JP2003507015A - Neutral brain sphingomyelinase - Google Patents

Abstract

(57) [Summary] The present invention relates to eukaryotic neutral brain sphingomyelinase (nSMase2) and uses thereof.

Description

Detailed Description of the Invention

The present invention relates to human or murine eukaryotic neutral brain sphingomyelinase (eukaryoti).
A nucleic acid encoding c neutral brain sphingomyelinase) and its use.

Sphingomyelin is an essential constituent of the plasma membrane. Degradation of sphingomyelin gives rise to many substances with potential second messenger properties, such as ceramide, sphingosine, sphingosine-1-phosphate. Two sphingomyelin-cleaving enzyme activities, namely a lysosomal acid sphingomyelinase enzyme activity and a membrane-bound neutral sphingomyelinase enzyme activity are known.

In humans and mammals, neutral sphingomyelinase activity is found primarily in the brain. Only the eukaryotic neutral sphingomyelinase characterized to date occurs in all types of mammalian tissues and does not significantly cause sphingomyelinase activity that can be measured in the brain.

The present invention provides for the first time nucleic acids encoding human and murine eukaryotic neutral brain sphingomyelinase. This is also called nSMase2. Eukaryotic neutral brain sphingomyelinase is enriched in the brain and is characterized by cleaving sphingomyelin into ceramide and phosphocholine, the activity of which depends on the addition of magnesium ions. It is a membrane bound enzyme. Its maximum activity is achieved in the neutral pH range.

Its molecular weight is in the range of 50-80 kD, preferably in the range of 70-75 kD.

FIG. 1 shows the results of Northern and Western blots obtained from various human tissues.

The nucleic acid according to the present invention has SEQ ID NO: 3 (SEQ. ID. NO. 3) and SEQ ID NO: 4
It is preferably a nucleic acid having (SEQ.ID.NO.4).

The nucleic acids according to the invention are suitable for the expression of eukaryotic neutral brain sphingomyelinase in prokaryotic or eukaryotic systems. Moreover, they are also suitable for the expression of nSMase2 in vivo in terms of gene therapy, or in particular in the form of fragments, nMase2.
It is also suitable for complementary structures such as antisense nucleotides to reduce the expression of ase2.

The nucleic acids according to the invention may be prepared by methods known per se to the person skilled in the art, by chemical synthesis or by amplification in genetically engineered organisms.

The invention also relates to a eukaryotic neutral brain sphingomyelinase obtainable by expression of the nucleic acid according to the invention.

[0011] This two partial _{motif, X 1 -X 2 -X 3 -X} 4 -D-Y-X 5 and X ₆ -X ₇ -
T-D-H-X ₈ where X ₁ , X ₆ = A or G; X ₂ , X ₃ = R or K; X ₄ ,
X ₅ , X ₇ , X ₈ = I or L or V or M.

The nSMase2 according to the invention can be prepared by expression in a genetically engineered organism. Eukaryotic expression systems are particularly suitable. Suitable eukaryotic expression system, eg pR
c / CMV (Stratagene) is known to those skilled in the art. Purification from a genetically engineered organism makes it possible to obtain directly the nSMase2 according to the invention, in particular in the case of overexpression, and further to isolate it in larger quantities.

The amino sequences of human and murine neutral brain sphingomyelinase are represented as SEQ ID NOS: 1 and 2 (SEQ.ID.NOS.1 and 2).

The molecular weights of human and murine sphingomyelinase 2 are about 71 k each.
It is Da. Compared to bacterial nSMases, the nSMase2 sequences of the invention do not contain a signal sequence at the N-terminus. From the hydrophobicity analysis, the two adjacent N-terminals
The two hydrophobic membrane domains may be considered separated by 35 amino acids. Thus, the protein appears to be a complete membrane protein with its catalytically active domain directed to the cytosol, while only a few enzymes appear to be in contact with the extracellular environment. This is in contrast to bacterial nSMase, a secreted soluble protein, which is consistent with previous work on the properties of mammalian neutral sphingomyelinase. However, the ubiquitous eukaryotic sphingomyelinase, nSMase1, also contains two hydrophobic membrane domains located at the C-terminus of the protein.

By Northern blot analysis, 6 kb mRNA of human nSMase2 is expressed mainly in the brain. In the liver and small intestine, Northern blots show even weaker signals, while cross-hybridizing (3.5 kB) of different sizes.
bridizing mRNA is expressed in the thymus.

