DE4438838C1 - DNA sequences for matrix metal proteases, their preparation and their use - Google Patents

Abstract

DNA sequences for human matrix metalloproteases are disclosed, as well as homologous DNA sequences homologous and derived therefrom. Also disclosed are the proteins and protein variants coded by these DNA sequences, their expression, preparation and use. The invention has applications in the fields of biomolecular, medical and pharmaceutical research, for medical diagnosis and therapy, and in the pharmaceutical and biotechnological industry.

Description

The invention relates to DNA sequences for human matrix metalloproteases as well as thereof derived and homologous sequences. It also affects those caused by the DNA sequences encoded proteins and protein variants, their expression, extraction and use. Areas of application are molecular biological, medical and pharmaceutical Research, medical diagnostics and therapy, pharmaceutical and biotechnology industry.

Matrix metalloproteases hydrolyze proteins from the extracellular matrix. You change that Matrix structure and affect cell-matrix interactions. To the Matrix-Me talloproteases include collagenases, gelatinases, stromelysins and metalloelastases / 1 /. The enzymes are u. a. involved in the following physiological processes: ovulation / 2 /, Embryo implantation in the uterus / 3 /, cell migration and tissue rearrangement during embryogenesis / 4 /, involution of the mammary gland / 5 / and uterus / 6 /, angiogenesis / 7 /. Matrix metalloproteases play an important role in wound healing and scar formation / 8 /, in the metastasis of tumor cells / 9, 10 /, in rheumatoid arthritis and in Osteoarthritis / 11, 12 /, for periodontal diseases / 13 /.

All matrix metalloproteases contain a Zn ²⁺ ion in the active center. The activation of the matrix metalloproteases synthesized in the form of inactive proenzymes requires the release of a bond between the Zn ²⁺ ion in the active center and a Cys residue in the N-terminal propeptide of matrix metalloproteases (cysteine switch) / 14 /. Matrix metal talloproteases consist of several protein domains that have homology to each other / 1, 14 /. While the protease Matrilysin consists of only one propeptide and the amino acid sequence of the catalytic domain, other matrix metalloproteases also contain a hemopexin-like sequence of approx. 200 amino acids. Gelatinases A and B contain additional amino acid sequences. Known human matrix metalloproteases, their molecular weights and their preferred substrates are listed in Table 1.

The different matrix metalloproteases are not only characterized by characteristic ones macromolecular specificity for matrix proteins. Your activity will be on different molecular and cellular levels controlled:

• 1. Regulation of the synthesis of matrix metalloproteases by growth factors, cytokines, Polypeptide hormones, prostaglandins, glucocorticoids, estrogen, progesterone and others Effectors / 1, 14 /.
• 2. Binding of matrix metalloproteases to membrane receptors / 15 /.
• 3. Activation of inactive proenzymes by specific hydrolysis of the respective propeptides / 16 / or by oxidation / 17 /.
• 4. inhibition of matrix metalloproteases by specific protein inhibitors such as TIMP-1, TIMP-2 and TIMP-3 (TIMP = Tissue Inhibitor of Matrix-Metalloproteases) / 16 /.
• 5. Proteolytic degradation of matrix metalloproteases.

Matrix metalloproteases are due to their important physiological functions and their role in the pathogenesis of diseases has been intensively studied. There is interest in the Discovery and characterization of further matrix metalloproteases.

The aim of the present invention is to develop novel and previously unknown matrix measurements human talloproteases for medical research, diagnostics and therapy open up. The task is to create DNA sequences for matrix metalloproteases identify and isolate, as well as the proteins encoded by the DNA sequences characterize.

The invention is implemented according to claims 1-13.

Novel matrix metalloproteases are determined using the following method:
Conserved amino acid sequences are selected in sequences of known matrix metalloproteases. Two suitable sequences are amino acids around a conserved Cys residue in the propeptide (cysteine switch) and amino acids that are involved in Zn ²⁺ binding in the active center of the enzymes. Degenerate oligonucleotides are synthesized for the selected peptides. Polymerase chain reactions (PCR) are carried out with the oligonucleotides and cDNA, which can be obtained by reverse transcription of mRNA from cells and tissues. Synthesized DNA fragments are cloned and sequenced. The determined sequences are compared with sequences in gene databases. PCR products with novel, previously unknown nucleotide sequences and homology to DNA sequences from matrix metalloproteases are used as probes for determining gene expression and for obtaining complete cDNA sequences from cDNA banks. The nucleotide sequences of complete cDNA are determined. The amino acid sequences of the associated proteins are derived by translation of the coding nucleotide regions and analyzed by known methods.

