JP2003512072A - Human sphingosine kinase gene - Google Patents

Abstract

(57) [Summary] The present invention relates to a human sphingosine kinase type 1 gene. More specifically, the present invention relates to a purified or isolated nucleic acid of the above sphingosine kinase, or a complementary sequence thereof, or a fragment thereof. The present invention encompasses oligonucleotides, recombinant polypeptides, recombinant vectors, recombinant host cells comprising the above nucleic acids, as well as antibody production, screening methods, antisense oligonucleotides, knockout mammals.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the human sphingosine kinase type 1 gene. More precisely,
The present invention relates to a purified or isolated nucleic acid of the above sphingosine kinase or a sequence complementary thereto, or a fragment thereof. The present invention includes oligonucleotides, recombinant polypeptides, recombinant vectors, recombinant host cells containing the above nucleic acids, as well as antibody production and screening methods, antisense oligonucleotides and knockout mammals.

BACKGROUND OF THE INVENTION Sphingosine-1-phosphate, a product of sphingosine kinase, is
It is an important signal molecule having intracellular and extracellular functions. A cDNA for mouse sphingosine kinase has been recently reported as described in patent application WO99 / 61581. Mouse SK1A and SK1B appear to be alternative splice forms. Differential splicing appears to result in two variants of the N-terminal peptide sequence, which is the result of the use of the alternative coding exons (Koham
a et al., 1998).

SUMMARY OF THE INVENTION The present invention relates to purified or isolated nucleic acid encoding human sphingosine kinase (henceforth hSK) cDNA or a sequence complementary thereto.

[0004] Oligonucleotide probes or primers that specifically hybridize to nucleic acids encoding hSK, fragments thereof or sequences complementary to them are part of the invention together with the methods of DNA amplification and detection using the above primers and probes. is there.

Another object of the invention relates to a recombinant vector comprising any of the nucleic acid sequences described herein, in particular a recombinant vector comprising a nucleic acid sequence encoding recombinant hSK.

The present invention also includes a recombinant expression vector comprising a nucleic acid sequence encoding a recombinant hSK.

The present invention also includes host cells and transgenic non-human mammals comprising the above nucleic acid sequences or recombinant vectors.

[0008]   The invention also relates to isolated recombinant hSK.

The present invention also relates to hSK polypeptides or peptide fragments thereof, as well as antibodies directed against the hSK peptides.

[0010]   The present invention also relates to a method of screening candidate molecules that are inhibitors of hSK.

The method comprises the steps of mixing recombinant hSK with sphingosine, labeled ATP and a candidate molecule of interest and determining the level of conversion of sphingosine to labeled sphingosine-1-phosphate (S1P).

The present invention also relates to a kit for screening a candidate molecule that is an hSK inhibitor.

[0013]   This kit is   Recombinant hSK, and in some cases   Labeled ATP and sphingosine Comprises.

The present invention provides an inhibitor of hSK1 obtained by the above screening method, structural analogues thereof, and degenerative disease states such as atherosclerosis and fibrosis; neurodegenerative diseases; atherosclerosis, Cardiovascular diseases such as thrombosis and dyslipidemia; diabetes mellitus such as type I and type II diabetes, especially type I diabetes; seizures; multiple sclerosis, psoriasis, dysplasia erythematosus and inflammatory arthritis Autoimmune and inflammatory diseases; T helper-1 related diseases; chronic obstructive pulmonary disease; asthma; cancer; congestion, stroke, coronary artery disease,
It also relates to their use in the treatment or prevention of one and / or several disease states selected from hematopoietic diseases such as leukemia, natural wound healing processes, myocardial infarction, embryogenesis.

Detailed Description of the Invention A) Human Sphingosine Kinase cDNA The first subject of the present invention is a purified or isolated nucleic acid encoding hSK, or a sequence complementary thereto.

Another subject of the invention is a nucleotide identity of at least 90%, preferably 95%, more preferably 98%, most preferably 99% with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A purified or isolated nucleic acid, or a sequence complementary thereto.

Another subject of the invention is at least 80%, preferably 90%, more preferably 95%, most preferably 98 or 99% of a human polypeptide of the amino acid sequence SEQ ID NO: 3 or a peptide fragment thereof. A purified or isolated nucleic acid encoding a polypeptide having the amino acid identity of, or a sequence complementary thereto.

The polypeptide having amino acid identity with hSK of the present invention has a high sequence identity between amino acid sequences, and thus has a hS of the amino acid sequence of SEQ ID NO: 3.
Polypeptides having a primary structure with respect to K; or these cognate polypeptides are recognized by antibodies specifically directed against the amino acid sequence of SEQ ID NO: 3, and / or these cognate polypeptides contain sphingosine. Includes polypeptides that are biologically related to the polypeptide of amino acid sequence SEQ ID NO: 3 because they have the same biological activity as the polypeptide of amino acid sequence SEQ ID NO: 3, such as the ability to convert to S1P. To do.

The term “isolated” as used herein requires that the material be removed from its original environment (eg, the natural environment when it is naturally produced).
For example, a naturally occurring polynucleotide or peptide contained in a living animal has not been isolated, but the same polynucleotide or peptide isolated from some or all of the coexisting materials in the natural system has been isolated. ing. Such polynucleotides can be part of the vector, and / or such polynucleotides or peptides can be part of the composition and can be further isolated. This is because the vector or composition is not part of the original environment of the nucleotide sequence it contains.

The term “purified” does not require absolute purity, rather it is for relative definition. Purification of the starting or natural material to the extent of at least one order, preferably of the order of 2 or 3, more preferably of the order of 4 or 5, is especially considered.

Throughout this specification the expression “nucleotide sequence” is used to describe a polynucleotide or nucleic acid inseparably. More precisely, a "nucleotide sequence"
The expression includes nuclear material and sequence information, and is not limited to sequence information that biochemically characterizes a particular DNA or RNA molecule (ie, a sequence of letters selected from the four base letters). .

The terms “oligonucleotide”, “nucleic acid” and “polynucleotide”, used interchangeably herein, refer to RNA, genomic DNA, any type of DNA such as cDNA, or single-stranded or double-stranded. It includes RNA / DNA hybrid sequences of two or more nucleotides in chain form.

In addition to its general meaning as understood by one of ordinary skill in the art, the term “nucleotide” is used herein to refer to (a) alternative linking groups, (b) analogs of purines, (c) analogs of pyrimidines. Or modified nucleotides such as (d) similar sugars, (e) modified nucleotides such as methylated, phosphorylated, ubiquitinated nucleotides:

For examples of similar linking groups, purines, pyrimidines, and sugars, see, for example, PCT Publication No. WO 95/04064.

The polynucleotide sequences of the invention can be prepared by any known method, such as synthetic, recombinant or combinations thereof, as well as any purification method known in the art.

B) Recombinant hSK Polynucleotides The present invention also includes polynucleotide fragments of the nucleic acid encoding hSK1 of the present invention. These fragments include, in particular, 1) a fragment encoding a polypeptide of hSK, which preferably retains its affinity for sphingosine, and 2) a nucleotide fragment useful as a nucleic acid primer or probe for amplification or detection, It is not limited to these.

Most preferred embodiment of the present invention for the fragment encoding the polypeptide of hSK is
This is a polynucleotide of the sequence of SEQ ID NO: 8 corresponding to the region of SK conserved between species. Indeed, we have shown that the 80 amino acid long region of hSK1 is conserved among species (FIG. 8).

Primers or Probes More particularly, the invention relates to at least 10 contiguous nucleotides of a nucleic acid encoding a hSK as described herein, preferably of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. It relates to a purified or isolated polynucleotide comprising at least 10 contiguous nucleotides or a sequence complementary thereto.

These nucleic acids have a length of 10, 12, 15, 18 or 20-25, 35, 40, 50, 70, 80, 100.
, Of a continuous span in the range of 250, 500 or 1000 nucleotides, or 10, 12
, 15, 18, 20, 25, 35, 40, 50, 100, 200, 250, 500 or 1000 nucleotides in length.

In a particular embodiment of the invention, these nucleic acids are present in the sample in the presence of at least one copy of the nucleotide sequence coding for hSK, more particularly the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. , Or a sequence complementary thereto, or a probe for detecting the presence of at least one copy of a fragment or variant thereof. The sequences of such nucleic acids are slightly modified (eg, by substituting one nucleotide for another) without substantially affecting the ability of the modified sequence to hybridize to the intended target sequence. can do.

[0031]   The most preferred probe is SK5 'end 49 (proximal gene) CTGGGTCTTGTAGAAGAGCAGCAAGTGCT (SEQ ID NO: 14) SK5 'end 48 (proximal gene) AGTTCACTGCAATCCTTTCTTATCTGGGTTCG (SEQ ID NO: 15) SK3 'end (distal gene) TTCTGTGGATGGAGAGCTGATGGTATGG (SEQ ID NO: 16) SK BOX (storage area) ATGAAGTGGTGAATGGGCTAATGGAACG (SEQ ID NO: 17) Is.

The nucleic acid probe of the present invention can also be used for the analysis of the expression level and pattern of hSK as described in PCT application WO 97/05277, the content of which is incorporated herein by reference. Incorporated herein by reference.

In another aspect of the invention, these nucleic acids are useful as primers.
The most preferred primers are: A = 5 'end TAT GCT AGC ATG GAT CCA GCG GGC GGC (SEQ ID NO: 4) B = 3' end AAT GAA TTC TCA TAA GGG CTC TTC TGG (SEQ ID NO: 5) C = 5 'end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ ID NO: 6) D = 3 ′ end ATT ATC GTC GAC TAA GGG CTC TTC TGG CGG (SEQ ID NO: 7) E = 5 ′ end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C ( SEQ ID NO: 10) F = 3 'end AGT CGA GGC TGA TCA GCG AG (SEQ ID NO: 11)

Hybridizing Polynucleotides The present invention also relates to purified or isolated nucleic acid sequences that hybridize to a polynucleotide encoding hSK under stringent hybridization conditions, or sequences complementary thereto.

A preferred embodiment of the present invention is a purified or isolated nucleic acid sequence which hybridizes under stringent conditions with a 270 nucleotide (SEQ ID NO: 22) nucleic acid encoding the 80 amino acid conserved region of hSK1.

