JP2014117191A - Phosphorylated neutral sphingomyelinase 1, antibody against phosphorylated neutral sphingomyelinase 1, mutant of neutral sphingomyelinase 1 and use thereof - Google Patents

Abstract

To elucidate the regulation mechanism of stress-induced apoptosis by neutral sphingomyelinase 1 and to provide an analysis tool thereof.
A phosphorylated neutral sphingomyelinase 1 polypeptide in which a serine residue that is a phosphorylation target by JN kinase is phosphorylated, or the phosphorylated serine residue, and has a length of 10 amino acids or more. Its partial polypeptide. An antibody that specifically recognizes the phosphorylated neutral sphingomyelinase 1 polypeptide. A neutral sphingomyelinase 1 mutant polypeptide in which a serine residue that is a phosphorylation target by JN kinase is substituted with an amino acid residue other than serine.
[Selection figure] None

Description

  The present invention relates to phosphorylated neutral sphingomyelinase 1, an antibody that specifically recognizes phosphorylated neutral sphingomyelinase 1, a method for detecting phosphorylated neutral sphingomyelinase 1, and the like. The present invention also provides a neutral sphingomyelinase 1 mutant in which a serine residue to be phosphorylated is substituted with another amino acid residue, a polynucleotide encoding the mutant, an expression vector comprising the polynucleotide, The present invention relates to a transformant containing an expression vector and uses thereof.

Neutral sphingomyelinase 1 is an enzyme that hydrolyzes sphingomyelin under neutral conditions in a Mg ²⁺ dependent manner to produce ceramide (Non-Patent Documents 1 to 4). Sphingomyelinase has been reported to have a large number of isozymes with different extracellular localization derived from microorganisms and intracellular localization of animal cells. Among these, five types of isozyme genes having gene numbers SMPD1 to SMPD5 have been reported in mammals and zebrafish (Non-Patent Documents 1 to 4). SMPD1 encodes acidic sphingomyelinase (P17405) derived from lysosomes, and SMPD2-5 encodes neutral sphingomyelinase, but these sphingomyelinases are different in their intracellular localization. Roles in the body are thought to be different. From the study using fish cells, neutral sphingomyelin phosphodiesterase 2, gene name SMPD2 is activated as a sphingomyelinase that is activated under stress conditions to release ceramide from sphingomyelin in the cell membrane and induce apoptosis. ) Was discovered (Non-Patent Documents 4 to 7). The mammalian sphingomyelinase corresponding to this has been isolated and identified from human and mouse gene sequences, but whether or not these mammalian neutral sphingomyelinase 1 is involved in stress-induced apoptosis is sufficient. Analysis has not been made, and activity regulation by phosphorylation is not known (Patent Document 1, Non-Patent Document 8). Currently, research in the field of cell biology activates this neutral sphingomyelinase 1 in response to growth factors, cytokines, chemotherapeutic drugs, irradiation, starvation, stress, etc., promotes ceramide synthesis, and induces apoptosis. Induction has been reported in many animal cells (Non-Patent Documents 5 to 7). However, it has been confirmed that neutral sphingomyelinase 1 triggers stress-induced apoptosis only from zebrafish cells, and apoptosis is induced by activation of neutral sphingomyelinase 1 in mammalian cells. Whether it is done is still unclear. In addition, the molecular mechanism by which neutral sphingomyelinase 1 is activated by stress stimulation to produce ceramide is unknown. Since the biological response in which neutral sphingomyelinase 1 is phosphorylated is not known, the presence of phosphorylated neutral sphingomyelinase 1 is not known, and its detection technique, antibody against the phosphorylated enzyme Has not been made so far.

International Publication No. 99/07855

Hofman, K et al., Proc. Natl. Acad. Sci. USA 2000; 97: 5895-5900. Krut O et al., J. Biol. Chem. 2006; 281: 13784-13793. Tomiuk S et al., Proc. Natl. Acad. Sci. USA 1998; 95: 3638-3643. Yabu T et al., J. Biol. Chem. 2008; 283: 29971-29982. Yabu T et al., Blood 2005; 106: 125-134. Yabu T et al., Fish. Sci. 2003; 69: 1218-1223. Kenji Tsuji, Noriaki Yamashita, Research Report of Fisheries Research Center 2008; 26: 15-21. Sawai H et al., J. Biol. Chem. 1999; 274: 38131-38139.

  An object of the present invention is to clarify the regulation mechanism of stress-induced apoptosis by neutral sphingomyelinase 1 and to provide an analysis tool therefor.

  As a result of intensive studies in view of the above object, the present inventors have found that neutral sphingomyelinase 1 is specifically phosphorylated by c-jun N-terminal kinase (JN kinase, JNK). The chemical reaction to be activated was found, and it was revealed that the specific phosphorylation site is a serine residue (Ser-270). By using a peptide having a specific amino acid sequence located in the vicinity of the phosphorylation site of sphingomyelinase 1 as an antigen, an antibody that specifically reacts with active neutral sphingomyelinase 1 was successfully produced. . Furthermore, by substituting Ser-270 with other amino acids, we succeeded in creating a dominant negative neutral sphingomyelinase 1 mutant and a permanently activated neutral sphingomyelinase 1 mutant. . The present inventors have further studied based on these findings and completed the present invention.

That is, the present invention relates to the following.
[1] The following polypeptide (1) or (2):
(1) a phosphorylated neutral sphingomyelinase 1 polypeptide in which a serine residue that is a phosphorylation target by JN kinase is phosphorylated, and (2) a partial amino acid sequence of the polypeptide of (1), A polypeptide comprising a serine residue which is a target for phosphorylation by a kinase, comprising a partial amino acid sequence having a length of 10 amino acids or more, and wherein the serine residue is phosphorylated.
[2] The polypeptide of [1], wherein the phosphorylated neutral sphingomyelinase 1 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 1, 2, or 3.
[3] A method for producing the polypeptide of [1], comprising phosphorylating the following polypeptide (1 ′) or (2 ′) with JN kinase:
(1 ′) Neutral sphingomyelinase 1 polypeptide and (2 ′) (1 ′) partial amino acid sequence comprising serine residue that is a phosphorylation target by JN kinase, and 10 amino acids A polypeptide comprising a partial amino acid sequence having the above length.
[4] An antibody that specifically recognizes the polypeptide of [1] or [2].
[5] A reagent for detecting phosphorylated neutral sphingomyelinase 1 polypeptide, comprising the antibody of [4].
[6] A method for detecting a phosphorylated neutral sphingomyelinase 1 polypeptide, comprising using the antibody of [4].
[7] A method for evaluating the activation state of a stress-induced apoptosis signal in a cell, comprising detecting the polypeptide of [1] in the cell.
[8] A neutral sphingomyelinase 1 mutant polypeptide in which a serine residue which is a phosphorylation target by JN kinase is substituted with an amino acid residue other than serine.
[9] The mutant polypeptide of [8], wherein the serine residue is substituted with an alanine residue.
[10] The mutant polypeptide of [8], wherein the serine residue is substituted with a glutamic acid residue.
[11] A polynucleotide encoding the mutant polypeptide according to any one of [8] to [10].
[12] An expression vector comprising the polynucleotide of [11].
[13] A transformant comprising the expression vector according to [12].
[14] A method for inhibiting stress-induced apoptosis of a cell, comprising introducing the mutant polypeptide of [9] or an expression vector thereof into the cell.
[15] A method for promoting stress-induced apoptosis of a cell, comprising introducing the mutant polypeptide of [10] or an expression vector thereof into the cell.

  By using phosphorylated neutral sphingomyelinase 1, anti-phosphorylated neutral sphingomyelinase 1 antibody and neutral sphingomyelinase mutant of the present invention, detailed mechanism analysis of the regulation mechanism of stress-induced apoptosis is possible. Become.

It is the result of having carried out the computer analysis of the amino acid sequence of neutral sphingomyelinase 1 polypeptide. It is the result of having analyzed the specificity of anti-phosphorylated neutral sphingomyelinase 1 antibody by ELISA. It is the result of detecting phosphorylated neutral sphingomyelinase 1 in human-derived cultured cell and flounder-derived cultured cell extracts by Western blot using an anti-phosphorylated neutral sphingomyelinase 1 antibody. This is a result of detecting phosphorylation of zebrafish neutral sphingomyelinase 1 by JN kinase by Western blotting using anti-phosphorylated neutral sphingomyelinase 1 antibody. Induction of phosphorylation of neutral sphingomyelinase 1 by stress on cultured zebrafish cells. Neutral sphingomyelinase 1 was phosphorylated by heat shock and ultraviolet irradiation (Example 5). Induction of apoptosis in zebrafish cultured cells by introducing an expression vector of wild-type neutral sphingomyelinase 1 or neutral sphingomyelinase 1 mutant. Detection of phosphorylated neutral sphingomyelinase 1 in human-derived cultured cells with anti-phosphorylated neutral sphingomyelinase 1 antibody.

