Use of ULIP proteins in the diagnosis and therapy of cancers and paraneoplastic neurological syndromes
The invention relates to the use of proteins designated ULIP/POP in the diagnosis and therapy of cancers and paraneoplastic neurological syndromes.
This application is a continuation of U.S. application Ser. No. 09/367,496, having a filing date of Nov. 24, 1999, which is a 371 of PCT/FR98/00328, filed Feb. 19, 1998.
The invention relates to the use of proteins designated ULIP/POP in the diagnosis and therapy of cancers and paraneoplastic neurological syndromes.
Paraneoplastic neurological syndromes (PNS) occur in the instance of a cancer, often before its discovery, and are not connected either to the tumour proliferation itself (direct invasion, metastases) or to the therapy. Their frequency is globally estimated at approximately 1% of cancers. Several clinical pictures have been individualized for a long time (encephalomyelitis, Denny-Brown's sensitive neuropathy, cerebellar atrophy, limbic encephalitis, opsoclonus, . . . ) corresponding in fact to the either elective or preferential attack of certain groups of neurons. The frequency of inflammatory cells in the neighbourhood of the lesions for numerous years brought to mind the possibility of an auto-immune or viral process. The more recent demonstration of auto-antibodies in the serum and the cerebrospinal fluid (CSF) of patients suffering from PNS, specific to the type of tumour and the type of neurons which degenerate, has revived the hypothesis of participation of auto-immunity in the genesis of this pathology (Graus et al., 1985; Greenlee et al., 1983).
Apart from the presence of a high titre of these antibodies in the blood and the CSF of patients, there are several arguments suggesting that PNS depend on auto-immune mechanisms. Thus the antigens recognized in the central nervous system are also present in the tumours of patients (Anderson et al., 1987). At the level of the tumour tissue, antibodies specifically directed against these antigens as well as B and T lymphocytes are found (Hetzel et al., 1990).
These data suggest that the auto-immune process could be triggered by the expression of tumour antigens. A cross-immunity process could provoke the lesions of the central nervous system. Other arguments additionally indicate that the cerebral lesions result from the auto-immune response. Thus, in the brain of the patients, the titre of specific antibodies is higher than that of the serum and the CSF (Dalmau et al., 1991). In addition, in the case of encephalomyelitis associated with anti-Hu antibodies, there is an intense lymphocytic reaction, made up of B and T cells, situated in proximity to neurons in the process of destruction (Dalmau et al., 1991; Graus et al., 1990).
Several types of auto-antibodies allowing precise syndromic groupings as a function of immunological, neurological and carcinogenic criteria have been described.
Thus, anti-Yo antibodies are found in the serum and the CSF of women having paraneoplastic cerebellar atrophy and a gynaecological cancer (ovary, breast or uterus) (Greenlee et al., 1983; Jaeckle et al., 1985).
These antibodies recognize two cytoplasmic proteins of 34 and 62 kDa specific to Purkinje cells of the cerebellum.
The anti-Ri antibodies are found in the serum and the CSF of patients (principally of women) having opso-myoclonus, cerebellar syndrome and breast cancer. These antibodies recognize two proteins of 50 and 80 kDa specific to neurons of the central nervous system (Luque et al., 1991).
Anti-Hu antibodies are most frequently found in the course of PNS. They are found in the serum and the CSF of patients having Denny-Brown's syndrome or encephalomyeloneuritis and small-cell lung cancer (Graus et al., 1985; Dalmau et al., 1992). These auto-antibodies recognize several proteins of 37 to 45 kDa expressed specifically by all the neurons of the nervous system.
Another type of auto-antibody has recently been identified in patients having PNS: anti-CV2 antibodies (Antoine et al., 1993; Honnorat et al., 1996). The latter are atypical, in the sense that the antigenic target recognized in adulthood is essentially non-neuronal, although the post-mortem analysis of the brain of four patients allows neuronal loss, gliosis and an inflammatory process characteristic of PNS to be objectivized.
The originality of the discovery of these auto-antibodies resides, on the one hand, in their demonstration. The latter escaped all the usual investigations which consisted in revealing the antigens recognized by immunohistochemistry on post-mortem brain. The antigen recognized is indeed soluble and disappears from post-mortem brain under the majority of fixation conditions. Only fixation of human post-mortem tissue by immersion in paraformaldehyde or in situ by perfusion of paraformaldehyde in animals has allowed the presence of these antibodies in the CSF or the serum of patients suffering from PNS to be revealed (Antoine et al., 1993; Honnorat et al., 1996).
The anti-CV2 auto-antibodies present in the sera of patients suffering from paraneoplastic neurological syndrome (PNS) have been defined by their capacity to recognize, by indirect immunohistochemistry, a cytoplasmic antigen expressed specifically, in adult rat brain, by a subpopulation of oligodendrocytes of the brain stem, the medulla and the cerebellum.
The originality of these auto-antibodies resides, on the other hand, in their diagnostic interest. Their presence in the serum or the CSF of patients is of diagnostic value because it allows the paraneoplastic origin of a neurological syndrome to be specified. The discovery of these antibodies, when it precedes that of cancer, directs the search to that and allows its discovery. Such was the case for six patients out of 19 having anti-CV2 antibodies. The clinical disorders were different according to the patients, certain of them having a picture of limbic encephalitis, others encephalomyeloneuritis and others Lambert-Eaton syndrome. Nevertheless, in more than 60% of the cases, the cerebellar syndrome was predominant. The most frequently associated tumour was small-cell lung cancer (60% of the cases).
Experiments on the brains of newborn rats showed that these anti-CV2 antibodies reacted with a protein of 66 kDa (Honnorat et al., 1996).
In the adult brain, this antigen is situated in a subpopulation of oligodendrocytes or in cells which retain differentiation capacities in the adult brain (olfactory bulb, dentate gyrus). The recognized antigen could play a role in neuronal survival, via Neuron/Oligodendrocyte interactions, as the loss of neurons observed in the post-mortem brain of patients suffering from PNS suggests.
Its very limited expression in adulthood contrasts with a very strong and transitory expression in the central and peripheral nervous system in development, suggesting the probable role of this antigen in the development of the nervous system.
The Applicant has characterized the target antigen of anti-CV2 antibodies, which corresponds to a protein designated below by “POP-66” for “paraneoplastic oligodendrocyte protein 66 kDa”.
Surprisingly, it has been discovered that the POP-66 protein belongs to the so-called ULIP family of proteins (for Unc-33-like phosphoprotein), involved in the control of neuronal development and axonal transport (T. Byk et al., 1996), and also studied in the form of CRMP proteins (Goshima et al., 1995, Wang et al., 1996), TOAD-64 (Minturn et al., 1995) and DRPs (Hamajima et al., 1996). More precisely, POP-66 has been identified as in fact being the human form of ULIP-4.
All of the data described below emphasize the complexity of this family of proteins, the existence of a very wide expression spectrum of members of this family in the brain in the course of ontogenesis, but a very limited spectrum in adults, as well as the specificity of the anti-CV2 antibodies for a member of this ULIP protein family, which is in fact POP-66.
Thus, the Applicant has shown that the protein recognized by the anti-CV2 antibodies of patients suffering from PNS is POP-66/ULIP-4 and has established the involvement of the ULIP proteins in paraneoplastic neurological syndromes and associated cancers. In addition to their role in cancers associated with PNS, the Applicant has likewise discovered that the proteins of the ULIP family could play a role in any other form of cancer not associated with PNS. More particularly, the ULIP proteins could especially be involved in cancers of tissues having a common embryonic origin with the central nervous system.
The present invention therefore relates to a purified ULIP polypeptide, derivative or polypeptide fragment of the said purified polypeptide, comprising an amino acid sequence selected from SEQ ID No. 2, No. 4, No. 6 and No. 8.
Preferentially, the present invention relates to a purified polypeptide, derivative, or biologically active polypeptide fragment of the said purified polypeptide, comprising the amino acid sequence SEQ ID No. 8, the said polypeptide being designated by “POP-66/ULIP-4”.
