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, there 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:
This application is a continuation of Ser. No. 08/704,711 filed Nov. 20, 1996, now U.S. Pat. No. 6,114,159 which is a 371 of PCT/DE95/00357 filed Mar. 17, 1995. 
    
    
     BACKGROUND OF THE INVENTION 
     The invention relates to DNA sequences for human matrix metalloproteases as well as to homologous sequences derived therefrom. It furthermore relates to the proteins and protein variants, coded by the DNA sequences, their expression, preparation and utilization. Areas of application are molecular biological, medical and pharmaceutical research, medical diagnosis and therapy and the pharmaceutical and biotechnological industry. 
     Matrix metalloproteases hydrolyze proteins of the extracellular matrix. They change the matrix structure and effect cell-matrix interactions. The matrix metalloproteases include collagenases, gelatinases, stromelysins and metalloelastases [1]. The following are some of the physiological processes, in which the enzymes participate: ovulation [2], embryo implantation in the uterus [3], cell migrations and tissue inversions during embryo genesis [4], involution of the mammary gland [5] and of the uterus [6] and angiogenesis [7]. Matrix metalloproteases play an important role in wound healing and scar formation [8], in metastasizing of tumors cells [9, 10], in rheumatic arthritis and osteoarthritis [11, 12] and in 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 pro-enzymes, requires the dissolution of a bond between the Zn 2+  ion in the active center and a Cys group in the it-terminal propeptide of matrix metalloproteases (cysteine switch) [14]. Matrix metalloproteases consist of several protein domains, which exhibit homology among members of the Protease family [1, 14]. Whereas the protease matrilysin consists only of a propeptide and of the amino acid sequence of the catalytic domain, other matrix metalloproteases contain, in addition, a hemopexin-like sequence of about 200 amino acids. The 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. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 MATRIX METALLOPROTEASES 
               
             
          
           
               
                   
                 Protease 
                 M r  (kDa) 
                 Substrate 
               
               
                   
                   
               
             
          
           
               
                   
                 Interstitial 
                 54.1 
                 Collagen I, II, III 
               
               
                   
                 Collagenase 
               
               
                   
                 (MMP-1) 
               
               
                   
                 Neutrophilic 
                 53.4 
                 Collagen I, II, III 
               
               
                   
                 Collagenase 
               
               
                   
                 (MMP-5) 
               
               
                   
                 Gelatinase A 
                 73.9 
                 Collagen IV, V, VII 
               
               
                   
                 (MMP-2) 
                   
                 Gelatin, Elastin 
               
               
                   
                 Gelatinase B 
                 78.4 
                 Collagen IV, V 
               
               
                   
                 (MMP-9) 
                   
                 Gelatin, Elastin 
               
               
                   
                 Stromelysin 1 
                 54 
                 Proteoglycans, 
               
               
                   
                 (MMP-3) 
                   
                 Fibronectin, 
               
               
                   
                   
                   
                 Laminin, Gelatin, 
               
               
                   
                   
                   
                 Collagen II, IV, V, 
               
               
                   
                   
                   
                 IX 
               
               
                   
                 Stromelysin 2 
                 54.1 
                 Proteoglycans, 
               
               
                   
                 (MMP-10) 
                   
                 Fibronectin, 
               
               
                   
                   
                   
                 Laminin, Gelatin, 
               
               
                   
                   
                   
                 Collagen II, IV, V, 
               
               
                   
                   
                   
                 IX 
               
               
                   
                 Matrilysin 
                 29.7 
                 Proteoglycans, 
               
               
                   
                 (MMP-7) 
                   
                 Fibronectin, 
               
               
                   
                   
                   
                 Gelatin, Elastin 
               
               
                   
                 Stromelysin 3 
                 54.6 
               
               
                   
                 Metalloelastase 
                 54 
                 Fibronectin, Elastin 
               
               
                   
                   
               
             
          
         
       
