MXPA02007027A - Human cystine knot polypeptide - Google Patents
Human cystine knot polypeptideInfo
- Publication number
- MXPA02007027A MXPA02007027A MXPA/A/2002/007027A MXPA02007027A MXPA02007027A MX PA02007027 A MXPA02007027 A MX PA02007027A MX PA02007027 A MXPA02007027 A MX PA02007027A MX PA02007027 A MXPA02007027 A MX PA02007027A
- Authority
- MX
- Mexico
- Prior art keywords
- seq
- polypeptide
- sequence
- polynucleotide
- protein
- Prior art date
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 35
- 229960003067 Cystine Drugs 0.000 title claims abstract 3
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Abstract
The invention relates to newly identified DNA sequences which code for a novel cystine knot polypeptide as well as to the encoded protein. The invention is useful in the field of fertility.
Description
POLI PÉPTI DO DE NU DO D E C IST1 NA H U MANA
The invention relates to a polynucleotide that encodes a novel polypeptide, the protein encoded by that polynucleotide as well as a recombinant cell that expresses this protein. Follicle Stimulation Hormone (FSH), Hormone
Luteinizing (LH) and Thyroid Stimulation Hormone of the pituitary, and human chorionic gonadotropin (hCG)) of the placenta belong to the family of glycoprotein hormones. These hormones have a heterodimeric structure, and contain two non-covalently linked subunits and β. The amino acid sequence of the subunits is identical, while the β subunits differ and confer biological specificity on the individual gonadotropins (Ulloa-Aguirre, 1988, 1995). The dimers are found to be biologically active. Both subunits a and β are glycosylated and contain chains of carbohydrates linked in N. HCG contains four additional O-linked carbohydrates on the terminal peptide in C. FSH, LH and TSH are present in most of the vertebrate species and synthesized and secreted by the pituitary.
GC has been found only in primates, including nutrians, and in horses and is synthesized by the placental tissue. Within a species, the a subunit is essentially identical for each member of the normonas family
glycoproteins; and it is also highly conserved from species to species. The ß subunits are different for each member, ie, CG, FSH, TSH and LH, but show considerable homology in the structure. In addition, also ß subunits are highly conserved from species to species. In humans, the mature subunit consists of 92 amino acid residues, while the β subunit varies in size for each member: 1 1 1 residues in hFSH, 121 residues in hLH, 1 18 residues in bTSH and 145 residues in hCG ( Co barnous, Y. (1992), Endocrine Reviews, 13, 670-691, Lustbader, JW et al. (1993), Endocrine Reviews, 14, 291-31 1). The β subunit of hCG is substantially larger than the other β subunits because it contains 34 additional amino acids in the C terms referred to herein as the carboxyl terminal protein (CTP). The two subunits of the heterodimer showed many retained intra-subunit disulfide bonds: five disulfide bridges in the a subunit and six disulfide bridges in the β subunit. The corresponding cysteine residues are completely conserved among all members of the gonadotropin family. In the subsurface ß of hCG the disulfide bridges are formed between the cysteines at positions 9-57; 23-72, 26-1 10, 34-88, 38-90 and 93-100. The x-ray structure of hCG shows that these disulfide bonds are involved in typical three-dimensional patterns called disulfide knots. Hormones have three or four asparagine residues that can
be glycosylated in N. In addition, the C-terminal peptide (CTP) of hCG can be glycosylated at O at four positions of serine. Glycoprotein hormones serve important functions in a variety of bodily functions including metabolism, temperature regulation and the reproductive process. The gonadotropin of the pituitary FSH for example plays a pivotal role in the stimulation, development and maturation of the follicle, while the
LH induces ovulation (Sharp, R.M. (1990), Clin Endocrinol; 33,
787-807; Dorrington and Armstrong (1979), Recent Prog. Horm. Beef; 35, 301-342). Currently, FSH is applied clinically, either alone or in combination with LH activity, for ovarian stimulation, ie ovarian hyperstimulation for in vitro fertilization (IVF) and induction of ovulation in vivo in infertile anovulatory women (Insler, V. (1998), I nt.J. Fertility, 33, 85-97, Navot and Rosenwaks (1998), J, Vitro Fert, Embryo Transfer, 5, 3-13), as well as for male hypogonadism. The objective of a controlled superovulation is to increase the number of mature oocytes that can be recovered for IVF and subsequent embryo transfers (ET). Generally, up to three embryos are replaced per transfer. As more than one treatment is usually necessary, in most infertility clinics, replacement embryos or fertilized oocytes are frozen and transferred in subsequent cycles. TSH can be used by patients who need thyroid hormone supplements, for example, for use in
patients with thyroid cancer who have had a partial or total removal of their thyroid gland. Genomic and cDNA clones have been prepared for all subunits and their primary structure has been resolved. In addition, Chinese Hamster Ovary (CHO) cells have been transfected with human gonadotropin subunit genes and these cells show that they are able to secrete intact dimers (eg, Keene et al. (1989), J. Biol. Chem. ., 264, 4769-4775, Van Wezenbeek and collaborators (1990), in From clone to Clinic (eds Crommelin D. J. A. and Schellekens H., 245-251) In principle, fertility regulation can be influenced in several stages, for example, the recruitment, folculogenesis, implantation and maintenance of the follicle of pregnancy, due to the selection of mechanisms, only one follicle of the group of follicles that leaves the primordial source, reaches the preovulatory state, that is, the dominant follicle , and provides a healthy and fertilizable oocyte, others become atheletic and degenerate.The mechanisms that control the selection of a dominant follicle are not completely understood, but the hypothesis has been made You know that the follicle most sensitive to FSH is the one that becomes dominant. It is well known that in addition to gonadotropins, other factors are necessary for optimal follicle and oocyte development. Follicular growth is controlled by growth factors such as IGF-1 and GDF-9, and in later stages by FSH and LH of gonadotropins, and by