A fragment of the polypeptide of sequence SEQ ID No. 8 of interest is, in particular, the antigenic fragment PARASCPGKIS (amino acids No. 517 to No. 527).
In the description of the invention, the following definitions are used:
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- derivative: any variant polypeptide of the polypeptide of sequence SEQ No. 2, No. 4, No. 6 or No. 8 or any other molecule resulting from a modification of genetic and/or chemical nature of the sequence SEQ ID No. 2, No. 4, No. 6 or No. 8, that is to say obtained by mutation, deletion, addition, substitution and/or chemical modification of a single or of a limited number of amino acids, as well as any isoform sequence, that is to say a sequence identical to the sequence SEQ ID No. 2, No. 4, No. 6 or No. 8, to one of its fragments or modified sequences, containing one or more amino acids in the D enantiomer form, the said modified or isoform variant sequences having conserved at least one of the properties making them biologically active.
- biologically active: having properties of induction and/or control of neuronal development and/or antigenic properties.
The invention likewise relates to an isolated nucleic acid sequence selected from SEQ ID No. 1, No. 3, No. 5 and No. 7 or a sequence derived from the sequences SEQ ID No. 1, No. 3, No. 5 and No. 7 on account of the degeneracy of the genetic code, or on account of mutation, of deletion or of insertion of at least one nucleotide, the said derived sequences having a biological activity virtually identical to that of the peptide encoded by the sequences SEQ ID No. 1, No. 3, No. 5 and No. 7.
The various nucleotide sequences of the invention can be of artificial or non-artificial origin. They can be DNA or RNA sequences, obtained by screening of banks of sequences by means of probes elaborated on the basis of sequences selected from SEQ ID No. 2, No. 4, No. 6 and No. 8. Such banks can be prepared by conventional techniques of molecular biology known to the person skilled in the art.
The nucleotide sequences according to the invention can likewise be prepared by chemical synthesis, or alternatively by mixed methods including the chemical or enzymatic modification of sequences obtained by screening of banks.
These nucleotide sequences allow the production of nucleotide probes capable of hybridizing strongly and specifically with a nucleic acid sequence of a genomic DNA or of a messenger RNA coding for a peptide according to the invention or a biologically active fragment of this. The appropriate hybridization conditions correspond to the conditions of temperature and of ionic strength usually used by the person skilled in the art (Sambrook et al., 1989), preferably to temperature conditions of between (Tm minus 5° C.) and Tm minus 30° C.) and more preferably to temperature conditions of between (Tm minus 5° C.) and (Tm minus 10° C.) (great stringency), Tm being the theoretical melting point, defined as being the temperature at which 50% of the paired strands separate. Such probes are likewise part of the invention. They can be used as a diagnostic tool in vitro for the detection, by hybridization experiments, of specific transcripts of polypeptides of the invention in biological samples or for the demonstration of aberrant syntheses or genetic anomalies resulting from polymorphism, mutations or bad splicing.
The probes of the invention contain at least 10 nucleotides, and at most contain the whole of a nucleotide sequence selected from SEQ ID No. 1, No. 3, No. 5 and No. 7 or of their complementary strand.
The in vitro diagnostic methods in which these nucleotide probes are employed for the detection of aberrant syntheses or genetic anomalies, such as the loss of heterozygosity and genetic rearrangement, at the level of nucleic sequences coding for a ULIP polypeptide according to the invention or a biologically active fragment are included in the present invention. Such a method type comprises:
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- the contacting of a nucleotide probe of the invention with a biological sample under conditions allowing the formation of a hybridization complex between the said probe and the abovementioned nucleotide sequence, optimally after a previous amplification step of the abovementioned nucleotide sequence;
- the detection of the hybridization complex optimally formed;
- optimally the sequencing of the nucleotide sequence forming the hybridization complex with the probe of the invention.
The cDNA probes of the invention can additionally be advantageously used for the detection of chromosomal anomalies.
The nucleotide sequences according to the invention are likewise useful for the production and use of sense and/or antisense oligonucleotide primers for sequencing reactions or specific amplification reactions according to the so-called PCR technique (polymerization chain reaction) or any other variant of this.
The nucleotide sequences according to the invention additionally have uses in the therapeutic field, for the production of antisense sequences capable of hybridizing specifically with a nucleic acid sequence, including a messenger RNA, which can be used in gene therapy. The invention thus relates to antisense sequences capable of inhibiting, at least partially, the production of a polypeptide according to the invention, such as defined above.
They are more particularly useful in the treatment of disorders of the central and peripheral nervous system and of vision, especially in the treatment of paraneoplastic neurological syndromes, as well as in anti-cancer treatment, especially of tumours associated with paraneoplastic neurological syndromes.
The nucleotide sequences according to the invention can additionally be used for the production of recombinant ULIP proteins according to the invention.
These proteins can be produced from nucleotide sequences defined above, according to techniques of production of recombinant products known to the person skilled in the art. In this case, the nucleotide sequence used is placed under the control of signals allowing its expression in a cell host.
An efficacious system of production of a recombinant protein necessitates having a vector, for example of plasmid or viral origin, and a compatible host cell.
The cell host can be selected from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect cells, CHO (Chinese hamster ovary) cells or any other system advantageously available. A preferred cell host for the expression of proteins of the invention is formed by the bacterium E. coli.
The vector must contain a promoter, translation initiation and termination signals, as well as the appropriate regions of transcription regulation. It must be able to be maintained stably in the cell and can possibly possess special signals specifying the secretion of the translated protein.
These different control signals are selected as a function of the cell host used. To this end, the nucleotide sequences according to the invention can be inserted in autonomous replication vectors within the selected host, or integrative vectors of the selected host. Such vectors will be prepared according to methods currently used by the person skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as, for example, electroporation.
The invention is additionally directed at the host cells transfected by these above vectors. These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector such as defined above, then the culturing of the said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence.
These cells can be used in a method of production of a recombinant polypeptide according to the invention or any fragment or biologically active derivative of this.
The method of production of a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transfected cells are cultured under conditions allowing the expression of a recombinant polypeptide according to the invention or of any fragment or biologically active derivative of this, and in that the said recombinant polypeptide is recovered.
The purification processes used are known to the person skilled in the art. The recombinant polypeptide can be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used separately or in combination, such as fractionation, chromatographic methods, immunoaffinity techniques with the aid of specific mono- or polyclonal antibodies, etc.
One variant consists in producing a recombinant polypeptide fused to a “carrier” protein (chimeric protein). The advantage of this system is that it allows a stabilization and a decrease in the proteolysis of the recombinant product, an increase in the solubility in the course of the in vitro renaturation and/or a simplification of the purification when the fusion component has an affinity for a specific ligand.
The exploitation of ULIP proteins, and in particular POP-66/ULIP-4, as well as antibodies directed against these proteins, is promising in various fields.
Thus, the detection of the anti-CV2 auto-antibody by immunofluorescence on fixed animal brain is currently used as a diagnostic test.
The production of POP-66/ULIP-4 recombinant protein according to the invention allows the production of a rapid and reliable test (of Elisa or Western Blot type) for detecting anti-CV2 antibodies.
Such tests already exist for anti-Hu, anti-Yo and anti-Ri antibodies. The test for detecting anti-CV2 in the serum of patients could be prescribed in the case of suspicion of paraneoplastic neurological syndrome and consequently could include anti-CV2 antibodies at the same titre as the other antibodies identified in the PNS such as mentioned above.
The invention is therefore likewise directed at a method for the diagnosis of paraneoplastic neurological syndromes and/or for the early diagnosis of the formation of tumours of cancerous origin, characterized in that auto-antibodies directed against a POP-66/ULIP-4 protein are demonstrated in a blood sample taken from an individual by
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- the contacting of a blood sample taken from an individual with a purified polypeptide (POP-66), derivative or biologically active polypeptide fragment of POP-66/ULIP-4 optionally attached to a support under conditions allowing the formation of specific immunological complexes between the said polypeptide and the auto-antibodies optionally present in the serum sample, and
- the detection of the specific immunological complexes optionally formed.
