WO2001031029A2 - Gene de la sphingosine kinase humaine - Google Patents

Gene de la sphingosine kinase humaine Download PDF

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WO2001031029A2
WO2001031029A2 PCT/EP2000/009498 EP0009498W WO0131029A2 WO 2001031029 A2 WO2001031029 A2 WO 2001031029A2 EP 0009498 W EP0009498 W EP 0009498W WO 0131029 A2 WO0131029 A2 WO 0131029A2
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seq
nucleic acid
sequence
recombinant
hsk
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PCT/EP2000/009498
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WO2001031029A3 (fr
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Janet Allen
Mark Gosink
Alirio J. Melendez
Laszlo Takacs
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Warner-Lambert Company
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Priority to AU10202/01A priority Critical patent/AU1020201A/en
Priority to JP2001533164A priority patent/JP2003512072A/ja
Priority to CA002389127A priority patent/CA2389127A1/fr
Priority to MXPA02004294A priority patent/MXPA02004294A/es
Priority to BR0015138-6A priority patent/BR0015138A/pt
Priority to EP00971299A priority patent/EP1228221A2/fr
Publication of WO2001031029A2 publication Critical patent/WO2001031029A2/fr
Publication of WO2001031029A3 publication Critical patent/WO2001031029A3/fr

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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • A—HUMAN NECESSITIES
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    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to the human sphingosine kinase type 1 gene. More precisely the invention concerns a purified or isolated nucleic acid of said sphingosine kinase or a sequence complementary thereto, or fragments thereof.
  • the invention includes oligonucleotides, recombinant polypeptides, recombinant vectors, recombinant host cells comprising said nucleic acid, as well as antibody production, methods of screening, antisense oligonucleotide, knock out mammals.
  • Sphingosine- 1 -phosphate the product of sphingosine kinase, is an important signaling molecule with intra- and extracellular functions.
  • the cDNA for the mouse sphingosine kinase has recently been reported as described in patent application number WO 99/61581.
  • the mouse SKI A and SK1B are presumably alternative splice forms. Differential splicing probably results in two variants of the N-terminal peptide sequence and it is the consequence of alternative coding exon usage (Kohama et al., 1998).
  • the invention concerns a purified or isolated nucleic acid encoding a human sphingosine kinase (hereinafter hSK) cDNA or a sequence complementary thereto.
  • hSK human sphingosine kinase
  • Oligonucleotide probes or primers specifically hybridizing to a nucleic acid encoding hSK, to fragments thereof or to a sequence complementary thereto are also part of the invention as well as DNA amplification and detection methods using said primers and probes.
  • a further object of the present invention concerns recombinant vectors comprising any of the nucleic acid sequences described herein, and in particular recombinant vectors comprising a nucleic acid sequence encoding a recombinant hSK.
  • the invention also includes recombinant expression vectors comprising a nucleic acid sequence encoding recombinant hSK.
  • the invention also encompasses host cells and transgenic non-human mammals comprising said nucleic acid sequences or recombinant vectors.
  • the invention further concerns an isolated recombinant hSK.
  • the invention also concerns a hSK polypeptide or a peptide fragment thereof as well as antibodies specifically directed against a peptide of hSK.
  • the invention further concerns a method for the screening of candidate molecules which are inhibitors of hSK.
  • the method comprises the steps of:
  • the invention also concerns a kit for the screening of candidate molecules which are inhibitors of hSK.
  • the kit comprises:
  • the invention also concerns inhibitors of hSKl obtained through the screening method described above, structural analogues thereof, and their use in the treatment or prevention of one and/or several disease states selected from: degenerative disease processes such as atherosclerosis and fibrosis; neurodegenerative disorders; cardiovascular diseases including atherosclerosis, thrombosis and dyslipidemia; diabetes including type I and type II diabetes and particularly type I diabetes; stroke; autoimmune and inflammatory diseases such as multiple sclerosis, psoriasis, epidermodysplasia verruciformis and inflammatory arthritis; T helper- 1 related diseases; chronic obstructive pulmonary disease; asthma; cancer; hemostatis, stroke, coronary artery disease, hematopoietic disorders such as leukemia, the natural wound healing processes, myocardial infarction, embryogenesis.
  • degenerative disease processes such as atherosclerosis and fibrosis
  • cardiovascular diseases including atherosclerosis, thrombosis and dyslipidemia
  • diabetes including type I and type II diabetes and particularly type I
  • Figure 1 illustrates the cDNA and predicted amino acid sequence of a human sphingosine kinase 1.
  • Figure 2A and 2B shows respectively the predicted secondary structure and the conserved regions of human sphingosine kinase type 1.
  • Figure 3 illustrates hSKl substrate recognition
  • Figures 4 A (4A1, 4A2) and 4B (4B1, 4B2) show that hSKl has high specificity for D-ervt ⁇ ro-sphingosine and illustrate that hSKl is inhibited by D,L-threo- dihydrosphigosine and N,N,diMethyl-sphingosine.
  • Figure 5 A describes the expression and cellular localisation of hSKl fused with EGPF at the N-terminal end.
  • Figure 5B illustrates the expression and cellular localisation of hSKl fused with EGPF at the C-terminal end.
  • Figure 6 shows the kinase activity of hSK fusion proteins.
  • Figure 7 describes the expression levels of hSK fusion proteins.
  • Figure 8 shows the tissue distribution of hSKl messenger RNA.
  • Figure 9 illustrates the comparison of hSK activity from different sources: CHO cells, Bacteria, partially purified hSKl from insect cells.
  • Figure 10 illustrates the comparison of hSKl activity from different sources: Cos7, bacteria, insect cells.
  • Figure 11 describes the bacterial growth conditions for optimization of actively expressed hSKl .
  • Figure 12 shows the comparison of hSKl activity expressed under different bacterial growth conditions and expressed in Cos cells.
  • the hSKl activity under optimal bacterial growth and induction conditions (50 ⁇ M 1PTG for 20hr) is 40% of the activity observed for the transfected Cos7 cells extract.
  • Figure 13 illustrates the physiological relevant role of hSKl proven by the use of an antisense oligonucleotide.
  • Figure 14 shows the vector for the construction of hSK-EGFP (N-terminal) fusion for expression in mammalian cells.
  • Figure 15 illustrates the vector for the construction of hSK-EGFP(C-terminal fusion) for expression in mammalian cells.
  • Figure 16 illustrates the vector for the construction of hSKl tagged with GST for expression in bacterial cells.
  • Figure 17 shows an electrophoresis gel of the partial purification of hSKl from Sf21 insect cells.
  • Figure 18 illustrates the antisense downregulation of hSKl protein levels.
  • a first object of the present invention is a purified or isolated nucleic acid encoding hSK, or a sequence complementary thereto.
  • Another object of the invention is a purified or isolated nucleic acid having at least 90%, preferably 95%, more preferably 98% and most preferably 99% nucleotide identity with the nucleotide sequence of SEQ ID N°l or of SEQ ID N°2, or a sequence complementary thereto.
  • a further object of the present invention is a purified or isolated nucleic acid encoding a polypeptide having at least 80%, preferably 90%, more preferably 95%, and most preferably 98 or 99% amino-acid identity with the human polypeptide of the amino-acid sequence of SEQ ID N°3 or with a peptide fragment thereof, or a sequence complementary thereto.
  • Polypeptides having amino-acid identity with the hSK of the invention encompass polypeptides:
  • N°3 such as for example the capacity to convert sphingosine into SIP.
  • isolated when used herein, requires that the material be removed from its original environment (e.g. the natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or a peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide can be part of a vector and/or such polynucleotide or peptide can be part of a composition, and still be isolated. This is so because the vector or composition is not part of the original environment of the nucleotide sequence it contains.
  • purified does not require absolute purity; rather, it is intended as a relative definition. Purification of starting materials or natural materials to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • nucleotide sequence is” used to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression “nucleotide sequence” encompasses the nucleic material and the sequence information and is not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.
  • sequence information i.e. the succession of letters chosen among the four base letters
  • oligonucleotides include RNA, any type of DNA such as genomic DNA, cDNA or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form.