The optimum pH of the neutral brain sphingomyelinase according to the present invention is in the range of 6-8. The activity depends on the presence of magnesium ions; when EDTA is added, nS
The Mase activity is inhibited, but it can be recovered by adding Mn ²⁺ or Mg ²⁺ ions. Activity is unaffected by treatment with DTT or 2-mercaptoethanol.

Variants of eukaryotic neutral brain sphingomyelinase are also claimed. The term "variant" refers to the naturally occurring form of the eukaryotic neutral brain sphingomyelinase.
Includes both allelic variants and proteins prepared by recombinant DNA technology whose expression subsequently occurs (especially by in vitro mutagenesis using chemically synthesized oligonucleotides), which proteins are biological and / or In terms of immunological activity, it corresponds to a eukaryotic neutral sphingomyelinase. This may include amino acid deletions, insertions or conservative substitutions.

By “conservative substitution” is meant that an amino acid is replaced by another amino acid with similar physicochemical properties.

Thus, for example, the following amino acids can be replaced with each other: serine and alanine; alanine and glycine; methionine and serine; lysine and arginine; lysine and serine.

In particular, the term “variant” also includes N-terminal and / or C-truncated proteins, as well as acetylated, glycosylated, amidated and / or phosphorylated derivatives. Compounds in which nSMase2 or variants thereof are linked to other molecules such as dyes, radionuclides or affinity moieties are also variants according to the invention.

Nucleic acids encoding eukaryotic neutral brain sphingomyelinase or nucleic acids complementary to such nucleic acids are also claimed. Nucleic acid is, for example, DNA, RNA, P
It can also be NA or a nuclease resistant analogue thereof. In particular, nuclease resistant analogs include those compounds that have a phosphodiester bond modified with a hydrolytically stable compound such as phosphothioates, methylphosphonates.

Particularly short nucleic acid fragments are suitable as antisense nucleotides.
They should preferably contain more than 6, more preferably more than 8 and most preferably more than 12 nucleotides for specificity. Diffusion
) And due to cost, their length is usually less than 30 nucleotides,
It is preferably 24 or less, and more preferably 18 or less nucleotides.

The present invention relates to derivatives of nucleic acids linked to other molecules, such as fluorescent dyes, radiolabels or affinity compounds, and fragments of the nucleic acids according to the invention, and to these nucleic acids for diagnostic or therapeutic purposes. It also relates to nucleic acids that are complementary, and variants of those nucleic acids.

As used herein, “fragment” means a nucleic acid that is truncated at the 5 ′ or 3 ′ or both ends. The term "variant" means that under stringent conditions these nucleic acids will hybridize with the nucleic acid according to the invention, or a nucleic acid complementary thereto. The term "stringent conditions" refers to hybridization under conditions (otherwise identical conditions) at temperatures up to 10 ° C lower than just sufficiently low to anneal nucleic acids that are exactly complementary. Is carried out. For example, if nucleic acids that are exactly complementary are annealed at a temperature of up to about 55 ° C. under the given conditions, then stringent conditions are temperatures above 45 ° C. The temperature range of stringent conditions is preferably within 5 ° C, more preferably within 3 ° C.

The invention further relates to an antibody directed to the nSMase2 according to the invention or the nucleic acid according to the invention. These substances are especially useful as agents for use in diagnostics, immunoassays known per se to the person skilled in the art, for histological studies and for treating conditions associated with overexpression of nSMase. Are suitable. 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, the nucleic acids or peptides according to the invention and the nucleic acid fragments in the presence of an adjuvant.

The invention further relates to a cell line which overexpresses nSMase2 according to the invention. Such cell lines can be obtained by transfection with a vector containing the nucleic acid according to the invention, which encodes nSMase. In the case of eukaryotic cell lines, for example, transfection may be electroporation.
ration). Preferably the cell line is stably transfected.

In this context, “overexpression” means that the cell line has a higher nSMase activity than a cell line which has not been transfected with the nucleic acid according to the invention. For example, suitable eukaryotic cell lines include the cell lines U937, HEK293 or Jukat.

In the experiment, the cell line was 0.1-10 μmol / mg protein per hour.
The specific nSMase activity was shown.

FIG. 1 shows Northern and Western blot analysis of nSMase expression in human tissues. Part A shows the results of Northern blot hybridization with nSMase2 probe. Part B shows the result of Northern blot hybridization using a probe specific for actin as a control. Part C shows a Western blot with an antibody specific for nSMase2 prepared by recombinant technology.

[0030]   2 to 5 show an arrangement according to the invention.