     
     The different matrix metalloproteases are distinguished not only by a characteristic, macromolecular specificity for matrix proteins. Their activity is controlled on different molecular and cellular levels: 
     1. Regulation of the synthesis of matrix metalloproteases by growth factors, cytokines, polypeptide hormones, prostaglandins, glucocorticoids, estrogen, progesterone and other effectors [1, 14]. 
     2. Binding of matrix metalloproteases to membrane a 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 being investigated intensively because of their important physiological functions and their role in the pathogenesis of diseases. There is interest in finding and characterizing further matrix metalloproteases. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to make accessible novel and previously unknown human matrix metalloproteases for medical research, diagnosis and therapy. The task consists of identifying and isolating DNA sequences for matrix metalloproteases and of characterizing the proteins coded by the DNA sequences. 
     Novel matrix metalloproteases are discovered by the following method. In sequences of known matrix metalloproteases, conserved amino acid sequences are selected. Two suitable sequences are amino acids about a conserved Cys group in the propeptide (cysteine switch) and amino acids, which participate in the Zn 2+  binding in the active center of the enzymes. 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 tissue. Synthesized DNA fragments are cloned and sequenced. The sequences determined are compared with sequences in gene data banks. PCR products with novel, previously unknown nucleotide sequences and homologous with DNA sequences of matrix metalloproteases are used as probes for determining the gene expression and for obtaining complete cDNA sequences from cDNA libraries. The nucleotide sequences of complete cDNA are determined. The amino acid sequences of the corresponding proteins are derived by translation of the coding nucleotide regions and analyzed by known methods. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows an agarose gel electrophoresis of PCR products, which were obtained with degenerated primers for matrix metalloproteases and cDNA of the human neuroblastoma cell line SK-N-SH (lane 1), cDNA of kidney carcinoma tissue (lane 2) and cDNA of osteosarcoma tissue (lane 3). 
     FIGS. 2A and 2B show the Northern Blot Analysis of mRNA for MMPm1 (FIG. 2A) and MMPm2 (FIG. 2B) in different human tissue. 
     FIGS. 3A-H show the homology comparison of MMPm1a (SEQ ID NO: 1), MMPm1b (SEQ ID NO:2) and MMPm2 (SEQ ID NO:3) with known human matrix metalloproteases. The comparison was carried out with the CLUSTAL program. Key for the abbreviations in the figures: hscollr.pep (SEQ ID NO:16)—interstitial collagenase, hsclgna.pep (SEQ ID NO: 17)—netrophile collagenases, PO8253, swisspro (SEQ ID NO:18)—gelatinase A, hs4cola.pep (SEQ ID NO:19)—gelatinase B, hsmmp3a.pep (SEQ ID NO:20)—stromelysin 1, hsstrom2.pep (SEQ ID NO:21)—stromelysin 2, hsstrol3.pep (SEQ ID NO:22)—stromelysin 3. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following cDNA sequences are found: 
     1. A cDNA sequence mmpm1a (Seq. ID No.:8) consisting of 
     a 5′ nontranslated region: base pairs 1 to 141 
     a coding region: base pairs 142 to 1881 
     a 3′ nontranslated region: base pairs 1882 to 3456 
     2. A cDNA sequence mmpm1b (Seq. ID No.:9) consisting of 
     a 5′ nontranslated region: base pairs 1 to 113 
     a coding region: base pairs 114 to 1862 
     a 3′ nontranslated region: base pairs 1863 to 3437 
     3. A cDNA sequence mmpm2 (Seq. ID No.:10) consisting of 
     a 5′ nontranslated region: base pairs 1 to 48 
     a coding region: base pairs 49 to 2058 
     a 3′ nontranslated region: base pairs 2059 to 3530 
