EP1021546A1 - Neutral sphingomyelinase - Google Patents

Neutral sphingomyelinase

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Publication number
EP1021546A1
EP1021546A1 EP98945230A EP98945230A EP1021546A1 EP 1021546 A1 EP1021546 A1 EP 1021546A1 EP 98945230 A EP98945230 A EP 98945230A EP 98945230 A EP98945230 A EP 98945230A EP 1021546 A1 EP1021546 A1 EP 1021546A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
neutral sphingomyelinase
eukaryotic
acid according
sphingomyelinase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98945230A
Other languages
German (de)
French (fr)
Inventor
Wilhelm Stoffel
Kay Institut füur Biochemie HOFMANN
Stephan Institut für Biochemie TOMIUK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Memorec Biotec GmbH
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Memorec Stoffel GmbH
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Publication date
Application filed by Memorec Stoffel GmbH filed Critical Memorec Stoffel GmbH
Publication of EP1021546A1 publication Critical patent/EP1021546A1/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04012—Sphingomyelin phosphodiesterase (3.1.4.12)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to nucleic acids encoding eukaryotic neutral sphingomyelinase and their use.
  • Sphingomyelin is an essential component of plasma membranes.
  • the breakdown of sphingomyelin gives a variety of substances that have potential second messenger properties, e.g. Ceramide, sphingosine, sphingosine-1-phosphate.
  • Two sphingomyelin-cleaving enzyme activities are known, firstly that of the lysosomal acidic sphingomyelinase and secondly that of the plasma membrane-bound neutral sphingomyelinase.
  • Bacterial neutral sphingomyelinase is a secreted, soluble protein.
  • the present invention for the first time makes nucleic acids coding for eukaryotic neutral sphingomyelinase available.
  • the eukaryotic neutral sphingomyelinase (nSMase) is characterized in that it cleaves sphingomyelin in ceramide and phosphocholine and the activity is dependent on the addition of magnesium ions. It is a membrane-bound enzyme. The maximum activity is achieved in the neutral pH range.
  • Figure 1 shows the gene sequence of the human neutral sphingomyelinase.
  • FIG. 2 shows the gene sequence of the murine neutral sphingomyelinase.
  • FIG. 3 shows the results of Northern and estern blots of nSMase overexpressing cell lines.
  • FIG. 4 shows the strategy for generating murine knockout mutants. The letters symbolize restriction interfaces.
  • FIG. 5 shows constructs for obtaining transgenic mouse mutants.
  • the nucleic acid according to the invention is preferably a nucleic acid which codes for the neutral sphingomyelinase of a mammal. It is particularly preferably the human and murine neutral sphingomyelinase.
  • the corresponding nucleic acid sequences are as Seq. ID. No. 3 and Seq. ID. No. 4 disclosed.
  • nucleic acid sequences correspond to the EST sequences AA028477 and AA013912 (murine) and W32352 and AA056024 (human).
  • the person skilled in the art can easily find the corresponding nucleic acids and proteins from other eukaryotes, taking into account the high homology between human and murine nSMase. For this purpose, he can use cross-reacting antibodies for a specific affinity-chromatographic purification, or he can synthesize oligonucleotide primers based on the nucleic acid sequence and amplify the nucleic acids in question in a cDNA library of the eukaryote using the polymerase chain reaction.
  • the corresponding cDNA library can be obtained in a manner known per se by isolating mRNA from a tissue sample and subsequent reverse transcription.
  • the amino acid sequence can be derived from the nucleic acid sequence using the genetic code. Alternatively, it is also possible to search for and combine homologous sequences in EST (Expressed Sequence Tags) databases.
  • the nucleic acids according to the invention are suitable for the expression of eukaryotic neutral sphingomyelinase in pro- or eukaryotic systems.
  • nSMase are also suitable for expression of the nSMase in vivo in the sense of a gene therapy or in particular in the form of fragments also in a complementary structure as antisense nucleotides for reducing the expression of the nSMase.
  • nucleic acids according to the invention can be produced by chemical synthesis or by duplication in genetically modified organisms by methods known per se to the person skilled in the art.
  • the invention also relates to the eukaryotic neutral sphingomyelinase obtainable by the expression of the nucleic acids according to the invention.
  • the nSMase according to the invention can be produced by expression in genetically modified organisms.
  • Eukaryotic expression systems are particularly suitable.
  • Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene). Purification from genetically modified organisms offers, especially in the case of overexpression,. easy and direct access to the nSMase according to the invention and also allows isolation in large quantities.
  • eukaryotic neutral sphingomyelinase of a mammal in particular human or murine neutral sphingomyelinase.
  • the amino acid sequences of human and murine neutral sphingomyelinase are as Seq. ID. Nos. 1 and 2 reproduced.
  • the molecular weights of human and murine sphingomyelinase are 47.6 and 47.5 kDa, respectively.
  • the mammalian nSMases according to the invention contain no signal sequence at the N-terminus. Due to the hydrophobizi- Analysis can be assumed that two adjacent hydrophobic membrane domains at the C-terminus are separated by eight amino acids. It therefore appears to be integral membrane proteins whose catalytically active domain points towards the cytosol, while only a small proportion of the enzymes are in contact with the extracellular environment.
  • nSMases which are secreted, soluble proteins
  • the 1.7 kb mRNA of the murine nSMase is expressed in all tissues according to Northern blot analysis.
  • Northern blot shows a strong signal in kidneys, brain, liver, heart and lungs, while expression in the spleen appears to be low. This measurement was not in agreement with the measured enzymatic activities of the corresponding tissues. This speaks for a post-transcriptional regulation of the nSMase.
  • the pH optimum of the neutral sphingomyelinase according to the invention is in the range from 6.5 to 7.5 with a P ⁇ value for C18 sphingomyelin in the range from 1.0 to 1.5 ⁇ 10 -5 M.
  • the activity is dependent on magnesium ions Addition of EDTA leads to an inhibition of SMase activity, but can be restored by adding Mn 2+ or Mg 2+ ions.
  • the addition of 0.3 to 0.5% Triton X-100 increases the enzyme activity. The activity is unaffected by treatment with DTT or 2-mercaptoethanol, whereas the addition of 20 mM glutathione led to the inhibition.
  • the activity of the nSMase is not limited to sphingomyelin, and the structurally related phosphatidylcholine was also cleaved with about 3% activity.
  • variants of eukaryotic neutral sphingomyelinase are also claimed.
  • the term "variants” includes both naturally occurring allelic variations of the eukaryotic neutral sphingomyelinase as well as by recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and other closing expression produced proteins which correspond to the eukaryotic neutral sphingomyelinase in terms of their biological and / or immunological activity.
  • Amino acids can be deleted, inserted or exchanged conservatively. Conservative exchange means that an amino acid is replaced by an amino acid that has similar physicochemical properties.
  • amino acids are interchangeable: serine for / against alanine, alanine for / against glycine, methionine for / against serine, lysine for / against arginine, lysine for / against serine.
  • variants also encompasses N- and / or C-terminal shortened proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives.
  • nSMase The activity of the nSMase appears to be at least partly in the C-terminal region, since the fragment 1 to 282 of the murine nSMase showed no increase in the sphingomyelinase activity when expressed in HEK293 cells.
  • C-terminal fragments of the nSMase are also the subject of this invention.
  • Compounds in which nSMase or its variants are coupled to further molecules such as dyes, radionuclides or affinity components also represent variants according to the invention.
  • Nucleic acids which code for eukaryotic neutral sphingomyelinase or which are complementary to these nucleic acids are also claimed.
  • the nucleic acids can be, for example, DNA, RNA, PNA or nuclease-resistant analogs.
