Rat C-type natriuretic peptide cDNA and precursor protein

Abstract: A cDNA coding for a rat derived CNP (rCNP: C-type natriuretic pepteide), and a rat derived CNP precursor protein and derivatives thereof are disclosed. The rat derived CNP precursor is represented by the following amino acid sequence. The rCNP precursor and its derivatives are novel and have natriuretic and hypotensive activities.

Description: This invention relates to a cDNA coding for a rat CNP (rat C-type natriuretic peptide which is hereunder abbreviated as rCNP), as well as the rCNP precursor protein (prepro rCNP) that is encoded by said cDNA. In recent years, various peptides collectively referred to as "natriuretic peptides" (NP) have been discovered from the atria and brains of various animals. Today those NPs can be classified into three types, A-type natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), depending on similarities in the primary amino acid sequence and the structure of their precursors. Among the three types, ANP and BNP were first isolated and identified from the atrium and brain and hence are sometimes called "atrial natriuretic peptide" and "brain natriuretic peptide", respectively (Matsuo, H. and Nakazato, H., Endocrinol. Metab. Clin. North Am., 16 , 43, 1987; Sudoh, T. et al., Nature, 332 , 78 , 1988). However, it is known today that ANP occurs not only in the atrium but also in the brain and that BNP occurs not only in the brain but also in the atrium. Further, both ANP and BNP exhibit significant natriuretic and hypotensive actions, so it has become clear that each of these peptides works not only as a hormone to be secreted from the atrium into blood but as a nerve transmitter in the brain, in either case helping regulate the homeostatic balance of body fluid volume and blood pressure in mammals. The CNP has very recently been isolated and identified from porcine brain by Sudoh et al. as a third type of NP that is assignable to neither ANP nor BNP (Sudoh. T. et al., Biochem. Biophys. Res. Commun., 168 , 863, 1990). The first discovered CNP consisted of 22 amino acid residues (this peptide is hereunder abbreviated as "pCNP-22"). Like ANP and BNP, CNP contained two cysteine residues which formed an intramolecular disulfide bond, producing a ring structure composed of 17 amino acid residues. Further, the primary amino acid sequence forming that ring structure in pCNP-22 was found to be highly homologous to that in ANP and BNP. However, pCNP-22 differs from ANP and BNP in that the latter have several amino acid residues additionally attached to the C-terminus of the ring structure whereas no such "tail structure" is present in pCNP-22. In other words, the C-terminus of pCNP-22 is terminated with a cysteine residue. On the basis of these facts, it has been found that the structure of pCNP-22 is similar to but clearly distinguishable from those of ANP and BNP. In addition, pCNP-22 exhibited natriuretic and hypotensive actions and it was also found to display a higher activity than ANP and BNP in relaxant activity tests on chick rectal samples. From these observations, pCNP-22 was found to be an NP assignable to a new type, the peptides of which were named "CNPs". Following pCNP-22, a second peptide assignable as CNP was isolated and identified from porcine brain by the present inventors (see Minamoto et al., BBRC, 170(2), 1990, p. 973-979). The peptide was found to consist of 53 amino acid residues with pCNP-22 present at the C-terminus (this peptide is hereunder abbreviated as pCNP-53). In other words, pCNP-53 was found to be a peptide that had 31 more amino acid residues attached to the N-terminus of pCNP-22. Interestingly enough, pCNP-53 has been found to occur in a greater amount than pCNP-22 in porcine brain (commonly assigned Japanese Patent Application No. 186582/1990). Another very recent study succeeded in identifying the structure of the precursor of pCNP-22 and pCNP-53 by gene analysis and this helped unravel the mechanism behind the biosynthesis of those peptides (commonly assigned Japanese Patent Application No. 186583/1990 and EP-A-0 466 175). The present inventors isolated and identified a porcine chromosomal gene and cDNA coding for pCNP-22 and pCNP-53; by their analysis, the structure of the porcine CNP precursor protein (prepro pCNP) was unravelled; at the same time, it was found that each of pCNP-22 and pCNP-53 was first translated from mRNA as a prepro pCNP composed of 126 amino acid residues and that the signal peptide present in the N-terminal region of the precursor protein was then cleaved in the process of secretion to be converted to pro pCNP, which was further cleaved specifically