     The invention also comprises variants of these sequences as well as homologous DNA sequences of man and other mammalian species, which are found by cross-hybridization with these sequences. The invention also comprises constructs, which consist of a vector for the gene transfer in prokaryotic or eukaryotic cells and one of the inventive sequences. 
     Different aspects of the biosynthesis of coded matrix metalloproteases can be investigated with the help of the sequences mmpm1a, mmpm1b and mmpm2. The structure of the genes, including flanking sequences, can be determined. cDNA sequences can be used as molecular probes for analyzing the gene expression in cells and tissue. 
     The cDNA sequences mmpm1a (SEQ ID NO:8), mmpm1b (SEQ ID NO:9) and mmpm2 (SEQ ID NO:10) code encode the protein with the following amino acid sequences MMPm1a (SEQ ID NO:1), MMPm1b (SEQ ID NO;2) and MMPm2 (SEQ ID NO:3), respectively. 
     The invention also comprises variants of these proteins, which are obtained by post-translational protein modifications, by chemical modifications of the proteins or by in vitro mutagenesis and expression of nucleotide sequences, as well as of homologous proteins of man and other mammalian species, which are identified by immunological cross-reactivity or comparable enzyme activity. The invention also comprises complexes of these proteins with one or several ligands. 
     MMPm1a (SEQ ID NO:1), MMpm1b (SEQ ID NO:2) and MMPm2 (SEQ ID NO:3) can also be isolated from natural sources. The proteins can also be synthesized by gene transfer and expression in prokaryotic and eukaryotic cells and obtained from the recombinant cells. The availability of the proteins MMPm1a, MMPm1b and MMPm2 enables their structure and function to be investigated. Methods for determining enzymatic activity and specificity can be worked out. beginning with the primary structure of the proteins MMPm1a, MMPm1b and MMPm2, monoclonal and polycolonal antibodies can be produced. The antibodies can be used for the 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. 
     The following additional statements can be made concerning the characterization of mmpm1a, mmpm1b and mmpm2 and of MMPm1a, MMPm1b and MMPm2. The DNA sequences mmpm1a and mmpm1b differ only in their 5′ nontranslated regions and in the immediately subsequent parts of their coding regions. starting with the nucleotides 363 (mmpm1a) and 344 (mmpm1b) respectively, the two sequences are identical. The mmpm1a sequence contains an open reading frame of 580 codons. The reading frame commences with the nucleotides A 142 TG. However, the surroundings of these nucleotides are unfavorable for a effective translation. On the other hand, the subsequent nucleotides A 154 TG in the reading frame are in an environment favoring a translation start. It is possible that the translation of mmpm1a commences only at the A 154 TG. Starting with A 114 TG, the mmp1b sequence has an open reading frame of 583 codons. The starting codon is within the nucleotide sequence ACCATGT, which favors an effective translation. The translation start of mmpm2 is found at A 49 TG. The open reading frame of mmpm2 contains 670 codons. 
     The proteins MMPm1a, MMPm1b and MMPm2, which are encoded by the cDNA sequences mmpm1a, mmpm1b and mmpm2, have calculated molecular weights of 65,591, 65,900 and 75,813. The primary sequences of MMPm1a, MMPm1b and MMPm2 are homologous with sequences of known matrix metalloproteases. Each of the three novel enzymes contains a signal peptide, a prosequence with the cysteine switch region PRCGVPD (Seq. ID No.:11) and a consensus sequence RRKRYA (Seq. ID No.:12). Catalytic domains with the specific arrangement of three histidine groups HELGHALGLEH (Seq. ID No.:13) and sequences homologous with hemopexin follow. In contrast to known matrix metalloproteases, MMPm1a, MMPm1b and MMPm2 furthermore