  • nucleotide is also used herein to encompass modified nucleotides which comprise at least one of the following modifications: (a) an alternative linking group, (b) an analogous form of purine,
  • modified nucleotides such as methylated, phosphorylated, ubiquitinated nucleotides.
  • analogous linking groups purines, pyrimidines, and sugars, see for example PCT publication N°WO 95/04064.
  • polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, "or a combination thereof as well as through any purification methods known in the art.
  • the invention also encompasses polynucleotide fragments of a nucleic acid encoding the hSKl of the invention. These fragments particularly include but are not restricted to 1) those fragments encoding a polypeptide of hSK which preferably retains its affinity for sphingosine and 2) nucleotide fragments useful, as nucleic acid primers or probes for amplification or detection purposes.
  • a most preferred embodiment of this invention for a fragment encoding a polypeptide of hSK is the polynucleotide of sequence SEQ ID NO: 8 corresponding to a region of SK conserved between species.
  • SEQ ID NO: 8 a region of SK conserved between species.
  • the inventors have shown that a 80 amino-acids long region of hsKl is conserved between species (figure ⁇ ).
  • the present invention concerns a purified or isolated polynucleotide comprising at least 10 consecutive nucleotides of a nucleic acid encoding the hSK described herein, preferably at least 10 consecutive nucleotides of the nucleotide sequence of SEQ ID N°l or of SEQ ID N°2, or a sequence complementary thereto.
  • nucleic acids consist of a contiguous span which ranges in length from 10, 12, 15, 18 or 20 to 25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides, or be specified as being 10, 12, 15, 18, 20, 25, 35, 40, 50, 100, 200, 250, 500 or 1000 nucleotides in length.
  • these nucleic acids are useful as probes in order to detect the presence of at least a copy of a nucleotide sequence encoding hSK, more particularly the presence of at least a copy of a nucleotide sequence of SEQ ID N°l or of SEQ ⁇ N°2 or a sequence complementary thereto or a fragment or a variant thereof in a sample.
  • the sequence of such nucleic acids could be slightly modified (for example by substituting one nucleotide for another) without substantially affecting the ability of such modified sequence to hybridize with the targeted sequence of interest.
  • the most preferred probes are the following: SK5 'end49 (gene proximal) CTGGGTCTTGTAGAAGAGCAGC AAGTGCT
  • nucleic acid probes of the invention may also be used for the analysis of the expression levels and patterns of hSK, such as described in the PCT Application N°WO 97/05 277, the entire contents of which is herein incorporated by reference.
  • these nucleic acids are useful as primers.
  • the most preferred primers are the following:
  • the invention also concerns purified or isolated nucleic acid sequences that ' hybridize, under stringent hybridization conditions, with a polynucleotide encoding hSK or a sequence complementary thereto.
  • a preferred embodiment of the invention is a purified or isolated nucleic acid sequence that hybridize, under stringent conditions, with the nucleic acid of 270 nucleotides (SEQ ID NO: 22) encoding the 80 amino acids conserved region of hSKl.
  • stringent hybridization conditions can be defined as follows:
  • the hybridization step is conducted at 65°C in the presence of 6 x SSC buffer, 5 x Denhardt's solution, 0.5 % SDS and lOO ⁇ g/ml of salmon sperm DNA.
  • the hybridization step is followed by four washing steps:
  • hybridization conditions defined above are suitable for nucleic acids of approximately twenty ' nucleotides in length and that these conditions may be also adapted for shorter or longer nucleic acids, according to techniques well known in the art, for example those described by Sambrook et al. (1989).
  • the appropriate length for probes under a particular set of assay conditions may be empirically determined by the one skilled in the art.
  • the probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as 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 the solid support method described in the application N°EP-0 707 792. The disclosures of all these documents are incorporated herein by reference.
  • nucleic acids of the present invention can be labelled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, autoradiographic, radiochemical, immunochemical, or chemical means.
  • useful labels include radio-active substances ( P, S, H, I), fluorescent dyes (5-bromodesoxyuridin, fluorecein, acetylaminofluoren, digoxygenin) or biotin. Examples of non-radioactive labelling of nucleic acid fragments are described in French Patent N°FR-78 10975 or by Urdea et al. (1988) or Sanchez-Pescador et al. (1988).
  • the probes according to the present invention may have structures and characteristics such that they allow signal amplification, such structural characteristics being, for example, those of branched DNA probes as described by Urdea et al. (1991).
  • nucleic acid probes of the invention can be conveniently immobilized on a solid support.
  • Solid supports are known those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitro-cellulose strips, membranes, microparticules such as latex particles, sheep red blood cells, duracytes and others.
  • nucleic acids of the invention and particularly the nucleotide probes described above can thus be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20 or 25 distinct nucleic acids of the invention to a single solid support.
  • a support on which nucleic acid probes of the invention are immobilized such a support may also contain other immobilized- probes, preferably probes that hybridize specifically with a nucleic acid encoding hSK, or a variant thereof, or a sequence complementary thereto, more preferably probes that hybridize specifically with the nucleic acid of 240 nucleotides (SEQ ED NO: 22) encoding the 80 amino acids conserved region of hSKl.
  • Another object of the invention consists of a method for the amplification of a nucleic acid encoding a hSK, said method comprising the steps of: (a) mixing a test sample suspected of containing the target hSK nucleic acid, a fragment or a variant thereof, or a sequence complementary thereto, with an amplification reaction reagent comprising a pair of amplification primers as disclosed herein which can hybridize under stringent conditions, the hSK nucleic acid region to be amplified, and
  • the nucleic acid encodes a hSK polypeptide of SEQ ED N°3.
  • the amplification product is detected by hybridization with a labelled probe having a sequence which is complementary to the amplified region.
  • the invention also concerns a kit for the amplification of a nucleic acid encoding hSK, a fragment or a variant thereof, or a complementary sequence thereto in a test sample, wherein said kit comprises:
  • a pair of oligonucleotide primers as disclosed in the present invention which can hybridize, under stringent conditions to the hSK nucleic acid to be amplified; (b) optionally, the reagents necessary for performing the amplification reaction.
  • the nucleic acid to be amplified encodes hSK polypeptide of SEQ ID N°3.
  • the amplification product is detected by hybridization with a labelled probe having a sequence which is complementary to the amplified region.
  • the present invention also encompasses a family of recombinant vectors comprising any one of the nucleic acids described herein. Firstly, the invention deals with a recombinant vector comprising a nucleic acid selected from the group consisting of:
  • a purified or isolated nucleic acid encoding hSK polypeptide and more preferably a polypeptide having at least 80% amino acid identity with the polypeptide of SEQ ED N°3, or a sequence complementary thereto; or (b) a purified or isolated polynucleotide comprising at least 10 consecutive nucleotides of a nucleic acid described in (a) or a sequence complementary thereto.
  • a recombinant vector of the invention is used to introduce the inserted polynucleotide derived from the nucleic acid encoding hSK polypeptide in a suitable host cell, this polynucleotide being amplified every time the recombinant vector replicates.
  • Recombinant expression vectors comprising a nucleic acid encoding hSK polypeptides that are described in the present specification are also part of the invention.
  • recombinant vectors consist of expression vectors comprising a nucleic acid encoding a hSK polypeptide of the invention, and more preferably a nucleic acid encoding a polypeptide having the amino acid sequence of SEQ TD N°3.
  • Preferred vectors comprises a nucleic acid sequence as shown in SEQ ID N° 1 or
  • expression vectors can be employed to express a recombinant hSK polypeptide which can then be purified and for example, be used as an immunogen in order to raise specific antibodies.
  • Preferred eukaryotic vectors of the invention are listed hereafter as illustrative but not limitative examples: pcDNA3, pFLAG, pCMV-Script, pIND, pMClNEO, pHIL, pGAPZA, pMT/V5-His-TOPO, pMT/V5-His, pAc5.1/V5- HisA, pDS47/V5-His, pcDNA4, pcDNA6, pEFl, pEF4, pEF6, pUB6, pZeoSV2, pRc/CMv2, pcDM8, pCR3.1, pDisplay, pSecTag2, pVP22, pEMZ, pCMV/Zeo, pSinRep5, pCEP,
  • Prefe ⁇ ed bacteriophage recombinant vectors of the invention are PI bacteriophage vectors such as described by Sternberg ⁇ .L. (1992;1994).