with processing enzymes to be converted to pCNP-53 or pCNP-22 as appropriate. On the basis of these observations, it has been found that as in the case of ANP and BNP, both pCNP-22 and pCNP-53 are secretory peptides that are biosynthesized from a common precursor protein (prepro pCNP). However, in sharp contrast with the peptides assignable to ANP and BNP which have been found by previous studies to work not only as hormones to be secreted from the atrium into blood but also as nerve transmitters in the brain, thereby regulating the homeostatic balance of body fluid volume and blood pressure, CNP remains unclear in many points as regards details of its distribution in vivo and physiological actions. As for ANP and BNP, their structures have been established in rats and the physiological actions in rats of rat ANP and BNP have also been unravelled, with the evaluation of their pharmaceutical efficacy being conducted with care being taken to eliminate possible effects due to differences in animal species. However, as of today, the structure of CNP has not been identified in rats. The present invention has been accomplished under these circumstances and has an object identifying the structures of CNPs in rats (rCNP), particularly the structure of their precursor protein which corresponds to prepro pCNP in porcine. Fig. 1a shows the restriction enzyme map for the chromosomal gene of rCNP precursor protein (BamHI DNA fragment) and the region whose base sequence was determined; Fig. 1b shows the determined base sequence and the primary amino acid sequence of rCNP precursor protein encoded by it; and Fig. 2 shows the whole base sequence of CNP cDNA and the primary amino acid sequence of rCNP precursor protein encoded by it. Noting that the amino acid sequence of ANP and the base sequence of the gene encoding it are both retained with high homology across animal species, the present inventors thought that there would be a high likelihood for the same situation to occur in CNP. Based on this assumption, the present inventors projected a program in which the cDNA of rCNP was isolated using the DNA probe (pDC-53) which they used previously in isolating the chromosomal gene or cDNA of porcine CNP and the isolated cDNA was analyzed to identify the structure of the precursor protein of rCNPs. In accordance with this program, the present inventors first screened a rat chromosomal gene library (λ-phage vector incorporating a chromosomal gene fragment) using pDC-53 as a DNA probe and obtained 5 clones that hybridized with pDC-53 under mild conditions. Analysis of one of those clones (λrCNP G3) showed that said clone harbored ca. 14 kbp of the rat chromosomal gene and that a BamHI DNA fragment of ca. 2.1 kbp of that 14 kbp hybridized with the pDC-53 DNA probe. When part of this BamHI DNA fragment of ca. 2.1 kbp was determined for its base sequence, it became apparent that the DNA of interest would code for the rCNP gene. The restriction enzyme map for the BamHI fragment and the region whose base sequence was determined (as indicated by arrows) are shown in Fig. 1a, and the determined base sequence and the amino acid sequence encoded by it are shown in Fig. 1b. In the next step, a library of rat brain cDNA was screened using as a probe a 150-bp DNA fragment coding for rCNP-22 that was sliced from the above-mentioned BamHI DNA fragment with restriction enzymes SmaI and MvaI (the 150-bp DNA fragment is hereunder referred to as rDC-22). As a result, 22 clones were obtained that would hybridize with rDC-22 and of those 22 clones, the one carrying the longest (ca. 1 kbp) cDNA was found to be λrCNP 21. On the basis of this result, the whole base sequence of the cDNA fragment composed of ca. 1 kbp that was carried by said λrCNP 21 was determined. As a result, it was found that the cDNA fragment coded for the whole amino acid sequence of rCNP precursor protein (see Fig. 2). First, the DNA fragment under discussion contained a long open reading frame starting with ATG that existed in positions 1 - 3 of the base sequence. Since this ATG was the first methionine codon that appeared in the cDNA and since the base sequence around that codon was in agreement with the consensus sequence of a translation initiation codon, A/G NNATG (N denotes either one of A, T, G and C), which is known to exist in eukaryotes, the present inventors estimated that the ATG of interest would be a translation initiation codon for the rCNP precursor. The open reading frame of interest was found to encode a polypeptide composed of 126 amino acid residues. It was also found that its primary amino acid sequence was in very good agreement with that of the