In an exemplary embodiment, stringent hybridization conditions can be defined as follows: The hybridization step comprises 6xSSC buffer, 5xDenhardt solution, 0.5% SDS, and the presence of 100 μg / ml salmon sperm DNA. Perform at 65 ° C below.

[0037]   After the hybridization step, four washing steps are performed:   Washing preferably in 2xSSC and 0.1% SDS buffer at 65 ° C for 5 minutes twice,   Washing preferably in 2xSSC and 0.1% SDS buffer at 65 ° C for 30 minutes once,   Wash once in 0.1x SSC and 0.1% SDS buffer, preferably at 35 ° C for 10 minutes.

The hybridization conditions defined above are suitable for nucleic acids of about 20 nucleotides in length, which conditions are well known in the art, eg Sambrook et al.
It can also be applied to shorter or longer nucleic acids by the procedure described by al (1989).

The appropriate length of a probe under a particular set of assay conditions can be empirically determined by one of ordinary skill in the art. Probes include, for example, cloning and restriction of appropriate sequences, and the phosphodiester method of Narang et al. (1979), the phosphodiester method of Brown et al. (1979), the diethylphosphoramidite method of Beaucage et al. (1981), and application number EP. -0 707 792 can be prepared by any suitable method such as direct chemical synthesis by methods such as the solid support method described in US Pat. The disclosures of all these documents are incorporated herein by reference.

Any of the nucleic acids of the invention may be labeled, if desired, by incorporating a label detectable by spectrophotometric, photochemical, biochemical, autoradiographic, radiochemical, immunochemical, or chemical means. You can For example, useful labels include
Examples include radioactive substances ( ³² P, ³⁵ S, ³ H, ¹²⁵ I), fluorescent dyes (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin), or biotin. Examples of non-radioactive labeling of nucleic acid fragments are described in French Patent FR-78 10975 or Urdea et al. (1988) or Sanchez-Pescador et al. (1988).

Advantageously, the probe according to the invention may have a structure and features capable of amplifying a signal, for example the structural features of the branched DNA probe described in Urdea et al. (1991). it can.

Any of the nucleic acid probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those of skill in the art and include reaction tray wells, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep red blood cells, dural cells, etc. Walls.

The nucleic acids of the invention, and in particular the nucleotide probes described above, are thus individually or on a single solid support at least 2, 5, 8, 10, 12, 15, 20, or 25 of the separate inventive compounds. Groups of nucleic acids can be bound or immobilized on a solid support.

In a specific embodiment of the support for immobilizing the nucleic acid probe of the present invention, such support is another fixed probe, preferably a probe that specifically hybridizes with a nucleic acid encoding hSK, or a variant thereof. , Or a sequence complementary thereto, more preferably
A probe that specifically hybridizes with a nucleic acid of 240 nucleotides (SEQ ID NO: 22) encoding the 80 amino acid conserved region of hSK1 can also be included.

C) Amplification of hSK cDNA Another object of the present invention is a method for amplifying a nucleic acid encoding hSK, which comprises (a) a target hSK nucleic acid, a fragment or variant thereof, or a sequence complementary thereto. Mixing a test sample suspected of containing with an amplification reaction reagent comprising a pair of amplification primers disclosed herein capable of hybridizing under stringent conditions to the hSK nucleic acid region undergoing amplification. , (B) optionally comprising the step of detecting an amplification product.

In a first preferred embodiment of the above method, the nucleic acid encodes the hSK polypeptide of SEQ ID NO: 3.

In a second preferred embodiment of the above amplification method, the amplification product is detected by hybridization with a labeled probe which has a sequence complementary to the amplified region.

The present invention provides a kit for amplifying a nucleic acid encoding hSK, a fragment or variant thereof, or a sequence complementary thereto in a test sample, comprising: (a) stringency of the hSK nucleic acid to be amplified. A pair of oligonucleotide primers disclosed in the present invention capable of hybridizing under various conditions, (b) optionally also comprising an amplification kit comprising reagents necessary for carrying out an amplification reaction. .

In a first preferred embodiment of the above kit, the nucleic acid undergoing amplification encodes the hSK polypeptide of SEQ ID NO: 3.

In the second preferred embodiment of the above amplification kit, the amplification product is detected by hybridization with a labeled probe having a sequence complementary to the amplification region.

D) Recombinant Vectors and Host Cells for Expression of Recombinant hSK 1) Recombinant Vectors The present invention also includes a family of recombinant vectors comprising any one of the nucleic acids described herein. Include. First, the invention provides (a) a purified or isolated nucleic acid encoding a hSK polypeptide, more preferably a polypeptide having at least 80% amino acid identity with the polypeptide of SEQ ID NO: 3, or its complement. Sequence, or (b) a purified or isolated polynucleotide comprising at least 10 consecutive nucleotides of the nucleic acid described in (a), or a nucleic acid selected from the group consisting of sequences complementary thereto. It relates to a recombinant vector comprising.

In a first preferred embodiment, the recombinant vector of the invention is used to introduce an inserted polynucleotide derived from a nucleic acid encoding a hSK polypeptide into a suitable host cell, which polynucleotide is recombinant. It is amplified each time the vector replicates.

Recombinant expression vectors comprising nucleic acids encoding the hSK polypeptides described herein are also part of this invention.

Another preferred embodiment of the recombinant vector according to the present invention comprises a nucleic acid encoding the hSK polypeptide of the present invention, more preferably a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 3. It consists of an expression vector.

A preferred vector comprises the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the expression vector can be used to express a recombinant hSK polypeptide, which can then be purified and used, for example, as an immunogen to raise specific antibodies.

Preferred eukaryotic vectors of the invention are given by way of illustration and not limitation: pcDNA3, pFLAG, pCMV-Script, pIND, pMC1NEO, pHIL, pGAPZA, pMT / V5-Hi.
s-TOPO, pMT / V5-His, pAc5.1 / V5-HisA, pDS47 / V5-His, pcDNA4, pcDNA6, pEF1,
pEF4, pEF6, pUB6, pZeoSV2, pRc / CMv2, pcDM8, pCR3.1, pDisplay, pSecTag2,
pVP22, pEMZ, pCMV / Zeo, pSinRep5, pCEP, pREP, pHook-1.

[0058]   Most preferred are the vectors pcDNA3, pFLAG, and pCMV (particularly pCMV5).

Preferred bacteriophage recombinant vectors of the present invention are, for example, Sternberg N.
P1 bacteriophage vector as described by L. (1992; 1994).

Suitable vectors for the expression of recombinant hSK are baculovirus vectors that can be propagated in insect cells and insect cell lines such as Sf9 and Sf21. Specific suitable host vectors include pFastBac-1, pIZ / V5-His, pBacMan-1, pBlueBac4.5,
Examples include pBlueBacHis2, pMelBacA, pVL1392, pVL1393, but are not limited thereto.

[0061]   A preferred baculovirus vector is pFastBacHTa.

[0062]   A preferred bacterial vector is pGEX.

A) Regulatory Expression Sequences Expression requires the supply of appropriate signals to the vector, which signals enhancers from viral and mammalian sources that drive the expression of the gene of interest in host cells. / Contains various regulatory elements such as promoters. The regulatory sequences of the expression vector of the invention are operably linked to the nucleic acid encoding the recombinant hSK.

The term “operably linked” as used herein refers to the association of polynucleotide elements in a functional relationship. For example,
If the promoter or enhancer affects the transcription of the coding sequence,
Operably linked to the coding sequence.

More precisely, two DNA molecules, such as a polynucleotide containing a promoter region and a polynucleotide encoding the desired polypeptide or polynucleotide, are linked to each other in a manner such that the two polynucleotides are linked. If the property does not (1) result in the introduction of a frameshift mutation, or (2) interfere with the ability of the polynucleotide comprising the promoter to direct the transcription of the encoding polynucleotide, then it is `` operably linked. It is said that

Recombinant expression vectors generally include an origin of replication, a selectable marker for transformation of host cells, and a promoter from a highly expressed gene that directs transcription of a downstream structural sequence. The heterologous structural sequence is assembled in frame with translation, initiation and termination sequences, preferably a leader sequence capable of directing the sequence of the translated protein into the periplasmic space or extracellular medium.

In certain aspects of adapting a vector for transfection and expression of a desired sequence in a eukaryotic host cell, a preferred vector comprises an origin of replication from the desired host, a suitable promoter, and enhancer, and is optional. It also comprises the necessary ribosome binding site, polyadenylation site, transcription termination sequence, and optionally 5'-flanking nontranscribed sequences.

DNA sequences from the SV40 viral genome, such as the SV40 origin early promoter, enhancer, and polyadenylation site, can be used to provide the necessary nontranscribed genetic elements, and another suitable promoter is It is a CMV promoter.

B) Promoter Sequence A suitable promoter region for use in the expression vector according to the invention is chosen in view of the host cell in which the heterologous nucleic acid has to be expressed.

A suitable promoter may be heterologous with respect to the nucleic acid for which the promoter controls expression, or alternatively may be endogenous to the original polynucleotide containing the coding sequence that effects expression.

Furthermore, the promoter is generally heterologous with respect to the recombinant vector sequence into which the structural promoter / coding sequence has been inserted.

2) Recombinant host cells Host cells described herein transformed or transfected with one of the nucleic acids described herein or one of the recombinant vectors, in particular recombinant expression vectors. Are also part of the present invention.

Also included are host cells transformed (prokaryotic cells) or transfected (eukaryotic cells) with a recombinant vector such as one of the above. A preferred host cell used as a recipient of the expression vector of the present invention is (1) a prokaryotic host cell: a bacterial cell, more specifically an Escherichia coli strain (that is, BL21, DH10 Bac strain) Bacillus subtilis, Salmonella typhimurium, and Pseud
strains derived from species such as omonas, Streptomyces and Staphylococcus, Sf-9 cells (ATCC No.
CRL 1711), Sf21 cells, (2) eukaryotic host cells: HeLa cells (ATCC No. CCL2; No. CCL2.1; No. CCL 2.2), Cv
1 cell (ATCC No. CCL70), COS cell (ATCC No. CRL 1650; No. CRL 1651), C127 cell (ATCC No. CRL-1804), 3T3 cell (ATCC No. CRL-6361), CHO cell (ATCC No. CCL-
61), human kidney 293 cells (ATCC No. 45504; No. CRL-1573), BHK (ECACC No. 84100 5)
01; No. 84111301), and hi-5 cells.

