Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ISOLATED POLYSIALYL TRANSF~R~C~, NUCLEIC
ACID MOLECULES CODING T~RFFOR, I-L-1~OVS
OF PRO~u~llON AND USE
Funds from NIH Grant CA 33895 were used in the
development o~ portions of the invention described herein.
Thus the U.S. government may have rights to portions of this
inventlon .
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent
application Serial No. 08/503,133 filed on July 17, 1995,
which is a continuation-in-part o~ PCT Application
PCT/EP94/04289 filed on December 22, 1994 designating the
United States. Thus, priority is claimed pursuant to 35
U.S.C. 365(a), (b) and (c).
FIELD OF THE lNV~NllON
This invention relates to the isolation and cloning o~
nucleic acid molecules which encode polysialyl transferases,
cell lines and vectors containing such molecules, and the uses
of these. Further the invention relates to the isolated
enzyme and its uses. Various diagnostic and therapeutic
applications are included as part of the field of the
invention, including stimulation of neurite outgrowth by
appropriate cells.
R~RouND AND PRIOR ART
Polysialic acid (PSA) is added to the neural cell
adhesion molecule, NCAM in a dynamically regulated,
postranslational process (S. Hoffman and G.M. Edelman, Proc.
Natl. Acad. USA 80: 5762-5766 (1983), U. Rutishauser et al.,
Science 240: 53-57 (1988)). The glycan structure is unusual
in vertebrates and has been shown to be attached to the fifth
immunoglobulin domain (K.L. Crossin et al., J. Cell Biol. 99:
1848-1855 (1984)). The presence of the large anionic
carbohydrate structure that is PSA modulates NCAM binding
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properties and, by increasing the intercellular space, also
influences interactions between other cell surface molecules
(S. Hoffman and G.M. Edelman, supra, U. Rutishauser et al.,
supra, A. Acheson et al., J. Cell Biol. 114: 143-153 (1991),
P. Yang et al., J. Cell. Biol. 116: 1487-1496 (1992), P.
Doherty et al , Neuron 5: 209-219 (1990)). In the course of
embryogenesis the expression o~ PSA underlies cell type and
developmental-specific
alterations (&.M. Edelman, Ann. Rev. Cell Biol. 2: 81-116
(1986)) and correlates with stages of cellular motility (G.M.
Edelman, supra (1986)), J. Tang et al., Neuron 8: 1031-1044
(1992), L. Landmesser, J. Neurobiol. 23: 1131-1139 (1992)).
In the adult, PSA is restricted to regions of permanent neural
plasticity and regenerated neural and muscle tissues (G.M.
Edelman. supra), R. Martini and M. Schachner, J. Cell Biol.
106: 1735-1746 (1988), J.K. Daniloff et al. J. Cell Biol. 103:
929-945 (1986)). Recent data implicate PSA in spatial learning
and memory (H. Cremer et al., Nature 367: 455-459 (1994), H.
Tomasiewicz et al., Neuron 11: 1163-1174 (1993)). Of utmost
clinical relevance are the observations that polysialylated
NCAM forms represent oncodevelopmental antigens in
neuroendocrine and hematolymphoid tumors (C.E.C.K. Moolenaar
et al., Canc. Res. 50: 1102-1106 (1990), K. Takamatzu et al.,
Canc. Res. 54: 2598-2603 (1994), P. Komminoth et al., Ann. J.
Patho. 139: 297-304 (1991), W.F. Kern et al., Leukemia &
Lymphoma 12: 1-10 (1993)). PSA expression enhances the
metastatic potential of these tumors and promotes an abnormal
localization of metastases (W.F. Kern et al., supra, E.P.
Scheidegger et al., J. Lab. Invest. 70: 45-106 (1994)).
Studies aimed at enlightening the biosynthetic
pathway and the regulation of PSA synthesis suggest the
concerted activity of several specific enzymes located within
the Golgi apparatus (R.D. McCoy et al., J. Biol. Chem. 260:
12659-12699 (1984), S. Kitazume et al., J. Biol. Chem. 269:
10330-10340 (1994)).
In recent years several m~mm~l ian sialyltransferases have
been cloned (K. Nara et al., Proc. Natl. Acad. Sci. USA 91:
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7952-7956 (1994), H. Kitagawa and J.C. Paulson, J. Biol. Chem.
269: 17875-17878 (1994), K. Sasaki et al., J. Biol. Chem. 269:
15950-15956 (1994) and literature cited therein). All enzymes
characterized to date are monosialyltransferases which are
specific for both the type of glycosidic linkage and the
acceptor structure to which the sialic acid is attached. In
fact, the synthesis of PSA in rainbow trout eggs has recently
been reported to involve the consecutive activity of several
specific enzymes (S. Kitazume, supra). Livingston and
Paulson, J. Biol. Chem. 268: 11504-11509 (1993) describe a rat
sialyl transferase "STX" which shows homology to the hamster
polysialyl transferase (PST) described herein, o~ about 59%.
The human STX sequence is shown in the GenBank~ EM3L Data Bank
under Accession Number L13445. However, STX does not catalyze
any polysialyl reaction.
Three bacterial polysialyl transferases are known (M.
Frosch et al., Mol. Microbiol. 5: 1251 (1991); C Weisgerber
et al., Glycobiol. 1: 357 (1991), and S.M. Steenbergen et al.,
J. Bacteriol. 174: 1099 (1992)). These polysialyl
transferases have a substrate and acceptor specificity which
is different from the specificity of the enzymes of the
invention and do not exhibit any sequence homology with the
polysialyl transferases, set forth in the disclosure which
follows.
In m~mm~ls, particularly humans, PSA is a critical
element in the modulation of NCAM binding activities. Thus,
there is a need to elucidate, to regulate, and to modulate PSA
synthesis, thereby modulating normal and pathological
processes which involve, inter alia, NCAM binding.
SUMMARY OF THE lNv~N-llON
The subject matter of the invention is the isolation and
molecular cloning of the key enzyme of eukaryotic PSA
synthesis, i.e., polysialyl transferase ("PST", hereafter),
and the isolation of nucleic acid molecules which encode this
enzyme. More specifically, the invention relates to those
nucleic acid molecules encoding mammalian PST enzymes,
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including hamster, human and other species. Especially
preferred are nucleic acid molecules which hybridize to SEQ ID
NOS: 1 and 7 and portions thereof, especially under stringent
conditions, as set forth below.
An additional aspect of the invention is a method of
detecting nucleic acid molecules which specifically code for
PST proteins. Preferably detected are cDNA and mRNA species.
Useful probes are nucleic acid molecules which bind
specifically under the conditions of the test method applied,
e.g. in situ hybridization, colony hybridization, Northern
hybridization, or related techniques.
A further aspect of the invention is an oligonucleotide
molecule which is complementary to a nucleic acid molecule
encoding mammalian PST. Such oligonucleotide molecules, which
are generally from 15 to 50 bases in length, are useful for
inhibiting expression of PST, preferably on the DNA or mRNA
level. Such oligonucleotide molecules are useful as antisense
agents in the therapy of pathological conditions involving a
tumor, especially in the treatment and prevention of
metastases.
Yet another aspect of the invention involves isolated PST
enzymes, such as m~mm~lian PST enzymes, which may be encoded
by the isolated nucleic acid molecules of the invention, the
production of said enzymes via recombinant methodologies, and
the use of the enzymes in promoting neurite growth in nerve
cells.
BRIEF DESCRIPTION OF THE FIGURES
Figure la FACS-analysis of CHO-wt and mutant clones.
Staining with the mouse-NCAM specific mAb H28
indicates that NCAM surface expression in the
mutant cell lines CHO-2A10 and CHO-lE3
(defective in sialic acid transport) is almost
identical to that observed in CHO-wt cells.
In contrast, the reactivity with the PSA
specific mAb 735 (M. Frosch et al., Proc.
Natl. Acad. Sci. USA 82: 1194-1998 (1985)) is
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restricted to the CHO-wt cells. In order to
obtain the hybrid cells CHO-2AlOxWT and
CHO-2AlOxCHO-lE3, a neomycin resistance gene
was introduced into clone CHO-2A10 and a
~ 5 hygromycin resistance gene was introduced into
CHO-wt and CHO-lE3 cells, respectively. Equal
amounts of the two relevant cell types were
plated into cell culture dishes and cell
fusion was induced by polyethylenglycol (50%
PEG 1500 in 50 mM Hepes buffer pH 7.4).
Double positive hybrids were selected with
G418 and hygromycin. While the NCAM signal in
hybrid cells was identical to that observed in
parental cells, both fusion products expressed
the 735 epitope.
Figure lb In order to determine whether
~-2,3-sialylation is a prerequisite in PSA
biosynthesis, CHO-wt cells and chemically
induced mutants were analyzed with Maackia
amurensis lectin ("MAA"), which speci~ically
recognizes sialic acid linkage to galactose
via an ~-2, 3 bond. Extracts are shown before
(-) and after (+) treatment with
exoneuraminidase. Due to polysialylation, both
MAA and the anti-NCAM mAb KDll recognized
microheterogenous bands in CHO-wt cells (lanes
1 and 2; wt-cells in two concentrations),
while discrete protein bands became visible in
PST-Cl mutants (lanes 4-6; 3 individual
mutants: 2A10, lH8, and 3A7). In contrast,
mutant CHO-lE3, which is defective in sialic
acid transport (lane 3) did not react with MAA
and, in accordance with the deficiency of
sialylated proteins, the polypeptide band
recognized by KDll in this mutant, showed a
significantly reduced Mr. Treatment with
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exoneuraminidase abolished MAA reactivity
completely and converted the protein bands
recognized by mab KDll into forms with
identical molecular weights.
Method: lx107 cells were harvested, membrane
proteins extracted with lysis buffer (20 mM
Tris/HCl, pH 8; 1% NP40) and NCAM was
immunopurified on a protein A bound anti-NCAM
antibody (mab KDll). Samples were separated
by 7.5% SDS-PAGE, blotted onto nitrocellulose
membranes, and in developed parallel with
either biotinylated MAA or anti-NCAM mab KDll.
Protein bands were visualized using alkaline
phosphatase conjugated streptavidin.
Figure 2 The PSA found on the surface of pEPST-ME7
transfectants is bound to NCAM. Cells with
the phenotype NCAM+/PSA- were transfected with
pEPST-ME7 or pCDM8 alone, as described infra,
harvested, and solubilized in lysis buffer,
NCAM was immunoprecipitated from the lysates
using an anti-NCAM serum, and the samples were
analyzed by Western blot with mAb 735.
Microheterogenous bands became visible in PST
transfectants (lane 1: CHO-2A10; lane 3:
NIH-3T3; lane 5 COS-hN-6) indicating that NCAM
in these cells is polysialylated. In
contrast, control transfection with pCDM8 did
not result in appearance of mab 735 epitopes
(lane 2: CHO-2A10; lane 4: NIH-3T3; lane 6:
COS-hN-6).
Figure 3a The Northern blot analysis of pEPST-ME7
transcripts revealed two bands of 5.1 kb and
2.1 kb in the PSA positive CHO-wt cells (lane
2), and in adult (lane 3) and postnatal day 1
mouse brain (lane 4). In accordance with the
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reduced PSA expression in adult brain, a very
faint band was obtained (lane 3). No signal
~ was found with PSA negative NIH-3T3 cells
(lane 1). In clonal sublines o~ the human
small cell lung cancer cell line HTB119 a
complex pattern with 6 hybridization signals
o~ about 6.1 kb; 3.8 kb; 3.3 kb; 3 kb; 1.7 kb;
and 1.3 kb appeared in the highly
polysialylated clone HTB119-54.2 (lane 5) and
in clone HTB119-38 (lane 6) which expresses
low levels of PSA, but was not visible in the
PSA-negative subline HTB119-45 (lane 7). The
probe used contains the entire coding region
of PST.
Figure 3b FACS analysis of sublines isolated by limiting
dilution from the small cell lung cancer cell
line HTB119. While staining with mab MB2
(specifically directed against human NCAM),
indicates that NCA~I expression on the 3
sublines is almost identical, the staining
with mAb 735 reveals large differences in the
amount o~ detectable PSA. HTB119-54.2 are
about 100% PSA positive, in the subline
HTB119-38 only 30% of the cells express PSA,
and in HTB119-45 the number of PSA expressing
cells is reduced to 2%. Immunostaining was
performed as described in Fig. la.
Method: Poly A+ RNA samples (3 ug each lane)
were blotted onto nylon membranes and analyzed
with a digoxigenin (Boehringer Mannheim)
labeled RNA-probe that contained the entire
coding region of pEPST-ME7. Final washing
conditions were 0.1 x SSC, 0.1% SDS, and 65~C.
Figures 4A-4G depict results obtained in various experiments
wherein eukaryotic cells were cotransfected
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with pH~A-NCAM and one of pcDNAI-PST or
pcDNAI. (These, and all other acronyms, are
elaborated upon in the examples). In figures
4A-F, COS-1 cells were co-transfected with
pcDNAI-PST (panels C-F) or pcDNAI (panels A
and B) and pH~A-NCAM. Sixty-four hours after
transfection, the cells were fixed and
examined by incubation with anti-PSA antibody
735 (panels B, D and F) or anti-NCAM antibody
(Dako Co. Carpenteria, CA)(panels A, C, and E)
followed by FITC-conjugated anti-mouse IgG.
Samples of COS-1 cells co-transfected with
pcDNAI-PST and pHgA-NCAM were also treated
with endo-N in the presence of protease
inhibitors before applying antibody 735
(panels E and F). Bar=20 ,um. In figure 4G,
cell lysates from 2.0x106 untransfected HeLa
cells (lane 1) or HeLa cells stably co-
transfected with pcDNAI-PST and pH~A-NCAM
(lanes 2 and 3) were subjected to sodium
dodecyl sulfate polyacrylamide (6.5%) gel
electrophoresis under reducing conditions,
transferred to nitrocellulose and incubated
with anti-NCAM antibody (Becton-Dickinson) as
described by Fredette et al [ (1993) . J. Cell
Biol. 123: 1867-1888]. The immuno reaction
was visualized by ECL Western Blotting
analysis system (Amersham, Arlington Heights,
IL). Lysates of transfected cells were either
treated with endo-N prior to electrophoresis
(lane 3) or untreated (lane 2).
