claim, or a part thereof, against which the amino acid sequence of a car can be directed antibodies,
• (b) a DNA hybridizing with the DNA of (a) or
• (c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.

3. Expression plasmid comprising the DNA of claim 2. 4. Transformant containing the expression plasmid according to claim 3. 5. A method for producing the autoantigen of claim 1 comprising culturing the transformant according to claim 4 under suitable Conditions. 6. Kit comprising the autoantigen of claim 1 and usual Additives.