EP0547074A1 - Enzyme de malate dependante du nad(p)?+ mitochondrial humain - Google Patents

Enzyme de malate dependante du nad(p)?+ mitochondrial humain

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Publication number
EP0547074A1
EP0547074A1 EP19910914750 EP91914750A EP0547074A1 EP 0547074 A1 EP0547074 A1 EP 0547074A1 EP 19910914750 EP19910914750 EP 19910914750 EP 91914750 A EP91914750 A EP 91914750A EP 0547074 A1 EP0547074 A1 EP 0547074A1
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Prior art keywords
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EP19910914750
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German (de)
English (en)
Inventor
Mark B. Dworkin
Gerhard Loeber
Edeltraud Krystek
Ingrid Maurer-Fogy
Bärbel FRÜHBEIS
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Boehringer Ingelheim International GmbH
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Boehringer Ingelheim International GmbH
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Priority claimed from DE19904028618 external-priority patent/DE4028618A1/de
Priority claimed from DE19914120178 external-priority patent/DE4120178A1/de
Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of EP0547074A1 publication Critical patent/EP0547074A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • the present invention relates to human mitochondrial NAD (P) + -dependent malate enzyme.
  • Malate enzyme catalyzes the oxidative decarboxylation of malate to pyruvate: malate + NAD (P) + -> pyruvate + NAD (P) H + + H +. It occurs in both eukaryotic and prokaryotic cells. So far, three different iso forms of the malate enzyme have been described in mammalian tissues: a strictly cytoplasmic NADP + -dependent enzyme, a NADP + -dependent mitochondrial iso-form, and a mitochondrial iso-enzyme that can use both NAD + and NADP +, but is generally preferred (Fraenkel, 1975). (In the following this isoform is called NAD (P) - »- dependent malate enzyme)
  • the mammalian isoforms are approximately 62-64 kDa (Moreadith and Lehninger, 1984b; Bagchi et al., 1987).
  • a native size of 240 kDA suggests a tetrameric structure for the active enzyme (Fraenkel, 1975; Moreadith and Lehninger, 1984b).
  • cytoplasmic malate enzyme The highest activities of the cytoplasmic malate enzyme are found in the liver and adipose tissue; there this iso form is connected with the formation of cytosolic NADPH for de novo fatty acid synthesis.
  • This isoenzyme is controllable via the food composition and can be induced by a carbohydrate-rich diet or by thyroid hormone (Fraenkel, 1975, Dozin et al., 1985).
  • NADP + -dependent mitochondrial malate enzyme activity can be demonstrated in many tissues (brain, heart and skeletal muscle tissue, adrenal gland) (Lin and Davis, 1974; Fraenkel, 1975; Nagel et al., 1980) where it may be associated with the production of NADPH for reductive biosynthesis (Simpson and Estabrook, 1969).
  • this special iso-form has not been characterized in detail so far.
  • Mitochondrial NAD (P) + -dependent malate enzyme activity can be detected in tissues that have high cell division rates, eg thymus or spleen, or in the basal cells of the small intestinal mucosa (Sauer et al., 1979); further in the first cell division cycles during the early Xenopus development (Dworkin and Dworkin-Rastl, 1990).
  • This isoform is missing or low in the brain, muscle tissue and normal and regenerating liver tissue of the rat (Nagel et al., 1980), but was found in the adrenal cortex of the rat (Sauer, 1973), in the skeletal muscle of the pigeon and humans (Lin and Davis, 1974; Taroni et al., 1988) and in the myocardium of various species (Lin and Davis, 1974; Liguori et al., 1989). It is expressed in many of the tumor cell lines examined to date.
  • NAD (P) + -dependent malate enzyme activity was detected in mitochondria of ascitic tumors (Sauer and Dauchy, 1978), hepatoma cells (Sauer et al., 1980) and various other tumors studied (Moreadith and Lehninger, 1984b). In the Morris hepatom series, the expression of NAD (P) + -dependent malate enzyme is progression-dependent (Sauer et al., 1980).
  • NAD (P) + -bound mitochondrial malate enzyme activity may be related to the conversion of amino acid carbon to pyruvate (Moreadith and Lehninger, 1984a; Sauer et al., 1980).
  • the task was made available for the first time in a homogeneous form of the human mitochondrial NAD (P) + -dependent malate enzyme (hereinafter referred to as "malate enzyme") and, on the basis thereof, to identify the DNA coding for it and to determine the nucleotide sequence.
  • malate enzyme human mitochondrial NAD + -dependent malate enzyme
  • the availability of the cDNA enables the expression of the enzyme in suitable host organisms.
  • a further object within the scope of the present invention was to provide onoclonal antibodies using the protein produced by the recombinant route.
  • the object was achieved by producing fractions with malate enzyme activity from the supernatant of a mitochondrial preparation from a transformed human T-lymphocyte cell line (cell line 1301; Bodo, 1981) and purifying and concentrating malate enzyme therefrom by means of ion exchange and ATP affinity chromatography has been.
  • This procedure provided malate enzyme with a purity of approx. 85 - 90% as the starting material for direct amino acid sequencing.
  • further cleaning steps can be used the homogeneous enzyme can be obtained.
  • further cleaning can be carried out using SDS gel electrophoresis and blotting on a membrane.
  • Affinity chromatography further chromatographic purification steps such as gel filtration and / or hydrophobic chromatography can be connected.
  • the homogeneous malate enzyme was obtained using FPLC.
  • the invention thus relates to human mitochondrial NAD (P) - »-dependent malate enzyme.