  • a suitable vector for the expression of a recombinant hSK is a baculovirus vector that can be propagated in insect cells and in insect cell-lines such as Sf9 and Sf21.
  • Specific suitable host vectors includes 1 , but are not restricted to pFastBac-1, pIZ/V5-His, pBacMan-1, pBlueBac4.5, pBlueBacHis2, pMelBacA, pVL1392, pVL1393
  • Preferred baculovirus vector is pFastBacHTa.
  • a preferred bacterial vector is pGEX.
  • Expression requires that appropriate signals are provided in the vectors, said signals including various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • the regulatory sequences of the expression vectors of the invention are operably linked to the nucleic acid encoding the recombinant hSK.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a promoter or an enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • two DNA molecules are said to be "operably linked” if the nature of the linkage between the two polynucleotides does not: (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide.
  • recombinant expression vectors include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in an appropriate frame with the translation, initiation and termination sequences, and preferably a leader sequence capable of directing sequences of the translated protein into the periplasmic space or the extra-cellular medium.
  • preferred vectors comprise an origin of replication from the desired host, a suitable promoter and an enhancer, and also any necessary ribosome binding sites, polyadenylation site, transcriptional termination sequences, and optionally 5 '-flanking non-transcribed sequences.
  • DNA sequences derived from the SV 40 viral genome, for example SV 40 origin early promoter, enhancer, and polyadenylation sites may be used to provide the required non-transcribed genetic elements, another suitable promoter is the CMV promoter.
  • Suitable promoter regions used in the expression vectors according to the invention are chosen taking into account the host cell in which the heterologous nucleic acids have to be expressed.
  • a suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression, or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the 5 recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
  • Host cells that have been transformed or fransfected with one of the nucleic acids described herein, or with one of the recombinant vector, particularly recombinant expression vector, described herein are also part of the present invention.
  • Preferred host cells used as recipients for the expression vectors of the invention are the following:
  • prokaryotic host cells bacterial cells and more particularly Escherichia coli, strains, (i.e. BL21, DH10 Bac strain) Bacillus subtilis, Salmonella typhimurium and strains from species such as Pseudomonas, 0 Streptomyces and Staphylococcus; Sf-9 cells (ATCC N°CRL 1711), Sf 21 cells.
  • HeLa cells ATCC N°CCL2; N°CCL2.1; N°CCL2.2
  • Cv 1 cells ATCC N°CCL70
  • COS cells ATCC N°CRL 1650; N°CRL 1651
  • C127 cells ATCC N°CRL-1804
  • 3T3 cells ATCC N°CRL- 6361
  • CHO cells ATCC N°CCL-61
  • human kidney 293 cells ATCC N° 45504; N°CRL-1573
  • BHK ECACC N°84100 501; N°84111301
  • expressions of the recombinant hSK of the invention in COS- 7 or in bacterial cells are preferred embodiment of the invention.
  • the results reported in the examples show that expression in Cos 7 and in bacteria are suitable for the production of an important amount of sphingosine kinase.
  • the present invention also concerns a method for producing one of the amino acid sequences described herein and especially the polypeptide having the amino acid sequence of SEQ ID N°3, wherein said method comprises the steps of: (a) inserting the nucleic acid encoding the desired amino acid sequence in an appropriate vector; or in a host cell;
  • step (b) culturing, in an appropriate culture medium, a host cell previously transformed or fransfected with the recombinant vector of step (a);
  • tags include, but are not limited to sequences encoding C-myc, FLAG, a sequence of histidine residues, heamaglutin A, V5, Xpress or GST. Most of these tags can be incorporated directly into the sequence, for instance through PCR amplification by incorporating the appropriate coding sequence in one of the PCR amplification primers.
  • FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp- Lys, SEQ ID NO: 23) which is used to express the recombinant hSK of the invention as a fusion protein. Both amino-terminal and carboxy-terminal FLAG fusion proteins fall within the scope of the present invention. In preferred embodiments, the FLAG fusion proteins are produced through vectors which are derivatives of the pCMN-5 vector.
  • a pFLAG-CMV-1 or pFLAG-CMV-2 vector can be used for amino-terminal tagging whereas a pFLAG-CMV-5a, -5b or 5c vector can be used for carboxy-terminal tagging.
  • the tag can also be introduced by other means such as covalent binding of the appropriate nucleic acid sequence encoding the tag moiety with the 3' or 5' end of the nucleic acid sequence encoding the polypeptide sequence. This is the case for GST.
  • Purification of the recombinant hSK according to the present invention is then carried out by passage onto a nickel or copper affinity chromatography column, such as a ⁇ i ⁇ TA column.
  • the polypeptide thus produced is further characterized, for example by binding onto an immuno-affinity chromatography column on which polyclonal or monoclonal antibodies directed to the hSK of interest have been previously immobilised.
  • the production rate is higher for bacterial expression- than for insect cells expression.
  • Another object of the present invention consists of a purified or isolated recombinant polypeptide comprising the amino acid sequence of hSK.
  • Preferred isolated recombinant polypeptides of the invention include those having at least 80%, preferably 90%, more preferably 95, and most preferably 98 or 99%, amino-acid identity with the polypeptide having the amino acid sequence of SEQ ID N°3.
  • Extract of infected insect cells expressing a tagged hSKl may be purified through resin column having affinity for the tag.
  • extract of infected insect cells expressing a 6His tagged hSKl are run through NI-NTA resin column.
  • extract ofinfected insect cells expressing a GST tagged hSKl are purified through glutathion resin.
  • the invention also relates to a recombinant hSK polypeptide comprising amino acid changes ranging from 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40 substitutions, additions or deletions of one amino acid as regards to polypeptides of anyone of the amino acid sequences of the present invention.
  • Preferred sequences are those of SEQ ED N°3.
  • Amino acid changes encompassed are those which will not abolish the biological activity of the resulting modified polypeptide.
  • These equivalent amino-acids may be determined either by their structural homoiogy with the initial amino-acids to be replaced, by the similarity of their net charge or of their hydrophobicity, and optionally by the results of the cross-immunogenicity between the parent peptides and their modified counterparts.
  • amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several consecutive or non-consecutive amino acids are replaced by "equivalent” amino acids.
  • amino acid substitution in the amino acid sequence of a polypeptide according to the invention, one or several consecutive or non-consecutive amino acids are replaced by "equivalent” amino acids.
  • the expression "equivalent” amino acid is used herein to designate any amino acid that may be substituted for one of the amino-acids belonging to the native protein structure without decreasing the binding properties of the corresponding peptides to the antibodies raised against the polypeptides of the invention.
  • the "equivalent" amino-acids are those which allow the generation or the synthesis of a polypeptide with a modified sequence when compared to the amino acid sequence of the recombinant hSK polypeptides of interest, said modified polypeptide being able to bind to the antibodies raised against the recombinant hSK of interest and/or to induce antibodies recognizing the parent polypeptide.
  • amino-acids containing one or several "equivalent" amino-acids must retain their specificity and affinity properties to the biological targets of the parent protein, as it can be assessed by a ligand binding assay or an ELISA assay.
  • amino-acids belonging to specific classes include Acidic (Asp,
  • a substitution of an amino acid in a recombinant hSK of the invention, or in a peptide fragment thereof, consists in the replacement of an amino acid of a particular class for another amino acid belonging to the same class.
  • an equivalent amino acid according to the present invention is also contemplated the replacement of a residue in the L-form by a residue in the D form or the replacement of a Glutamic acid (E) residue by a Pyro-glutamic acid compound.
  • the synthesis of peptides containing at least one residue in the D- form is, for example, described by Koch (1977).
  • a specific embodiment of a modified peptide of interest according to the present invention includes, but is not limited to, a peptide molecule, which is resistant to proteolysis.
  • the invention also encompasses a recombinant hSK in which at least one peptide bond has been modified as described above.
  • the polypeptides according to the invention may also be prepared by the conventional methods of chemical synthesis, either in a homogenous solution or in solid phase. As an illustrative embodiment of such chemical polypeptide synthesis techniques, it may be cited the homogenous solution technique described by Houbenweyl (1974).