estrogen Regulatory factors are also involved in the control of follicular arrest, early follicular recruitment, follicular growth, antral formation and the ovulation process. Also, these regulatory factors influence the process of implantation of the embryo in the uterus and is involved in the regulation of spermatogenesis in the male. There is a need to identify the factors involved in the different stages of female and male fertility. Such factors can also be used in therapeutic protocols in vivo or in vitro. The present invention provides such a factor. More specifically, the present invention provides a polynucleotide sequence comprising the coding SEQ I D NO: 1. The complete genetic sequence can be used in the preparation of vector molecules for expression of the protein factor in suitable host cells. The complete genes or their variants can be derived from cDNA or genomic DNA from natural sources or synthesized using known methods. The invention also includes the entire mRNA sequence as indicated in SEQ ID NO: 1. The mRNA contains an open reading frame corresponding to the nucleotide sequence 101 -490 of SEQ ID NO: 1. This sequence encodes a 130 amino acid precursor protein (SEQ ID NO: 2). In addition, to accommodate (to codon variability, the invention also includes sequences that code for
the same amino acid sequences as the sequences described herein. Also portions of coding sequences that code for a functional polypeptide are part of the invention as well as its allelic and species variations. Sometimes, a gene is expressed in a certain tissue as a spliced variant, resulting in an altered 5 'or 3' mRNA or in the inclusion or exclusion of one or more exon sequences. These sequences as well as the proteins encoded by these sequences are expected to all perform the same or similar functions and also form part of the invention. In particular, SEQ ID NO: 3 represents a specific binding variant which differs from SEQ ID NO: 1 in that an insertion of 128 nucleotides is present. The transfer of this binding variant leads to a truncated version of the protein in SEQ ID NO: 2, as shown in SEQ 1D: 4. It has now been found that these sequences are specifically expressed in the pituitary and in the endometrium. The sequence of information as provided herein may not be constructed as closely as required by the exclusion of misidentified bases. The specific sequence described herein can be quickly used to isolate the complete genes of several species. Thus, in one aspect, the present invention provides isolated polynucleotides that encode a novel protein hormone.
The term "isolated" denotes that the polynucleotide has been removed from its natural environment and is thus in a form suitable for use within production systems of genetically engineered proteins. The DNA according to the invention can be obtained from cDNA. The tissues are preferably of human origin. Preferably the ribonucleic acids are isolated from the pituitary, placenta or endometrium. Alternatively, the coding sequence could be genomic DNA or prepared using DNA synthesis techniques. The polynucleotide can also be in the form of RNA. If the polynucleotide is DNA, it can be in the form of simple helices or double helices. The simple helix could be the encoding helix or the non-coding helix (anti-sense). The polypeptide according to the present invention can exist as a monomer. However, also dimeric forms of the peptide are part of the invention. Such dimers are consistent nomodimers of two identical polypeptides or, alternatively, heterodimer complexes. Preferably, such a dimer consists of the polypeptide according to the invention combined with the common subunit of the gonadotropin hormone family. As an alternative also chimeric proteins are contemplated comprising the functional part of the polypeptide sequence according to the invention. Such chimeric construction can be easily prepared by linking e! DNA encoding fas subunits of the neterodimeric complex coupled by a link
as described in the PCT application W096 / 05224. Similarly, bifunctional glycoproteins can be prepared wherein the subunit of the present invention is covalently linked by linkers to other members of the glycoprotein hormone family. Examples of such constructions are described in PCT application W099 / 25849. The present invention also relates to polynucleotides that have slight variations or that have polymorphic sites. Polynucleotides that have slight variations encode polypeptides which retain the same function or biological activity as the mature, natural protein. Polymorphic sites are useful for diagnostic purposes. Such polynucleotides can be identified by hybridization preferably under highly stringent conditions. According to the present invention the term "rigorous" means washing conditions of 1 x SSC, 0. 1% SDS at a temperature of 65 ° C, highly stringent conditions refers to a reduction in SSC towards 0.3 x SSC, more preferably at 0.1 x SSC. Preferably the first two washings are carried out subsequently every two times for 15-30 minutes. If there is a need to wash under highly stringent conditions, an additional wash is performed with 0.1 x SSC once for 15 minutes. Hybridization can be carried out, for example, at night in a 0.5 M phosphate retardant solution, pH 7.5 / 7% SDS at 65 ° C.