  • oligonucleotides were synthesized and used in a polymerase chain reaction (PCR) in order to amplify a DNA fragment specific for malate enzyme under conditions of low stringency directly from the supernatant of a cDNA library (a fibrosarcoma library was used) .
  • PCR polymerase chain reaction
  • a single band was obtained from the PCR amplification and subcloned (UC vector). Sequence analysis of the amplified DNA showed significant homology with other known malate enzyme sequences.
  • the clone obtained was used for a plaque hybridization of the fibrosarcoma library used for the cDNA amplification, and a plaque obtained in this way was isolated and purified with an insert of 1923 bp. Since the insert contained internal EcoRI interfaces, a partial digestion was carried out and the complete insert was cloned into the EcoRI site of a suitable vector (Bluescript KS +). From this clone became the nucleotide sequence certainly; 3 shows the entire nucleotide sequence of the isolated cDNA clone.
  • the cDNA clone with a total length of 1923 bp and a poly (A) tail contains a long open reading frame from position 90 (ATG) to position 1841 (TAG) which codes for 584 amino acids.
  • the 5 'untranslated region is extremely G / C rich (76.3% G / C; 61 of the first 80 nucleotides are G / C); the nucleotides in the vicinity of the start signal agree relatively well with the Kozak consensus sequence (Kozak, 1987).
  • the 3 'untranslated region is 79 bp long, followed by the poly (A) section (26 consecutive A residues were found in the isolated cDNA clone).
  • 13 bp before the poly (A) section is a sequence element (AGATAA) which has a certain homology with the consensus poly-adenylation signal (AATAAA) (Birnstiel et al., 1985).
  • AATAAA consensus poly-adenylation signal
  • the molecular weight of the protein with the amino acid sequence derived from the nucleotide sequence is 65.4 kD (including the mitochondrial signal sequence).
  • the first 20 amino acids of the derived protein show the characteristics of a typical mitochondrial signal sequence: a possible amphipathic alpha helix, which has a secondary structure with non-polar amino acids on one side of the helix and basic amino acids on the other side (von Heijne, 1986; Allison and Schatz, 1986) (the portion of the mitochondrial signal sequence may, for scientific interest or if necessary, exactly determined by N-terminal sequencing of the processed protein).
  • 67 of the 584 amino acids of the entire protein are acidic, 78 are basic, 11 are cysteines.
  • a theoretically possible N-glycosylation site is at position 421 of the protein (Asn-Xaa-Thr).
  • the amino acid Xaa is Pro in this case.
  • glycosylation is therefore not to be assumed as probable.
  • the lack of glycosylation is further corroborated by the finding that human mitochondrial malate enzyme expressed in E. coli is fully functional.
  • the NAD (P) - »- dependent mitochondrial malate enzyme should therefore not be glycosylated in human cells.
  • the eukaryotic malate enzymes have low but significant homology with the Bacillus stearothermophilus enzyme; they all show about 27% Identity with this enzyme.
  • the homology among the different malate enzymes is not evenly distributed across the protein sequence; “Homologous sections are interrupted by longer segments that have little or no homology. It is noteworthy that longer stretches of human, rat and maize malate enzymes are almost identical, while other regions show little or no similarity.
  • the regions between amino acids 160 to 189, 251 to 294 and 411 to 452 are particularly conserved.
  • the region between amino acids 111 and 119 is similar to the proposed NADP + binding sites of the goose fatty acid synthetase and the human glyceraldehyde-3-phosphatase (Poulouse and Kolattukudy, 1983).
  • a segment between amino acids 311 and 343 comprises a motif which also has a certain homology with a proposed nicotinamide-coenzyme binding site (Scrutton et al., 1990), the coenzyme specificity not being readily deduced from the sequence.
  • Both the NADP + -dependent rat and the human NAD (P) + -dependent malate enzyme have alanines at positions characteristic of NADP + binding proteins.
  • NAD + binding proteins tend to uncharged or acidic amino acids at this point.
  • the human malate enzyme according to the invention predominantly has acidic amino acids in this section (positions 334, 335 and 341), while the NADP + -dependent rodent malate enzymes at this point have more amino acids with basic side chains. All of these putative dinucleotide binding domains are highly conserved between the various malate enzymes described to date, including cinnamyl alcohol dehydrogenase. Three cysteines at positions 120, 198 and 428 are also conserved within these different enzymes and are possible sites for disulfide bridges.
  • the malate enzyme according to the invention has only 33-36% identity with the other eukaryotic NADP + -dependent malate enzymes which have a degree of similarity of 47-67% with respect to this region. From this it can be assumed that this section of the protein is responsible for some of its specific allosteric properties (stimulation by fumarate, inhibition by ATP).
  • the cDNA coding for the polypeptide from amino acid 19 to amino acid 584 was cloned into a suitable bacterial expression plasmid (pRH281). Induction of expression in E. coli resulted in 1-2 mg of active soluble human malate enzyme; the bacterial malate enzyme was separated by means of DEAE ion exchange chromatography. The recombinant malate enzyme obtained was examined for its substrate and allosteric properties, which on the one hand made it possible to differentiate it from the endogenous bacterial malate enzyme activity and on the other hand from the other isoforms.
  • the bacterial enzyme was found to effectively use both NAD + and NADP + as the electron acceptor, while the recombinant human enzyme with NADP + is virtually inactive; A corresponding preference for NAD + was also found for the enzyme isolated from lymphocytes. Fumarate similarly activated the recombinant and natural human malate enzyme, but had no effect on the endogenous bacterial enzyme. ATP inhibited the activity of human and recombinant malate enzyme, but not that of the endogenous bacterial enzyme. Overall, it was found that the recombinant malate enzyme practically does not differ from the natural enzyme in terms of its allosteric and kinetic properties.