Figure 5 sets forth results obtained following Northern
Blot analysis of various human tissues, to
determine expression of PST. Each lane
contained 2 ug of poly(A) RNA. The blots ~or
the first two lanes at the far left were
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prepared separately and contained less RNA
than the blots of the remaining lanes. The
~ blots were hybridized with [32P]-labeled human
PST cDNA, as well as $-actin cDNA as a
control.
Figures 6A-6D show results obtained ~rom immunohistochemical
analysis of various human fetal, newborn, and
adult tissues to determine if PSA was
expressed therein. Para~in-embedded sections
of tissue were stained with antibody 735 by an
avidin-biotin-peroxidase complex method: (A)
~etal cerebral cortex, ( B) ~etal lung, (C)
newborn thymus and (D) adult liver. B=100 um.
Figures 7A-7F set ~orth the result of experiments designed
to determine neurite outgrowth in confluent
HeLa cell substrata. Neurons from embryonic
day 10 chick dorsal root ganglia (panels A, B
C) or embryonic day 6 ventral spinal cord
(panes D, E and F) were seeded on HeLa cell
substrata and cultured for 15 hours. Neurons
shown in panels A and D were grown on
untrans~ected HeLa cells. Neurons shown in
panels B and E were grown on HeLa cells stably
transfected with N-CAM-encoding DNA only.
Neurons shown in panels C and F were grown on
HeLa cells cotransfected with DNAs encoding N-
CAM and human PST. Neurites were visualized
by immuno~luorescent neuro~ilament-staining.
Bar=100 ~um.
DE~TT~T~.n DESCRIPTION OF PR~UK~ E~rBODlL~ S
In reconstitution experiments it has been shown that PST
is able to induce PSA synthesis in all NCAM expressing cell
lines tested. Furthermore, the soluble, recombinant PST is
active in vitro. The data presented herein show that the
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polycondensation of ~-2,8-linked sialic acids in m~mm~ls is
the result of the activity of a single enzyme. Furthermore,
since Northern blot analysis confirmed the close correlation
between cell surface expression of PSA and the presence of PST
mRNA, this enzyme provides a useful target structure for
cancer therapy, as do the nucleic acid molecules, when
presented, e.g., as antisense oligonucleotides.
The nucleotide sequences according to the invention
encode a protein that functions in the final step of PSA
biosynthesis. The close correlation between cell surface
expression of PSA and the occurrence of the PST specific mRNA
implies that the regulation of PST occurs predominantly on the
mRNA level. PST seems to be the primary factor involved in
polycondensation of sialic acids during mammalian PSA
synthesis and is unique in PSA positive cells. Confirmation
for this assumption comes from the fact that expression in the
NCAM positive cell lines tested resulted in biosynthesis of
PSA. These data together with the observation that PSA
surface expression enhances the metastatic potential of
neuroendocrine tumors (W.F. Kern et al., supra, E.P.
Scheidegger et al., supra) show that PST may be used as a
target for tumor therapy and diagnosis. Additional data
presented herein suggest the use of PST to stimulate neurite
outgrowth on neural cells.
Example 1
This set of experiments determined how many enzymatic
activities are involved in transferring PSA to its acceptor
molecule NCAM. CHO cells, which are positive for NCAM and PSA
(Fig. la), were used to perform a complementation analysis.
After chemical treatment, PSA deficient CHO mutants were
negatively selected by panning (B. Seed and A. Aruffo, Proc.
Natl. Acad. Sci. USA 84: 3365-3369 (1987)) on the PSA specific
monoclonal antibody 735 (M. Frosch et al., supra). All
mutants positive for NCAM surface expression and
~-2,3-sialylation (Fig. lb) were subsequently used in fusion
experiments. Surprisingly, only one complementation class
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(PST-Cl) could be identified, with 40 individual clones
selected from 9 independent mutagenesis experiments. In order
~ to rule out the possibility that the mutation introduced in
these clones is of a d~m;n~nt phenotype, control fusions were
carried out between the PST-Cl clone CHO-2A10 and either
CHO-wild-type (CHO-wt) cells or the clone CHO-lE3 which is
de~ective in sialic acid transport and therefore does not
express sialylated proteins. In hybrid cells from both
fusions, PSA-surface expression was detectable (Fig. la).
These data strongly suggest that the polycondensation of
~-Z,8-linked sialic acids in m~mm~ls is mediated by a single
enzyme, which is mutated in clones of the complementation
class PST-Cl.
~.~le 2
In order to isolate the defective gene that is in PST-Cl
clones, the mutant clone CHO-2A10 was used for expression
cloning. Samples of CHO-2A10 cells were transfected, via
electroporation, with a complementary DNA (cDNA) library
prepared from RNA obtained from CHO-wt cells (in pCDM8) and
vector pPSVEl-PyE carrying the polyoma large T-antigen (M.F.A.
Bierhuizen et al., Genes Dev. 7: 468-478 (1993)). PSA-
positive transfectants were collected by panning (B. Seed and
A. Aruffo, supra) on mAb 735, and plasmid DNA was subsequently
extracted and amplified in E. coli. Mclo6l/p3 following Seed
et al, supra. After 3 cycles of transfection and panning, the
cDNA encoding PST was enriched to about 1 : 103 and could be
finally isolated by sibling selection. The cDNA clone
pEPST-ME7 (for eukaryotic polysialyl transferase clone ME7)
was isolated.
pEPST-ME7 contained an insert of 2026 base pairs (bp) and
an open reading frame of 1080 bp (SEQ ID NO: 1), potentially
encoding a protein of 359 amino acids with a predicted Mr of
41.2 kDa. The amino acid sequence (SEQ ID NO: 2) showed the
characteristic features of the sialyltransferase family,
including the two sialylmotifs (K. Drickamer, Glycobiol. 3: 2-
3 (1993)), as found at amino acids 141 to 183 and 258 to 300.
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The requirements of a type II transmembrane protein (P. Klein
et al., Biochem. Biophys. Acta 815: 468-471 (1985)) are only
partially fulfilled, however. A stretch of 13 hydrophobic
amino acids (at least 16 are required) was found within the
N-terminal domain of the molecule. These are amino acids 8 to
20 of SEQ ID NO: 2. Nevertheless, it is likely that this
truncated hydrophobic motif represents a Golgi-retention-
signal, since the cationic borders characteristic of type II
transmembrane proteins were also found.
A vector which contains SEQ ID NO: 1 (pME7/PST-l) was
deposited under the Budapest Treaty on 15 December 1994 at
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
(DSM), Mascheroder Weg lb, D-38124 Braunschweig and was
accorded the
Accession Number DSM 9582. Comparison of the deduced PST
primary sequence with the primary sequences of known sialyl
transferases revealed that the similarity is predomin~ntly
concentrated to the sialylmotifs L and S (K. Drickamer,
Glycobiol. 3: 2-3 (1993)). Analysis according to Higgins and
Scharp, supra, yielded a dendrogram. The highest degree of
homology is found with STX (59%), a sialyl transferase (H.
Kitagawa et al, J. Biol. Chem. 269: 17872-17878 (1994)) and
with the recently cloned GD3 synthase (28%), the only
~-2,8-sialyltransferase identified so far (K. Nara et al.,
Proc. Natl. Acad. Sci. USA 91: 7952-7956 (1994), K. Sasaki et
al., J. Biol. Chem. 269: 15950-15956 (1994)).
Example 3
Transient expression of pEPST-ME7 cDNA in the mutant
clone CHO-2A10, in an NCAM-positive subline of NIH-3T3, and in
a COS-M6 clone stably transfected with human NCAM-140, i.e.,
DNA encoding human NCAM protein (COS-hN-6), respectively,
resulted in surface expression of PSA. As shown in Fig. 2, PSA
was attached to NCAM immunoprecipitated by anti-NCAM serum
after expression of pEPST-ME7 in cell lines with the phenotype
NCAM+/PSA-.
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In standard Northern blot analysis, a PST specific cDNA
probe recognized two bands of about 2.8 kb and about 5.1 kb in
~ CHO-wt cells, and in both embryonic, and adult mouse brain
(Fig. 3a; lanes 2-4). However, according to the restricted
expression of PSA, both signals are drastically reduced in
adult mouse brain. NIH-3T3 cells, which are negative for PSA,
gave no hybridization signal (lane 1). These results imply
that PST activity is regulated at the transcriptional level.
Recently, it has been shown that differential expression
of PSA in sublines of the small cell lung cancer cell line
HTB119 modulates potential malignancy (E.P. Scheidegger et
al., J. Lab. Invest. 70: 95-106 (1994)). The expression of
PST was investigated in clonal sublines derived from this
tumor cell line. By limiting dilution, 3 sublines were
obtained which, although identical in
NCAM expression, varied in the amount of PSA present. In FACS
analysis 100% of the cells in subline HTB119-54.2, 30% in
subline HTB119-38, and only 2% in subline HTB119-45 were
positive with mAb 735 (Fig. 3b). Poly (A)+ RNA from these
cell lines was isolated and analyzed in Northern blot with a
probe containing the entire coding region of pEPST-M~7. The
results are shown in Fig. 3a (lanes 5-7). At least 6 bands
were stained in highly polysialylated HTB119-54.2 and in
HTB119-38 which express low amounts of PSA. Again, an
extremely good correlation between surface expression and the
level of PST mRNA was observed. These data suggest that PST
is the cellular control element regulating PSA synthesis and
furthermore indicate that the metastatic efficiency of these
cells might be influenced by the abundance of PST mRNA.
Southern blot analysis of hamster and mouse genomic DNA
using the PST cDNA as a probe, revealed a restriction pattern
for the PST
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14
gene consistent with the presence of a single copy of the
gene.
ExamDle 4
Preparation of antisense DNA
The fact that PSA can affect the metastatic potential of
tumors suggests the desirability of modulating synthesis of
the molecule by regulating PST expression. This approach is
seen as being useful as a method for treating pathological
conditions, such as tumor associated diseases characterized by
excess or inappropriate expression of PST, and/or
inappropriate level of PSA. Three sets of experiments were
carried out to test the ability of antisense oligonucleotides
to inhibit expression of PST, and hence of PSA activity.
First, phosphothioate oligonucleotide molecules
complementary to nucleotide sequences of SEQ ID NO: 1 were
synthesized with phosphorothioate linkages on an automated
nucleotide sequencer following M. Matsukara et al., Proc.
Natl. Acad Sci. USA 84: 7705-7710 (1987). As a control,
random oligonucleotide molecules having the same length were
prepared in identical fashion. The purity o~
oligonucleotide molecules was determined by electrophoresis
through 15% polyacrylamide gels stained with etidium bromide.
The oligonucleotide molecules prepared are complementary
to sequence sections of SEQ ID NO: 1 as follows:
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oligo 1 : Oligonucleotides 325-345 (21mer) (SEQ ID NO: 3)
oligo 2 : Oligonucleotides 522-539 (18mer) (SEQ ID NO: 4)
Oligo 3 : Oligonucleotides 846-862 (17mer) (SEQ ID NO: 5)
Oligo 4 : Oligonucleotides 1356-1373 (18mer) (SEQ ID NO: 6)
PSA-positive CHO-wt cells were incubated with 3umol of each of
the oligonucleotides. PSA activity was measured in a kinetic
test, since PSA has a hal~ e longer than 24 hours. Cells
were analyzed by FACS analysis with mAb 735 according to
Example 3 a~ter 20, 40, 60 and 80 hours.
In further experiments, the oligodeoxynucleotides, and
polylysine modified conjugates were prepared according to J.P.
Leonetti et al., Gene 72 (1988) 232-332, incorporated by
reference, but summarized herein.
First, a solution of N~, 2~-(3')o-dibenzoyl-5~-dimethoxy-
trityl-adenosine (2.15 g, 2.76 mmol) (Kempe et al., Nucleic
Acids Res. 10 (1982) 6695-6714) and dimethylamino-4-pyridine
(0.506 g, 4.15 mmol) in methylene chloride (12.3 ml) were
combined with succinic anhydride (0.415 mmol) and
triethylamine (0.58 ml, 4.15 mmol). The mixture was stirred
for 2.5 hours, poured into 1 M aqueous triethylammonium
hydrogen carbonate (120 ml), and the resulting products were
extracted with methylene chloride (3 x 150 ml). The combined
organic layers were washed with water, dried over anhydrous
sodium sul~ate and evaporated to dryness. The residue was
fractionated by silica gel chromatography using methanol-
triethylamine-methylene chloride (0:1:99 to 2:1:97, v/v/v) as
eluent. Fractions containing the pure-o-hemisuccinate were
combined and evaporated to dryness. The residue was dissolved
in 1.2-dimethoxyethane, pentachlorophenol (0.783g, 2.94 mmol),
~ollowed by addition o~ N,N'-dicyclohexylcarbodimide (0.606 g,
2.94 mmol). The solution was stirred for 24 hours and
evaporated to dryness. The resulting residue was purified by
silica gel chromatography using acetone-methylene chloride
(5.95 to 8.92, v/v) as eluent. Fractions containing pure
succinate diester were combined and evaporated to dryness.
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16
Precipitation of the residue from petroleum ether resulted in
a colorless powder (1.69 g) at a yield of 56%.