The invention likewise relates to a kit for the diagnosis of paraneoplastic neurological syndromes and for the early diagnosis of the formation of tumours from a biological sample, comprising:
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- at least one purified POP-66/ULIP-4 polypeptide, derivative or biologically active polypeptide fragment of POP-66/ULIP-4, optionally attached to a support,
- means of visualization of the formation of specific antigen/antibody complexes between an anti-POP-66 auto-antibody and the said purified POP-66 polypeptide, derivative or polypeptide fragment and/or means of quantification of these complexes.
The invention likewise relates to mono- or polyclonal antibodies or their fragments, chimeric or immunoconjugated antibodies obtained from a purified ULIP polypeptide comprising an amino acid sequence selected from SEQ ID No. 2, No. 4, No. 6 and No. 8, derivative or biologically active polypeptide fragment of ULIP and their use for the purification or the detection of a ULIP protein in a biological sample.
Polyclonal antibodies can be obtained from the serum of an animal immunized against the protein, produced, for example, by genetic recombination according to the method described above, according to the usual working methods.
The monoclonal antibodies can be obtained according to the conventional method of hybridoma culture described by Köhler and Milstein.
The antibodies can be chimeric antibodies, humanized antibodies, Fab and F(ab′)2 fragments. They can likewise be present in the form of immunoconjugates or labeled antibodies.
The invention likewise relates to the use of antibodies directed against a protein of the ULIP family for the demonstration of a ULIP protein in neoplasms, and paraneoplastic neurological syndromes for diagnostic purposes.
Preferentially, the invention relates to the use of monoclonal antibodies obtained from polyclonal anti-CV2 serum of patients by immortalization of lymphocytes, according to the usual techniques known to the person skilled in the art.
Thus, the antibodies directed against a protein of the ULIP family are useful for detecting abnormal expression of ULIP protein in patients having neurological syndromes, in whom cancer has not been diagnosed by the conventional methods. This abnormal expression of ULIP protein will be able to be correlated with the existence of a cancer which had not been spotted. Thus, the antibodies directed against a ULIP protein, especially against POP-66/ULIP-4, are useful for the early diagnosis of cancer.
The invention likewise relates to a method of determination of an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic anomaly of the POP-66/ULIP-4 gene, situated on chromosome 10 in the 26q region and which can be involved in pathologies, characterized in that it employs at least one nucleotide sequence SEQ ID No. 7. Amongst the methods of determination of an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic anomaly of the POP-66/ULIP-4 gene, a method comprising at least one PCR amplification step of the nucleic sequence of POP-66/ULIP-4 capable of having a polymorphism, a mutation, a deletion or an insertion with the aid of pairs of primers of nucleotide sequences, a step in the course of which amplified products are treated with the aid of appropriate restriction enzymes and a step in the course of which at least one of the products of the enzymatic reaction is detected or determined is preferred.
Advantageously, it is possible to search for the mutations associated with the said chromosome 10 in relation to cancer, especially peripheral cancerous tumours and primitive cerebral tumours of glial origin, for example.
The invention likewise relates to a pharmaceutical composition comprising at least one purified protein of the ULIP family, polypeptide fragment or biologically active derivative of this, a nucleotide sequence or nucleotide sequence fragment coding for the said protein, an antisense sequence capable of hybridizing specifically with a nucleotide sequence coding for the said protein, or an antibody directed against the said protein, combined with a pharmaceutically acceptable vehicle.
The invention preferentially comprises pharmaceutical compositions comprising as active principle a purified POP-66 polypeptide, derivative or polypeptide fragment of POP-66, preferentially in soluble form, combined with a pharmaceutically acceptable vehicle.
Such compositions offer a new approach to treating disorders of the central and peripheral nervous system and of vision, and especially paraneoplastic neurological syndromes. In addition, they are useful for treating neurological disorders connected with a neuronal loss and/or an underexpression of ULIP proteins in the nervous system.
Thus, POP-66/ULIP-4 is also of interest in neurodegenerative pathologies such as multisystemic atrophies which are conditions similar to those of PNS and for which an anomaly of an oligodendrocytic subpopulation has been detected (Papp et al., 1992).
The compositions according to the invention are additionally useful in anticancer therapy.
The antibodies directed against one or more ULIP proteins can be combined with antineoplastic agents, thus allowing the targeting of medicaments towards the tumour cells.
They can additionally be combined with a hydrophilic chemical group chosen in such a way so as to cross or not to cross the blood-brain barrier, according to the type of tumour.
The ULIP proteins and in particular POP-66 as well as the nucleotide sequences coding for the said proteins and the antisense sequences or oligonucleotides can be useful in the therapy of any type of cancer in which a gene coding for a ULIP protein is involved. Amongst examples of cancers, it is possible to mention peripheral tumours, such as small-cell lung cancer, thymoma, cancer of the breast and of the ovary, as well as cerebral tumours, preferably primitive cerebral tumours of glial origin. The expression of POP-66 in the non-proliferative cells of normal brain, its absence in normal tissues such as lung or thymus, for example, its differential reexpression during tumorigenesis of these tissues and the modulation of its expression in a tumour line in the course of differentiation suggest in this respect that POP-66 could be a tumour suppressor gene.
Preferentially, the pharmaceutical compositions according to the invention can be administered by the systemic route, preferably by the intravenous route, by the intramuscular route, intradermally or by the oral route.
Their modes of administration, dosages and optimal pharmaceutical forms can be determined according to the criteria generally taken into account in the establishment of a therapeutic treatment adapted to a patient, such as, for example, the age or the body weight of the patient, the seriousness of his/her general condition, the tolerance to the treatment and the secondary effects noted, etc.
The invention likewise comprises the use of a purified protein of the ULIP family, polypeptide fragment or biologically active derivative of this, a nucleotide sequence or nucleotide sequence fragment coding for the said protein, an antisense sequence capable of hybridizing specifically with a nucleotide sequence coding for the said protein, or an antibody directed against the said protein, combined with a pharmaceutically acceptable vehicle, for the production of a medicament intended for treating neurodegenerative illnesses and neoplasms.
The invention finally relates to a method of treatment of neurodegenerative illnesses and neoplasms, comprising the administration to a subject requiring such a treatment of a therapeutically efficacious quantity of a purified protein of the ULIP family, polypeptide fragment or biologically active derivative of this, a nucleotide sequence or nucleotide sequence fragment coding for the said protein, an antisense sequence capable of hybridizing specifically with a nucleotide sequence coding for the said protein, or an antibody directed against the said protein, combined with a pharmaceutically acceptable vehicle.
The examples and the figures whose legends are presented below are given by way of illustration.
LEGEND TO THE FIGURES
A: silver staining of all of the proteins.
B: immunoblot with the anti-CV2 serum of patients.
The arrows indicate the spots corresponding to POP-66, revealed with anti-CV2 antibodies.
Immunoblot with A-antipeptide antibody 3 and B-anti-CV2 antibody.
Immunoblot with a: preimmune serum for peptide 3
Immunoblot with b: anti-peptide serum 3
Immunoblot with c: anti-peptide serum 4
Immunoblot with d: preimmune serum for peptide 4.
A: anti-CV2 serum of a patient suffering from PNS
B: rabbit serum with anti-peptide 3 antibodies
C: rabbit serum with anti-peptide 4 antibodies.
A: Staining with toluidine blue; ge=external granular layer; m=molecular layer (×400).
B: Immunolabelling after incorporation of BrdU (bromodeoxyuridine). The cells which have incorporated BrdU are virtually all situated in the most external zone of the external granular layer (ge). Some positive cells are situated in the internal granular layer (×400).
