WO2001012818A1 - Neutral cerebral-sphingomyelinase - Google Patents
Neutral cerebral-sphingomyelinase Download PDFInfo
- Publication number
- WO2001012818A1 WO2001012818A1 PCT/EP2000/007889 EP0007889W WO0112818A1 WO 2001012818 A1 WO2001012818 A1 WO 2001012818A1 EP 0007889 W EP0007889 W EP 0007889W WO 0112818 A1 WO0112818 A1 WO 0112818A1
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- WO
- WIPO (PCT)
- Prior art keywords
- sphingomyelinase
- nucleic acid
- brain
- eukaryotic
- neutral
- Prior art date
Links
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Classifications
-
- 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)
-
- 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
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
Definitions
- the present invention relates to nucleic acids coding for human or murine eukaryotic neutral brain 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, sphingosin, sphingosine-1-phosphate.
- Two sphingomyelin-cleaving enzyme activities are known, firstly that of the lysosomic acidic sphingomyelinase and secondly that of the membrane-bound neutral sphingomyelinase.
- neutral sphingomyelinase In humans and in mammals, the activity of neutral sphingomyelinase is preferentially in the brain. The only eukaryotic neutral sphingomyelinase so far characterized occurs in mammals in all tissue types and is not significantly responsible for the sphingomyelinase activity that can be measured in the brain.
- the present invention makes nucleic acids coding for human or murine eukaryotic neutral brain sphingomyelinase available for the first time. It is also known as nSMase2.
- the eukaryotic neutral brain sphingomyelinase is characterized by the fact that it is enriched in the brain, splits sphingomyelin into 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.
- the molecular weight is in the range from 50 to 80 kD, preferably in the range from 70 to 75 kD.
- Figure 1 shows the results of Northern and Western blots from various human tissues.
- the nucleic acid according to the invention is preferably nucleic acids with the Seq. ID. No. 3 and Seq. ID. No. 4.
- nucleic acids according to the invention are suitable for the expression of the eukaryotic neutral brain sphingomyelinase in pro- or eukaryotic systems.
- they are also suitable for the expression of nSMase2 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 nSMase2.
- 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 brain sphingomyelinase obtainable by the expression of the nucleic acids according to the invention.
- the nSMase2 according to the invention can be produced by expression in genetically modified organisms.
- eukary ontic expression systems suitable.
- Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene).
- the purification from genetically modified organisms offers, especially in the case of overexpression, easy and direct access to the nSMase2 according to the invention and also allows isolation in large quantities.
- amino acid sequences of human and murine neutral brain sphingomyelinase are as Seq. ID. Nos. 1 and 2 reproduced.
- the molecular weights of human and murine sphingomyelinase 2 are approximately 71 kDa in each case.
- the nSMase2 sequences according to the invention contain no signal sequence at the N-terminus. Based on the hydrophobicity analysis, it can be assumed that two neighboring hydrophobic membrane domains at the N-terminus are separated by 35 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 have contact with the extracellular environment.
- nSMasen which are te is sekretier-, is soluble proteins, but is in compliance 'with previous studies of the properties of the neutral sphingomyelinase of mammals.
- the ubiquitous eukaryotic sphingomyelinase nSMsel also has two hydrophobic transmembrane domains, but these are located at the C-terminus of the protein.
- the 6 kb mRNA of human nSMase2 is preferably expressed in the brain.
- the Northern blot shows a weaker signal in the liver and small intestine, while a cross-hybridizing mRNA of different sizes (3.5 kB) is expressed in the thymus.
- the pH optimum of the neutral brain sphingomyelinase according to the invention is in the range from 6 to 8.
- the activity is dependent on magnesium ions, the addition of EDTA leads to an inhibition of the nSMase activity, but can be achieved by adding Mn 2+ or Mg 2 + - Ions are restored. Activity is unaffected by treatment with DTT or 2-mercaptoethanol.
- variants of eukaryotic neutral brain sphingomyelinase are also claimed.