More specifically, expression of the recombinant hSK of the invention in COS-7 or bacterial cells is a preferred aspect of the invention.

The results described in the examples show that Cos 7 and bacterial expression are suitable for the production of significant amounts of sphingosine kinase.

E) Production of Recombinant hSK The present invention provides a method of producing a polypeptide having one of the amino acid sequences described herein, in particular SEQ ID NO: 3, which comprises (a) suitable A vector or a host cell into which a nucleic acid encoding a desired amino acid sequence is inserted, (b) a step of culturing a host cell previously transformed or transfected with the recombinant vector of step (a) in a suitable medium. , (C) collecting the medium thus obtained, or lysing the host cells by, for example, sonication or osmotic shock, (d) a host cell lysis product produced from or produced in the above medium And a step of separating or purifying the recombinant polypeptide of interest thus produced from the pellet of.

In some cases, it may be necessary to tag the recombinant hSK prior to purification.
This tag is then most often encoded in the nucleotide sequence required to express the polypeptide. Examples of such tags are C-myc, FLAG,
Examples include, but are not limited to, sequences of histidine residues, sequences encoding hemagglutin A, V5, Xpress or GST. Most of these tags can be incorporated directly into the sequence, such as by PCR amplification by incorporating the appropriate coding sequence in one of the PCR amplification primers.

One preferred tag is the FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, SEQ ID NO: 23) used to express the recombinant hSK of the invention as a fusion protein. Is. Both amino-terminal and carboxy-terminal FLAG fusion proteins are within the scope of the invention. In a preferred embodiment, the FLAG fusion protein is pCMV
-5 produced by a vector that is a derivative of the vector. More specifically, pFLA
Amino-terminal tagging can be performed using the G-CMV-1 or pFLAG-CMV-2 vector, and carboxy-terminal tagging can be performed using the pFLAG-CMV-5a, -5b or -5c vector. .

However, the tag may also be introduced by other means such as covalent attachment of the appropriate nucleic acid sequence encoding the tag moiety to the 3'or 5'end of the nucleic acid sequence encoding the polypeptide sequence. This is true for GST.

Purification of the recombinant hSK according to the invention is then carried out by passing through a nickel or copper affinity chromatography column such as a Ni NTA column.

In another embodiment of the above method, the polypeptide thus produced is bound to an immunoaffinity chromatography column pre-immobilized with a polyclonal or monoclonal antibody directed against the target hSK, for example. Further characterize.

According to these results, the production rate is higher for bacterial expression than for insect cell expression.

F) Purified Recombinant hSK Another subject of the invention consists of a purified or isolated recombinant polypeptide comprising the amino acid sequence of hSK.

A preferred isolated recombinant polypeptide of the invention comprises at least 80%, preferably 90%, more preferably 95% of the polypeptide having the amino acid sequence of SEQ ID NO: 3.
, And most preferably those having 98 or 99% amino acid identity.

Extracts of infected insect cells expressing tagged hSK1 can be purified by a resin column having affinity for the tag.

In a specific embodiment, the extract of infected insect cells expressing 6His-tagged hSK1 is NI-N.
Pass through the TA resin column.

In another embodiment, the extract of infected insect cells expressing GST-tagged hSK1 is
Purify in glutathione resin.

G) Modified Recombinant hSK The present invention relates to any one of the amino acid sequences of the present invention with 1, 2, 3, 4, 5, 1
It also relates to a recombinant hSK polypeptide comprising amino acid changes ranging from 0, 20, 25, 30, 35, 40 substitutions, 1 amino acid additions or deletions.

The amino acid changes included are those that do not destroy the biological activity of the resulting modified polypeptide. These equivalent amino acids are derived from their structural homology to the first amino acid making the substitution, their net charge or their hydrophobic similarity, and, in some cases, the parent peptides and their modified homologs. It can be determined by the result of cross immunogenicity with the object.

Alternatively, in the case of amino acid substitutions in the amino acid sequence of the polypeptides according to the invention, one or several contiguous or non-contiguous amino acids are replaced by "equivalent" amino acids. The expression "equivalent" amino acid replaces herein one of the amino acids belonging to the original protein structure without diminishing the binding properties of the corresponding peptide to the antibody raised against the polypeptide of the invention. Used to represent any amino acid that can be. In other words, an "equivalent" amino acid is one that is capable of producing or synthesizing a polypeptide having a modified sequence when compared to the amino acid sequence of the recombinant hSK polypeptide of interest, wherein the modified polypeptide Can bind to an antibody raised against the target recombinant hSK and / or can induce an antibody that recognizes the parent polypeptide.

A peptide containing one or several “equivalent” amino acids must retain the specificity and affinity of the parent protein for a biological target, as can be assessed by a ligand binding assay or an ELISA assay. I won't.

Examples of amino acids belonging to a specific class include acidic (Asp, Glu) and basic (Lys,
Arg, His), non-polar (Ala, Val, Leu, Ile, Pro, Met, Phe, Trp), or uncharged polar (Gly, Seu, Thr, Lys, Tyr, Asn, Gln) amino acids.

Preferably, the amino acid substitution in the recombinant hSK or peptide fragment thereof of the present invention is to replace an amino acid of a particular class with another amino acid belonging to the same class.

By the equivalent amino acid according to the present invention, the substitution of the L-type residue with the D-type residue or the substitution of the glutamic acid (E) residue with the pyro-glutamic acid compound is possible. The synthesis of peptides containing at least one D-type residue is described, for example, by Koch (1977).

[0095]   Specific embodiments of the target modified peptide according to the present invention include proteolysis.
Peptide molecules that are resistant to A. niger, but are not limited thereto. This is -CONH-
The peptide bond is modified and (CH₂(NH) reduced bond, (NHCO) inversion bond, (CH₂-
O) methyleneoxy bond, (CH₂S) thiomethylene bond, (CH₂CH₂) Carba bond, (CO-CH ₂ ) Cetomethylene bond, (CHOH-CH₂) Hydroxyethylene bond, (N-N) bond, E-al
It is a peptide substituted by a sen bond or a -CH = CH bond.

The present invention also includes a recombinant hSK in which at least one peptide bond has been modified as described above.

The polypeptides according to the invention can be prepared by conventional chemical synthesis methods, either in homogeneous solution or in solid phase. An exemplary embodiment of such a chemical polypeptide synthesis procedure is the homogeneous solution method described by Houbenweyl (1974).

Recombinant hSK or fragments thereof of interest may be chemically synthesized in liquid or solid phase, eg by sequential coupling of various amino acid residues to be incorporated (in the liquid phase from the N-terminus to the C-terminus, or In the solid phase, from the C-terminal to the N-terminal), the N-terminal and the reactive side chain can be prepared by a method in which the N-terminal and the reactive side chain are previously blocked by a usual group.

For solid-phase synthesis, the procedure described by Merrifield (1965a; 1965b) can be used in particular.

H) Antibody Production The recombinant hSK of the present invention and the peptide fragment of interest can be used for the preparation of antibodies.

Polyclonal antibodies are obtained by affinity chromatography in which a polypeptide used as an antigen is pre-immobilized after immunization of a mammal, in particular rabbit, sheep, donkey, horse, or goat with a polypeptide according to the present invention conjugated with an adjuvant for immunization. It can be prepared by purifying the specific antibody contained in the serum of the immunized animal on the chromatography column.

Monoclonal antibodies of mammalian, in particular mouse or rat origin are described by Kohler and Mil.
It can be prepared from hybridomas by the method described by Stein (1975).

The present invention also relates to antibodies produced by the trioma method and the human B-cell hybridoma method as described by Kozbor et al. (1983).

As the antibody of the present invention, a chimeric single chain Fv antibody fragment (US Pat. No. 4,946,778; Martineau et al., (1998), Phage Display Library (Ridder et al., 1995)
And the humanized antibody (Leger et al., 1997).

I) Assays for screening for inhibitors of hSK Sphingosine kinase converts the substrate sphingosine to sphingosine-1-phosphate (S1P). S1P is thought to play several roles in physiological processes. Some of the possible physiological roles of S1P are: 1) intracellular release of calcium from depots, activation of cyclin-dependent kinases, key signal intermediates in the Fc receptor-initiated cascade. , FMLP-induced enzyme release, TNF-α-induced (endothelial cell) adhesion molecule expression, and decreased excitability in ventricular myocytes.

Sphingosine kinase regulates Fc by regulating the balance of counterregulatory lipids.
ξ It appears to play a pivotal role in activation of the RI-initiated signal cascade (Prieschl et al., 1999).

Furthermore, PDGF (platelet-derived growth factor) induces high levels of sphingosine kinase activity and S1P development in platelets (Yatomi et al., 1997; Yatomi et al., 1995).

In human umbilical vein endothelial cells, the pleiotropic cytokine TNFα induces sphingosine kinase activation and S1P development, which in turn mediates TNFα-induced endothelial cell activation and adhesion molecule expression. Can act as a second messenger (Xia et al., 1998).

In osteoblasts, sphingosine monophosphate also plays a second messenger role for TNFα-induced IL-6 (interleukin 6) synthesis (Tokuda et al., 1999). These properties suggest a potentially important role for sphingosine kinase itself in S1P, and thus pain, as well as inflammation, especially following inflammation.

S1P has also been shown to protect from apoptosis. More specifically, S1P prevents the appearance of intranucleosome DNA divisions and morphological changes that are a major feature of apoptosis (Spiegel et al., 1998).

Furthermore, S1P is a key mediator of the mitogenic effect of oxLDL (oxidized low density lipoprotein), which is involved in various biological events leading to atherosclerotic lesions (Auge et al., 1999).

As a result, sphingosine kinase may play a role in conditions such as congestion, thrombosis, stroke, atherosclerosis, coronary artery disease, and dyslipidemia.

High cell concentrations of sphingosine act as potent inhibitors of immunoglobulin (Ig) E + antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. To do. In contrast, high intracellular concentrations of sphingosine-1-phosphate, which are also secreted by mast cells stimulated by allergy, activate the mitogen-activated protein kinase pathway, releasing hexose aminidase and leukotrienes. Or in combination with ionomycin to produce cytokines. Therefore,
The balance of sphingosine and S1P regulates allergic responsiveness of mast cells (Prieschl et al., 1999).