The following cDNA sequences were determined:

• 1. cDNA sequence mmpm1a consisting of
• - 5′-non-translated region: base pairs 1 to 141
• - coding region: base pairs 142 to 1881
• - 3′-non-translated region: base pairs 1882 to 3456 (see Attachment 1)
• 2. cDNA sequence mmpm1b consisting of
• - 5′-non-translated region: base pairs 1 to 113
• coding region: base pairs 114 to 1862
• - 3'-untranslated region: base pairs 1863 to 3437 (see Appendix 2)
• 3. cDNA sequence mmpm2 consisting of
• - 5′-non-translated region: base pairs 1 to 48
• - coding region: base pairs 49 to 2058
• - 3′-non-translating region: base pairs 2059 to 3530 (see Appendix 3)

The invention also includes variants of these sequences and homologous DNA sequences of the Humans and other mammalian species by cross hybridization with these sequences be found. The invention also includes constructs that consist of a vector for gene transfer into prokaryotic or eukaryotic cells and one of the invention Sequences exist.

With the help of the sequences mmpm1a, mmpm1b and mmpm2 different aspects of the Biosynthesis of encoded matrix metalloproteases will be investigated. They can Structural genes including flanking sequences can be determined. cDNA sequences can be used as molecular probes to analyze gene expression in cells and tissues be used.

The cDNA sequences mmpm1a, mmpm1b and mmpm2 encode the following Amino acid sequences:

MMPm1a

MMPm1b

MMPm2

The invention also encompasses variants of these proteins by post-translational Protein modification, by protein chemical modifications or by in vitro mutagenesis and expression of nucleotide sequences and homologous proteins of humans and other mammalian species by immunological cross-reactivity or equivalent Enzyme activity can be identified. The invention also includes complexes of these proteins with one or more ligands.

MMPm1a, MMPm1b and MMPm2 can be isolated from natural sources. The Proteins can also be generated by gene transfer and expression in prokaryotic and eukaryotic cells are synthesized and obtained from the recombinant cells. The availability of the proteins MMPm1a, MMPm1b and MMPm2 enables Studies of their structure and function. There can be methods of determining the enzymatic activity and specificity are worked out. Starting from the The primary structure of the proteins MMPm1a, MMPm1b and MMPm2 can be monoclonal and polyclonal antibodies are produced. The antibodies can be used for diagnostic analysis of MMPm1a, MMPm1b and MMPm2. MMPm1a, MMPm1b and MMPm2 can be used as test structures for finding natural and synthetic Activators and inhibitors of matrix metalloproteases can be used.

For the characterization of mmpm1a, mmpm1b and mmpm2 as well as MMPm1a, MMPM1b and MMPm2 the following further details are given:
The DNA sequences mmpm1a and mmpm1b differ only in their 5′-untranslated regions and in the immediately following parts of their coding regions. Starting with nucleotides 363 (mmpm1a) and 344 (mmpm1b), both sequences are identical. The mmpm1a sequence contains an open reading frame of 580 codons. The reading frame begins with the nucleotides A₁₄₂TG. However, the area surrounding these nucleotides is unfavorable for effective translation. In contrast, the following nucleotides A₁₅₄TG in the reading frame are in an environment preferred for a translation start. It is possible that the translation of mmpm1a does not start until the second ATG. The sequence mmp1b has an open reading frame of 583 codons starting with A₁₁₄TG. The start codon is located within the ACCATGT nucleotide sequence, which favors effective translation.

The translation start of mmpm2 is at A₄₉TG. The open reading frame from mmpm2 contains 670 codons.