contain C-terminal sequences with characteristic sequences of hydrophobic amino acid groups. MMPm1a and MMPm1b have the hydrophobic amino acid sequence AAAVVLLLLLVLAVGLAV (amino acid positions 536-556 in MMPm1a, amino acid positions 539-559 in MMPm1b). An analogous sequence in MMPm2 is VVMVLVPLLLLLCVLGLTY (amino acid groups 626-645). The hydrophobic sequences, which go beyond the positions given, are flanked by charged amino acid groups. N-terminally, negatively charged glutamine and aspartate groups predominate and C-terminally, positively charged arginine and lysine groups predominate. 
     The presence of the hydrophobic sequences in MMPm1a, MMPm1b and MMPm2 permits the conclusion to be drawn that MMPm1a, MMPm1b and MMPm2, contrary to known soluble matrix metalloproteases (Table 1), are membrane-associated enzymes. It follows from the primary sequences that propeptides, catalytic domains and domains of the proteases, homologous with hemopexin, are localized extracellularly. The outermost C terminal of the proteins, on the other hand, should be located in the cytosol of cells expressing MMPm1a, MMPm1b and MMPm2. 
     MMPm1a, MMPm1b and MMPm2 contain a potential glycosylating site at. 
     MMPm1a and MMPm1b differ only in their signal and prosequences. The different structure of the prosequences implies different activation mechanisms of MMPm1a and MMPm1b. Since the prosequences are cleaved off by hydrolysis in the course of the activation of matrix metalloproteases, the activation of MMPm1a and MMPm1b should lead to an identical, active enzyme. 
     As shown in FIG. 2A and 2B, Northern blot analyses of mRNA of human tissue confirm that MMPm1 and MMPm2 are expressed differently. Matrix metalloproteases of the MMPm1 type are expressed primarily in lung, placenta, kidney, ovary, prostate, small intestine, spleen, thymus and testicle tissue. Their expression is clearly less in heart and pancreas tissue and hardly detectable in the brain, liver and skeletal muscles. MMPm2 is expressed in the placenta, heart, liver, skeletal muscles, kidneys, pancreas, lung, testicle, colon and small intestine. 
     In summary, it is noted that the invention makes available novel, previously not known matrix metalloproteases of man. A knowledge of the cDNA and protein sequences of the matrix metalloproteases permits the biosynthesis, structure and function of the enzymes to be investigated further. Inherited and acquired mutations can be found by analyzing gene sequences. Diagnostic information can be obtained from determining the concentration and activity of matrix metalloproteases in cells, tissues and exoreta. The enzymes can be used advantageously as test structures for discovering new pharmaceutical drugs, including activators and inhibitors of matrix metalloproteases. 
     The invention is described further by the following examples: 
     1. Identification of Novel DNA Sequences for Matrix Metalloproteases 
     For finding cDNA sequences, which code for matrix metalloproteases, mRNA was isolated from human cell and tumor tissue, including neuroblastoma cells, kidney carcinoma and osteosarcoma tissue. The mRNA was transcribed into cDNA with reverse transcriptase. 
     Two preserved amino acid sequences were selected from the protein sequences of known matrix metalloproteases. 
     1. A sequence about a characteristic Cys group in propeptides in matrix metalloproteases 
     P-R-C-G-V/N-P-D (Seq. ID No.:4) 
     2. A sequence with three His groups 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 (Seq. ID No.:5) 
     Corresponding to the amino acid sequences, degenerate oligo-nucleotide primers, which take into consideration the variation of the amino acids in the two conserved sequences, as well as the degeneracy of the genetic code, were synthesized: 
     1. Propeptide primer (Seq. ID No.:6) 
     