Long-chain alkylamine controlled pore glass (5 g from
Sigma) was activated with triethylamine (0.3 ml, 1.5 mmol) in
pyridine (10.5 ml). After evaporation of the solvent, the
residue was suspended in a solution of succinate diester (1.69
g, 2.5 mmol) in dry pyridine (15 ml). After the mixture was
stirred gently for 3 days at room temperature, the solid
material was collected by suction, washed thoroughly with
pyridine and methylene chloride, and dried. The glass beads
were suspended in a capping solution 24 mol) made from acetic
anhydride (0.186 ml), 19.98 mmol), 2.6-lutidine (0.198 ml,
1.71 mmol) and 4-dimethylaminopyridine (1.8 g, 14.7 mmol) in
anhydrous tetrahydrofuran (30 ml). The mixture was stirred
for 10 min. and the glass beads were collected by suction,
washed with tetrahydrofuran (2 x 20 ml) and methylene chloride
(4 x 20 ml) and then dried. Spectrophotometric measurement
of the amount of dimethoxytrityl cation liberated by treating
a portion of the functionalized adenosine-derivatized glass
beads with 0.1 M toluene-sulfonic acid in acetonitrile
indicated a loading of 24 ,umol g.
(~)-Anomeric oligonucleotides, i.e., SEQ ID NOS: 3-
6, were synthesized on a Biosearch Cyclone DNA synthesizer
using the well known phosphoramide method. The synthesis was
carried out on an adenosine derivatized support prepared as
described supra. Samples of (~)-anomeric oligos (80 nmol) in
100 um, of 20 mM Na acetate (pH 4.4) were oxidized with 4.6
~umol Na metaperiodate for 30 minutes at O~C in the dark. An
equal volume of polylysine (PLL) (mean 14-kDa, Sigma) 80 nmol
in 2 M NaCl, 0.2 M Na borate buffer (pH 8.4) and 100 umol
sodium cyanoborohydride were added. The mixture was incubated
overnight at 20~C and then loaded on Sephadex G-50 column
equilibrated with 0.5 M NaCL, 20 mM Na acetate buffer (pH
6.0). Each fraction was assayed for its oligo-PLL content by
absorbance at 260 nm and by the BCA protein assay (from
Pierce). The conjugates were stored at -80~C.
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PSA positive CHO-wt cells were incubated with 0.9 ug
oligo-PLL conjugate, and activity was measured as above.
In a third set of experiments, 3'-cholesterol-modified
oligo-deoxynucleotides were prepared according to Chou, J.H.
et al., Cancer Research 54 (1994) 5783-5787 and Reed, M.W., et
al., Bioconjugate Chem. 2 (1991) 217-225, both of which are
incorporated by reference.
Cholesterol-modified CPG (controlled pore glass) was used
as support. The oligonucleotides were prepared from 2 ,umol
columns of these supports on an Applied siosystems Model 394
DNA Synthesizer using the 1 ~mol protocol supplied by the
manu~acture. 3'-cholesterol-modified oligonucleotides with
C6-linker were prepared ~rom a commercially available 1 ~mol
CPG column (Clontech, Palo Alto, CA). Standard reagents for
~-cyanoethylphosphoramidite coupling chemistry were used.
After ammonia deprotection, the oligonucleotides were HPLC-
purified, detritylated and precipitated from butanol as
described in Reed, M.W., et al., supra. The oligonucleotides
were at least 90% pure (HPLC).
PSA-positive CHO-wt cells (10,000/well) were plated in
24-well plates and incubated with 3 ,umol oligonucleotide per
well in 0.5 ml culture medium, and analyzed as above.
le 5
Recombinant expression of fusion-free PST in Escherichia coli
The DNA sequence coding for PST is modified in a way
which allows for efficient expression in E. coli.
For expression, an expression plasmid is transfected into
a suitable E. coli strain using standard methodologies. Such
strains are, in the case o~ the use of an expression plasmid
under the control of lac repressor such as the expression
plasmid pll379, strains which possess a sufficiently high
intracellular concentration of lac repressor. These kinds of
strains can be prepared by transfection of a second plasmid
such as pREP4 (Diagen GmbH), pUBS 500 or pUBS520 (Brinckmann
et al., Gene 85 109-114 (1989)). The E. coli strains
employed should preferably have low protease activity, as is
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18
the case, for instance, with E. coli UT5600 (Earhart et al.,
FEMS Microbiol. ~ett. 6: 277-280 (1979)), E. coli BL21
(Grodberg and Dunn, J. Bacteriol. 170: 1245-1253 (1988)) or E.
coli B. Expression cultivation is accomplished in a fashion
according to the state of the art. For recovery, PST obtained
as a protein aggregate from E. coli is processed, e.g.,
according to the procedures described in EP 0 241 022, EP 0
364 926, EP 0 219 874 and DE-A 40 37 196 all of which are
incorporated by reference. An example of such a protocol is
set forth.
PST-containing protein aggregates from E. coli
fermentation (so called "inclusion bodies") are solubilized in
6 M guanidinium hydrochloride, 100 mM Tris-HCl at pH 8, 1 mM
EDTA, subsequently
adjusted to a pH of 3 to 4 and dialyzed against 4 M
guanidinium hydrochloride at pH 3.5. The renaturing of the
solubilized protein is then carried out in 1 M arginine at pH
8, 1 mM EDTA, 5 mM GSH (glutathione, reduced) and 0.5 mM GSSG
(glutathione, oxidized). From the renaturing preparation, PST
can be obtained, for instance, after addition of 1.4 M
ammonium sulfate by adsorption to hydrophobic gel matrices
such as Fractogel TS~ Butyl and subse~uent elution in 20 mM
TrisHCl at pH 7.
Exam~le 6
Recombinant expression of PST in -- -lian cells
In order for recombinant PST to be expressed in
heterologous m~mm~lian cells, DNA from a first m~mm~lian
species which encodes a PST is ligated into a vector and is
then transcribed in cells of a second mammalian species. This
approach is used, e.g., to produce human PST in CHO cells,
although it should be understood that a coding sequence ~rom
a mammalian species can also be transfected into cells of the
same species. A strong promoter-enhancer system can be used,
and in the case of genomic PST fragments, this step is
required because the promoters of PST are active only in
certain cell types (e.g. melanomas). Thus endogenous
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19
promoters may not be suitable for general recombinant
expression in all cell types. Expression can also be
accomplished by homologous recombination n vitro, whereby a
suitable exogenous promoter is used in combination with a PST
coding region. Such exogenous promoters and enhancers are
generally ~rom viruses such as SV40, hCMV, polyoma, or
retroviruses. Alternatively, one can use promoter enhancer
systems which are specific to a certain cell type or tissue
type, such as WAP- or immune globulin promoters, or systems
which are inducible, such as metallothionein and MMTV
promoters. These constructs supplement the PST cDNA with donor
and acceptor signals for RNA processing as well as a signal
for poly-A-addition. For example, pCMX-pLl (Umesono et al.,
Cell 65: 1255-1266 (1991)) is a suitable vector containing a
CMV promoter. The PST cDNA is provided with EcoRI linkers and
then ligated into the vector's single EcoRI cleavage site and,
by using the other known cleavage sites in the polylinker of
this vector, the PST cDNA is oriented in the proper reading
frame for promotion of expression by the CMV promoter. An
analogous procedure is applied when cloning into other
vectors, such as pCDNA3 (Invitrogen, San Diego/USA) or pSG5
(Stratagene, LaJolla/USA). The DNA of these expression
plasmids is from E. coli and may be transfected into a
m~mm~lian cell sample of choice, applying standard techniques.
See, e.g. Methods of Enzymology 185 (Gene Expression
Technology), ed. David V. Goeddel, Academic Press 1991,
section V. After transfection, the cells may be cultured in
minimum essential medium (MEM) without addition of fetal calf
serum, whereby PST is detectible in the cell culture
supernatant after 48 hours.
An example of such a system is the expression of
pEPST-ME7 cDNA in the mutant clone CHO-2A10, in NCAM-positive
subclones of NIH-3T3 cells, and in COS-hN-6. In each case
surface expression of PSA was shown by immunofluorescence
using mAb 735 as is now explained. The presence of PSA in
these cells indicates that PST is being expressed, because the
cells normally do not produce PSA.
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Tissue culture dishes were seeded with freshly
trypsinized cells 18 hours before transfection with 2mg of
pEPST-ME7, or an equal amount of the base vector pCDM8
described su~ra, using lipofectamin (Gibco BRL). 62 hours
later, transfected cells were incubated with the PSA specific
mab 735 for 30 minutes at room temperature, and any mab bound
to target was detected with fluorescein conjugated, anti-
mouse Fab fragments.
Example 7
Digoxigenin(DIG~-labelled RNA probes
For preparing a probe, a nucleic acid fragment to be
employed for hybridization is cloned in a suitable
transcription vector (with a T3, T7 or SP6 promoter). For
labelling, 1 to 2 ug of the plasmid were linearized, purified
by means of phenol/chloroform extraction and precipitated with
ethanol. 1 to 2 ,ug DNA (dissolved in DEPC-treated H~O), 2 ,ul
10 x transcription buffer, 40 U RNA poIymerase (T3, T7 or
SP6), 2 ,ul NTP/DIG-UTP mixture (10 mM
ATP, CTP, GTPi 6.5 mM UTP; 3.5 mM DIG-UTP) and 1 ,ul RNase
inhibitor (20 U/,ul) were augmented with H2O to a volume of 20
,ul .
This preparation was incubated for two hours at 37~C.
Subsequently the DNA is removed by the addition of 2 ,ul
RNase-free DNase I (10 U/,ul) and by further incubation for 15
minutes at 37~C. The reactions were stopped by adding 1 ,ul
0.5 M EDTA (pH 8.0), and the synthesized RNA was precipitated
with 0.1 vol. 3 M sodium acetate (pH 5.2) and 2.5 vol.
ethanol. The RNA was washed once with 70% ethanol, dried and
then dissolved in 100 ,ul H2O (DEPC-treated). To determine
labelling efficiency a dilution series of the labelled sample
and of a labelled control RNA are fixed on a nylon membrane
and subsequently developed with the anti-DIG-Fab-AP conjugate
as described in Example 8, in~ra.
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~m~le 8
Hybridization
RNA or DNA immobilized on nylon membranes was hybridized
with DIG-labelled RNA probes. Hybridization was carried out
under identical conditions, except that lower hybridization
temperatures were used for DNA-RNA hybridizations.
The membranes were first pre-hybridized in hybridi~ing
buffer (50% formamide, 50 mM sodium phosphate pH 7.0, 7% SDS,
0.1% N-lauroylsarcosine, 5xSSC, 2% blocking reagent) for 1 to
2 hours (65~C in the case of RNA-RNA and 50~C in the case of
DNA-RNA hybridizations). Hybridization with DIG-labelled
probe was
carried out ~or 16 to 90 hours and, at 65~C and 50~C,
respectively. Immediately beforehand, the probe was heated to
98~C for 5 minutes and subsequently cooled on ice. After
hybridization, the membrane was washed, twice for 5 minutes in
2xSSC, 0.1% SDS (at ambient temperature) and twice for 15
minutes in O.lxSSC, 0.1% SDS (at 65~C).
The detection of the hybridized probe was carried out
applying the following procedure:
1. Wash in buffer 1 (150 mM NaCl, 100 mM maleic acid pH 7.5)
for 2 minutes;
2. Saturate the membrane in buffer 2 (1% blocking reagent in
buffer l) for 30 to 60 minutes;
3. Incubate with anti-DIG-Fab-AP conjugate (1:10,000 diluted
in buffer 2; 75 mU/ml) for 30 minutes;
4. Wash twice in buffer 1 with 0.3% Tween-20 for 15 minutes;
5. Wash in buffer 3 (100 mM NaCl, 100 mM Tris/HCl pH 9.5)
for 2 minutes.
The membrane, together with the substrate solution (CSPD:
[di-sodium-3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)t
ricyclo[3.3.1.13,7]decan}-4-yl)phenylphosphate] diluted at a
ratio of 1:100 in buffer 3), was wrapped in plastic ~oil and
incubated for 30 minutes at 37~C. Thereafter, the substrate
solution was removed from the foil, the foil was sealed, and
the signals detected by exposure to X-ray, and development on
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film. The optimum exposure times were between 30 minutes and
2 hours.
~mrlle 9
As a first step in isolating a coding sequence for human
PST, an appropriate eukaryotic cell system was needed. COS
and CHO cells are used, frequently, as recipients for
eukaryotic DNA, and were tested to determine if they were
appropriate.
Samples of both COS-1 cells, and CHO cells, were
transfected with plasmid pH~A-NCAM. This plasmid encodes a
transmembrane form of the neural cell adhesion molecule
("NCAM"), which has a molecular weight of about 140 kDa. See
Dickson, et al., Cell 50: 1119-1130 (1987), incorporated by
reference. Both before and after transfection, the recipient
cells were tested in an indirect immunofluorescence assay for
polysialic acid, using monoclonal antibody 735, which is
specific for this molecule. See Frosch, et al., Proc. Matl.
Acad. Sci. USA 82: 1194-1198 (1985), incorporated by
reference. The results of the assay showed that CHO cells
were an inappropriate choice, because they expressed PSA
before transfection. In contrast, COS-1 cells were negative
both before and after transfection. Thus, COS-1 cells were
used in the experiments which follow.
EXamD1e 10
A human fetal brain cDNA library (Invitrogen, San Diego,
CA) (40 ug) constructed in plasmid pcDNAI was cotransfected
into 2.4x107 COS-1 cells, together with an e~ual amount of
pH~A-NCAM, using lipofectamine.
Forty-eight hours later, cells were tested via
fluorescence activated cell sorting, using mAb 735, described
supra, following Hayrinen, et al., Mol. Immunol. 26: 523-529
(1989), incorporated by reference. This methodology permitted
separation of "mAb 735 positive" cells.