The proteins MMPm1a, MMPm1b and MMPm2, which are composed of the cDNA sequences mmpm1a, mmpm1b and mmpm2 derive have calculated molecular weights of 65 591, 65 900 and 75 813. The primary sequences of MMPm1a, MMPm1b and MMPm2 are homologous to Sequences of known matrix metalloproteases. The three novel enzymes each contain one Signal peptide, a prosequence with the cysteine switch region PRCGVPD and one RRKRYA consensus sequence. Sequences of catalytic domains follow with the specific arrangement of three histidine residues HELGHALGLEH and hemopexin homologue Sequences. In contrast to known matrix metalloproteases, MMPm1a contain MMPm1b and MMPm2 also have C-terminal sequences with characteristic sequences hydrophobic amino acid residues. MMPm1a and MMPm1b have the hydrophobic Amino acid sequence AAAVVLPVLLLLLVLAVGLAV (amino acid positions 536-556 in MMPm1a, amino acid positions 539-559 in MMPm1b). An analog sequence in MMPm2 is VVMVLVPLLLLLCVLGLTY (amino acid residues 626-645). The hydrophobic Sequences that go beyond the specified positions are loaded by Flanked by amino acid residues. Negatively charged glutamine and Aspartate residues, C-terminally positively charged arginine and lysine residues.

The presence of the hydrophobic sequences in MMPm1a, MMPm1b and MMPm2 allowed the conclusion that MMPm1a, MMPm1b and MMPm2 - in contrast to known ones soluble matrix metalloproteases (Table 2) - are membrane-associated enzymes. From the Primary sequences follow that propeptides, catalytic domains and hemopexin homologous Domains of the proteases are located extracellularly. The outermost C-termini of the proteins cells should express themselves in the cytosol of MMPm1a, MMPm1b or MMPm2 expressing cells are located.

In contrast to MMPm1a and MMPm1b, MMPm2 contains a potential  Glycosylation site at N₁₄₀YT.

MMPm1a and MMPm1b differ only in their signal and prosequences. The different structure of the pro sequences implies different activation mechanisms of MMPm1a and MMPm1b. As the pro sequences in the course of activation are separated from matrix metalloproteases by hydrolysis, the activation of MMPm1a and MMPm1b lead to an identical active enzyme.

Northern blot analyzes of human tissue mRNA show a different Expression of MMPm1 and MMPm2. MMPm1 type matrix metalloproteases especially in lung, placenta, kidney, ovary, prostate, small intestine, spleen, thymus, and Testis tissue expressed. Their expression is significantly lower in heart and pancreatic tissue, hardly detectable in the brain, liver and skeletal muscles. MMPm2 is in placenta, heart, Liver, skeletal muscle, kidney, pancreas, lungs, testis, colon and small intestine expressed.

In summary, it is found that the invention is novel, previously unknown Matrix-Me human talloproteases. Knowledge of cDNA and Protein sequences of the matrix metalloproteases allow further investigation of Biosynthesis, structure and function of the enzymes. By analyzing the gene sequences you can inherited and acquired mutations can be found. The determination of concentration and activity of the matrix metalloproteases in cells, tissues and body waste enables diagnostic statements. The enzymes can advantageously be used as test structures for Discovery of new drugs, including activators and inhibitors of Matrix metalloproteases can be used.

The invention is further illustrated by the following application examples:

1. Identification of novel DNA sequences for matrix metalloproteases

To find cDNA sequences that code for matrix metalloproteases, was mRNA from human cells and tumor tissues, including neuroblastoma cells, Renal carcinoma and osteosarcoma tissues isolated. The mRNA was reversed Transcriptase rewritten in cDNA.

Two were conserved in the protein sequences of known matrix metalloproteases Selected amino acid sequences.

• 1. A sequence around a characteristic Cys residue in propeptides of matrix metalloproteases (cysteine switch):
  P - R - C - G - V / N - P - D
• 2. A sequence with three His residues in the catalytic protein domain of matrix metalloproteases (Zn ²⁺ binding region):
  H - E - L / I / F - G - H - S / V / A - L / M - G

According to the amino acid sequences, degenerate oligonucleotide primers, both the variation of the amino acids in the two conserved sequences, as well as the Consider degeneration of the genetic code, synthesized:

1. Propeptide primer

2. Zn binding region primer

Both primers each contain four deoxyinosine nucleotides and additional nucleotides for the recognition sites of the restriction endonucleases XbaI (propeptide primer) and BamHI (Zn ²⁺ binding region primer).