  • Nuclease-resistant analogs are in particular those compounds in which the phosphodiester bond is modified by compounds which are stable to hydrolysis, for example phosphothioates, methylphosphonates or the like.
  • Short fragments of the nucleic acids are particularly suitable for antisense nucleotides. For reasons of specificity, these should preferably be more than 6, more preferably more than 8 and am most preferably have more than 12 nucleotides. For diffusion and cost reasons, they are typically less than 30 nucleotides in length, preferably 24 or less, and even more preferably 18 or fewer nucleotides in length.
  • the invention also relates to derivatives of nucleic acids which are coupled with other molecules for diagnostic or therapeutic purposes, for example with fluorescent dyes, radioactive markers or affinity components, and fragments of the nucleic acids according to the invention and of the nucleic acids complementary to these nucleic acids, and variants of the nucleic acids.
  • Fragments refer to nucleic acids that are shortened on the 5 'or 3' or on both sides.
  • variants is understood to mean that these nucleic acids hybridize under stringent conditions with the nucleic acid according to the invention or nucleic acids complementary thereto.
  • stringent conditions is understood to mean that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (under otherwise identical conditions) under which exactly complementary nucleic acids would just just hybridize. For example, if a precisely hybridizing nucleic acid hybridizes under given conditions up to a temperature of approx. 55 ° C, then stringent conditions are temperatures equal to or higher than 45 ° C.
  • the preferred temperature range for stringent conditions is 5 ° C, more preferably 3 ° C.
  • the invention relates to antibodies which are directed against the nSMase according to the invention or the nucleic acids according to the invention. These substances are particularly suitable for use in diagnostics, immunoassays known to those skilled in the art, for histological examination and as a medicament for the treatment of conditions which are associated with overexpression of the nSMase.
  • Such antibodies according to the invention can be carried out using methods known to those skilled in the art Immunization with nSMase, nucleic acids according to the invention or peptide and nucleic acid fragments can be obtained in the presence of auxiliary reagents.
  • the invention furthermore relates to cell lines which overexpress the nSMase according to the invention.
  • Such cell lines are obtainable by transfection with vectors which contain the nucleic acids according to the invention which code for nSMase.
  • the transfection can be carried out, for example, by electroporation.
  • the cell lines are preferably stably transfected.
  • overexpression means that this cell line has a higher activity of the nSMase than the cell lines which were not transfected with the nucleic acids according to the invention.
  • Suitable eukaryotic cell lines are, for example, the cell lines U937, HEK 293 or Jurkat.
  • the cell lines showed a specific nSMase activity between 0.3 and 10 ⁇ mol / mg protein / hour.
  • FIG. 3 shows the Northern and Western blot analysis of the nSMase expression in transfected cell lines.
  • Part A shows the result of an RT-PCR of the total cell RNA with primers that hybridize with human and murine nSMase cDNA.
  • Part B shows the T-PCR of the total RNA as a control with primers which hybridize to human ⁇ -actin cDNA.
  • Part C shows the Western blot of the plasma membrane protein extract from various HEK 293 cell lines after SDS polyacrylamide gel electrophoresis and hybridization with the polyclonal anti-nSMase antibodies.
  • the invention further relates to a transgenic mammal which has an overexpression (gain of function) or a gene definition. ciency or a gene defect (loss of function) for the nSMase according to the invention.
  • the mammal is preferably a rodent, in particular a mouse.
  • These transgenic mammals can be obtained by methods known per se to the person skilled in the art and are particularly suitable for elucidating the function of the neutral sphingomyelinase.
  • defined gene constructs are injected by DNA microinjection into the fore nucleus (pronucleus) of a fertilized egg cell at the single-cell stage in order to achieve expression of the additional gene.
  • the function of a gene is switched off by targeted modification of a gene in the genome of ES cells, which are subsequently injected into blastocysts.
  • FIGS. 4 and 5 The strategy and constructs for generating the mouse mutants are shown in FIGS. 4 and 5.
  • the transgenic animals are preferably animals in which the gene can be switched on and off inductively from the outside in a time-specific and tissue-specific manner.
  • Corresponding transgenic mammals are particularly suitable for elucidating the metabolic and signal transduction pathways associated with the nSMase according to the invention, which in turn open up diagnostic or therapeutic applications.
  • the transgenic mammals are particularly suitable for screening active pharmaceutical ingredients.
  • the eukaryotic neutral sphingomyelinase according to the invention, the nucleic acids according to the invention and the antibodies according to the invention can optionally be contained in medicaments and diagnostic agents together with further auxiliaries.
  • These medicinal and diagnostic agents are suitable for the diagnosis and treatment of diseases which are based on over- or under-expression and / or an increased or reduced activity of the eukaryotic neutral sphingomyelinase and / or on disorders of cell proliferation, cell differentiation and / or apotosis.
  • diseases in which inflammatory processes, cell growth disorders and metabolic disorders play a role can be, for example, cancer or disorders of cholesterol homeostasis (arteriosclerosis).
  • a pharmaceutical screening method according to the invention is based on changing the expression or activity of the nSMase according to the invention in nSMase-overexpressing cell lines when at least one potentially pharmaceutically active substance is added.
  • the cell lines are therefore particularly suitable for the development and testing of pharmaceutical lead structures.
  • nucleic acids coding for the neutral sphingomyelinase according to the invention were cloned into the NotI interfaces of the cloning site of the eukaryotic expression vector pRc / CMV (Stratagene). The sequences obtained were obtained by sequencing with a Perkin-Elmer DNA sequencer 377A.
  • RNA was isolated from various organs from eight three-week-old CD1 mice by known methods and poly (A + ) RNA was isolated by affinity purification on oligo (dT) cellulose (Boehringer Mannheim Germany) according to standard methods.
  • poly (A + ) RNA was isolated by affinity purification on oligo (dT) cellulose (Boehringer Mannheim Germany) according to standard methods.
  • U937 cells grew in RPMI 1640 medium with 10% fetal calf serum, 1 ⁇ g / ml penicillin / streptomycin and 0.03% glutamine at 37 ° C and 5% CO 2 .
  • 5 ⁇ 10 6 cells were transfected with 1 ⁇ g of linearized plasmid DNA which coded for the nSMase according to the invention by electroporation with a “gene pulser” (company Bio-Rad).
  • Stable clones were selected under 1 mg / ml geneticin (G418, Life Technologies, Gaithersburg, MD).
  • the nSMase purified from the cell lines showed a specific activity between 0.3 and 10 ⁇ mol / mg protein / hour.
  • the pH optimum was 6.5 and 7.5.
  • the K ⁇ value for C18 sphingomyelin was 1.0 to 1.5 x 10 ⁇ 5 M.
  • the activity was dependent on the presence of magnesium ions; the addition of EDTA inhibited the activity.
  • the enzymatic activity was examined in cells and mouse tissue.
  • the cells were washed twice with ice-cold PBS and sedimented at 1,000 g.
  • the pellet was resuspended in lysis buffer and the cells were destroyed by repeated freezing and thawing. After centrifugation for 2 min at 2500 g, followed by extraction with lysis buffer with 0.2% Triton X-100. This is followed by centrifugation at 100,000 g for 15 min.
  • Tissue from three week old mice was homogenized in cold lysis buffer.
  • the amount of protein or homogenized tissue to be examined was incubated with 10 nm (80,000 dpm) [N- 14 CH 3 ] sphingomyelin for 30 min at 37 ° in a total volume of 200 ⁇ l.
  • 100 ⁇ l of water were added and the unreacted substrate was removed by extraction with chloroform-methanol (2: 1, v / v).
  • the radioactivity of the aqueous phase, the containing the enzymatically released phosphocholine was measured in a sintillation counter.