Once the positives were isolated, plasmid DNA in the
positive cells was itself isolated, following the classic
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procedure of Hirt, J. Mol. Biol. 26: 365-369 (1967),
incorporated by re~erence. The isolated plasmids were then
amplified in host bacteria MC 1061/P3, using both ampicillin
and tetracycline. This was possible because starting plasmid
pcDNAI contains supF suppressor tRNA, and thus confers
resistance to both antibiotics. In contrast, the pHi~-A-NCAM
plasmid confers resistance to ampicillin, but not to
tetracycline. In this assay, the isolated plasmids were
divided into 23 plates, each plate containing about 500
colonies of the bacteria. Plasmid DNA was prepared ~rom each
plate. As a result, the procedure results in rescue of
positive library clones, but not others.
The recovered plasmid DNA was trans~ected into COS-1
cells with pHi~A-NCAM and then subjected to sibling selection,
which is a standard technique, resulting in sequentially
smaller, active pools, activity being determined in an
immunoassay, using mAb 735, described supra, and mAb 1263, the
latter of these being used only in the analysis following
cloning. In this way, a single plasmid, i.e., "pcDNAI-PST"
was identified which, upon expression in host cells resulted
in the appearance of PSA on the host cell surface. The
presence of PSA in COS-1 cells co-transfected with pcDNAI-PST
and pHi~A-NCAM was detected by irrununostaining with monoclonal
antibody 735, as shown in figure 4D. The staining with
monoclonal antibody 735 was abolished by pretreatment with
endo-N, which hydrolyzes PSA. This is shown in figure 4F.
In order to confirm that the NCAM molecule contains
polysialic acid (PSA), HeLa cells were cotransfected, using
lipofectamine, with pH~A-MCAM alone, or pHi~A-MCAM and pcDNAI-
PST, in the same manner described su~ra, on COS-1 cells.
Following transfection and incubation, the HeLa cells were
- subjected to Western Blotting. To perform the blotting, the
lysates obtained from 2X106 cells (untransfected parent or
cotransfected cells), were subjected to SDS gel
electrophoresis, transferred to nitrocellulose, and incubated
with anti-NCAM antibody (Becton-Dickinson). As shown in
figure 4G, lysates of untransfected HeLa cells showed only
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24
barely detectable cross-reactivity of an ~100-kDa protein
with anti-NCAM antibody (lane 1). In contrast, lysates of
HeLa cells co-transfected with pcDNAI-PST and pH$A-NCAM
displayed multiple bands of immunoreactivity corresponding to
species of 140 kDa and greater molecular mass (see smear in
lane 2). Lysates of the co-transfected cells that were
treated with endo-N prior to electrophoresis displayed a
single sharp band of immunoreactivity corresponding to a 140-
kDa protein. These results confirm that pcDNAI-PST encodes a
protein involved in synthesis of PSA, and that the PSA is
attached to NCAM.
Example 11
In view of these results, the sequence of the insert in
pcDNAI-PST was deduced, using the classic Sanger methodology
(Sanger, et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467
(1977)), and is presented as SEQ ID NO: 7. This insert
contains an open reading frame which encodes a protein with
predicted 359 amino acid residues, (SEQ ID NO: 8) and a
deduced molecular mass of 41,279, or about 41-42 kDa.
A hydropathy plot was prepared from the deduced amino
acid sequence, using well known techniques. When analyzed,
the plot suggests type II transmembrane topology with a short
cytoplasmic sequence at the -NH2 terminus, followed by a
transmembrane domain, a so-called stem region, and a large
catalytic domain, which one presumes resides in the Golgi
lumen. This structure appears to be in agreement with all
m~mm~l ian glycotransferases cloned thus far (see Schacter, in
Fukuda, ed. Molecular Glycobiology Oxford University Press,
1994, pp. 88-162).
Analysis of the deduced amino acid sequence (SEQ ID NO:
8), shows 27.0% identity with GD3 synthase, an ~-2, 8
sialytransferase (Nara, et al., supra; Sasaki, et al., supra)
and 58.2% with "STX", an enzyme of unknown specificity
(Kitagawa, et al., supra). There is also much less homology
(9.7%) with Gal$1-3GalNAc ~-2,3-sialyltransferase, taught by
Lee, et al., Eur. J. Biochem. 216: 377-385 (1993).
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Moving to a more in depth comparison, the highest degree
of homology is found in a portion of the catalytic ~o~in, the
sialyl motif "L", (amino acids 141 to 185 in SEQ ID NO: 8)
shared by all sialyl transferases cloned to date (Sasaki, et
al, suprai Wen, et al., J. Biol. Chem. 267: 21011-21019
(1992); Datta, et al., J. Biol. Chem. 270: 1497-1500 (1995).
Datta, et al., identify this motif as being the binding site
for enzyme substrate CMP-NeuNAc. The predicted sialyl motif
"S" is located at amino acids 288-300 in SEQ ID NO: 8.
Upstream from motif L, the predicted amino acid sequence
shows a cluster o~ basic amino acids (Arg Arg Arg), at
residues 114-116, and one at residues 137-140, i.e., Arg Arg
Phe Lys. These clusters are either completely absent or
incomplete in the corresponding sequences of GD3 synthase and
STX. These basic amino acid clusters in PST may be critical
for PST's binding to the acceptor containing multiple
negatively charged sialic acid residues. A consensus sequence
for polyadenylation was not found in the 3'-flanking sequence
of the cloned cDNA; however, one can posit with some assurance
that, during construction of the library, PST cDNA synthesis
was started at a nucleotide sequence rich in adenine, i.e.,
nucleotides 1672-1682.
Exam~le 12
The distribution of PST mRNA in human tissues was
determined via Northern Blotting.
Human fetal and adult brain poly(A)+ RNA was obtained,
and electrophoresed in 1.2% agarose gel containing 2.2 M
formaldehyde, followed by transfer to a nylon filter.
Similarly, Northern blots of human multiple tissue poly(A)~
RNAs were purchased (Clontech, Palo Alto CA). The RNAs were
- hybridized with a gel purified cDNA insert of pcDNAI-PST,
which had been labelled with ~[32p] -dCTP via random
oligonucleotide priming (Feinberg, et al., Anal. Biochem. 132:
6-13 (1983)), in accordance with Bierhuizen, et al., Genes and
Dev. 7: 468-478 (1993).
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26
Results, set ~orth in figure 5 show a strong, 6. 5 kb band
and a weak, 3.7 kb band in poly(A) RNA from fetal brain. The
size difference is probably due to alternative usage of
polyadenylation sites.
When compared to fetal brain, the signal in adult brain
was weaker. The signal from fetal lung and fetal kidney was
strong, while the fetal liver signal was not. With respect to
adult tissues, PST transcripts were detected strongly in
heart, spleen, and thymus tissue, and moderately in brain,
placenta, lung, large intestine, small intestine, and
peripheral blood leukocytes.
Different parts of adult human brain showed differing
albeit weak levels of expression. Substantial amounts of PST
mRNA were found in thalamus, subthalamic nucleus, substantia
nigra, and cerebral cortex, with moderate amounts in amygdala,
caudate nucleus, corpus callosum, hippocampus, and putamen.
Generally, PST was expressed more in forebrain derivatives
than in midbrain, hindbrain, and caudal neural tube
derivatives.
EXaUnD1e 13
In order to determine whether or not expression of PST
mRNA is solely responsible for expression of polysialic acid,
immuno-histochemical analysis was carried out.
For these experiments, samples of tissue were fixed for
24 hours in cold, 4% paraformaldehyde in O.lM phosphate
buffer, (pH 7.4), followed by embedding in paraffin, and
sectioning ( 3 um thickness).
Tissue sections were deparaffinized, hydrated, and then
immersed in absolute methanol containing 0. 3% H2O, for 30
minutes.
Following this, mAb 735 described supra was used to
determine presence of PSA, using the avidin-biotin-peroxidase
methodology taught by Hsu, et al., J. Histochem. Cytochem. 29:
577-580 (1981). Briefly, biotinylated mAb 735 was contacted
to the sections, followed by avidin-peroxidase complexes, and
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then a peroxidase substrate which yields a color upon reaction
with the enzyme.
Figures 6A to 6D show strong presence of polysialic acid
in neurons of fetal cerebral cortex, bronchial epithelia of
fetal lung (arrows in figure 6B), Hassall's corpuscles of the
thymus (see the arrows in figure 6C), and epithelial cells of
the thymus (arrowheads in figure 6C). In contrast, tissues
lacking PST mRNA were also negative for polysialic acid
staining. These results suggest, strongly, that PST alone is
responsible for biosynthesis of polysialic acid.
~m~le 14
A study was carried out to determine what effect, i~ any,
polysialic acid expression in living subs~rates had on neurite
outgrowth. It has been reported that neural cell migration
and axon outgrowth were influenced by polysialic acid
expressed on either neural cells, or crude membrane substrates
prepared from chick tectum. See Tang, et al., Neuron 13: 405-
414 (1994). Doherty, et al., Neuron 5: 209-219 (1990);
Boisseau, et al., Development 112: 69-82 (1991). In one of
these studies (Doherty, et al), neurite outgrowth was observed
in neural cells on substrate cultures of 3T3 cells which had
been transfected to express NCAM. HeLa cells, cotransfected
as described in Example 10, supra, were used as substrate for
growth of sensory neurons from dorsal root ganglia of 10 day
old chick embryos, and neurons from ventral portions of spinal
cords from six day old chick embryos, which predo~;n~ntly
contained motor neurons. Prior work by Doherty, et al.,
Neuron 5: 209-219 (1990) has shown that both of these cell
types express both NCAM and polysialic acid.
Cells were trypsinized (0.5% trypsin), counted, seeded at
low density over monolayers of HeLa cells, and cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum. The sensory neuron culture had nerve growth factor
added to it as well.
Cultures were grown up for 15 hours, after which they
were fixed with 4% formaldehyde, in phosphate buffered saline.
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28
The fixed cells were then stained with anti neurofilament
antibody RM 0270 (Lee, et al., J. Neurosci. 7: 3474-3488
(1987)), followed by fluorescein isothiocyanate conjugated
mouse anti-IgG antibody. The length of the neurites were
measured, using the standard "JAVA" morphometric system
(Jandel Scientific), via epifluorescence. The longer neurite
of each neuron was measured for 30 neurons within adjacent
fields, in duplicate experiments. Only neurons whose neurites
did not overlap others were included. Mean neurite lengths,
and the number of neurite branches per neuron occurring on
three different substrates (untransfected HeLa cells, those
transfected by N-CAM cDNA, and cells transfected by both N-CAM
cDNA, and PST cDNA), were counted, and compared by Student's
T test.
Figures 7A, B and C show these results. Those neurons
derived from dorsal root ganglia (almost exclusively sensory
neurons), showed modest neurite outgrowth on confluent layers
of untransfected HeLa cells (mean length; 196.3 ~m), as in
figure 7A and HeLa cells expressing M-CAM (171.3 ,um), as in
figure 7B. Those cells cultured on the cotransfectants,
however, grew neurites with a mean length of 253.6 ~m. See
figure 7C. The branching on these neurons (mean: 4.0
branches/neuron) was significantly higher than those grown on
cells transfected with NCAM only (2.3 branches/neuron). The
same pattern was found with the spinal cord derived neurons,
which are mainly motor neurons (figure D, E and F).
The work set forth herein shows that polysialic acid is
a critical regulator of neurite outgrowth on living cells.
ExamDle 15
Isolation o~ Nucleic Acids Encodin~ Mouse PST
1. Isolation of Mouse PST cDNA
To isolate nucleic acids encoding mouse PST, cDNAs were
prepared using RNA isolated from neonatal brain of BALB/c mice
as the template, following standard procedures. The cDNAs
were subjected to nucleic acid amplification (performed
according to standard procedures) using oligonucleotides based
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29
on the human PST cDNA (see Sequence ID NO: 7) as primers.
Specifically, the first primer corresponded to nucleotides
206-225 in SEQ ID NO: 7 and the second primer corresponded to
the complement of nucleotides 1284-1299 in SEQ ID NO: 7. The
product of this amplification procedure, which is similar in
length to the cDNA encoding human PST, is purified, ligated
into a plasmid such as pBluescript II, and characterized by
DNA sequence analysis.
2. Isolation of Genomic DNA Encoding Mouse PST
Genomic DNA was isolated from mouse cell line I29SVJ
according to standard procedures and ligated into the Lambda
FIX II cloning vector. The resulting genomic DNA library was
screened using conditions as described supra for hybridization
to an oligonucleotide probe corresponding to nucleotides 1-557
of SEQ ID NO: 7. Hybridizing plaques were purified and
characterized by DNA sequence analysis. DNA sequence analysis
of the genomic DNA thus isolated revealed that it contains at
least two exons: one corresponding to an extreme 5'domain of
the human PST cDNA (i.e., nucleotides 1-325 of SEQ ID NO: 7)
and a second corresponding to a more downstream location of
the human PST cDNA (i.e., nucleotides 458-715 of SEQ ID NO:
7). Through such analysis of the complete isolated DNA, the
genomic structure of the gene encoding mouse PST can be
elucidated The resulting information is particularly useful
in designing gene llknock-out" experiments in mice, wherein the
mouse PST gene in mouse embryos is rendered nonfunctional.
The resulting transgenic mice are then analyzed for the
effects of a lack of PST activity.
EXAMPLE 16
PST Is Capable of Catalyzing The Synthesis of
In Vivo In The Absence of N-CAM
The results set forth in examples 1-15 established
that PST, when expressed together with N-CAM in cell lines
such as HeLA, CHO-2A10, and NIH/3T3, results in the production
of polysialic acid (PSA). To ascertain whether PST can
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catalyze synthesis of PSA in cells which do not express N-CAM,
COS-l cells were transiently transfected with cDNA encoding
PST, without cotransfection with cDNA encoding an N-CAM
substrate. Transfection was carried out using the plasmid
pcDNAI-PST, described supra, using lipofectamine. After 48
hours, the transfected COS-l cells were subjected to
immunoflourescent staining, using murine monoclonal antibodies
M6703 (Nakayama, et al, J. Histochem Cytochem 41:1563-1572
(1993)) 12E3 (Seki, et al. Anat. Embryol 184: 395-401 (1991)),
and 735 (Frosch, et al, Proc. Natl. Acad. Sci. USA 82: 1194-
1198 (1985)). All of these references are incorporated by
reference. The mAb M6703 is known to react with oligosialic
acids which contain 2 or 3 ~-2,8 linked sialic acid residues,
12E3 reacts with polysialic acids containing 6 or more
residues of ~-2,8 linked sialic acid, and 735 with polysialic
acids containing 8 or more of these residues.