C: Indirect immunoperoxidase with a patient serum containing an anti-CV2 antibody (×400). The immunoreactivity is concentrated in the internal part of the external granular layer (future molecular layer (m)). Some cells are immunoreactive in the internal granular layer. The Purkinje cells (p) are negative as well as the cells of the external part of the external granular layer (ge).
D: Indirect immunoperoxidase with a patient serum containing an anti-CV2 antibody (×1000). Above all, the immunolabelling is concentrated in the internal part of the external granular layer (future molecular layer (m)). A reactive cell is noted in the internal granular layer (gi) (arrow).
A: brain of control patient,
B: brain of patient having limbic encephalitis, and circulating anti-CV2 antibody. It is possible to note the disappearance of the granular cells.
The proteins are revealed:
a) by autoradiography to locate the proteins translated in vitro (translation);
b) by immunoblotting with the anti-CV2 serum.
lanes 1-3: small-cell lung tumour
lane 2: small-cell lung tumour with anti-CV2 serum
lane 4: control cDNA.
lane 5: medulloblastoma treated by HTLV1 infection
lanes 6-7: medulloblastoma
lane 8: C6 line of glial cells in mice
lane 9: control
lane 10: nothing
lane 11: kb scale.
The black arrows correspond to POP-66; the white arrows correspond to the molecular weight standard.
An erroneous stop codon in the human ULIP-4 sequence (asterisk) arises from a fault of the reverse transcriptase in the production of the bank. By comparing with ULIP-4 of rats and of mice, it is almost certain that the TAG sequence coding for a stop is in fact an AAG codon, coding for a lysine as in rats and mice. In addition, the region around this amino acid is entirely conserved in the three species.
The amino acid sequence has been completed in SEQ ID No. 8 by 15 C-terminal amino acids (No. 554 to No. 568). This C-terminal region which is missing in
The purification of POP-66 is carried out according to the material and the methods described in the article of Honnorat et al., 1996, incorporated by reference, starting from serum of patients suffering from PNS.
To identify the protein POP-66, a purification strategy was chosen which allows a partial sequencing to be obtained. The screening of an expression bank of brain cDNA or the immunoaffinity purification of the protein were excluded because of the limited quantities of sera linked to the death of the patients. It was possible to develop a method of biochemical purification starting from brains of newborn rats on account of the anti-CV2 human sera, which allowed each purification step to be monitored.
The tissues, preserved at −70° C. before use, were treated with a solution containing 0.2 M DTT (dithiothreitol) (Sigma) 2% Ampholine 3-10 (Pharmacia), 2% Triton X-100 (Merck) and placed at 2-4° C. Immediately before use, solid urea (Pharmacia) was added to obtain an 8M solution.
The POP-66 protein is soluble, at least in part, and precipitates entirely at a concentration of 40% ammonium sulphate.
Centrifugation at 100,000 (times) g and ammonium sulphate precipitation (eliminating the proteins precipitating below 20% and above 40% ammonium sulphate) allows protein extracts enriched in POP-66 to be obtained. The proteins of this extract are then separated, after dialysis, by isofocusing on agarose gel (Peltre et al., 1982).
After transfer to a membrane, the anti-CV2 antibodies recognize several bands of isoelectric points of between 5.85 and 6.55. All of these bands correspond to the POP-66 protein recognized by the anti-CV2 antibodies. This spectrum suggests the possibility of transcriptional modifications (phosphorylations and/or glycosylations) of the protein.
The zone of proteins of pI between 5.85 and 6.55 from the agarose gel is used for a new electrophoretic migration in denaturating medium on polyacrylamide gel previously equilibrated with an equilibration solution (0.05 mol/1 Tris/HCl, pH 6.8, 6M urea, 30% glycerol, 1% weight/volume SDS for 2×10 minutes) to which is added DTT (0.25% weight/volume) and bromophenol blue.
Two methods of detection are used:
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- silver staining. Immediately after the end of the migration, the gel is immersed in a fixing solution (40% ethanol, 10% acetic acid) for 30 minutes; it is then placed in an incubation solution (30% ethanol, 7% weight/volume of sodium acetate, 0.1% glutaraldehyde, 0.2% weight/volume of sodium thiosulphate) for 30 minutes or one night. After washing, the gel is placed in a silver solution (0.1% weight/volume of silver nitrate+formaldehyde) and developed (2.5% weight/volume of sodium carbonate +formaldehyde). The reaction is stopped with Na2 EDTA (1.5% weight/volume). The gels are preserved in a glycerol solution.
- transfer to a PVDF membrane (Immobilon-P®, Millipore). The separated proteins are transferred to a PVDF membrane using a 100 mM CAPS buffer (Sigma) of pH 11. The transfers are incubated for one hour in TBS buffer (Tris buffer saline) with 5% of casein (milk) and 18 hours in TBS buffer (+1% of casein) containing antibody ( 1/500 anti-CV2 serum). After washing with TBS-casein (15 minutes), visualization is carried out by incubating the transfers for 1 and a half hours with biotinylated anti-IgG antibodies ( 1/1000) and for 1 and a half hours with the streptavidin-peroxidase complex ( 1/2000). The transfers are then visualized with DAB (0.06% weight/volume diaminobenzidine in 0.05 M Tris) and with H2O2 (0.02 μg/ml).
A single band corresponding to a protein of 66 kDa is visible. This is specifically labeled with anti-CV2 antibodies (
Seven peptides, having the following sequences, were obtained:
- X: corresponds to a non-determined amino acid,
- (?): corresponds to a probable but uncertain amino acid.
According to the analysis of databanks available in 1994, no known protein corresponded to these sequences.
EXAMPLE 2 Cloning of the cDNA of POP-66 or of Related ProteinsThe cloning of the cDNA of the POP-66 protein or of related proteins was carried out by using degenerate oligonucleotide probes obtained from fragments of two peptides:
- Ile-Ile-Ala-Glu-Glu-Gln
- Tyr-Ala-Asp-Ile-Tyr-Met-Glu-(Asp ?)
Four sets of degenerate oligonucleotide primers (sense/antisense) are therefore determined
- (AT(C/T)ATTGC(T/A)GA(A/G)CA;TG(C/T)TC(T/C)AC(T/A)GCAT(A/G)AT;
- TATGC(A/T)GA(C/T)AT(C/T)ATGGA; TCCAT(G/A)TA(G/A)CT(T/A)GCATA, and used for a PCR amplification.
The matrix is prepared in the form of double-stranded cDNA (Promega kit) from poly(A+)RNA extracted from the brain of rats 10 days old (Zivic-Miller, USA) using the Fast Track mRNA isolation kit (Invitrogen).
The conditions of amplification by PCR are as follows: 35 cycles at 94° C., 1 minute for denaturation, 55° C., 1 minute for hybridization and 72° C., two minutes, for extension.
The PCR products are analysed by 1% agarose gel electrophoresis, electroeluted, cloned in a TA cloning vector (Invitrogen) and sequenced using the primer sites of the T7 and SP6 promoters.
The sequence of amino acids inferred from the MFB-17 clone agrees with the sequences of the two original peptides of POP-66 determined by the analysis of the amino acid sequence.
A comparative analysis of the nucleic acid sequences using the Genbank and EMBL databases reveals that MFB-17 is a partial cDNA with a nucleotide sequence identical to that of a segment of TOAD-64, a rat neuronal protein (Minturn et al., 1995).
The amino acid sequence inferred in the cDNA of TOAD-64 agrees with the sequences of the seven peptides determined by partial sequence analysis of the protein recognized by the anti-CV2 antibodies after purification by electrophoresis.
The molecular weight, the isoelectric point, the immunohistochemical profile and the regulation of TOAD-64 are similar to those of the POP-66 antigen.
Since the MFB-17 clone did not have the complete coding region, it was necessary to produce an intact recombinant protein to continue the research concerning the CV2 protein.
