Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN SPHINGOSINE HINASE GENE
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
Sphingosine-1-phosphate, the product of sphingosine kinase, is an important
signaling molecule with infra- 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 SK1A 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).
SUMMARY OF THE INVENTION
The invention concerns a purified or isolated nucleic acid encoding a human
sphingosine kinase (hereinafter hSK) cDNA or a sequence complementary
thereto.
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.
CONFIRMATION COPY
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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:
- mixing a recombinant hSK with sphingosine, labelled ATP and a
candidate molecule of interest; and
- measuring the level of conversion of sphingosine to labelled-
sphingosine-1-phosphate (S1P).
The invention also concerns a kit for the screening of candidate molecules
which
are inhibitors of hSK.
The kit comprises:
- recombinant hSK; and, optionally,
- labelled ATP and sphingosine.
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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 obstcucfiive
pulinonary disease; asthma; cancer; hemostatis, stroke, coronary artery
disease,
hematopoietic disorders such as leukemia, the natural wound healing processes,
myocardial infarction, embryogenesis.
BRIEF DESCRIPTION OF THE FIGURES
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-erythro-sphingosine and illustrate that hSKI 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 SB illustrates the expression and cellular localisation of hSK1 fused
with
EGPF at the C-terminal end.
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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 hSKI activity from different sources:
Cos7, bacteria, insect cells.
Figure 11 describes the bacterial growth conditions for optimization of
actively
expressed hSKI.
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
(SO~M IPTG for 20hr) is 40% of the activity observed for the transfected Cos7
cells extract.
Figure 13 illustrates the physiological relevant role of hSKI 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.
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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
5 Sf21 insect cells.
Figure 18 illustrates the antisense downregulation of hSKl protein levels.
DETAILED DESCRIPTION OF THE INVENTION
A) Human sphin~osine kinase cDNA
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 n7 N°1 or of
SEQ B7
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 m 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:
-that have primary structures which are related to the hSK of the amino-
acid sequence of SEQ >D N°3, due to the high sequence identity between
the
amino-acid sequences; or
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-that are biologically related to the polypeptides of the amino-acid
sequence of SEQ ID N°3, either because these homologous polypeptides
are
recognized by antibodies specifically directed against the amino-acid sequence
of SEQ m N°3 and/or because these homologous polypeptides have the same
biological activity as the polypeptides of the amino-acid sequence of SEQ m
N°3, such as for example the capacity to convert sphingosine into
S 1P.
The term "isolated", when used herein, requires that the material be removed
from its. original environment (e.g. the natural environment if it is
naturally
occurring). For example, 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.
The term "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.
Throughout the present specification, the expression "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.
As used interchangeably herein, the terms "oligonucleotides", "nucleic acids"
and "polynucleotides" 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.
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Further to its general meaning understood by one skilled in the art, the term
"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,
(c) an analogous form of pyrimidine, or
(d) an analogous sugar;
(e) modified nucleotides such as methylated, phosphorylated, ubiquitinated
nucleotides.
For examples of analogous linking groups, purines, pyrimidines, and sugars,
see
for example PCT publication N°WO 95/04064.
The 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.
B) Recombinant hSK polynucleotides
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 B7 NO: 8
corresponding to a region of SK conserved between species. In fact the
inventors
have shown that a 80 amino-acids long region of hsKl is conserved between
species (figure8).
_ ~ Primers or probes
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More particularly, 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° 1 or of SEQ Il7
N°2, or a
sequence complementary thereto.
These 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.
In one particular embodiment of this invention 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 ~ N°1 or of SEQ >D-~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) CTGGGTCTTGTAGAAGAGCAGCAAGTGCT
(SEQ B7 NO: 14)
SK5'end48 (gene proximal)
AGTTCACTGCAATCCTTTCTTATCTGGGTTCG (SEQ B7 NO: 15)
SK3'end (gene distal) TTCTGTGGATGGAGAGCTGATGGTATGG (SEQ
m NO: 16)
SK BOX (conserved region) ATGAAGTGGTGAATGGGCTAATGGAACG
(SEQ )D NO: 17)
The 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
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N°WO 97/05 277, the entire contents of which is herein
incorporated by
reference.
In another embodiment of the invention these nucleic acids are useful as
primers.
The most preferred primers are the following:
A= 5'end TAT GCT AGC ATG GAT CCA GCG GGC GGC (SEQ >D NO:
4)
B= 3'end AAT GAA TTC TCA TAA GGG CTC TTC TGG (SEQ m NO:
5)
C= 5'end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ
~ NO: 6)
D= 3'end ATT ATC GTC GAC TAA GGG CTC TTC TGG CGG (SEQ m
NO: 7)
E= 5'end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ
m NO: 10)
F= 3'end AGT CGA GGC TGA TCA GCG AG (SEQ m NO: 11)
Hybridizing_polynucleotides
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 B7 NO: 22) encoding the 80 amino acids conserved region of
hSKl .
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As an illustrative embodiment, 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 100~.g/ml of salinon sperm DNA.
5 The hybridization step is followed by four washing steps:
~ two washings during 5 minutes, preferably at 65°C in a 2 x SSC and
0.1% SDS buffer;
~ one washing during 30 minutes,.preferably at 65°C in a 2 x SSC and
0.1 % SDS buffer;
10 ~ one washing during 10 minutes, preferably at 35°C in a 0.1 x SSC
and
0.1 % SDS buffer,
It being understood that the 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.
Any of the 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. For
example, useful labels include radio-active substances (3zP, 3sS, 3H~ lzsn~
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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).
Advantageously, 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).
Any of the 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.
The 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 specific embodiment of a support on which nucleic acid probes of the
invention are immobilized, such a support may also contain other immobilizeda
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
m NO: 22) encoding the 80 amino acids conserved region of hSKI.
C) Amplification of the hSK cDNA
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:
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(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
(b) optionally, detecting the amplification products.
In a first preferred embodiment of the above method, the nucleic acid encodes
a
hSK polypeptide of SEQ 1D N°3.
~ a second preferred embodiment of the above amplification method, 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) 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.
In a first preferred embodiment of the kit described above, the nucleic acid
to be
amplified encodes hSK polypeptide of SEQ ID N°3.
In a second preferred embodiment of the above amplification kit, the
amplification product is detected by hybridization with a labelled probe
having a
sequence which is complementary to the amplified region.
D) Recombinant vectors and hosts cells for the expression of a recombinant
hSK
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1) Recombinant vectors
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) 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 >D 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.
In a first preferred embodiment 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.
Another preferred embodiment of the recombinant vectors according to the
invention 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 >D N°3.
Preferred vectors comprises a nucleic acid sequence as shown in SEQ >D
N° 1 or
SEQ >D N°2.
Within certain embodiments, 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.