1. Phosphorylated neutral sphingomyelinase 1 polypeptide
(1) A phosphorylated neutral sphingomyelinase 1 polypeptide (phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention) in which a serine residue that is a phosphorylation target by JN kinase is phosphorylated, or (2) A partial amino acid sequence of the polypeptide of (1), comprising a serine residue that is a phosphorylation target by JN kinase, and comprising a partial amino acid sequence having a length of 10 amino acids or more, and the serine residue is phosphorous Oxidized polypeptide (phosphorylated partial polypeptide of the present invention)
I will provide a. The above (1) and (2) may be collectively referred to as the phosphorylated polypeptide of the present invention.

  In a preferred embodiment, the phosphorylated polypeptide of the present invention can induce specific antibody production against the phosphorylated polypeptide when administered to a non-human mammal (eg, rabbit). The epitope of the specific antibody includes a phosphorylated serine residue that is a target for phosphorylation by JN kinase.

Neutral sphingomyelinase 1 is a known enzyme that hydrolyzes sphingomyelin under neutral conditions to produce ceramide in a Mg ²⁺ dependent manner, and is also called neutral sphingomielin phosphodiesterase 2 (SMPD2).

  Herein, the neutral sphingomyelinase 1 polypeptide may be derived from any eukaryotic organism. Examples of eukaryotes include mammals, birds, reptiles, amphibians, fish, insects, yeasts, etc., but mammals or fish are preferred. Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. The mammal is preferably a laboratory animal (eg, a mouse) or a primate (eg, a human). Although it does not specifically limit as fish, For example, as an edible, the fish species (for example, flounder fish (flounder etc.), flounder, flounder, turbot etc.), Thai fish (red sea bream, sea bream etc.) ), Yellowtail, mullet, ayame, tuna, tilapia, salmon, carp, trout, rainbow trout, yamame, amago, eel and the like. Appreciation applications include carp, funa, medaka, goldfish and the like. Furthermore, zebrafish, medaka, goldfish, loach, etc. are mentioned as research or experiment use. The fish is preferably for research or experiment, preferably zebrafish.

  In the present specification, for a polypeptide, “organism X (derived) polypeptide Y” refers to the amino acid sequence of the polypeptide Y that is naturally expressed in the organism X (wild-type amino acid sequence). ) Or the same or substantially the same amino acid sequence. Similarly, in the present specification, for a gene (polynucleotide), “organism X (derived) gene Z” means that the nucleotide sequence of gene Z (preferably a cDNA sequence) is naturally expressed in organism X. It means having the same or substantially the same nucleotide sequence as the nucleotide sequence of the gene (wild type nucleotide sequence) (preferably a cDNA sequence). “Substantially the same” means that the focused amino acid sequence or nucleotide sequence is 70% or more (preferably 80% or more, preferably) of the amino acid sequence or nucleotide sequence of a factor (wild type factor) naturally expressed in the organism X More preferably 90% or more, still more preferably 95% or more, most preferably 99% or more), and the function (activity) of the protein or gene is maintained.

  As used herein, “identity” of amino acid sequences refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm is an optimal alignment). Is the ratio of the same amino acid residue to the total overlapping amino acid residues in which one may consider the introduction of a gap into one or both of the sequences.

  The identity of amino acid sequences in the present specification can be determined by, for example, using the homology calculation algorithm NCBI blastp / Blast 2 sequences (National Center for Biotechnology Information Basic Local Alignment Search Tool) published on the NCBI Internet homepage as follows: It can be calculated by the conditions (Short queries = off / Expect threshold = 10 / Matrix = BLOSUM62 / Gap Costs = Existence: 11 Extension: 1 / Compositional adjustments = Conditional compositional score matrix adjustment / filter = off / Mask = off) .

The activity of neutral sphingomyelinase 1 (ie, the activity of hydrolyzing sphingomyelin under neutral conditions to produce ceramide in a Mg ²⁺ -dependent manner) can be achieved under neutral conditions (for example, by the method described in WO 99/07855) In Example, pH 7.0-7.5), the polypeptide to be evaluated was incubated with [N- ¹⁴ CH ₃ ] sphingomyelin at 37 ° C. for 30 minutes, and the unreacted substrate was chloroform-methanol (2: 1. v / v) It can be evaluated by measuring the radioactivity of an aqueous phase containing choline phosphate produced enzymatically by removing it by extraction and measuring with a scintillation counter. In addition, fluorescent C ₆ -NBD sphingomyelin was used as a substrate and incubated with a polypeptide to be evaluated at 37 ° C. for 30 minutes, and then C ₆ -NBD ceramide produced by hydrolysis was converted into chloroform-methanol (2 : 1, v / v) concentrated and recovered by extraction, separated by thin phase chromatography (developing solvent chloroform: methanol: 12 mM MgCl ₂ aqueous solution, 65: 25: 4) and neutralized by measuring fluorescence The activity of sphingomyelinase 1 can also be analyzed (Non-patent Document 4).

  For many eukaryotes, the amino acid sequence of neutral sphingomyelinase 1 is known. For example, a representative amino acid sequence of zebrafish neutral sphingomyelinase 1 is registered as GenBank accession number NP_001025390.1 (updated on August 25, 2012) (SEQ ID NO: 1). A typical amino acid sequence of human neutral sphingomyelinase 1 is registered as GenBank accession number NP_003071.2 (updated on November 11, 2012) (SEQ ID NO: 2). A typical amino acid sequence of mouse neutral sphingomyelinase 1 is registered as GenBank accession number NP_033239.1 (updated on June 28, 2012) (SEQ ID NO: 3).

  The phosphorylated polypeptide of the present invention is characterized in that a serine residue which is a phosphorylation target by JN kinase in neutral sphingomyelinase 1 is phosphorylated.

  JN kinase (c-Jun N-terminal kinase) is a well-known kinase and is also called MAPK (mitogen-activated protein kinase) -8 to 10 or stress activated protein kinase (SAPK). The JN kinase is preferably JNK1. As used herein, JN kinase can be derived from any eukaryotic organism. Examples of eukaryotes include those listed above. The eukaryote is preferably a fish (eg, zebrafish) or a mammal (eg, human, mouse). For many eukaryotes, the amino acid sequence of JN kinase is known. For example, a representative amino acid sequence of zebrafish JN kinase is registered as GenBank accession number NP_001103859.1 (updated on August 27, 2012). A representative amino acid sequence of human JN kinase is registered as GenBank accession number NP_620637 (updated on November 25, 2012). A representative amino acid sequence of mouse JN kinase is registered as GenBank accession number NP_057909 (updated on November 24, 2012).

  In one embodiment, the animal species from which the neutral sphingomyelinase 1 polypeptide is derived and the animal species from which the JN kinase is derived are the same. For example, when the phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention is derived from human, the phosphorylated serine residue contained in the polypeptide is a phosphorylation target by human JN kinase.

  In another embodiment, the animal species from which JN kinase is derived is human.

The serine residue, which is a phosphorylation target by JN kinase, is obtained by incubating neutral sphingomyelinase 1 polypeptide with JN kinase in the presence of the required substrate (eg, ATP) to leave phosphorylated serine or threonine residues. The group can be identified by mass spectrometry analysis or by analyzing a radiolabeled serine or threonine residue using [γ- ³² P] -labeled ATP. In addition, in zebrafish neutral sphingomyelinase 1 polypeptide (SEQ ID NO: 1), human neutral sphingomyelinase 1 polypeptide (SEQ ID NO: 2) and mouse neutral sphingomyelinase 1 polypeptide (SEQ ID NO: 3), In any case, the 270th serine residue (Ser270) corresponds to a serine residue which is a phosphorylation target by JN kinase. Therefore, more simply, the amino acid sequence of the neutral sphingomyelinase 1 polypeptide to be analyzed is aligned with SEQ ID NOs: 1, 2 and 3 using sequence analysis software, and the serine residue corresponding to Ser270 is defined as JN. It can be specified as a serine residue which is a phosphorylation target by a kinase.