HEK2 overexpressing nSMase2 upon addition of 0.5 mM arachidonic acid
NSMase activity in the extract of 93 cells increased 3-fold. Addition of phosphatidylserine increased nSMase activity 6-fold in extracts of HEK293 cells overexpressing nSMase2.

The invention further relates to a transgenic mammal exhibiting overexpression (gain of function) or a genetic defect or deficiency (loss of function) for the nSMase2 according to the invention. The mammal is preferably a rodent, especially a mouse. Such transgenic mammals can themselves be obtained by methods known to those skilled in the art and are particularly suitable for elucidating the function of neutral sphingomyelinase. In the case of transgenic mammals, certain gene constructs are injected into the pronucleus of single-cell stage fertilized egg cells by DNA microinjection to express additional genes. Next, by selectively changing the gene in the genome of ES cells injected into the blastocyst, the function of the gene is stopped (swi
tched off).

The transgenic animal is preferably an animal in which the gene can be temporarily switched on and off by a tissue-specific method by external induction. Such transgenic mammals are particularly suitable for elucidating the metabolic and signal transduction pathways associated with nSMase according to the present invention; which may be used in turn, diagnostic or therapeutic. it can. In particular, transgenic mammals are suitable for screening pharmaceutically active substances.

The eukaryotic neutral sphingomyelinase according to the present invention, the nucleic acid according to the present invention and the antibody according to the present invention can be included in drugs and diagnostic agents, optionally together with further adjuvants. Such agents and diagnostic agents are diagnostics for diseases based on overexpression or underexpression, and / or elevated or decreased eukaryotic neutral sphingomyelinase activity, and / or cell proliferation, impaired cell differentiation, and / or apoptosis. And suitable for treatment.

In particular, these are diseases involving inflammatory processes, impaired cell growth and metabolic disorders. For example, they may be cancer or cholesterol homeostasis (atherosclerosis).

The pharmaceutical screening method according to the invention relies on a change in the expression or activity of nSMase2 according to the invention in a cell line which overexpresses nSMase2 when at least one potential pharmaceutically active substance is added. The cell line is thus particularly suitable for developing and testing pharmaceutically important structures.

[0037]

【Example】

The invention is further described by the following examples. Example 1 Nucleic Acid Cloning The inventive nucleic acid encoding the neutral brain sphingomyelinase was cloned into the NotI restriction site of the cloning site of the eukaryotic expression vector pRc / CMV (Stratagene). The sequence of the resulting DNA was obtained by sequencing using a Perkin-Elmer DNA sequencer 377A.

Example 2 RNA Cloning Total RNA was isolated from different organs of eight 3 week old CD1 mice by known methods and oligo (dT) cellulose (Boehringer Mannheim (Boehringer) was used by standard methods. Poly (A ⁺ ) RNA was isolated by affinity purification in Mannheim), Germany).

Example 3 Overexpression of Cell Lines 10% fetal bovine serum, 1 μg / ml penicillin / streptomycin and 1 m
HEK in DMEM medium containing M-pyruvate at 37 ° C. and 5% CO ₂ .
293 cells were cultured. 5 × 10 ⁶ cells were electroporated with Gene Pulser (Bio-Rad) using 1 μg of linearized plasmid DNA encoding the nSMase according to the invention. Transfected. Selection of stable clones was carried out with 1 mg / ml of geneticin (G418, Life Technologies (L
if Technologies, Gaithersburg, MD
ersburg, MD)).

NSMase purified from the cell line was 0.1 mg / mg protein in 1 hour.
It showed a specific activity of 2-1 μmol. Its optimum pH was 6-8. The activity was dependent on the presence of magnesium ions; the addition of EDTA inhibited the activity.

Example 4 Measurement of nSMase activity Enzyme activity in cell and murine tissues was tested. Cells were washed twice with ice cold PBS and pelleted at 1,000 xg. The pellet was resuspended in lysis buffer and cells were disrupted by repeated freeze and thaw cycles. After centrifugation at 2,500 xg for 2 minutes, extraction was performed with a lysis buffer containing 0.2% Triton X-100, and then centrifugation at 100,000 xg for 15 minutes. It was

Tissues from 3 week old mice were homogenized in cold lysis buffer. The protein to be tested or homogenized tissue was treated with 10 nM (80,000 dpm) [N- ¹⁴ C
H ₃ ] sphingomyelin was incubated for 30 minutes at 37 ° C. in a total volume of 200 μl. Then add 100 μl of water and add chloroform-methanol (
Unreacted substrate was removed by extraction with 2: 1, v / v). The radioactivity of the aqueous phase containing the phosphocholine released by the enzyme was measured with a scintillation counter.