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Preferred eukaryotic vectors of the invention are listed hereafter as
illustrative
but not limitative examples: pcDNA3, pFLAG, pCMV-Script, . pIND,
pMCINEO, pHIL, pGAPZA, pMT/VS-His-TOPO, pMT/VS-His, pAc5.1/VS-
HisA, pDS47/VS-His, pcDNA4, pcDNA6, pEFI, pEF4, pEF6, pUB6, pZeoSV2,
pRc/CMv2, pcDMB, pCR3.l, pDisplay, pSecTag2, pVP22, pEMZ, pCMV/Zeo,
pSinRepS, pCEP, pREP, pHook-1.
The vectors pcDNA3, pFLAG, and pCMV (particularly pCMVS) are most
preferred.
Preferred bacteriophage recombinant vectors of the invention are P1
bacteriophage vectors such as described by Sternberg N.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 S~ and
SfZl. Specific suitable host vectors includes; but are not restricted to
pFastBac-1,
pIZ/VS-His, pBacMan-l, pBlueBac4.5, pBlueBacHis2, pMelBacA, pVL1392,
pVL1393
Preferred baculovirus vector is pFastBacHTa.
A preferred bacterial vector is pGEX.
a) ReEUlatory expression seguences
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.
As used herein, the term "operably linked" refers to a linkage of
polynucleotide
elements in a functional relationship. For instance, a promoter or an enhancer
is
operably linked to a coding sequence if it affects the transcription of the
coding
sequence.
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More precisely, two DNA molecules (such as a polynucleotide containing a
promoter region and a polynucleotide encoding a desired polypeptide or
polynucleotide) 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.
Generally, 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
10 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.
In a specific embodiment wherein the vector is adapted for transfecting and
15 expressing desired sequences in eukaryotic host cells, 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.
b) Promoter seguences
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.
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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
recombinant vector sequences within which the construct promoter/coding
sequence has been inserted.
2) Recombinant host cells
Host cells that have been transformed or transfected 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.
Are included host cells that are transformed (prokaryotic cells) or are
transfected
(eukaryotic cells) with a recombinant vector such as one of those described
above. Preferred host cells used as recipients for the expression vectors of
the
invention are the following:
(1) 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,
Streptomyces and Staphylococcus; Sf 9 cells (ATCC N°CRL 1711), Sf 21
cells.
(2) eukaryotic host cells: HeLa cells (ATCC N°CCL2; N°CCL2.1;
N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC
N°CItL 1650;
N°CRI, 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)
and hi-5
cells.
More particularly, expressions of the recombinant hSK of the invention in COS-
7 or in bacterial cells are preferred embodiment of the invention.
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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.
E) Production of recombinant hSK
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;
(b) culturing, in an appropriate culture medium, a host cell previously
transformed or transfected with the recombinant vector of step (a);
(c) harvesting the culture medium thus obtained or lyse the host cell, for
example by sonication or osmotic shock;
(d) separating or purifying, from said culture medium, or from the pellet
of the resultant host cell lysate, the thus produced recombinant polypeptide
of
interest.
In some instances, it may be required to tag the recombinant hSK prior to
purification. The tag is then in most instances encoded into the nucleotide
sequence that is needed to express the polypeptide. Examples of such 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.
One preferred tag is the FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-
Lys, SEQ )D NO: 23) which is used to express the recombinant hSK of the
invention as a fusion protein. Both amino-terminal and carboxy-terminal FLAG
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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 pCMV-5 vector. More particularly, a pFLAG-CMV-1 or
pFLAG-CMV-2 vector can be used for amino-terminal tagging whereas a
pFLAG-CMV-Sa, -Sb or Sc vector can be used for carboxy-terminal tagging.
However, 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.
20
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 Ni NTA column.
In another embodiment of the above method, 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.
According to the results the production rate is higher for bacterial
expressions
than for insect cells expression.
F~ Purified recombinant hSK
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
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or 99%, amino-acid identity with the polypeptide having the amino acid
sequence of SEQ >D N°3.
Extract of infected insect cells expressing a tagged hSKI may be purified
through resin column having affinity for the tag.
In a particular embodiment, extract of infected insect cells expressing a 6His
tagged hSKl are run through NI-NTA resin column.
In another embodiment, extract ofrinfected insect cells expressing a GST
tagged
hSKl are.purified through glutathion resin.
G) Modified recombinant hSK
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 m 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 homology 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.
Alternatively, in the case of an 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. In other
words,
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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.
The peptides 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.
10 Examples of amino-acids belonging to specific classes include Acidic (Asp,
Glu), 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, 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
15 amino acid of a particular class for another amino acid belonging to the
same
class.
By 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
20 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. This is a peptide in which the -CONH- peptide bond is modified
and
replaced by a (CHZNH) reduced bond, a (NHCO) retro inverso bond, a (CHZ-O)
methylene-oxy bond, a (CHzS) thiomethylene bond, a (CHZCHZ) carba bond, a
(CO-CHZ) cetomethylene bond, a (CHOH-CH2) hydroxyethylene bond), a (N-N)
bound, a E-alcene bond or also a -CH=CH-bond.
The invention also encompasses a recombinant hSK in which at least one peptide
bond has been modified as described above.
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The polypeptides according to the invention may also be prepared by the
conventional methods of chemical synthesis, either iri 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.
For solid phase synthesis, the technique described by Mernfield (1965a; 1965b)
may be used in particular.
H) Antibody production
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
Milstein (1975)).
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).
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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).)
I) Assay for the screeninE of inhibitors of hSK
Sphingosine kinase converts the substrate sphingosine to sphingosine-1-
phosphate (51P). 51P is believed to play several roles in physiological
processes.
Some of the potential physiological roles of S 1P include:
1 ) Within cells:
Release of calcium from stores;
Activation of cyclin-dependent kinases;
Key signalling intermediate in Fc receptor initiated cascades;
flVILP induced enzyme release;
TNF-a 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
signaling cascade initiated at Fc~ RI by modulating the balance of the
counterregulatory lipids. (Prieschl et al., 1999)
Furthermore, PDGF (platelet derived growth factor) induces high levels of
sphingosine kinase activity and S1P generation in platelets. (Yatomi et al. ,
1997;
Yatomi et al., 1995)
~ h~~ ~bilical vein endothelial cells, TNFa, a pleiotropic cytokine, induces
activation of sphingosine kinase and generation of S1P which turn may serve as
a second messenger to mediate TNFa induced endothelial cell activation and
adhesion molecule expression. (Xia et al., 1998)
Also, in osteoblast sphingosine monophosphate plays a role of second messenger
for TNFa induced IL-6 (interleukine 6) synthesis. (Tokuda et al., 1999) These
properties strongly indicate a potentially important role of S1P and hence of
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sphingosine kinase itself in pain, as well as in inflammation, particularly
inflammation following injury.
