WO2001012818A1 - Sphingomyelinase cerebrale neutre - Google Patents

Sphingomyelinase cerebrale neutre Download PDF

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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
Application number
PCT/EP2000/007889
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German (de)
English (en)
Inventor
Kay Hofmann
Original Assignee
Memorec Stoffel Gmbh Medizinisch-Molekulare Entwicklung
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from DE19938671A external-priority patent/DE19938671A1/de
Application filed by Memorec Stoffel Gmbh Medizinisch-Molekulare Entwicklung filed Critical Memorec Stoffel Gmbh Medizinisch-Molekulare Entwicklung
Priority to EP00956442A priority Critical patent/EP1204757A1/fr
Priority to JP2001516905A priority patent/JP2003507015A/ja
Publication of WO2001012818A1 publication Critical patent/WO2001012818A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—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

Acides nucléiques codant pour de la sphingomyélinase cérébrale neutre eucaryote (nSMase2), qui possèdent les motifs de séquence X1-X2-X3-X4-D-Y-X5 et X6-X7-T-D-H-X8, dans lesquels X1, X6 représentent A ou G, X2, X3 représentent R ou K et X4, X5, X7, X8 représentent I ou L ou V ou M.
PCT/EP2000/007889 1999-08-14 2000-08-12 Sphingomyelinase cerebrale neutre WO2001012818A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP00956442A EP1204757A1 (fr) 1999-08-14 2000-08-12 Sphingomyelinase cerebrale neutre
JP2001516905A JP2003507015A (ja) 1999-08-14 2000-08-12 中性脳スフィンゴミエリナーゼ

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19938671A DE19938671A1 (de) 1999-08-14 1999-08-14 Neutrale Hirn-Sphingomyelinase
DE19938671.4 1999-08-14
EP00100873 2000-01-18
EP00100873.9 2000-01-18

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WO2001012818A1 true WO2001012818A1 (fr) 2001-02-22

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003106704A1 (fr) * 2002-06-14 2003-12-24 Memorec Biotec Gmbh Procede d'identification de modulateurs du transport intracellulaire par mesure de l'expression ou de l'activite de la sphingomyelinase neutre

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028445A1 (fr) * 1996-12-24 1998-07-02 The Johns Hopkins University N-smases recombinees et acides nucleiques les codant
WO1998037094A2 (fr) * 1997-02-24 1998-08-27 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO1999007855A1 (fr) * 1997-08-11 1999-02-18 Memorec Stoffel Gmbh Sphingomyelinase neutre

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028445A1 (fr) * 1996-12-24 1998-07-02 The Johns Hopkins University N-smases recombinees et acides nucleiques les codant
WO1998037094A2 (fr) * 1997-02-24 1998-08-27 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO1999007855A1 (fr) * 1997-08-11 1999-02-18 Memorec Stoffel Gmbh Sphingomyelinase neutre

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAYASHI ET AL: "cca1 is required for formation of growth-arrested confluent monolayer of rat 3Y1 cells", JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 272, no. 29, 1997, pages 18082 - 18086, XP002140656, ISSN: 0021-9258 *
HOFMANN ET AL.: "Cloning and characterization of the mammalian brain-specific, Mg21-dependent neutral sphingomyelinase", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, USA, vol. 97, no. 11, 23 May 2000 (2000-05-23), pages 5895 - 5900, XP002157877 *
ITO M ET AL: "A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms", JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 270, no. 41, 13 October 1995 (1995-10-13), pages 24370 - 24374, XP002140657, ISSN: 0021-9258 *
LIU BIN ET AL: "Purification and characterization of a membrane bound neutral pH optimum magnesium-dependent and phosphatidylserine-stimulated sphingomyelinase from rat brain", JOURNAL OF BIOLOGICAL CHEMISTRY,US,THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC.,, vol. 273, no. 51, 18 December 1998 (1998-12-18), pages 34472 - 34479, XP002140655, ISSN: 0021-9258 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003106704A1 (fr) * 2002-06-14 2003-12-24 Memorec Biotec Gmbh Procede d'identification de modulateurs du transport intracellulaire par mesure de l'expression ou de l'activite de la sphingomyelinase neutre

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JP2003507015A (ja) 2003-02-25

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