In the phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention, the serine residue which is a phosphorylation target by JN kinase is phosphorylated, and the activity of the neutral sphingomyelinase 1 (ie, Mg ²⁺ dependently depends on the intermediate). The amino acid sequence of wild-type neutral sphingomyelinase 1 has a deletion, substitution and / or addition of one or more amino acid residues as long as it has the activity of hydrolyzing sphingomyelin under sexual conditions to produce ceramide) It may be. The number of amino acid residues to be deleted, substituted or added is not limited as long as the activity of neutral sphingomyelinase 1 is maintained. For example, about 1 to 30, preferably about 1 to 20, more Preferably about 1-10, even more preferably about 1-5, most preferably 1 or 2. The site where amino acid residues are deleted, substituted or added is not particularly limited as long as the polypeptide maintains the activity of neutral sphingomyelinase 1.

In a preferred embodiment, the phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention is neutralized depending on its activity (ie, Mg ²⁺ -dependent neutralization) by serine residue phosphorylation target by JN kinase being phosphorylated. The activity to hydrolyze sphingomyelin under conditions to produce ceramide) is higher compared to the non-phosphorylated control neutral sphingomyelinase 1 polypeptide.

  The phosphorylated partial polypeptide of the present invention includes a partial amino acid sequence of the polypeptide of (1) above, which includes a partial amino acid sequence including a serine residue that is a phosphorylation target by JN kinase.

  When the phosphorylated partial polypeptide of the present invention is administered to a non-human mammal, the length of the partial amino acid sequence is determined based on a specific antibody against the phosphorylated partial polypeptide (phosphorylated target by JN kinase). Is not particularly limited as long as it induces the production of a serine residue that is an epitope), but is usually 10 amino acids or more, preferably 12 amino acids or more. The upper limit of the length of the partial amino acid sequence is not particularly limited as long as it does not exceed the full length amino acid sequence of neutral sphingomyelinase 1, but if the partial amino acid sequence is too long, it is caused by JN kinase phosphorylated to an epitope. Since the efficiency of inducing an antibody containing a serine residue that is a phosphorylated target is decreased, the length of the partial amino acid sequence is usually 30 amino acids or less, preferably 20 amino acids or less, more preferably 15 amino acids or less.

  The site of the partial amino acid sequence contains a serine residue that is a phosphorylation target by JN kinase, and is specific to the phosphorylated partial polypeptide when the phosphorylated partial polypeptide of the present invention is administered to a non-human mammal. As long as it induces the production of a target antibody (containing a serine residue that is a phosphorylated target of phosphorylation by JN kinase as an epitope). Preferably, the site of the partial amino acid sequence is set so that the serine residue which is a phosphorylation target by JN kinase does not become the N-terminal or C-terminal of the partial amino acid sequence. In one embodiment, the partial amino acid sequence includes a contiguous sequence of 3 amino acids or more, preferably 4 amino acids or more on the N-terminal side of the serine residue that is a phosphorylation target by JN kinase. In one embodiment, the partial amino acid sequence includes a contiguous sequence of 6 amino acids or more, preferably 7 amino acids or more on the C-terminal side of a serine residue that is a phosphorylation target by JN kinase. In a preferred embodiment, the partial amino acid sequence includes a contiguous sequence of 3 amino acids or more, preferably 4 amino acids or more on the N-terminal side of the serine residue that is a phosphorylation target by JN kinase, and C of the serine residue. An adjacent sequence of 6 amino acids or more, preferably 7 amino acids or more is included on the terminal side.

  In one embodiment, the phosphorylated partial polypeptide of the present invention comprises the amino acid sequence represented by SEQ ID NO: 4, and the serine residue contained in the amino acid sequence is phosphorylated. The amino acid sequence represented by SEQ ID NO: 4 is a partial amino acid sequence of zebrafish neutral sphingomyelinase 1.

  When the full length of the phosphorylated partial polypeptide of the present invention is administered to a non-human mammal, a specific antibody against the phosphorylated partial polypeptide (phosphorylated serine residue which is a target for phosphorylation by JN kinase) There is no particular limitation as long as it induces the production of (including the epitope). However, when the phosphorylated partial polypeptide is too long, the efficiency of inducing an antibody containing a serine residue that is phosphorylated by an epitope and phosphorylated by JN kinase is reduced. The total length of the partial polypeptide is preferably 30 amino acids or less, more preferably 20 amino acids or less, and even more preferably 15 amino acids or less.

  The phosphorylated polypeptide of the present invention has one or two or more (for example, 1 to 500) in addition to the amino acid sequence that is the same as or substantially the same as the amino acid sequence of wild-type neutral sphingomyelinase 1, or a partial sequence thereof. May be included, preferably 1-100, more preferably 1-15, most preferably 1 or 2). The amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of the polypeptide. As tags, Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (for example, GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. In addition, examples of added amino acids include amino acids that mediate conjugation with other proteins. Examples of the amino acid include cysteine.

  The polypeptide of the present invention may be further modified in addition to phosphorylation of a serine residue that is a phosphorylation target by JN kinase. Examples of such modifications include lipid chain addition (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.

The phosphorylated polypeptide of the present invention contains an appropriate labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase). , Peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin) Etc.).

  The phosphorylated polypeptide of the present invention includes a salt form thereof. As the salt, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal salt) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The phosphorylated polypeptide of the present invention is preferably isolated or purified. “Isolation or purification” means that an operation to remove factors other than the target component has been performed, and the state existing in nature has been removed. The purity of “isolated or purified phosphorylated polypeptide” (percentage of the desired phosphorylated polypeptide in the total polypeptide weight) is usually 70% or more, preferably 80% or more, more preferably 90%. As mentioned above, it is most preferably substantially 100%.

  There is no restriction | limiting in particular about the manufacturing method of the phosphorylated polypeptide of this invention, You may manufacture according to a well-known peptide synthesis method, and you may manufacture using a well-known gene recombination technique. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.

In one aspect, the phosphorylated polypeptide of the present invention comprises
A partial amino acid sequence of (1 ′) neutral sphingomyelinase 1 polypeptide or (2 ′) (1 ′) polypeptide, comprising a serine residue that is a phosphorylation target by JN kinase, and 10 amino acids A polypeptide containing a partial amino acid sequence having the above length can be produced by phosphorylation with JN kinase. The present invention also provides a method for producing such a phosphorylated polypeptide of the present invention.

  Methods for in vitro phosphorylation of polypeptides by JN kinase are known (see, for example, Cell, vol. 76, no. 6, pages 1025-1037, 1994), for example, as described in (1 ′) or (2 ′) above. By incubating the polypeptide (preferably isolated or purified) with JN kinase (preferably isolated or purified) in the presence of the required substrate (eg ATP) The serine residue which is a phosphorylation target by JN kinase contained in the polypeptide is phosphorylated.

  The isolation or purification of the phosphorylated polypeptide of the present invention from the reaction mixture can be carried out by, for example, subjecting a cell lysate or culture supernatant to a plurality of chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. This can be achieved by subjecting to the following. Alternatively, the phosphorylated polypeptide of the present invention can be isolated or purified from the reaction mixture by an antibody column using an antibody (described later) that specifically recognizes phosphorylated neutral sphingomyelinase 1.

  The phosphorylated polypeptide of the present invention is an antigen for producing an antibody specifically recognizing a phosphorylated neutral sphingomyelinase 1 polypeptide in which a serine residue that is a phosphorylation target by JN kinase is phosphorylated. It is useful as an immunogen).

2. Antibody that specifically recognizes phosphorylated neutral sphingomyelinase 1 polypeptide and uses thereof The present invention provides an antibody that specifically recognizes the phosphorylated polypeptide of the present invention (the antibody of the present invention).

  “Specific recognition” of antigen X by an antibody means that the binding affinity of an antibody to antigen X in an antigen-antibody reaction is higher than the binding affinity of a non-specific antigen (eg, BSA).

  In a preferred embodiment, the epitope of the antibody of the present invention includes a phosphorylated serine residue that is a phosphorylated target of JN kinase. Therefore, in this embodiment, the binding affinity of the antibody of the present invention to the phosphorylated polypeptide of the present invention is determined by comparing the corresponding non-phosphorylated control polypeptide (the serine residue that is phosphorylated by JN kinase is phosphorylated). Except for the above, the binding affinity for the phosphorylated polypeptide of the present invention to be compared and a polypeptide having the same constitution) is higher.