To obtain a complete TOAD-64 protein, the ds-cDNA matrix of rat brains was amplified with two sets of primers situated at the 5′ and 3′ extremities of the coding regions
This approach allowed two different clones to be produced, one corresponding to the TOAD-64 sequence and the other to a clone designated by C-22.
EXAMPLE 3 Comparison of the Amino Acid Sequence Inferred From C-22 with the ULIP ProteinsThe amino acid sequence inferred from the open reading frame indicates that this C-22 clone belongs to the superfamily of ULIP genes represented by several genes of EST sequences.
The amino acid sequence inferred from C-22 has a homology of 30% with the amino acid sequence of the unc-33 protein of Caenorhabditis elegans.
Recently, four different homologous genes in the unc-33 protein have been described in mammals and the chicken.
An analysis of the sequences by the Genbank databases and protein banks has allowed a classification of the unc-33-like (ULIP) proteins into four different subgroups to be proposed (Byk et al. 1996).
However, as the real functions of these proteins are not clearly known, the proposed classification is simply based on the percentage of identity of amino acids. ULIP-1 is represented by a mouse “unc-33-like” phosphoprotein which has a homology of 76% with TOAD-64, Crmp-62 and Munc, a mouse sequence recently available from Genbank.
ULIP-2 is composed of TOAD-64, Crmp-62 and Munc which between them have a 97% amino acid identity.
The partial human EST sequences, that is to say hcrmp-1, which have a 75% identity with ULIP-1 or ULIP-2, have been found. They belong to a third group called ULIP-3. The last group identified called ULIP-4 comprises r-CRMP-3 in the rat and the forms ULIP-4 in the mouse and POP-66/ULIP-4 in man.
The comparison of the amino acid sequence of the three ULIP genes, namely TOAD-64 in the rat, Crmp-62 in the chicken and ULIP-1 in the mouse, with the amino acid sequence inferred from the open reading frame of the present C-22 clone, using the Clustal V alignment software program reveals that C-22 has an identity of 74% with ULIP-1, 77% with Crmp-62 and 76% with TOAD-64.
The nucleotide sequence C-22 has an identity of 97% with the partial sequence EST, hCrmp-1, and thus defines the third member of the ULIP-3 group. The TOAD-64, Crmp-62 and C-22 genes each code for a protein of 572 amino acids in length, whereas the amino acid sequence inferred from ULIP-1 gives a protein of 570 amino acids.
The analysis of the amino acid sequence of C-22 does not show any signal sequence or transmembrane domain suggesting that the product(s) of the C-22 gene could be localized in the cytoplasm of the cells.
Several consensus sites of phosphorylation by the kinase C protein (S/T X R/K) appear along the length of the product of the C-22 gene. These observations suggest that C-22 is a phosphoprotein and that the slight differences in the phosphorylation could dictate the activity or the role of different members of this family of proteins throughout the cell cycle.
The evaluation of alterations in the expression of the C-22 gene could have considerable significance for the knowledge of the functional aspects of the C-22 protein.
Consequently, the Applicant studied the possible regulation of the expression of the C-22 gene in the course of development. The total RNA is extracted and separated by electrophoresis on 1% agarose gel and transferred to Nytran membrane (Duchemin et al. 1987). The transfers are hybridized with a C-22 coding sequence labelled with 32P, a 0.5 mM phosphate buffer and 5% SDS at 65° C. for 16 hours.
At the end of the hybridization, the transfers are washed successively three times with 2×SSC, 0.1% SDS at ambient temperature, then 1×SSC, 0.1% SDS at 65° C. for 60 minutes, and exposed to X-rays.
Under the conditions used, a single band at 3.8 kb was detected representing the C-22 mRNA which is also the smallest transcript of the unc-33 family of genes of vertebrates. The size of the transcript remains the same during the pre- and post-natal periods.
The kinetics of the C-22 gene in the brain of rats in the course of development shows that the messenger is detectable in the course of the embryonic period on day E17. The quantity of C-22 transcripts increases up to day 7 post-natally then decreases rapidly from the second week after birth to a virtually undetectable level in the adult.
Around birth, a still unknown regulation signal is probably received, which increases the expression of the C-22 gene, this signal being temporarily linked to neuronal differentiation and to axonal development.
The mRNA of C-22 has not been able to be detected by Northern Blot analysis in several regions of the brain such as the frontal cortex, the midbrain and the thalamus in adults and rats more than two years old.
In addition, it has not been possible to detect the mRNA of C-22 in non-neuronal tissues, such as the heart, the lung, the liver and the kidney in one-week old rats and adult rats.
The data on the expression profile of the mRNA of C-22 suggests a decisive role of the C-22 protein in the development of the brain.
EXAMPLE 5 Immunoblotting of POP-66A—Materials and Methods
Transfection of the ULIPs in E. coli
The full-length cDNAs of rat ULIP-2 and ULIP-3 and mouse ULIP-1 and ULIP-4 were subcloned directionally in the E. coli pET-21a(+) expression vector after introduction of a 5′ Nde I site and of a 3′ EcoRI site by PCR, and the four constructs were resequenced. The expression of the target gene induced by IPTG was carried out according to the protocol of the manufacturer (Novagen).
Production of Anti-ULIP Antibodies
Rabbit antibodies (anti-Pep3) are directed against the peptide ITGPEGHVLSRPEEVE (amino acids 217-232 of the sequence SEQ ID No. 8), synthesized in a multiple peptide synthesis apparatus using F-moc (432A Peptide Synthesizer SYNERGY, Applied Biosystems). The purity was checked by analysis of the sequence by HPLC and mass spectrometry. 1 mg of the synthetic peptide conjugated to limpet haemocyanin, in complete Freund's adjuvant, was used to immunize rabbits with a booster dose of 0.5 mg of bound peptide in complete Freund's adjuvant after 4 weeks. The anti-Pep3 antibodies recognized the four recombinant ULIP proteins expressed in E. coli.
Labelling with the anti-Pep3 antibodies was removed after preincubation with peptide 3. Controls with pre-immune rabbit sera were negative.
Anti-peptide 4 antibodies directed against the peptide LEDGTLHVTEGS were produced according to the same protocol.
B—Results
Antibodies against four of the sequenced peptides were products. Two of the sera turned out to be of particular interest.
One contains antibodies (Ab anti-pep3) which recognize several members of the ULIP family on two-dimensional electrophoresis of protein extracts of newborn rat brain (
Immunohistochemistry
The tissue preparations for immunohistochemistry are obtained from newborn rat brains and from post-mortem human brains, fixed at 4° C. in 4% paraformaldehyde and 0.2% picric acid diluted in phosphate buffer (0.1 M, pH=7.4), then cryoprotected.
Immunocytochemistry can be carried out by the indirect immunofluorescence technique. Sections of 12 μm in thickness are prepared in a cryostat and then mounted on gelatin-covered slides, treated with 0.1% Triton X100 for 2 hours in PBS buffer and 1% bovine serum albumin (BSA) and incubated for 12 h with anti-CV2 serum of patients in PBS/1% BSA at ambient temperature ( 1/100 dilution of the serum). After several washes with PBS/1% BSA, the sections are incubated for 2 h with a rabbit anti-human antiserum conjugated to fluoroscein, diluted to 1% (Dakopatts) in PSB/1% BSA. After washing in PBS, the slides are examined under the microscope. The control sections are incubated either with anti-human IgG antiserum conjugated to fluoroscein alone, or PBS/1% BSA alone, or the patient serum alone, or finally the control serum (patients not suffering from PNS) and antibodies conjugated to fluoroescein at the same dilution.