It has been further shown that S1P protects from apoptosis. More particularly,
S1P prevents the appearance of intranucleosomal DNA fragmentation and
morphological changes which are main features of apoptosis. (Spiegel et al.,
1998)
Furthermore, it has also been demonstrated that S1P 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. (Auge et al., 1999)
As a result, 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. In contrast, high intracellular levels of sphingosine-1-phosphate,
also
secreted by allergically stimulated mast cells, activate the mitogen-activated
protein kinase pathway, resulting in hexosaminidase and leukotriene release
or,
in combination with ionomycin, cytokine production. Hence, the balance
between sphingosine and S1P modulates the allergic responsiveness of mast-
cells. (Prieschl et al., 1999)
As a result, inhibitors of sphingosine kinase may be useful in preventing
allergy
reactions.
It has been previously shown that 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
sph~gosine kinase prevented the survival effect of 1 a,25-dihydroxyvitamin D3
(1,25-(OH)2D3), a cytoprotective agent, on human promyelocytic leukemia HL-
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60 cells. (Kleuser et al., 1998) Thus, sphingosine kinase inhibitors may be
useful
in the prevention and treatment of proliferative diseases including cancer,
hematopoietic disorders such as leukemia.
2) When released from the cells:
Proliferation;
Chemotaxis (attraction and activation of macrophages);
Cytoskeletal changes (Stress fiber formation and cell shape contraction,
aggregation and secretion);
Mediates attachment: Fibronectin matrix assembly; and
Assembly and phosphorylation of paxillin and p125-FAK.
More particularly, sphingosine kinase plays a role in Caz+ release through
GPCRs (G-protein-coupled receptors) induced Ca2+ signaling. (Meyer et al,
1998)
S1P is released from activated platelets in large amounts. (Yatomi et al.,
1995)
This could indicate a potential role of S1P in thrombosis,.hemostasis, the
natural
wound healing processes, atherosclerosis, stroke, myocardial infarction.
Furthermore, S1P stimulates the binding of fibronectin or its N-terminal 70-
kd~i
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)
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More particularly, S1P could have a likely role in early atherogenesis and
fibrosis. As a consequence, 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 arid
inflammatory arthritis, T helper-1 related diseases, chronic.- obstructive
pulmonary disease, asthma, cancer and neurodegenerative disorders.
10 The isolation of a nucleotide sequence encoding human sphingosine kinase is
useful in that it allows the skilled person to screen for suitable 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.
The
term "structural analogue" is intended to designate compounds which have a
15 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.
Several assay formats can be used to carry out the method of the present's
invention. Preferred assay formats include scintillation assays such as the
scintillation proximity assay (SPA) or the 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)
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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
3H,
~ZSh iaC~ 3sS or 33P that emit low-energy radiation, the energy of which is
easily
dissipated in an aqueous environment. For example, the electrons emitted by 3H
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 interactiowvith 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 flashplafe surface, its radiation
energy is
1 S . 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.
In the context of the present invention, 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 S1P 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
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and a signal resulting from the interaction between labelled S1P and the
flashplate or SPA bead will be recorded.
J1 Antisense
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. Thus the inhibition of sphingosine kinase expression permits the
study of the effect of hSKI in cells, tissues or animals.
K) Knock Out animals
The determination by the inventors of the 80 amino acids between species
conserved region present in SK now allows the design of polynucleotide
constructs wherein the nucleic acid portion encoding the 80 amino acids
conserved region; or a portion of it has been deleted.
In a preferred embodiment 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",
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.
According to this specific embodiment, 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.
In a first embodiment of the nucleic acid above, 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.
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In a second embodiment of the nucleic acid above, the heterologous
polynucleotide comprises a selection marker.
In a third embodiment of the nucleic acid above, 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 by
separated by a 8 by 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).
In the specific embodiment of the 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.
In a further aspect of the invention, a nucleic acid which encodes for a
polypeptide as defined above is operably linked to a regulatory sequence.
Preferably, the regulatory sequence consists of a inducible promoter.
Most preferably, the regulatory sequence consists of a promoter inducible by
Ponasterone.
EXAMPLES
Material and methods
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Growth medium including all supplements were purchased from Gibco BRL
(Paris, France). Transfection reagents were from QIAGEN (Paris, France). All
lipids were purchased from Sigma-Aldrich (Paris, France). [a3zP]ATP and
[y33P]ATP were from Amersham Pharmacia Biotech (Amersham, Paris, France).
Human poly A+ 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 penicilli~i and 10 mg/ml streptomycin at
37°C,
6.5% carbon dioxide in a water saturated atmosphere.
Example 1: Human Sphin~osine Kinase (hSKl) cDNA isolation.
Searches using the recently cloned marine sphingosine kinase cDNA sequence
(Kohama et al., 1998) identified a human Est cluster and several human Est
sequences in public (Unigene) and local (Compugen) cDNA cluster databases.
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 SKla
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 IMAGE 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).
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The cDNA sequence and peptide sequences of hSKI 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).
5 The PCR primers are the following
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:
S)
Primers C and D:
C= S' end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ
117 NO: 6)
D= 3'end ATT ATC GTC GAC TAA GGG CTC TTC TGG CGG (SEQ 1D
NO: 7)
These primers are used for the cDNA amplification such as PCR amplification.
Example 2: Sphin~osine kinase characterization
The predicted peptide sequence is 384 as (seq ID N°3), with a predicted
mass of
42.5 kD and pI of 6.9 at neutral pH. Similarity to the mouse SKIa is 85%
(Needleman Wunsch similarity index). With the exception of the C terminal,
similarity with the mouse SK is contiguous.
Peptide similarity searches identify a 80 as conserved region (Argl6 -Pro95)
(SEQ ID N° 8) present in various known and hypothetical peptides from
bacteria
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to human. (CAB14972 Bacillus subtilis; CAA18718 Arabidobsis thaliana;
CAB11477 Saccaromyces pombe; 551398 Saccaromyces cerevisae; SS67059
Saccaromyces cerevisae; CAA91259 Caenorhabditis elegans; AAC67466
Caenorhabditis elegans; Mouse SKla (Mouse SPHK la); hSKI (huSPHK)). The
conserved amino acids are shown in black (Figure 2B)
This sequence includes a region distantly related to a short signature
peptide,
LVRSEELGRWDALVVM (SEQ >D N°9) of NADPH dependent aldo-keto
reductase family of enzymes. Within the 80 as conserved region, highly
conserved residues mark seemingly characteristic and predictable features of
the
secondary peptide structure in three blocks.(Figure 2A) Conserved Asn22-Pro23
and G1y26 residues present a probable beta turn and a coil structure, proximal
to
the GGKGK sequence (SEQ m NO: 24) which may be part of the ATP binding
site also suggested for the mouse SK1 (KQh ama et al., 1998). His59-A1a60 are
indicated to be exposed on the surface, while G1y80-Asp81-G1y82 suggest the
presence of a flexible region. Spacing of Asn22-Pro23, and G1y26, in block
one, Thr50, His59-A1a60, in block two and G1y80-Asp81-G1y82 , G1u86 and
G1y90 residues in block three of the conserved region is identical from
Bacillus
subtilis to human.
Example 3: Transfection of hSKl.