Following staining with the murine mAbs, fluoresein
isothiocyanate (FITC) conjugated goat-antimouse IgG antibody
F(ab')~ fragments were added to detect M6703 and 735, while
FITC conjugated goat-antimouse IgM antibody F(ab' )2 fragments
were used to detect 12E3. The IFA protocol was identical to
that described supra.
The strongest staining pattern obtained was that
using 12E3. These results indicate that the majority of PSAs
formed by PST were those containing 6 or more ~-2,8 linked
sialic acid residues.
It was confirmed that N-CAM was absent, because
there was no staining of the transfectants with an anti N-CAM
antibody. Thus, PST does not require N-CAM in order to
produce PSAs.
These results were confirmed, using HeLa as the host
cell line. In these experiments, parallel experiments, HeLa
cells were cotransfected with pcDNAI-PST and pSV2neo, and
selected with antibiotic G418, using standard protocols, i.e.,
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those described in the prior examples. Following the
selection, immunofluorescent staining was carried out in the
same manner described for the COS-1 cells.
Transfected HeLa cells were stained with mAb 735.
When the transfectants were treated with the enzyme endo-N,
which is known to cleave PSA, no staining was detected by
antibody 735. The data generated and described herein show
that N-CAM is not required for production of PSA, when PST is
present.
E~MPLE 17
PST Adds PSA To Molecules Other Than N-CAM In
Vitro: Construc~ion of Vectors Encodina Fusion Proteins
The data from the example 16 suggest that PST can
add PSA to molecules other than N-CAM. A series of
experiments were carried out to further demonstrate PSA
synthesis via PST in vitro, beginning with the following.
A fusion protein was produced, using a putative
catalytic domain for PST, a signal peptide, and an IgG binding
domain. The signal peptide was derived from human granulocyte
colony stimulating factor (G-CSF), and the IgG binding domain
was that of the S. aureus protein A.
The fusion protein was prepared using cDNA
sequences, and the well known polymerase chain reaction ("PCR"
herea~ter). The pcDNAI-PST plasmid was used as a template.
The upstream and downstream primers used were:
5'-CGGGATCCGG GTGAATTGTC TTTGAGTCGG T-3'
(SEQ ID NO:9)
~ and
5'-GGGGTACCTC AAAATGTGCT TTATTGCTTT ACAC-3'
(SEQ ID NO:10).
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IN SEQ ID NO:9, the underlined portion is a BamHI
restriction site, while the underlined portion of SEQ ID NO:10
is a KpnI restriction site.
Following standard PCR techniques, the resulting
product encompassed nucleotides 118 (codon 40) through 1092
(12 nucleotides downstream of the stop codon. Thus, amino
acids 40-359 of SEQ ID NO:8 constitute a soluble, PST active
molecule.
Parallel to this procedure, the plasmid pAMoA-GD3,
described by Sasaki, et al., J. Biol. Chem 22:15950-15956,
(1994) incorporated by reference, was digested with BamHI and
KpnI, with the result that pAMoA was formed, which contained
only DNA encoding the human G-CSF signal sequence and a
sequence encoding S. aureus protein A.
The PCR product referred to herein was then digested
with the same two endonucleases, and cloned into pAMoA, using
standard methods. The resulting plasmid has been named p~MoA-
PST.
The plasmid pAMoA-PST was then digested with SalI
and Asp718, resulting in excision of cDNA encoding a fusion
protein consisting of the human G-CSF signal peptide, S.
aureus protein A, and the PST sequence referred to, supra.
The released cDNA insert was filed in using Klenow fragment of
DNA polymerase I, and cloned into pcDNAI which had been
digested previously with EcoRV. The resulting plasmid was
named pcDNAI-A PST. A control vector was also prepared,
consisting of only signal peptide and protein A cDNA. This
vector was named pcDNAI-A. Standard methodologies were used
to confirm that the vectors were in proper 5'-3' orientation.
EXAMPLE 18
PST Adds Sialic Acid To Molecules Other Than N-CAM
The vectors pcDNAI-A PST and pcDNAI-A were
transfected, separately, into COS-l cells, using the
- ~ ~ ~
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lipofectamine method described, supra. The transfected cells
were cultured for 62 hours, after which any fusion protein
secreted into culture medium was adsorbed to IgG-Sepharose
6FF, essentially following the methods o~ Kukows~a-Latallo, et
al., Genes Dev 4: 1288-1303 (1990~, and Bierhuizen, et al.,
Proc. Natl. Acad. Sci USA 89:9326-9330 (1992), both o~ which
are incorporated by reference. Resin was collected via
centrifugation, washed nine times with 50mM Tris-HCl, pH7.5
containing 1% bovine serum albumin followed by two washes with
20mM Tris-HCl, pH7.5, containing 7.5 mM CaCl2, and 0.05% Tween
20. Finally, the resin was suspended in an equal volume of
serum free medium opti-~M1. This entire procedure is akin to
that described by Kukowska - Latallo, et al., supra Sasaki, et
al., J. Biol. Chem 22:15950-15956 (1994), incorporated by
reference.
The enzyme activity of the fusion protein was tested
[following Sasaki, et al., supra, and Kojima, et al., FEBS
Lett 360:1-4 (1995), incorporated by reference]. Briefly, a
substrate solution was prepared, which contained lOO~g of a
substrate sialylated glycoprotein (elaborated upon infra),
dissolved in 50ul of O.lM sodium cacodylate buffer (pH 6.0),
which contained 20mM MnCl2, 1% Triton CF-54, and 2.4 nmoles of
CMP-[l4C] NeuNAc, which served as a donor substrate. An
aliquot of 50ul of the solution containing the fusion protein
was added, mixed, and the resulting mixture was incubated at
37~C, for either 4 hours, or 24 hours. Following incubation,
the reaction mixture was centrifuged. Samples (20,ul), of the
resulting supernatant were mixed with 20,ul of the sample
buffer for SDS-gel electrophoresis, described by Lemmli,
Nature 227:680-685 (1970). These mixtures were heated at 85~C
for 3 minutes. The r~m~;n;ng sample was stored at -70~C until
used.
The products contained in the sample buffer were
subjected to SDS polyacrylamide gel electrophoresis (7.5%
acrylamide gel), and any sialic acids incorporated therein
were determined fluorographically. The fluorography was
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34
carried out by fixing the gels in 40% methanol, for 30
minutes, followed by two 15 minute soaks in dimethylsulfoxide
followed by soaking in dimethylsulfoxide containing 2,5 -
diphenyloxazole (18.5g in 88ml of DMSO) in accordance with
Bonner, et al., Eur. J. Biochem 46:83-88 (1974), incorporated
by reference.
The sialylated glycoproteins referred to supra were
rat ~1~ - acid glycoprotein, fetuin, and huma~ ~ , - acid
glycoprotein, all of which had been purchased from Sigma.
In order to analyze the linkages of any incorporated
sialic acids, 90% ethanol was added to the reaction mixtures,
followed by centrifugation. The products were washed, once in
90% ethanol, and the samples were then digested with one of
NANase I (0.17 units/ml), NANase II (5 units/ml), or NANase
III (1.7 units/ml), all of which are commercially available.
The digestion was at 37~C for 19 hours, in accordance with the
supplier's instructions. NANase I cleaves ~-2,3 linked sialic
acids. NANase II cleaves ~-2,3 linked sialic acids as well as
~-2,6 linked sialic acids. NANase III cleaves all of ~-2,3
linked sialic acids, ~-2,6 linked sialic acids, and ~-2,8
linked sialic acids. Also, reaction mixtures were digested
for 36 hours with either of N-Glycanase, following the
manufacturer's instructions, or with endoneuraminidase ("endo-
N"), in accordance with Hallenback, et al., J. Biol. Chem
262:3553-3561 (1987), incorporated by reference. Following
digestion, the materials were subjected to SDS-polyacrylamide
gel electrophoresis and fluorography, as described herein.
When fetuin was incubated for 24 hours in the manner
described, a wide, high molecular weight band (100-170kd), was
formed. While some small amounts of wide and high molecular
weight bands were found for the two ~ acid glycoproteins, the
majority of the radioactivity from the donor subst~ate moved
very close to the position of untreated glycoproteins (~48kd
for rat, and 44kd for human).
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The products formed by PST were not susceptible to
NANase II digestion, but were digested by NANase III, which
differs from NANase II in its ability to cleave ~-2,8 - linked
sialic acid. Eurther, the products were susceptible to
cleavage by both endoneuriminadase, and N-Glycanase.
The conclusion drawn from this is that the wide,
high molecular weight bands represent glycoproteins containing
polysialic acid side chains in N-Glycans. The digestion
pattern with the NANases and N-Glycanase proves that the
attachment was through ~-2,8 linkages.
When fetuin product was treated with endo-N, it
migrated at about 90kd, which is larger than the untreated
molecule. This can probably be attributed to the inability of
endo-N to cleave polysialic acid chains which are shorter than
lS 5 or 6 sialic acid residues in length, as was explained,
supra. The product following endo-N treatment most likely
represents fetuin containing polysialic acid ch~ln.~ consisting
of 6 or fewer ~-2,8 sialic acid residues in a side chain.
Spiro, et al., J. Biol Chem 263: 18253-18268 (1988), show that
fetuin contains both N- and 0- linked oligosaccharides. Since
N-Glycanase treatment removed almost all of the incorporated
sialic acid, it can be concluded that polysialylation took
place on N-glycans.
EXAMPLE 19
Requirements of a PST Acceptor
In a further set of experiments, investigations were
carried out to determine what requirements had to be satisfied
by a molecule for it to act as a PST acceptor. Since the
previous example showed that fetuin did act as a PST acceptor,
it was digested with various neuraminidases and with N-
Glycanase, to yield a series o~ digestion products. These
products were then used in acceptor experiments. Briefly, the
fetuin was digested with NANase I, II, III or N-Glycanase, in
the same manner as was described, supra. Following digestion,
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36
90% ethanol was added to the mixture, mixed well, digested
substrate was recovered by centrifugation, and washed with 90%
ethanol. These products were desialylated (NANase treatment),
or deglycosylated (N-Glycanase treatment). They were then
used in precisely the same way that the rat ~1- acid
glycoproteins were used, supra. The results, show that once
~-2,3-linked sialic acids were removed from fetuin, PST did
not add sialic acid residues, whether or not ~-2,6 linked
sialic acid residues were present (see the two left most lanes.
of the figure). When N-glycans were removed, there was no
incorporation either (see last lane from the left).
These studies confirm that PSA is attached to an ~-
2,3 linked sialic acid on a glycoconjugate template and that
PST alone can add the first ~-2,8 ~ linked sialic acid to a
precursor containing ~2,3- linked sialic acid and then add
multiple ~-2,8- linked sialic residues to the acceptor,
yielding PSA. The experiment also established an in vitro
assay system for PSA synthesis.
The foregoing examples set forth, as one aspect of
the invention, isolated nucleic acid molecules which encode
polysialic acid transferases. These transferases may be
eukaryotic proteins, more preferably mammalian, and most
preferably human. Among the non-human mammalian species
embraced by the invention are various rodent species, such as
mouse, rat, rabbit, and guinea pig "PSTs~. Especially
preferred are isolated nucleic acid molecules comprising or
consisting essentially of SEQ ID NOS: 1 or 7, as well as their
complementary se~uences, as well as isolated nucleic acid
molecules which encode proteins consisting of SEQ ID NO: 2 or
SEQ ID NO: 8. Also preferred are isolated nucleic acid
molecules consisting of nucleotides 301-1377 of SEQ ID NO: 1,
and consisting of nucleotides 213-1289 of SEQ ID NO: 7. Also
included are isolated nucleic acid molecules which (i) code
for PST proteins as described herein and which hybridize with
one or both of nucleotides 721-1200 of SEQ ID NO: 1, and
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nucleotides 633 to 1112 of SEQ ID NO: 7 under stringent
conditions. The term stringent conditions is discussed below.
Proteins such as those coded by the sequences set forth
supra, and all isolated protein having PST activity are also
a feature of the invention. These proteins may have
particular amino acid se~uences, such as that of SEQ ID NO: 2
or SEQ ID NO: 8. The PST proteins can occur in natural
allelic variations which differ from individual to individual.
Such variations of the amino acids are usually amino acid
substitutions. However, they may also be deletions,
insertions or additions of amino acids to the total se~uence.
The PST protein according to the invention - depending, both
in respect of the extent and type, on the cell and cell type
in which it is expressed - can be in glycosylated or non-
glycosylated form.
The term "polysialyl transferase activity" denotes the
protein's capability of catalyzing the polycondensation of
~-2,8-linked sialic acids in vivo and/or in vitro. The number
of sialic acids that are being condensed is dependent on the
surrounding conditions, including, e.g., the cell type
in which condensation takes place, and on the CMP-activated
substrate (sialic acid or derivatives, e.g.
N-glycolylneuraminic acid or N-acetylneuraminic acid). The
number of condensed sialic acids may vary widely and ranges
between but a few (e.g. 10) and several hundreds or thousands
of such monomers. Preferably, N-acetylneuraminic acid
residues are condensed. PST activity may also include
induction of PSA synthesis in NCAM expressing cell lines as
well as cell-free synthesis of oligosaccharides. Expression
of PST in tumorigenic cells can influence metastatic
efficiency of the cells, and expression of PST in substrate
cells and/or nerve cells can also regulate neurite outgrowth,
as was shown, supra.