pCNP precursor. On the basis of these findings, the present inventors were convinced that the cDNA obtained was definitely a cDNA coding for rCNP. As described above, the present inventors isolated rCNP cDNA and analyzed it, thereby successfully identifying the rCNP precursor protein as a polypeptide composed of 126 amino acid residues having the primary amino acid sequence shown in Fig. 2. In the C-terminal region of the thus identified primary amino acid sequence of the rCNP precursor protein (which is hereunder abbreviated as prepro rCNP), rat CNP-22 that was the sequence corresponding to pCNP-22 whose structure was first determined from porcine brain (rCNP-22: positions 105 - 126 of the amino acid sequence shown in Fig. 2) and rat CNP-53 that was the sequence corresponding to pCNP-53 whose structure was also first determined from porcine brain (rCNP-53: positions 74 - 126 of the amino acid sequence shown in Fig. 2) and, in addition, pCNP-22 and pCNP-53 were in complete agreement with rCNP-22 and rCNP-53, respectively, in terms of amino acid sequence. For synthesis of those peptides, it would be indispensable that their precursor be cleaved with processing enzymes. Since the amino acid sequences of prepro rCNP in areas near positions 22 and 53 from the C-terminus of prepro rCNP where the processing would take place were in complete agreement between rat and porcine, the possibility that rCNP-22 or rCNP-53 is synthesized in rat's brain would be extremely high. Further, as in the case of porcine, a region rich in hydrophobic amino acid residues (in positions 10 - 16 of the primary amino acid sequence shown in Fig. 2) was present in the N-terminal region of prepro rCNP; hence, in view of this fact, there is a high possibility that the signal peptide necessary for secretion will exist in the N-terminal region of prepro rCNP. With the above-discussed facts taken into consideration, rCNP-22 and rCNP-53 are presumably biosynthesized by the following pathway. First, prepro rCNP composed of 126 amino acid residues is translated from mRNA. Then, the signal peptide present in the N-terminal region of the prepro CNP is cleaved for conversion to pro rCNP in the process of secretion. Further, the pro rCNP is cleaved by processing enzymes at specific positions (between positions 73 and 74 of the primary amino acid sequence that is shown in Fig. 2 and between positions 104 and 105 of the same sequence) to be converted to rCNP-53 and rCNP-22. In summary, the present inventors isolated cDNA coding for the precursor protein of rCNPs (rCNP-22 and rCNP-53) and analyzed them to identify the primary amino acid sequence of the rCNP precursor protein. At the same time, they successfully identified the structures of rat CNPs (rCNP-53 and rCNP-22) corresponding to porcine CNPs (pCNP-53 and pCNP-22). The present invention has been accomplished under these circumstances. The following examples are provided for the purpose of further illustrating the present invention but are in no way to be taken as limiting. Example 1: Isolation of chromosomal gene coding for rat CNP precursor protein E. coli strain K12 derived LE 392 was infected with a porcine chromosomal gene phage DNA library (product of Clonetech Co.) stored at 4°C. The cells were plated on an LB medium (10 g, bactotryptone; 5 g, yeast extract; 5 g, NaCℓ; 1.5%, bactoagar; total volume, 1 ℓ) and cultivated overnight at 37°C. The plate was cooled at 4°C for 30 min and a nitrocellulose filter (product of Shleicher & Schnell Co.) was left to stand on the phage plaque for 5 minutes. Subsequently, the filter was stripped from the plate, dried with air, immersed in an alkaline denaturation solution (0.5 M NaOH and 1.5 M NaCℓ) for 1 minute, and then immersed in a neutralizing solution (0.5 M Tris-HCℓ; pH 7.0; 1.5 M NaCℓ) for 1 minute. Thereafter, the nitrocellulose filter was rinsed with a 3 × SSC solution (20 × SSC NaCℓ, 175.3 g; trisodium citrate, 88.2 g; total volume, 1 ℓ), dried with air and heat-treated under vacuum at 80°C for 120 min. Using the thus prepared nitrocellulose filter, plaque hybridization was performed under the following conditions. First, a prehybridization solution [3 × SSC; 5 × Denhardt's solution (consisting of albumin, polyvinyl pyrrolidone and Ficoll, each weighing 1 mg/mℓ); salmon sperm DNA, 100 µg/mℓ; 30% formamide; 0.1% SDS] was added to the nitrocellulose filter and prehybridization was conducted at 37°C for 2 hours. Then, using 10 6 cpm of the pDC-53 DNA probe (the method of its preparation is described by the present inventors in commonly assigned Japanese Patent Application No. 186583/1990) and 1 mℓ of the prehybridization solution for two sheets