In the present specification, antibodies include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb); and recombinant antibodies (for example, single-chain Fv fragments (scFv), bispecific, etc.) that can be produced using gene recombination techniques. -chimeric scFV (χ-scFv), tandem scFV (scFv) ₂ , bispecific- (scFv) ₂ , disulfide-linked scFv, disulfide-stabilized Fv fragments (dsFv), diabody, single-chain diabody (scDb), bivalent diabody, bispecific diabody, knob-into-hole stabilized diabody, disulfide-stabilized diabody, triabody, tetrabody, trispecific triabody, CL-dimerized scFv, CH1-CL-dimerized scFv, CH3-dimerized scFv, knob-into-hole CH3-dimerized scFv, CH3-dimerized bivalent diabody, Fc-dimerized scFv, Fab-scFv fusions, Ig-scFv fusions, leucine-zipper stabilized scFv dimers, helix-stabilized scFv dimers, 4 helix-bundle stabilized scFv tetramers, streptavidin-scFv, intrabody, Fab ' fragments, F (ab ') fragments, Fv fragments (Fv), key La antibodies, humanized antibodies, single chain antibodies, etc.), but human antibody, and the like that can be produced using human antibody-producing transgenic animal, and the like. As the antibody, a polyclonal antibody or a monoclonal antibody is preferably used from the viewpoint of ease of production. The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE.

Further, in the present invention, “antibody” is a concept including a binding fragment thereof. The antibody-binding fragment means a partial region of the above-mentioned antibody, specifically, for example, F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), scFv, dsFv (disulfide stabilized) Fv), dAb (single domain antibody), etc. (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996), antibody fragments produced by the Fab expression library, etc. Illustrated.

  The antibody of the present invention immunizes a non-human mammal with the phosphorylated polypeptide of the present invention or a conjugate of the phosphorylated polypeptide and a carrier protein, and specifically the phosphorylated polypeptide of the present invention. It can be produced by isolating the recognizing antibody.

  As the carrier protein, hemocyanin (keyhole limpet hemocyanin, hereinafter referred to as KLH), bovine serum albumin, egg albumin, and the like can be used, and KLH is preferably used. When a phosphorylated polypeptide having a cysteine added to the C-terminus is used as the phosphorylated polypeptide of the present invention, the phosphorylated peptide and the carrier protein are bound using antigenic acid-N-hydroxysuccinimide ester (MBS) as an antigen. This can be carried out by crosslinking the carrier protein to the thiol group of the cysteine residue added to the C-terminus of the peptide. In addition, there are a method of binding the phosphorylated polypeptide of the present invention and a carrier protein using other cross-linking agents such as glutaraldehyde and carbodiimide, and a method of enhancing antigenicity without using a carrier protein by multiple antigen peptides (MAP). Available.

  In the case of producing a monoclonal antibody, the phosphorylated polypeptide of the present invention or the above-mentioned conjugate is administered to a non-human mammal with itself or a carrier, a diluent, etc. Immunize non-human mammals. In administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be used in order to enhance antibody production ability. The administration is usually performed once every 2 to 6 weeks, and about 2 to 10 times in total. Examples of the non-human mammal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice, rats or chickens are preferably used.

  Selecting an individual with an antibody titer from an immunized mammal, collecting spleen or lymph nodes 2 to 5 days after the final immunization, and fusing the antibody-producing cells contained therein with myeloma cells Thus, a monoclonal antibody-producing hybridoma can be produced. The fusion operation can be performed according to known methods, for example, the method of Kohler and Milstein (Nature, 256, 495 (1975)) and known variations thereof. The monoclonal antibody-producing hybridoma is cultured according to a method known per se or a method analogous thereto. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine).

  Subsequently, a hybridoma producing a monoclonal antibody that specifically recognizes the phosphorylated polypeptide of the present invention is selected from the monoclonal antibody-producing hybridomas. Various methods can be used for the selection. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which the phosphorylated polypeptide of the present invention is adsorbed by ELISA, and then radioactive material is added. An anti-immunoglobulin antibody labeled with or an enzyme (horseradish peroxidase, alkaline phosphatase, etc.) (if the spleen cells used for cell fusion are derived from a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, For example, a method for detecting a monoclonal antibody bound to s. Thus, a hybridoma that produces an antibody that specifically recognizes the phosphorylated polypeptide of the present invention can be obtained.

  In order to select an antibody containing a serine residue as an epitope phosphorylated by JN kinase in the phosphorylated polypeptide of the present invention, an antigen to be adsorbed on a solid phase in the above ELISA is used as the antigen of the present invention. And a corresponding non-phosphorylated control polypeptide in parallel to produce a hybridoma that produces an antibody that has a higher binding affinity for the phosphorylated polypeptide of the invention than the control polypeptide. It is preferable to select.

  Subsequently, a monoclonal antibody that specifically recognizes the phosphorylated polypeptide of the present invention is isolated from the culture supernatant of the hybridoma or ascites obtained by transferring the hybridoma to a pristane-treated nude mouse. Monoclonal antibodies are isolated by methods known per se, such as immunoglobulin separation and purification methods (eg, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Adsorption / desorption method, ultracentrifugation method, gel filtration method, specific purification method in which only antibody is collected with an antigen-binding solid phase or active adsorbent such as protein A or protein G, and the antibody is obtained by dissociating the binding. Can do.

  When producing a polyclonal antibody specifically recognizing the phosphorylated polypeptide of the present invention, the phosphorylated polypeptide of the present invention or the above conjugate is itself or a carrier, diluted as in the case of the monoclonal antibody. It is administered together with an agent and the like, and a mammal is immunized with the conjugate. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and rabbits, sheep, goats and the like are preferably used.

  Polyclonal antibodies can be isolated from the serum, preferably ascites of mammals immunized by the above method, preferably from serum. Polyclonal antibodies can be isolated according to the same immunoglobulin separation and purification method as the above monoclonal antibody isolation. Preferably, the polyclonal antibody is purified by separating an antibody fraction that specifically recognizes the phosphorylated polypeptide of the present invention by affinity chromatography using a column to which the phosphorylated polypeptide of the present invention is bound. The

  Further, by treating the antibody fraction with a column to which a non-targeted non-phosphorylated control polypeptide is bound, the antibody fraction having cross-reactivity with the non-phosphorylated control polypeptide is removed. In the phosphorylated polypeptide of the present invention, a phosphorylated antibody containing a serine residue which is a phosphorylation target by JN kinase as an epitope can be obtained.

  Alternatively, the antibody fraction that is cross-reactive with the non-phosphorylated control polypeptide can be removed by first treating the antibody fraction in the antiserum with a column to which the non-phosphorylated control polypeptide is bound. Thereafter, affinity purification may be performed using a column to which the phosphorylated polypeptide of the present invention is bound.

  Using the antibody of the present invention, phosphorylated neutral sphingomyelinase 1 polypeptide can be specifically detected by immunological techniques. Therefore, the present invention provides a method for detecting phosphorylated neutral sphingomyelinase 1 polypeptide, comprising using the antibody of the present invention.

  More specifically, a sample containing the phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention is contacted with the antibody of the present invention, and the phosphorylated neutral sphingomyelinase 1 polypeptide and the antibody of the present invention are contacted. The antigen-antibody complex is formed, and the formed antigen-antibody complex is quantified to detect and quantify the phosphorylated neutral sphingomyelinase 1 polypeptide in the sample.

  Examples of immunochemical methods include flow cytometry analysis, radioisotope immunoassay (RIA method), ELISA method (Methods in Enzymol. 70: 419-439 (1980)), Western blotting, immunohistochemical staining, etc. However, it is not limited to these.

  As shown in Examples described later, stress (heat, radiation, ultraviolet rays, peroxide (hydrogen peroxide, etc.), FAS agonist (FASL, anti-FAS antibody, etc.), inflammatory cytokine (IL1β, IFNγ, etc.) When apoptosis is induced by starvation, chemotherapeutic drugs, etc., phosphorylation of neutral sphingomyelinase 1 by JN kinase is mediated as an important regulatory mechanism. That is, when a cell is stress-stimulated, the serine residue (in the case of zebrafish, human and mouse, Ser-270) which is a phosphorylation target of JN kinase in neutral sphingomyelinase 1 is phosphorylated by JN kinase. Oxidized to activate neutral sphingomyelinase 1 and induce apoptosis by hydrolyzing sphingomyelin to produce ceramide. Therefore, by detecting the phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention in the cell, the activation state of the stress-induced apoptosis signal in the cell can be evaluated.