The polysialyltransferases of the invention catalyze the
polycondensation of sialic acids on acceptor structures like
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38
NCAM but do not use isolated poly- or oligomeric N-
acetylneuraminic acid substrates like colominic acid or
capsular polysaccharides from E.coli K1, E.coli K92 or
Neisseria meningitidis sero group B. In contrast to this, the
bacterial polysialyltransferases use such substrates from
E.coli Kl (PST of C.Weisgerber et al., Glycobiol. 1 (1991)
357) E.coli K92 (PST of S.M. Steenbergen et al., J. Bacteriol.
174 (1992) 1099), or from Neisseria meningitidis sero group B
(M. Frosch et al., Mol. Microbiol. 5 (1991) 1251). Therefore,
the polysialyltransferases according to the invention show a
different acceptor specificity than bacteria polysialylic
transferases.
The term "do not use isolated poly- or oligomeric N-
acetylneuraminic acid substrates" means that a conversion of
these substrates cannot be observed within the detection limit
in an appropriate detection method. Such methods of detection
are well known to one skilled in the art and are described,
for instance, in Cho and Troy, Proc. Natl. Acad. Sci. USA 91
(1994) 11427-11431.
"Isolated" and/or "purified" as used herein indicates
that the material modified thereby has been modified from its
native, in vivo cellular environment. As a result of this
modification, the recombinant DNAs, RNAs, polypeptides and
proteins of the invention are useful in ways that the DNAs,
RNAs, polypeptides or proteins as they naturally occur are
not, such as in the isolation of nucleic acids encoding
related proteins and in the treatment of certain pathological
conditions.
"Stringent conditions" as used herein, refers to
hybridization in the presence of lM NaCl, 1% SDS, and 10%
dextran sulfate, followed by two washes of a filter at room
temperature for 5 minutes, in 2xSSC, and one final wash for 30
minutes. This final wash may be at 0. 5xSSC, 0.1% SDS, more
preferably at 0. 2xSSC, 0.1% SDS, and most preferably at
O.lxSSC, 0.1% SDS, final wash taking place at 65~C. Those of
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39
ordinary skill in the art will recognize that other conditions
will afford the same degree of stringency, and are encompassed
by the phraseology "under stringent conditions", and are
encompassed herein.
With the aid of the nucleic acid molecules provided by
the invention, and techniques well known to the skilled
artisan, the PST gene or its variants in genomes of any
m~mm~l ian cells and tissue, preferably in such cells or tissue
which are positive for PSA may be isolated. Such processes
and suitable standard stringent hybridization conditions are
known to a person skilled in the art and are described for
example by J. Sambrook, Molecular Cloning: A Laboratory
Manual (1989) and B.D. Hames, et al., Nucleic Acid
Hybridization: A Practical Approach (1985). In this case the
standard protocols described in these publications are usually
used for the experiments. In particular section IX of Hames,
"Hybridization of radiolabeled probes to immobilized nucleic
acidll, page 947-962 with regard to the hybridization of
nucleic acid molecules, and to section XI, page 1145-1161,
"Conditions ~or hybridization o~ oligonucleotide probes" with
regard to the hybridization of oligonucleotide probes, both
~uotations beings incorporated herein by reference. Standard
stringent conditions are also described, ~or example, by
Holtke and Kessler, "The Dig System User's Guide For Filter
Hybridization" (1990).
~ells and ti~sue -~hich are p~siti~e f~r ~SA ~an be
identified either by an antibody which is specific for
polysialic acids and/or by the use of endoneuraminidase ME.
Such specific antibodies can be easily produced according to
~ 30 M. Frosch et al., supra or according to Moolenaar, et al.,
supra, both of which are incorporated herein by reference.
The isolation of endoneuraminidase NE is described in S.
Tomlinson and P.W. Taylor, J. Virol. 55: 374-378 (1985), which
is incorporated herein by reference. Endoneuraminidase NE
degrades ~-2,8-linked sialic acids with at least 8 sialic acid
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residues and releases in the cell or tissue supernatant
monomeric sialic acids.
For the detection of these free neuraminic acids (sialic
acids), an assay is described by L. Warren, J. Biol. Chem.
234: 1971-1975 (1959), which is incorporated herein by
reference. With this thiobarbituric acid assay (TBA assay)
free reducing ends (also oligomers as are obtained in the
endoneuraminadase NE digest, 3 to 8 residues) are detectable.
Thus it is possible to detect polysialic acid in cells by
harvesting the cells, washing in PBS, digesting with
endoneuraminidase NE (in PBS), employing the supernatant with
released sialic acid oligomers in the TBA assay and in the
colorimetric detection.
For example, nucleic acids encoding mammalian proteins
having PST activity may be isolated by screening suitable cDNA
or genome libraries under suitable hybridization conditions
with nucleic acids disclosed herein (including nucleic acids
derived from any of SEQ ID NOS: 1 or 7). The library can be
screened with a portion of the disclosed nucleic acids
including substantially the entire coding sequence thereof or
with a suitable oligonucleotide probe based on a portion of
the nucleic acids. As used herein, a probe is a single-
stranded DNA or RNA that has a sequence of nucleotides that
includes at least about 10-50 contiguous bases, preferably 16-
40, most preferably 25-30, that are the same as (or the
complement of) any contiguous bases set forth in any of SEQ ID
NOS: 1 or 7. Preferred regions from which to construct probes
include sequences predicted to encode transmembrane domains,
catalytic domains, sialylmotifs and the like.
Either the full-length cDNA clones, fragments thereof or
oligonucleotides based on portions of the cDNA clones can be
used as probes, preferably labeled with suitable label means
for ready detection. Non-radioactive labels are preferred.
These probes can be used for identification and isoIation of
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41
additional nucleic acids encoding proteins having PST
activity.
Thus, in accordance with another embodiment of the
present invention, there is provided a method for identifying
nucleic acids encoding proteins having PST activity said
method comprising: contacting m~mm~lian DNA with a nucleic
acid probe as described above, wherein said contacting is
carried out under conditions ~avorable to the hybridization o~
the probe to its complement, and identifying nucleic acid
molecules which hybridize to the probe.
After screening the library, positive clones are
identified by detecting a hybridization signal; the identified
clones are characterized by DNA sequence analysis and then
examined by comparison with sequences set ~orth herein to
ascertain whether they encode a complete PST protein. The
cDMA clones can be incorporated into expression vectors and
expressed in suitable host cell lines as described herein to
determine if the corresponding protein product displays PST
activity as also described herein.
Alternatively, nucleic acid amplification techniques
which are well known to those o~ skill in the art, can be used
to isolate nucleic acids encoding proteins with PST activity.
This is encompassed by employing oligonucleotides based on the
sequences disclosed herein in SEQ ID NOS: 1, or 7 as primers
for amplifying m~mm~lian RNA or DNA.
Once nucleic acid encoding a particular protein with PST
activity has been isolated, ribonuclease (RNase) protection
assays and i situ hybridization assays can be employed to
determine which cells and tissues express mRNA encoding the
protein. These assays provide a sensitive means for detecting
and quantifying an RNA species in a complex mixture of total
or cellular RNA.
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42
The use o~ recombinant DNA technology enables the
production of numerous PST protein derivatives. Such
derivatives can for example be modified in individual or
several amino acids by substitution, deletion or addition.
The derivatization can for example be carried out by means of
site directed mutagenesis. Such variations can be easily
carried out by a person skilled in the art (J. Sambrook,
su~ra, B.D. Hames, supra). It merely has to be ensured that
the characteristic properties of the PST protein (polysialyl
transferase activity) are preserved.
As PST is an intracellular (Golgi-resident) enzyme which
may be present in the cell in a dimeric form usually linked
via the N-terminus, dimerization in the cell is essentially
carried out via the transmembrane region (approximately amino
acid Nos. 8 to 20 of SEQ ID NOS: 2 and 8, e.g.). Even when in
monomeric form, however, the enzyme will display activity. A
soluble enzyme which is monomeric and in which the
transmembrane region and, thus, approximately the first 20 to
30 amino acids, preferably 25 amino acids, are absent, is
preferred. A soluble enzyme of this type is also active in
vitro and catalyzes the polycondensation of ~-2,8-linked
sialic acids.
The invention thus also comprises an isolated PST protein
which is a product of prokaryotic or eukaryotic expression of
an exogenous DNA as described herein.
The invention additionally concerns PST proteins and
nucleic acid molecules from other cells and tissue such as
m~mm~lian cells and tissues, including mouse, rat, bovine,
sheep, etc., which exhibit polysialyl transferase activity in
an essentially analogous manner. These proteins can be
obtained in a manner well known to the art. For example,
given the nucleotide sequences disclosed herein, one screens
a library, such as a cDNA library of a particular m~mm~l or
other eukaryote, for sequences which hybridize with the
complements of disclosed sequences. These ~targets~ as it
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43
were presumably encode functional equivalents of the PST
proteins encoded by the disclosed sequences. Transfection
with these isolated targets is expected to lead to expression
of the PST protein.
With the aid of the nucleic acid molecules of the
invention, the proteins according to the inven~ion can be
obtained in a reproducible manner and in large amounts. The
nucleic acid molecules are integrated into suitable expression
vectors, such as exogenous nucleic acid molecules, according
to methods familiar to a person skilled in the art, and are
introduced into a prokaryotic host cell or a eukaryotic host
cell. Such an expression vector preferably contains a
regulatable/inducible promoter to which the coding sequence is
operably linked. These recombinant vectors are then
introduced into suitable host cells such as, e.g., E. coli
(prokaryote) or Saccharomyces cerevisiae, Terato carcinoma
cell line PA-1 sc 9117 (Buttner et al., Mol. Cell Biol. 11:
3573-3583 (1991)), insect cells, such as Sf9, and all insect
cells transfected with baculovirus vector, and also CHO or COS
cells. The transformed or transduced host cells are cultured
under conditions which allow for expression of the
heterologous or homologous gene. The isolation of the protein
can be carried out according to known methods from the host
cell or from the culture supernatant of the host cell. Such
methods are described for example by Ausubel, et al., Curr.
Prof. Mol. Biol. John Wiley & Sons (1992). Also, n vitro
reactivation of the protein may be necessary and/or useful
using art recognized techni~ues. Thus, the invention provides
methods of producing recombinant PST that can be used, for
example, in cell-~ree synthesis of oligosaccharides, in the
~ study of PST structure and function, and in screening
compounds as potential inhibitors of PST enzymatic activity.
- Soluble PST enzyme is preferred for these uses. For
recombinant production of soluble PST, it is preferable to
utilize a DNA molecule comprising or consisting of PST coding
sequences (i.e., nucleotides 301-1377 in SEQ ID NO: 1 or
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44
nucleotides 213-1289 in SEQ ID NO: 7) lacking nucleotides
which code for the first 20-30 or more amino acids of the N-
terminus. Particularly preferred are DNA molecules comprising
nucleotides 330- 1289 of SEQ ID NO: 7 and the equivalent
section of SEQ ID NO: 1. Expression of recombinant PST may be
further enhanced by including in the PST DNA at positions
immediately upstream of the first codon and down of the
translation termination codon, respectively, 5' and 3'
untranslated sequence (e.g., 5' untranslated sequence
approximating a consensus sequence [see, e.g., Kozak (1991) J.
Biol. Chem. 266: 19867-19870] such as nucleotides 207-212 in
SEQ ID NO: 7 and 3' untranslated sequence comprising
nucleotides 1293-1304 in SEQ ID NO: 7).
It is further preferred to utilize a host cell that
facilitates secretion of the soluble enzyme. For secretion of
soluble PST from host cells, the nucleic acid encoding soluble
PST is linked, at the 5' end, to nucleic acid encoding a
signal peptide capable of directing secretion of proteins in
the selected host cell. For example, nucleic acids encoding
the protein A signal sequence can be linked to the 5' end of
nucleic acid encoding PST for secretion of PST from
appropriate host cells, in a manner well known to the skilled
artisan.
It is possible to provide a test based upon nucleic acid
molecules encoding PST protein, which are then used to detect
nucleic acid molecules which code specifically for PST
proteins. Such a test can be carried out, e.g., in cells or
cell lysates. Such a test can be carried out in accordance
with standard nucleic acid diagnostic methods. In such cases,
the sample to be examined is brought into contact with a probe
which hybridizes with the nucleic acid molecules coding for
the PST protein of interest such as nucleotides 301-720 of SEQ
ID NO: 1 or sequences specific for 1201 to 1377 of SEQ ID NO:
7. Hybridization between the probe and nucleic acid molecules
from the sample indicates the presence of expressed PST
proteins. Such methods are known to a person skilled in the
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art and are for example described in WO 89/06698, EP-A 0 200
362, USP 4,915,082, EP-A 0 063 879, EP-A 0 173 251, EP-A 0
128 018. In pre~erred embodiments o~ the invention, the
nucleic acid molecule of the sample which codes for a PST
protein is amplified before testing, e.g. by the well-known
PCR technique. A derivatized (labelled) nucleic acid probe is
normally used. This probe is brought into contact with a
carrier-bound denatured DNA or RNA from the sample and in this
process the temperature, ionic strength, pH value and other
buffer conditions are selected in such a way that - depending
on the length of the nucleic acid molecule sample and the
resulting melting temperature o~ the expected hybrid - the
labelled DNA or RMA can bind to homologous DNA or RNA
(hybridization, see also J. Mol. Biol. 98: 503 (1975),; Proc.
Natl. Acad. Sci. USA 76: (1979). Suitable carriers are
membranes or carrier materials based on nitrocellulose
rein~orced or bound nitrocellulose in a powder ~orm or nylon
membranes derivatized with various functional groups (e.g.
nitro group).
The hybridized DNA or RNA is then detected by incubating
the carrier, a~ter thorough washing and saturation to prevent
unspecific binding, with an antibody or antibody fragment.