[Sequence list]

[Brief description of drawings]

FIG. 1 shows the results of Northern and Western blots obtained from various human tissues.

FIG. 2 shows a sequence according to the invention.

FIG. 3 shows a sequence according to the invention.

FIG. 4 shows a sequence according to the invention.

FIG. 5 shows a sequence according to the invention.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] November 19, 2001 (2001.11.19)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

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Claims (24)

[Claims] 1. Sequence motif X₁-X₂-X₃-X_Four-D-Y-X_FiveAnd X₆-X₇-T-
D-H-X₈(X here₁, X₆= A or G; X₂, X₃= R or K; X_Four, X_Five, X₇, X ₈ = I or L or V or M), eukaryotic neutral brain sphingomyelinase (
A nucleic acid encoding nSMase2). 2. Nucleic acid according to claim 1, characterized in that it encodes a mammalian neutral sphingomyelinase, in particular a murine or human brain sphingomyelinase. 3. The nucleic acid according to claim 1, wherein the neutral sphingomyelinase cleaves sphingomyelin into ceramide and phosphocholine, and its activity depends on the addition of magnesium ion. 4. A sequence according to SEQ ID NO: 3 or SEQ ID NO: 4.
3. The nucleic acid according to at least one of 3 to 3. 5. Nucleic acid according to at least one of claims 1 to 4, characterized in that it is DNA, RNA, PNA or a nuclease-resistant analogue thereof. 6. Nucleic acid according to at least one of claims 1 to 5, characterized in that it is mRNA, cDNA or genomic DNA. 7. A nucleic acid, which is complementary to the nucleic acid according to at least one of claims 1 to 6. 8. A eukaryotic neutral brain sphingomyelinase obtainable by expression of the nucleic acid according to claims 1 to 7, in particular having a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2. 9. An antibody which is directed against the eukaryotic neutral brain sphingomyelinase according to claim 8 or the nucleic acid according to at least one of claims 1 to 7. 10. A cell line characterized by overexpressing the eukaryotic neutral brain sphingomyelinase according to claim 8. 11. Cell line according to claim 10, characterized in that it expresses a eukaryotic neutral brain sphingomyelinase and is based on the cell line U937, HEK293 or Jurkat. 12. A transgenic mammal exhibiting overexpression (gain of function) or genetic defect or deficiency (loss of function) with respect to eukaryotic neutral brain sphingomyelinase. 13. The transgenic mammal according to claim 12, which is a rodent. 14. A 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 a further auxiliary agent. Drug. 15. A eukaryotic neutral brain sphingomyelinase according to claim 8, the nucleic acid according to at least one of claims 1 to 7 and / or the antibody according to claim 9 together with a further auxiliary agent. Diagnostic agent. 16. In the diagnosis and treatment of diseases based on overexpression or underexpression, and / or increased or decreased activity of eukaryotic neutral brain sphingomyelinase, and / or cell proliferation, impaired cell differentiation, and / or apoptosis. ,
Use of the agent according to claim 14 or the diagnostic agent according to claim 15. 17. Use according to claim 16, characterized in that the disease is an inflammatory process, impaired cell growth, cancer and / or metabolic disorders such as cholesterol homeostasis disorder (atherosclerosis). 18. A change in the expression or activity of eukaryotic neutral brain sphingomyelinase upon addition of at least one potential pharmaceutically active substance is measured with the cell line according to claim 10. Active substance screening method. 19. Use of the cell line according to claim 10 for the development and testing of pharmaceutically important structures. 20. A method of preparing a eukaryotic neutral brain sphingomyelinase according to claim 8 by expression in a genetically engineered organism, in particular in a eukaryotic expression system, or by chemical peptide synthesis. 21. A method for preparing a nucleic acid according to at least one of claims 1 to 7 by amplification in a genetically engineered organism or by chemical synthesis. 22. A gene for eukaryotic neutral brain sphingomyelinase, which comprises a non-coding region (intron) in addition to a coding region (exon), and at least one of claims 1 to 7. The nucleic acid according to item 1. 23. A variant of the eukaryotic neutral brain sphingomyelinase according to claim 8. 24. Nucleic acid according to at least one of claims 1 to 7 or claim 22, characterized in that it is a derivative, fragment or variant of such a nucleic acid.