  Detection of phosphorylated neutral sphingomyelinase 1 polypeptide in cells can be carried out by the method for detecting phosphorylated neutral sphingomyelinase 1 polypeptide of the present invention.

  As a result of the detection, an increase in phosphorylated neutral sphingomyelinase 1 polypeptide was detected when phosphorylated neutral sphingomyelinase 1 polypeptide was detected, or compared to control cells not subjected to stress stimulation. In some cases, it can be evaluated that the stress-induced apoptosis signal is activated in the cell. On the other hand, when phosphorylated neutral sphingomyelinase 1 polypeptide is not detected, or when phosphorylated neutral sphingomyelinase 1 polypeptide of the same level as that of control cells not subjected to stress stimulation is detected, It can be evaluated that the stress-induced apoptosis signal is not activated.

  The present invention also provides a phosphorylated neutral sphingomyelinase 1 polypeptide detection reagent comprising an antibody that specifically recognizes the above-mentioned phosphorylated neutral sphingomyelinase 1 polypeptide. The reagent of the present invention may be a kit for detecting phosphorylated neutral sphingomyelinase 1 polypeptide. By using the reagent of the present invention, the phosphorylated neutral sphingomyelinase 1 polypeptide can be easily detected by the above-described method, and the activation state of the stress-induced apoptosis signal in the cell can be evaluated.

  An antibody that specifically recognizes phosphorylated neutral sphingomyelinase 1 polypeptide is dissolved in water or an appropriate buffer (eg, TE buffer, PBS, etc.) to an appropriate concentration, and is about -20 ° C. Can be stored at ~ 4 ° C.

  The reagent of the present invention may further contain other components necessary for carrying out the method as a component, depending on the type of immunological technique employed.

  For example, the reagent of the present invention can further contain a labeled secondary antibody, a chromogenic substrate, a blocking solution, a washing buffer, an ELISA plate, a blotting membrane and the like.

3. Neutral sphingomyelinase 1 mutant polypeptide The present invention relates to a neutral sphingomyelinase 1 mutant polypeptide in which a serine residue that is a phosphorylation target by JN kinase is substituted with an amino acid residue other than serine (the present invention). Mutant polypeptides). The mutant polypeptide of the present invention comprises the amino acid sequence of wild-type neutral sphingomyelinase 1, provided that a serine residue that is a phosphorylation target by JN kinase in the amino acid sequence is substituted with an amino acid residue other than serine. Has been.

  As described above, when cells are stress-stimulated, the serine residue (in the case of zebrafish, human and mouse, Ser-270) which is a phosphorylation target of JN kinase in neutral sphingomyelinase 1 is caused by JN kinase. Phosphorylated and activated neutral sphingomyelinase 1 induces apoptosis by hydrolyzing sphingomyelin to produce ceramide. Therefore, when a serine residue that is a phosphorylation target by JN kinase is substituted with an amino acid residue other than serine, it is not controlled by phosphorylation by JN kinase, so by using the mutant polypeptide of the present invention, It is possible to intentionally inhibit or activate stress-induced apoptotic signals in cells. Therefore, the mutant polypeptide of the present invention is useful as a tool for analyzing stress-induced apoptosis signals in cells.

  For example, the mutant polypeptide of the present invention comprises the amino acid sequence represented by SEQ ID NO: 1, 2, or 3, provided that the 270th serine residue (Ser270) in the amino acid sequence is an amino acid residue other than serine. Has been replaced.

  Examples of amino acids other than serine include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, and valine. it can.

In one embodiment, the mutant polypeptide of the invention is a dominant negative mutant. Dominant negative mutants can inhibit the activity of wild-type neutral sphingomyelinase 1 in cells by introducing mutations into the amino acid sequence of wild-type neutral sphingomyelinase 1. Say things. The dominant negative mutant competes with the wild-type neutral sphingomyelinase 1 in the cell, thereby indirectly causing the activity of the wild-type neutral sphingomyelinase 1 (ie, in neutral conditions depending on Mg ^2+). Activity of hydrolyzing sphingomyelin to produce ceramide), and as a result, it can inhibit stress-induced apoptosis in cells. Substitutions that enable dominant negative mutants include amino acids other than acidic amino acids of serine residues that are phosphorylation targets by JN kinase (ie, alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, isoleucine, leucine). , Lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine). Preferably, a serine residue which is a phosphorylation target by JN kinase is substituted with an alanine residue.

  In a preferred embodiment, the dominant negative mutant polypeptide of the present invention comprises the amino acid sequence represented by SEQ ID NO: 1, 2, or 3, provided that the 270th serine residue (Ser270) in the amino acid sequence contains an alanine residue. Substituted on the group.

In one embodiment, the mutant polypeptide of the present invention is an activated mutant. An activated mutant is a wild-type neutral sphingomyelinase 1 that is constitutively introduced by introducing a mutation into the amino acid sequence of wild-type neutral sphingomyelinase 1 without requiring phosphorylation by JN kinase. A polypeptide that has acquired activity exceeding that (activity of hydrolyzing sphingomyelin and generating ceramide under neutral conditions in an Mg ²⁺ -dependent manner). The activated mutant in cells, constitutively, the Mg ²⁺ dependent manner sphingomyelin in neutral conditions to hydrolyze to generate ceramide, a result, without requiring a stress stimuli, apoptosis of the cell Can be induced. Examples of substitutions that enable activated mutants include substitution of serine residues, which are phosphorylation targets by JN kinase, with acidic amino acid (aspartic acid, glutamic acid) residues. Preferably, a serine residue which is a phosphorylation target by JN kinase is substituted with a glutamic acid residue.

  In a preferred embodiment, the activated mutant polypeptide of the present invention comprises the amino acid sequence represented by SEQ ID NO: 1, 2 or 3, provided that the 270th serine residue (Ser270) in the amino acid sequence is glutamic acid. It is substituted with a residue.

  In addition to the amino acid sequence of wild-type neutral sphingomyelinase 1 (however, a serine residue that is a phosphorylation target by JN kinase is substituted with an amino acid residue other than serine) of the mutant polypeptide of the present invention, One or more (for example, 1 to 500, preferably 1 to 100, more preferably 1 to 15, most preferably 1 or 2) additional amino acids may be included. The amino acid sequence to be added is not particularly limited, and examples thereof include a tag for facilitating detection and purification of the polypeptide. As tags, Flag tag, histidine tag, c-Myc tag, HA tag, AU1 tag, GST tag, MBP tag, fluorescent protein tag (for example, GFP, YFP, RFP, CFP, BFP, etc.), immunoglobulin Fc tag, etc. It can be illustrated. In addition, examples of added amino acids include amino acids that mediate conjugation with other proteins. Examples of the amino acid include cysteine.

  The mutant polypeptide of the present invention may be modified. Examples of such modifications include lipid chain addition (aliphatic acylation (palmitoylation, myristoylation, etc.), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue) Phosphorylation), acetylation, addition of sugar chain (N-glycosylation, O-glycosylation) and the like.

The mutant polypeptide of the present invention contains a suitable labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase). , Peroxidase, malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin) Etc.).

  The mutant polypeptide of the present invention includes a salt form thereof. As the salt, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal salt) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  The variant polypeptides of the present invention are preferably isolated or purified. “Isolation or purification” means that an operation to remove factors other than the target component has been performed, and the state existing in nature has been removed. The purity of the “isolated or purified variant polypeptide” (percentage of the target variant polypeptide weight in the total polypeptide weight) is usually 70% or more, preferably 80% or more, more preferably 90%. As mentioned above, it is most preferably substantially 100%.

  The method for producing the mutant polypeptide of the present invention is not particularly limited, and the polypeptide may be produced according to a known peptide synthesis method or may be produced using a known gene recombination technique. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.

  When the polypeptide of the present invention is produced using gene recombination technology, first, a polynucleotide encoding the polypeptide of the present invention as described below is obtained, and a host is transformed with an expression vector containing the polynucleotide. The polypeptide can be produced by culturing the obtained transformant.

4). Polynucleotide Encoding Neutral Sphingomyelinase 1 Variant Polypeptide The present invention provides a polynucleotide encoding the mutant polypeptide of the present invention. The polynucleotide may be DNA, RNA, or a DNA / RNA chimera, preferably DNA. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.