The antibody or antibody fragment is directed towards the
substance incorporated into the nucleic acid probe during the
derivatization. The antibody is in turn labelled. It is,
however, also possible to use a directly labelled DNA. After
incubation with the antibodies, it is washed again in order to
only detect specifically bound antibody conjugates. The
determination is then carried out via the label of the
antibody or antibody fragment according to well-known methods.
The detection of the PST expression can be carried out
for example as:
- in situ hybridization with immobilized whole cells using
immobilized tissue smears and isolated metaphase
chromosomes,
CA 0220829l l997-06-l9
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46
- colony hybridization (cells) and plaque hybridization
(phages and viruses),
- Northern hybridization (RNA detection),
- serum analysis (e.g. cell type analysis of cells in serum
by slot-blot analysis),
- after amplification (e.g. PCR technique).
Since PST preferably is regulated on the mRNA level, it
is preferred to carry out, for detecting the PST expression,
a hybridization with the mRNA of the cell to be examined.
The invention therefore includes a specific method for
the detection of nucleic acid molecules which code for a PST
protein which is characterized in that the sample to be
examined is incubated with a nucleic acid probe which may be
selected from the group comprising nucleic acid molecules or
specific oligo- nucleotides which hybridize with the
nucleotides 301 to 720 and/or 1201 to 1377 of SEQ ID NO:1, 633
to 1112 of SEQ ID NO: 7 and their complementary sequences.
The nucleic acid probe is incubated with the nucleic acid
molecules from the sample and the hybridization of the nucleic
acid molecules in the sample and nucleic acid probe is
detected, if desired, via a further binding partner.
Thus, PST and its expression is a valuable prognostic
marker in tumor diagnostics (metastasis ! progress).
Another feature of the invention is oligonucleotide
molecules, such as SEQ ID NOS: 3, 4, 5, and 6, as well as (a)
nucleotides 237-257, (b) nucleotides 434-451, (c) nucleotides
759-775, and (d) nucleotides 1268-1285 of SEQ ID NO: 7, and
their complementary sequences which hybridize to PST coding
sequences and may specifically inhibit the expression of PST
in m~mm~lian cells. It has been found that when an
oligonucleotide ~olecule reaches a length which is more than
about 15 to 17 nucleotides the sequence is unique relative to
the entire human genome. Thus, these "15+ meres" are specific
to particular regions of the genome. It has been shown that
short antisense oligo- nucleotides can be imported into cells
CA 02208291 1997-06-19
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47
and inhibit expression of a gene to which they are targeted
(zamecnik et al., Proc. Natl. Acad. USA 83: 4143 (1986)). In
particular, oligonucleotide molecules having 15 to 50,
preferably 15 to 25, bases are suitable for use.
Therefore, such oligonucleotide molecules which is
complementary to, and designed on the basis of portions of the
PST genes described herein are useful for inhibiting the
expression of PST.
A further embodiment of the invention is an
oligonucleotide molecule which hybridizes in a manner which is
specific for nucleic acid molecules of m~mm~lian polysialyl
transferase, e.g. with a part or all of SEQ ID NO: 1, or SEQ
ID NO: 7.
The term "hybridizes in a manner which is specific for a
nucleic acid molecule of m~mm~lian polysialyl transferase"
means that such a nucleic acid molecule or oligonucleotide
molecule, when transfected into m~mm~lian cells such as human
cells, binds to the nucleic acid moiecules which code for a
polysialyl transferase in said cells. Specific binding occurs
if these nucleic acid molecules inhibit the expression of PST
in a considerable manner (more than 50%, preferably more than
80%, or more than 90%) and in such fashion that the other
metabolism processes of the cell are not impaired.
As is shown by comparing SEQ ID NOS: 2 and 8 and the
known sequences of sialyl transferase (Sasaki et al., J. Biol.
Chem. 269: 15950-15956 (1994)) and literature cited therein),
the region of approx.0 amino acid 141 to approximately amino
acid 300 of SEQ ID NOS: 2 and 8 are similar to sialyl motifs
of known monosialyl transferases. Accordingly, an
oligonucleotide molecule or a nucleic acid molecule which is
to hybridize specifically with the PST nucleic acid molecule
will at least in its essential part be complementary to
sequences in the other regions of SEQ ID NOS: 1, or 7
especially in the coding region of nucleotides 301 to 720 and
CA 0220829l l997-06-l9
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48
1201 to 1377 of SEQ ID NO: 1, 213-632 and 1113-1289 of SEQ ID
NO: 7. Especially preferred are oligonucleotides which bind
to the PST gene at or in the vicinity of the start codon or
the promoter region. Standard hybridization conditions are
described in Sambrook et al., supra.
A preferred oligonucleotide molecule interferes in a
sequence-specific manner with processes such as the
translation of PST mRNA into the protein by binding to PST
mRNA. Further oligonucleotide molecules which are suitable
for use are oligo- nucleotide molecules which are
complementary to genomic DNA which can interact in forming a
triple helical structure (M. Cooney et al., Science 241: 456
(1988) and Duvall-Valentine et al., Proc. Natl. Acad. Sci. USA
89: 50~-508 (1992)). Formation of a triple helix prevents
expression of the gene normally encoded by the double helix.
Preferred oligonucleotide molecules include
oligonucleotide derivatives, such as phosphotriester,
methylphosphonates, phosphorothioates or substituted
oligonucleotides, such as acridine, interchalating coupled
oligonucleotides, or ~-anomers or $-anomers (J.J. Toulmé and
C. Hélène, Gene 72: 51-58 (1988)). Phosphorothioates and
methylphosphonates are specifically preferred. Such oligo
derivatives can be synthesized according to the state of the
art, for example by automated technology (S. Beaucage and M.
Caruthers, Tetrahedron Lett. 37: 3556 (1981); G. Zon and T.
Geiser; Anticanc. Drug Des. 6: 539 (1991); C.A. Stein et al.,
Pharmacol. Ter. 52: 365 (1991); P. Miller. Biotechnology 9:
358 (1991)). Especially preferred are phosphothioates and
methylphosphonates, because they are resistant against serum
and intracellular nucleases. Further useful antisense
oligonucleotides which are nuclease-resistant are described in
P.S. Miller et al., Nucleosides and Nucleotides 6: 769-776
t1985).
It is, in principle, possible to use "naked'l antisense
oligonucleotide molecules according to the invention, because
CA 0220829l l997-06-l9
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49
these oligonucleotides can be taken up by cells
non-specifically. However, such oligonucleotides are
integrated in the cells at a low efficiency. Therefore, in
general, a delivery system for antisense oligos is preferred.
5Another aspect of the invention is a soluble complex for
targeting an antisense oligonucleotide molecule according to
the invention which hybridizes in a manner which is speci~ic
for nucleic acid molecules encoding m~mm~lian polysialyl
transferase with, e.g., a part or all of SEQ ID NO: 1, or SEQ
10ID NO: 7 complexed with an oligonucleotide molecule binding
agent, which complexes the oligonucleotide molecule under
extracellular conditions and releases said oligonucleotide
molecule under intracellular conditions as an oligonucleotide
molecule speci~ically bindable to polysialyl transferase
15encoding nucleic acid molecule.
Such delivery systems are well-known in the state of the
art. For example, oligonucleotides are covalently coupled to
polycations, such as polylysine (M. Lemaitre et al., Proc.
Natl. Acad. Sci. USA 84: 648-652 (1387)). Further delivery
20systems using polycation conjugates (e.g. transferrin) are
described in WO 92/20316, USP 5,166,320, WO 92/19281, WO
92/13570, EP-A 0 388 758, WO 93/07283, WO 92/17210, WO
91/17773, WO 93/04701 and transfer peptides as specified in
PCT/EP94/01147 are also suitable.
25The efficacy of the internalization of the nucleic acid
molecules according to the invention in cells can be improved
by binding the nucleic acid molecule to amphiphilic molecules,
such as polyethylene glycol in a complex. Further preferred
are the use of transfection reagents, such as DOTMA (WO
3091/06309 and WO 91/17424). Liposomes and dendromers are also
useful.
In order to attain cell specificity, the nucleic acid
molecules according to the invention can be coupled
non-covalently to conjugates from a DNA-binding substance
CA 0220829l l997-06-l9
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(e.g. polycations) and/or a cell-specific ligand (e.g.
protein, preferably PSA specific antibody) (Wu, et al. J.
Biol. Chem. 4429-4432 (1987); Wu et al. (1988)). Further,
internalization of the nucleic acid molecules in cells is
accomplished by means of a soluble DNA carrier system
consisting of a chemically synthesized conjugate comprising
mannose and lactose as the ligands (P. Midoux et al., Nucl.
Acids Res. 21: 871-878 (1993)). EP-A O 388 758 discloses
chemically synthesized transferrin polycation conjugates which
form complexes with polyanionic nucleic acid molecules. By
means of the binding to the transferrin receptor these
complexes can be internalized in the target cells.
It is also known to use conjugates of polylysine and
asialoglycoprotein (Wu et al., J. Biol. Chem. 263: 14621-14624
(1988)) or with a galactose ligand (Plank et al., Bioconjugate
Chem. 3: 533-539 (1992)) in the complexes. As ligands there
were also employed inactivated adenoviruses (Cotten et al.,
Proc. Natl. Acad. Sci. USA 89: 6094-6098 (1992); Wagner et
al., Proc. Natl. Acad. Sci. USA 89: 6099-6103 (1992)) or
hemagglutinin infusion peptides (Wagner et al., Proc. Natl.
Acad. Sci. USA 89: 7934-7938 (1992)). WO 93/07283 also
describes, with regard to non-viral gene transfer, a
"2-ligand-system" comprising a DNA-binding (polycationic)
portion (substance with affinity towards nucleic acid
molecules) and an internalization factor for the take-up of
DNA in the cell. To release the complexes from the endosomes
into cytoplasm, there may be added to these complexes, as
described in WO 93/07283, a socalled endosomolytic agent which
corresponds, for example, to a virus or a virus component
(e.g. adenovirus or influenza hemagglutinin).
If the carrier for the oligonucleotide molecule is a
conjugate from a cell-specific ligand and a DNA binding
substance, these substances are preferred to be covalently
linked and the linkage typically is a peptide bond. This can
be formed, for example, with a water-soluble carbodiimide as
CA 02208291 1997-06-19
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described by G. Jung et al., Biochem. Biophys. Res. Comm. 101:
599-606 (1981). An alternative linkage is a disulfide bond.
A cell-specific agent, which can be a natural or
synthetic ligand (for example, a protein polypeptide,
glycoprotein, etc.) or it can be an antibody or an analogue
thereof which specifically binds to a cellular surface
structure which then mediates internalization of the bound
complex can be, and is preferably used in these systems. Such
antibodies are described in Frosch et al., Proc. Natl. Acad.
Sci. USA 82: 1194-1198 (1985) and in Molenaar, et al., Canc.
Res. 50: 1102-1106 (1990). The antibodies described in
Molenaar recognize epitopes on all NCAM isoforms. After
binding, these antibodies are internalized.
Typically, the cell-specific binding agent is a ligand
which binds to a cell-surface receptor. Preferably receptors
are employed which are specific for such tissue cells from
which the tumor to be treated originates.
It is specifically preferred to use the antisense oligos
for the treatment of tumor diseases involving a high
metastasis potential (small cell lung carcinoma,
medulloblastoma, Wilms'tumor and lymphoid tumor. See Kern, et
al., Leukemia and Lymphoma 12: 1-10 (1993)). Tumor antigens
that are suitable for use as surface receptors are, for
instance, the tumor antigens.
It is further preferred that the employed cell-specific
ligand acts as a substance which facilitates the
internalization of the oligonucleotide molecules according to
the invention. Such internalization factors are, for example,
transferrin or anti-CD4-antibody.
r 30 The optimal ratio of the cell-specific binding agent to
the oligonucleotide molecule and the oligonucleotide binding
agent in the complexes can be determined empirically. When
polycations are used, the molar ratio of the components will
CA 02208291 1997-06-l9
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vary depending on the size of the polycation and the size of
the oligonucleotide. To form the complex, the oligonucleotide
molecules and carriers are mixed and incubated under
conditions conductive to complexation. For example, the
oligonucleotide molecule and carrier can be mixed at the
appropriate ratio in 2 mol/l NaCl and the solution can be
diluted to 0.15 mol/l and filtered to provide an
administerable composition.
The oligonucleotide molecules or the molecular complexes
of this invention can be administered parenterally.
Preferably, it is injected intravenously. The complex is
administered in solution in a physiologically acceptable
vehicle.
A further object of the invention therefore is the use of
a nucleic acid molecule or oligonucleotide molecule or complex
according to the invention, for the production of a
therapeutic agent for the treatment of tumor therapy,
especially for prevention or inhibition of metastasis tumors.
Accordingly, a further aspect of the invention provides
methods of retarding or preventing tumor metastasis comprising
inhibition of PST activity within the tumor cells. Preferred
methods of retarding or preventing tumor metastasis comprise
introducing into tumor cells oligonucleotides that
speci~ically bind to all or a portion(s) of nucleic acid
encoding proteins having psm activity, most preferably
oligonucleotides complementary to all or portions of the
sequences set forth in SEQ ID NOS: 1 or 7. Preferably, the
tumor chosen is melanoma, small cell lung cancer, or lymphoma.
Other tumors which are neuro- ectodermal in nature which
express inappropriate levels of PST may also be so treated.
In another aspect, the invention involves methods of
modulating cellular interactions and adhesion comprising
recombinant expression of exogenous PST in appropriate host
cells expressing adhesion proteins, e.g., N-CAM. For this
CA 02208291 1997-06-19
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purpose, it is preferably to utilize nucleic acids comprising
or consisting of PST coding sequences (i.e., nucleotides 301-
1377 in SEQ ID NO: l or nucleotides 213-1289 in SEQ ID NO: 7).