  • the present invention therefore also relates to the DNA coding for human mitochondrial NAD (P) - »-dependent malate enzyme.
  • recombinant malate enzyme can be found in suitable prokaryotic or eukaryotic host organisms, for example E. coli, Sacch. cerevisiae or cells of higher organisms are expressed.
  • the choice of the host organism depends on the intended use of the malate enzyme in the context of the scientific question, for the clarification of which the recombinant enzyme or the cells expressing the enzyme are used.
  • the cDNA coding for malate enzyme is the prerequisite for accessing the genomic DNA and thus for obtaining information about the 5 'non-coding region of the malate enzyme gene.
  • a hybridization probe can be constructed with which a genoic DNA library is screened.
  • the clones obtained are then examined to determine whether they contain the required regulatory sequence elements in addition to the coding regions, for example by fusing the DNA sections obtained with coding regions of suitable reporter genes and checking for promoter function. With the help of the regulatory elements, the expression of malate enzyme can be examined for tissue specificity and for whether it responds to known expression-modulating factors, for example growth factors or hormones.
  • the invention relates to recombinant DNA molecules containing the DNA coding for malate enzyme or sections thereof and the host organisms transformed therewith.
  • the invention therefore also relates to the recombinant human NAD (P) + -dependent malate enzyme and to processes for its preparation.
  • “recombinant malate enzyme” is also to be understood as meaning modified proteins which have changes in the amino acid sequence, in particular in certain domains which may be essential for the biological activity. This includes e.g. the dinucleotide binding site or the substrate binding site which can be identified, for example by targeted mutagenesis.
  • the malate enzyme according to the invention can For example, to investigate the carbon metabolism in rapidly dividing cells, in particular to investigate the formation of pyruvate from amino acids.
  • the present invention thus relates to monoclonal antibodies against malate enzyme, hybridoma cells which produce such antibodies, and methods for their production.
  • These include hybridoma cell lines or the monoclonal antibodies produced by them which react specifically with NAD (P) + -dependent malate enzyme, ie have little or no cross-reactivity with the other isoforms of malate enzyme.
  • the term "antibodies which react specifically with malate enzyme” within the scope of the present invention also includes antibodies which are clear with mitochondrial NAD (P) + -dependent malate enzyme, but with the other malate enzyme isoforms show only low reactivity, ie only weakly cross-react.)
  • the process for the production of monoclonal antibodies against malate enzyme is characterized in that animals, preferably mice, are immunized with malate enzyme, F-lymphocytes from the immunized animals are fused with myeloma cells, the hybridomas obtained are examined for production of anti-malate enzyme antibodies, the positive cultures cloned, cultured and isolated from cultures antibody.
  • animals preferably mice
  • F-lymphocytes from the immunized animals are fused with myeloma cells
  • the hybridomas obtained are examined for production of anti-malate enzyme antibodies
  • the positive cultures cloned cultured and isolated from cultures antibody.
  • mice female, approx. 6 week old BALB / c mice were produced three times in succession at intervals of approx. 4 weeks each time by injection of recombinant purified malate enzyme (in each case approx. 10 ⁇ g, once) to produce monoclonal anti-malate enzyme antibodies immunized with complete Freund's Ad uvans, the second time with incomplete Freund's adjuvant and the third time without adjuvant). After a further 11 days, blood was drawn from the mice (after this period, a clear antibody titer can generally be detected in the blood serum). In these sera, the antibody titer was determined in an ELISA (Enzyme Linked Immunosorbent Assay) in comparison to normal mouse serum, which showed no reaction.
  • ELISA Enzyme Linked Immunosorbent Assay
  • the recombinant malate enzyme was bound directly to the surface of the microtiter plate, then the correspondingly diluted mouse sera were added and their binding with antibodies against mouse Ig, coupled to horseradish peroxidase, was detected.
  • the antibody titer was determined by reaction with a suitable substrate (1,2-phenylenediamine) using a color reaction.
  • the spleen was removed from the mouse with the highest antibody titer and the spleen cells were fused with myeloma cells of the P3X63 Ag 8.653 line (Kearney et al., 1979), which could be selected using HAT medium.
  • Suitable myeloma cells are, for example, those with the designation X-63-Ag8, MPC-11, NSI-Ag4 / l, MOPC-21 NS / 1 or SP 2/0.
  • the fusion was carried out according to the von Köhler and Milstein, 1975 described method by mixing the B-lymphocytes and the myeloma cells with the addition of the cell fusion agent polyethylene glycol 4000.
  • suitable cell fusion agents are, for example, Sendai virus, calcium chloride or lysolecithin.
  • the use of selective culture medium ensured that only fused cells, the so-called hybridomas, survived in culture.
  • the hybridomas were cultured (in RPMI 1640 with usual additives) and their culture supernatants were examined for anti-malate enzyme antibodies using the ELISA described above.
  • the 5 positive polyclones obtained were recloned by the so-called "limiting dilution” method, the resulting 5 monoclones were cultured again and tested for reaction with NAD (P) - »- dependent malate enzyme; all 5 monoclones showed a positive reaction.
  • the monoclones obtained were cultured in large amounts in vivo and the antibodies produced were purified from ascites and characterized.
  • the monoclonal anti-malate enzyme antibodies according to the invention which can be obtained according to the described protocol (which can be modified where appropriate) can be used for the preparation of immunoaffinity chromatography columns for the purification of malate enzyme preparations or, provided that they do not cross-react with the other malate enzyme preparations. Show isoforms for the separation of mitochondrial NAD (P) + - dependent malate enzyme from other isoforms.