To confirm the immunofluorescence, it is possible to use indirect labelling by immunoperoxidase. The frozen tissue sections fixed with paraformaldehyde are incubated with 0.3% H2O2 (to destroy the intrinsic peroxidase activity) and 10% normal rabbit serum (to avoid the non-specific binding of the rabbit IgG) or 1% BSA. After incubation for 12 h with patient sera diluted to 1/1000 and washing, the sections are incubated for 2 h with biotinylated rabbit anti-human IgG antiserum diluted to 1/1000 in PBS/1% BSA. The bound human IgGs are visualized by incubation with an avidin-biotin-peroxidase complex (Vectastain ABC complex, Vector) and developed with 0.05% DAB (Sigma). The control sections are obtained with sera of 15 patients without PNS according to the same protocol.
A—Localization of Proteins of the ULIP Family With the Aid of Antipeptide Antibodies:
Immunohistochemical labelling was carried out on sections of newborn and adult rat brains. The antipeptide-3 antibody recognizes (an) antigen(s) present in several cell types on sections of newborn and adult rat brains (
B—Expression of POP-66 in the Course of the Normal Development of the Brain:
Materials and Methods
a) Partial Purification of ULIP-1
Partially purified ULIP-1 was obtained from newborn mouse brains by three purification steps. These brains were homogenized in 4 volumes of homogenization buffer (25 mM sodium phosphate, pH 7.8, 1 mM EGTA, 10 μg/ml of leupeptin, 25 μg/ml of aprotinin, and 10 μg/ml of pepstatin. The homogenates were centrifuged for 10 minutes at 400× g. The plugs were resuspended in 2 volumes of homogenization buffer, homogenized and centrifuged again. The supernatants from two centrifugations were collected, sonicated and centrifuged for 1 hour at 100,000×g. The supernatant (S2) was loaded onto a column of DEAE-Sepharose CL-6B (1.75 cm2×26 cm) equilibrated with 100 ml of buffer A (25 mM sodium phosphate, pH 7.8, 1 mM EGTA) at a flow of 30 ml per hour. The proteins were eluted in 300 ml of a 0-250 mM linear gradient of sodium chloride in buffer A and 5 ml samples were collected. The fractions containing ULIP were collected and solid ammonium sulphate was added to 20% saturation. This pool was loaded onto a column of phenyl-Sepharose CL-4B (1.75 cm2×22 cm) which had been previously equilibrated with 100 ml of buffer B (10 mM sodium phosphate, pH 7.8, 1 mM EGTA) containing 20% of saturated ammonium sulphate. The proteins were eluted in a linear gradient decreasing from 20 to 0% of saturated ammonium sulphate in buffer B. The fractions containing ULIP were collected and dialysed twice against 20 volumes of buffer A. The proteins were concentrated in a small (10 ml) column of DEAE-Sepharose CL-6B and eluted with 400 mM sodium chloride in buffer A. The eluate was desalted on a Sephadex G-25 (NAP-10) column and concentrated to a final volume of 0.5 ml by evaporation. In the last purification step, the concentrated fraction was chromatographed in three successive steps, on two Superose 12 FPLC (Fast Protein Liquid Chromatography) columns mounted in series, in buffer C (50 mM sodium phosphate, pH 7.2, 150 mM sodium chloride) at a rate of 0.3 ml/minute. The fractions (0.6 ml) were collected and the fractions enriched in ULIP were analysed. The presence of ULIP in the successive purification steps was tested by a one-dimensional Western Blot using an anti-stathmin antibody capable of cross-reactivity. The proteins were quantified according to the method of Bradford.
b) Migration on Electrophoresis Gel:
A one-dimensional electrophoresis was carried out on 13% polyacrylamide gels according to the method of Laemmli. The two-dimensional PAGE electrophoreses were carried out as described above. The isoelectro-focusing gels contained 2% of total ampholines, pH 6-8 and 3-10 in a ratio of 4:1. The second dimension had been carried out on 10% acrylamide gels. The proteins had been either subjected to immunoblotting or stained with silver.
c) Western Blot Analysis:
The proteins were transferred from gels to nitrocellulose in buffer containing 48 mM Tris, 39 mM glycine and 5% of methanol. The membrane was saturated with casein (2.5%) in the immunoblotting solution (12 mM Tris-HCl, pH 7.4, 160 mM NaCl, 0.1% Triton X-100) and tested with an antiserum directed against the peptide I of rat stathmin ( 1/10,000 dilution) or an antiserum directed against the recombinant ULIP protein (dilution 1/20,000) diluted in an immunoblotting solution containing 1% of casein. The bound antibodies were detected either with a protein A labelled with 125I, and autoradiographed or with anti-rabbit antibodies bound to peroxidase using the ECL kit (Amersham).
d) Analysis of the Protein Sequence:
The fractions enriched in ULIP were separated on polyacrylamide gels in two dimensions. The gels are fixed for 30 minutes in 25% ethanol and 10% acetic acid and stained for 3 minutes in 0.1% amido black in 1% acetic acid and 40% methanol. The gels were decolourized in 1% acetic acid and the spots corresponding to the principal form of ULIP were cut out in these three gels, collected and digested with 2 mg/ml of Lys C endoprotease. The peptides eluted from the gel were then separated by HPLC on a DEAE-C18 column with a gradient of 0-55% of acetonitrile in 0.1% trifluoro-acetic acid. The peptides were then sequenced according to the Edman automatic degradation.
e) Expression In Vitro in a Mammal
1 μg of the Bluescript plasmid containing the entire cDNA coding for ULIP-1, ULIP-2, ULIP-3 or ULIP-4 was used to carry out the transcription and translation in vitro with the “Reticulocyte lysate” system (Promega) according to the protocol described by the manufacturer. 5 μg of the total transcription/translation mixture of 25 μl were analysed on electrophoresis gel in two dimensions.
Results
Neither the recombinant protein ULIP-1, nor the recombinant proteins TOAD-64 (ULIP-2) and C-22 (ULIP-3) were recognized by the anti-CV2 sera. In addition, the distribution profile of the spots corresponding to POP-66 recognized by the anti-CV2 antibodies on electrophoresis in two dimensions does not correspond to the spots recognized by the anti-ULIP-1 antibodies. However, POP-66 is a member of the ULIP family since the three POP-66 spots are recognized by the anti-pep3 Ab. POP-66 therefore corresponds to a member of the family of more basic pHi.
After translation in vitro of the four proteins (ULIP-1, 2, 3 and 4), it was shown that ULIP-4 has the same 2D electrophoretic profile as POP-66 and is recognized by the anti-CV2 antibodies (
For this, the ULIP-4 protein and, as control, the ULIP-2 protein were translated in vitro in the presence of 35S methionine from cDNA clones coding for the entire proteins. The proteins were separated by two-dimensional electrophoresis (in the presence of a brain extract providing the essential references), transferred to nitrocellulose and visualized:
-
- by autoradiography to localize the proteins translated in vitro (translation);
- by immunoblotting with the CV2 serum.
The CV2 serum therefore specifically recognizes ULIP-4.
This allowed POP-66 to be identified like ULIP-4.
EXAMPLE 9 Chromosomal Localization of the POP-66/ULIP-4 ProteinHaving cloned the cDNA of human ULIP-4, it is then possible to determine the chromosomal localization of the POP-66/ULIP-4 gene by genetic mapping by in situ isotopic hybridization (Levy and Mattei et al., 1995).
In situ hybridization is carried out on preparations of chromosomes obtained from human lymphocytes stimulated by phytohaemagglutinin cultured for 72 hours. 5-Bromodeoxyuridine was added during the last 7 hours of culture (60 μg/ml of medium) to ensure a post-hybridization image of chromosome bands of good quality. The clone containing an insert of 1300 base pairs coding for ULIP-4 in the Bluescript vector is labelled with tritium by nick translation with a specific activity of 1×108 dpm. μg−1 The radiolabelled probe was hybridized in the metaphase stage at a final concentration of 200 ng per ml of hybridization solution. After covering with a Kodak NTB2 emulsion, the slides were exposed for 20 days at +4° C. and then developed. To avoid the shifting of the silver grains during the process, the chromosome blots were previously labelled with a Giemsa buffer solution and the metaphases were photographed. The visualization of the bands was carried out by the “Giemsa fluorochrome photolysis” (FPG) method and the metaphases were rephotographed before analysis. Of the 100 cells in metaphase examined after hybridization in situ, 246 silver grains associated with the chromosomes were counted and 54 amongst these (21.9%) were localized on chromosome 10. The distribution of the grains on this chromosome was not random: 39 out of 54 (72.2% of the latter) were localized on the q25.2-q26 region of the long arm of chromosome 10.