The PST-encoding nucleic acid can be introduced into the cells
through transfection of the cells with PST-encoding DNA or
injection of the cells with PST mRNA. In a particular
embodiment, such methods include methods of promoting neurite
outgrowth on nerve cells and/or substrate cells on which nerve
cells are grown. Accordingly, in a further aspect, the
invention involves methods of promoting neuroregeneration
comprising transplantation of recombinant PST-expressing
neuroepithelial cells into tissues exhibiting
neurodegeneration.
In accordance with yet another embodiment of the
invention, there are provided antibodies generated against the
PST proteins disclosed herein. Such antibodies can be
employed for studying PST tissue localization, structure of
functional domains, purification of PST as well as in
diagnostic and therapeutic applications and the like.
Preferably, for therapeutic applications, the antibodies
employed will be monoclonal antibodies.
The antibodies can be prepared employing standard
techniques, as are well known to those of skill in the art,
using the invention PST proteins or portions thereof as
antigens for antibody production. Both anti-peptide and anti-
fusion protein antibodies can be used [see, for example,
~urrent Protocols in Molecular Biologv (Ausubel et al. eds.)
John Wiley and Sons, N.Y. (1989)]. Factors to consider in
selecting portions of the PST protein for use as immunogen (as
either a synthetic peptide or a recombinantly produced
bacterial fusion protein) include antigenicity, accessibility
(i.e., cytoplasmic domains), uniqueness, etc.
The availability of PST-specific antibodies makes
possible the application of the technique of
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54
immunohistochemistry to monitor the distribution and
expression density of the PST protein. In addition PST-
specific antibodies can be employed in methods of treating as
indicated supra.
Due to the fact that the occurrence of minor errors in
the sequencing of DNA sequences cannot be ruled out, it is
pointed out that in the event of any discrepancy between the
sequence contained in the vector pME7/PST-l and the sequence
stated in SEQ ID NO: 1, the sequence accessible, to the person
skilled in the art, from pME7/PST-l should be resorted to.
Other features of the invention will be clear to the skilled
artisan an-d are not presented here.
The terms and expressions which have been employed are
used as _erms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described
or portions thereof, it being recognized that various
modifications are possible within the scope of the invention.
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SE~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Gerardy-Schahn, Rita; Fukuda, Minoru;
Nakayama, Jun; Eckhardt, Matthias;
(ii) TITLE OF INVENTION: Isolated Polysialyl Trans~erases,
Nucleic Acid Molecules Coding There~or, Methods o~
Production and Us e
(iii NUMBER OF SEQUENCES:
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Felfe & Lynch
(B) STREET: 805 Third Avenue
(C) CITY: New York City
(D) STATE: New York
(E) COUNTRY: USA
(F) ZIP: 10022
(v) COM~U'l~:~ READABLE FORM:
(A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage
(B) COMPUTER: IBM PSi2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/EP94/04289
(B) FILING DATE: 22-DEC-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NUMBER: 30,946
(C) REFERENCE/DOCKET NUMBER: BOER 1050
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200
(B) TELEFAX: (212) 838-3884
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56
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 202 6 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:301..1377
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GCGACCGCCA CCTCCAATGC ACAAGGTGTC ACATTTGAAA AGAAACCTGA GCCCGGGGGA 60
GAAGGCGCTG AGAGACCCTG GCCTGGCTAG TGCAAACTGC GCGGCAGGGC GCTGGGCAGC 120
CTGGAGAACC CAGAGAGCTC CACCGCAGAC CATCCTAGCG ACCTGATTCT GGGATCTCGG 180
CTCCACGCTC CCTTCGCAAT TTTCAGATTT CTCTCCCTCG ATTATTTCCC CAAAACGGAA 240
CCTTTATACC AAGAGAAGGT GCCGGAGCTG GGGCAACCAG GACTTTCCCG GGCACCCAAG 300
ATG CGC TCC ATT AGA AAA CGG TGG ACC ATC TGC ACT ATA AGT CTA CTT 348
Me~ Arg Ser Ile Arg Lys Arg Trp Thr Ile Cys Thr Ile Ser Leu Leu
1 5 10 15
CTG ATC TTT TAT AAG ACA AAA GAG ATA GCC AGA ACT GAG GAG CAC CAA 396
Leu Ile Phe Tyr Lys Thr Lys Glu Ile Ala Arg Thr Glu Glu His Gln
GAG ACG CAA CTC ATC GGA GAT GGT GAA TTG TGT TTG AGC AGA TCA CTT 444
Glu Thr Gln Leu Ile Gly Asp Gly Glu Leu Cys Leu Ser Arg Ser Leu
GTC AAC AGC TCT GAT AAA ATC ATT CGG AAG GCT GGC TCA ACC ATC TTC 492
Val Asn Ser Ser Asp Lys Ile Ile Arg Lys Ala Gly Ser Thr Ile Phe
CAA CAT TCT GTA CAA GGC TGG AGA ATC AAT TCT TCT TTA GTC CTG GAG 540
Gln His Ser Val Gln Gly Trp Arg Ile Asn Ser Ser Leu Val Leu Glu
ATA CGG AAG AAC ATT CTC CGT TTC TTA GAT GCT GAA CGT GAT GTC TCT 588
Ile Arg Lys Asn Ile Leu Arg Phe Leu Asp Ala Glu Arg Asp Val Ser
GTG GTC AAG AGC AGC TTC AAG CCT GGT GAT GTC ATC CAC TAT GTG TTG 636
Val Val Lys Ser Ser Phe Lys Pro Gly Asp Val Ile His Tyr Val Leu
100 105 '110
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GAC AGA CGC CGG ACG CTA AAT ATT TCC CAT GAT CTG CAC AGC CTC CTG 684
Asp Arg Arg Arg Thr Leu Asn Ile Ser His Asp Leu His Ser Leu Leu
115 120 125
CCT GAA GTT TCA CCA ATG AAA AAC CGC AGG TTT AAG ACC TGT GCT GTT 732
Pro Glu Val Ser Pro Met Lys Asn Arg Arg Phe Lys Thr Cys Ala Val
130 135 140
GTT GGA AAC TCT GGC ATT CTA CTA GAC AGT GGA TGT GGC AAG GAG ATT 780
Val Gly Asn Ser Gly Ile Leu Leu Asp Ser Gly Cys Gly Lys Glu Ile
145 150 155 160
GAC AGT CAC AAT TTT GTA ATC AGG TGC AAT CTA GCT CCT GTG GTG GAG 828
Asp Ser His Asn Phe Val Ile Arg Cys Asn Leu Ala Pro Val Val Glu
165 170 175
TTT GCT GCG GAT GTG GGG ACT AAA TCA GAT TTT ATT ACC ATG AAC CCA 876
Phe Ala Ala Asp Val Gly Thr Lys Ser Asp Phe Ile Thr Met Asn Pro
180 185 190
TCA GTT GTG CAG AGA GCA TTT GGA GGC TTT CGG AAT GAG AGT GAC AGA 924
Ser Val Val Gln Arg Ala Phe Gly Gly Phe Arg Asn Glu Ser Asp Arg
195 200 2G5
GCA AAA TTT GTG CAT AGA CTT TCC ATG CTG AAT GAC AGT GTC CTT TGG 972
Ala Lys Phe Val His Arg Leu Ser Met Leu Asn Asp Ser Val Leu Trp
210 215 220
ATC CCC GCT TTC ATG GTC AAA GGA GGA GAG AAG CAC GTG GAA TGG GTT 1020
Ile Pro Ala Phe Met Val Lys Gly Gly Glu Lys His Val Glu Trp Val
225 230 235 240
AAT GCA TTA ATC CTT AAG AAC AAG CTG AAA GTG CGA ACT GCC TAT CCA 1068
Asn Ala Leu Ile Leu Lys Asn Lys Leu Lys Val Arg Thr Ala Tyr Pro
245 250 255
TCA CTG AGA CTT ATT CAT GCT GTC AGA GGT TAC TGG CTG ACC AAC AAA 1116
Ser Leu Arg Leu Ile His Ala Val Arg Gly Tyr Trp Leu Thr Asn Lys
260 265 270
GTG CCC ATC AAA AGA CCC AGC ACA GGC CTC CTC ATG TAC ACA CTG GCC 1164
- Val Pro Ile Lys Arg Pro Ser Thr Gly Leu Leu Met Tyr Thr Leu Ala
275 280 285
ACC AGA TTT TGT GAT GAA ATT CAC CTG TAT GGG TTC TGG CCC TTC CCT 1212
Thr Arg Phe Cys Asp Glu Ile His Leu Tyr Gly Phe Trp Pro Phe Pro
290 295 300
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58
AAG GAT TTG AAT GGA AAA GCT GTG AAA TAT CAT TAC TAC GAT GAC TTG 1260
Lys Asp Leu Asn Gly Lys Ala Val Lys Tyr His Tyr Tyr Asp Asp Leu
305 310 315 320
AAA TAT AGA TAC TTT TCC AAC GCA AGC CCT CAC AGA ATG CCA TTA GAA 1308
Lys Tyr Arg Tyr Phe Ser Asn Ala Ser Pro His Arg Met Pro Leu Glu
325 330 335
TTC AAA ACC CTG AAT GTG CTA CAC AAC AGA GGA GCA CTA AAA CTG ACC 1356
Phe Lys Thr Leu Asn Val Leu His Asn Arg Gly Ala Leu Lys Leu Thr
340 345 350
ACA GGG AAG TGC ATG AAG CAA TAAAGCACAT ATTGAAGGAT CAAAACTGGA 1407
Thr Gly Lys Cys Met Lys Gln
355
TAGAAACTTT TTCTAAAGAT GCTTCTGGAG ATTTAGAAAC AGGATCCAAA ACAAGGCTGG 1467
GGTTCAGCAT CCACACTGAC TGAATAGCTG AAATGGAAGT CCATGGGAAT CCACCACCAG 1527
CTGATGAAAT ACCTGCCAAG TGCTCTAACT ATAAAATATT CTGACTTCAA GGGTCCTAGT 1587
AAGTGCCACT TCCACGAAGA ATACAGTTTG AATGTATTAT CAGTAGTGTT TACAAGATCC 1647
AACAGTGCAC TCATCATTAA TTAGCAAAGC AAATATGTTC GTCACTGTGG GGCAGCCGCT 1707
GTAATGCCAA GCACACTGGA AGAGGAACTC AGGAGCATCA CGACTCGGAG CTTGGGAAAT 1767
TAACATCCTT ATCCGCAGAA ATGAAGAAGA AAAAGAATTC AAACAGTGAA ATCCATGAGA 1827
TGAAGTAACT TGAAGGAATG TCTTCAGTCA GGACACTGAG AGTGATCATG TGTGTGTTTT 1887
GCTTGTGTTT TTGTTTGTCT TCTGAAACTT GTTTTCTTTT GGGTATGGGG TGAATAGAAA 1947
TTCATCTGAG GTACAGAAAT GGGAAATACA TGACAGAGAA AAATAAACAT CAAACAGTCA 2007
2}~AP}~ P}~AP}~ 2026
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Arg Ser Ile Arg Lys Arg Trp Thr Ile Cys Thr Ile Ser Leu Leu
1 5 10 15
Leu Ile Phe Tyr Lys Thr Lys Glu Ile Ala Arg Thr Glu Glu His Gln
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59
Glu Thr Gln Leu Ile Gly Asp Gly Glu Leu Cys Leu Ser Arg Ser Leu
Val Asn Ser Ser Asp Lys Ile Ile Arg Lys Ala Gly Ser Thr Ile Phe
Gln His Ser Val Gln Gly Trp Arg Ile Asn Ser Ser Leu Val Leu Glu
Ile Arg Lys Asn Ile Leu Arg Phe Leu Asp Ala Glu Arg Asp Val Ser
Val Val Lys Ser Ser Phe Lys Pro Gly Asp Val Ile His Tyr Val Leu
100 105 110
Asp Arg Arg Arg Thr Leu Asn Ile Ser His Asp Leu His Ser Leu Leu
115 120 125
Pro Glu Val Ser Pro Met Lys Asn Arg Arg Phe Lys Thr Cys Ala Val
130 135 140
Val Gly Asn Ser Gly Ile Leu Leu Asp Ser Gly Cys Gly Lys Glu Ile
145 150 155 160
Asp Ser His Asn Phe Val Ile Arg Cys Asn Leu Ala Pro Val Val Glu
165 170 175
Phe Ala Ala Asp Val Gly Thr Lys Ser Asp Phe Ile Thr Met Asn Pro
180 185 190
Ser Val Val Gln Arg Ala Phe Gly Gly Phe Arg Asn Glu Ser Asp Arg
195 200 205
Ala Lys Phe Val His Arg Leu Ser Met Leu Asn Asp Ser Val Leu Trp
210 215 220
Ile Pro Ala Phe Met Val Lys Gly Gly Glu Lys His Val Glu Trp Val
225 230 235 240
Asn Ala Leu Ile Leu Lys Asn Lys Leu Lys Val Arg Thr Ala Tyr Pro
245 250 255
Ser Leu Arg Leu Ile His Ala Val Arg Gly Tyr Trp Leu Thr Asn Lys
260 265 270
Val Pro Ile Lys Arg Pro Ser Thr Gly Leu Leu Met Tyr Thr Leu Ala
- 275 280 285
Thr Arg Phe Cys Asp Glu I 1 e Hi s Leu Tyr Gly Phe Trp Pro Phe Pro
- 290 295 300
Lys Asp Leu Asn Gly Lys Ala Val Lys Tyr His Tyr Tyr Asp Asp Leu
305 310 315 320
Lys Tyr Arg Tyr Phe Ser Asn Ala Ser Pro His Arg Met Pro Leu Glu
325 330 335
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Phe Lys Thr Leu Asn Val Leu His Asn Arg Gly Ala Leu Lys Leu Thr
Thr Gly Lys Cys Met Lys Gln
355