The degenerate primers were used in the PCR together with cDNA.  

The reaction mixture contained:

100ng cDNA
1 µg propeptide primer
1 µg Zn ²⁺ binding region primer
2.5 U / 100 µl DNA polymerase AmpliTaq
100 µM dNTP
0.01% gelatin
50 mM KCl
1.5 mM MgCl₂
10 mM Tris-HCl, pH 8.3

30 reaction cycles of the sequence 50 sec 94 °, 1 min 56 °, 2 min 72 ° were carried out. The amplified DNA was extracted with phenol / chloroform and then cleaved with the restriction enzymes XbaI and BamHI. DNA fragments of the size 350-500 base pairs were isolated by agarose gel electrophoresis ( FIG. 1) and cloned in the plasmid pBluescript SK (Stratagene). Individual clones were sequenced with T3 and T7 primers and Sequenase 2.0 (USB / Amersham Life Science). The sequences obtained were compared with DNA sequences in the Genbank and EMBL databases. The sequence comparison was carried out using the FASTA program of the HUSAR program package (GCG Package, Copyright Genetics Computer Group, Inc.). Starting from renal carcinoma cDNA z. B. several hundred clones with amplified DNA obtained, 50 of which were sequenced. The evaluation revealed both known and novel DNA sequences. Among the former were sequences for human interstitial collagenase and for matrilysin. Among the latter were two sequences with homology to human matrix metalloproteases. The two new sequences, designated PCRmmpm1 and PCRmmpm2, contained the following nucleotides:

PCRmmpm1

PCRmmpm2

2. Northern blot analysis

The expression of matrix metalloproteases in human tissues was determined using the Fragments PCRmmpm1 and PCRmmpm2 examined. The fragments became radioactive labeled (Multiprime DNA Labeling Kit, Amersham Life Science) and with mRNA on nylon (Multiple Tissue Blot, Clontech) hybridized. The hybridizations and subsequent Washes were carried out under standard conditions / 18 /.

Both PCRmmpm1 and PCRmmpm2 hybridize with RNA of approximately 3.6 kb. However, the experiments demonstrated a different expression of the mRNA hybridizing with PCRmmpm1 and PCRmmpm2 ( Fig. 2). Specific transcripts that hybridize with PCRmmpm1 are particularly found in lung, placenta, kidney and kidney carcinoma tissues (not shown). They are present in a significantly small amount in pancreas and heart tissue, in the liver, skeletal muscles and brain.

Messenger RNA that hybridizes with PCRmmpm2 is particularly found in placental tissue synthesized. Their expression is in the heart, liver, skeletal muscle, kidney, pancreas and lungs  however comparable. It is significantly lower in brain tissue.

3. Isolation and sequencing of cDNA mmpm1 and mmpm2

A lung cDNA library in the vector λgt 11 (Clontech) was analyzed by phage transfer to nylon and hybridization with the radioactively labeled probes PCRmmpm1 and PCRmmpm2 / 18 /. The hybridizations were carried out for 16 h at 40 ° in 50% formamide, 5 × SSPE, 5 × Denhardt, 0.5% SDS, 50 μg / ml denatured herring sperm DNA. After the hybridization, the filters were washed at room temperature and at 65 ° in 2 × SSC, 0.1% SDS and then evaluated in a bio-imaging analyzer (BAS 2000, Fuji Photo Film Co, LTD). Phage clones that hybridized with PCRmmpm1 and PCRmmpm2 were identified using specifically bound radioactivity. The phages found were gradually separated by dilution. The DNA of individual phages was isolated (Quiagen Lambda Kit, Diagen GmbH) and the cDNA inserts contained were inserted into the plasmid vector pBluescript SK (Stratagene). The inserts were then broken down into partial fragments (Erase-a-Base system, Promega) and sequenced (Sequenase 2.0, USB / Amersham Life Science). Determined DNA sequences were analyzed with the aid of the program packages DNA-STAR (DNA-STAR. Inc) and HUSAR (GCG Package, Copyright Genetics Computer Group, Inc). The translation of the coding sequences and the comparison of the amino acid sequences obtained with known matrix metalloproteases showed that the novel sequences belong to the family of matrix metalloproteases ( Fig. 3).