COS7 cells were transiently transfected with the vector pcDNA3 alone or vector
containing the human sphingosine kinase cDNA, using the Qiagen reagent,
SuperFect. Cells were seeded 5 X 106 per well, in 6 wells plates. After 24
hrs,
cells were transfected with 10 ~g of vector (pcDNA3) mixed with 20 ~,1
SuperFect, or with 10 pg vector containing the human sphingosine kinase cDNA
(pcDNA3-hSKl) mixed with 20 ~1 SuperFect.
Example 4: Sphineosine kinase activity and specificity assay.
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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 p,M sphingosine,
0.25% Triton
X-100, and [33P]ATP (10 ~Ci, 1 mM), and MgCl2 (10 mM). The kinase activity
was expressed as nanomoles of SPP/min/mg.
~ hSKl activity and substrate characterization.
To ensure that the hSK was indeed a functional sphingosine kinase, COS7 cells
were transfected with the vector-pcDNA3-containing the hSKl cDNA and, after
48 hours, sphingosine kinase activity was measured. Low levels of endogenous
sphingosine kinase activity were present in control cells (either
untransfected or
transfected with vector alone). However, cells transfected with hSK (with 10
p,g
DNA) generated over 107-fold increased sphingosine kinase activity (Figure 3).
Figure 3 shows that hSKl specifically phosphorylates n-erythro-sphingosine (D-
erythro-SPH), and to a lesser extent n>L-erythro-dihydrosphingosine (D,L-
erythro-DHS). This kinase does not phosphorylate: any of the "threo"isoforms
of
dihydrosphingosine (D,L-treo-dihydrosphingosine; L-threo-dihydrosphingosine;
L-threo-dihydrosphingosine); ceramides (hydroxy-ceramide; non-hydroxy-
ceramide); diacylglycerol (DAG); phosphatidylinositol (PI);
phosphatidylinositol-4-phosphate (PIP); or phosphatidylinositol-4,5-
bisphosphate (PIP2).
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.
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~ Substrate~ecificity and competitive inhibition of the hSKl.
The expressed hSKl shows typical Michaelis-Menten kinetics (vMax = 56
nMole/min/mg and Km = S~.M) (Figures 4A (4A1, 4A2) and 4B (4B 1, 4B2)).
D,L-threo-dihydrosphingosine (on figure 4A (4A1, 4A2), DHS) and N,N-
diMethyl-sphingosine (N,NdiMS) are known inhibitors of sphingosine kinase
(Kohama et al., 1998; Olivera et al., 1998). In agreement to this, we show
here
that both these compounds inhibit expressed hSKl activity. The kinase is
inhibited by D,L-threo-dihydrosphigosine (Ki = 3~M), and N,N,diMethyl-
sphingosine (Ki = S~M).
Example 5: hSK constructs fused to EGFP (Enhanced Green Fluorescence
Protein
In order to characterise and understand the potential mechanisms that regulate
hSKl activity and cellular localisation, we have made two hSK constructs fused
to EGFP (Enhanced Green Fluorescence Protein), at either end of the kinase.
~ Construction of EGFP-hSKl. (N-terminal fusion) mammalian expression.
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 (NheI - EcoRI) in
order
to align the EGFP with the hSKI and make the fusion protein in frame. The
constructs carries the EGFP at the N-terminus of the hSKl .
Primers A and B:
A= S' end TAT GCT AGC ATG GAT CCA GCG GGC GGC (SEQ B? NO:
4)
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B= 3' end AAT GAA TTC TCA TAA GGG CTC TTC TGG (SEQ ID
NO:S)
~ Construction of EGFP-hSKl . (C-terminal fusion) mammalian expression.
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 - SaII) 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 .
' 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 B7
NO: 7)
~ Transfection of hSKI .
COS7 cells were transiently transfected with the vector pcDNA3 alone or vector
containing the human sphingosine kinase cDNA, using the Qiagen reagent,
SuperFect as described in example 4.
~ Transfection of EGFP-hSKI . (1V-terminal fusion)
COS7 cells were transiently transfected with the vector pCI-EGFP1 alone or
vector containing the human sphingosine kinase cDNA (see figure 14), using the
Qiagen reagent, SuperFect. Cells were seeded 5 X 106 per well, in 6 wells
plates.
After 24 hrs, cells were transfected with 10 ~g of vector (pCI-EGFP1) mixed
with 20 ~1 SuperFect, or with 10 pg vector containing the human sphingosine
kinase cDNA (pCI-EGFPI-hSKI) mixed with 20 ~1 SuperFect.
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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.XbaI (sens) and
5 STOP.EGFP (antisens) primers, cut XBAI/XhoI and subcloned into pCI cut by
NheI and XhoI. EGFP STOP codon was deleted. The frame for subcloning a
sequence of interest with EGFP fused to the N-terminus is shown at the bottom
of the figure.
10 ~ Transfection of hSKI-EGFP. (C-terminal fusion)
COS7 cells were transiently transfected with the vector pCI-EGFP-2 alone or
vector containing the human sphingosine kiilase cDNA (see figure 15), using
the
Qiagen reagent, SuperFect. Cells were seeded 5 X 106 per well, in 6 wells
plates.
15 After 24 hrs, cells were transfected with 10 p,g of vector (pCI-EGFP-2)
mixed
with 20 ~,1 SuperFect, or with 10 pg vector containing the human sphingosine
kinase cDNA (pCI-EGFP-2-hSKl) mixed with 20 p.1 SuperFect.
Figure 15 illustrates the vector for the construction of hSK-EGFP (C-terminal
20 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 XhoI/NotI and subcloned into pCI cut
by SaII and NotI. A new SaII site was included into the PCR product. The frame
for subcloning a sequence of interest with EGFP fused to the C-terminus is
25 shown at the bottom of the figure.
The two fusion proteins express well in COS7 cells, the EGFP/hSKI fusion
protein expresses primarily as a soluble cytosolic protein. (Figures SA and
SB)
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Figure 5 A describes the expression and cellular localisation of hSKl fused
with
EGPF at the N-terminal end. EGFP-hSKl (N-terminal fusion) is expressed in a
cytosolic manner when transfected into Cos7 cells, as shown by the green
colour
(lightest colours on figure 5A).
Whereas, the hSKI-EGFP appear to be partially localised in a granular form,
although, general cytosolic expression is also observed (Figure 5B).
Figure SB illustrates the expression and cellular localisation of hSKl fused
with
EGPF at the C-terminal end. hSKI-EGFP (C-terminal fusion) is expressed
primarily in a cytosolic manner, with some granular localisation when
transfected into Cos 7 cells, as shown by the green colour (lightest colours
on
figure 5B).
Kinase assays, of cell extracts from cells transfected with either contract,
show
that the EGFP-hSKl fusion protein is more active than the hSKl-EGFP one.
(Figure 6)
Figure 6 shows the lcinase activity of hSK fusion proteins. Overexpression of
hSK-EGFP (N-terminal fusion) (EGFP-hSKl) has similar activity as the
overexpressed unfused untagged protein. On the other hand, the hSK-EGFP C-
terminal fusion (hSKl-EGFP) shows 40% less activity than the unfussed or N-
terminal fusion proteins.