of the nitrocellulose filter, hybridization was performed overnight at 37°C. Subsequently, the filter was washed three times with a 3 × SSC solution containing 0.1% SDS, two washings done at 40°C for 40 minutes and one washing at 55°C for 30 minutes; the washed filter was dried with air and subjected to autoradiography at -80°C for 24 h. By screening ca. 5 × 10 5 clones in this way, eight clones hybridizing with the pDC-53 DNA probe were obtained. One of those clones was named "λrCNP G3" and subjected to analyses in the subsequent stages. Example 2: Analysis of λrCNP G3 phage and determination of its base sequence A. analysis of λrCNP G3 phage DNA DNA was prepared from λrCNP G3 phage in the usual manner. Subsequently, the phage DNA was cleaved with restriction enzymes BamHI, EcoRI and PstI and the resulting DNA fragments were separated and analyzed by electrophoresis on an agarose gel. The λrCNP G3 was found to be a phage containing a ca. 14-kbp rat chromosomal gene. Analysis by Southern blotting using the pDC-53 DNA probe showed that each of BamHI DNA fragment of ca. 2.1 kbp, EcoRI DNA fragment of ca. 19 kbp and PstI DNA fragment of ca. 1.65 kbp hybridized with the pDC-53 DNA probe. The base sequence of part of the BamHI DNA (2.1 kbp) which hybridized with the pDC-53 DNA probe was determined by the following method. B. Determining the base sequence of BamHI DNA fragment In order to determine the base sequence of the BamHI DNA fragment, the latter was first subcloned in a plasmid vector pUC 118 (Takara Shuzo Co., Ltd.) at the BamHI site. From the resulting plasmid (pUC rCNP G3), a SmaI-XbaI DNA fragment (450 bp) hybridizing with the pDC-53 DNA probe was isolated and subcloned to phage vectors M13 mp18 and 19. Using a universal primer, with the resulting single-stranded DNA fragment being used as a template, the DNA base sequence of the BamHI DNA fragment of interest was determined by the dideoxy method with SEQUENASE (United States Biochemical Corporation) and a sequencer kit of Takara Shuzo Co., Ltd. The base sequence of the BamHI DNA fragment of interest which was determined by the above-described method and the amino acid sequence as predictable from that base sequence are shown in Fig. 1b. Example 3: Preparation of DNA probe (rDC-22) The DNA probe (rDC-22) to be used for cloning cDNA coding for the rCNP precursor protein was prepared by the method consisting of cleaving the aforementioned plasmid pUC rCNP G3 with restriction enzymes SmaI and MvaI to isolate a 14-bp DNA fragment and then marking the DNA with [α- 32 P] dCTP using a multi-prime labelling kit of Takara Shuzo Co., Ltd. Example 4: Isolation of rat CNP cDNA A. Preparation of λgt10 cNDA library Using a guanidine-thiocyanate method, 1295 µg of total RNA was extracted and isolated from 2.7 g of rat's brain. Then, using an oligo(dT)-cellulose column, ca. 84 µg of poly(A) + RNA was prepared from 1295 µg of the total RNA. Subsequently, using 4 µg of poly (A) + RNA, a double-stranded cDNA was prepared by the method of Gubler and Hoffman (Gubler, U. et al., Gene, 25 , 263, 1983). A 13-bp EcoRI adapter was ligated to the resulting cDNA and the product was subjected to size fractionation by 1% agarose gel electrophoresis. The thus obtained cDNA composed of 300 - 1500 bp was ligated to a phage λgt10 arm and by subsequent in vitro packaging, a cDNA library was prepared that consisted of 4.7 - 11.1 x 10 6 independent clones per microgram of poly(A) + RNA. B. Screening the cDNA library About 6 x 10 5 clones of the cDNA library obtained in step A were screened using the rDC-22DNA probe prepared in Example 4. The screening method was the the same as adopted in Example 2, except that c600hfl was used as cells and that the concentration of formamide in the hybridization solution was 50%, with 5 × SSPE (20 × SSPE, 3 M NaCℓ, 0.2 M sodium primary phosphate and 0.02 M EDTA). Two washings were conducted with 2 × SSC containing 0.1% SDS at 65°C for 40 min each time. As a result of the screening, a clone was obtained that hybridized with the rDC-22 DNA probe and it was designated λrCNP 21. C. Analysis of λcCNP phage and determination of its base sequence First, DNA was prepared from the λrCNP 21 phage in the usual manner. Cleavage of this DNA with a restriction enzyme EcoRI showed that λrCNP 21 contained ca. 1 kbp of cDNA. For final analysis of the cDNA, the 1-kbp DNA fragment was first subcloned to M 13 phage and then the base sequence of the DNA was determined by a dideoxy method. The thus determined base sequence of the cDNA and the primary amino acid sequence as predictable from it are shown in Fig. 2. As described on the foregoing pages, the present inventors