Abstract

The invention relates to eukaryontic neutral sphingomyelinase (nSMase) and the use thereof.

Description

Neutrale Sphingomyelinase Neutral sphingomyelinase
Die vorliegende Erfindung betrifft Nukleinsäuren, die für eukaryontische neutrale Sphingomyelinase codieren, und ihre Anwendung .The present invention relates to nucleic acids encoding eukaryotic neutral sphingomyelinase and their use.
Sphingomyelin ist eine wesentliche Komponente von Plasmamembranen. Der Abbau des Sphingomyelins gibt eine Vielzahl von Substanzen, die potentielle second messenger Eigenschaf en haben, z.B. Ceramid, Sphingosin, Sphingosin-1-phosphat . Es sind zwei sphingomyelinspaltende Enzymaktivitäten bekannt, zum einen die der lysosomalen sauren Sphingomyelinase und zum anderen die der plasmamembran-gebundenen neutralen Sphingomyelinase.Sphingomyelin is an essential component of plasma membranes. The breakdown of sphingomyelin gives a variety of substances that have potential second messenger properties, e.g. Ceramide, sphingosine, sphingosine-1-phosphate. Two sphingomyelin-cleaving enzyme activities are known, firstly that of the lysosomal acidic sphingomyelinase and secondly that of the plasma membrane-bound neutral sphingomyelinase.
Die bakterielle neutrale Sphingomyelinase ist ein sezerniertes, lösliches Protein.Bacterial neutral sphingomyelinase is a secreted, soluble protein.
Durch die vorliegende Erfindung- werden erstmals Nukleinsäuren, codierend für eukaryontische neutrale Sphingomyelinase, verfügbar gemacht. Die eukaryontische neutrale Sphingomyelinase (nSMase) ist dadurch charakterisiert, daß sie Sphingomyelin in Ceramid und Phosphocholin spaltet und die Aktivität von der Zugabe von Magnesiumionen abhängig ist. Es handelt sich um ein membrangebundenes Enzym. Die maximale Aktivität wird im neutralen pH-Bereich erzielt .The present invention for the first time makes nucleic acids coding for eukaryotic neutral sphingomyelinase available. The eukaryotic neutral sphingomyelinase (nSMase) is characterized in that it cleaves sphingomyelin in ceramide and phosphocholine and the activity is dependent on the addition of magnesium ions. It is a membrane-bound enzyme. The maximum activity is achieved in the neutral pH range.
Figur 1 zeigt die Gensequenz der humanen neutralen Sphingomyelinase.Figure 1 shows the gene sequence of the human neutral sphingomyelinase.
Figur 2 zeigt die Gensequenz der murinen neutralen Sphingomyelinase .FIG. 2 shows the gene sequence of the murine neutral sphingomyelinase.
Figur 3 zeigt die Ergebnisse von Northern- und esternblots nSMase-überexprimierender Zellinien. Figur 4 zeigt die Strategie zur Erzeugung von murinen Knockout- Mutanten. Die Buchstaben symbolisieren Restriktionsschnittstellen.Figure 3 shows the results of Northern and estern blots of nSMase overexpressing cell lines. FIG. 4 shows the strategy for generating murine knockout mutants. The letters symbolize restriction interfaces.
Figur 5 zeigt Konstrukte zur Gewinnung transgener Mausmutanten.FIG. 5 shows constructs for obtaining transgenic mouse mutants.
Bevorzugt handelt es sich bei der erfindungsgemäßen Nukleinsäure um eine Nukleinsäure, die für die neutrale Sphingomyelinase eines Säugetiers codiert. In besonders bevorzugter Weise handelt es sich dabei um die humane und murine neutrale Sphingomyelinase. Die entsprechenden Nukleinsäuresequenzen sind als Seq. ID. Nr. 3 und Seq. ID. Nr. 4 offenbart.The nucleic acid according to the invention is preferably a nucleic acid which codes for the neutral sphingomyelinase of a mammal. It is particularly preferably the human and murine neutral sphingomyelinase. The corresponding nucleic acid sequences are as Seq. ID. No. 3 and Seq. ID. No. 4 disclosed.
Teile der Nukleinsäuresequenzen stimmen mit der EST-Sequenzen AA028477 und AA013912 (murin) und W32352 und AA056024 (human) überein.Parts of the nucleic acid sequences correspond to the EST sequences AA028477 and AA013912 (murine) and W32352 and AA056024 (human).
Bei Kenntnis der Aminosäure- und Nukleinsäurestruktur der humanen und murinen neutralen Sphingomyelinase kann der Fachmann unter Berücksichtigung der hohen Homologie zwischen der humanen und murinen nSMase die entsprechenden Nukleinsäuren und Proteine aus anderen Eukaryonten leicht auffinden. Dazu kann er zum einen kreuzreagierende Antikörper für eine spezifische affinitätschro- atographische Aufreinigung einsetzen, oder er kann auf der Grundlage der Nukleinsäuresequenz Oligonukleotidprimer synthetisieren und die gesuchten Nukleinsäuren mit Hilfe der Polymerase- kettenreaktion in einer cDNA-Bank des Eukaryonten amplifizieren. Die entsprechende cDNA-Bank kann durch Isolierung von mRNA aus einer Gewebeprobe und anschließende Reverse-Transkription in an sich bekannter Weise erhalten werden. Aus der Nukleinsäuresequenz kann mit Hilfe des genetischen Codes die Aminosäuresequenz abgeleitet werden. Alternativ ist es hierzu auch möglich, homologe Sequenzen in EST (Expressed Sequence Tags) -Datenbanken zu suchen und zu kombinieren. Die erfindungsgemäßen Nukleinsäuren eignen sich zur Expression der eukaryontisehen neutralen Sphingomyelinase in pro- oder eukaryontisehen Systeme. Darüber hinaus sind sie auch zur Expression der nSMase in vivo im Sinne einer Gentherapie oder insbesondere in Form von Fragmenten auch in komplementärer Struktur als Antisense-Nukleotide zur Verringerung der Expression der nSMase geeignet .If the amino acid and nucleic acid structure of human and murine neutral sphingomyelinase is known, the person skilled in the art can easily find the corresponding nucleic acids and proteins from other eukaryotes, taking into account the high homology between human and murine nSMase. For this purpose, he can use cross-reacting antibodies for a specific affinity-chromatographic purification, or he can synthesize oligonucleotide primers based on the nucleic acid sequence and amplify the nucleic acids in question in a cDNA library of the eukaryote using the polymerase chain reaction. The corresponding cDNA library can be obtained in a manner known per se by isolating mRNA from a tissue sample and subsequent reverse transcription. The amino acid sequence can be derived from the nucleic acid sequence using the genetic code. Alternatively, it is also possible to search for and combine homologous sequences in EST (Expressed Sequence Tags) databases. The nucleic acids according to the invention are suitable for the expression of eukaryotic neutral sphingomyelinase in pro- or eukaryotic systems. In addition, they are also suitable for expression of the nSMase in vivo in the sense of a gene therapy or in particular in the form of fragments also in a complementary structure as antisense nucleotides for reducing the expression of the nSMase.
Die erfindungsgemäßen Nukleinsäuren können durch chemische Synthese oder durch Vervielfältigung in gentechnisch veränderten Organismen nach dem Fachmann an sich bekannten Verfahren hergestellt werden.The nucleic acids according to the invention can be produced by chemical synthesis or by duplication in genetically modified organisms by methods known per se to the person skilled in the art.
Gegenstand der Erfindung ist auch die durch die Expression der erfindungsgemäßen Nukleinsäuren erhältliche eukaryontische neutrale Sphingomyelinase.The invention also relates to the eukaryotic neutral sphingomyelinase obtainable by the expression of the nucleic acids according to the invention.