This is not a problem with transfection efficiency, since Western blots
(Figure
7), as well as the confocal images (figures 5A and SB), indicate that the
levels of
expression for the two proteins is similar.
Figures 5A and SB show similar green fluorescence intensity suggesting that
the
expression levels for both C-terminal and N-terminal fusion proteins are
similar.
Figure 7 is a Western blot analysis with anti-EGFP Antibody. Figure 7
demonstrates that both C-terminal (hSKl-EGFP) and N-terminal (EGFP-hSKl)
hSKIEGFP fusion proteins are expressed to similar levels in Cos7 cells.
Example 6: Sphin~osine kinase localisation in tissues
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A Northern blot containing approximately 1 p,g of poly A+ RNA per lane from
12 different human tissues, was hybridised with the 1.7 kb insert of pcDNA3-
hSKl, purified from the gel and labeled with [3zP]aATP using random primer
labeling kit. The hybridization using ExpressHybTM buffer (CLONTECH), was
carried out according to the manufacturer instructions. The bands were
visualized by autoradiography and quantified by densitometer.
The tissue distribution of hSKI mRNA expression in human tissues was
analyzed by Northern blotting (Figure 8).
Figure 8 shows the tissue distribution of hSKI messenger RNA. Premade
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). 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. In addition, data base searches, with the stSG2854 marker
suggest expression in endothelial cells, retinal pigment epithelium, and
senescent
fibroblasts.
Examt~le 7: Genomic localisation of sphin~osine kinase, related diseases:
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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
UniGene cluster.
The cloning of the hSKl is an important step towards the elucidation of the
role
this enzyme plays in signal transduction pathways mediated by a wide range of
receptor coupled mechanisms. Moreover, several members of the Unigene
cluster Hs68061 have been mapped. An approximately 50 cM region, on 17q25,
which harbors the stSG2854 has been implicated in several autoimmune and
inflammatory diseases, such as multiple sclerosis (Kuokkanen et al., 1997),
psoriasis and epidermodysplasia verruciformis (Hair et al., 1977; Tomfohrde et
al., 1994; Enlund et al., 1999; Ramoz et al., 1999), and by synteny homology,
in
a rat model of inflammatory arthritis (Remmers et al., 1996). Linkage in
psoriasis has been reported by multiple independent groups. Together, these
data
identify a shared autoimmune / inflammatory region described recently by
Becker et. al. (Becker et al., 1998). Because of its expression pattern and
biology, SK is a possible disease susceptibility gene candidate in this
region. As
a result, 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 SK1 to the DNA
sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and determining the presence of
single nucleotide polymorphism or polymorphic region in said mammal's coding
sequence encoding SKl.
Example 8: Sphin~osine kinase expression in insect cells
~ Isolation of recombinant Bacmid DNA preparation
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Sphingosine kinase cDNA was cloned into a pFasbac HT donor plasmid
according the manufacturers instructions (Gibco BRL,Gaithersburg,MD).
FastBac plasmid DNA and sphingosine kinase cDNA was prepared by digesting
1 pg DNA with the selected resfiriction endonucleases under appropriate
conditions. The insert fragment was ligated into the prepared pFasbac HT
vector
downstream from histidine tag under appropriate conditions. 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).
~ Recombinant protein expression in Sf21 cells-
Sf21 cells were transfected with the recombinant bacmid DNA in presence of
Cellfectin reagent. Cell culture, recombinant virus purification and titration
of
the viruses were performed according the manufacturers instructions (Gibco
BRL,Gaithersburg,MD). For protein expression, cells at a density of 2x106/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 IOmM, 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.
~ Partial purification
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HSK1 was subcloned into the Baculovirus shuttle vector pFastBacHTa which
incorporates the sequence for a 6x histidine affinity tag onto the N-terminus.
This Baculovirus construct was mixed with viral DNA and introduced into Sf21
insect cells. New recombinant Baculovirus was isolated by plaque purification
5 and five isolates examined for protein producation. The best isolate was
chosen
and high titre viral stock generated. 5f21 insect cells were infected and the
cells
harvested 60 hours post infection. Expression of hSKI was confirmed by
Westernblot. Infected Sf21 insect cells from a culture were used for partial
purification of hSKl using Nickel beads.
10 hSKI was sub-cloned into pFastBacHTa (Life technologies Cat No 50322, lot
No KDW704) using restriction site BamHI and PstI. This construct was
confirmed by sequencing.
The recombinant hSKl in pFastBacHTa was used to transform DHlOBac E. Coli
15 (which contains the bacmid shuttle vector bmon14272). Transposition from
donor plasmid to acceptor shuttle plasmid was detected by blue/white colony
selection on X-gal/IPTG 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 (NERC,Oxford) were grown in 47.5% TC100 (fromw -
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
(lot No 9N3936) were transfected with purified recombinant bacmid DNA in the
presence of Lipofectin (Life Technologies) using standard methods (King et al.
,1992). The culture medium was collected 7 days post-transfection.
A monolayer of Sf21 cells (3.5x106 cells/60mm dich) was infected with serial
dilutions of the viral stocks from the transfection mix described above,
overlaid
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with a mixture of Graces Insect Medium (2x; Life T'chnologies), 10% FBS (Life
Technologies; heat incativated North american; cat No 10085-140; lot
No06Q6073A); 1.5%SeaPlaque Agarose (lowgen) solution and stained with
neutral red (6% in PBS; Sigma) 4 days post-infection. (King et al, 1992)
Figure 17 shows an electrophoresis gel of the partial purification of hSKl
from
Sfzl insect cells.
150m1 of Sf21 insect cells were infected at an MOI 10 and cells collected 60hr
post infection. Samples were prepared and binding to alVi column carned out.
lOpl from column sample eluates were mixed with SX reduscing SDS/PAGE
buffer and 15p,1 loaded per well on 4-12% NuPAGE Bis-Tris Gel. The
electrophoresis gel was stained with Coomassie Blue.
1=Total cell lysate
2=Lysate following low speed spin
3=Flowthrough column
4-6=Column wash fractions 1-3
7-11=Column elution fractions 1-5
The single band of line 8 corresponds to the predicted molecular weight of
hSKI
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
~ Construction of GST-hSKI. ~N-terminal fusion~bacterial expression.
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 - Xho1), 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 hSKI.
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Primers E and F:
E= 5'end TTA GAA TTC CAC CAT GGA TCC AGC GGG CGG C (SEQ
m NO: 10)
F= 3'end AGT CGA GGC TGA TCA GCG AG (SEQ m NO: 11)
Figure 16 illustrates the vector for the construction of hSKI 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 cells
E. Coli competent cells (strain BL21 ) were vpurchased from Promega
Bacterial transformation was carried out as per supplyer Standard
Transformation Protocol.