Thus, also functional equivalents which is SEQ I D NO: 1 comprising polypeptides or parts thereof which have sequence variations while still maintaining functional characteristics, are included in the invention. The DNA according to the invention will be very useful for expression in vivo or in vitro for the novel protein according to the invention in sufficient quantities and in substantially pure form. The variations that can occur in a sequence can be demonstrated by a difference (s) of amino acids in the total sequence or by deletions, substitutions, insertions, inversions or additions of an amino acid (s) in said sequence. . Substitutions of amino acids that are expected not to alter essentially the biological and immunological activities have been described. The amino acid replacements between relative amino acids or replacements which have occurred frequently in evolution are, among others, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, lie / Val (see Dayhof, MD, Atlas of protein sequence and structure, Nat. Biomed, Res. Found., Washington DC, 1978, vol.5, sup.3) ). Based on this information Lipman and Pearson developed a method for comparing fast and sensitive proteins (Science, 1985, 227, 1435-1441) and to determine the functional similarity between homologous polypeptides. It will be clear that also polynucleotides encoding such variants are part of the invention.
Thus, in another aspect of the invention SEQ ID NO: 2 or SEQ I D NO: 4 are provided which comprise polypeptides, but also polypeptides with a similarity of 70%, preferably 90%, more preferably 95%, even more preferably 98%. NCBI-BLASTX 2.0.4 [Feb-24-1998] (Altschul, Stephen F.,
Thomas L. Madden, Alejandro A. Schaffer, J inghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1 997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402) is used to search for sequence alignments using default positions . For amino acid alignments the BLOSUM62 matrix is used as an omission and the similarity is indicated as the number of positives. Filtering or low complexity of composition is not included.
Preferably, the polypeptide comprises cysteine residues at positions corresponding to amino acid positions 36, 50, 60 and 64 of SEQ ID NO: 2 or SEQ JD NO: 4. Even more preferably, cysteine residues are present in positions corresponding to amino acid positions 84, 99, 1 15, 17, 120, and 127 of SEQ ID NO: 2. The correspondence with a certain position indicates the position in a second sequence that is aligned with the reference sequence as indicated in SEQ ID NO: 2 or SEQ ID NO: 4 when the sequences are optimally aligned. Thus, the polypeptide is capable of forming all the disulfide bridges at the corresponding positions as compared to the β subunit of the hormone family of the
glycoprotein with the exception of the so-called seat belt disulfide bond (in the corresponding positions 26-1 10 of ß hCG). The protein as indicated in SEQ I D NO: 2 or SEQ I D NO: 4 is a precursor protein and is subjected during secretion to a proteolytic cleavage. Mature proteins are also part of the invention. The protein as indicated in SEQ I D NO: 2 or SEQ ID NO: 4 as well as the mature protein may be subject to post-translational modifications, for example glycosylation. Such modified proteins are also part of the invention. It is understood that portions of such polypeptides still capable of conferring biological effects are also included. Especially portions which are linked even to objectives are part of the invention. Such proteins or functional parts thereof may be functional in themselves, for example in solubilized form or they may be linked to other polypeptides (for example CTP, WO90 / 09800), either by known biotechnological means or by chemical synthesis, to obtain chimeric proteins. Such proteins could also be useful as a therapeutic agent preventing the target from interacting with the natural proteins in the body. Thus, such altered proteins could be used as an agonist or antagonist of their natural function. In this regard also antibodies against the protein according to the invention form part of the invention. Such antibodies can be prepared by conventional hybridoma technology or technologies
of recombinant DNA (Antibodies, A laboratory manual, 1 988, Cold Spring Harbor Laboratory). Alternatively, deregulation of the expression level of the protein can be obtained using anti-sense nucleic acids through triple-helix formation (Cooney et al., 1988 Science, 241, 456-459) or by binding to mRNA. This in itself could also guide the regulation of fertility, that is, contraception or infertility treatment. The present invention comprises all isolated polynucleotides which comprise in their coding sequence the polypeptides as indicated above. A wide variety of combinations of host cells and cloning vehicles can be usefully employed in the cloning of the nucleic acid sequence encoding the polypeptide according to the invention. Suitable expression vectors are, for example, bacterial or yeast plasmids, plasmids of a wide range of hosts and vectors derived from combinations of plasmid DNA and phage or virus. Also included are vectors derived from chromosomal DNA. In addition, an origin of replication and / or a dominant selection marker may be present in the vector according to the invention. The vectors according to the invention are suitable for transforming a host cell. In the case of dimeric proteins, similar cloning vehicles can be used for insertion of a second subunit to the
host cell. The subunits could be coded by different vectors as well as by a single vector. The vehicles for use in expressing the protein or its parts of the present invention will further comprise control sequences operably linked to the nucleic acid sequence encoding the protein. Such control sequences generally comprise a promoter sequence and sequences, which regulate and / or enhance expression levels. Of course the control sequences and others may vary depending on the selected host cell. Recombinant expression vectors comprise the DNA of the invention as well as cells transfected with said DNA or said expression vector, whether transient or stable, also form part of the present invention. Suitable host cells according to the invention are host cells Bacterial, yeast and other fungi, plants or animal hosts such as Chinese Hamster Ovary cells or monkey cells. Thus, a host cell comprising the DNA or expression vector according to the invention is also within the scope of the invention. The manipulated host cells can be cultured in a conventional nutrient medium which can be modified for example for proper selection, amplification or induction of transcription. The culture conditions such as temperature, pH, nutrients, etc. , are well known to all those ordinarily skilled
in the technique. Techniques for the preparation of the DNA or vector according to the invention as well as the transformation or transfection of a host cell with said DNA or vector are standard and well known in the art, see for example Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. The culture host cells comprising vectors encoding the polypeptide according to well-known methods and recovering the polypeptide of interest can produce the polypeptide according to the invention. The dimeric proteins can be isolated in a similar way from the culture cells transfected with an additional vector encoding the second protein or by culturing cells transfected with a single vector encoding both subunits-EJ by peptide according to the invention can be recovered and purified from recombinant cell cultures by common biochemical purification methods (as described in Guide to Protein purification, edited by Murray P. Deutscher (1 990) Methods in Enzymology, Vol 182. Academic Press, Inc. San Diego CA 92101. Harcourt Brace Jovanovich, Publishers, including ammonium sulfate precipitation, extraction, chromatography such as hydrophobic interaction chromatography, cation or anion exchange chromatography or affinity chromatography and high performance liquid chromatography.