- the term "variants” includes both naturally occurring allelic variations of the eukaryotic neutral brain sphingomyelinase as well as recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and subsequent expression of proteins that are biological and / or correspond to the immunological activity of the eukaryotic neutral sphingomyelinase.
- 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 includes N- and / or C-terminal shortened proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives.
- Compounds in which nSMase2 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 brain 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 hydrolysis-stable compounds, 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 have more than 6, more preferably more than 8 and most preferably more than 12 nucleotides. For reasons of diffusion and cost, they are usually less than 30 nucleotides in length, preferably 24 or less and even more preferably 18 or fewer nucleotides.
- 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 means that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (at otherwise identical conditions), in which exactly complementary nucleic acids would 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 further relates to antibodies which are directed against the nSMase2 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 obtained by methods known per se to the person skilled in the art by immunization with nSMase, nucleic acids according to the invention or peptide and nucleic acid fragments in the presence of auxiliary reagents.
- the invention furthermore relates to cell lines which overexpress the nSMase2 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. In experiments, the cell lines showed a specific nSMase activity between 0.1 and 10 ⁇ mol / mg protein / hour.
- Figure 1 shows the Northern and Western blot analysis of nSMase2 expression in human tissues.
- Part A shows the result of a Northern blot hybridization with an nSMase2 probe.
- Part B shows the result of a Northerblot hybridization with a probe specific for actin as a control.
- Part C shows a Western blot with an antibody specific for recombinantly produced nSMase2.
- FIGS 2 to 5 show the sequences according to the invention.
- the invention further relates to a transgenic mammal which has an overexpression (gain of function) or a gene deficiency or a gene defect (loss of function) for the nSMase2 according to the invention.
- the mammal is preferably a rodent, in particular a mouse.
- These transgenic mammals are obtainable by methods known per se to the person skilled in the art and are particularly suitable for the functional elucidation 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 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 diagnosing and treating diseases which are based on over- or under-expression and / or an increased or reduced activity of eukaryotic neutral sphingomyelinase and / or on disorders of cell proliferation, cell differentiation and / or apoptosis.
- these are diseases in which inflammatory processes, cell growth disorders and metabolic disorders play a role.
- 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).
- a pharmaceutical screening method according to the invention is based on changing the expression or activity of the nSMase2 according to the invention in nSMase2 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.
- the nucleic acids coding for the neutral brain 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 of 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.
- HEK 293 cells grew in DMEM medium with 10% fetal calf serum, 1 ⁇ g / ml penicillin / streptomycin and ImM pyruvate at 37 ° C and 5% CO2. 5 ⁇ 10 6 cells were transfected with 1 ⁇ g of linearized plasmid DNA which encoded the nSMase according to the invention by electroporation with a “gene pulser” (company Bio-Rad). The selection of stable clones was carried out under 1 mg / ml Geneticin (G418, Life Technologies, Gaithersburg, MD).
- nSMase purified from the cell lines showed a specific activity between 0.2 and 1 ⁇ mol / mg protein / hour.
- the pH optimum was 6 and 8.
- 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 J sphingomyelin for 30 min at 37 ° C. 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 containing the enzymatically released phosphocholine was measured in a scintillation counter.
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Abstract
Nucleic acids which code for eucaryontic neutral cerebral-sphingomyelinase n (nSMase2) have the following sequence motifs: X1-X2-X3-X4-D-Y-X5 and X6-X7-T-D-H-X8, whereby X1, X6 = A or G; X2, X3 = R or K; X4, X5, X7, X8 = I or L or V or M.
Description
Neutrale Hirn-Sphinαomvelinase Neutral brain sphinαomvelinase
Die vorliegende Erfindung betrifft Nukleinsäuren, die für humane oder murine eukaryontische neutrale Hirn-Sphingomyelinase codieren, und ihre Anwendung.The present invention relates to nucleic acids coding for human or murine eukaryotic neutral brain sphingomyelinase and their use.