  • the recombinant hSK of interest, or a fragment thereof may thus be prepared by chemical synthesis in liquid or solid phase by successive couplings of the different amino acid residues to be incorporated (from the N-terminal end to the C-terminal end in liquid phase, or from the C-terminal end to the N-terminal end in solid phase) wherein the N-terminal ends and the reactive side chains are previously blocked by conventional groups.
  • the recombinant hSK of the invention and its peptide fragments of interest can be used for the preparation of antibodies.
  • Polyclonal antibodies may be prepared by immunization of a mammal, especially a rabbit, a sheep, a donkey, a horse or a goat with a polypeptide according to the invention that is combined with an adjuvant of immunity, and. then by purifying the specific antibodies contained in the serum of the immunized animal on an affinity chromatography column on which has previously been immobilized the polypeptide that has been used as the antigen.
  • Monoclonal antibodies from mammals especially from mouse or rat may be prepared from hybridomas according to the technique described by Kohler and
  • the present invention also deals with antibodies produced by the trioma technique and by the human B-cell hybridoma technique, such as described by Kozbor et al. (1983).
  • Antibodies of the invention also include chimeric single chain Fv antibody fragments (US Patent N° 4,946,778; Martineau et al., (1998), antibody fragments obtained through phage display libraries Ridder et al. (1995) and humanized antibodies (Leger et al., 1997).)
  • Sphingosine kinase converts the substrate sphingosine to sphingosine- 1- phosphate (SIP).
  • SIP is believed to play several roles in physiological processes. o Some of the potential physiological roles of S IP include:
  • TNF- ⁇ induced (endothelial cells) adhesion molecule expression TNF- ⁇ induced (endothelial cells) adhesion molecule expression; and Depression of excitability in ventricular myocytes.
  • Sphingosine kinase appears to play a pivotal role in the activation of the 0 signaling cascade initiated at Fc ⁇ RI by modulating the balance of the counterregulatory lipids. (Prieschl et al., 1999)
  • PDGF platelet derived growth factor
  • TNF ⁇ a pleiotropic cytokine
  • SIP protects from apoptosis. More particularly, SIP prevents the appearance of intranucleosomal DNA fragmentation and morphological changes which are main features of apoptosis.
  • SIP is a key mediator of the mitogenic effect of oxLDL (oxidized low density lipoprotein) which have been implicated in diverse biological events leading to development of atherosclerotic lesions.
  • sphingosine kinase may play a role in conditions such as hemostatis, thrombosis, stroke, atherosclerosis, coronary artery disease and dyslipidemia.
  • a high cellular concentration of sphingosine acts as a potent inhibitor of the immunoglobulin (Ig)E+ antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway.
  • high mtracellular levels of sphingo sine- 1 -phosphate also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release or, n combination with ionomycin, cytokine production.
  • the balance between sphingosine and SIP modulates the allergic responsiveness of mast cells.
  • inhibitors of sphingosine kinase may be useful in preventing allergy reactions.
  • sphingosine kinase activity is stimulated by tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate. Hence, one may infer that excessive stimulation of sphingosine kinase activity could lead to the development of proliferative diseases. On the other hand, inhibition of sphingosine kinase prevented the survival effect of l ,25-dihydroxyvitamin D3
  • sphingosine kinase inhibitors may be useful in the prevention and treatment of proliferative diseases including cancer, hematopoietic disorders such as leukemia.
  • Chemotaxis attraction and activation of macrophages
  • Cytoskeletal changes Stress fiber formation and cell shape contraction, aggregation and secretion
  • Mediates attachment Fibronectin matrix assembly
  • G GPPCC ⁇ Rs G-protein-coupled receptors
  • SIP is released from activated platelets in large amounts. (Yatomi et al., 1995) This could indicate a potential role of SIP in thrombosis, hemostasis, the natural wound healing processes, atherosclerosis, stroke, myocardial infarction.
  • SIP stimulates the binding of fibronectin or its N-terminal 70-kd* fragment to cells.
  • Organization of fibronectin into extracellular matrix is a tigthly regulated process, mediated by initial reversible binding by the 70-kd N-terminal region of fibronectin to specific cells surface binding sites, followed by insolubilization into fibrils.
  • the adhesive information present after insolubilization of fibronectin is postulated to play a central role in various physiological and pathophysiological processes, including embryogenesis, wound-healing, inflammation, and degenerative disease processes such as atherosclerosis and fibrosis.(Windh et al., 1999) More particularly, SIP could have a likely role in early atherogenesis and fibrosis.
  • suitable sphingosine kinase inhibition could be useful in the treatment of cardiovascular diseases including atherosclerosis, thrombosis and dyslipidemia, diabetes including type I and type II diabetes and particularly type I diabetes, stroke, autoimmune and inflammatory diseases such as multiple sclerosis, psoriasis, epidermodysplasia verruciformis and inflammatory arthritis, T helper- 1 related diseases, chronic obstructive pulmonary disease, asthma, cancer and neurodegenerative disorders.
  • sphingosine kinase inhibitors include sphingosine kinase inhibitors.
  • These inhibitors or structural analogues thereof can be used to treat or prevent one and/or several of the -disease states refered to above.
  • structural analogue is intended to designate compounds which have a common chemical backbone with the initial inhibitors identified through the screening assays of the invention but which bare substituents which have been modified to improve or enhance properties of the initial inhibitors such as biological activity, reduced side effects, enhanced solubility, enhanced bioavailability and the like.
  • assay formats can be used to carry out the method of the present- invention.
  • Preferred assay formats include scintillation assays such as the scintillation proximity assay (SPA) or the flashplate assay.
  • SPA scintillation proximity assay
  • flashplate assay Other assay formats well known to those skilled in the arts such as the filter binding assay and the centrifugation assay are also contemplated in the present invention.
  • SPA and flashplate assays are preferred assay formats for the present invention. Additional details on these assays are provided below.
  • Scintillation assay technology either involves the use of scintillant beads (for the SPA assay) or plates (for the flashplate assay).
  • SPA beads are usually made from either cerium-doped yttrium ion silicate (y2SiO5:Ce) or polyvinyltoluene (PVT) containing an organic scintillant such as PPO.
  • Flashplates commonly used are those such as Ni chelate flashplates although other flashplates can also be used.
  • Assays are usually carried out in aqueous buffers using radioisotopes such as 3 H, 125 I, 14 C, 35 S or 33 P that emit low-energy radiation, the energy of which is easily dissipated in an aqueous environment.
  • radioisotopes such as 3 H, 125 I, 14 C, 35 S or 33 P that emit low-energy radiation, the energy of which is easily dissipated in an aqueous environment.
  • the electrons emitted by H have an average energy of only 6 keV and have a very short path length (-1 ⁇ tm) in water. If a molecule labelled with one of these isotopes is bound to the bead or flashplate surface, either directly or via interaction with another molecule previously coupled to the bead or flashplate, the emitted radiation will activate the scintillant and produce light.
  • the amount of light produced which is proportional to the amount of labelled molecules bound to the beads, can be measured conveniently with a liquid scintillation (LS) counter. If the labelled molecule is not attached to the bead or a flashplate surface, its radiation energy is absorbed by the surrounding aqueous solvent before it reaches the bead, and no light is produced. Thus, bound ligands give a scintillation signal, but free ligands do not, and the need for a time- consuming separation step, characteristic of conventional radioligand binding assays, is eliminated. The manipulations required in the assays are reduced to a few simple pipetting steps leading to better precision and reproducibility.
  • LS liquid scintillation
  • one of the preferred embodiments of the assay includes the binding of sphingosine to SPA beads or flashplates.
  • the binding is preferably carried out through BSA although other binding means could be contemplated.
  • the assay medium comprises recombinant hSK and labelled ATP. What is measured is the ability of the candidate ligand to prevent conversion of sphingosine to labelled SIP by phosphorylation of sphigosine using recombinant hSK through labelled ATP. If the candidate ligand inhibits recombinant hSK, conversion of sphingosine will not occur and a signal not substantially different from the background noise signal will be recorded. On the other hand, if no hSK inhibition occurs, sphingosine conversion will take place and a signal resulting from the interaction between labelled SIP and the flashplate or SPA bead will be recorded.
