WO1991014777A1 - Isoforme de chaine a de facteur de croissance derive de plaquettes (pdgf) - Google Patents

Isoforme de chaine a de facteur de croissance derive de plaquettes (pdgf) Download PDF

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Publication number
WO1991014777A1
WO1991014777A1 PCT/JP1991/000381 JP9100381W WO9114777A1 WO 1991014777 A1 WO1991014777 A1 WO 1991014777A1 JP 9100381 W JP9100381 W JP 9100381W WO 9114777 A1 WO9114777 A1 WO 9114777A1
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WIPO (PCT)
Prior art keywords
dna
pdgf
growth factor
platelet
amino acid
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PCT/JP1991/000381
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English (en)
Japanese (ja)
Inventor
Ryozo Nagai
Kenichi Nakahara
Makoto Kuroo
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Yamasa Shoyu KK
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Yamasa Shoyu KK
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Publication of WO1991014777A1 publication Critical patent/WO1991014777A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/49—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to platelet-derived growth factor A protein (Platelet).
  • PDGF-AJ -Derived growth factor A Chain
  • Platelet-derived growth factor is a protein purified from platelet suspension as a growth factor for smooth muscle and fibroblasts released from platelets, and contains two proteins called A-chain and B-chain. It has a structure in which the peptide chains are linked by S—S bonds.
  • PDGF-A a component of PDGF, has potent growth factor activity on mesenchymal cells and is thought to play an important role in the development of atherosclerosis.
  • the cDNA of PDGF-A has been cloned from human glioma cells, human endothelial cells and ovule cells, and the nucleotide sequence of the cDNA encoding PDGF-A and The amino acid sequence of PDGF-A has been elucidated (Nature, 32, 695-6999 (19986), Nature, 32, 619-62) (1989), Science, 241, 1223 to 1225 (19888), etc.).
  • PDGF-A As a result of comparing the amino acid sequences of the revealed PDGF-A, PDGF-A cloned from human glial cells and PDGF-A cloned from human endothelial cells were compared.
  • the amino acid sequence from the amino terminus to the 107th amino acid is the same, and the amino acid sequence near the C-terminal part from the 108th position is different.
  • PDGF-A from endothelial cells with short carboxy terminus is abbreviated as PDGF-A1
  • PDGF-A from glioma cells with long carboxy terminus is abbreviated as PDGF-A2.o
  • PDGF-A showed high homology in its amino acid sequence to PDGF-A2 derived from human glioma, and was included in the group of PDGF-A2.
  • the growth factor activity of PDGF-A on mesenchymal cells depends on the structure of the carboxyl terminus. This part (the part after the 108th position) is short, and PDGF-A1 is long. It is known to have a remarkably low activity as compared to (Nature, 328, 621-1624 (19987)).
  • An object of the present invention is to provide a novel isoform of PDGF-A having a structure different from that of PDGF-A and having strong growth factor activity.
  • Another object of the present invention is to provide a DNA encoding a novel isoform of PDGF-A.
  • the present invention relates to a novel PDGF-A isoform (PDGF-A3) and a precursor protein of the PDGF-A isoform (pro and prebu oral bodies) It is.
  • the present invention also relates to a DNA containing a DNA sequence encoding the PDGF-A isoform of the present invention and a precursor protein (pro-form and prebu-form) of the PDGF-A isoform. It is.
  • FIG. 1 shows an outline of P1 (PDGF-A1) cDNA and P3 (PDGF-A3) cDNA cloned from the fetal avian aorta.
  • the 222 bp cDNA shows the same DNA sequence in P1 and P3 that encode PDGF-A, and the 8 'bp of P1 and the 110 bp of P3 on the 3' end side.
  • DNA shows the DNA sequence of the non-common part.
  • the 60 bp and 204 bp of the 5 'end of P1 are a DNA sequence encoding a signal peptide and a DNA sequence encoding a propeptide, respectively ⁇ Show.
  • Figure 2 is coded by PIc DNA
  • the white triangle indicates the connection site between the signal peptide and the propeptide
  • the black triangle indicates the connection site between the propeptide and PDGF-A1.
  • the single underline indicates a portion that differs from the amino acid sequence of the previously reported propeptide.
  • the double underline indicates the carboxy terminus different from PDGF-A3.
  • Fig. 3 is coded by P3cDNA.
  • This figure shows the amino acid sequence and base sequence of the PDGF-A isoform (PDGF-A3) precursor protein.