       
         
               
             
           
               
                 5′- NN TCT AGA CCC AGI TGT GGI GTI CCI GA -3′ 
               
               
                                          C     AA 
               
             
          
         
       
     
     2. Zn binding: region primer (Seq. ID No.:7) 
     
       
         
               
             
           
               
                 5′- NN GGA TCC CC CAT IGA ATG ICC IAI TTC ATG -3′ 
               
               
                                     G CC  G           C   G 
               
             
          
         
       
     
     Both primers contain four desoxyinosine nucleotides as well as additional nucleotides for identification sites of the restriction endonucleases XbaI (propeptide primer) and BamHI (zn 2+  binding region primer). 
     The degenerated primers were used together with cDNA in the PCR. 
     The reaction mixture contained: 
     100 ng cDNA 
     1 μg propeptide primer 
     1 μg Zn 2  bonding region primer 
     2.5U/100 μL DNA polymerase AmpliTaq 
     100 μM dNTP 
     0.01% gelatin 
     50 mM KCl 
     1.5 mM MgCl 2    
     10 mM Tris HCl, pH 8.3 
     In all, 30 reaction cycles of the sequence, 50 seconds at 94° C., 1 minute at 56° C. and 2 minutes at 72° C., were carried out. The amplified DNA was extracted with phenol/chloroform and subsequently treated with the restriction enzymes XbaI and BamHI. DNA fragments, ranging in size from 350 to 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 sequence 2.0 (USB/Amersham Life Science). The sequences obtained were compared with DNA sequences in the Genbank and EMBL data banks. The sequences were compared with the FASTA program of the HUSAR (GCG Package, Copyright Genetics Computer Group, Inc.) program package. Beginning with the kidney carcinoma cDNA, for example, several hundred clones with amplified DNA were obtained, of which 50 were sequenced. The evaluation revealed known as well as novel DNA sequences. The former included sequences for human interstitial collagenase and for matrilysin. The latter included two sequences homologous to human matrix metalloproteases. The two novel sequences, which were named PCRmmpm1 and PCRmmpm2, contain the nucleotides, of SEQ ID NO:14 and SEQ ID NO:15, respectively. 
     2. Northern Blot Analysis 
     The expression of matrix metalloproteases in human tissue was investigated with the help of the PCRmmpm1 and PCRmmpm2 fragments. The fragments were labeled radioactively (Multiprime DNA-Labeling Kit, Amersham Life Science) and hybridized with mRNA on nylon (Multiple Tissue Blot, Clontech). The hybridizations and subsequent washings took place under standard conditions [18]. 
     PCRmmpm1 and PCRmmpm2 hybridized with RNA of about 3.6 kb. However, experiments confirm a different 43 expression of the mRNA hybridizing with PCRmmpm1 and PCRmmpm2 (FIGS.  2 A and  2 B). Specific transcripts, which hybridize with PCRmmpm1, are contained particularly in lung, placenta, kidney and kidney carcinoma tissue (not shown). They are represented in clearly lesser amounts in pancreas and heart tissue and hardly at all in the liver, skeletal muscle and brain. 
     Messenger RNA, which hybridizes with PCRmmpm2, is synthesized particularly in placenta tissue. Its expression, however, is comparable in the heart, liver, skeletal muscle, kidneys, pancreas and lung and clearly less in brain tissue. 
     3. Isolation and sequencing of cDNA mmpm1 and mmpm2 
     A lung cDNA bank in the λgt 11 vector (Clontech) was analyzed by means of phage transfer on nylon and hybridization with the radioactively labeled PCRmmpm1 and PCRmmpm2 probes [18]. The hybridizations were carried out for 16 hours at 40° C. in 50% formamide, 5×SSPE, 5×Denhardt, 0.5% SDS and 50 μg/ml denatured herring sperm DNA. After the hybridization, the filters were washed at room temperature and at 65° C. in 2×SSC, 0.1% SDS and subsequently evaluated in a Bio-Imaging-Analyzer (BAS 2000, Fuji Photo Film Co., LTD). Phage clones, which hybridized with PCRmmpm1 and PCRmmpm2, were identified by means of specifically bound radioactivity. The phages found were isolated stepwise by dilution. The DNA of isolated phages was isolated (Quiagen Lambda Kit, Diagen GmbH) and the cDNA inserts contained were inserted into the plasmid vector pbluescript SK (Stratagene). The inserts subsequently were divided into partial fragments (Erase-a-Base-System, Promega) and sequenced (Sequence 2.0, USB/Amersham Life Science). DNA sequences detected were analyzed with the help of the DNA-STAR (DAN-STAR. Inc) and HUSAR (GCT Package, Copyright Genetics Computer Group, Inc) program packages. The translation of the coding sequences and the comparison of the amino acid sequences obtained with known matrix metalloproteases confirmed-that the novel sequences belong to the family of matrix metalloproteases (FIGS.  3 A- 3 D). 
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