  • Oligonucleotides ie RNA, DNA such as: genomic DNA, cDNA or RNA/DNA hybrid sequences, comprising the antisense strand of the human sphingosine kinase type 1 are used to inhibit in vitro or in vivo the sphingosine kinase expression.
  • RNA DNA
  • cDNA or RNA/DNA hybrid sequences comprising the antisense strand of the human sphingosine kinase type 1 are used to inhibit in vitro or in vivo the sphingosine kinase expression.
  • the polynucleotide construct as defined above contains a genomic polynucleotide encoding a SK from which at least a part of the nucleic acid portion encoding the 80 amino acids conserved region has been deleted and wherein the deleted nucleic acid portion is replaced by a deliberately heterologous polynucleotide sequence.
  • Said constructs may be included in vectors in order to replace a portion of the naturally occurring sphingosine kinase sequence within the genome of a mammal by homologous recombination.
  • such a recombinant vector of the invention may be used to generate knock-out animals, preferably knock-out mammals, most preferably knock-out mice and rats.
  • the genomic polynucleotide encodes a human, a mouse or a rat SK from which the nucleic acid portion encoding the 80 amino acids conserved region or a portion of it has been deleted.
  • the heterologous polynucleotide comprises a selection marker.
  • the heterologous polynucleotide comprises at least a loxP sequence at its 5' end and at least a loxP sequence at its 3' end.
  • the loxP sequence is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (HOESS et al., 1986).
  • the recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.
  • the Cre-loxP system used in combination with a homologous recombination technique is described by GU et al. (1993, 1994).
  • the vector containing the genomic SK sequence in which the sequence encoding the 80 amino acids conserved region or a portion of it has been deleted is designed in such a way that selectable markers are flanked by loxP sites of the same orientation. It is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while relocating the hSK genomic polynucleotide of interest that has been inserted by a homologous recombination event. Two selectable markers are needed: a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-loxP system are described by ZOU et al. (1994).
  • nucleic acids of the invention wherein said nucleic acid comprises the genomic polynucleotide encoding the mouse SK in which the nucleic acid portion encoding the 80 amino acids conserved region or a portion of it has been deleted, the person skilled in the art may advantageously refer to the examples below.
  • a nucleic acid which encodes for a polypeptide as defined above is operably linked to a regulatory sequence.
  • the regulatory sequence consists of a inducible promoter.
  • the regulatory sequence consists of a promoter inducible by Ponasterone.
  • RNA multiple tissue Northern blots and the pcDNA3 mammalian expression vector were purchased from CLONTECH (Palo Alto, CA, USA.). Restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA). EST-IMAGE clone 1946069 was purchased from UK HGMP Resource Centre (Hinxton Cambridge, UK.)
  • COS7 cells (Monkey fibroblast cells) were grown in Dulbecco's modified Eagle's medium containing 4,500 mg/L Glucose supplemented with 10% fetal calf serum, 2 mM glutamine, 10 IU/ml penicillin and 10 mg/ml streptomycin at 37°C, 6.5%o carbon dioxide in a water saturated atmosphere.
  • Example 1 Human Sphingosine Kinase (hSKl) cDNA isolation.
  • the insert of the IMAGE clone 1946069 a member of the cluster was sequenced * and subcloned into the pcDNA3 mammalian expression vector.
  • the 1.7 kb insert showed high level of similarity (76%) to the mouse SKI a cDNA and covered the entire coding region.
  • Peptide sequence alignment of mouse and human sequences and the biological activity of the expressed enzyme suggest that the insert of the EMAGE clone 1946069 harbors the coding region of the human SK cDNA. This is in agreement with human partial peptide sequences, deduced from Est sequences by Kohama et al (Kohama et al., 1998).
  • the cDNA sequence and peptide sequences of hSKl are shown in Figure 1.
  • the open reading frame of the cDNA is 1155 nt.
  • the translational initiator ATG is in a partial Kozak consensus (Kozak, 1987).
  • the PCR primers are the following :
  • primers are used for the cDNA amplification such as PCR amplification.
  • the predicted peptide sequence is 384 aa (seq ID N°3), with a predicted mass of
  • This sequence includes a region distantly related to a short signature peptide, LVRSEELGRWDALVVM (SEQ ED N°9) of NADPH dependent aldo-keto reductase family of enzymes. Within the 80 aa conserved region, highly conserved residues mark seemingly characteristic and predictable features of the secondary peptide structure in three blocks.
  • Figure 2 A conserved Asn22-Pro23 and Gly26 residues present a probable beta turn and a coil structure, proximal to the GGKGK sequence (SEQ ED NO: 24) which may be part of the ATP binding site also suggested for the mouse SKI (Kohama et al., 1998).
  • His59-Ala60 are indicated to be exposed on the surface, while Gly80-Asp81-Gly82 suggest the presence of a flexible region. Spacing of Asn22-Pro23, and Gly26, in block one, Thr50, His59-Ala60, in block two and Gly80-Asp81-Gly82 , Glu86 and Gly90 residues in block three of the conserved region is identical from Bacillus subtilis to human.
  • COS7 cells were transiently fransfected with the vector pcDNA3 alone or vector containing the human sphingosine kinase cDNA, using the Qiagen reagent, SuperFect. Cells were seeded 5 X 10 6 per well, in 6 wells plates. After 24 hrs, cells were fransfected with 10 ⁇ g of vector (pcDNA3) mixed with 20 ⁇ l
  • Example 4 Sphingosine kinase activity and specificity assay. Sphingosine kinase activity was assayed as previously described (Kohama et al., 1998). Briefly, sphingosine kinase activity was determined, by incubating cell extracts for 30 min at 37°C, in the presence of 50 ⁇ M sphingosine, 0.25% Triton X-100, and [ 33 P]ATP (10 ⁇ Ci, 1 mM), and MgC12 (10 mM). The kinase activity was expressed as nanomoles of SPP/min/mg.
  • COS7 cells were fransfected with the vector-pcDN A3 -containing the hSKl cDNA and, after
  • Figure 3 shows that hSKl specifically phosphorylates D-erytAro-sphingosine (D- erythro-SPH), and to a lesser extent D-L-erytAro-dihydrosphingosine (D,L- erythro-DHS).
  • This kinase does not phosphorylate: any of the "t/jreo"isoforms of dihydrosphingosine (D,L-freo-dihydrosphingosine; L-threo-dihydrosphingosine; L-threo-dihydrosphingosine); ceramides (hydroxy-ceramide; non-hydroxy- ceramide); diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-phosphate (PEP); or phosphatidylinositol-4,5- bisphosphate (PEP2).
  • D,L-freo-dihydrosphingosine L-threo-dihydrosphingosine
  • L-threo-dihydrosphingosine L-threo-dihydrosphingosine
  • ceramides hydroxy-ceramide; non-hydroxy-
  • the substrate specificity of the expressed hSK was found to be similar to purified rat sphingosine kinase (Olivera et al., 1998), and to the recently cloned mouse sphingosine kinase (Kohama et al., 1998).
  • the best substrate was D-(+)- erythro-sphingosine, followed by the D,L-erythro-dihydrosphingosine, which was phosphorylated to 50% of the observed phosphorylation levels achieved for D-(+)-erythro-sphingosine.
  • Substrate specificity and competitive inhibition of the hSKl was found to be similar to purified rat sphingosine kinase (Olivera et al., 1998), and to the recently cloned mouse sphingosine kinase (Kohama et al., 1998).
  • the best substrate was D-(+)- ery
  • D,L-threo-dihydrosphingosine on figure 4A (4A1, 4A2), DHS
  • N,N- diMethyl-sphingosine N,NdiMS
  • sphingosine kinase Kohama et al., 1998; Olivera et al., 1998.
  • hSKl activity we show here that both these compounds inhibit expressed hSKl activity.
  • a PCR reaction was carried out using the pcDNA3-hSKl construct as a template, and primers designed to amplify the coding sequence of hSKl at the same time inserting cloning restriction sites at both ends (Nhel - EcoRI) in order to align the EGFP with the hSKl and make the fusion protein in frame.
  • the constructs carries the EGFP at the N-terminus of the hSKl.
  • 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)
  • a PCR reaction was carried out using the pcDNA3-hSKl construct as a template, and primers designed to amplify the coding sequence of hSKl at the same time inserting cloning restriction sites at both ends (EcoRI - Sail) in order to align the hSKl sequence with the EGFP and make the fusion protein in frame.