Die erfindungsgemäße nSMase läßt sich durch Expression in gentechnisch veränderten Organismen herstellen. Insbesondere sind eukaryontische Expressionssysteme geeignet. Entsprechende eukaryontische Expressionssysteme sind dem Fachmann bekannt wie beispielsweise pRc/CMV (Firma Stratagene) . Die Aufreinigung aus gentechnisch veränderten Organismen bietet, insbesondere im Falle der Überexpression, . ein leichten und direkten Zugang zur erfindungsgemäßen nSMase und erlaubt darüber hinaus die Isolierung in größeren Mengen.The nSMase according to the invention can be produced by expression in genetically modified organisms. Eukaryotic expression systems are particularly suitable. Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene). Purification from genetically modified organisms offers, especially in the case of overexpression,. easy and direct access to the nSMase according to the invention and also allows isolation in large quantities.
Bevorzugt handelt es sich um die eukaryontische neutrale Sphingomyelinase eines Säugetiers, insbesondere um humane oder murine neutrale Sphingomyelinase. Die Aminosäuresequenzen der humanen und murinen neutralen Sphingomyelinase sind als Seq. ID. Nr. 1 und 2 wiedergegeben.It is preferably the eukaryotic neutral sphingomyelinase of a mammal, in particular human or murine neutral sphingomyelinase. The amino acid sequences of human and murine neutral sphingomyelinase are as Seq. ID. Nos. 1 and 2 reproduced.
Die Molekulargewichte der humanen bzw. murinen Sphingomyelinase beträgt 47,6 bzw. 47,5 kDa . Im Gegensatz zu den bakteriellen nSMasen enthalten die erfindungsgemäßen nSMasen von Säugetieren keine Signalsequenz am N-Terminus. Aufgrund der Hydrophobizi- tätsanalyse kann davon ausgegangen werden, daß zwei benachbarte hydrophobe Membrandomänen am C-Terminus durch acht Aminosäuren getrennt sind. Es scheint sich daher um integrale Membranproteine zu handeln, deren katalytisch aktive Domäne zum Cytosol zeigt, während nur ein geringer Anteil der Enzyme Kontakt zur extrazellulären Umgebung hat. Dies ist im Gegensatz zu den bakteriellen nSMasen, bei denen es sich um sekretierte, lösliche Proteine handelt, ist aber in Übereinstimmung mit bisherigen Untersuchungen zu den Eigenschaften der neutralen Sphingomyeli- nasen von Säugetieren. Die 1,7 kb mRNA der murinen nSMase wird gemäß Northern Blot Analyse in allen Geweben exprimiert . In Nieren, Hirn, Leber, Herz und Lunge zeigt der Northern Blot ein starkes Signal, während die Expression in der Milz gering zu sein scheint. Diese Messung war nicht in Übereinstimmung mit den gemessenen enzymatischen Aktivitäten der entsprechenden Gewebe. Dies spricht für eine posttranskriptionale Regulation der nSMase.The molecular weights of human and murine sphingomyelinase are 47.6 and 47.5 kDa, respectively. In contrast to the bacterial nSMases, the mammalian nSMases according to the invention contain no signal sequence at the N-terminus. Due to the hydrophobizi- Analysis can be assumed that two adjacent hydrophobic membrane domains at the C-terminus are separated by eight amino acids. It therefore appears to be integral membrane proteins whose catalytically active domain points towards the cytosol, while only a small proportion of the enzymes are in contact with the extracellular environment. This is in contrast to the bacterial nSMases, which are secreted, soluble proteins, but is in agreement with previous studies on the properties of the neutral sphingomyelinases in mammals. The 1.7 kb mRNA of the murine nSMase is expressed in all tissues according to Northern blot analysis. Northern blot shows a strong signal in kidneys, brain, liver, heart and lungs, while expression in the spleen appears to be low. This measurement was not in agreement with the measured enzymatic activities of the corresponding tissues. This speaks for a post-transcriptional regulation of the nSMase.
Das pH-Optimum der erfindungsgemäßen neutralen Sphingomyelinase liegt im Bereich von 6,5 bis 7,5 mit einem P^-Wert für C18 Sphingomyelin im Bereich von 1,0 bis 1,5 x 10~5 M. Die Aktivität ist magnesiumionenabhängig, die Zugabe von EDTA führt zu einer Inhibierung der SMase-Aktivität , kann jedoch durch Zugabe von Mn2+- oder Mg2+-Ionen wiederhergestellt werden. Die Zugabe von 0,3 bis 0,5% Triton X-100 erhöht die Enzymaktivität. Die Aktivität ist unbeeinflußt durch Behandlung mit DTT oder 2-Mercapto- ethanol, wohingegen die Zugabe von 20 mM Glutathion zur Inhibierung führte. Die Aktivität der nSMase ist nicht auf Sphingomyelin limitiert, auch das strukturell verwandte Phosphatidylcholin wurde mit etwa 3% Aktivität gespalten.The pH optimum of the neutral sphingomyelinase according to the invention is in the range from 6.5 to 7.5 with a P ^ value for C18 sphingomyelin in the range from 1.0 to 1.5 × 10 -5 M. The activity is dependent on magnesium ions Addition of EDTA leads to an inhibition of SMase activity, but can be restored by adding Mn 2+ or Mg 2+ ions. The addition of 0.3 to 0.5% Triton X-100 increases the enzyme activity. The activity is unaffected by treatment with DTT or 2-mercaptoethanol, whereas the addition of 20 mM glutathione led to the inhibition. The activity of the nSMase is not limited to sphingomyelin, and the structurally related phosphatidylcholine was also cleaved with about 3% activity.
Weiterhin beansprucht werden Varianten der eukaryontischen neutralen Sphingomyelinase. Unter den Begriff "Varianten" fallen sowohl natürlich vorkommende allelische Variationen der eukaryontischen neutralen Sphingomyelinase sowie durch rekombinante DNA-Technologie (insbesondere durch in vitro Mutagenese mit Hilfe von chemisch synthetisierten Oligonukleotiden) und an- schließende Expression erzeugte Proteine, die hinsichtlich ihrer biologischen und/oder immunologischen Aktivität der eukaryonti- sehen neutralen Sphingomyelinase entsprechen. Dabei können sowohl Aminosäuren deletiert, eingefügt oder konservativ ausgetauscht werden. Konservativer Austausch bedeutet, daß eine Aminosäure durch eine Aminosäure ersetzt wird, die ähnliche physikalisch-chemische Eigenschaften aufweist.Variants of eukaryotic neutral sphingomyelinase are also claimed. The term "variants" includes both naturally occurring allelic variations of the eukaryotic neutral sphingomyelinase as well as by recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and other closing expression produced proteins which correspond to the eukaryotic neutral sphingomyelinase in terms of their biological and / or immunological activity. Amino acids can be deleted, inserted or exchanged conservatively. Conservative exchange means that an amino acid is replaced by an amino acid that has similar physicochemical properties.
So sind beispielsweise folgende Aminosäuren austauschbar: Serin für/gegen Alanin, Alanin für/gegen Glycin, Methionin für/gegen Serin, Lysin für/gegen Arginin, Lysin für/gegen Serin.For example, the following amino acids are interchangeable: serine for / against alanine, alanine for / against glycine, methionine for / against serine, lysine for / against arginine, lysine for / against serine.
Insbesondere umfaßt der Begriff Varianten auch N- und/oder C- terminale verkürzte Proteine sowie acetylierte, glykosylierte, amidierte und/oder phosphorylierte Derivate.In particular, the term variants also encompasses N- and / or C-terminal shortened proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives.