TABLES AND FIGURES Table 1

Human matrix metalloproteases. The molecular weights given are from the cDNA sequences of the enzymes calculated.

Fig. 1:
Agarose gel electrophoresis of PCR products obtained with degenerate primers for matrix metal talloproteases and cDNA of the human neuroblastoma cell line SK-N-SH (lane 1), cDNA from renal carcinoma tissue (lane 2) and cDNA from osteosarcoma tissue (lane 3) were.

Fig. 2:
Northern blot analysis of mRNA for MMPm1 (top) and MMPm2 (bottom)

Fig. 3:
Homology comparison of MMPm1a, MMPm1b and MMPm2 with known matrix metal oproteases in humans. The comparison was carried out with the CLUSTAL program. The abbreviations mean: hscollr. pep - Interstitial collagenase, hsclgna.pep - Neutrophil collagenase, P08253.swisspro - Gelatinase A, hs4cola.pep - Gelatinase B, hsmmp3a.pep - Stromelysin 1, hsstrom2.pep - Stromelysin 2, hsstro13.pep - Stromelysin

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Table 1

Matrix metalloproteases

Claims (16)

1. DNA sequences mmpm1a, mmpm1b, mmpm2 and amino acid sequences derived therefrom for human matrix metalloproteases, characterized in that the cDNA sequence mmpm1a (Appendix 1) from

• - 5′-nichuran-translated region: base pairs 1 to 141
• coding region for the matrix metalloprotease MMPm1a: base pairs 142 to 1881
• - 3′-non-translated region: base pairs 1882 to 3456

consists,
the cDNA sequence mmpm1b (Appendix 2)

• - 5′-non-translated region: base pairs 1 to 113
• coding region for the matrix metalloprotease MMPm1b: base pairs 114 to 1862
• - 3′-non-translated region: base pairs 1863 to 3437

exists and
the cDNA sequence mmpm2 (Appendix 3)

• - 5′-non-translated region: base pairs 1 to 48
• coding region for the matrix metalloprotease MMPm2: base pairs 49 to 2058
• - 3′- non-translated region: base pairs 2059 to 3530

consists, the annexes 1, 2 and 3 components of this Are demanding. 2. DNA sequences according to claim 1, characterized in that these in Human genes as well as homologous Genes from other mammalian species are included. 3. Variants of the sequences mmpm1a, mmpm1b and mmpm2, as well as homologous DNA sequences of humans and other mammalian species, which by cross-hybridization with the in the sequences listed in claim 1 are obtained. 4. Constructs made from a vector for gene transfer to prokaryotic or eukaryotic Cells and one of the nucleotide sequences according to claims 1 to 3 exist.   5. The matrix metalloprotease MMPm1a of the following primary structure: 6. The matrix metalloprotease MMPm1b of the following primary structure: 7. The matrix metalloprotease MMPm2 of the following primary structure: 8. Variants of the proteins MMPm1a, MMPm1b and MMPm2 according to claims 5-7, through post-translational protein modification, through protein chemical modifications or by in vitro mutagenesis and expression of nucleotide sequences according to claims 1 and 2 are obtained, as well as homologous proteins of humans and other mammalian species that have immunological cross-reactivity or comparable enzyme activity.   9. Proteins according to claims 5-8, characterized in that these with one or several ligands are present as a complex. 10. Recombinant prokaryotic and eukaryotic cells generated by transfer of DNA sequences according to claims 1 to 3 are obtained in cells and the proteins according to the Express claims 5-8. 11. Method of obtaining the proteins MMPm1a, MMPm1b, MMPm2 and variants said proteins from natural sources and from recombinant cells according to claim 10 or from culture supernatants of recombinant cells according to claim 10. 12. Use of matrix metalloproteases MMPm1a, MMPm1b and MMPm2 according to Claims 5-8 and of cells that express these enzymes for generation proteolytic activity. 13. Use of matrix metalloproteases MMPm1a, MMPm1b, MMPm2 according to Claims 5-8 and of cells expressing MMPm1a, MMPm1b and MMPm2 to Screening protease effectors.