Sphingomyelin ist eine wesentliche Komponente von Plasmamembranen. Der Abbau des Sphingomyelins gibt eine Vielzahl von Substanzen, die potentielle second messenger Eigenschaften haben, z.B. Ceramid, Sphin- gosin, Sphingosin-1-phosphat. Es sind zwei sphingomyelinspaltende Enzymaktivitäten bekannt, zum einen die der lysosomaien sauren Sphingo- myelinase und zum anderen die der membran-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, sphingosin, sphingosine-1-phosphate. Two sphingomyelin-cleaving enzyme activities are known, firstly that of the lysosomic acidic sphingomyelinase and secondly that of the membrane-bound neutral sphingomyelinase.
Im Menschen und in Säugetieren liegt die Aktivität der neutralen Sphingomyelinase bevorzugt im Gehirn vor. Die einzige bisher charakterisierte eukaryontische neutrale Sphingomyelinase kommt in Säugern in allen Gewebetypen vor und ist nicht maßgeblich für die im Gehirn messbare Sphingomyelinase-Aktivität verantwortlich .In humans and in mammals, the activity of neutral sphingomyelinase is preferentially in the brain. The only eukaryotic neutral sphingomyelinase so far characterized occurs in mammals in all tissue types and is not significantly responsible for the sphingomyelinase activity that can be measured in the brain.
Durch die vorliegende Erfindung werden erstmals Nukleinsäuren, codierend für humane oder murine eukaryontische neutrale Hirn- Sphingomyelinase, verfügbar gemacht. Sie wird auch als nSMase2 bezeichnet. Die eukaryontische neutrale Hirn-Sphingomyelinase ist dadurch charakterisiert, dass sie im Gehirn angereichert ist, 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.
Das Molekulargewicht liegt im Bereich von 50 bis 80 kD, vorzugsweise im Bereich von 70 bis 75 kD.The present invention makes nucleic acids coding for human or murine eukaryotic neutral brain sphingomyelinase available for the first time. It is also known as nSMase2. The eukaryotic neutral brain sphingomyelinase is characterized by the fact that it is enriched in the brain, splits sphingomyelin into 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. The molecular weight is in the range from 50 to 80 kD, preferably in the range from 70 to 75 kD.
Figur 1 zeigt die Ergebnisse von Northern- und Westernblots aus verschiedenen humanen Geweben.Figure 1 shows the results of Northern and Western blots from various human tissues.
Bevorzugt handelt es sich bei der erfindungsgemäßen Nukleinsäure um Nukleinsäuren mit der Seq. ID. Nr. 3 und Seq. ID. Nr. 4.The nucleic acid according to the invention is preferably nucleic acids with the Seq. ID. No. 3 and Seq. ID. No. 4.
Die erfindungsgemäßen Nukleinsäuren eignen sich zur Expression der eukaryontischen neutralen Hirn-Sphingomyelinase in pro- oder eukary- ontischen Systemen. Darüber hinaus sind sie auch zur Expression der nSMase2 in vivo im Sinne einer Gentherapie oder insbesondere in Form von Fragmenten auch in komplementärer Struktur als Antisense- Nukleotide zur Verringerung der Expression der nSMase2 geeignet.The nucleic acids according to the invention are suitable for the expression of the eukaryotic neutral brain sphingomyelinase in pro- or eukaryotic systems. In addition, they are also suitable for the expression of nSMase2 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 nSMase2.
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 Hirn- Sphingomyelinase.The invention also relates to the eukaryotic neutral brain sphingomyelinase obtainable by the expression of the nucleic acids according to the invention.
Sie weist zwei Teilmotive X1-X2-X3-X4-D-Y-X5 und X6-X7-T-D-H-X8, wobei Xi, X6 = A oder G; X2, X3 = R oder K; X4, X5, X7, Xs = I oder L oder V oder M ist, auf.It has two sub-motifs X1-X2-X3-X4-DY-X5 and X 6 -X7-TDHX 8 , where Xi, X 6 = A or G; X 2 , X 3 = R or K; X 4 , X 5 , X7, Xs = I or L or V or M.