  • the constructs carries the EGFP at the C-terminus of the hSKl.
  • D 3'end ATT ATC GTC GAC TAA GGG CTC TTC TGG CGG (SEQ ID NO: 7)
  • COS7 cells were transiently fransfected with the vector pcDNA3 alone or vector containing the human sphingosine kinase cDNA, using the Qiagen reagent, SuperFect as described in example 4.
  • COS7 cells were transiently fransfected with the vector pCI-EGFPl alone or vector containing the human sphingosine kinase cDNA (see figure 14), using the Qiagen reagent, SuperFect.
  • Cells were seeded 5 X 10 6 per well, in 6 wells plates. After 24 hrs, cells were fransfected with 10 ⁇ g of vector (pCI-EGFPl) mixed with 20 ⁇ l SuperFect, or with 10 ⁇ g vector containing the human sphingosine kinase cDNA (pCI-EGFPl-hSKl) mixed with 20 ⁇ l SuperFect.
  • Figure 14 shows the vector for the construction of hSK-EGFP (N-terminal) fusion for expression in mammalian cells.
  • pCI-EGFP size is 4724bp.
  • EGFP coding sequence (716pb) was amplified with EGFP.Xbal (sens) and STOP.EGFP (antisens) primers, cut XBAI/XhoI and subcloned into pCI cut by
  • COS7 cells were transiently fransfected with the vector pCI-EGFP-2 alone or vector containing the human sphingosine kinase cDNA (see figure 15), using the Qiagen reagent, SuperFect.
  • Cells were seeded 5 X 10 6 per well, in 6 wells plates. After 24 hrs, cells were fransfected with 10 ⁇ g of vector (pCI-EGFP-2) mixed with 20 ⁇ l SuperFect, or with 10 ⁇ g vector containing the human sphingosine kinase cDNA ( ⁇ CI-EGFP-2-hSKl) mixed with 20 ⁇ l SuperFect.
  • Figure 15 illustrates the vector for the construction of hSK-EGFP (C-terminal fusion) for expression in mammalian cells.
  • pCI-EGFP2 size is 4733 bp.
  • EGFP coding sequence (725bp) was amplified by PCR with EGFP2-TOP (sens) and EGFP2-BOTTOM (antisens) primers, cut Xhol/Notl and subcloned into pCI cut by Sail and Notl. A new Sail site was included into the PCR product.
  • the frame for subcloning a sequence of interest with EGFP fused to the C-terminus is shown at the bottom of the figure.
  • Figure 5 A describes the expression and cellular localisation of hSKl fused with EGPF at the N-terminal end.
  • EGFP-hSKl N-terminal fusion
  • EGFP-EGFP N-terminal fusion
  • the hSKl-EGFP appear to be partially localised in a granular form, although, general cytosolic expression is also observed ( Figure 5B).
  • Figure 5B illustrates the expression and cellular localisation of hSKl fused with EGPF at the C-terminal end.
  • hSKl-EGFP C-terminal fusion
  • hSKl-EGFP C-terminal fusion
  • Figure 6 shows the kinase activity of hSK fusion proteins.
  • Overexpression of hSK-EGFP (N-terminal fusion) (EGFP-hSKl) has similar activity as the overexpressed unfused untagged protein.
  • the hSK-EGFP C- terminal fusion (hSKl-EGFP) shows 40% less activity than the unfussed or N- terminal fusion proteins.
  • Figure 7 is a Western blot analysis with anti-EGFP Antibody.
  • Figure 7 demonstrates that both C-terminal (hSKl-EGFP) and N-te ⁇ ninal (EGFP-hSKl) hSK/EGFP fusion proteins are expressed to similar levels in Cos7 cells.
  • Example 6 Sphingosine kinase localisation in tissues
  • Figure 8 shows the tissue distribution of hSKl messenger RNA.
  • Northern blot containing approximately 1 ⁇ g of poly A+ RNA per lane from 12 different human tissues, was hybridized as described under methods. The numbers at the bottom of each line indicate the expression levels relative to background, and were quantified by densitometry. This revealed highest expression in adult lung (46 fold over background) and spleen (38 fold), followed by peripheral blood leukocytes (30 fold), thymus (28 fold) and kidney (24 fold), it is also expressed in brain (12 fold), and heart (11.5 fold). Low levels of expression are observed in skeletal muscle (2.6 fold), colon (2 fold), liver (1.8 fold), small intestine (1.2 fold), and placenta (1.3 fold).
  • hSKl mRNA The tissue distribution and expression levels of hSKl mRNA are overall very similar to that reported for the murine homologue (Kohama et al 1998). However, in both mouse and human, mRNA levels in the liver are low, and this contrasts to the finding that in the rat liver SK enzyme activity is twofold elevated compared to the brain (Olivera et al 1998). However, mRNA levels for SK have not been reported in the rat. En addition, data base searches, with the stSG2854 marker suggest expression in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.
  • Example 7 Genomic localisation of sphingosine kinase, related diseases: Several members of the Unigene cluster Hs.68061 have been mapped. Sequence identity of these Ests with hSK and the mapping data indicate that the gene is located in chromosome 17q25.2 band in a 9 cM interval between microsatellite markers D17S785 and D17S836 (104.7 and 114 cM respectively). The interval includes an STS (stSG2854), identical with Est sequences of the Hs.68061
  • the invention also concerns a method for detecting a mammal's susceptibility to develop auto-immune and inflammatory diseases which comprises comparing said mammal's DNA sequence encoding SKI to the DNA sequence of SEQ ED NO: 1 or SEQ ED NO: 2 and determimng the presence of single nucleotide polymorphism or polymorphic region in said mammal's coding sequence encoding SKI.
  • Example 8 Sphingosine kinase expression in insect cells
  • the recombinant plasmid pFastBac donor plasmid was transformed into DHlOBac (instructions (Gibco BRL,Gaithersburg,MD) for transposition into the bacmid. Isolation of recombinant bacmid DNA was selected by PCR of the sequence desired on whites colonies. Preparation of DNA bacmid was performed under conditions specifically developed for isolating large plasmids (> 100 Kb) and adapted for isolating bacmid DNA (Quiagen).
  • Sf21 cells were fransfected with the recombinant bacmid DNA in presence of Cellfectin reagent.
  • Cell culture, recombinant virus purification and tifration of the viruses were performed according the manufacturers instructions (Gibco BRL,Gaithersburg,MD).
  • Gibco BRL,Gaithersburg,MD For protein expression, cells at a density of 2xl06/ml were infected with the recombinant virus at an MOI of 5 to 10.
  • Three days post infection cells were pelleted by centrifugation and harvested in homogenization., buffer (Bis Tris 20mM (pH6.5), EDTA lOmM, DTT 2.5 M) supplemented with a mixture of protease inhibitors (Boerhinger).
  • Glycerol was added to a final concentration of 20-30% to all homogenates that were then stored at - 20°C in aliquots.
  • the gene has been cloned into a pFastbac HT expression vector, the expressed protein will contain 6X his at its amino terminus allowing the desired protein to be purified.
  • the fusion protein was purified with a appropriate based buffer system using NI-NTA resin.
  • DHlOBac E. Coli which contains the bacmid shuttle vector bmonl4272.
  • Transposition from donor plasmid to acceptor shuttle plasmid was detected by blue/white colony selection on X-gal/EPTG plates. White colonies were selected, grown up and recombinant Bacmid purified bac-to-Bac Baculovirus expression systems. (Instruction manual Gibco BRL Life Technologies)
  • Sf21 cells for viral stocks were grown in 47.5% TC100 (from Life Technologies; cat No 13055-025; lotNO 3031505)+5% heat-inactivated North American foetal bovine serum (FBS; from Life technologies; cat NO 10085-140; lot No 06Q6073A) as suspension cultures in shaker flasks and attached using standard procedures (King et al. ,1992).
  • Sf21 cells in ExCell 401 were grown in 47.5% TC100 (from Life Technologies; cat No 13055-025; lotNO 3031505)+5% heat-inactivated North American foetal bovine serum (FBS; from Life technologies; cat NO 10085-140; lot No 06Q6073A) as suspension cultures in shaker flasks and attached using standard procedures (King et al. ,1992).