  • the monoclonal antibodies according to the invention is in the immunological detection of malate enzyme and thus the determination of malate enzyme expression. If the monoclonal antibodies according to the invention have no or a significantly lower reactivity with the other malate enzyme isoforms, they can be used to distinguish the mitochondrial NAD (P) + -dependent malate enzyme from other isoforms.
  • the immunoassays for the detection of malate enzyme which can be used in the context of the present invention are based on standard methods which are known to the person skilled in the art in the relevant field and of which a wide range is available. These methods are based on the formation of a complex between the antigenic substance to be determined and one or more antibodies. One or more of the complex partners is marked so that the antigen can be detected and / or determined quantitatively.
  • the marking can e.g. in the form of a coupled enzyme, radioisotope, metal chelate, or a fluorescent, chemiluminescent or bioluminescent substance.
  • the antigen in the sample to be analyzed competes with a known amount of labeled antigen for binding to the antibody binding sites.
  • the amount of labeled antigen bound to the antibody is therefore indirectly proportional to the amount of antigen in the sample.
  • the amount of labeled bound antibody is directly proportional to the amount of antigen.
  • Antibody / antigen / antibody complex based, using two antibodies that do not interfere with each other in the binding to the antigen are referred to as "sandwich" immunoassays.
  • Lanes A, B Natural enzyme, purified from lymphocytes by DEAE cellulose chromatography (A) and by ATP agarose affinity column chromatography (B).
  • C The recombinant protein, purified from induced E.coli XL1 blue cells.
  • Nucleotide and amino acid sequence of the human malate enzyme The entire nucleotide sequence of the isolated cloned cDNA is shown. Coding regions are given in capital letters, 5 'and 3' untranslated regions in lower case. The of the DNA sequence-derived amino acid sequence is given below the nucleotide sequence.
  • MEM-3 specifically recognizes human mitochondrial
  • examples 3 - 6 used standard molecular biological methods which can be found in the relevant manuals or which correspond to the conditions recommended by the manufacturers.
  • Restriction endonucleases refers to the catalytic cleavage of the DNA by means of restriction endonucleases (restriction enzymes) at these specific sites (restriction sites). Restriction endonucleases are commercially available and are sold under the conditions recommended by the manufacturers (buffer, bovine serum albumin (BSA) Carrier protein, dithiothreitol (DTT) used as oxidation protection). Restriction endonucleases are identified with an uppercase letter, usually followed by a lowercase letter and usually a Roman numeral. The letters are derived from the microorganism from which the restriction endonuclease in question was isolated (for example: Sma I: Serratia marcescens).
  • DNA is cut with one or more units of the enzyme in about 20 ⁇ l of buffer solution.
  • An incubation period of 1 hour at 37 ° C is normally used, but can be varied according to the manufacturer's instructions.
  • the 5'phosphate group is sometimes removed from calf intestine (CIP) by incubation with alkaline phosphatase. This serves to prevent an undesired reaction of the specific site in a subsequent ligase reaction (for example circularization of a linearized plasmid without inserting a second DNA fragment).
  • CIP calf intestine
  • DNA fragments are usually not dephosphorylated after cutting with restriction endonucleases.
  • Reaction conditions for incubation with alkaline phosphatase can be found, for example, in the M13 cloning and sequencing manual (Cloning and Sequencing Handbook, Fa. Amersham, PI / 129/83/12). After the incubation, protein is removed by extraction with phenol and chloroform and the DNA precipitated from the aqueous phase by adding ethanol.
  • Isolation of a particular DNA fragment means the separation of the DNA fragments obtained by the restriction digest, for example on a 1% agarose gel. After electrophoresis and visualization of the DNA in UV light by staining with ethidium bromide (EtBr) the desired fragment is localized on the basis of molecular weight markers applied and bound to DE 81 paper (Schleicher and Schull) by further electrophoresis.
  • the DNA is precipitated by adding ethanol.
  • Transformation means the introduction of DNA into an organism so that the DNA can be replicated there, either extrachromosomally or chromosomally integrated. Transformation of E.coli follows the method given in the M13 Cloning and Sequencing Handbook (Cloning and Sequencing Handbook, Amersham, PI / 129/83/12).
  • “Sequencing" a DNA means determining the nucleotide sequence.
  • the DNA to be sequenced is first cut with various restriction enzymes, and the fragments are introduced into appropriately cut M13 mp8, mp9, mpl8 or mpl9 double-stranded DNA, or the DNA is fragmented using ultrasound, the ends are repaired and the size-selected fragments in Sma I cut, dephosphorylated M13 mp8 DNA introduced (shotgun method). After the transformation of E.
  • Another sequencing method consists of cloning the DNA to be sequenced into a vector which, among other things, carries an origin of replication of a single-stranded DNA phage (M13, fl) (e.g. Bluescribe or Bluescript M13 from Stratagene).
  • M13, fl single-stranded DNA phage
  • the transformants can be transformed with a helper phage, e.g. M13K07 or R408 from Promega).
  • the result is a mixture of helper phage and packaged, single-stranded recombinant vector.
  • the sequencing template is processed in analogy to the M13 method. Double-stranded plasmid DNA was denatured according to the sequencing manual given above by alkali treatment and sequenced directly.
  • Ligase T4-DNA ligase
  • Preparation of DNA from transformants means the isolation of the plasmid DNA from bacteria using the alkaline SDS method, modified according to Birnboim and Doly, omitting the lysozyme.
  • the bacteria from 1.5 to 50 ml of culture are used.