Die erfindungsgemäße nSMase2 lässt sich durch Expression in gentechnisch veränderten Organismen herstellen. Insbesondere sind eukary-
ontische 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, einen leichten und direkten Zugang zur erfindungsgemäßen nSMase2 und erlaubt darüber hinaus die Isolierung in größeren Mengen.The nSMase2 according to the invention can be produced by expression in genetically modified organisms. In particular, eukary ontic expression systems suitable. Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene). The purification from genetically modified organisms offers, especially in the case of overexpression, easy and direct access to the nSMase2 according to the invention and also allows isolation in large quantities.
Die Aminosäuresequenzen der humanen und murinen neutralen Hirn- Sphingomyelinase sind als Seq. ID. Nr. 1 und 2 wiedergegeben.The amino acid sequences of human and murine neutral brain sphingomyelinase are as Seq. ID. Nos. 1 and 2 reproduced.
Die Molekulargewichte der humanen bzw. murinen Sphingomyelinase 2 beträgt jeweils etwa 71 kDa. Im Gegensatz zu den bakteriellen nSMasen enthalten die erfindungsgemäßen nSMase2 Sequenzen keine Signalsequenz am N-Terminus. Aufgrund der Hydrophobizitätsanalyse kann davon ausgegangen werden, dass zwei benachbarte hydrophobe Membrandomänen am N-Terminus durch 35 Aminosäuren getrennt sind. Es scheint sich daher um integrale Membranproteine zu handeln, deren ka- talytisch 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 sekretier- te, lösliche Proteine handelt, ist aber in Übereinstimmung' mit bisherigen Untersuchungen zu den Eigenschaften der neutralen Sphingomyelinasen von Säugetieren. Die ubiquitäre eukaryontische Sphingomyelinase nSMa- sel besitzt ebenfalls zwei hydrophobe Transmembrandomänen, die sich jedoch am C-Terminus des Proteins befinden.The molecular weights of human and murine sphingomyelinase 2 are approximately 71 kDa in each case. In contrast to the bacterial nSMases, the nSMase2 sequences according to the invention contain no signal sequence at the N-terminus. Based on the hydrophobicity analysis, it can be assumed that two neighboring hydrophobic membrane domains at the N-terminus are separated by 35 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 have contact with the extracellular environment. This is in contrast to the bacterial nSMasen, which are te is sekretier-, is soluble proteins, but is in compliance 'with previous studies of the properties of the neutral sphingomyelinase of mammals. The ubiquitous eukaryotic sphingomyelinase nSMsel also has two hydrophobic transmembrane domains, but these are located at the C-terminus of the protein.
Die 6 kb mRNA der humanen nSMase2 wird gemäß Northern Blot Analyse bevorzugt im Gehirn exprimiert. In Leber und Dünndarm zeigt der Northern Blot ein schwächeres Signal, während in Thymus eine kreuzhybridisierende mRNA von unterschiedlicher Größe (3.5 kB) exprimiert wird.
Das pH-Optimum der erfindungsgemäßen neutralen Hirn- Sphingomyelinase liegt im Bereich von 6 bis 8. Die Aktivität ist mag- nesiumionenabhängig, die Zugabe von EDTA führt zu einer Inhibierung der nSMase-Aktivität, kann jedoch durch Zugabe von Mn2+- oder Mg2+- Ionen wiederhergestellt werden. Die Aktivität ist unbeeinflusst durch Behandlung mit DTT oder 2-Mercaptoethanol.According to Northern blot analysis, the 6 kb mRNA of human nSMase2 is preferably expressed in the brain. The Northern blot shows a weaker signal in the liver and small intestine, while a cross-hybridizing mRNA of different sizes (3.5 kB) is expressed in the thymus. The pH optimum of the neutral brain sphingomyelinase according to the invention is in the range from 6 to 8. The activity is dependent on magnesium ions, the addition of EDTA leads to an inhibition of the nSMase activity, but can be achieved by adding Mn 2+ or Mg 2 + - Ions are restored. Activity is unaffected by treatment with DTT or 2-mercaptoethanol.