Die Aktivität der nSMase scheint zumindest zum Teil im C-ter- minalen Bereich zu liegen, da das Fragment 1 bis 282 der murinen nSMase bei Expression in HEK293 Zellen keine Erhöhung der Sphin- gomyelinase-Aktivität zeigte. C-terminale Fragmente der nSMase sind ebenfalls Gegenstand dieser Erfindung. Auch Verbindungen, bei denen nSMase oder seine Varianten mit weiteren Molekülen wie Farbstoffe, Radionukliden oder Affinitätskomponenten gekoppelt sind, stellen erfindungsgemäße Varianten dar.The activity of the nSMase appears to be at least partly in the C-terminal region, since the fragment 1 to 282 of the murine nSMase showed no increase in the sphingomyelinase activity when expressed in HEK293 cells. C-terminal fragments of the nSMase are also the subject of this invention. Compounds in which nSMase or its variants are coupled to further molecules such as dyes, radionuclides or affinity components also represent variants according to the invention.
Beansprucht werden auch Nukleinsäuren, die für eukaryontische neutrale Sphingomyelinase codieren bzw. komplementär zu diesen Nukleinsäuren sind. Bei den Nukleinsäuren kann es sich beispielsweise um DNA, RNA, PNA oder um nukleaseresistenter Analoga handeln. Nukleaseresistente Analoga sind insbesondere solche Verbindungen, in denen die Phosphodiesterbindung durch hydrolysestabile Verbindungen modifiziert sind, beispielsweise Phospho- thioate, Methylphosphonate o.a.Nucleic acids which code for eukaryotic neutral sphingomyelinase or which are complementary to these nucleic acids are also claimed. The nucleic acids can be, for example, DNA, RNA, PNA or nuclease-resistant analogs. Nuclease-resistant analogs are in particular those compounds in which the phosphodiester bond is modified by compounds which are stable to hydrolysis, for example phosphothioates, methylphosphonates or the like.
Für Antisensenukleotide sind insbesondere kurze Fragmente der Nukleinsäuren geeignet. Diese sollten aus Gründen der Spezifität bevorzugt mehr als 6, noch mehr bevorzugt mehr als 8 und am meisten bevorzugt mehr als 12 Nukleotide aufweisen. Aus Gründen der Diffusion und der Kosten haben sie üblicherweise eine Länge von weniger als 30 Nukleotiden, bevorzugt 24 oder weniger und noch mehr bevorzugt 18 oder weniger Nukleotide.Short fragments of the nucleic acids are particularly suitable for antisense nucleotides. For reasons of specificity, these should preferably be more than 6, more preferably more than 8 and am most preferably have more than 12 nucleotides. For diffusion and cost reasons, they are typically less than 30 nucleotides in length, preferably 24 or less, and even more preferably 18 or fewer nucleotides in length.
Gegenstand der Erfindung sind auch Derivate von Nukleinsäuren, die für diagnostische oder therapeutische Zwecke mit anderen Molekülen gekoppelt sind, beispielsweise mit Fluoreszenzfarbstoffen, radioaktiven Markern oder Affinitätskomponenten, sowie Fragmente der erfindungsgemäßen Nukleinsäuren und der zu diesen Nukleinsäuren komplementären Nukleinsäuren sowie Varianten der Nukleinsäuren .The invention also relates to derivatives of nucleic acids which are coupled with other molecules for diagnostic or therapeutic purposes, for example with fluorescent dyes, radioactive markers or affinity components, and fragments of the nucleic acids according to the invention and of the nucleic acids complementary to these nucleic acids, and variants of the nucleic acids.
Fragmente bezeichnet dabei Nukleinsäuren, die am 5' oder 3' oder an beiden Seiten verkürzt sind. Unter dem Begriff "Varianten" wird verstanden, daß diese Nukleinsäuren unter stringenten Bedingungen mit der erfindungsgemäßen Nukleinsäure bzw. dazu komplementären Nukleinsäuren hybridisieren. Unter dem Begriff "stringente Bedingungen" wird verstanden, daß die Hybridisierung bei Bedingungen durchgeführt wird, bei der die Temperatur noch bis zu 10 °C unter der Temperatur liegt (bei sonst identischen Bedingungen) , bei der exakt komplementäre Nukleinsäuren gerade noch hybridisieren würden. Wenn beispielsweise eine exakt hybrisierende Nukleinsäure unter gegebenen Bedingungen bis zu einer Temperatur von ca. 55°C hybridisiert, dann sind stringente Bedingungen Temperaturen gleich oder höher 45°C. Bevorzugt ist der Temperaturbereich für stringente Bedingungen von 5°C, noch mehr bevorzugt von 3°C.Fragments refer to nucleic acids that are shortened on the 5 'or 3' or on both sides. The term “variants” is understood to mean that these nucleic acids hybridize under stringent conditions with the nucleic acid according to the invention or nucleic acids complementary thereto. The term "stringent conditions" is understood to mean that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (under otherwise identical conditions) under which exactly complementary nucleic acids would just just hybridize. For example, if a precisely hybridizing nucleic acid hybridizes under given conditions up to a temperature of approx. 55 ° C, then stringent conditions are temperatures equal to or higher than 45 ° C. The preferred temperature range for stringent conditions is 5 ° C, more preferably 3 ° C.
Desweiteren betrifft die Erfindung Antikörper, die gegen die erfindungsgemäße nSMase oder die erfindungsgemäßen Nukleinsäuren gerichtet sind. Diese Substanzen eignen sich insbesondere zum Einsatz in der Diagnostik, dem Fachmann an sich bekannten Immunoassays, zur histologischen Untersuchung sowie als Arzneimittel zur Behandlung von Zuständen, die mit einer Überexpression der nSMase verbunden sind. Solche erfindungsgemäßen Antikörper können mit dem Fachmann an sich bekannten Verfahren durch Immunisierung mit nSMase, erfindungsgemäßen Nukleinsäuren oder Peptid- und Nukleinsäurenfragmenten in Gegenwart von Hilfsreagenzien erhalten werden.Furthermore, the invention relates to antibodies which are directed against the nSMase according to the invention or the nucleic acids according to the invention. These substances are particularly suitable for use in diagnostics, immunoassays known to those skilled in the art, for histological examination and as a medicament for the treatment of conditions which are associated with overexpression of the nSMase. Such antibodies according to the invention can be carried out using methods known to those skilled in the art Immunization with nSMase, nucleic acids according to the invention or peptide and nucleic acid fragments can be obtained in the presence of auxiliary reagents.
Weiterhin sind Gegenstand der Erfindung Zellinien, die die erfindungsgemäße nSMase überexprimieren. Solche Zellinien sind erhältlich durch Transfektion mit Vektoren, die die erfindungsgemäßen Nukleinsäuren, die für nSMase kodieren, enthalten. Im Falle von eukaryontischen Zellinien kann die Transfektion beispielsweise durch Elektroporation erfolgen. Die Zellinien sind dabei vorzugsweise stabiltransfiziert .The invention furthermore relates to cell lines which overexpress the nSMase according to the invention. Such cell lines are obtainable by transfection with vectors which contain the nucleic acids according to the invention which code for nSMase. In the case of eukaryotic cell lines, the transfection can be carried out, for example, by electroporation. The cell lines are preferably stably transfected.
Überexpression bedeutet in diesem Zusammenhang, daß diese Zellinie eine höhere Aktivität der nSMase aufweisen als die Zellinien, die nicht mit den erfindungsgemäßen Nukleinsäuren transfiziert wurden. Geeignete eukaryontische Zellinien sind beispielsweise die Zellinien U937, HEK 293 oder Jurkat .In this context, overexpression means that this cell line has a higher activity of the nSMase than the cell lines which were not transfected with the nucleic acids according to the invention. Suitable eukaryotic cell lines are, for example, the cell lines U937, HEK 293 or Jurkat.