Frozen competent cells were thawed on ice for 5 minutes, 100,1 was transfered
to a child culture tube. SOng of hSKI-cI7NA 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. 9001 of cold SOC
medium was added to the transformation reaction and incubated for 60 minutes
~a
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: Sphin~osine kinase optimized source for the screening assays
~ Sphingosine kinase source choice
Several sources of hSK have been tested. Figure 9 illustrates the comparison
of
hSK activity. from different sources: CHO cells, Bacteria, partially purified
hSKI
from insect cells. Similar levels are observed in mammal (Cho) and bacterial
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(BL21) cell extracts. There is a dose response effect of increasing levels of
partial purified hSK from insect cell transfections.
As in figure 9 (Y axis is the enzyme activity in SPA cpm), Z~g total protein
from
bacteria (B1 21 ) and 0.1 pg 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.
Experiments have been carned out in an effort to identify the best possible
solution for the generation of enough recombinant hSK to run a High
Throughput Screening (HTS). Since the mammalian transient expression
presents many difficulties to generate enough enzyme for the entire HTS,
different bacterial expression systems have.-been tried, as well as, the
baculovirus
expression system in insect cells. The results are expressed in Figure 10.
Figure 10 illustrates the comparison of hSKI activity from different sources:
Cos7, bacteria, insect cells. BL21 Transf. Basal means BL21 transfected
without
IPTG induction. BL21 Trnas. Induced means BL21 transfected with IPTG
induction. P.Pur.rSPHK means partial purified recombinant hSKI.
40p,g of the total cells extract from transfected cos7 cells shows 50% more
activity than 40p,g of total transfected bacterial cell extract. 40p,g of
insect cell
extract shows minimal hSKl activity over basal levels (Cos7 basal). However 6
~g of partially purified hSKI from insect cell shows a 3 fold increase over
the
transfected 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. However, with similar amounts of total cell extracts, the
bacterial extracts that overexpress the hSK gives between 40% to 50% of the
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total activity observed with the mammalian system, which renders this system
as
the most attractive solution.
~ ~hin~osine kinase bacterial source optimization
Experiments have been carried out in order to generate enough recombinant
human sphingosine kinase for the HTS. Transfected BL21 bacteria, grown at
25°C overnight, yielded active recombinant hSKl, as showw by enzymatic
activity of total protein extract. The production has been scaled up to
generate
over 400mg of total bacterial protein that shows very good levels of SK
activity.
In order to solve the solubility/expression problem observed for the
recombinant
GST-tagged hSKl, we set up a wide range of conditions for bacterial growth and
for the induction of protein expression. Thus, growing the bacteria at
RT° for 20
hours, and inducing protein expressiowvith 50 p,M IPTG, appear to be the
optimal condition for the expression of significant amounts of active/soluble
recombinant GST-tagged- hSK. (Figure 11)
Figure 11 describes the bacterial growth conditions for optimization of
actively
expressed hSKl. Different concentration of IPTG for induction, different
temperatures of growth (R°T means room temperature),different
incubation
times are tested.
Furthermore, the bacterial cell extract under optimal bacterial growth and
induction conditions (SOE.~M IPTG for 20hr) has 40% activity of the maximalr
activity observed for transfected mammalian cells (Cos cells) (Figure 12)
Figure 12 shows the comparison of hSKI activity expressed under different
bacterial growth conditions and expressed in Cos cells.
Example 11: Sphingosine kinase antisense oli~onucleotides
In order to demonstrate the physiological role of sphingosine kinase 1 in
intracellular signalling pathways in immune cells, 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
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transfected 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. Here we show that, in the antisense treated cells the release
of
calcium from intracellular stores is impaired, demonstrating that sphingosine
5 kinase does indeed play a significant role in receptor-coupled triggered
physiological responses.
~ Construction of an antisense oligonucleotide against the hSKI
10 An antisense sequence to the first 24 nucleotides (coding for the first 8
amino
acids) of the hSKI-cDNA, (said antisense having the sequence: GGG GCC GCC
CGC CGC TGG ATC CAT, SEQ m NO: 12), was synthesised and protected at
both ends with Phosphorothioate linkages -for the first and last two
nucleotide
pairs.
15 A control "scrambled oligo" (CTGGTGGAAGAAGAGGACGTCCAT, SEQ m
N0:13) was synthesised and protected at both ends with Phosphorothioate
linkages for the first and last two nucleotide pairs.
Transfection of antisense oli~onucleotide to hSKl.
U937 cells were transiently transfected 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 106
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 p,1 SuperFect.
~ Protein analysis of hSKl in U937 cells and the effect of the antisense.
Figure 13 illustrates the physiological relevant role of hSKl proven by the
use of
an antisense oligonucleotide.
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FCyRI triggered calcium signal in U937 cells (control) was inhibited in cells
treated for 48 hrs with an antisense against hSKl.
Figure 18 illustrates the antisense downregulation of hSKl protein levels.
The blot has been probed with a polyclonal antibody against hSKI (Ab 0144).
1= Background
2= U937 untreated cell extract 100mg (taken as 100% of expression)
3= U937 antisense treated cell extract 100 mg (12% compared to untreated
cells)
This figure shows that the antisense reduces SK1 protein expression level by
88%.
Example 12 : Sphingosine kinase knock out Mouse
A high density filter set of BAC colonies from-the 129 mouse strain BAC
library
RPC22 (Research Genetics) has been screened with the following radiolabelled
oligonucleotide probes.
SKS'end49 (gene proximal) CTGGGTCTTGTAGAAGAGCAGCAAGTGCT
(SEQ ID NO: 14)
SKS'end48 (gene proximal)
AGTTCACTGCAATCCTTTCTTATCTGGGTTCG (SEQ ID NO: 15)
SK3'end (gene distal) TTCTGTGGATGGAGAGCTGATGGTATGG (SEQ
>D NO: 16)
SK BOX (conserved region) ATGAAGTGGTGAATGGGCTAATGGAACG
(SEQ >D NO: 17)
The oligonucleotide probes were derived from the mouse SKl cDNA sequence
(Kohama et al., 1998). Based on multiple alignments of SK1 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 .
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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.
Positive clones (with conserved region probes)
83B4,442C20,424E5,46M1,270B3,225D6.
3'end (gene distal probes) positive clones: 61 K 3 , 166 L 16 , 126 H 16 , 69
D 8 ,
2406,203P6,224A21,84A12,387G19,545H5,431019,224A23
The catalytic domain of the enzyme presumably lies in the highly conserved
region (see SED m N°8), which is between aal6 and aa95 in the peptide
sequence (SEQ ~ 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). In alternative experiments (B) 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' homology arms for targeting. Appropriate
restriction sites are inserted in order to create an optimal situation for the
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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 electroporation or other
methods. Neomycin resistant colonies are screened for the identification of
specific targeting events. In a possible variant of the experiment 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, gennline transmitting mice will be
selected
by breeding and tail DNA testing. 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 IRL press/Oxford, 1993). In 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
Example 13 : hSK polyclonal antibodies
Four peptide sequences were selected for their apparent hydrophobisity
properties, and
synthesised.