Weiterhin beansprucht werden Varianten der eukaryontischen neutralen Hirn-Sphingomyelinase. Unter den Begriff "Varianten" fallen sowohl natürlich vorkommende allelische Variationen der eukaryontischen neutralen Hirn-Sphingomyelinase sowie durch rekombinante DNA-Technologie (insbesondere durch in vitro Mutagenese mit Hilfe von chemisch synthetisierten Oligonukleotiden) und anschließende Expression erzeugte Proteine, die hinsichtlich ihrer biologischen und/oder immunologischen Aktivität der eukaryontischen 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 physikalischchemische Eigenschaften aufweist.Variants of eukaryotic neutral brain sphingomyelinase are also claimed. The term "variants" includes both naturally occurring allelic variations of the eukaryotic neutral brain sphingomyelinase as well as recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and subsequent expression of proteins that are biological and / or correspond to the immunological activity of the eukaryotic neutral sphingomyelinase. 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. Auch Verbindungen, bei denen nSMa- se2 oder ihre Varianten mit weiteren Molekülen wie Farbstoffen, Radionukliden oder Affinitätskomponenten gekoppelt sind, stellen erfindungsgemäße Varianten dar.
Beansprucht werden auch Nukleinsäuren, die für eukaryontische neutrale Hirn-Sphingomyelinase codieren bzw. komplementär zu diesen Nukleinsäuren sind. Bei den Nukleinsäuren kann es sich beispielsweise um DNA, RNA, PNA oder um nukleaseresistente Analoga handeln. Nukleasere- sistente Analoga sind insbesondere solche Verbindungen, in denen die Phosphodiesterbindung durch hydrolysestabile Verbindungen modifiziert sind, beispielsweise Phosphothioate, Methylphosphonate o.a.In particular, the term variants also includes N- and / or C-terminal shortened proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives. Compounds in which nSMase2 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 brain 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 hydrolysis-stable compounds, 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 Nukleoti- den, 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 have more than 6, more preferably more than 8 and most preferably more than 12 nucleotides. For reasons of diffusion and cost, they are usually less than 30 nucleotides in length, preferably 24 or less and even more preferably 18 or fewer nucleotides.
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 Afflnitätskomponenten, 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" means that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (at otherwise identical conditions), in which exactly complementary nucleic acids would 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 nSMase2 oder die erfindungsgemäßen Nukleinsäuren gerichtet sind. Diese Substanzen eignen sich insbesondere zum Einsatz in der Diagnostik, dem Fachmann an sich bekannten Immunoassays, zur histologi- schen 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.The invention further relates to antibodies which are directed against the nSMase2 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 obtained by methods known per se to the person skilled in the art by immunization with nSMase, nucleic acids according to the invention or peptide and nucleic acid fragments in the presence of auxiliary reagents.
Weiterhin sind Gegenstand der Erfindung Zellinien, die die erfindungsgemäße nSMase2 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 nSMase2 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.
Die Zellinien zeigten in Experimenten eine spezifische nSMase-Aktivität zwischen 0,1 und 10 μmol/mg Protein/Stunde.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. In experiments, the cell lines showed a specific nSMase activity between 0.1 and 10 μmol / mg protein / hour.
Figur 1 zeigt die Northern- und Westernblot Analyse der nSMase2- Expression in humanen Geweben. Teil A zeigt dabei das Ergebnis eine Northernblot Hybridisierung mit einer nSMase2 Sonde. Teil B zeigt als Kontrolle das Ergebnis einer Northerblot Hybridisierung mit einer Sonde spezifisch für Actin. Teil C zeigt einen Westernblot mit einem Antikörper spezifisch für rekombinant hergestellte nSMase2.Figure 1 shows the Northern and Western blot analysis of nSMase2 expression in human tissues. Part A shows the result of a Northern blot hybridization with an nSMase2 probe. Part B shows the result of a Northerblot hybridization with a probe specific for actin as a control. Part C shows a Western blot with an antibody specific for recombinantly produced nSMase2.