necessary, protein redobling steps can also be included. Alternatively, the protein can be expressed and purified as a fusion protein containing ("tags") which can be used for affinity purification. The polypeptide according to the invention is useful for the control of follicular arrest and recruitment. Inhibition of recruitment can be used to delay (premature) menopause or as a contraception. In addition, this polypeptide can be used for the in vitro maturation and growth of follicles, for example from the tissues of frozen ovaries. The polypeptides of the invention are also useful in the detection and purification of receptors to which the proteins are linked. For example, the polypeptides can be coupled to solid supports and used in the affinity chromatographic preparation of anti-hormone receptors or antibodies. The receptors are useful in themselves to evaluate the hormonal activity for candidate drugs in research tests for therapeutic candidates. Such candidate drugs could behave as agonists or antagonists of the polypeptide according to the invention and as such could improve the efficiency of embryo implantation or prevent implantation. The invention also provides the formulation of a pharmaceutical composition comprising mixing the protein according to the invention with a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers are well known to those skilled in the art and include, for example, sterile salines, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and Water. Furthermore, the pharmaceutical composition according to the invention may comprise one or more stabilizers such as, for example, carbohydrates including sorbitol, mannitol, starch, sucrosedextrin and glucose, proteins such as albumin or casein, retarding solutions such as alkaline phosphates. Methods for making intravenous preparations and mixtures are described in Remingtons's Pharmaceutical Sciences, pp. 1463-1497 (16th ed 1980, Mack Publ Co., Easton, Pa., USA). Therapeutic dosages will generally be in the range of 0.1-100 μg / kg of patient weight per day, preferably 0.5-20 μg / kg per day. Thus, the protein according to the invention is useful in the preparation of a pharmaceutical product. The pharmaceutical product is to be used in disorders related to fertility or in contraception.
Legends of Figures Figure 1 RT PCR using SEQ ID NO: 5 and SEQ I D NO: 6 primaries using human pituitary cDNA as a standard.
Figure 2 Alignment of SEQ I D NO: 2 with partial sequences derived from monkey, pig and rabbit, respectively. The dashes indicate that sequence information is not available.
Figure 3 General view of an array section of human tissue stained with H &E (hematoxylin-eosin).
Figure 4 Hybridization in situ. to. Hybridized section of endometrium (secretory phase) with antisense probe b. Hybridized section of endometrium (secretory phase) with sense probe. c. Hybridized pituitary section (secretory phase) with antisense probe. d. Hybridized section of endometrium (secretory phase) with sense probe.
Examples Example 1: Sequence identification. Using parts of the DNA sequence and / or protein sequence of the beta subunit of FSH humaho (ßFSH) we have purified several databases for the presence of sequences
related A human genomic clone was identified which contains a region with a low degree of total homology. However, the genomic sequence predicted in an open reading frame where a number of cysteine residues were present with a spacing that was very similar to that of ß FSH and related proteins such as J3LH, J3hCG and ßTSH. To obtain a DNA fragment corresponding to the novel gene, a PCR was performed on human genomic DNA using SEQ JD NO: 5 and SEQ ID NO: 6 primaries. A fragment with the expected size of 142 base pairs was obtained, cloned into the PCR2.1 vector and sequenced. The sequence was identical to the part of the genomic clone and corresponds to nucleotide 337 to 478 in SEQ ID NO: 1. In order to clone full-length cDNA spanning the complete open reading frame (ORF), we developed 5 'and 3' RACE PCR experiments (rapid amplification of cDNA terminals). As a standard we used Marathon cDNA ready derived from human pituitary (Clontech cat # 7424-1). For RACE 5 ', in the first PCR, the primary AP1 (SEQ ID NO: 7) of the package was used in conjunction with the SEQ ID NO: 6 of the specific gene using 5 microliters of pituitary cDNA as a model. For RACE 3 ', similarly, the first reaction was performed using SEQ 1D NO: 7 with SEQ ID NO: 5 primaries. The PC R protocol was as follows: 5 min. 94 ° C; 5 cycles 5 sec. 94 ° C / 4 min. 72 ° C; 5 cycles 5 sec. 94 ° C / 4 min. 70 ° C; 25 cycles 5 sec. 94 ° C / 4 min. 68 ° C; 5 min 72 ° C; storing at 4o C.