  • No06Q6073A 1.5%SeaPlaque Agarose (lowgen) solution and stained with neutral red (6% in PBS; Sigma) 4 days post-infection.
  • Figure 17 shows an electrophoresis gel of the partial purification of hSKl from
  • the single band of line 8 corresponds to the predicted molecular weight of hSKl ie around 43kD.
  • the addition of the column elution fractions 7 and 8 represents the partial purified hSKl .
  • Example 9 Sphingosine kinase expression in bacteria
  • a PCR reaction was carried out using the pcDNA3-hSKl construct as a template, and primers designed to amplify the coding sequence of hSKl at the same time inserting cloning restriction sites at both ends (EcoRI - Xhol), in order to align the PGEX vector containing GST with the hSKl and make the fusion protein in frame (figure 16).
  • the construct carries the GST at the N-terminus of the hSKl.
  • Primers E and F :
  • Figure 16 illustrates the vector for the construction of hSKl tagged with GST for expression in bacterial cells.
  • the PGEX-SX-3 vector (from Promega) was used to construct and express a hSKl-GST fusion protein in bacteria.
  • E. Coli competent cells (strain BL21) were purchased from Promega
  • Bacterial transformation was carried out as per supplyer Standard Transformation Protocol.
  • Frozen competent cells were thawed on ice for 5 minutes, lOO ⁇ l was transfered to a child culture tube. 50ng of hSKl-cDNA was added and mixed by flicking the tube. The tubes were returned to ice for 10 minutes, after which a heat-shock was performed by placing the tubes in a water bath at 42°C for 45 seconds. Immediately the tubes were placed on ice for 2 minutes. 900 ⁇ l of cold SOC medium was added to the transformation reaction and incubated for 60 minutes at 37°C with shaking. Aliquotes of cells were plated on antibiotic containing plates and incubated at 37°C for 12-14 hours.
  • Example 10 Sphingosine kinase optimized source for the screening assays
  • Figure 9 illustrates the comparison of hSK activity from different sources: CHO cells, Bacteria, partially purified hSKl from insect cells. Similar levels are observed in mammal (Cho) and bacterial (BL21) cell exfracts. There is a dose response effect of increasing levels of partial purified hSK from insect cell transfections.
  • Y axis is the enzyme activity in SPA cpm
  • 2 ⁇ g total protein from bacteria (BI 21) and 0.1 ⁇ g of partially-purified baculovirus/insect cells hSK gave a good signal to noise ratio (around 12 fold).
  • the total amount of protein needed is 400 mg for 2000 plates, which represent around 10 liters of transformed and induced bacteria.
  • FIG. 10 illustrates the comparison of hSKl activity from different sources:
  • BL21 Transf. Basal means BL21 fransfected without IPTG induction.
  • BL21 Trnas. Induced means BL21 fransfected with IPTG induction.
  • P.Pur.rSPHK means partial purified recombinant hSKl .
  • 40 ⁇ g of the total cells extract from fransfected cos7 cells shows 50% more activity than 40 ⁇ g of total fransfected bacterial cell extract.
  • 40 ⁇ g of insect cell- extract shows minimal hSKl activity over basal levels (Cos7 basal).
  • 6 ⁇ g of partially purified hSKl from insect cell shows a 3 fold increase over the fransfected COS7 cell extract.
  • Transfected COS7 represent our positive control for optimal activity.
  • the partial purified enzyme from the baculovirus system gives the maximal activity observed thus far.
  • the bacterial extracts that overexpress the hSK gives between 40% to 50% of the total activity observed with the mammalian system, which renders this system as the most attractive solution.
  • Figure 11 describes the bacterial growth conditions for optimization of actively expressed hSKl. Different concentration of EPTG for induction, different temperatures of growth (R°T means room temperature),different incubation times are tested.
  • the bacterial cell extract under optimal bacterial growth and induction conditions (50 ⁇ M IPTG for 20hr) has 40% activity of the maximal" activity observed for fransfected mammalian cells (Cos cells) (Figure 12)
  • Figure 12 shows the comparison of hSKl activity expressed under different bacterial growth conditions and expressed in Cos cells.
  • Example 11 Sphingosine kinase antisense oligonucleotides
  • an antisense oligonucleotide corresponding to the first 21 coding nucleotides of the hSKl, was designed in an attempt to downregulate the protein and hence its activity.
  • U937 cells were fransfected with the antisense oligo, and calcium signals were analysed in a receptor coupled model in which we have previously shown sphingosine kinase to be activated.
  • the release of calcium from infracellular stores is impaired, demonstrating that sphingosine kinase does indeed play a significant role in receptor-coupled triggered physiological responses.
  • An antisense sequence to the first 24 nucleotides (coding for the first 8 amino acids) of the hSKl-cDNA was synthesised and protected at both ends with Phosphorothioate linkages -for the first and last two nucleotide pairs.
  • a control "scrambled oligo" (CTGGTGGAAGAAGAGGACGTCCAT, SEQ ED
  • U937 cells were transiently fransfected with an antisense oligonucleotide against * the first coding 21 nucleotides (coding for the first 7 amino-acids) of the human sphingosine kinase cDNA, using the Qiagen reagent, SuperFect.
  • Cells 1 X 10 6 per ml, in 10 ml. After 24 hrs, cells were transfected with 2 ⁇ g of scrambled antisense oligo (control) mixed with 20 ⁇ l SuperFect, or with 2 ⁇ g antisense oligo against human sphingosine kinase mixed with 20 ⁇ l SuperFect.
  • Figure 18 illustrates the antisense downregulation of hSKl protein levels.
  • the blot has been probed with a polyclonal antibody against hSKl (Ab 0144).
  • RPC22 Research Genetics
  • oligonucleotide probes were derived from the mouse SKI cDNA sequence (Kohama et al., 1998). Based on multiple alignments of SKI related cDNA sequences (Melendez et al., 2000), oligonucleotides were selected from the two ends of the cDNAs (gene proximal and gene distal probes) and from the conserved region . Positive BAC clones were purchased (Research Genetics) and have been re- screened with gene distal and conserved region probes. No clones were found positive with both conserved region and 3' (gene distal) radiolabelled oligonucleotide probes in hybridization experiments.
  • the catalytic domain of the enzyme presumably lies in the highly conserved region (see SED ID N°8), which is between aal6 and aa95 in the peptide sequence (SEQ ID NO: 3) downstream of the presumed alternative first exon coded sequences, therefore, this highly conserved region will be targeted in ES cells.
  • Catalysis critical region of the human SK are determined by 5' and 3' truncations and internal deletions.
  • Mouse BAC clones are identified by screening BAC libraries. Mini-libraries are prepared from verified positive clones and these libraries are screened with oligonucleotide probes to obtain genomic fragments that code for the catalytic domain. Sequencing verifies the presence of catalysis critical exons on one genomic fragment. 5' and 3' flanking genomic fragments with appropriate size (2.5-5 Kb) are cloned with oligo-probes, or are PCR amplified with appropriate primers from the cDNA. These fragments are inserted into the pSV-loxP targeting vector, in reverse orientation to the NEO transcription unit (experiment A).
  • loxP sites are inserted flanking the catalysis critical exon containing genomic fragments and these is also cloned immediately adjacent to the Neo transcriptional unit, the region is flanked with the 5' and 3' homoiogy arms for targeting.
  • Appropriate restriction sites are inserted in order to create an optimal situation for the detection of recombination mediated replacement of the wild type catalysis critical region by the loxP site flanked fragment.
  • the targeting vectors are introduced into ES cells by elecfroporation or other methods. Neomycin resistant colonies are screened for the identification of specific targeting events.
  • transient Cre recombinase expression in ES cells are used to remove the loxP flanked Tn-5 Neomycin resistance gene from the targeted allele in experiment A. Once ES cell colonies with targeted alleles are identified blastocysts will be injected with ES cells from these colonies. Mice with high degree of ES cell contribution are screened by coat colour examination, germline transmitting mice will be selected by breeding and tail DNA testing.
  • mice Once hemizygous targeted mice (SK -/+) are obtained, they are tested in biological experiments together with homozygous null allele (SK -/-) mice (if these are viable) are generated by breeding (Gene targeting, Ed. A.L. Joyner ERL press/Oxford, 1993).