Die Zellinien zeigten in Experimenten eine spezifische nSMase- Aktivität zwischen 0,3 und 10 μmol/mg Protein/Stunde.In experiments, the cell lines showed a specific nSMase activity between 0.3 and 10 μmol / mg protein / hour.
Figur 3 zeigt die Northern und Western Blot Analyse der nSMase- Expression in transfizierten Zellinien. Teil A zeigt dabei das Ergebnis einer RT-PCR der Gesamtzelle RNA mit Primern, die mit humaner und muriner nSMase cDNA hybridisieren. Teil B zeigt als Kontrolle die T-PCR der Gesamt-RNA mit Primern, die zu humanem ß-Actin cDNA hybridisieren. Teil C zeigt den Westernblot des Plasma Membran Proteinextrakts von verschiedenen HEK 293 Zellinien nach SDS Polyacrylamid-Gelelektrophorese und Hybridisierung mit dem polyklonalen Anti-nSMase-Antikörpern.FIG. 3 shows the Northern and Western blot analysis of the nSMase expression in transfected cell lines. Part A shows the result of an RT-PCR of the total cell RNA with primers that hybridize with human and murine nSMase cDNA. Part B shows the T-PCR of the total RNA as a control with primers which hybridize to human β-actin cDNA. Part C shows the Western blot of the plasma membrane protein extract from various HEK 293 cell lines after SDS polyacrylamide gel electrophoresis and hybridization with the polyclonal anti-nSMase antibodies.
Die Zugabe von 0,5 mM Arachidonsäure führte zu einer dreifachen Erhöhung der nSMase-Aktivität in den überexprimierenden HEK- Zellen.The addition of 0.5 mM arachidonic acid resulted in a three-fold increase in the nSMase activity in the overexpressing HEK cells.
Gegenstand der Erfindung ist weiterhin ein transgenes Säugetier, das eine Überexpression (gain of function) oder eine Gendefi- zienz bzw. einen Gendefekt (loss of function) für die erfindungsgemäße nSMase aufweist . Bevorzugt handelt es sich bei dem Säugetier um ein Nagetier, insbesondere eine Maus. Diese trans- genen Säugetiere sind durch für den Fachmann an sich bekannte Verfahren erhältlich und eignen sich insbesondere zur Funktionsaufklärung der neutralen Sphingomyelinase. Für transgene Säugetiere werden definierte Genkonstrukte durch DNA-Mikroinjektion in den Vorkern (Pronukleus) einer befruchteten Eizelle im Einzellstadium injiziert, um die Expression des zusätzlichen Gens zu erreichen. Durch zielgerichtete Veränderung eines Gens im Genoms von ES-Zellen, die nachfolgend in Blastozysten injiziert werden, wird die Funktion eines Gens ausgeschaltet.The invention further relates to a transgenic mammal which has an overexpression (gain of function) or a gene definition. ciency or a gene defect (loss of function) for the nSMase according to the invention. The mammal is preferably a rodent, in particular a mouse. These transgenic mammals can be obtained by methods known per se to the person skilled in the art and are particularly suitable for elucidating the function of the neutral sphingomyelinase. For transgenic mammals, defined gene constructs are injected by DNA microinjection into the fore nucleus (pronucleus) of a fertilized egg cell at the single-cell stage in order to achieve expression of the additional gene. The function of a gene is switched off by targeted modification of a gene in the genome of ES cells, which are subsequently injected into blastocysts.
Die Strategie und Konstrukte zur Generierung der Mausmutanten sind in Figur 4 und 5 gezeigt.The strategy and constructs for generating the mouse mutants are shown in FIGS. 4 and 5.
Bevorzugt handelt es sich bei den transgenen Tieren um Tiere, bei denen das Gen zeitlich und gewebsspezifisch von außen induzierbar ein- bzw. ausgeschaltet werden kann. Entsprechende transgene Säugetiere eignen sich insbesondere zur Aufklärung der mit der erfindungsgemäßen nSMase im Zusammenhang stehenden Stoffwechsel- und Signaltransduktionswegen, die wiederum diagnostische oder therapeutische Anwendungen eröffnen. Insbesondere eignen sich die transgenen Säugetiere zum Screening von pharmazeutischen Wirkstoffen.The transgenic animals are preferably animals in which the gene can be switched on and off inductively from the outside in a time-specific and tissue-specific manner. Corresponding transgenic mammals are particularly suitable for elucidating the metabolic and signal transduction pathways associated with the nSMase according to the invention, which in turn open up diagnostic or therapeutic applications. The transgenic mammals are particularly suitable for screening active pharmaceutical ingredients.
Die erfindungsgemäße eukaryontische neutrale Sphingomyelinase, die erfindungsgemäßen Nukleinsäuren sowie die erfindungsgemäßen Antikörper können in Arzneimitteln und Diagnostikmitteln gegebenenfalls zusammen mit weiteren Hilfsstoffen enthalten sein. Diese Arznei- und Diagnostikmittel eigenen sich zur Diagnose und Behandlung von Erkrankungen, die auf einer Über- oder Unterexpression und/oder einer erhöhten oder verminderten Aktivität der eukaryontisehen neutralen Sphingomyelinase und/oder auf Störungen der Zellproliferation, Zelldifferenzierung und/oder Apotose beruhen. Insbesondere sind dies Erkrankungen, bei denen Entzündungsprozesse, Zellwachstumstörungen und StoffWechselstörungen eine Rolle spielen. Dies können beispielsweise Krebserkrankungen oder Störungen der Cholesterinhomöostase (Arteriosklerose) sein.The eukaryotic neutral sphingomyelinase according to the invention, the nucleic acids according to the invention and the antibodies according to the invention can optionally be contained in medicaments and diagnostic agents together with further auxiliaries. These medicinal and diagnostic agents are suitable for the diagnosis and treatment of diseases which are based on over- or under-expression and / or an increased or reduced activity of the eukaryotic neutral sphingomyelinase and / or on disorders of cell proliferation, cell differentiation and / or apotosis. In particular, these are diseases in which inflammatory processes, cell growth disorders and metabolic disorders play a role. These can be, for example, cancer or disorders of cholesterol homeostasis (arteriosclerosis).
Ein erfindungsgemäßes pharmazeutisches Screening-Verfahren beruht auf der Veränderung der Expression oder Aktivität der erfindungsgemäßen nSMase in nSMase-überexprimierenden Zellinien bei Zugabe von mindestens einer potentiell pharmazeutisch wirksamen Substanz. Die Zellinien eignen sich somit insbesondere zur Entwicklung und Prüfung von pharmazeutischen LeitStrukturen.A pharmaceutical screening method according to the invention is based on changing the expression or activity of the nSMase according to the invention in nSMase-overexpressing cell lines when at least one potentially pharmaceutically active substance is added. The cell lines are therefore particularly suitable for the development and testing of pharmaceutical lead structures.
Die Erfindung soll durch die folgenden Beispiele weiter erläutert werden.The invention is illustrated by the following examples.
Beispiel 1example 1
Klonierung der NukleinsäureCloning of the nucleic acid
Die erfindungsgemäßen für die neutrale Sphingomyelinase kodierenden Nukleinsäuren wurden in die Notl Schnittstellen der Klonierungsstelle des eukaryontisehen Expressionsvektors pRc/CMV (Stratagene) kloniert . Die erhaltenen Sequenzen wurden durch Sequenzierung mit einem Perkin-Elmer DNA-Sequenzer 377A erhalten.The nucleic acids coding for the neutral sphingomyelinase according to the invention were cloned into the NotI interfaces of the cloning site of the eukaryotic expression vector pRc / CMV (Stratagene). The sequences obtained were obtained by sequencing with a Perkin-Elmer DNA sequencer 377A.