As a result, inhibitors of sphingosine kinase may be useful in preventing allergic reactions.

It has previously been shown that sphingosine kinase activity is stimulated by tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate.
Therefore, excessive stimulation of sphingosine kinase activity may cause proliferative disorders. On the other hand, inhibition of sphingosine kinase prevented the survival effect of 1α, 25-dihydroxyvitamin D3 (1,25- (OH) 2D3), a cytoprotective agent on human promyelocytic leukemia HL-60 cells ( Kleuser et al., 1998). Therefore, sphingosine kinase inhibitors may be useful for the prevention and treatment of proliferative diseases such as cancer and hematopoietic diseases such as leukemia.

[0116] 2) When released from cells,   Proliferation,   Chemotaxis (attraction and activation of macrophages),   Cytoskeletal changes (stress fiber formation and cell shape contraction, aggregation and secretion);   Mediates adhesion: fibronectin matrix assembly, and   The assembly of baxillin and p125-FAK.

[0117] More specifically, sphingosine kinase, GPCRs - plays a role in the Ca ²⁺ release by Ca ²⁺ signaling induced by (G-protein coupling and receptor) (Meyer et al., 1998).

S1P is released in large amounts from activated platelets (Yatomi et al., 1995). this is,
It suggests a potential role for S1P in thrombosis, congestion, natural wound healing processes, atherosclerosis, stroke, and myocardial infarction.

Furthermore, S1P stimulates the binding of fibronectin or its N-terminal 70-kd fragment to cells. The organization of fibronectin into the extracellular matrix is a tightly regulated process, mediated by the reversible binding of fibronectin's 70-kd N-terminal region to its initial specific cell surface binding site. Insolubilized in fibrils. The adhesion information that exists after insolubilization of fibronectin plays a central role in various physiological and pathophysiological processes such as embryogenesis, wound healing, inflammation, and degenerative disease processes such as atherosclerosis and fibrosis. Is assumed (Windh et al., 1999).

More specifically, S1P may have a suitable role in early atherosclerosis and fibrosis. As a result, suitable sphingosine kinase inhibitors are suitable for cardiovascular diseases such as atherosclerosis, thrombosis and dyslipidemia, diabetes such as type I and type II diabetes, especially type I diabetes, stroke, multiple sclerosis, psoriasis. , Erythema epidermophytosis and inflammatory arthritis, T helper-1 related diseases, chronic obstructive pulmonary disease, asthma,
It may be useful in the treatment of cancer and autoimmune and inflammatory diseases such as neurodegenerative diseases.

Isolation of the nucleotide sequence encoding human sphingosine kinase is useful in allowing the skilled practitioner to screen for suitable sphingosine kinase inhibitors. These inhibitors or structural analogs thereof can be used to treat or prevent one and / or some of the above disease states. The term “structural analog” has a chemical backbone in common with the initial inhibitor identified by the screening assays of the invention, but which has a biological activity, reduced side effects, improved solubility, improved bioavailability, etc. It is intended to represent compounds with substituents that have been modified in order to improve or enhance the properties of such early inhibitors.

Several assay formats can be used to perform the methods of the invention.
A preferred assay format is scintillation proximity assay (scint
illation proximity assay (SPA) or flashplate assay (flashplate)
scintillation assay such as assay). Other assay formats well known to those of skill in the art, such as filter binding assays and centrifugation assays,
It is considered in the present invention. SPA and flash plate assays are the preferred assay formats of the present invention. Further details of these assays are provided below.

The scintillation assay involves the use of scintillant beads (for SPA assay) or plates (for flash plate assay). SPA beads are usually cerium-doped yttrium ion silicate (y2SiO5: Ce),
Alternatively, it is manufactured from polyvinyltoluene (PVT) containing an organic scintillant such as PPO. Commonly used flash plates are like Ni chelate flash plates, but other flash plates can be used.

Assays typically use radioactive isotopes, such as ³ H, ¹²⁵ I, ¹⁴ C, ³⁵ S, or ³³ P, which emit low-energy radiation, the energy of which is readily dissipated in aqueous environments. Performed in an aqueous buffer. For example, the electrons emitted by ³ H only have an average energy of 6 KeV, and the path length in water is very short (-1 to tm). When a molecule labeled with one of these isotopes binds directly to the bead or flashplate surface or through interaction with other molecules previously coupled to the bead or flashplate, the radiation activates the scintillant, which causes photoluminescence. Cause The amount of light produced, which is proportional to the amount of labeled molecule bound to the beads, can be conveniently measured with a liquid scintillation (LS) counter. When the labeled molecule does not bind to the beads or flash plate surface, its radiant energy is absorbed by the surrounding aqueous solvent before reaching the beads and no light is generated. Thus, the bound ligand gives a scintillation signal, whereas the free ligand does not give a scintillation signal, obviating the need for the time consuming separation steps characteristic of conventional radioligand binding assays. The manipulations required for these assays are reduced to a few simple pipetting steps, further improving accuracy and reproducibility.

In the context of the present invention, one of the preferred embodiments of this assay includes the binding of sphingosine to SPA beads or flash plates. The coupling is preferably done via BSA, but other coupling means are also conceivable. The assay medium is
It comprises recombinant hSK and labeled ATP. What is measured is recombinant hSK via labeled ATP
Is the ability of the candidate ligand to prevent the conversion of sphingosine into labeled S1P by phosphorylation of sphingosine with. When the candidate ligand inhibits recombinant hSK, sphingosine conversion does not occur and a signal is recorded that is not substantially different from the background noise signal. On the other hand, when hSK inhibition does not occur, sphingosine conversion occurs and the signal resulting from the interaction of labeled S1P with the flashplate or SPA beads is recorded.

J) Antisense An oligonucleotide comprising an antisense strand of human sphingosine kinase type 1, ie RNA such as genomic DNA, cDNA or RNA / DNA hybrid sequences, D
NA is used to inhibit sphingosine kinase expression in vitro or in vivo. Therefore, inhibition of sphingosine kinase expression allows the study of the effects of hSK1 in cells, tissues or animals.

K) Knockout animal SK determined by the inventors of the region conserved between the 80 amino acids contained in SK, the nucleic acid part or part thereof encoding the conserved region of 80 amino acids. It is possible to design a polynucleotide structure in which is deleted.

In a preferred embodiment, the polynucleotide structure as described above has a deletion of at least a portion of the nucleic acid portion encoding a region of conserved 80 amino acids and the deleted nucleic acid portion is a heterologous polynucleotide. It includes a genomic polynucleotide encoding SK that has been replaced by a sequence.

The above structure may be included in a vector for the purpose of replacing a portion of the naturally occurring sphingosine kinase sequence within the mammalian genome by homologous recombination.

According to this embodiment, such recombinant vectors of the invention can be used to generate knockout animals, preferably knockout mammals, most preferably knockout mice and rats.

[0131] In the first embodiment of the above-mentioned nucleic acid, the genomic polynucleotide is human, mouse or rat SK, in which a nucleic acid portion encoding a region conserved of 80 amino acids or a part thereof is deleted. Code things.

In a second aspect of the above nucleic acid, the heterologous polynucleotide comprises a selectable marker.

In a third aspect of the above nucleic acid, the heterologous polynucleotide is at least at its 5'end.
It comprises one loxP sequence and at least one loxP sequence at its 3'end. The loxP sequence consists of two 13-bp palindromic sequences separated by an 8-bp conserved sequence (HOE
SS et al., 1986).

Recombination by the Cre enzyme between two loxP sites with the same orientation results in the deletion of the DNA fragment. The Cre-loxP system used in combination with the homologous recombination method is described by GU et al. (1993, 1994).
It is described by.

Vectors containing a genomic SK sequence that lacks a sequence encoding a region of 80 amino acids conserved, or a portion thereof, are designed so that the selectable marker is flanked by loxP sites in the same orientation. It HS of interest inserted by homologous recombination event
While rearranging the K genomic polynucleotide, it is possible to remove the selectable marker by treatment with the Cre enzyme.

Two selectable markers are required: a positive selectable marker to select for recombination events and a negative selectable marker to select for homologous recombination events.
Vectors and methods using the Cre-loxP system are described by ZOU et al. (1994).

A particular feature of a nucleic acid of the invention, wherein the nucleic acid comprises a genomic polynucleotide encoding mouse SK lacking a portion of the nucleic acid encoding a region conserved of 80 amino acids or a portion thereof. In aspects, one of skill in the art may advantageously refer to the following examples.

In a further aspect of the invention, a nucleic acid encoding a polypeptide as defined above is operably linked to regulatory sequences.

[0139]   Preferably, the regulatory sequence consists of an inducible promoter.

Most preferably, the regulatory sequence consists of a promoter inducible by ponasterone.

Examples Materials and Methods Growth medium with all supplements was purchased from Gibco BRL (Paris, France).
Transfection reagent was from QIAGEN (Paris, France). All lipids were purchased from Sigma-Aldrich (Paris, France). [α ³² P] ATP and [γ ³³ P] ATP
Was from Amersham Pharmacia Biotech (Amersham, Paris, France). Human poly A + RNA multiple tissue northern blotting, and pcDNA3 mammalian expression vector
Purchased from LONTECH (Palo Alto, California, USA). Restriction enzymes were purchased from New England Biolabs (Beverley, Massachusetts, USA). EST-IMAGE clone 1946069 was purchased from the UK HGMP Resource Center (Hinxton Cambridge, UK).

COS7 cells (monkey fibroblasts) were treated with 10% at 37 ° C., 6.5% carbon dioxide in a water saturated atmosphere.
Growing in Dulbecco's modified Eagle medium containing 4500 mg / L glucose supplemented with fetal bovine serum, 2 mM glutamine, 10 IU / ml penicillin, and 10 mg / ml streptomycin.

Example 1: Isolation of human sphingosine kinase (hSK1) cDNA The recently cloned rodent sphingosine kinase cDNA sequence (Kohama et al., 199).
A study with 8) identified human Est clusters and several human Est sequences in the public (Unigene) and local (Compugen) cDNA cluster databases. The insert of IMAGE clone 1946069, a member of the cluster, was sequenced and pc
Subcloned into DNA3 mammalian expression vector.