  • experiment B homozygous insertion positive mice are generated and crossed with tissue specific Cre recombinase expressing transgenic mice. The result of this experiment is tissue specific deletion of the SK gene. If the Cre recombinase is controlled by an inducible promoter, deletion of SK is inducible
  • Peptide 1 FTLMLTERRNHARELVRSEE SEQ ED NO: 18
  • Peptide 3 ADVDLESEKYRRLGEMRFTL (SEQ ED NO:20)
  • Peptide 4 SGCVEPPPSWKPPQQMPPPEE (SEQ ED NO:21)
  • Peptide 1 Serum # 0140 (rabbit 1); Serum # 0141 (rabbit 2).
  • Peptide 2 Serum # 0142 (rabbit 1); Serum # 0143 (rabbit 2).
  • Peptide 3 Serum # 0144 (rabbit 1); Serum # 0145 (rabbit 2).
  • Peptide 4 Serum # 0146 (rabbit 1); Serum # 0147 (rabbit 2).

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Abstract

L'invention concerne un gène de type 1 de la sphingosine kinase. Plus précisément, l'invention concerne un acide nucléique de ladite sphingosine kinase purifié ou isolé, ou une séquence complémentaire de celle-ci, ou des fragments de celle-ci. L'invention concerne des oligonucléotides, des polypeptides recombinants, des vecteurs recombinants, des cellules hôtes recombinantes comprenant ledit acide nucléique, ainsi que la production d'anticorps, des méthodes de criblages, des oligonucléotides antisens et des mammifères 'knock out'.
PCT/EP2000/009498 1999-10-28 2000-10-27 Gene de la sphingosine kinase humaine WO2001031029A2 (fr)

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AU10202/01A AU1020201A (en) 1999-10-28 2000-10-27 Human sphingosine kinase gene
JP2001533164A JP2003512072A (ja) 1999-10-28 2000-10-27 ヒトスフィンゴシンキナーゼ遺伝子
CA002389127A CA2389127A1 (fr) 1999-10-28 2000-10-27 Gene de la sphingosine kinase humaine
MXPA02004294A MXPA02004294A (es) 1999-10-28 2000-10-27 Gen de esfingosina cinasa de humano.
BR0015138-6A BR0015138A (pt) 1999-10-28 2000-10-27 Gene da esfingosina quinase humana
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WO2001085953A1 (fr) * 2000-05-11 2001-11-15 Medvet Science Pty. Ltd. Sphingosine kinase et ses utilisations
WO2002000887A1 (fr) * 2000-06-28 2002-01-03 Medvet Science Pty Ltd Nouveaux variants moleculaires et leurs utilisations
WO2002028406A2 (fr) * 2000-10-05 2002-04-11 Novartis Ag Initiation de la formation de vaisseaux sanguins par administration de polynucleotides codant pour des sphingosine kinases
US6858383B2 (en) 2000-12-22 2005-02-22 Medlyte, Inc. Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
EP1517989A2 (fr) * 2002-01-17 2005-03-30 Children's Hospital & Research Center at Oakland Compositions et procedes permettant de moduler le metabolisme des sphingolipides et/ou la signalisation par les sphingolipides
US7674580B2 (en) 2002-01-17 2010-03-09 Children's Hospital & Research Center At Oakland Compositions and methods for the modulation of sphingolipid metabolism and/or signaling
US7829674B2 (en) 2006-10-27 2010-11-09 Lpath, Inc. Compositions and methods for binding sphingosine-1-phosphate
US7973143B2 (en) 1997-09-29 2011-07-05 Children's Hospital & Research Center At Oakland Sphingosine-1-phosphate lyase polypeptides, polynucleotides and modulating agents and methods of use therefor
US8871202B2 (en) 2008-10-24 2014-10-28 Lpath, Inc. Prevention and treatment of pain using antibodies to sphingosine-1-phosphate
US9217749B2 (en) 2006-05-31 2015-12-22 Lpath, Inc. Immune-derived moieties reactive against lysophosphatidic acid
US9274129B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Methods and reagents for detecting bioactive lipids
US9274130B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Prevention and treatment of pain using antibodies to lysophosphatidic acid

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CN109355269B (zh) * 2018-10-21 2020-03-24 北京双因生物科技有限公司 鞘氨醇激酶1及其融合蛋白及其用途
CN113295793B (zh) * 2021-05-20 2022-11-04 复旦大学附属中山医院 预测早期糖尿病以及糖尿病发生的生物标志物、其检测方法与应用

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7973143B2 (en) 1997-09-29 2011-07-05 Children's Hospital & Research Center At Oakland Sphingosine-1-phosphate lyase polypeptides, polynucleotides and modulating agents and methods of use therefor
WO2001060990A2 (fr) * 2000-02-14 2001-08-23 Curagen Corporation Nouvelles kinases de sphingosine
WO2001060990A3 (fr) * 2000-02-14 2002-03-21 Curagen Corp Nouvelles kinases de sphingosine
WO2001085953A1 (fr) * 2000-05-11 2001-11-15 Medvet Science Pty. Ltd. Sphingosine kinase et ses utilisations
WO2002000887A1 (fr) * 2000-06-28 2002-01-03 Medvet Science Pty Ltd Nouveaux variants moleculaires et leurs utilisations
WO2002028406A3 (fr) * 2000-10-05 2002-06-20 Novartis Ag Initiation de la formation de vaisseaux sanguins par administration de polynucleotides codant pour des sphingosine kinases
WO2002028406A2 (fr) * 2000-10-05 2002-04-11 Novartis Ag Initiation de la formation de vaisseaux sanguins par administration de polynucleotides codant pour des sphingosine kinases
US6881546B2 (en) 2000-12-22 2005-04-19 Medlyte, Inc., Sdsu Heart Institute Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
US7169390B2 (en) 2000-12-22 2007-01-30 Lpath Therapeutics, Inc. Compositions and methods for the treatment and prevention of cancer, angiogenesis, and inflammation
US7901682B2 (en) 2000-12-22 2011-03-08 Lpath, Inc. Compositions and methods for the treatment and prevention of cancer, angiogenesis, and inflammation
US6858383B2 (en) 2000-12-22 2005-02-22 Medlyte, Inc. Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor
EP1517989A2 (fr) * 2002-01-17 2005-03-30 Children's Hospital & Research Center at Oakland Compositions et procedes permettant de moduler le metabolisme des sphingolipides et/ou la signalisation par les sphingolipides
EP1517989A4 (fr) * 2002-01-17 2006-07-12 Childrens Hosp & Res Ct Oak Compositions et procedes permettant de moduler le metabolisme des sphingolipides et/ou la signalisation par les sphingolipides
US7674580B2 (en) 2002-01-17 2010-03-09 Children's Hospital & Research Center At Oakland Compositions and methods for the modulation of sphingolipid metabolism and/or signaling
US9217749B2 (en) 2006-05-31 2015-12-22 Lpath, Inc. Immune-derived moieties reactive against lysophosphatidic acid
US9274130B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Prevention and treatment of pain using antibodies to lysophosphatidic acid
US9274129B2 (en) 2006-05-31 2016-03-01 Lpath, Inc. Methods and reagents for detecting bioactive lipids
US7956173B2 (en) 2006-10-27 2011-06-07 Lpath, Inc. Nucleic acids coding for humanized antibodies for binding sphingosine-1-phosphate
US8067549B2 (en) 2006-10-27 2011-11-29 Lpath, Inc. Humanized antibodies and compositions for binding sphingosine-1-phosphate
US8025877B2 (en) 2006-10-27 2011-09-27 Lpath, Inc. Methods of using humanized antibodies and compositions for binding sphingosine-1-phosphate
US8026342B2 (en) 2006-10-27 2011-09-27 Lpath, Inc. Compositions and methods for binding sphingosine-1-phosphate
US7829674B2 (en) 2006-10-27 2010-11-09 Lpath, Inc. Compositions and methods for binding sphingosine-1-phosphate
US8871202B2 (en) 2008-10-24 2014-10-28 Lpath, Inc. Prevention and treatment of pain using antibodies to sphingosine-1-phosphate

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