Die Figuren 2 bis 5 zeigen die erfindungsgemäßen Sequenzen.Figures 2 to 5 show the sequences according to the invention.
Die Zugabe von 0,5 mM Arachidonsäure führte zu einer dreifachen Erhöhung der nSMase-Aktivität in einem Extrakt aus HEK 293 Zellen, die nSMase2 überexprimieren. Die Zugabe von Phosphatidylserin führte zu einer sechsfachen Erhöhung der nSMase-Aktivität in einem Extrakt aus HEK 293 Zellen, die nSMase2 überexprimieren.The addition of 0.5 mM arachidonic acid resulted in a three-fold increase in nSMase activity in an extract from HEK 293 cells that overexpress nSMase2. The addition of phosphatidylserine led to a six-fold increase in nSMase activity in an extract from HEK 293 cells which overexpress nSMase2.
Gegenstand der Erfindung ist weiterhin ein transgenes Säugetier, das eine Überexpression (gain of function) oder eine Gendefizienz bzw. einen Gendefekt (loss of function) für die erfindungsgemäße nSMase2 aufweist. Bevorzugt handelt es sich bei dem Säugetier um ein Nagetier, insbesondere eine Maus. Diese transgenen 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 deficiency or a gene defect (loss of function) for the nSMase2 according to the invention. The mammal is preferably a rodent, in particular a mouse. These transgenic mammals are obtainable by methods known per se to the person skilled in the art and are particularly suitable for the functional elucidation 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. Targeted modification of a gene in the genome of ES cells that are subsequently injected into blastocysts, the function of a gene switched off.
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 Signaltransduk- tionswegen, 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 eukaryontischen neutralen Sphingomyelinase und/oder auf Störungen der Zellproliferation, Zelldifferenzierung und/oder Apoptose beruhen.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 diagnosing and treating diseases which are based on over- or under-expression and / or an increased or reduced activity of eukaryotic neutral sphingomyelinase and / or on disorders of cell proliferation, cell differentiation and / or apoptosis.
Insbesondere sind dies Erkrankungen, bei denen Entzündungsprozesse, Zellwachstumstörungen und Stoffwechselstörungen eine Rolle spielen. Dies können beispielsweise Krebserkrankungen oder Störungen der Cho- lesterinhomöostase (Arteriosklerose) sein.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 erfindungsgemä- ßen nSMase2 in nSMase2-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 nSMase2 according to the invention in nSMase2 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 Hirn-Sphingomyelinase kodierenden Nukleinsäuren wurden in die Notl Schnittstellen der Klonie- rungsstelle des eukaryontischen Expressionsvektors pRc/CMV (Stratagene) Moniert. Die erhaltenen Sequenzen wurden durch Sequenzierung mit einem Perkin-Elmer DNA-Sequenzer 377A erhalten.The nucleic acids coding for the neutral brain 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)cellulose (Boehringer Mannheim Deutschland) gemäß Standardmethoden isoliert.The total RNA was isolated from various organs of 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.
Beispiel 3Example 3
Überexprimierende ZellinienOverexpressing cell lines
HEK 293 Zellen wuchsen in DMEM Medium mit 10% fötalem Kälberserum, 1 μg/ml Penicillin/Streptomycin und ImM Pyruvat bei 37°C und 5% CO2. 5xl06-Zellen wurden mit 1 μg linearisierter Plasmid-DNA, die für die erfindungsgemäße nSMase kodierte durch Elektroporation mit einem "gene pulser" (Firma Bio-Rad) transfiziert. Die Selektion stabiler Klone
erfolgte unter 1 mg/ml Geneticin (G418, Life Technologies, Gaithersburg, MD).HEK 293 cells grew in DMEM medium with 10% fetal calf serum, 1 μg / ml penicillin / streptomycin and ImM pyruvate at 37 ° C and 5% CO2. 5 × 10 6 cells were transfected with 1 μg of linearized plasmid DNA which encoded the nSMase according to the invention by electroporation with a “gene pulser” (company Bio-Rad). The selection of stable clones was carried out under 1 mg / ml Geneticin (G418, Life Technologies, Gaithersburg, MD).