The 1.7 kb insert showed a high level of similarity to the mouse SK1a cDNA (76%),
Covered the whole code area. Peptide sequence alignment of mouse and human sequences and biological activity of the expressed enzyme was determined by IMAGE clone 1946069 insert human SKcD.
It is suggested to include the coding region of NA. This is based on Kohama et al. (Kohama et al. 1998
3) is identical to the partial human peptide sequence derived from the Est sequence.

The cDNA and peptide sequences of hSK1 are shown in FIG. The open reading frame of cDNA is 1155 nt. The translation initiator ATG is a partial Kozak consensus (Kozak, 1987).

[0146]   PCR primers are as follows.   Primers A and B: A = 5 'end TAT GCT AGC ATG GAT CCA GCG GGC GGC (SEQ ID NO: 4) B = 3 'end AAT GAA TTC TCA TAA GGG CTC TTC TGG (SEQ ID NO: 5)   Primers C and D: C = 5 'end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ ID NO: 6) D = 3 ′ end ATT ATC GTC GAC TAA GGG CTC TTC TGG CGG (SEQ ID NO: 7).

[0147]   These primers are used for cDNA amplification such as PCR amplification.

Example 2: Characterization of sphingosine kinase The predicted peptide sequence is 384aa (SEQ ID NO: 3), the expected mass is 42.5kD and the pI at neutral pH is 6.9. The similarity to mouse SK1a is 85% (Needleman
Wunsch similarity index). Except for the C-terminus, the similarity with mouse SK is continuous.

Peptide similarity studies identified an 80aa conserved region (Arg16-Pro95) (SEQ ID NO: 8) contained in various known and hypothetical peptides from bacteria against humans (CA
B14972 Bacillus subtilis; CAA18718 Arabidobsis thaliana; CAB11477 Saccar
omyces pombe; S51398 Saccaromyces cerevisae; SS67059 Saccaromyces cerevi
sae; CAA91259 Caenorhabditis elegans; AAC67466 Caenorhabditis elegans; M
ouse SK1a (Mouse SPHK 1a); hSK1 (huSPHK)). Conserved amino acids are shown in black (
(Figure 2B).

This sequence encompasses the distance-separated regions for the short signal peptide LVRSEELGRWDALVVM (SEQ ID NO: 9) of the NADPH-dependent aldo-ketoreductase family of enzymes. Within the 80aa conserved region, highly conserved residues mark the apparently unique and predictable features of secondary peptide structure in 3 blocks (Fig. 2A). The conserved Asn22-Pro23 and Gly26 residues are promising proximal to the GGKGK sequence (SEQ ID NO: 24) that may be part of the suggested ATP binding site for mouse SK1 (Kohama et al., 1998). The β-turn and coil structure are shown. It has been pointed out that His59-Ala60 is exposed on the surface, and Gly80-Asp81-Gly82 suggests the presence of a flexible region. Asn22-Pro23 and Gly26 in block 1 of storage area, Thr50, H in block 2
The spatial arrangement of is59-Ala60 and Gly80-Asp81-Gly82, Glu86 and Gly90 residues in block 3 is identical from Bacillus subtilis to human.

Example 3: Transfection of hSK1 COS7 cells were transiently transfected with vector pcDNA3 alone or vector containing human sphingosine kinase cDNA using Qiagen reagent SuperFect. Cells were seeded in ^6- well plates at 5x10 ⁶ cells / well. After 24 hours, add 20 μl of cells
Transfection was performed with 10 μg of vector (pcDNA3) mixed with SuperFect or 10 μg of vector containing human sphingosine kinase cDNA (pcDNA3-hSK1) mixed with 20 μl of SuperFect.

Example 4: Sphingosine Kinase Activity and Specificity Assay Sphingosine kinase activity was assayed as previously described (Kohama et al., 1998).
Was analyzed. Briefly, sphingosine kinase activity was measured using 50 μM sphingosine, 0.25% Triton X-100, and [ ³³ P] ATP (10 μCi, 1 mM), and MgCl ₂ (10 mM).
) Was measured by incubating the cell extract for 30 minutes at 37 ° C. Kinase activity was expressed as nanomoles of SPP / min / mg.

Characterization of hSK1 Activity and Substrate To confirm that hSK is indeed a functional sphingosine kinase, COS7
Cells were transfected with vector pcDNA3 containing hSK1 cDNA, and after 48 hours, sphingosine kinase activity was measured. Only low levels of endogenous sphingosine kinase activity were present in control cells (untransfected or vector-only transfected). However, hSK (with 10 μg DNA)
The cells transfected with A.p. produced 107-fold or more increased sphingosine kinase activity (Fig. 3).

FIG. 3 shows that hSK1 specifically phosphorylates D-erythro-sphingosine (D-erythro-SPH) and lowers D, L-erythro-dihydrosphingosine (D, L-erythro-DHS) to a lesser extent. It shows that only phosphorylation occurs. This kinase is a "threo" isoform of dihydrosphingosine (D, L-threo-dihydrosphingosine, L-threo-dihydrosphingosine, L-threo-dihydrosphingosine), ceramide (hydroxy-ceramide, non-hydroxy-ceramide), diacyl. Glycerol
(DAG), phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), or phosphatidylinositol-4,5-bisphosphate (PIP2)
Neither of them is phosphorylated.

The substrate specificity of the expressed hSK was determined by the purified rat sphingosine kinase (Oliver
a et al., 1998) and recently cloned mouse sphingosine kinase (Kohama
Et al., 1998). The best substrate was D-(+)-erythro-sphingosine, followed by D, L-erythro-dihydrosphingosine, which was the phosphorylation achieved for the observed D-(+)-erythro-sphingosine. Phosphorylated to 50% of level.

Substrate specificity and competitive inhibition of hSK1 Expressed hSK1 has a typical Michaelis-Menten kinetics (vMax = 56 nM / min / mg,
And Km = 5 μM) (FIGS. 4A (4A, 4A2) and 4B (4B1, 4B2)). D, L-threo-dihydrosphingosine (Fig.4A (4A, 4A2), DHS) and N, N-dimethyl-sphingosine (N, NdiMS)
Is a known inhibitor of sphingosine kinase (Kohama et al., 1998; Olivera et al.
, 1998). In this regard, the present inventors have shown that hSK1 expressed by any of these compounds
It shows that the activity is inhibited. The kinase is D, L-threo-dihydrosphingosine (
Ki = 3 μM) and N, N-dimethylsphingosine (Ki = 5 μM).

Example 5: hSK Structure Fused to EGFP (Enhanced Green Fluorescent Protein) To characterize and understand the possible mechanisms that regulate hSK1 activity and cellular localization, we present the kinase Two types of hSK structures fused to EGFP (enhanced green fluorescent protein) at either end were created.

Construction of EGFP-hSK1 (N-terminal fusion). In order to align the EGFP and hSK1 in a mammalian expression PCR reaction and create a fusion protein in frame, pcDNA3-hSK1 structure as a template and cloning restriction sites (NheI-Eco
RI) was inserted at the same time and primers were designed to amplify the coding sequence of hSK1. The structure has EGFP at the N-terminus of hSK1. Primers A and B: A = 5 ′ end TAT GCT AGC ATG GAT CCA GCG GGC GGC (SEQ ID NO: 4) B = 3 ′ end AAT GAA TTC TCA TAA GGG CTC TTC TGG (SEQ ID NO: 5)

Construction of EGFP-hSK1 (C-terminal fusion). The mammalian expression PCR reaction was performed by aligning the hSK1 sequence with EGFP and creating a fusion protein in frame by using pcDNA3-hSK1 structure as a template and cloning restriction sites (NheI
-EcoRI) was inserted at the same time and primers were designed to amplify the coding sequence of hSK1. The structure has EGFP at the C-terminus of hSK1. Primers C and D: C = 5 ′ end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ ID NO: 6) D = 3 ′ end ATT ATC GTC GAC TAA GGG CTC TTC TGG CGG (SEQ ID NO: 7)

Transfection of hSK1 COS7 cells into the vector pcDN using the Qiagen reagent SuperFect described in Example 4.
A3 alone or transiently transfected with a vector containing human sphingosine kinase cDNA.

EGFP-hSK1 Transfection (N-Terminal Fusion) COS7 cells were transiently transfected with vector pCI-EGFP1 alone or vector containing human sphingosine kinase cDNA using SuperFect, Qiagen reagent ( (See Figure 14). Cells were seeded in ^6- well plates at 5x10 ⁶ cells / well. After 24 hours, cells were mixed with 20 μl SuperFect and 10 μg vector (pCI-EGFP1) or 20 μg
10 μg of vector containing human sphingosine kinase cDNA (pCI-EGFP1-hSK1) mixed with l SuperFect was transfected.

FIG. 14 shows a vector for the construction of hSK-EGFP (N-terminal) fusions for expression in mammalian cells. The size of pCI-EGFP is 4724 bp. EGFP coding sequence (716 bp), amplified using EGFP.XbaI (sense) and STOP.EGFP (antisense) primer,
XbaI / XhoI digested and subcloned into pCI digested with NheI and XhoI. The EGFP.STOP codon was deleted. The frame for subcloning the sequence of interest with EGFP fused to the N-terminus is shown at the bottom of the figure.

Transfection of hSK1-EGFP (C-Terminal Fusion) COS7 cells were transiently transfected with vector pCI-EGFP-2 alone or with vector containing human sphingosine kinase cDNA using SuperFect which is Qiagen reagent. (See Figure 15). Cells were seeded in 6-well plates at 5x10 ⁶ cells / well. After 24 hours, cells were mixed with 20 μl SuperFect and 10 μg of vector (pCI-EGFP-2
) Or 20 μl of SuperFect mixed human sphingosine kinase cDNA (pCI-EGFP-
Transfected with 10 μg of vector containing 2-hSK1).

FIG. 15 shows a vector for the construction of hSK-EGFP (C-terminal fusion) for expression in mammalian cells. The size of pCI-EGFP2 is 4733 bp. The EGFP coding sequence (725 bp) was amplified by PCR using EGFP2-TOP (sense) and EGFP2-BOTTOM (antisense) primers, XhoI / NotI digested and subcloned into pCI digested with SalI and NotI. A new SalI site was included in the PCR product. E fused to the C-terminus
The frame for subcloning the sequence of interest in GFP is shown at the bottom of the figure.

The two fusion proteins are well expressed in COS7 cells and the EGFP / hSK1 fusion protein is expressed primarily as a soluble cytoplasmic protein (FIGS. 5A and 5B).