  The polynucleotide of the present invention is a DNA clone that encodes wild-type neutral sphingomyelinase 1 by designing an appropriate primer by utilizing known sequence information or sequence information described in the sequence listing of the present specification. Can be obtained by introducing a mutation such as PCR using cDNA or cDNA as a template. Alternatively, a polynucleotide encoding a mutant polypeptide may be synthesized by a polynucleotide synthesizer based on the sequence information.

  The obtained polynucleotide of the present invention can be used as it is or after digestion with a restriction enzyme or addition of a linker, if desired. The polynucleotide may have ATG as a translation initiation codon on the 5 'end side, and may have TAA, TGA or TAG as a translation stop codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

  The polynucleotide of the present invention is preferably isolated or purified.

  The polynucleotide of the present invention is useful for producing the mutant polypeptide of the present invention.

5. Expression vector The present invention also provides an expression vector comprising the polynucleotide of the present invention. The expression vector can be produced by functionally linking the polynucleotide of the present invention downstream of a promoter that can function in a host cell in an appropriate expression vector. Examples of the vector include plasmid vectors and virus vectors, and can be appropriately selected depending on the host to be used.

  Examples of the host include Escherichia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacillus subtilis, etc.), yeasts (Saccharomyces cerevisiae, etc.), insect cells (night stealing) Larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), insects (larvae of silkworms), mammalian cells (rat neurons, monkey cells (COS-7, etc.), Chinese hamster cells (CHO cells, etc.) Etc.) is used.

  Examples of mammals include, for example, laboratory animals such as rodents and rabbits such as mice, rats, hamsters, and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, Primates such as monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.

  As plasmid vectors, plasmid vectors derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmid vectors derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmid vectors (eg, pSH19, pSH15), etc. And can be appropriately selected according to the type of host used and the purpose of use.

  The type of viral vector can be appropriately selected according to the type of host used and the purpose of use. For example, when insect cells are used as the host, baculovirus vectors can be used. When mammalian cells are used as hosts, Moloney murine leukemia virus vectors, lentivirus vectors, Sindbis virus vectors and other retrovirus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, Vaccinia virus vectors, Sendai virus vectors, and the like can be used.

  In addition, a promoter that can initiate transcription in the host can be selected according to the type of host to be used. For example, when the host is Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. When the host is a mammalian cell, a subgenomic (26S) promoter, CMV promoter, SRα promoter and the like are preferable.

  In addition to the promoter, the expression vector of the present invention can function, if desired, an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori), and the like. You may contain.

6). Transformant The above-described expression vector of the present invention is converted into a gene transfer method known per se (for example, lipofection method, calcium phosphate method, microinjection method, proplast fusion method, electroporation method, DEAE dextran method, gene gun transfer method) Etc.), a transformant into which the expression vector has been introduced (transformant of the present invention) can be produced. By using an expression vector as the introduced vector, the transformant can express the polypeptide of the present invention. The transformant of the present invention is useful for the production of the mutant polypeptide of the present invention.

  The transformant of the present invention is cultivated by a method known per se according to the type of host, and the mutant polypeptide of the present invention is isolated from the culture to produce the mutant polypeptide of the present invention. I can do it. For isolation and purification of the polypeptide of the present invention from the culture, for example, the cell lysate and the culture supernatant are subjected to multiple chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. Can be achieved.

7). Method for Inhibiting or Promoting Stress-Induced Apoptosis The present invention includes a method for inhibiting stress-induced apoptosis of cells (inhibiting method of the present invention), which comprises introducing the above-described dominant negative mutant polypeptide of the present invention or an expression vector thereof. )I will provide a. When the dominant negative mutant polypeptide of the present invention or an expression vector thereof is introduced into a cell, the dominant negative mutant polypeptide competes with wild type neutral sphingomyelinase 1 indirectly, thereby inducing wild type neutrality. It can inhibit the activity of sphingomyelinase 1 (ie, the activity of hydrolyzing sphingomyelin and generating ceramide under neutral conditions in a Mg ²⁺ -dependent manner), and as a result, it can inhibit stress-induced apoptosis in cells. it can.

The present invention also provides a method for promoting stress-induced apoptosis of the cells (the promotion method of the present invention), which comprises introducing the above-mentioned activated mutant polypeptide of the present invention or an expression vector thereof. When the activated mutant polypeptide of the present invention or the expression vector thereof is introduced into a cell, the activated mutant polypeptide is sphingomyelin constantly and neutrally in Mg ²⁺ dependently in the cell. Is hydrolyzed to produce ceramide, which promotes stress-induced apoptosis of the cell.

  In a preferred embodiment, the animal species from which the amino acid sequence of neutral sphingomyelinase 1 contained in the mutant polypeptide used in the inhibition and promotion methods of the present invention is the same as the animal species from which the cell to be introduced is derived. is there. For example, in the case of inhibiting and promoting stress-induced apoptosis of human cells, human neutral sphingomyelinase 1 dominant negative mutant polypeptide and activated mutant polypeptide are used, respectively.

  Any eukaryotic cell can be used as the cell used in the inhibition method and the promotion method of the present invention. Examples of eukaryotes include mammals, birds, reptiles, amphibians, fish, insects, yeasts, etc., but mammals or fish are preferred. Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Examples include, but are not limited to, primates such as monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. The mammal is preferably a laboratory animal (eg, a mouse) or a primate (eg, a human). Although it does not specifically limit as fish, For example, as an edible, the fish species (for example, flounder fish (flounder etc.), flounder, flounder, turbot etc.), Thai fish (red sea bream, sea bream etc.) ), Yellowtail, mullet, ayame, tuna, tilapia, salmon, carp, trout, rainbow trout, yamame, amago, eel and the like. Appreciation applications include carp, funa, medaka, goldfish and the like. Furthermore, zebrafish, medaka, goldfish, loach, etc. are mentioned as research or experiment use. The fish is preferably for research or experiment, preferably zebrafish.

  Introduction of the mutant polypeptide into the cell can be carried out using a known method for introducing protein into the cell. Examples of such a method include a method using a protein introduction reagent, a method using a protein introduction domain (PTD) fusion protein, and a microinjection method. Protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systems), cationic lipid-based BioPOTER Transfection Reagent (PIERCE) and ProVctin (IMGENEX), and lipid-based Profect-1 (Targeting Systems), Penetratin Peptide (Q Biogene), Chariot Kit (Active Motif) and the like based on a membrane-permeable peptide are commercially available. The introduction can be carried out according to the protocol attached to these reagents, but the general procedure is as follows. The mutant polypeptide of the present invention is diluted in an appropriate solvent (for example, a buffer solution such as PBS, HEPES, etc.), an introduction reagent is added, and the mixture is incubated at room temperature for 5 to 15 minutes to form a complex. Add to the medium and incubate the cells in the medium at 37 ° C. for 1 to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium.

  As PTDs, those using cell-passing domains of proteins such as Drosophila-derived AntP, HIV-derived TAT, and HSV-derived VP22 have been developed. A fusion protein expression vector incorporating the cDNA encoding the mutant polypeptide of the present invention and a PTD sequence is prepared and recombinantly expressed, and the fusion protein is recovered and used for introduction. The introduction is the same as above except that no protein introduction reagent is added.

  Microinjection is a method in which a protein solution is poured into a glass tube having a tip of about 1 μm and puncture is introduced into the cell, and the protein can be reliably introduced into the cell.

  Expression vectors can be introduced into cells by a method known per se according to the type of vector. For example, in the case of a viral vector, a plasmid containing the nucleic acid is introduced into an appropriate packaging cell (eg, Plat-E) or a complementary cell line (eg, 298 cell), and a viral vector produced in the culture supernatant is obtained. It collect | recovers and it infects a cell with this vector by an appropriate method according to each viral vector. On the other hand, in the case of a plasmid vector, the vector can be introduced into cells using lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method, etc. .

  After the introduction, if desired, stress stimulation (eg, heat shock, chemotherapeutic drug, irradiation, starvation) may be applied to the cells.

  Therefore, the dominant negative mutant polypeptide of the present invention or an expression vector thereof is used as an inhibitor of cell stress-induced apoptosis, and the activated mutant polypeptide of the present invention or the expression vector thereof is a cell stress-inducible. Each is useful as an apoptosis promoter.