Die aus den Zellinien aufgereinigte nSMase zeigte eine spezifische Aktivität zwischen 0,2 und 1 μmol/mg Protein/Stunde. Das pH-Optimum lag bei 6 und 8. 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 0.2 and 1 μmol / mg protein / hour. The pH optimum was 6 and 8. The activity was dependent on the presence of magnesium ions; the addition of EDTA inhibited the activity.
Beispiel 4Example 4
Messung der nSMase2-AktivitätMeasurement of nSMase2 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 Lysepuffer 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-14CH3J- Sphingomyelin für 30 min bei 37°C 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 Szintillationszähler 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 J sphingomyelin for 30 min at 37 ° C. 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 containing the enzymatically released phosphocholine was measured in a scintillation counter.
Claims
1. Nukleinsäure kodierend für eukaryontische neutrale Hirn- Sphingomyelinase (nSMase2) mit den Sequenzmotiven X1-X2-X3- X4-D-Y-X5 und X6-X7-T-D-H-X8, wobei X X6 = A oder G; X2, X3 = R oder K; X4, X5, X7, Xs = I oder L oder V oder M ist.1. Nucleic acid coding for eukaryotic neutral brain sphingomyelinase (nSMase2) with the sequence motifs X 1 -X 2 -X 3 - X4-DY-X5 and X 6 -X 7 -TDHX 8 , where XX 6 = A or G; X 2 , X 3 = R or K; X 4 , X5, X7, Xs = I or L or V or M.
2. Nukleinsäure nach Anspruch 1, dadurch gekennzeichnet, dass es sich um neutrale Hirnsphingomyelinase eines Säugetiers, insbesondere um murine oder humane Hirnsphingomyelinase handelt.2. Nucleic acid according to claim 1, characterized in that it is neutral mammalian brain spingomyelinase, in particular murine or human brain spingomyelinase.
3. Nukleinsäure gemäß Anspruch 1 oder 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 claim 1 or 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 bis 3 mit der Sequenz gemäß Seq. ID. Nr. 3 oder Seq. ID. Nr. 4.4. Nucleic acid according to at least one of claims 1 to 3 with the sequence according to Seq. ID. No. 3 or Seq. ID. No. 4.
5. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 4 dadurch gekennzeichnet, dass es sich um DNA, RNA, PNA oder nuk- leaseresistente Analoga handelt.5. Nucleic acid according to at least one of claims 1 to 4, characterized in that it is DNA, RNA, PNA or nuclease-resistant analogs.
6. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 5 dadurch gekennzeichnet, daß es sich um mRNA, cDNA oder genomische DNA handelt. 6. Nucleic acid according to at least one of claims 1 to 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 Hirnsphingomyelinase erhältlich durch Expression der Nukleinsäure gemäß Anspruch 1 bis 7, insbesondere mit der Sequenz gemäß Seq. ID. Nr. 1 oder Seq. ID. Nr. 2.8. Eukaryotic neutral brain spingomyelinase obtainable by expression of the nucleic acid according to claims 1 to 7, 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 Hirnsphingomyelinase 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 brain spingomyelinase according to claim 8 or a nucleic acid according to at least one of claims 1 to 7.
10. Zellinie, dadurch gekennzeichnet, dass sie eukaryontische neutrale Hirnsphingomyelinase gemäß Anspruch 8 überexprimiert.10. cell line, characterized in that it overexpresses eukaryotic neutral brain spingomyelinase according to claim 8.