Subsequently, nested PCR reactions were performed using 1% of the volume of the first PCR as a model. Here, primary AP2 (SEQ ID NO: 8) of the package was used in combination with SEQ ID NO: 9 primary for RACE 5 '. For RACE 3 'primary SEQ I D NO: 8 was used in combination with SEQ I D NO: 10. Nested reactions were performed using the Advantage 2 cDNA polymerase package (Clontech) with the following protocol: 5 min. 94 ° C; 20cycles 5 sec. 94 ° C / 4 min. 68 ° C; 5 min 68 ° C; storing at 4 ° C. The PCR products were analyzed on a 1.2% agarose gel, the gel was bottled overnight in nitrocellulose 20 x SSC on Hybond N +. The DNA was interlaced by baking for 2 hours at 80 ° C. The stain was hybridized (overnight at 65 ° C in 0.5 molar phosphate retardant solution with pH 7.5 / 7% SDS) with the PCR fragment from specific gene of 142 base pairs described above. The filters were washed in 0.3 x SSC / 0.1% SDS at 65 ° C and subsequently in 0.1 x SSC / 0.1% SDS at 65 ° C. A hybridization fragment of approximately 480 base pairs was cut that originated from the RACE 5 'reaction of the gel was purified using a Qiaquick gel extraction pack (Qiagen) according to the manufacturers' instructions. Similarly, a hybridization band of approximately 650 base pairs was isolated for the 3 'RACE reactions. Both fragments were cloned into the pCR2.1 vector and sequenced. The resulting 5 'and 3' RACE fragments revealed
overlapping sequences as expected. The sequence of the 5 'fragment corresponds to nucleotide 1 to 499 in SEQ I D NO: 1. The sequence of the 3 'fragment corresponds to nucleotide 377 to 917 in SEQ I D NO: 1, followed by a stretch of A-residues. The sequence AP2 as well as the majority of the stretching of poly-A is omitted in SEQ ID NO: 1. To verify the sequences that were obtained, a PCR was performed to amplify the region covering the ORF using two primaries: one upstream of the ATG tracing initiation codon (SEQ ID NO: 1 1) and the other downstream of the codon of stop (SEQ ID NO: 12). An expected fragment of approximately 530 base pairs was obtained as a larger band using pituitary cDNA as a model (see Figure 1). The sequence of this fragment corresponds to nucleotides 23 to 548 of SEQ JD NO; 1 and was identical to that of (part of) the combined RACE fragments. The sequence SEQ ID NO: 1 contains an open reading frame (nucleotides 101 to 490) coding for 130 amino acids. Upstream of the ATG translation initiation codon a stop codon inter-frame (nucleotides 44 to 46) is presented. A polyadenylation signal (ATTAAA, nucleotides 894 to 899) is followed somewhat downstream by a stretch of poly A, which is only partially included in the sequence SEQ ID NO: 1. The open reading frame contains 10 cysteine residues with a spacing that is extremely similar as it is in ßFSH, ßLH, ßhCG and ßTS H. The amino terminal region of the reading frame
probably corresponds to a sequence signal. A number of features can be noted, for example, the presence of stretches of hydrophobic residues as well as the presence of a basic amino acid that follows the amino terminal methionine. Comparison of the complete sequence of SEQ ID NO: 1 with human genomic DNA sequences revealed that the novel gene consists of three exons. Exon 1 corresponds to nucleotides 1 to 99, exon 2 corresponds to nucleotides 100 to 304 and exon 3 corresponds to nucleotides 305 to 91 1. Figure 1 shows that in addition to the expected fragment of approximately 530 base pairs a second fragment is obtained which is somehow longer (approximately 660 base pairs). This fragment was cloned and sequenced and established to correspond to a binding variant containing sequences of an intron (corresponding to SEQ ID NO: 3). The encoded protein is shown in the sequence SEQ JD NO: 4.
Example 2: Evolutionary Conservation To establish whether the novel gene is retained in evolution, the primary SEQ ID NO: 5 and SEQ ID NO: 6 sequences were also used for the PCR reactions using pig, monkey and rabbit genomic DNA. Fragments of the expected size were obtained and analyzed by the cloning of the purified fragments in pCR2.1 and the nucleotide sequencing. The three sequences are extremely homologous to the human sequence. When are they
omitted the sequences of the primaries used for PCR, an alignment of the deduced amino acid sequences shows a high degree of sequence conservation (see Figure 2).
Example 3 in vitro hybridization. Arrangements of human tissue. Tissue arrays were obtained from Superbiochips Laboratories (Seoul, Korea, FH-A 1 and FH-A2, Figure 3). Briefly, the tissue arrangements used consisted of 60 different cylinders of normal human tissue of 4 mm in diameter. Each cylinder was taken from a specimen that had previously been fixed in formalin and routinely embedded in paraffin. The 60 cylinders were assembled in a single block of paraffin. Then sections of 5 μm were cut and collected on RNase-free objects.