FIG. 5A illustrates the expression and cellular localization of hSK1 fused to EGPF at the N-terminus.
EGFP-hSK1 (N-terminal fusion) is expressed cytoplasmically when transfected into Cos7 cells as indicated by the green color (lightest color in Figure 5A).

On the other hand, hSK1-EGFP appears to be partially localized in the form of granules, but general cytoplasmic expression is also observed (FIG. 5B).

FIG. 5B illustrates expression and cell localization of hSK1 fused to EGPF at the C-terminus.
hSK1-EGFP (C-terminal fusion) was expressed predominantly cytoplasmically with some granule localization when transfected into Cos7 cells, as indicated by the green color (lightest color in Figure 5B). Can be seen.

Kinase assays of cell extracts from cells transfected with either construct show that the EGFP-hSK1 fusion protein is more active than the hSK1-EGFP fusion protein (FIG. 6).

FIG. 6 shows the kinase activity of hSK fusion protein. hSK-EGFP (N-terminal fusion) (EGF
Overexpression of P-hSK1) has similar activity to the overexpressed unfused untagged protein. On the other hand, hSK-EGFP C-terminal fusion (hSK1-EGFP) is unfused or N-
40% lower than the activity of the terminal fusion protein.

Western blots (FIG. 7) and confocal images (FIGS. 5A and 5B) show similar levels of expression for the two proteins, which is a problem for transfection efficiency. is not.

5A and 5B show similar green fluorescence intensities, suggesting similar expression levels for C-terminal and N-terminal fusion proteins.

FIG. 7 is a Western blot analysis with anti-EGFP antibody. Figure 7 shows the C-terminal (
Both hSK1-EGFP) and N-terminal (EGFP-hSK1) hSK / EGFP fusion proteins have been shown to be expressed to similar levels in Cos7 cells.

Example 6: Localization of sphingosine kinase in tissues Northern blots containing about 1 μg / lane per lane of poly A + RNA from 12 different human tissues were hybridized with a 1.7 kb insert of pcDNA3-hSK1 and gel And was labeled with [ ³² P] αATP using a random primer labeling kit. Ex
Hybridization with pressHyb ™ buffer (CLONTECH) was performed according to the manufacturer's instructions. Bands were visualized by autoradiography and quantified by densitometer.

The tissue distribution of hSK1 mRNA expression in human tissues was analyzed by Northern blotting (FIG. 8).

FIG. 8 shows the tissue distribution of hSK1 messenger RNA. Prefabricated Northern blots containing approximately 1 μg / lane of poly A + RNA from 12 different human tissues were hybridized as described below. The numbers at the bottom of each line indicate the level of expression over background and were quantified by densitometry.

It shows maximal expression in adult lung (46-fold above background) and spleen (38-fold), followed by peripheral blood leukocytes (30-fold), thymus (28-fold), and kidney (24-fold). , This is the brain (12
Fold) and heart (11.5 fold). Low levels of expression were found in skeletal muscle (2.6x), colon (2
Fold), liver (1.8 fold), small intestine (1.2 fold), and placenta (1.3 fold). The tissue distribution and expression levels of hSK1 mRNA are generally quite similar to those reported for rodent analogs (Kohama et al., 1998). However, liver mRNA levels were low in both mice and humans, contrary to the finding that SK enzyme activity is 2-fold higher in rat liver compared to brain (Olivera et al., 1998). However, SK
MRNA levels for A. cerevisiae have not been reported in rats. In addition, database searches using the stSG2854 marker suggest expression in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

Example 7: Genomic Localization of Sphingosine Kinase, Associated Diseases Several members of the Unigene cluster Hs.68061 were mapped. These E
Sequence identity and mapping data between sts and hSK suggest that the gene is located on chromosome 17q25.2 band at 9 cM intervals between microsatellite markers D17S785 and D17S836 (104.7 and 114 cM respectively). . This interval is Hs.68061
The STS (stSG2854) identical to the Est sequence of the UniGene cluster is included.

Cloning of hSK1 is an important step in elucidating the role of this enzyme in the signaling pathway transduced by a wide range of receptor coupling mechanisms. In addition, we mapped some members of the Unigene cluster Hs68061.
A region of about 50 cM on 17q25 with stSG2854 shows multiple sclerosis (Kuokkanen et al., 1997).
, Psoriasis and erythematous epidermophytia (Nair et al., 1977; Tomfohrde et al., 1994; Enlu
nd et al., 1999; Ramoz et al., 1999), and several rat models of inflammatory arthritis due to syntenic homology (Remmers et al., 1996). Multiple independent groups have reported associations in psoriasis. Also,
These data are shared by the Becker et al.
d) Identify autoimmune / inflammatory areas (Becker et al., 1998). Due to its expression pattern and biology, SK is a possible susceptibility gene candidate in this region. as a result,
The present invention provides a method for detecting the susceptibility of a mammal expressing autoimmune and inflammatory diseases, wherein the mammalian DNA sequence encoding SK1 is SEQ ID NO: 1 or SEQ ID NO:
A method comprising comparing a DNA sequence of: 2 with a DNA sequence of: 2 to determine the presence of a single nucleotide polymorphism or polymorphic region in the mammalian coding sequence encoding SK1.

Example 8: Sphingosine Kinase Expression in Insect Cells Isolation of Recombinant Bacmid DNA Preparation Sphingosine kinase cDNA was isolated from the manufacturer (Gibco BRL, Gaithersburg (Gai
(Thersburg), Maryland) and cloned into the pFasbac HT donor plasmid. FastBac plasmid DNA and sphingosine kinase cDNA, 1
Prepared by digesting μg of DNA with the selected restriction endonuclease under appropriate conditions. The insert fragment was ligated downstream from the histidine tag into the prepared pFasbac HT vector under appropriate conditions. Recombinant plasmid pFas
The tBac donor plasmid was transformed into DH10Bac for transposition into the bacmid (instructions for use (Gibco BRL, Gaithersburg, MD). Isolation of recombinant bacmid DNA was performed to isolate the desired sequence on white colonies. Selection by PCR The preparation of DNA bacmid was developed specifically for the purpose of isolating large plasmids (> 100 Kb) and bacmidD
It was performed under conditions suitable for the isolation of NA (Quiagen).

Recombinant protein expression in Sf21 cells Sf21 cells were transfected with recombinant bacmid DNA in the presence of Cellfectin reagent. Manufacturers for cell culture, recombinant virus purification, and virus titration
(Gibco BRL, Gaithersburg, MD). For protein expression, cells at a density of 2x106 / ml were infected with recombinant virus at MOI 5-10. Three days after infection, cells were pelleted by centrifugation and homogenization buffer (Bis Tris 20) supplemented with a mixture of protease inhibitors (Boerhinger).
It was recovered in mM (pH 6.5), EDTA 10 mM, DTT 2.5 M). Glycerol was added to a final concentration of 20-30% of the total homogenate, which was then aliquoted and stored at -20 ° C. The gene is cloned into the pFastbac HT expression vector and the expressed protein contains 6X his at its amino terminus and the desired protein can be purified.
The fusion protein was purified in a suitable base buffer system using NI-NTA resin.

Partially purified HSK1 was subcloned into the baculovirus shuttle vector pFastBacHTa which incorporates a sequence for the 6x histidine affinity tag at the N-terminus. This baculovirus construct was mixed with viral DNA and introduced into Sf21 insect cells. Novel recombinant baculovirus was isolated by plaque purification and 5 isolates examined for protein production. The best isolate was selected to produce a high titer virus stock. Sf21 insect cells were infected and cells were harvested 60 hours post infection. Expression of hSK1 was confirmed by Western blot. Infected Sf21 insect cells from culture were used for partial purification of hSK1 with nickel beads.

HSK1 was cloned into pFastBacHTa (Life technologies Cat) using the restriction sites BamHI and PstI.
No. 50322, lot No. KDW704). This structure was confirmed by sequencing. Recombinant hSK1 in pFastBacHTa was used to transform DH10Bac E. coli (containing the bacmid shuttle vector bmon14272). Translocation of the donor plasmid to the acceptor shuttle plasmid was detected by selecting blue / white colonies on X-gal / IPTG plates. White colonies are selected, grown and recombinant Ba
The bac-to-Bac baculovirus expression system was purified by cmid (instructions for use Gibco BRL
Life Technologies).

St21 cells (NERC, Oxford) for virus stocks were grown in shake flasks using 47.5% TC100 (Life Technologies; Catalog No. 13055-025) as a suspension culture.
; Lot No. 3031505) + 5% heat inactivated North American Fetal Bovine Serum (FBS; Life Technologies
Cat.No. 10085-140; Lot No. 06Q6073A) and standard procedures (Kin
g et al., 1992). Sf21 cells in ExCell 401 (lot number 9N3936)
Transfection with recombinant bacmid DNA purified in the presence of Lipofectin (Life Technologies) using standard methods.

[0185]   The medium was harvested 7 days after transfection.

Monolayers of Sf21 cells (3.5 × 10 6 cells / 60 mm dish) were infected with serial dilutions of virus stocks from the transfection mix described above, and the gray tin insect medium (Gra
ces Insect Medium) (2x; Life Technologies), 10% FBS (Life Technologies; heat inactivated North American species; Catalog No. 10085-140; Lot No. 06Q6073A); 1.5% SeaPlaque Agarose (lowgen) solution Was overlaid and stained 4 days after infection with neutral red (6% in PBS; Sigma) (King et al., 1992).

[0187]   FIG. 17 shows a partially purified electrophoretic gel of hSK1 from Sf21 insect cells.

150 ml of Sf21 insect cells were infected with MOI 10 and cells were harvested 60 hours after infection. Samples were prepared and bound to a Ni column. 5 μl of 10 μl from column sample eluate
x 15 μl per well mixed with reducing SDS / PAGE buffer and loaded on 4-12% NuPAGE Bis-Tris Gel. The electrophoresis gel was stained with Coomassie blue. 1 = total cell lysate 2 = lysate after slow spin 3 = flow-through column 4-6 = column wash fractions 1-3 3 7-11 = column elution fractions 1-5

The single band in line 8 corresponds to the expected molecular weight of hSK1, ie about 43 kD. Addition of column elution fractions 7 and 8 represent partially purified hSK1.