11. Zellinie gemäß Anspruch 10, dadurch gekennzeichnet, dass es sich um eine eukaryontische neutrale Hirn-Sphingomyelinase exprimie- rende 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 cell line expressing eukaryotic neutral brain sphingomyelinase, 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 Hirn-Sphingomyelinase.12. Transgenic mammal with overexpression (gain of function) or gene deficiency or gene defect (loss of function) for eukaryotic neutral brain 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 Hirn-Sphingomyelinase gemäß Anspruch 8, eine Nukleinsäure gemäß min- destens einem der Ansprüche 1 bis 7 und/oder einen Antikörper gemäß Anspruch 9 zusammen mit weiteren Hilfsstoffen.14. Medicament containing eukaryotic neutral brain sphingomyelinase according to claim 8, a nucleic acid according to min 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 Hirn-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 brain 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 eukaryontischen neutralen Hirn-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 reduced activity of the eukaryotic neutral brain sphingomyelinase and / or on disorders of cell proliferation, Cell differentiation and / or apotosis are based.
17. Verwendung gemäß Anspruch 16, dadurch gekennzeichnet, dass es sich bei den Erkrankungen um Entzündungsprozesse, Zellwachstumstörungen, Krebs und/oder Stoffwechselstörungen wie Störungen der Cholesterinhomöostase (Arteriosklerose) handelt.17. Use according to claim 16, characterized in that the diseases are inflammatory 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 eukaryontischen neutralen Hirn-Sphingomyelinase in Zellinien gemäß Anspruch 10 bei Zugabe von mindestens einer möglichen pharmazeutisch wirksamen Substanz gemessen wird.18. A method for screening active ingredients, characterized in that the change in expression or activity of the eukaryotic neutral brain 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 eukaryontischen neutralen Hirn- Sphingomyelinase gemäß Anspruch 8 durch chemische Peptid- synthese oder durch Expression in gentechnisch veränderten Organismen, insbesondere in eukaryontischen Expressionssystemen.20. A method for producing the eukaryotic neutral brain sphingomyelinase according to claim 8 by chemical peptide synthesis or by expression in genetically modified organisms, especially 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 mindestens einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass es sich um das Gen für eukaryontische neutrale Hirn-Sphingomyelinase handelt und neben codierenden Bereich (Exons) nicht codierende Bereiche (Introns) aufweist.22. Nucleic acids according to at least one of claims 1 to 7, characterized in that it is the gene for eukaryotic neutral brain sphingomyelinase and in addition to coding region (exons) has non-coding regions (introns).
23. Varianten der eukaryontischen neutralen Hirn-Sphingomyelinase gemäß Anspruch 8.23. Variants of the eukaryotic neutral brain 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.
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| JP2001516905A JP2003507015A (en) | 1999-08-14 | 2000-08-12 | Neutral brain sphingomyelinase |
| EP00956442A EP1204757A1 (en) | 1999-08-14 | 2000-08-12 | Neutral cerebral-sphingomyelinase |
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| DE19938671A DE19938671A1 (en) | 1999-08-14 | 1999-08-14 | New nucleic acid encoding cerebral sphingomyelinase, useful e.g. for diagnosis and treatment of inflammation and cancer |
| DE19938671.4 | 1999-08-14 | ||
| EP00100873.9 | 2000-01-18 | ||
| EP00100873 | 2000-01-18 |
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| 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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| WO1998028445A1 (en) * | 1996-12-24 | 1998-07-02 | The Johns Hopkins University | Recombinant n-smases and nucleic acids encoding same |
| WO1998037094A2 (en) * | 1997-02-24 | 1998-08-27 | Genetics Institute, Inc. | Secreted proteins and polynucleotides encoding them |
| WO1999007855A1 (en) * | 1997-08-11 | 1999-02-18 | Memorec Stoffel Gmbh | Neutral sphingomyelinase |
-
2000
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- 2000-08-12 WO PCT/EP2000/007889 patent/WO2001012818A1/en active Search and Examination
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| WO1998028445A1 (en) * | 1996-12-24 | 1998-07-02 | The Johns Hopkins University | Recombinant n-smases and nucleic acids encoding same |
| WO1998037094A2 (en) * | 1997-02-24 | 1998-08-27 | Genetics Institute, Inc. | Secreted proteins and polynucleotides encoding them |
| WO1999007855A1 (en) * | 1997-08-11 | 1999-02-18 | Memorec Stoffel Gmbh | Neutral sphingomyelinase |
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| 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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