Generation of sense and antisense RNA test. With specifically designed primer packages containing either an RNA T7 and SP6 polymerase site, a unique part of the gene was amplified. Using this approach, both sense and antisense probes could be generated from a single PCR fragment. The PCR mixture contained SP6 front primer (2 ng / μl) (SEQ ID NO: 13), T7 reverse primer (2 ng / μl) (SEQ ID NO: 14), 1 x PCR retarder solution (Pharmacia, with 15 mM) of MgCL2), mixture of dNTP (0.2 mM / dNTP), Taq polymerase (0.02 U / μl)
and DNA standard (0.5 ng / μl). The PCR reaction consisted of an initial denaturation (5 min 95 ° C), 8 cycles at low annealing temperature (0.5 min 95 ° C, 0.5 min 55 ° C, 1 min 72 ° C), and 30 high temperature annealing cycles (0.5 min 95 ° C, O.dmin 60 ° C, 1 min 72 ° C), and 5 min. at 72 ° C. 5-10 μl of PCR product was run on a 2% agarose gel to confirm the yield and correct amplification of the expected DNA fragment. The PCR product was ethanol precipitated overnight, centrifuged (14,000 rpm), washed in 70% ethanol and subsequently resuspended in H2O. After purification on GFX columns (Pharmacia) the concentration of the probe was calculated based on OD260 / OD280 values and diluted to a final concentration of 100 ng / μl. The RNA probes were generated starting with 500 ng of standard (according to the protocol supplied by the manufacturer, Boehringer-Roché) in the presence of DIG labeling mixture (D1G-UTP, unlabeled nucleotides, blocking agents), transcription, 10mM DTT, 1 U / μk of Rnase inhibitor and 2-4 U / μl of the appropriate RNA polymerase. Incubations were carried out at 37 ° C for 2 hours and stopped by adding approximately 25 nM EDTA (pH 8.0) 400 mM LiCl and excess 100% ethanol. The labeled product was precipitated overnight, centrifuged, washed in 70% ethanol and subsequently resuspended in H2O with RNase inhibitor. After in vitro transcription, a small sample was analyzed
amount of the probe on a 1.5% agarose gel to confirm the success of in vitro transcription. Probe concentrations were estimated according to a protocol of Boehringer-Roche and using the advised reagents. Serial DIG-RNA dilutions labeled with probes and control of known concentration (10-0.01 ng / μl) were located on a Hybond N + membrane (Amersham). The membrane was subjected to microwave for 2 min. After being blocked for specific agglutination the membrane was incubated with Fab 'fragments of anti-DIG alkaline phosphatase (anti-D IG-AP) for 30 min. Spotting was initiated by adding NBT / BC IP substrate and continued until sufficient spotting was seen at the lowest concentration in the control series. The concentration of the recently labeled probe was estimated by comparing the intensity of spot-spots with those of the control series. In situ hybridization Tissue sections were baked at 60 ° C for two hours, dextriated in xylene and rehydrated in decreasing concentrations of ethanol. Subsequently the sections were treated for 20 min. in 0.2M HCl, washed in MilliQ treated with DEPC and digested with proteinase K (1 μg / ml) in digestion retarder solution (100 mM Tris, 50 mM EDTA pH 8) for 30 min. at 37 ° C. The digestion was stopped in glycine pre-cooled to 0.2% in PBS for 10 min. at room temperature (RT). The samples were acetylated for 5 min. with 0.25% acetic acid anhydride in
0.1 M triethanolamine retarder solution, followed by two washes in Milli Q treated with DEPC. The sections were prehybridized at hybridization temperature in a humid chamber with pre-hybridization mixture, containing 52% formamide, 21 mM Tris, 1 mM EDTA, 0.33 M NaCl, 10% dextran sulfate, 1 x Denhardt's solution, 100 μg / ml of salmon sperm DNA, 100 μg / ml of tRNA and 250 μg / ml of total R NA of yeast. The slides were covered with a glass cover. After two hours the pre-hybridization mixture was replaced with probe hybridization mixture containing pre-hybridization mixture with the following additions: 0.1 mM DTT, 0.1% sodium thiosulfate, 0.1% SDS and a variable amount of labeled DJG probe. Hybridization was carried out overnight (16 hours) in a humid chamber at 50 ° C. Plates were washed in 2 x SSC for 15 min., followed by washes in 2 x SSC, 1 x SSC and 0, 1 x SSC each for 15 mip. at annealing temperature. The sections were treated with ribonuclease A (20 μg / ml) in Rnasa retardant solution (0.6 M NaCl, 20 mM Tris, 1.0 mM EDTA) for one hour at 37 ° C. After two washes (5 min RT) in pre-cooled PBS and one wash in retarder solution 1 (100 mM maleic acid, 150 mM NaCl) the sections were incubated for 30 min. with blocking solution (1 g / ml of blocking reagent in retarder solution 1). After the sections were incubated with anti-DIG-AP (Boehringer / Roche), diluted in 1: 500 blocking solution, during
one hour to .RT. After two washes in retarder solution 1 (1 5 min RT) the plates were carefully dry cleaned around the tissue and the sections were circulated with DAKO-pen® (DAKO). The sections were covered with reagent to develop N BT / BCI P color (Boehringer / Roche) and incubated in a humid chamber at RT. After two hours the sections were rinsed in water and optionally counter-stained with 0. 1% methyl green for 30 seconds. The plates were mounted in gelatin-glycerol from Kaiser. In all the experiments, both anti-sense and sense probes were used at different concentrations (200 and 1000 ng / ml). The temperatures used for hybridization were 50 ° C.