Example 9: Construction of sphingosine kinase expressing GST-hSK1 (N-terminal fusion) in bacteria . In the bacterial expression PCR reaction, the PGEX vector containing GST was aligned with hSK1 and the pcDNA3-hSK1 structure as a template and cloning restriction sites (EcoRI-XhoI) were inserted at both ends simultaneously to create a framed fusion protein. , HSK1 using primers designed to amplify the coding sequence (FIG. 16). The construct has GST at the N-terminus of hSK1. Primers E and F: E = 5 'end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ ID NO: 10) F = 3' end AGT CGA GGC TGA TCA GCG AG (SEQ ID NO: 11)

FIG. 16 shows a vector for the construct of GSK-tagged hSK1 for expression in bacterial cells. The hSK1-GST fusion protein was constructed and expressed in bacteria using the PGEX-SX-3 vector (Promega).

E. coli cells E. coli competent cells (BL21 strain) were purchased from Promega. Bacterial transformation was performed according to the supplier's standard transformation protocol.

Frozen competent cells were thawed on ice for 5 minutes and 100 μl were transferred to a chilled culture tube. 50 ng of hSK1-cDNA was added and mixed by flicking the tube. After returning the test tube to ice for 10 minutes, heat shock was performed by placing the test tube in a 42 ° C. water bath for 45 seconds. Immediately, the test tube was placed on ice for 2 minutes. 900 μl cold SOC medium
Was added to the transformation reaction and incubated at 37 ° C with shaking for 60 minutes. Some of the cells were plated on antibiotic-containing plates and incubated at 37 ° C for 12-14 hours.

Example 10: Optimized Sources of Sphingosine Kinase for Screening Assays Selection of Sphingosine Kinase Sources Several sources of hSK were tested. FIG. 9 shows a comparison of hSK activity from various sources: partially purified hSK1 from CHO cells, bacteria, insect cells. Similar levels are observed with mammalian (Cho) and bacterial (BL21) cell extracts. Increased levels of partially purified hSK from insect cell transfection have a dose response effect.

As in FIG. 9 (Y-axis is enzymatic activity at SPA cpm), 2 μg of total protein from bacteria (BL21) and 0.1 μg of partially purified baculovirus / insect cells hSK showed good signal to noise. The ratio (about 12 times) was given.

The total amount of protein required is 400 mg for 2000 plates, which is approximately 10 liters of transformed and induced bacteria.

Experiments were conducted in an effort to identify the best possible solution to generate sufficient recombinant hSK to perform high throughput screening (HTP). Since mammalian transient expression causes many difficulties to produce sufficient enzyme for whole HTS, various bacterial as well as baculovirus expression systems were tested in insect cells. The result is shown in Figure 10.
Shown in.

FIG. 10 shows a comparison of hSK1 activity from various sources: Cos7, bacteria, insect cells.
BL21 Transf. Basal means BL21 transfected without IPTG induction. BL21 Trnas. Induced is BL21 transfected using IPTG induction.
Means P. Pur. RSPHK means partially purified recombinant hSK1.

40 μg of whole cell extract from transfected cos7 cells show 50% greater activity than 40 μg of transfected whole bacterial cell extract. Insect cell extract 40 μg shows minimal hSK1 activity compared to basal levels (Cos7 basal). However, 6 μg of partially purified hSK1 from insect cells was transfected with COS7.
It shows a 3-fold increase compared to the cell extract.

Transfected COS7 represents a positive control for optimal activity. The partially purified enzyme from the baculovirus system exhibits the maximum activity observed to date. However, for the same amount of whole cell extract, bacterial extracts overexpressing hSK represent 40% -50% of the total activity found in mammalian systems, making this system the most attractive solution.

Optimization of Sphingosine Kinase Bacterial Source Experiments were performed to generate sufficient recombinant human sphingosine kinase for HTS. Transfected BL21 bacteria were grown overnight at 25 ° C., yielding active recombinant hSK1 as indicated by the enzymatic activity of the total protein extract. Production was scaled up to produce over 400 mg of total bacterial protein with very good levels of SK activity. To solve the solubility / expression problems seen with recombinant GST-tagged hSK1, a wide range of conditions were set for the induction of bacterial growth and protein expression. Thus, 20 h bacterial growth at room temperature and induction of protein expression by 50 μM IPTG appear to be optimal conditions for expression of significant amounts of active / soluble recombinant GST-tagged-hSK (FIG. 11). .

FIG. 11 shows optimized bacterial growth conditions for actively expressed hSK1. Different IPTG concentrations for induction, different growth temperatures (R ° T means room temperature), different incubation times are tested.

Furthermore, bacterial cell extracts under optimal bacterial growth and induction conditions (50 μM IPTG, 20 h) have an activity of 40% of the maximal activity seen for transfected mammalian cells (Cos cells) (FIG. 12).

FIG. 12 shows a comparison of hSK1 activity expressed under various bacterial growth conditions and in Cos cells.

Example 11: Sphingosine Kinase Antisense Oligonucleotides To illustrate the physiological role of sphingosine kinase 1 in intracellular signaling pathways in immune cells, antisense oligonucleotides corresponding to the first 21 coding nucleotides of hSK1. Was designed to try to suppress the protein and thus its activity. U937 cells were transfected with antisense oligos and the calcium signal was analyzed in a model in which the activating sphingosine kinase was coupled to a previously indicated receptor. Here we show that in antisense-treated cells, calcium release from intracellular stores is impaired and that sphingosine kinase actually plays a key role in receptor-induced physiological responses. ing.

Construction of Antisense Oligonucleotides for hSK1 Antisense sequences for the first 24 nucleotides (encoding the first 8 amino acids) of hSK1-cDNA (the above antisense sequences are sequences GGG GCC GCC CGC CGC T
(GG ATC CAT, having SEQ ID NO: 12) was synthesized and the ends were protected with phosphorothioate linkages for the first and last two nucleotide pairs.

A control “scrambled oligo” (CTGGTGGAAGAAGAGGACGTCCAT, SEQ ID NO: 13) was synthesized and protected at both ends with phosphorothioate linkages for the first and last two nucleotide pairs.

Transfection of antisense oligonucleotides against hSK1 U937 cells were antisense to the first 21 coding nucleotides (encoding the first 7 amino acids) of human sphingosine kinase cDNA using SuperFect, a Qiagen reagent. Transiently transfected with oligonucleotides. 1x10 ⁶ pieces / ml, in 10 ml. Twenty-four hours later, cells were transfected with 2 μg of scrambled antisense oligo (control) mixed with 20 μl SuperFect or 2 μg antisense oligo to human sphingosine kinase mixed with 20 μl SuperFect.

Protein analysis of hSK1 in U937 cells and effect of antisense FIG. 13 illustrates the physiologically relevant role of hSK1 revealed by using antisense oligonucleotides.

FCγRI-induced calcium signals in U937 cells (control) were inhibited in cells treated with antisense to hSK1 for 48 hours.

[0211]   Figure 18 shows antisense suppression of hSKl protein levels.

[0212]   Blots were probed with a polyclonal antibody against hSK1 (Ab 0144).   1 = background   2 = U937 untreated cell extract 100 mg (expression is 100%)   3 = U937 antisense treated cell extract 100 mg (12% compared to untreated cells)

This figure shows that antisense reduces SK1 protein expression levels by 88%.

Example 12: Sphingosine Kinase Knockout Mouse 129 Mouse Strain BAC Library A high density filter set of BAC colonies from RPC22 (Research Genetics) was screened with the following radiolabeled oligonucleotide probes. SK5 'end 49 (gene proximal) CTGGGTCTTGTAGAAGAGCAGCAAGTGCT (SEQ ID NO: 14) SK5' end 48 (gene proximal) AGTTCACTGCAATCCTTTCTTATCTGGGTTCG (SEQ ID NO: 15) SK3 'end (gene distal) TTCTGTGGATGGAGAGCTGATGGTATGG (SEQ ID NO: 16) SKBOX Storage area) ATGAAGTGGTGAATGGGCTAATGGAACG (SEQ ID NO: 17)

Oligonucleotide probes were derived from the mouse SK1 cDNA sequence (Kohama et al.,
1998). Based on the multiple sequences of the SK1-related cDNA sequences (Melendez et al., 2000), oligonucleotides were selected from the two ends of the cDNA (gene proximal and gene distal probes) and a conserved region.

Positive BAC clones were purchased (Research Genetics) and rescreened with gene distal and conserved region probes. Using a conserved region in the hybridization experiment and 3 '(distal gene) radiolabeled oligonucleotide probe,
Neither clone was positive.

Positive clones (using conserved region probes): 83 B 4, 442 C 20, 424 E 5, 46 M 1, 270 B 3, 225 D 6. 3'end (distal gene probe) Positive clone: 61 K 3, 166 L 16, 126 H 16
, 69 D 8, 24 O 6, 203 P 6, 224 A 21, 84 A 12, 387 G 19, 545 H 5, 431 O 1
9, 224 A 23.

The catalytic domain of the enzyme appears to be in a highly conserved region (see SEQ ID NO: 8), which is a peptide sequence downstream of the sequence encoded by the hypothesized alternative first exon (SEQ ID NO: 8). : 3) aa16 to aa95, thus targeting this highly conserved region with ES cells.

The catalytic critical region of human SK is determined by 5'and 3'point truncations and internal deletions. Mouse BAC clones are identified by screening BAC libraries. Mini-libraries were prepared from proven positive clones and these libraries were screened with oligonucleotide probes to obtain genomic fragments encoding the catalytic domain. Sequencing reveals the presence of catalytic critical exons in one genomic fragment. Suitable size (2.5
5'and 3'flanking genomic fragments with -5 Kb) were cloned with oligoprobes or PCR amplified with appropriate primers from cDNA. These fragments are inserted into the pSV-loxP targeting vector in the reverse orientation with respect to the NEO transcription unit (Experiment A). In another experiment (B), loxP sites were inserted next to the catalytic critical exon containing the genomic fragment, and they were also cloned immediately adjacent to the Neo transcription unit, and the regions were 5'and 3'homology homologous for targeting. Adjacent to the arm. Appropriate restriction sites are inserted to create optimal positions for detection of recombination-mediated substitutions of the wild-type catalytic critical region with the loxP site flanking fragment.