  All references cited in this specification, including publications, patent documents, etc., are cited by reference as if they were individually incorporated by reference specifically and in their entirety. To the same extent, it is incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

[Example 1]
1. Preparation of antigen A peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 and a cysteine residue introduced at the C-terminus thereof was chemically synthesized by Nippon Bioservice Co., Ltd. and used as an antigen. Using KLH as a carrier, a thiol group at the cysteine residue at the C-terminal of the antigen peptide and KLH were bound using a crosslinking reagent MBS (manufactured by PIERCE) to obtain an immunogen (T. Kitagawa and T. Aikawa., J. Biochem., 79, 233-236, 1976; Shinobu Oumi, Kunio Tsujimura, Masaki Inagaki: Anti-peptide antibody experimental protocol, Shujunsha, pp. 1-312, 1994).

2. Preparation of antibody Antigen protein solution (protein concentration 2 mg / mL) conjugated with KLH using a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in which the serine residue is phosphorylated and hapten antigen as a hapten antigen. The resulting emulsion was mixed with adjuvant (PIERCE) and the resulting emulsion was immunized to rabbits once every two weeks. After 133 days, venous blood was collected to obtain antiserum. The Sepharose 4B gel in which the rabbit antiserum thus obtained was diluted twice with PBS and immobilized with non-phosphorylated neutral sphingomyelinase 1 peptide antigen (peptide consisting of the amino acid sequence represented by SEQ ID NO: 4) The mixture was reacted by shaking at 37 ° C. for 1 hour. The non-adsorbed fraction was eluted, and the eluate was immobilized with phosphorylated neutral sphingomyelinase 1 peptide antigen (a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in which the serine residue was phosphorylated). The mixture was reacted with 4B gel by shaking at 37 ° C. for 1 hour. The gel was then loaded onto the column and unreacted components were washed away with PBS containing 0.1% Tween 20 and PBS. While monitoring the elution position of the protein at 280 nm, the antibody was eluted from the column with 0.1 M glycine-hydrochloric acid buffer (pH 2). The eluted antibody solution was neutralized by adding 1 M Tris-HCl buffer (pH 8). Thus, 34.4 μg of affinity purified antibody (protein concentration of 34.4 μg / mL) was obtained.

[Example 2]
Confirmation of antibody specificity It was examined by ELISA whether the antibody obtained in Example 1 specifically reacts with neutral sphingomyelinase 1 in which the serine residue (Ser270) was phosphorylated. That is, a diluted solution of peptide antigen (peptide consisting of the amino acid sequence represented by SEQ ID NO: 4) is dispensed into a 96-well microplate, the peptide antigen is adsorbed to the well, and then blocked with a BSA blocking solution. Antigen peptide was immobilized on the well. The wells were washed, diluted with the antibody obtained in Example 1 as a primary antibody, reacted, and then washed with PBS. Further, a peroxidase-labeled secondary antibody was added to each well for reaction, and then washed with PBS. A TMBZ (3,3 ′, 5,5′-tetramethylbenzidine) substrate was added to each well for color development, and the absorbance was measured at 450 nm. As a result, as shown in FIG. 2, all of the purified antibodies reacted with the phosphopeptide antigen. On the other hand, in the ELISA analysis using a peptide antigen that was not phosphorylated, the reaction of the antibody obtained in Example 1 was weak. From these results, it was confirmed that the antibody obtained in Example 1 has specificity for the neutral sphingomyelinase 1-derived polypeptide in which the serine residue (Ser270) is phosphorylated.

[Example 3]
Method for measuring phosphorylated neutral sphingomyelinase 1 (antibody analysis by Western blotting)
Using the anti-phosphorylated neutral sphingomyelinase 1 antibody prepared in Example 1 by Western blotting, Shinobu Oumi, Kunio Tsujimura, Masaki Inagaki: Anti-peptide antibody experimental protocol, Shujunsha, pp. 1 -312, 1994).

  In other words, the common soleus muscle was homogenized with 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM magnesium chloride, 25 mM potassium chloride, protease inhibitor cocktail (Complete Mini, Roche), 1 mM sodium fluoride and 1 mM sodium vanadate. And centrifuged at 8,000 × g for 10 minutes. A sample buffer (10 mM Tris-HCl buffer (pH 6.8) containing 2% SDS and 0.1% bromophenol blue) was added to the resulting supernatant to prepare a sample for electrophoresis. Further, human kidney-derived cultured cells HEK293 were cultured at 37 ° C. in a DMEM medium (Invitrogen) containing fetal bovine serum (5%), penicillin (5 units / mL) and streptomycin (50 μg / mL) in a petri dish having a diameter of 6 cm. did. The cells were fixed by adding 2 ml of 10% trichloroacetic acid to each petri dish and peeled and collected with a scraper. After removing trichloroacetic acid by centrifugation, the above sample buffer containing 1 mM sodium fluoride, 1 mM sodium vanadate and protease inhibitor cocktail (Complete Mini, Roche) is added to each cell pellet, and then 2 MTris is added to The cells were lysed by heat treatment at 100 ° C. for 2 minutes to prepare a sample for electrophoresis. These samples were separated by 10% SDS-polyacrylamide gel electrophoresis and then electrically transferred to a PVDF membrane (GE Healthcare Bioscience). A PVDF membrane was used as a primary antibody, a diluted solution (500 ng / ml) of the anti-phosphorylated neutral sphingomyelinase 1 antibody prepared in Example 1, and a peroxidase-labeled anti-rabbit IgG antibody (DakoCytomation) 2 as a secondary antibody. The reaction was carried out stepwise with a 000-fold dilution. After washing with TBST, chemiluminescence was generated by an antigen-antibody reaction using a chemiluminescence analysis reagent Chemilmiwan L (Nacalai Tesque) and detected with LAS-1000mini (Fujifilm). The molecular weight marker was a protein molecular weight marker (TEFCO) and stained with Coomassie Brilliant Blue R250.

  As a result, as shown in FIG. 3, by using the anti-phosphorylated neutral sphingomyelinase 1 antibody prepared in Example 1, phosphorous having a molecular weight of 50,000 is contained in extracts of cultured human-derived cells and Japanese flounder-derived cultured cells. The presence of oxidized neutral sphingomyelinase 1 was confirmed. When an absorption test is performed in which the antibody-phosphorylated neutral sphingomyelinase 1 antibody is treated with a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 in which the serine residue is phosphorylated, phosphorylated neutral sphingomyelinase in human cells Since the signal for RNase 1 disappeared, it was found that phosphorylation of the serine residue at position 270 also occurs specifically under stress conditions in neutral sphingomyelinase 1 derived from human cells.

[Example 4]
Phosphorylation of neutral sphingomyelinase 1 by JN kinase A recombinant protein of zebrafish-derived SMase 1 consisting of the amino acid sequence represented by SEQ ID NO: 1 was prepared by the method of Non-Patent Document 4 using an E. coli expression system. In addition to wild-type SMase1, a mutant SMase1 (S270A) in which the phosphorylation site of Ser-270 was substituted with alanine was prepared. A commercially available JN kinase (JNK1, manufactured by Cell Signaling) was allowed to act on wild-type SMase1 and mutant SMase1 (S270A), and then the reaction solution was separated by SDS-polyacrylamide gel electrophoresis using a 10% gel. It was electrically transferred to a membrane (GE Healthcare Bioscience). A PVDF membrane was used as a primary antibody, a diluted solution (500 ng / ml) of the anti-phosphorylated neutral sphingomyelinase 1 antibody prepared in Example 1, and a peroxidase-labeled anti-rabbit IgG antibody (DakoCytomation) 10,10 as a secondary antibody. The reaction was carried out stepwise with a 000-fold dilution. After washing with TBST, chemiluminescence by an antigen-antibody reaction was generated using a chemiluminescence analysis reagent Chemilmiwan L (Nacalai Tesque), and the film was exposed to hyperfilm ECL (GE Healthcare Japan) and detected. The molecular weight marker was a protein molecular weight marker (TEFCO) and stained with Coomassie Brilliant Blue R250.

  As a result, it was confirmed that the SMase1 recombinant protein was phosphorylated by JN kinase and the enzyme activity was increased about 4 times (FIG. 4). The anti-phosphorylated neutral sphingomyelinase 1 antibody did not react with the mutant SMase1 (S270A), and no signal was obtained. Moreover, when JN kinase inhibitor SP600125 (Sigma Aldrich Japan) was added, phosphorylation was inhibited.

  From the above results, it was shown that the phosphorylated neutral sphingomyelinase 1 of the present invention can be prepared by phosphorylating the neutral sphingomyelinase 1 with JN kinase. The anti-phosphorylated neutral sphingomyelinase antibody prepared in Example 1 binds to phosphorylated zebrafish-derived wild-type neutral sphingomyelinase 1, but the serine residue at position 270 was substituted with alanine. It was shown not to bind to the mutant molecule.