Microscopic evaluation EJ analysis of in situ hybridization revealed that two tissues showed significant staining with the antisense probe compared to the sense probe. These tissues were the endometrium (see Figure 4a and 4b) and pituitary (see Figure 4c and 4d). All other tissues were negative.
LISTING OF SECUENC IAS < 1 10 > Akzo Nobel N.V. < 120 > Novel pituitary hormone < 130 > 2000527 < 140 >
< 141 > < 160 > 14 < 170 > Patentln Ver. 2.1 < 210 > 1 < 21 1 > 917 < 212 > DNA < 213 > Homo sapiens
< 400 > 1 gacatttacc cagggcaaac ttctaccatt cattgtgact tcctgaaatc ttagtgcaag 60 tttcagctct aaaagaagag tgggctcctg caagattagc atgaagcígg cattcctctt 120 ccttggcccc atggccctcc tccttctggc tggctatggc tgtgtcctcg gtgcctGcag 180 tgggaacctg cgcacctttg tgggctgtgc cgtgagggag tttacíttcc tggccaagaa 240 gccaggctgc aggggccttc ggatcaccac ggatgcctgc tggggtcgct gtgagacctg 300 ggagaaaccc attctggaac ccccctatat tgaagcccaí catcgagíct gtacctacaa 360 cgagaccaaa caggtgactg tcaagctgcc caactgtgcc ccgggagtcg accccttcta 420 cacctatccc tggccatcc gctgtgactg cggagcctgc tccactgcca ccacggagtg 480 tgagaccatc tgaggccgct agctgctctc tgcagacccg cctgtgtgag cagcacatgc 540 agttatactt cctggatgca agactgttta atttcgacca cacccatgga ggaggttacc 600
tgtcgcccct taggtccagc tcaggcaaaa ggcccaaatg cagcctactt atgctaaaag 660 ttcaaaacaa tattcgtgcc ttcaccaaaa taatttctcc agctcacata cctgcaaatt 720 aatttttctt tgccttgagt cttggaacat aatttgtgta tcacaatcct cccccaattt 780 ggacttataa tatgctaatg atttaaacac atgggatgta attaggatat ggggctggaa 840 agtctttaaa ttctcatgtt ctatttaacc tctgatctcc aaccggattt atgattaaag gaaaaaa ggctagaaat 900 917
< 210 > 2 < 211 > 130 < 212 > PRT < 213 > Homo sapiens < 400 > 2 < 400 > 2 Met Lys Leu AJa Phe Leu Phe Leu Gly Pro Met Wing Leu Leu Leu Leu 1 5 10 1 5 Wing GJy Tyr GJy Cys Val Leu Gly Wing Being Ser Gly Asn Leu Arg Thr 20 25 30 Phe Val Gly Cys Wing Val Arg Glu Phe Thr Phe Leu Wing Lys Lys Pro 35 40 45 Gly Cys Arg Gly Leu Arg He Thr Thr Asp Wing Cys Trp Gly Arg Cys 50 55 60 Glu Thr Trp Glu Lys Pro He Leu Glu Pro Pro Tyr He Glu Wing His 65 70 75 80 His Arg Val Cys Thr Tyr Asn Glu Thr Lys Gln Val Thr Val Lys Leu 85 90 95
Pro Asn Cys Wing Pro Gly Val Asp Pro Phe Tyr Thr Tyr Pro Val Wing
100 105 1 1 0 He Arg Cys Asp Cys Gly Wing Cys Ser Thr Wing Thr Thr Glu Cys Glu 1 15 120 125 Thr l ie 1 30
< 210 > 3 < 21 1 > 1045 < 212 > DNA < 213 > Homo sapiens
< 400 > 3 gacatttacc cagggcaaac ttctaccatt cattgtgact tcctgaaatc ttagtgcaag 60 tttcagctct aaaagaagag tgggctcctg caagattagc atgaagctgg cattcctctt 120 ccttggcccc atggccctcc tccttctggc tggctatggc tgtgtcctcg gtgcctccag 180 tgggaacctg cgcacctttg tgggctgtgc cgtgagggag tttactttcc tggccaagaa 240 gccaggctgc aggggccttc ggatcaccac ggatgcctgc tggggtcgct gtgagacctg 300 ggagcttttg tcaagatgtc gtgtatgaac aaggcattca atacacattt gttggttgac 360 tgggatggac ctccccctgg agctgtagat cctccagcct aatggaaggc catttagaat 420 cacacttgca ctaaacccat tctggaaccc ccctatattg aagcccatca tcgagtctgt 480 acctacaacg agaccaaaca ggtgactgtc aagctgccca actgtgcccc gggagtcgac 540 cccttctaca cctatcccgt ggccatccgc tgtgactgcg gagcctgctc cactgccacc 600 acggagtgtg agaccatctg aggccgctag ctgctctctg cagacccgcc tgtgtgagca 660 gcacatgcag ttatacttcc tggatgcaag actgtttaat ttcgaccaca cccatggagg 720
aggttacctg tcgcccctta ggtccagctc aggcaaaagg cccaaatgca gcctacttat 780 gctaaaagtt caaaacaata ttcgtgcctt caccaaaata atttctccag ctcacatacc 840 tgcaaattaa tttttctttg ccttgagtct tggaacataa tttgtgtatc acaatcctcc 900 cccaatttgg acttataata tgctaatgat ttaaacacat gggatgtaat taggatatgg 960 ggctggaaag tctttaaatt ctcatgttct atttaacctc tgatctccaa ccggatttat 1020 ctagaaatga gattaaaggg aaaaa 1045
< 210 > 4 < 211 > 75 < 212 > PRT < 213 > Homo sapiens
< 400 > 4 Met Lys Leu Ala Phe Leu Phe Leu Gly Pro Met Ala Leu Leu Leu Leu 1 5 10 15