The POP-66/ULIP-4 gene is therefore found to be situated on chromosome 10 in the q25.2-q26 region. The loci of neurodegenerative diseases and of suppressor genes of tumours involved in different types of cancer have been localized in this chromosome region. The locus of a brain disease of early origin (infantile onset spinocerebellar ataxia) was identified in the 10q24-26 region (Varilo et al., 1996; Nikali et al., 1995). The symptoms of this recessive hereditary degenerative disease which is characterized by ataxia, neuropathy and visual atrophy are similar to those observed in patients suffering from paraneoplastic neurological syndromes with circulating anti-CV2 auto-antibodies (Honnorat et al., 1996). On the other hand, 80% of glioblastomas have mutations in this chromosome region and several suppressor loci involved in different types of tumours (prostate, kidney, small-cell lung cancer and endometrial carcinomas) are localized in this chromosome region. These data support the possibility that POP-66/ULIP-4 plays a crucial role in neurodegeneration and tumorigenesis.
In this respect, it is notable that the expression of ULIP-1 is regulated upwards in neuroblastoma cells differentiated by retinoic acid and that ULIP-1 and ULIP-3 are regulated upwards but ULIP-4 is regulated downwards in differentiated PC12 cells in the presence of NGF, suggesting that the stop in cell growth can be linked to expression levels of the ULIP proteins.
EXAMPLE 10Expression of ULIP Proteins in Transfected HeLa Cells
A—Materials and Methods
A flag (EcoRI-ATGGACTACAAGGACGACGATGACAAGG-BamHI) sequence (Kodak) was cloned in the EcoRI site of pSG5 followed by ULIP-1 (EMBEL X87817), base pairs: 309-2023), ULIP-2 (Y10339, base pairs: 23-1741), ULIP-3 (Y09080, base pairs: 269-1991) or ULIP-4 (Y09079, base pairs: 102-1820), respectively. The HeLa cells were cultured in DMEM media (Gibco) to which 10% of foetal calf serum (v/v) was added. The transfections were carried out by calcium phosphate precipitation (Maniatis et al., 1978). The HeLa cells were mixed with 5 μg of pSG5flag-ULIP-1, 2, 3 and 4 plasmids and 10 μg of pUC18. Twenty-four hours after the transfection, the HeLa cells were fixed with 4% paraformaldehyde and immunolabelled with different human sera (dilution 1/300), visualized by human anti-IgG antibodies conjugated to FITC (Biosys), or anti-flag antibodies (M2, Kodak) (dilution 1/1000), visualized by anti-rabbit antibodies conjugated to Texas red (Vector).
Double immunolabelling was carried out on the HeLa cells transfected with ULIPs using anti-flag and anti-Pep3 antibodies. In the cells transfected by any cDNA, 10 to 20% among them showed immunolabelling with the anti-flag antibodies visualized by the anti-mouse antibodies conjugated to Texas red.
All the transfected cells were doubly labelled by antibodies directed against Pep3 and a peptide common to the four ULIPs is visualized by rabbit anti-IgG antibodies conjugated to fluorescein.
Double immunolabelling was likewise carried out on HeLa cells transfected with ULIPs using anti-flag and anti-CV2 antibodies. The human sera of patients suffering from PNS with circulating anti-CV2 auto-antibodies labelled the cells transfected by ULIP-4, and an anti-CV2 serum likewise labelled the cells transfected by ULIP-3. No labelling of the cells transfected by ULIP-4 was detected in the control sera of patients without cancer or neurological disease.
B) Results
After transfection of HeLa cells with cDNAs labelled by the flags of ULIP-4, 10 to 20% of the cells were strongly reactive with anti-flag antibodies and anti-Pep3 antibodies which recognize the ULIP-4s of mammals. The transfected cells were not immunolabelled with control serum of 10 neurological patients without PNS nor with rabbit pre-immune serum. On the other hand, the cells transfected with small cDNA of ULIP-4 showed an intense immunoreactivity with all the 7 tested sera of patients with circulating anti-CV2 auto-antibodies. These sera are negative on cells transfected with cDNAs of other ULIPs, with the exception of a sera which also recognized the cells transfected with ULIP-3 and a serum which also recognized the cells transfected with ULIP-1, 3 and 4. No labelling was observed on non-transfected HeLa cells, with an anti-CV2 serum.
Table 1 below shows the results of indirect immunofluorescence with different sera on HeLa cells by labelled cDNAs of members of the ULIP family.
PCD: paraneoplastic cerebellar degeneration;
LE: lymbic encephalitis;
PEM: paraneoplastic encephalomyelitis;
UC: undifferentiated carcinoma;
SCLC: small-cell lung carcinoma.
A—Expression of ULIP-2 and ULIP-3 in Cancers:
1) Materials and Methods: RT-PCR Experiments:
The total RNA was extracted using 1 ml of RNAZOL™B (Bioprobe) according to the method of Chomczynski and Sacchi. The quantity of RNA was determined by optical density measured at 260 nm and its purity was determined from the ratio of the absorbances measured at 260 and 280 nm (ratios 1.8-2.0). The integrity of the RNA preparations was additionally checked by electrophoresis on 1% agarose gel in TBE (0.45 M Tris-borate, 10 mM EDTA, pH 8). The specificity of the primers was analyzed by comparing their sequences with the various gene databanks (EMBL and FASTA). For a relative quantification, the gene coding for G3PDH (glyceraldehyde-3-phosphate dehydrogenase, Clontech), a ubiquitous gene expressed in numerous tissues including the brain, was co-amplified with the mRNA tested as an internal standard to check the uniformity of the quantities of RNA in the samples and to test the efficacy of the reverse transcription step for different RNA samples. The primers 5′, 3′ and the oligonucleotides of internal probes of G3PDH were synthesized and purified by Eurogentec. The total mRNA (1 μg) was denatured (15 minutes at 65° C.) and transcribed in single-stranded cDNA (1 and a half hours, 42° C.) in a final volume of 20 μl of buffer (50 mM Tris HCl, 75 mM KCl, pH 8.3, Gibco BRL) containing 5 ng per μl of oligo-dT 12-18 primer (Pharmacia Biotech), 40 units of reverse transcriptase of the Moloney murine leukaemia virus (Mu-LV) (Gibco BRL), 40 units of RNAsine (Promega), 10 mM DTT (Gibco BRL) and 0.5 mM of each of the triphosphate deoxynucleotides (Promega). The cDNA samples were diluted to 1/10 in distilled water and the PCR reactions were carried out using 1 μl, 4 μl or 2 μl of cDNA sample for the messenger RNA of ULIP-2 and ULIP-3, in a buffer (50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X100, 0.4% glycerol and 800 μM NaCl, pH 9), to which was added 40 μmol of DTT, 3 mmol of MgCl2, 0.2 mmol of each dNTP, 0.4 μM of each selected primer and 2 units of AmpliTaq DNA polymerase (Promega) in a final volume of 50 μl). The samples were then placed in a thermocycler (Biomed-Hybaid), denatured at 95° C. for 5 minutes and amplified for 35 cycles (one cycle=95° C. denaturation for 65 seconds, 60° C. hybridization of the primers for 45 seconds, 72° C. extension for 4 minutes and 15 minutes final elongation at 72° C. The products were separated by electrophoresis on 1% Seakem agarose gel and the test bands of the RT-PCR products of expected size as well as the molecular weight marker scale (100 base pairs) (Promega) were visualized using ethidium bromide staining.