  • Oligonucleotides are short polydeoxynucleotides that are chemically synthesized.
  • the Applied Biosystems Synthesizer Model 381A was used for this.
  • the oligonucleotides were processed according to the Model 381A User Manual (Applied Biosystems). (Sequence primers are used directly without further purification.
  • Polyacrylamide gel electrophoresis (6% acrylamide, 0.15% bisacrylamide, 6 M urea, TBE buffer) was cleaned and, after elution from the gel, desalted using a G-25 Sepharose column. )
  • PCR polymerase chain reaction
  • oligonucleotide primers and heat-stable DNA polymerase (Taq polymerase).
  • Taq polymerase heat-stable DNA polymerase
  • Two oligonucleotide primers are used for the reaction, which flank the section of the DNA to be amplified and which hybridize with opposite strands.
  • the hybridized primers are arranged with their 3 'ends opposite one another in such a way that the synthesis by DNA polymerase extends over the region of the DNA template between the primers.
  • each primer Since each primer is complementary to one of the newly synthesized strands, each new strand takes as a template in subsequent cycles of primer extension and Sequence amplification part. Therefore, in each cycle consisting of the steps of denaturation, primer hybridization and enzymatic extension, the amount of DNA from the previous cycle is doubled (Erlich et al., 1988; Mullis and Faloona, 1987). Within a short time, this method has wide application in the analysis or detection of known sequences (e.g.
  • prenatal diagnosis, forensic medicine in the direct sequencing of non-cloned material, in the production of hybridization probes, in the field of DNA manipulation (introduction of restriction sites, directed mutagenesis, addition of promoters, etc.) and in the analysis and cloning of (partially) unknown sequences (e.g. based on the cDNA determination of the position and size of introns in chromosomal DNA, use of degenerate oligonucleotides, starting from the peptide sequence cDNA).
  • lymphocytes human transformed T-lymphocyte cell line 1301; Bodo, 1981
  • buffer containing 82.5 mM NaCl, 2.5 mM KC1, 1 mM Na2HP04, 5 mM HEPES pH 7.9 rinsed and homogenized in 400 ml buffer A (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.25 M sucrose).
  • Cell nuclei and cell debris were centrifuged in a Beckmann JA21-250 ml rotor at 2,500 rpm for 10 minutes.
  • the mitochondria were isolated in the same rotor after centrifugation at 10,000 rpm for 15 minutes.
  • the mitochondrial fraction was washed twice with buffer A and then in 50 ml buffer B (5mM HEPES pH 7.6, 50mM KCl, 0.1mM EDTA, 0.5mM DTT and 0.2% Lubrol PX (Sigma).
  • the mitochondria were broken up by ultrasound treatment (6 x 30 s sonication at maximum power on ice in a Branson ultrasound generator, 2 minutes Cooling periods between the sonication times) Glycerol was added to the broken up mitochondrial fraction to a final concentration of 15%, centrifuged for 30 min at 40,000 rpm in a Beckmann T150.2 rotor at 4 ° C and with the same volume of buffer D (30mM Tris-HCl pH 7.4, 1.5 mM MgCl2, 0.5 mM EDTA, 0.2 mM DTT and 20% glycerol) The sample was applied to a DEAE ion exchange column (12x2.5 cm DE52, Whatman) and washed with 200 ml buffer D.
  • buffer D 30mM Tris-HCl pH 7.4, 1.5 mM MgCl2, 0.5 mM EDTA, 0.2 mM DTT and 20% glycerol
  • the malate enzyme was then washed from the column with a KCl concentration gradient (40-120 mM KCl in buffer D) and eluted from the column at a concentration of 80-90 mM KCl. Active fractions of the eluate were pooled, with the same volume Buffer D diluted and brought to ImM fumarate and ImM MnCl2.
  • Malate enzyme activity was determined spectrophotometrically at 340 nm as described by Mandella and Sauer, 1975.
  • the reaction mixture contained 20 mM Tris pH 7.4, 1 mM MnCl2, 0.12 mM NAD + and 2.5 mM fumarate. The determinations were carried out at 37 ° C. carried out in a Beckman DU 64 spectrophotometer equipped with temperature-controlled cuvette holders.
  • the sample was applied to a 5 ml ATP affinity column (type II ATP agarose affinity column, Pharmacia). Highly purified malate enzyme was eluted from the ATP column with buffer D + 100 mM KCl + 4 mM NAD +, diluted with the same volume of buffer D and in one 2 ml DEAE column concentrated.
  • the first DEAE column delivered 6,400 mE NAD + -dependent malate enzyme
  • Fig. 1 shows the result of the gel electrophoresis carried out with a part of the sample (10% SDS-polyacrylamide gel, Laemmli, 1970, the staining was carried out using the Biorad silver staining kit); the malate enzyme obtained in this step was over 80% pure. (In a final purification step, the malate enzyme was purified to homogeneity by means of FPLC, the parameters used in example 7 for the purification of recombinant malate enzyme being selected.)
  • the majority of the malate enzyme purified according to Example 1 (80% pure) was dialyzed extensively against 2.5 mM Tris pH 7.5, lyophilized to dryness, dissolved in SDS application buffer and further separated by gel electrophoresis. For this purpose, the sample was applied in 2 traces of a 10% polyacrylamide gel after reduction with mercaptoethanol.
  • the "Mighty-Small" system (Hoefer) was used, separation distance: 5.5 cm, gel thickness: 0.75 mm, running conditions: 150 V constant over 100 minutes.
  • the gel was blotted onto a Immobilon P membrane (Millipore) with the appropriate size using the semi-dry method with a discontinuous buffer system.