Ala Gly Tyr Gly Cys Val Leu Gly Wing Ser Gly Asn Leu Arg Thr 20 25 30 Phe Val Gly Cys Ala Val Arg Glu Phe Thr Phe Leu Ala Lys Lys Pro 35 40 45 Gly Cys Arg G1y Leu Arg He Thr Thr Asp Ala Cys Trp Gly Arg Cys 50 55 60 Glu Thr Trp Glu Leu Leu Ser A-rg Cys Arg Val 65 70 75
< 210 > 5
< 211 > 26 < 212 > DNA < 213 > Homo sapiens
< 400 > 5 ccatcatcga gtctgtacct acaacg 26
< 210 > 6 < 211 > 23 < 212 > DNA < 213 > Homo sapiens
< 400 > 6 ctccgtggtg gcagtggagc agg 23
< 210 > 7 < 211 > 27 < 212 > DNA < 213 > Homo sapiens
< 400 > 7 ccatcctaat acgactcact atagggc 27
< 210 > 8 < 211 > 2. 3
< 212 > DNA < 213 > Homo sapiens
< 400 > 8 actcactata gggctcgagc ggc 23
< 210 > 9 < 211 > 25 < 212 > DNA < 213 > Homo sapiens
< 400 > 9 agtcacagcg gatggccacg ggate 25
< 210 > 10 < 21 1 > 25 < 212 > DNA < 213 > Homo sapiens
< 400 > 10 actgtcaagc tgcccaactg tgccc 25
< 210 > 1 1 < 211 > 23 < 212 > DNA
< 213 > Homo sapiens
< 400 > 1 1 ctaccattca ttgtgacttc ctg 23
< 210 > 12 < 211 > 23 < 212 > DNA < 213 > Homo sapiens
< 400 > 12 gtataactgc atgtgctgct cac 23
< 210 > 13 < 21 1 > 40 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence: primary
< 400 > 13 cgatttaggt gacactatag gcatgaagct ggcattcctc 40
< 210 > 14
< 21 1 > 42 < 212 > DNA < 213 > Artificial Secuance
< 220 > < 223 > Description of Artificial Sequence: primary
< 400 > 14 cgtaatacga ctcactatag gggtctgcag agagcagcta ge 42
Claims (13)
- RE IVINDICATIONS 1 . An isolated polynucleotide encoding a polypeptide that is at least 70% similar to SEQ I D NO: 2 or SEQ ID NO: 4.
- 2. An isolated polynucleotide encoding a mature polypeptide that is at least 70% similar to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
- The polynucleotide of claim 1 or 2 which is at least 90, preferably 95% similar to SEQ ID NO: 2 or SEQ ID NO: 4.
- 4. The polynucleotide of claims 1-3, said polypeptide comprising the amino acid Cis at positions corresponding to amino acid positions 36, 50, 60 and 64 of SEQ JD NO: 2 or SEQ JD NO; 4-
- 5. EJ polynucleotide of claim 4 with the amino acid Cis in positions corresponding to amino acid positions 84, 99, 1 15, 117, 120 and 127 of SEQ ID NO: 2.
- 6. Polynucleotide EJ according to claim 5, said polynucleotide comprising the sequence SEQ ID NO: 1 or the sequence extending from the nucleoijds 101-490 of SEQ JD NO: 1.
- The polynucleotide according to claim 4, said polynucleotides comprising the sequence SEQ ID NO.3 or the sequence extending from the nucleotides 101-325 of SEQ ID NO: 3.
- 8. A recombinant expression vector comprising the DNA according to claims 1-7.
- 9. A polypeptide encoded by the polynucleotide according to claims 1-7 or the expression vector according to claim 8.
- 10. A cell transfected with DNA according to claims 1-7 or the expression vector according to with claim 8.
- 1 1. A cell according to claim 10 which is a transfected cell expressing the protein according to claim 9.
- 12. A method for producing the polypeptide of claim 9 is the method comprising culturing the cells of claim 1 under conditions of wherein said protein is produced and recovered said culture protein.
- 13. A pharmaceutical composition comprising a polypeptide according to claim 9 in admixture with a pharmaceutically acceptable carrier. ek / * 37 SUMMARY The invention relates to newly identified DNA sequences that encode a novel cystine knot polypeptide as well as the encoded protein. The invention is useful in the field of fertility.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00200185.7 | 2000-01-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA02007027A true MXPA02007027A (en) | 2003-11-07 |
Family
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