[Example 5]
Stress test by irradiation with ultraviolet rays on zebrafish cultured cells Zebrafish embryo-derived ZE cells were treated with Leibovitz L. cerevisiae containing 2% fetal bovine serum. The culture was performed at 28 ° C. using 15 medium. The cultured cells were subjected to heat shock or ultraviolet irradiation to examine the phosphorylation response of neutral sphingomyelinase 1 under stress conditions (FIG. 5). The heat shock treatment was performed by culturing at 38 ° C. for 1 hour. The ultraviolet treatment was performed by irradiating with 5 mJ / cm ² of ultraviolet rays using an ultraviolet irradiation device (UV crosslinker, manufactured by Funakoshi, CL-1000). Cells subjected to stress treatment by heat shock or ultraviolet irradiation were returned to normal culture conditions at 28 ° C. and cultured. Untreated cultured cells and cultured cells stress-treated by heat shock or ultraviolet irradiation were collected and separated by 10% gel SDS polyacrylamide gel electrophoresis, and then the anti-phosphorylated neutral sphingomyeli prepared in Example 1 was used. Phosphorylated neutral sphingomyelinase 1 was detected by Western blotting using the enzyme 1 antibody in the same manner as in Example 4. As a control experiment, neutral sphingomyelinase 1 was detected using a normal anti-neutral sphingomyelinase 1 antibody (Non-patent Document 4) that does not recognize the presence or absence of phosphorylation. In addition, an actin band was detected by protein staining using Coomassie Brilliant Blue R-250.

  As a result of this experiment, the signal of phosphorylated neutral sphingomyelinase 1 increased in 1 hour after the start of stress treatment, but the signal intensity of neutral sphingomyelinase 1 and actin did not change (FIG. 5). From these results, it became clear that phosphorylation of neutral sphingomyelinase 1 is induced by stress conditions.

[Example 6]
A recombinant protein of a neutral sphingomyelinase 1 mutant in which the serine residue at position 270, which is a phosphorylation site in the amino acid sequence of zebrafish-derived neutral sphingomyelinase 1 (SEQ ID NO: 1), was modified to alanine or glutamic acid It was prepared by the method of Non-Patent Document 4 using an expression system. The activity of the purified enzyme was measured using C ₆ -NBD sphingomyelin as a substrate (FIG. 6). As a result, wild-type neutral sphingomyelinase 1 and neutral sphingomyelinase 1 mutant (S270A) in which the serine residue at position 270 was substituted with alanine were hardly detected. Neutral sphingomyelinase 1 mutant (S270E) in which the phosphorylation site at position 270 was replaced with glutamic acid showed strong enzyme activity. Moreover, p3xFLAG-CMV-14 vector (Sigma Aldrich Japan) was prepared from the expression vector containing cDNA encoding these wild type and mutant neutral sphingomyelinase 1 by the method of Non-Patent Document 4. The expression vector was introduced into ZE cells, and after 24 hours of culture, dead cells were counted according to the method described in Non-Patent Document 4 to examine apoptosis induction. In the empty vector (Mock) containing no neutral sphingomyelinase 1 cDNA, apoptosis was hardly induced (FIG. 6). Cell death occurred with an expression vector (wild-type) containing neutral sphingomyelinase 1 cDNA (FIG. 6). On the other hand, in the neutral sphingomyelinase 1 mutant cDNA expression vector (S270A) in which the serine residue at position 270 was substituted with alanine so that phosphorylation did not occur, induction of apoptosis was suppressed. In addition, the neutral sphingomyelinase 1 mutant cDNA expression vector (S270E) in which the phosphorylation site at position 270 was replaced with glutamic acid induced apoptosis more strongly than the wild-type molecule (FIG. 6).

  From these results, when the serine residue at the 270th phosphorylation site of neutral sphingomyelinase 1 is replaced with an alanine residue that is not phosphorylated, it is converted into a dominant negative inactive enzyme that is not activated by stress. These results suggest that substitution with glutamic acid having an acidic amino acid side chain results in conversion to an active enzyme having the same activity as a phosphorylated serine residue.

[Example 7]
Detection of phosphorylated neutral sphingomyelinase 1 derived from humans Phosphorylation of neutral sphingomyelinase 1 in mammalian cells was examined. Human T cell leukemia-derived Jurkat cells (ATCC TIB-152) were cultured in RPMI 1640 medium containing 2% fetal calf serum at 37 ° C. As conditions for inducing apoptosis, heat shock at 43 ° C. for 1 hour, irradiation with ultraviolet light (254 nm) at 5 mJ / cm ² , treatment with 1 mM hydrogen peroxide or anti-fas antibody (Roche, 50 ng / ml) The cells were cultured, and the cells were collected over time. Western blotting was performed in the same manner as in Example 3 using the antibody against phosphorylated neutral sphingomyelinase 1 prepared in Example 1 (FIG. 7). In addition, phosphorylation (S257 / T261) antibody (manufactured by Cell Signaling) of phosphorylase MKK4 that activates JN kinase, phosphorylation antibody of JN kinase (Phosphorylated JNK1 / 2) (manufactured by Cell Signaling), JN kinase JN kinase antibody that does not distinguish phosphorylation (Total JNK1 / 2) (manufactured by Cell Signaling), phosphorylated antibody of c-jun which is a substrate protein of JN kinase (S73) (manufactured by Cell Signaling), actin antibody as a control experiment Using (Actin) (manufactured by Cell Signaling), the response of each molecule to stress was detected. In cells subjected to stress treatment, a phosphorylation signal of serine at position 270 was detected, but no phosphorylation signal was detected under normal culture conditions before stress treatment (FIG. 7). This neutral sphingomyelinase 1 phosphorylated MKK4 (S257 / T261), JN kinase (JNK1 / 2) and c-jun in response to phosphorylation. From these results, it was found that neutral sphingomyelinase 1 is phosphorylated by stress-responsive phosphorylation signal of JN kinase in mammalian cells as well.

  By using phosphorylated neutral sphingomyelinase 1, anti-phosphorylated neutral sphingomyelinase 1 antibody and neutral sphingomyelinase mutant of the present invention, detailed mechanism analysis of the regulation mechanism of stress-induced apoptosis is possible. Become.

Claims (15)

The following polypeptide (1) or (2):
(1) a phosphorylated neutral sphingomyelinase 1 polypeptide in which a serine residue that is a phosphorylation target by JN kinase is phosphorylated, and (2) a partial amino acid sequence of the polypeptide of (1), A polypeptide comprising a serine residue which is a target for phosphorylation by a kinase, comprising a partial amino acid sequence having a length of 10 amino acids or more, and wherein the serine residue is phosphorylated.   The polypeptide according to claim 1, wherein the phosphorylated neutral sphingomyelinase 1 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 1, 2, or 3. The method for producing a polypeptide according to claim 1, comprising phosphorylating the following polypeptide (1 ') or (2') with JN kinase:
(1 ′) Neutral sphingomyelinase 1 polypeptide and (2 ′) (1 ′) partial amino acid sequence comprising serine residue that is a phosphorylation target by JN kinase, and 10 amino acids A polypeptide comprising a partial amino acid sequence having the above length.   An antibody that specifically recognizes the polypeptide according to claim 1 or 2.   A reagent for detecting phosphorylated neutral sphingomyelinase 1 polypeptide, comprising the antibody according to claim 4.   A method for detecting a phosphorylated neutral sphingomyelinase 1 polypeptide, comprising using the antibody according to claim 4.   A method for evaluating an activation state of a stress-induced apoptosis signal in a cell, comprising detecting the polypeptide according to claim 1 in the cell.   A neutral sphingomyelinase 1 mutant polypeptide in which a serine residue that is a phosphorylation target by JN kinase is substituted with an amino acid residue other than serine.   The mutant polypeptide according to claim 8, wherein the serine residue is substituted with an alanine residue.   The mutant polypeptide according to claim 8, wherein the serine residue is substituted with a glutamic acid residue.   A polynucleotide encoding the mutant polypeptide according to any one of claims 8 to 10.   An expression vector comprising the polynucleotide according to claim 11.   A transformant comprising the expression vector according to claim 12.   A method for inhibiting stress-induced apoptosis of a cell, comprising introducing the mutant polypeptide according to claim 9 or an expression vector thereof into the cell.   A method for promoting stress-induced apoptosis of a cell, comprising introducing the mutant polypeptide according to claim 10 or an expression vector thereof into the cell.