Peptide 1: FTLMLTER;I~7VHARELVRSEE (SEQ ID NO: 18)
Peptide 2: VNGLMERPDWETAIQKPLCS (SEQ ID N0:19)
Peptide 3: ADVDLESEKYRRLGEMRFTL (SEQ 117 N0:20)
Peptide 4: SGCVEPPPSWKPPQQMPPPEE (SEQ ID N0:21)
Two rabbits were immunised for each peptide giving rise to eight peptide
derived polyclonal antibodies (two for each peptide).
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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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~ Tomfohrde et al (1994) Science 264, 1141-S.
~ Nair et al (1977} Hum. Mol. Genet. 8, 1349-56.
~ Remmers et al (1996). Nat. Genet. 14, 82-5.
~ Ramoz et al. (1999) J. Invest. Dermatol. 112, 259-63
~ Becker et al. (1998) Proc. Natl. Acad. Sci. USA. 95, 9979-84.
~ King et al. (1992) The Baculovirus expression system Chapman and Hall.
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
1
SEQUENCE LISTING
<110> Warner-Lambert
$ <120> Human sphingosine kinase gene
<130> A000198PCT
<140>
<141>
<160> 24 -
<170> PatentIn Ver. 2.1
1$
<210> 1
<211> 1719
<212> DNA
<213> Homo Sapiens
<400> 1
gtcccgggat ttagtcgggcgctcccacctctggcagctgcggccccggactccgccagc
60
gctgtcttct ctccctcaggtccagccgccgcagggaatgacaccggtgctcctacagcc
2$ lzo
acggctccgg gcgggaagggagccccacagcggccctgcgacgcccgcctgggcaggacc
180
gataaggaac tgaaggcaggagccgccgccacggcagcgcccccacagcgccagggaccc
240
cctggcagcg ggagccgcgggtcgaggttatggatccagcgggcggcccccggggcgtgc
300
tcccgcggcc ctgccgcgtgctggtgctgctgaacccgcgcggcggcaagggcaaggcct
360
tgcagctctt ccggagtcacgtgcagccccttttggctgaggctgaaatctccttcacgc
3$ 4zo
tgatgctcac tgagcggcggaaccacgcgcgggagctggtgcggtcggaggagctgggcc
480
gctgggacgc tctggtggtcatgtctggagacgggctgatgcacgaggtggtgaacgggc"
540
tcatggagcg gcctgactgggagaccgccatccagaagcccctgtgtagcctcccagcag
600
gctctggcaa cgcgctggcagcttccttgaaccattatgctggctatgagcaggtcacca
660
atgaagacct cctgaccaactgcacgctattgctgtgccgccggctgctgtcacccatga
4$ 720
acctgctgtc tctgcacacggcttcggggctgcgcctcttctctgtgctcagcctggcct
780
ggggcttcat tgctgatgtggacctagagagtgagaagtatcggcgtctgggggagatgc
840
gcttcactct gggcaccttcctgcgtctggcagccctgcgcacctaccgcggccgactgg
900
cctacctccc tgtaggaagagtgggttccaagacacctgcctcccccgttgtggtccagc
.960
agggcccggt agatgcacaccttgtgccactggaggagccagtgccctctcactggacag
$$ 1020
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
2
tggtgcccga cgaggacttt gtgctagtcc tggcactgct gcactcgcac ctgggcagtg
1080
agatgtttgc tgcacccatgggccgctgtgcagctggcgtcatgcatctgttctacgtgc
1140
gggcgggagt gtctcgtgccatgctgctgcgcctcttcctggccatggagaagggcaggc
1200
atatggagta tgaatgcccctacttggtatatgtgcccgtggtcgccttccgcttggagc
1260
ccaaggatgg gaaaggtgtgtttgcagtggatggggaattgatggttagcgaggccgtgc
1~ 1320
agggccaggt gcacccaaactacttctggatggtcagcggttgcgtggagcccccgccca
1380
gctggaagcc ccagcagatgccaccgccagaagagccctta.tgacccctgggccgcgttg
1440
15 tgccttagtg tctacttgcaggacccttcctccttccctagggctgcagggcctgtccac
1500
agctcctgtg ggggtggaggagactcctctggagaagggtgagaaggtggaggctatgct
1560
ttggggggac aggccagaatgaagtcctgggtcaggagcccagctggctgggcccagctg
2~ 1620
cctatgtaag gccttctagtttgttttgagacccccaccccacgaaccaaatccaaataa
1680
agtgacattc ccaaaaaaaaaaaaaaaaaaaa~aaaaaa
1719
<210> 2
<211> 1155
<212> DNA
3~ <213> Homo sapiens
<400> 2
atggatccag cgggcggcccccggggcgtgctcccgcggccctgccgcgtgctggtgctg
60
ctgaacccgc gcggcggcaagggcaaggccttgcagctcttccggagtcacgtgcagccc
120
cttttggctg aggctgaaatctccttcacgctgatgctcactgagcggcggaaccacgcg
180
cgggagctgg tgcggtcggaggagctgggccgctgggacgctctggtggtcatgtctgga
z4o
gacgggctga tgcacgaggtggtgaacgggctcatggagcggcctgactgggagaccgcc
300
atccagaagc ccctgtgtagcctcccagcaggctctggcaacgcgctggcagcttccttg
360
aaccattatg ctggctatgagcaggtcaccaatgaagacctcctgaccaactgcacgcta
420
ttgctgtgcc gccggctgctgtcacccatgaacctgctgtctctgcacacggcttcgggg
480
ctgcgcctct tctctgtgctcagcctggcctggggcttcattgctgatgtggacctagag
540
agtgagaagt atcggcgtctgggggagatgcgcttcactctgggcaccttcctgcgtctg
600
gcagccctgc gcacctaccgcggccgactggcctacctccctgtaggaagagtgggttcc
660
aagacacctg cctcccccgttgtggtccagcagggcccggtagatgcacaccttgtgcca
720
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
3
ctggaggagc cagtgccctc tcactggaca gtggtgcccg acgaggactt tgtgctagtc
780
ctggcactgc tgcactcgca cctgggcagt gagatgtttg ctgcacccat gggccgctgt
840
gcagctggcg tcatgcatct gttctacgtg cgggcgggag tgtctcgtgc catgctgctg
900
cgcctcttcc tggccatgga gaagggcagg catatggagt atgaatgccc ctacttggta
960
tatgtgcccg tggtcgcctt ccgcttggag cccaaggatg ggaaaggtgt gtttgcagtg
1020
gatggggaat tgatggttag cgaggccgtg cagggccagg tgcacccaaa ctacttctgg
1080
atggtcagcg gttgcgtgga gcccccgccc agctggaagc cccagcagat gccaccgcca
1140
gaagagccct tatga
1155
<210> 3
<211> 384
<212> PRT
<213> Homo Sapiens
<400> 3
Met Asp Pro Ala Gly Gly Pro Arg Gly Val Leu Pro Arg Pro Cys Arg
1 5 10 15
Val Leu Va1 Leu Leu Asn Pro Arg Gly Gly Lys Gly Lys Ala Leu Gln
20 25 30
Leu Phe Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala Glu Ile Ser
40 45
Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg Glu Leu Val
35 50 55 60
Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met Ser Gly
65 70 75 80
Asp Gly Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro Asp
85 90 95
Trp Glu Thr Ala Ile Gln Lys Pro Leu Cys Ser Leu Pro Ala Gly Ser
100 105 110