  • a transfer Buffer was used anodically 0.3 M or "0.025 M Tris, pH 10.4, each with 20% methanol, cathodically
  • the electroblot on the membrane is shown in Fig. 2.
  • the main band at about 64 kd is homogeneous
  • Lane B at about 64 kd, only one band is visible on the gel.
  • the membrane pieces were then blocked with 0.5% Tween 20 in 100 mM acetic acid at 37 ° C for 30 min. The excess Tween 20 was removed in several washing steps with distilled water.
  • the blotted malate enzyme was cleaved with trypsin in an Eppendorf tube at 37 ° C. (24 hours).
  • the membrane pieces were incubated in 200 ⁇ l 100 mM Tris buffer, pH 8.2 with 5% acetonitrile with the addition twice of 2% w / w (based on malate enzyme) trypsin (Boehringer-Mannheim, sequencing grade). The separation of the resulting tryptic cleavage peptides was carried out by reverse phase HPLC, using a Delta Pak C18 column (Waters, 3.9 x 150 mm, 5 ⁇ m
  • oligonucleotides Complementary degenerate (64- and 96-fold) deoxyoligonucleotides (hereinafter referred to as "oligonucleotides”) were prepared for partial areas of tryptic peptides (peptides 56 and 64), which were equipped with an EcoRI restriction site to facilitate subsequent subcloning.
  • the peptide sequences were selected because in one case (peptide 56, EBI 2824) they were very similar to a region of the cytoplasmic malate enzyme and in the other case (peptide 64, EBI 2834) they had a relatively low degree of degeneration.
  • oligonucleotide EBI 2824 is oriented to the 3 'end of the sequence; it is identical to the coding strand of the malate enzyme gene.
  • a cDNA library of the human fibrar sarcoma cell line HS 913 was used for the PCR amplification of malate enzyme-specific DNA.
  • the cDNA library was produced according to the method described in EP-Al-0293 567 for the human placental cDNA library, with the difference that 109 fibrosarcoma cells from the HS 913 T cell line, which were stimulated with human TNF- ⁇ (10 ng / ml) had been grown. Instead of ⁇ gtlO ⁇ gtll was used (cDNA synthesis: Amersham RPN 1256; EcoRI digested ⁇ gtll arms: Promega Biotech; in vitro packaging of the ligated DNA: Gigapack Plus, Stratagene).
  • the amplification by means of PCR was carried out in 100 ml reaction volume containing 5 ⁇ l of the phage supernatant which had been heat-denatured at 95 ° C. (without further purification), 50 mM KCl, 10 mM Tris pH 8.3, 1.5 mM MgCl 2, 0.01% gelatin, 0.2 mM of the deoxynucleotides dATP, dCTP, dGTP and dTTP, each containing 200 pmol of the two primers and 2 units of Taq polymerase (Boehringer Mannhein). The reaction mixture was protected against evaporation by overlaying with 0.1 ml paraffin. The PCR was then carried out in a thermal cycler from Perkin Elmer in 30 amplification cycles, the following parameters being used: 45 sec at 95 ° C. (denaturation);
  • Example 4 Since, based on homology comparisons with the cytoplasmic malate enzyme from rat liver, the larger of the fragments obtained in Example 4 appeared to be the desired one, this was eluted from the agarose gel.
  • This DNA fragment was ligated with EcoRI-cut plasmid pUC18 (Pharmacia) and E. coli JM101 was transformed with the ligation mixture.
  • the mini-preparation plasmids were characterized by cutting with EcoRI and subsequent electrophoresis in agarose gels to determine the length of the insert.
  • the insert was then sequenced from both sides in a double strand with the aid of two pUC-specific primers, the first 250 to 300 bp being determined from both sides in this step; the desired clone was designated pUC * hmtMEA.
  • the filter hybridization method was used to search for a full-length cDNA clone (Benton and Davis, 1977).
  • the malate enzyme cDNA fragment obtained from Example 4 was used as the hybridization probe.
  • Approximately 200,000 phages from the HS913T cDNA library were plated on E.coli Y1088 on 15 cm LB agar petri dishes in LB top agarose (0.7% LB agarose) (approx. 20,000 phage plaques / petri dish). The plates were incubated at 37 ° C for about 6 hours until the plaques reached the size of a pinhead.
  • Two nitrocellulose filter prints (Schleicher and Schull, BA85 filter) were made from each plate.
  • the filters were placed in denaturation buffer (0.5 M NaOH, 1.5 M NaCl) for 1 min, in renaturation buffer (0.5 M Tris pH 7.5, 3 M NaCl) for 5 min and in 20 x SSC (3 M NaCl, 0.3 M Na citrate) for 1 min. treated and then baked in a vacuum oven at 80 ° C for 1 hour.
  • denaturation buffer 0.5 M NaOH, 1.5 M NaCl
  • renaturation buffer 0.5 M Tris pH 7.5, 3 M NaCl
  • 20 x SSC 3 M NaCl, 0.3 M Na citrate
  • radioactive hybridization probe The subcloned malate enzyme-specific fragment obtained from the PCR was cut out of the pUC18 vector with EcoRI and eluted in a low melting point agarose gel (FMC Seaplaque). 100 ng of this fragment were denatured at 100 ° C. for 5 minutes and subjected to radioactive labeling by "random priming". The following were contained in 50 ⁇ l reaction volume:
  • Hybridization of the nitrocellulose replicas The filters were placed in 200 ml prehybridization buffer (3xSSC, 1 X Denhard's solution (Maniatis et al., 1982), 50% formamide, 0.5% SDS and 100 ⁇ g / ml denatured DNA from herring sperm) in a shaking water bath for 2 hours Incubated at 43 ° C. The prehybridization buffer was then exchanged for 200 ml of hybridization buffer (corresponds to the prehybridization buffer plus 1 x 108 cpm of radioactivity; specific activity 500,000 cpm / ml). Hybridization was carried out in a shaking water bath at 43 ° C. for 20 hours.