Composition of the oligonucleotide probes used for ULIP-3 PCR
Composition of the oligonucleotide probes used for ULIP-2 PCR
2) Results
The RT-PCR experiments show that TOAD-64 (ULIP-2) and C-22 (ULIP-3) are expressed in certain small-cell lung tumours (cf.
B—Expression of ULIP-4 in Cancers
1) Materials and Methods
Preparation of the RNA and RT-PCR
The total RNAs are extracted from cerebral tumours preserved in liquid nitrogen according to the conventional RNAZOL™ technique (Bioprobe, France). Reverse transcription was carried out using oligo(dt)18 on 1 μg of total RNA and the PCR was carried out with 1/20 of the volume of the mixture for the reverse transcription (RT mix). The primers used for ULIP-4 are:
- 5′CATCTGGCTGTCGCTGCAC3′, 5′GCCGCCCCTACCAGAGACC3′, and for GAPDH: 5′GGAGATTCAGTGTGGTGG3′, 5′GGCTCTCCAGAACATCATCC3′. The cDNA was denatured at 95° C. for five minutes. PCR amplification was carried out for 30 cycles. ULIP-4: 95° C., 45 sec; 62° C., 45 sec; 72° C., 45 sec. GAPDH: 95° C., 45 sec; 55° C., 45 sec; 72° C., 45 sec. The final extension was carried out at 72° C. for 5 minutes.
2) Results
Of the 8 glioblastoma extracts studied, 4 (50%) expressed the messenger RNA of ULIP-4. Conversely, of the 10 oligodendroglioma extracts tested, none expressed the messenger RNA of ULIP-4. This differential expression, as a function of the primitive cerebral tumour type, is in favour of a potential role of ULIP-4 in the cell proliferation of these tumours.
The protein POP-66/ULIP-4 as well as the proteins of the ULIP family could be expressed in the peripheral tumours (small-cell lung tumour, thymoma, cancer of the breast and of the ovary). Their presence could therefore be correlated with a prognosis. The localization of the POP-66/ULIP-4 gene on the distal part of chromosome 10 confirms this in the case of cerebral tumours.
Thus, the differential expression of members of the ULIP family in tumours such as small-cell lung cancer, although the corresponding ULIP gene is absent in a healthy tissue, as well as the modulation of the expression of members of the ULIP family obtained during differentiation by the HTLV1 human retrovirus of a medulloblastoma line, suggest the involvement of ULIPs in cancerous tumours.
EXAMPLE 12 Production of Specific Antibodies of Each of the Human ULIP ProteinsSpecific peptides of each member of the ULIP family were synthesized on a multiple peptide synthesis apparatus using F-moc (432A Peptide Synthesizer SYNERGY, Applied Biosystems). The purity was checked by sequence analysis by HPLC and mass spectrometry.
These peptides are:
- Specific peptide of ULIP-1: G S A R G S P T R P N (11 amino acids)
- Specific peptide of ULIP-2: S S A K T S P A K Q Q A (12 amino acids)
- Specific peptide of ULIP-3: P S A K S S P S K H Q (11 amino acids)
- Specific peptide of ULIP-4: P A R A S C P G K I S (11 amino acids).
1 mg of the synthetic peptide conjugated to limpet haemocyanin, in complete Freund's adjuvant, was used to immunize rabbits with a booster dose of 0.5 mg of bound peptide in complete Freund's adjuvant after 4 weeks.
The antibodies obtained specifically recognize each member protein of the ULIP family.
EXAMPLE 13 Production of Transgenic Animals Expressing ULIP-4Drosophila fruit flies were transformed by the cDNA of human ULIP-4.
The cDNA of ULIP-4, previously cloned in pbluescript SK-phagemid, was excised by Kpn1 and Xba1 enzymatic double digestion. After electrophoresis on agarose gel, the cDNA fragment was purified and then cloned in pUAST, originating from pCaSpeR3, digested by the restriction enzymes Kpn1 and Xba1. The 10-C plasmid results from the directional cloning of the cDNA of ULIP-4 in pUAST associated with the mini-white reporter gene. The 10-C plasmid was injected with a p-delta-2-3 helper plasmid coding for the transposase of the P element active in the germinal line.
The transformed fruit flies are identified by their red eyes resulting from the expression of the mini-white gene. These lines transformed by the cDNA of ULIP-4 under the control of UASGAL4 regulatory sequences allow a targeted expression of the cDNA of ULIP-4.
This production of transformed fruit flies allows the role of ULIP-4 to be studied specifically in different cells and its involvement in human pathologies to be understood.
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Claims
1. A purified ULIP polypeptide comprising the amino acid sequence of SEQ ID No. 8.
2. A composition comprising a purified polypeptide comprising amino acid sequence SEQ ID No. 8, or a purified fragment thereof, wherein the purified polypeptide or fragment binds to anti-CV2 antibodies.
3. A method for detecting the presence of anti-CV2 antibodies in a biological sample, comprising:
- contacting a biological sample with a purified ULIP polypeptide comprising SEQ ID No. 8, or a fragment thereof that binds to anti-CV2 antibodies; and
- detecting specific immunological complexes optionally formed, the specific immunological complexes being indicative of the presence of anti-CV2 antibodies.
4. A kit for detection of anti-CV2 antibodies in a biological sample, said kit comprising:
- at least one purified ULIP polypeptide comprising SEQ ID No. 8, or a fragment thereof that binds to anti-CV2 antibodies, said polypeptide or fragment optionally attached to a support, and
- means of visualization of the formation of specific antigen/antibody complexes between an anti-POP-66 auto-antibody and the purified ULIP polypeptide or fragment and/or means of quantification of these complexes.
5. A method of detecting anti-CV2 antibodies in a subject, said method comprising the steps of:
- contacting a sample from the subject with a purified polypeptide comprising SEQ ID No. 8, or a fragment thereof that binds to anti-CV2 antibodies, said contacting carried out under conditions sufficient to allow the formation of specific immunological complexes between the polypeptide or fragment thereof and anti-CV2 antibodies present within the sample; and
- detecting the specific immunological complexes formed;
- wherein the presence of immunological complexes is indicative of the presence of anti-CV2 antibodies in said subject.
6. The method of claim 5, wherein the polypeptide consists of SEQ ID No. 8.
7. The method of claim 5, comprising contacting the sample from the subject with a fragment of the purified polypeptide, said polypeptide consisting of amino acid sequence SEQ ID No. 8, wherein said fragment binds to anti-CV2 antibodies.
8. A reagent for identifying anti-CV2 antibodies to a polypeptide in a sample from a subject, said reagent comprising
- a purified peptide comprising a fragment of the polypeptide of claim 1 wherein the fragment binds to anti-CV2 antibodies, said fragment attached to a solid support.
9. A kit for identifying antibodies in a sample from a subject to a polypeptide comprising amino acid sequence of SEQ ID No. 8, said kit comprising a fragment of said polypeptide that binds to anti-CV2 antibodies.
10. The kit of claim 9, wherein the kit further comprises means of visualizing formation of complexes between said fragment and antibodies to the polypeptide comprising amino acid sequence of SEQ ID No. 8.
11. The kit of claim 9, wherein the fragment of said polypeptide is purified.
12. A reagent for identifying anti-CV2 antibodies in a sample from a subject, said reagent comprising:
- a purified polypeptide comprising amino acid sequence SEQ ID NO:8, said polypeptide attached to a solid support.
Type: Application
Filed: Dec 20, 2006
Publication Date: May 24, 2007
Inventors: Michele Aguera (Caluire), Marie-Francoise Belin (Lyon), Jerome Honnorat (Bron), Pappachan Kolattukudy (Colombus, OH), ThanhTam Quach (Lyon), Tamara Byk (Creteil), Andre Sobel (Paris), Dominique Aunis (Strasbourg), Jean-Christophe Antoine (Chalin Le Comtal)
Application Number: 11/641,878
International Classification: G01N 33/574 (20060101); C07K 14/705 (20060101);