isolated part of the rCNP gene from a rat chromosomal gene library using pDC-53 as a probe. With the isolated rCNP gene being used as a probe, a cDNA coding for the whole region of the rCNP precursor was successfully synthesized and isolated from mRNA derived from the rat brain. As a result, it was verified that a gene coding for CNP also existed in rats as well as in pigs. The present inventors further determined the whole structure of the rCNP precursor protein. As in pigs, the rCNP precursor protein in rats was composed of 126 amino acid residues and contained at the C-terminus the amino acid sequence that corresponded to pCNP-53 and pCNP-22 isolated from porcine brain. Since the amino acid sequences of those peptides at the processing site were in complete agreement between porcine and rat, it is predicted that those peptides would also be biosynthesized in rats through a pathway similar to that involved in pigs and that they would work as hormones or nerve transmitters that regulate the homeostatic balance of body fluid volume and blood pressure in vivo. If the cDNA coding for the rCNP precursor is expressed in animal cells and if the protein or peptide secreted out of the cells is isolated and identified, the physiological activities of three novel peptides that are biosynthesized from the rCNP precursor but which cannot be synthesized from the pCNP precursor (i.e., modified species of prepro rCNP that are specifically cleaved at the C-terminus of lysine residues at positions 24 and 30 of the primary amino acid sequence and arginine residue at position 33) can be investigated. What is worth particular note in the present invention is that the structures of rCNP-53 and rCNP-22 were identified as being entirely the same as those in pigs. This attests to the fact that the physiological activities of pCNP which have so far been evaluated with rats reflect on the correct measurement not influenced by differences in animal species. Further, the sites of expression of CNPs in rats can be examined correctly by using the cDNA coding for the rat CNPs obtained in the present invention. The information obtained by the present invention concerning the cDNA of the rCNP precursor protein and its primary amino acid sequence will make great contributions not only to future studies for unravelling the mechanism behind the biosynthesis and physiological actions of CNPs in mammals but also to the efforts to establish pharmaceutical applications of peptides assignable to the CNP family.

Claims: Claims for the following Contracting States : AT, BE, CH, LI, DE, DK, FR, GB, GR, IT, LU, NL, SE A polypeptide having the following amino acid sequence: A polypeptide according to Claim 1 which is deficient of the peptide at the N-terminus because it is cleaved between any two adjacent amino acid residues in said amino acid sequence at positions of 1 - 48 of the N-terminus. A DNA coding for a polypeptide having the following amino acid sequence: A DNA according to Claim 3 which comprises the following base sequence: A DNA coding for a portion of a polypeptide which has the following amino acid sequence, said portion being one that is left after deleting the peptide at the N-terminus by cleavage between any two adjacent amino acid residues in said amino acid sequence at positions of 1 - 48 of the N-terminus: A DNA having the following base sequence: Claims for the following Contracting State : ES A method for the preparation of polypeptide having the following amino acid sequence: A method according to Claim 1 in which the polypeptide is deficient of the peptide at the N-terminus because it is cleaved between any two adjacent amino acid residues in said amino acid sequence at positions of 1 - 48 of the N-terminus. A method for the production of a DNA coding for a polypeptide having the following amino acid sequence: A method according to Claim 3 in which the DNA comprises the following base sequence: A method for the production of a DNA coding for a portion of a polypeptide which has the following amino acid sequence, said portion being one that is left after deleting the peptide at the N-terminus by cleavage between any two adjacent amino acid residues in said amino acid sequence at positions of 1 - 48 of the N-terminus: A method for the production of a DNA having the following base sequence:

Inventors: KANGAWA KENJI, KOJIMA MASAYASU, MATSUO HISAYUKI, MINAMINO NAOTO, KANGAWA, KENJI, KOJIMA, MASAYASU, MATSUO, HISAYUKI, MINAMINO, NAOTO
Applicants: MATSUO HISAYUKI, SUNTORY LTD, MATSUO, HISAYUKI, SUNTORY LIMITED
Application year: 1991
Main classification: C12N15
IPC codes: C12N15, A61P9, C07K14, A61K38, C07H21, C12P21, A61P7