  • prehybridization buffer 3xSSC, 1 X Denhard's solution (Maniatis et al., 1982), 50% formamide, 0.5% SDS and 100 ⁇ g / ml denatured DNA from herring sperm
  • the filters were washed 4 ⁇ 15 minutes at 65 ° C. in 2 ⁇ SSC, 0.5% SDS, dried and exposed overnight with Amersham hyper film and intensifying film (Dupont Cronex). Two clearly positive signals were obtained and the plaques were excised precisely.
  • the phages were suspended in 500 ⁇ l 50 mM Tris pH 7.5, 10 mM MgCl2, plated in various dilutions on 9 cm petri dishes and incubated overnight at 37 ° C. From plates that have about 100 plaques filter triggers were made which were hybridized as described above. This process was repeated once until clear signals could be assigned to well-isolated plaques.
  • a size determination by means of PCR using the lambda gtll-specific primers EBI 1880 and 1913 showed that one of the clones obtained had an insert with a length of about 2000 bp, which corresponded to the length expected for the full length clone.
  • the DNA of this clone was isolated and cut with EcoRI. It was found that the insert had internal EcoRI restriction sites. The DNA was therefore partially digested with EcoRI and a fragment the size of the total insert was eluted from a low melting point agarose gel. This fragment was cloned into the EcoRI site of the Bluescript KS + vector (Stratagene, La Jolla). Restriction mapping identified 2 clones (BS * hmtME-forw and hmtME-rev) that contained the insert in opposite directions.
  • the clones BS * hmtME-forw and -vv obtained after a) were examined by sequencing using the universal sequencing primer (EBI 1513) according to the dideoxy chain termination method. For this purpose, 19 insert-specific primers oriented in both directions were produced on the basis of the sequence information obtained and in this way the clone was completely sequenced in both orientations, overlapping.
  • the complete nucleotide sequence and the deduced amino acid sequence are given in FIG. 3.
  • the sequenced clone contains an open reading frame encoding 584 amino acids and a 26 bp poly A tail at the 3 'end.
  • the expression vector pRH281 (a derivative of the plasmid pER103, (EP-A-0 115 613)), described in DE-A-38 10474, which contains the inducible tryptophan operon, was used to express the human mitochondrial malate enzyme. Two constructs were produced: one clone contained the complete coding sequence of the malate enzyme, a second the coding for amino acids 19 to 584, i.e. the region shortened by the presumptive mitochondrial signal sequence.
  • the expression plasmids were prepared as follows:
  • Two constructs were produced by PCR with purified BS * hmtME DNA, the middle of the coding region (with or without the sequence coding for the mitochondrial leader peptide), a Hind HI site at the 3 'end (EBI 2982) and am 5 'end of an Xhol cleavage site, a Shine-Dalgarno sequence (ribosome binding site) and 20 base pairs of the sequence, specific for the 5' end of the peptide (EBI 2980: with leader; EBI 2981: without leader).
  • the PCR was carried out in the same buffer system as indicated in Example 4.
  • the PCR products obtained were cut with Xhol and HindIII and in this way provided expression cassettes for malate enzyme with or without a mitochondrial leader sequence, which were ligated into the expression vector pRH 281.
  • clones which contained the corresponding plasmid were isolated by means of ampicillin selection.
  • the plasmid which contained the malate enzyme sequence without a mitochondrial leader was designated pRH281 * hmtME.
  • the correctness of the sequence was confirmed by sequencing (Sanger et al., 1977).
  • Clones were obtained from both constructs, which produce the corresponding protein inducibly and in high yield. However, the enzyme was obtained in inactive form from the construct which contains the sequence coding for the mitochondrial leader. The reason for this may be either the insolubility of the unprocessed precursor protein or the fact that the mitochondrial leader interferes with the activity of the protein.
  • E. Coli XL1 blue (Stratagene, La Jolla) cells, transformed with pRH281 * hmtME, were raised in 1 1 Luria Broth incl. 100 ⁇ g / ml ampicillin until approximately the middle of the logarithmic growth phase (OD600 0.3-0.4) and then induced with 50 ⁇ g / ml indolacrylic acid and grown for a further 3 h.
  • the bacterial suspension was centrifuged in a Beckmann JA-10 rotor at 4,000 rpm and in 10 ml of buffer A (20mM Tris pH7.4, 1mM EDTA, 0.25M sucrose) added.
  • the cells were broken up by ultrasound treatment (2 x 30 sec sonication at maximum power in a Branson ultrasound generator, with 2 minutes cooling periods on ice between sonication times).
  • Glycerol was added to the broken-up bacteria to a final concentration of 15%, centrifuged for 30 min at 40,000 rpm in a Beckmann Ti50 rotor at 4 ⁇ C and with the same volume of buffer F (30 mM Tris pH7.4, 20 mM KCl, 3 mM MgCl2, 0.2 mM DTT, 0.2 mM EDTA).
  • buffer F (30 mM Tris pH7.4, 20 mM KCl, 3 mM MgCl2, 0.2 mM DTT, 0.2 mM EDTA).
  • the sample was applied directly to a DEAE ion exchange column (20 x 3 cm, Whatman DE52 cellulose, equilibrated with buffer F) and washed with 400 ml buffer F.