The targeting vector is introduced into ES cells by electroporation or other methods. Neomycin resistant colonies are screened to identify specific targeting events. In another possible morphology experiment, transient Cre in ES cells
Using recombinase expression, lox from the targeted allele in Experiment A
Remove the P-flanking Tn-5 neomycin resistance gene. Once we identified ES cell colonies with the targeted allele, placental vesicles from these colonies were identified.
Inject with ES cells. Mice with a high degree of ES cell contribution are screened by the coat color test, and germline-transmitted mice are selected by rearing and tail DNA tests. Once hemizygous targeted mice (SK − / +) were obtained, they were treated with homozygous nulls (n
(ull) allele (SK-/-) tested in biological experiments with mice ("Gene targeting", supervised by AL Joyner, IRL Publishing / Oxford, 19
1993). In experiment B, homozygous insertion positive mice were generated and tissue-specific Cre
It is crossed with a transgenic mouse expressing recombinase. The result of this experiment is
It is a tissue-specific deletion of the SK gene. Deletion of SK is inducible when controlling Cre recombinase by an inducible promoter.

Example 13: hSK Polyclonal Antibodies Four peptide sequences were selected for their apparent hydrophobicity and synthesized. Peptide 1: FTLMLTERRNHARELVRSEE (SEQ ID NO: 18) Peptide 2: VNGLMERPDWETAIQKPLCS (SEQ ID NO: 19) Peptide 3: ADVDLESEKYRRLGEMRFTL (SEQ ID NO: 20) Peptide 4: SGCVEPPPSWKPPQQMPPPEE (SEQ ID NO: 21)

Two rabbits were immunized for each peptide and raised polyclonal antibodies from 8 peptides (2 for each peptide). Peptide 1: Serum number 0140 (rabbit 1); Serum number 0141 (rabbit 2) Peptide 2: Serum number 0142 (rabbit 1); Serum number 0143 (rabbit 2) Peptide 3: Serum number 0144 (rabbit 1); Serum number 0145 (Rabbit 2) Peptide 4: Serum No. 0146 (Rabbit 1); Serum No. 0147 (Rabbit 2)

[0223]

[Sequence list]

[Brief description of drawings]

[Figure 1]   1 is the cDNA and predicted amino acid sequence of human sphingosine kinase 1.

FIG. 2A and B show predicted secondary structure and conserved region of human sphingosine kinase type 1, respectively.

[Figure 3]   hSK1 substrate recognition.

FIG. 4 shows that hSK1 is highly specific for D-erythro-sphingosine and that hSK1 is inhibited by D, L-threo-dihydrosphingosine and N, N-dimethyl-sphingosine.

FIG. 5A   Figure 7 shows the expression and cellular localization of hSK1 fused to EGPF at the N-terminus.

FIG. 5B   Figure 7 shows the expression and cellular localization of hSK1 fused to EGPF at the C-terminus.

[Figure 6]   The kinase activity of hSK fusion protein is shown.

[Figure 7]   The expression level of hSK fusion protein is shown.

[Figure 8]   The tissue distribution of hSK1 messenger RNA is shown.

FIG. 9. Comparison of hSK activity of various sources: partially purified hSK1 from CHO cells, bacteria, insect cells.

[Figure 10]   Comparison of hSK1 activity from various sources: Cos7, bacteria, insect cells.

FIG. 11   Bacterial growth conditions for optimization of positively expressed hSK1.

FIG. 12 shows a comparison of hSK1 activity expressed under various bacterial growth conditions and expressed in Cos cells. The hSK1 activity under optimal bacterial growth and induction conditions (50 μM IPTG, 20 h) is 40% of that found in the transfected Cos7 cell extracts.

FIG. 13 shows the physiologically relevant role of hSK1 demonstrated by the use of antisense oligonucleotides.

FIG. 14 shows a vector for constructing hSK-EGFP (N-terminal) fusions for expression in mammalian cells.

FIG. 15   1 shows a vector for constructing hSK-EGFP (C-terminal) for expression in mammalian cells.

FIG. 16   1 shows a vector for constructing GSK-tagged hSK1 for expression in bacterial cells.

FIG. 17   Fig. 7 shows an electrophoretic gel of partially purified hSK1 derived from Sf21 insect cells.

FIG. 18   7 shows antisense suppression of levels of hSK1 protein.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C12N 1/21 C12N 9/12 4C084 5/10 C12Q 1/48 Z 4H045 9/12 1/68 A C12Q 1 / 48 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 M 33/50 33/566 33/53 A61K 45/00 33/566 A61P 3/06 // A61K 45/00 3 / 10 A61P 3/06 7/02 3/10 9/02 7/02 9/10 9/02 101 9/10 103 101 11/00 103 11/06 11/00 17/00 11/06 17/02 17 / 00 17/06 17/02 19/02 17/06 25/00 19/02 25/28 25/00 29/00 25/28 35/00 29/00 37/02 35/00 43/00 111 37/02 C12N 15/00 ZNAA 43/00 111 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , N , PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM , HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Gothink, Mark USA Washington, Bothell, NY One Hundred and Six Tea Force Street 12138 (72) Inventor Melendez, Alirio, Jay Singapore Singapore, Gilman Heights Condominium Block 1 E, number 06-42 (72) Inventor Takaku, Laszlo France Bourg-la-Rahrain, Luder Roger, 12- 16 F Term (reference) 2G045 AA35 BB20 BB24 CB01 CB17 DA13 FB02 FB03 FB05 FB09 GC10 4B024 AA01 AA11 BA10 CA04 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA01 Q06Q01Q01Q01Q01Q06Q01Q06Q01 CC01 CC03 DD0307 QR08 QR33 QR42 QR59 QR62 QR74 QR80 QS05 QS25 QS36 QX02 4B065 AA01X AA57X AA87X AA93Y AB01 AB02 BA01 BA08 CA25 CA29 C A44 CA46 4C084 AA02 AA17 BA44 CA62 NA14 ZA022 ZA152 ZA362 ZA402 ZA452 ZA542 ZA592 ZA892 ZA962 ZB072 ZB112 ZB262 ZB272 ZC202 ZC332 ZC352 4H045 AA11 AA20 AA30 BA10 CA40 DA76 EA74 EA50 FA72 FA72 FA72

Claims (28)

[Claims] 1. A purified or isolated nucleic acid encoding human sphingosine kinase. 2. The purified or isolated according to claim 1, which comprises a polynucleotide having at least 90% identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence complementary thereto. Nucleic acid. 3. A purified or isolated polynucleotide encoding human sphingosine kinase having at least 80% amino acid identity with the sequence of SEQ ID NO: 3. 4. A purified or isolated polynucleotide of SEQ ID NO: 22, or a polynucleotide which hybridizes therewith. 5. At least the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2
A purified or isolated polynucleotide comprising 10 consecutive nucleotides. 6. A purified or isolated polynucleotide comprising at least 10 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 22. 7. SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, The purified or isolated polynucleotide of claim 5, which comprises the sequence of SEQ ID NO: 17. 8. A recombinant vector comprising the nucleic acid according to any one of claims 1-7. 9. A recombinant vector comprising the nucleic acid of claim 8 which is a bacterial vector. 10. A nucleic acid according to claim 9, which is a pGEX vector.
Recombinant vector. 11. A recombinant vector comprising a nucleic acid according to claim 10 which is a baculovirus vector, preferably pFastBacHTa. 12. A recombinant vector comprising the nucleic acid of claim 8 which is a eukaryotic vector. 13. A recombinant vector comprising the nucleic acid of claim 12 selected from pcDNA3, pFLAG and pCMV. 14. A recombinant host cell comprising the nucleic acid of any one of claims 1-7. 15. A recombinant host cell comprising a recombinant vector according to any one of claims 8-13. 16. An oligonucleotide comprising an antisense strand of the nucleotide according to any one of claims 1-7. 17. The oligonucleotide according to claim 16, which has the sequence of SEQ ID NO: 12. 18. A transgenic mammal comprising a nucleic acid according to any one of claims 1-7. 19. A transgenic mouse comprising the nucleic acid according to any one of claims 1 to 7. 20. A purified or isolated recombinant polypeptide comprising the amino acid sequence of human sphingosine kinase according to any one of claims 1-7. 21. The recombinant polypeptide of claim 20, which has at least 80% amino acid identity with the polypeptide of SEQ ID NO: 3 or a sequence complementary thereto. 22. A purified or isolated recombinant polypeptide comprising the amino acid sequence of SEQ ID NO: 8. 23. A method for amplifying a nucleic acid encoding hSK according to any one of claims 1 to 7, comprising: (a) a target hSK nucleic acid, a fragment or variant thereof, or a sequence complementary thereto. A test sample suspected of containing a pair of amplification primers according to any one of claims 4 to 7 capable of hybridizing to the hSK nucleic acid region to be amplified under stringent conditions. The above method, which comprises a step of contacting with a reaction reagent, and (b) optionally detecting an amplification product. 24. (a) A pair of oligonucleotide primers according to any one of claims 4 to 7, capable of hybridizing to an hSK nucleic acid to be amplified under stringent conditions, (b) optionally Is a kit for amplification, which comprises reagents necessary for carrying out an amplification reaction. 25. (a) Any one of claims 1 to 7 which encodes a desired amino acid sequence in a suitable vector according to any one of claims 8 to 13 or a host cell according to claims 14 or 15. Step of inserting the nucleic acid according to any one of (1), (b) a step of culturing a host cell previously transformed or transfected with the recombinant vector of step (a) in an appropriate medium, (c) The resulting medium is harvested, or the host cells are lysed, for example, by sonication or osmotic shock, and (d) thus produced from the medium or from the resulting pellet of host cell lysates. A method for producing an amino acid sequence, preferably the sequence of SEQ ID NO: 3, which comprises a step of separating or purifying the finally tagged recombinant polypeptide of interest. 26. An antibody directed against the polypeptide of any one of claims 20-22. 27. A method for screening a candidate molecule which is an inhibitor of hSK, wherein the recombinant polypeptide according to any one of claims 20 to 22 is sphingosine,
The above method, which comprises a step of mixing with labeled ATP and a candidate molecule of interest, and a step of measuring the conversion rate of sphingosine into labeled sphingosine-1-phosphate (S1P). 28. A kit for screening a candidate molecule that is an inhibitor of hSK, comprising the recombinant polypeptide according to any one of claims 20 to 22, and optionally a labeled ATP and sphingosine. A kit as described above, comprising.