Beispiel 2Example 2
Klonierung der RNACloning the RNA
Die Gesamt-RNA wurde nach bekannten Methoden aus verschiedenen Organen von acht drei Wochen alten CD1 Mäusen isoliert und Poly(A+)-RNA wurde durch Affinitätsreinigung an Oligo (dT) cel- lulose (Boehringer Mannheim Deutschland) gemäß Standardmethoden isoliert . Beispiel 3The total RNA was isolated from various organs from eight three-week-old CD1 mice by known methods and poly (A + ) RNA was isolated by affinity purification on oligo (dT) cellulose (Boehringer Mannheim Germany) according to standard methods. Example 3
Überexprimierende ZellinienOverexpressing cell lines
U937 Zellen wuchsen in RPMI 1640 Medium mit 10% fötalem Kälberserum, 1 μg/ml Penicillin/Streptomycin und 0,03% Glutamin bei 37°C und 5% C02. 5xl06-Zellen wurden mit 1 μg linearisierter Plasmid-DNA, die für die erfindungsgemäße nSMase kodierte durch Elektroporation mit einem "gene pulser" (Firma Bio-Rad) transfi- zier . Die Selektion stabiler Klone erfolgte unter 1 mg/ml Geneticin (G418, Life Technologies, Gaithersburg, MD).U937 cells grew in RPMI 1640 medium with 10% fetal calf serum, 1 μg / ml penicillin / streptomycin and 0.03% glutamine at 37 ° C and 5% CO 2 . 5 × 10 6 cells were transfected with 1 μg of linearized plasmid DNA which coded for the nSMase according to the invention by electroporation with a “gene pulser” (company Bio-Rad). Stable clones were selected under 1 mg / ml geneticin (G418, Life Technologies, Gaithersburg, MD).
Die aus den Zellinien aufgereinigte nSMase zeigte eine spezifische Aktivität zwischen 0,3 und 10 μmol/mg Protein/Stunde. Das pH-Optimum lag bei 6,5 und 7,5. Der K^-Wert für C18 Sphingomyelin betrug 1,0 bis 1,5 x 10~5 M. Die Aktivität war von der Anwesenheit von Magnesiumionen abhängig; die Zugabe von EDTA inhibierte die Aktivität.The nSMase purified from the cell lines showed a specific activity between 0.3 and 10 μmol / mg protein / hour. The pH optimum was 6.5 and 7.5. The K ^ value for C18 sphingomyelin was 1.0 to 1.5 x 10 ~ 5 M. The activity was dependent on the presence of magnesium ions; the addition of EDTA inhibited the activity.
Beispiel 4Example 4
Messung der nSMase -Aktivi tätMeasurement of nSMase activity
Die enzymatische Aktivität wurde in Zellen und Mäusegewebe untersucht. Die Zellen wurden zweimal mit eiskaltem PBS gewaschen und bei 1.000 g sedimentiert . Das Pellet wurde in Lysepuf- fer resuspendiert und die Zellen wurden durch wiederholtes Einfrieren und Auftauen zerstört. Nach Zentrifugation für 2 min bei 2.500 g gefolgt von einer Extraktion mit Lysepuffer mit 0,2% Triton X-100. Anschließend erfolgt eine Zentrifugation für 15 min bei 100.000 g.The enzymatic activity was examined in cells and mouse tissue. The cells were washed twice with ice-cold PBS and sedimented at 1,000 g. The pellet was resuspended in lysis buffer and the cells were destroyed by repeated freezing and thawing. After centrifugation for 2 min at 2500 g, followed by extraction with lysis buffer with 0.2% Triton X-100. This is followed by centrifugation at 100,000 g for 15 min.
Gewebe von drei Wochen alten Mäusen wurde in kaltem Lysepuffer homogenisiert . Die zu untersuchende Menge an Protein oder homogenisiertem Gewebe wurde mit 10 nm (80.000 dpm) [N-14CH3] - Sphingomyelin für 30 min bei 37° in einem Gesamtvolumen von 200 μl inkubiert. Dann wurden 100 μl Wasser zugesetzt und unreagiertes Substrat durch Extraktion mit Chloroform-Methanol (2:1, v/v) entfernt. Die Radioaktivität der wäßrigen Phase, die das enzymatisch freigesetzte Phosphocholin enthielt, wurde in einem Sintillationszähler gemessen.Tissue from three week old mice was homogenized in cold lysis buffer. The amount of protein or homogenized tissue to be examined was incubated with 10 nm (80,000 dpm) [N- 14 CH 3 ] sphingomyelin for 30 min at 37 ° in a total volume of 200 μl. Then 100 μl of water were added and the unreacted substrate was removed by extraction with chloroform-methanol (2: 1, v / v). The radioactivity of the aqueous phase, the containing the enzymatically released phosphocholine was measured in a sintillation counter.
Beispiel 5Example 5
Polyklonale AntikörperPolyclonal antibodies
Kaninchen wurden mit dem synthetischen Peptide CDPHSDKPFSDHE (entsprechend den Aminosäuren 261 bis 273 der murinen nSMase) gekoppelt an Keyhole-Limpit-Hemocyanin immunisiert. Das polyklonale Antikörperserum wurde durch Chromatographie an Hydroxy- apatit und Affinitätschromatographie an einer Säule, an der das oben genannte synthetische Peptide gebunden war, gereinigt. Rabbits were immunized with the synthetic peptide CDPHSDKPFSDHE (corresponding to amino acids 261 to 273 of the murine nSMase) coupled to keyhole limpit hemocyanin. The polyclonal antibody serum was purified by chromatography on hydroxyapatite and affinity chromatography on a column to which the above-mentioned synthetic peptide was bound.

Claims

Patentansprüche claims
1. Nukleinsäure kodierend für eukaryontische neutrale Sphingomyelinase .1. Nucleic acid coding for eukaryotic neutral sphingomyelinase.
2. Nukleinsäure gemäß Anspruch 1, dadurch gekennzeichnet, daß es sie für die neutrale Sphingomyelinase eines Säugetiers, insbesondere für humane oder murine neutrale Sphingomyelinase kodiert .2. Nucleic acid according to claim 1, characterized in that it codes for the neutral sphingomyelinase of a mammal, in particular for human or murine neutral sphingomyelinase.
3. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 2, dadurch gekennzeichnet, daß die neutrale Sphingomyelinase Sphingomyelin in Ceramid und Phosphocholin spaltet und ihre Aktivität von der Zugabe von Magnesiumionen abhängig ist .3. Nucleic acid according to at least one of claims 1 to 2, characterized in that the neutral sphingomyelinase cleaves sphingomyelin in ceramide and phosphocholine and its activity is dependent on the addition of magnesium ions.
4. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis4. Nucleic acid according to at least one of claims 1 to
3 mit der Sequenz gemäß Seq. ID. Nr. 3 oder Seq. ID. Nr. 4.3 with the sequence according to Seq. ID. No. 3 or Seq. ID. No. 4.
5. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis5. Nucleic acid according to at least one of claims 1 to
4 dadurch gekennzeichnet, daß es sich um DNA, RNA, PNA oder nukleaseresistente Analoga handelt.4 characterized in that it is DNA, RNA, PNA or nuclease-resistant analogs.
6. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis6. Nucleic acid according to at least one of claims 1 to
5 dadurch gekennzeichnet, daß es sich um mRNA, cDNA oder genomische DNA handelt.5 characterized in that it is mRNA, cDNA or genomic DNA.
7. Nukleinsäure dadurch gekennzeichnet, daß sie komplementär zur Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 6 ist.7. Nucleic acid, characterized in that it is complementary to the nucleic acid according to at least one of claims 1 to 6.