Gly Asn Ala Leu Ala Ala Ser Leu Asn His Tyr Ala Gly Tyr Glu Gln
115 120 125
Val Thr Asn Glu Asp Leu Leu Thr Asn Cys Thr Leu Leu Leu Cys Arg
130 135 140
Arg Leu Leu Ser Pro Met Asn Leu Leu Ser Leu His Thr Ala Ser Gly
145 150 155 160
$5 Leu Arg Leu Phe Ser Val Leu Ser Leu Ala Trp Gly Phe Ile Ala Asp
165 170 175
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
4
Val Asp Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met Arg Phe
180 185 190
$ Thr Leu GlyThrPhe LeuArgLeu Ala LeuArgThrTyr ArgGly
Ala
195 200 205
Arg Leu AlaTyrLeu ProValGly Val GlySerLysThr ProAla
Arg
210 215 220
Ser Pro ValValVal GlnGlnGly Val AspAlaHisLeu ValPro
Pro
225 230 235 240
Leu Glu GluProVal ProSerHis Thr ValValProAsp GluAsp
Trp
245 250 _ 255
Phe Val Leu Val Leu Ala Leu Leu His Ser His Leu Gly Ser Glu Met
260 265 270
2~ Phe Ala AlaProMet GlyArgCys AlaAla GlyValMet HisLeuPhe
275 280 285
Tyr Val ArgAlaGly ValSerArg Ala-Met LeuLeuArg LeuPheLeu
290 295 300
2$
Ala Met GluLysGly ArgHisMet GluTyr GluCysPro TyrLeuVal
305 310 315 320
Tyr Val ProValVal AlaPheArg LeuGlu ProLysAsp GlyLysGly
325 330 335
Val Phe AlaValAsp GlyGluLeu MetVal SerGluAla ValGlnGly
340 345 350
3$ Gln Val HisProAsn TyrPheTrp MetVal SerGlyCys ValGluPro
355 360 365
Pro Pro SerTrpLys ProGlnGln MetPro ProProGlu GluProLeu
370 375 380
4$ <210> 4
<211> 27
<212> DNA
<213> Artificial Sequence
$Q <220>
<223> Description of Artificial Sequence:primer
<400> 4
tatgctagca tggatccagc gggcggc
$$ 27
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
<210> 5
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 5
aatgaattct cataagggct cttctgg
27
IS <210> 6
<211> 31
<212> DNA
<213> Artificial Sequence
2~ <220>
<223> Description of Artificial Sequence: primer
<400> 6
ttagaattcc accatggatc cagcgggcgg c
25 31
<zlo> 7
<211> 30
3~ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 7
attatcgtcg actaagggct cttctggcgg
40
<210> a
<211> so
<212> PRT
<213> Homo sapiens
<400> s
Arg Val Leu Val Leu Leu Asn Pro Arg Gly Gly Lys Gly Lys Ala Leu
1 5 10 15
Gln Leu Phe Arg Ser His Val Gln Pro Leu Leu Ala Glu Ala Glu Ile
20 25 30
Ser Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg Glu Leu
35 40 45
Val Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met Ser
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
6
50 55 60
Gly Asp Gly Leu Met His Glu Val Val Asn Gly Leu Met Glu Arg Pro
65 70 75 80
<210> 9
<211> 16
<212> PRT
<213> Artificial Sequence
1$ <220>
<223> Description of Artificial Sequence: peptide
<400> 9
Leu Val Arg Ser Glu Glu Leu Gly Arg Trp Asp Ala Leu Val Val Met
1 5 10 15
<210> 10
<211> 31
2$ <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> to
ttagaattcc accatggatc cagcgggcgg c
31
3$
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 11
4$ agtcgaggct gatcagcgag
<210> 12
$0 <211> 21
<212> DNA
<213> Artificial Sequence
<220>
$5 <223> Description of Artificial Sequence:oligonucleotide
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
7
<400> 12
ggggccgccc gccgctggat c
21
<zlo> 13
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:oligonucleotide
<400> 13
ctggtggaag aagaggacgt ccat
24
<210> 14
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: probes
<400> 14
ctgggtcttg tagaagagca gcaagtgct
29
<210> 15
<211> 32
<212> DNA
3S <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: probes
<400> 15
agttcactgc aatcctttct tatctgggtt cg
32
45 <210> 16
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
probes
<400> 16.
ttctgtggat
ggagagctga
tggtatgg
55 28
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
8
<210> 17
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: probes
<400> 17
atgaagtggt gaatgggcta atggaacg
28
<210> is
<211> zo
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: peptide
<400> 18
Phe Thr Leu Met Leu Thr Glu Arg Arg Asn His Ala Arg Glu Leu Val
1 5 10 15
Arg Ser Glu Glu
30
<210> 19
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: peptide
<400> 19
4~ Val Asn Gly Leu Met Glu Arg Pro Asp Trp Glu Thr Ala Ile Gln Lys
1 5 10 15
Pro Leu Cys Ser
45
<210> 20
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: peptide
5s <400> 20
Ala Asp Val Asp Leu Glu Ser Glu Lys Tyr Arg Arg Leu Gly Glu Met
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
9 -
1 5 10 15
Arg Phe Thr Leu
5
<210> 21
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: peptide
15 <400> 21
Ser Gly Cys Val Glu Pro Pro Pro Ser Trp Lys Pro Pro Gln Gln Met
1 5 10 15
Pro Pro Pro Glu Glu
20 20
<210> zz
<211> 240
<212> DNA
<213> Homo sapiens
<400> 22
cgcgtgctgg tgctgctgaa cccgcgcggc ggcaagggca aggccttgca gctcttccgg
so
agtcacgtgc agcccctttt ggctgaggct gaaatctcct tcacgctgat gctcactgag
120
cggcggaacc acgcgcggga gctggtgcgg tcggaggagc tgggccgctg ggacgctctg
180
gtggtcatgt ctggagacgg gctgatgcac gaggtggtga acgggctcat ggagcggcct
240
<210> 23
4~ <211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: peptide
<400> 23
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 24
<211> 5
<212> PRT
SS <213> Artificial Sequence
CA 02389127 2002-04-26
WO 01/31029 PCT/EP00/09498
<220>
<223> Description of Artificial Sequence: peptide
<400> 24
Gly Gly Lys Gly Lys
1 5