  • the recombinant malate enzyme was then mixed with a KCl concentration gradient in this buffer (40-170 mM KCl ) from the column and eluted from the column like the native protein from human cells at a concentration of 80 mM KCl.Endogenic bacterial malate enzyme eluted at a concentration of 150-170 M KCl.Active fractions of the eluate were combined and with the same volume of buffer F diluted to approximately 100 ml (the determination of malate enzyme activity was carried out as indicated in Example 1.) The sample was brought to a concentration of 1 mM MnCl2, to a
  • mice 3 approximately 6-week-old female BALB / c mice were immunized with the malate enzyme obtained according to Example 7 and purified to homogeneity according to the following scheme: 1. Immunization: approx. 10 ⁇ g malate enzyme per mouse in complete Freund's adjuvant, intraperitoneally
  • Serum samples were taken from the mice 11 days later, so that an ELISA test could be set up and the serum titers determined. (The ELISA test was carried out as described in Example 9a), the test sera were applied in dilutions of 1:103 to 1:106.) The mouse with the highest titer was boosted with 3 ⁇ g malate enzyme for three consecutive days; the spleen cells of this mouse were then used for the fusion with hybridoma cells.
  • mice spleen was removed sterile, mechanically crushed and washed with serum-free culture medium (RPMI 1640 with the addition of 1% antibiotics and 10% L-glutamine). Approximately 108 spleen cells were fused with approx. 108 myeloma cells of the line P3X63 Ag 8.653 (Kearney et al., 1979) according to the method of Köhler and Milstein, 1975, in the presence of PEG 4000 (40% in serum-free culture medium).
  • HAT selection medium RPMI 1640 - completed with 1% antibiotics, 10% L-glutamine and 20% FCS - with 10-4 M hypoxanthine, 4x10-7 M aminopterin and 1.6x10-5 thymidine
  • RPMI 1640 - completed with 1% antibiotics, 10% L-glutamine and 20% FCS - with 10-4 M hypoxanthine, 4x10-7 M aminopterin and 1.6x10-5 thymidine
  • 96-well microtiter plates of the type Costar 3590 were overnight with 100 ⁇ l PBS (phosphate-buffered saline) with a content of 1 ⁇ g / ml malate enzyme (this amount had proven to be optimal in preliminary tests) incubated at 4 ° C or 1 h at 37 °. After 3 washes with 200 ⁇ l PBS / 0.05% Tween each, the mixture was blocked for 1 h at 37 ° with 200 ⁇ l PBS / 0.5% BSA (bovine serum albumin) / 0.05% Tween and then washed again 3 times.
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • test sera in dilutions of 1: 103 to 1: 106, supernatants of hybridoma cultures in dilutions of 1: 1 to 1: 100 were then applied (100 ⁇ l each).
  • BSA and normal mouse serum (1:103) served as negative controls. After incubation at 37 ° for one hour, washing was carried out again. The mixture was then incubated for 1 h at 37 ° C. with 100 ⁇ l of peroxidase-coupled rabbit anti-mouse immunoglobulin (1: 4000, DAKO, P 161).
  • the molecular weight determination of the purified monoclonal antibodies obtained according to Example 8c) was carried out by means of 7.5% SDS-PAGE according to the method of Laemmli, 1973; was colored with 0.1% Coomassie Brillant Blue.
  • the immunoglobulin was characterized using a subclass test kit (Serotec) according to the manufacturer's instructions.
  • the western blot is shown in FIG. 5:
  • the monoclonal antibody MEM-3 reacts specifically with the mitochondrial NAD (P) + -dependent malate enzyme from 1301 cells.
  • P mitochondrial NAD
  • HS913T cells the activity of this isoform is below the detection limit; the other isoforms are produced by these cells with significant activity.
  • the weak band visible in the HS913T lane is likely due to a weak cross-reactivity of the monoclonal antibody with another malate enzyme isoform.
  • MEM-3 monoclonal antibody 100 ⁇ g each of total lysates (cytoplasm a + mi ochondria) from 1301 cells, Xenopus laevis oocytes and CVI cells (African Green Monkey Kidney cells, ATCC No. CCL70) were, as indicated under c), on a 10% strength SDS gel separated and blotted. Immunodetection was carried out with the hybridoma supernatant of MEM-3 (undiluted), and the color reaction was carried out as described above.

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Abstract

Enzyme de malate dépendente du NAD(P)+ mitochondrial humain, son ADN de codage et enzyme recombinante de malate, ainsi qu'anticorps anti-enzyme de malate.
EP19910914750 1990-09-08 1991-08-23 Enzyme de malate dependante du nad(p)?+ mitochondrial humain Withdrawn EP0547074A1 (fr)

Applications Claiming Priority (4)

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DE4028618 1990-09-08
DE19904028618 DE4028618A1 (de) 1990-09-08 1990-09-08 Humanes mitochondriales nad(p)(pfeil hoch)+(pfeil hoch)-abhaengiges malatenzym
DE4120178 1991-06-19
DE19914120178 DE4120178A1 (de) 1991-06-19 1991-06-19 Humanes mitochondriales nad(p)(pfeil hoch)+(pfeil hoch)-abhaengiges malatenzym

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WO2003040296A2 (fr) * 2001-11-08 2003-05-15 DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung Proteine men, gst2, rab-rp1, csp, proteine a f-box lilina/fbl7, abc50, coronine, sec61 alpha ou vhappa1-1 ou proteines homologues jouant un role dans la regulation de l'homeostasie energetique

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