8. Eukaryontische neutrale Sphingomyelinase erhältlich durch Expression der Nukleinsäure gemäß Anspruch 1 bis 6, insbesondere mit der Sequenz gemäß Seq. ID. Nr. 1 oder Seq. ID. Nr. 2. 8. Eukaryotic neutral sphingomyelinase obtainable by expression of the nucleic acid according to claims 1 to 6, in particular with the sequence according to Seq. ID. No. 1 or Seq. ID. No. 2.
9. Antikörper, dadurch gekennzeichnet, daß sie gegen eukaryontische neutrale Sphingomyelinase gemäß Anspruch 8 oder eine Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 gerichtet sind.9. Antibodies, characterized in that they are directed against eukaryotic neutral sphingomyelinase according to claim 8 or a nucleic acid according to at least one of claims 1 to 7.
10. Zellinie, dadurch gekennzeichnet, daß sie neutrale Sphingomyelinase gemäß Anspruch 8 überexprimiert .10. cell line, characterized in that it overexpresses neutral sphingomyelinase according to claim 8.
11. Zellinie gemäß Anspruch 10 dadurch gekennzeichnet, daß es sich um eine eukaryontische neutrale Sphingomyelinase exprimierende Zellinie handelt, die auf den Zellinien U937, HEK 293 oder Jurkat beruht.11. Cell line according to claim 10, characterized in that it is a eukaryotic neutral sphingomyelinase expressing cell line, which is based on the cell lines U937, HEK 293 or Jurkat.
12. Transgenes Säugetier mit Überexpression (gain of function) oder Gendefizienz oder Gendefekt (loss of function) für eukaryontische neutrale Sphingomyelinase.12. Transgenic mammal with overexpression (gain of function) or gene deficiency or gene defect (loss of function) for eukaryotic neutral sphingomyelinase.
13. Transgenes Säugetier gemäß Anspruch 12 dadurch gekennzeichnet, daß es ein Nagetier ist.13. Transgenic mammal according to claim 12, characterized in that it is a rodent.
14. Arzneimittel enthaltend eukaryontische neutrale Sphingomyelinase gemäß Anspruch 8, eine Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 und/oder einen Antikörper gemäß Anspruch 9 zusammen mit weiteren Hilfsstoffen.14. Medicament containing eukaryotic neutral sphingomyelinase according to claim 8, a nucleic acid according to at least one of claims 1 to 7 and / or an antibody according to claim 9 together with further auxiliaries.
15. Diagnostikmittel enthaltend eukaryontische neutrale Sphingomyelinase gemäß Anspruch 8, eine Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 und/oder einen Antikörper gemäß Anspruch 9 zusammen mit weiteren Hilfsstoffen.15. Diagnostic agent containing eukaryotic neutral sphingomyelinase according to claim 8, a nucleic acid according to at least one of claims 1 to 7 and / or an antibody according to claim 9 together with other auxiliaries.
16. Verwendung der Arzneimittel gemäß Anspruch 14 oder der Diagnostikmittel gemäß Anspruch 15 zur Diagnose und Behandlung von Erkrankungen, die auf einer Über- oder Unterexpression und/oder einer erhöhten oder verminderten Aktivität der eukaryontisehen neutralen Sphingomyelinase und/oder auf Störungen der Zeilproliferation, Zelldifferenzierung und/oder Apotose beruhen.16. Use of the medicament according to claim 14 or the diagnostic agent according to claim 15 for the diagnosis and treatment of diseases based on over- or under-expression and / or an increased or decreased activity of the eukaryotic neutral sphingomyelinase and / or based on disorders of cell proliferation, cell differentiation and / or apotosis.
17. Verwendung gemäß Anspruch 15, dadurch gekennzeichnet, daß es sich bei den Erkrankungen um Entzüngsdungsprozesse, Zellwachstumstörungen, Krebs und/oder Stoffwelchselstörun- gen wie Störungen der Cholesterinhomöostase (Arteriosklero- se) handelt.17. Use according to claim 15, characterized in that the diseases are inflammation processes, cell growth disorders, cancer and / or metabolic disorders such as disorders of cholesterol homeostasis (arteriosclerosis).
18. Verfahren zum Screening von Wirkstoffen dadurch gekennzeichnet, daß die Veränderung der Expression oder Aktivität der eukaryontisehen neutralen Sphingomyelinase in Zellinien gemäß Anspruch 10 bei Zugabe von mindestens einer möglichen pharmazeutisch wirksamen Substanz gemessen wird.18. A method for screening active substances, characterized in that the change in expression or activity of the eukaryotic neutral sphingomyelinase in cell lines according to claim 10 is measured with the addition of at least one possible pharmaceutically active substance.
19. Verwendung der Zellinie gemäß Anspruch 10 zur Entwicklung und Prüfung von pharmazeutischen LeitStrukturen.19. Use of the cell line according to claim 10 for the development and testing of pharmaceutical lead structures.
20. Verfahren zur Herstellung der eukaryontisehen neutralen Sphingomyelinase gemäß Anspruch 8 durch chemische Peptid- synthese oder durch Expression in gentechnisch veränderten Organismen, insbesondere in eukaryontisehen Expressionssys- temen.20. A method for producing the eukaryotic neutral sphingomyelinase according to claim 8 by chemical peptide synthesis or by expression in genetically modified organisms, in particular in eukaryotic expression systems.
21. Verfahren zur Herstellung einer Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 durch chemische Synthese oder durch Vervielfältigung in gentechnisch veränderten Organismen.21. A method for producing a nucleic acid according to at least one of claims 1 to 7 by chemical synthesis or by duplication in genetically modified organisms.
22. Nukleinsäuren gemäß Anspruch 5, dadurch gekennzeichnet, daß es sich um das Gen für eukaryontische neutrale Sphingomyelinase handelt und neben codierenden Bereich (Exons) nicht codierende Bereiche (Introns) aufweist, insbesondere ein Gen mit der Sequenz gemäß Seq. ID. Nr. 5 und Seq. ID. Nr. 6. 22. Nucleic acids according to claim 5, characterized in that it is the gene for eukaryotic neutral sphingomyelinase and in addition to coding region (exons) has non-coding regions (introns), in particular a gene with the sequence according to Seq. ID. No. 5 and Seq. ID. No. 6.
23. Varianten der eukaryontischen neutralen Sphingomyelinase gemäß Anspruch 8.23. Variants of the eukaryotic neutral sphingomyelinase according to claim 8.
24. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 oder 22, dadurch gekennzeichnet, daß es sich um Derivate, Fragmente oder Varianten der Nukleinsäuren handelt . 24. Nucleic acid according to at least one of claims 1 to 7 or 22, characterized in that it is derivatives, fragments or variants of the nucleic acids.
EP98945230A 1997-08-11 1998-08-11 Neutral sphingomyelinase Withdrawn EP1021546A1 (en)

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EP1204757A1 (en) * 1999-08-14 2002-05-15 Memorec Stoffel GmbH Medizinisch-Molekulare Entwicklung Neutral cerebral-sphingomyelinase
GB0005326D0 (en) * 2000-03-06 2000-04-26 Deutsches Krebsforsch Enzymes and uses relating thereto
US6881546B2 (en) 2000-12-22 2005-04-19 Medlyte, Inc., Sdsu Heart Institute Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
JP2002253225A (en) * 2001-02-14 2002-09-10 Sk Corp Cell surface sphingomyelinase derived from bovine brain, method for isolating the same and anti-sphingomyelinase monoclonal antibody
WO2003106704A1 (en) * 2002-06-14 2003-12-24 Memorec Biotec Gmbh Method for identifying intracellular transport modulators by measuring the expression or activity of neutral sphingomyelinase
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