  • the white triangle indicates the connection site between the signal peptide and the propeptide
  • the black triangle indicates the connection site between the propeptide and PDGF-A3.
  • the single underline indicates a portion that differs from the amino acid sequence of the previously reported propeptide.
  • the double underlined part is a carboxy terminal different from PDGF-A1.
  • Fig. 4 shows the same PDGF-A gene in vivo.
  • FIG. 3 schematically shows a process in which PDGF-A1 and PDGF-A3 are prepared by selective plating, respectively.
  • FIG. 5 shows the C-terminus of PDGF-A3 of the present invention, human glioma PDGF-A and PDGF-A This is a comparison of amino acid sequences near the part.
  • amino acid is represented by a three-letter code
  • DNA base is represented by a one-letter code
  • the PDGF-A isoform of the present invention has an amino acid sequence of the following formula [I] at the carboxyl terminal or a sequence identical to a part of the amino acid sequence. And a peptide having a function substantially the same as that of the amino acid sequence of the following formula [I] at the carboxyl terminal.
  • ProG1yG1yVa1HisPr0G1nG1yCysLeuArgA1aHisAsG1yCysG1nSerSerArgA sn H is M et G 1 n A 1 a L eu G 1 y T r PL ys Lys Lys M et CI
  • the polypeptide represented by the amino acid sequence of the above formula [I] binds to the amino acid at the 108th position or later counted from the terminal. What is being done is preferred.
  • the amino acid sequence other than the carboxy terminus may be a known amino acid sequence of PDGF-A derived from human endothelial cells, human glioma cells or ovule cells [Nature, 320, 6 9 5-6 9 9 (1 9 8 6), Nature, 3 2 8, 6 1 9-6 2 1 (1 9 8 7);
  • aminoic acid derived from the fetal aorta of the egret embryo cloned according to the present invention may be an array.
  • L eu G 1 u G 1 u H is L e ⁇ G 1 u C ys A 1 a C ys A 1 a A 1 a Serser A 1 a G 1 y P r 0 G 1 ⁇ H is
  • the cysteine residue may exist in a reduced state, or may be in a oxidized state, that is, a molecule between two cystine residues by disulfide bond. Of these, an S-S bond may be formed.
  • one or more independent or continuous amino acids may be deleted, inserted or substituted. Good.
  • the peptide in the precursor protein of the PDGF-A isoform of the present invention is not particularly limited, and this probeptide and the PDGF-A isoform of the present invention are referred to.
  • the PDGF-A isoform of the present invention is converted from the precursor protein of the PDGF-A isoform by the action of the protease. Anything that can guide the team will do.
  • such a peptide is represented by the following formula:
  • Examples include polypeptides consisting of 68 amino acids represented by [III], but are not particularly limited to these polypeptides, as long as they have the same function.
  • one or more independent or continuous amino acids near the amino terminal site may be deleted, inserted or substituted.
  • the signal peptide in the precursor protein of the PDGF-A isoform of the present invention has a function of promoting secretion of the PDGF-A isoform into and out of a host cell.
  • a host cell There is no particular limitation.
  • a polypeptide consisting of 20 amino acids represented by the following formula [IV] is exemplified. Also has a similar function As long as it has, one or more independent or continuous amino acids may be deleted, inserted or substituted.
  • propeptides and signal peptides bind to the amino acid terminus of PDGF-A isoform to form a precursor protein of PDGF-A isoform.
  • the PDGF-A isoform and the propeptide combine to form a pro-PDGF-A isoform
  • the pro-PDGF-A isoform and the signal The peptides combine to form a pre-pro PDGF-A isoform.
  • the PDGF-A isoform of the present invention and a DNA containing a DNA sequence encoding a precursor protein thereof The PDGF-A isoform of the present invention is coded.
  • DNA containing a DNA sequence having a PDGF-A isoform having the amino acid sequence of the above formula [I] at the carboxy terminus is a DNA containing a DNA sequence encoding a PDGF-A isoform.
  • DNA sequence encoding the PDGF-A isoform at the carboxy terminus Contains DNA. This DNA encodes the PDGF-A isoform of the present invention and can express the PDGF-A isoform of the present invention when introduced into a suitable host cell. Any DNA may be used.
  • CTGCCATCTGCAAGACCAGGA CGGTCATTTACGAGATACCTC GGAGTCAGGTGGACCCCACGT CGGCC AA CTTCCTGATCTGGCCGCCGTG CGTGGAGGTCAAGCGCTGCAC CGGCTGTTGCAACACCAGCAG CGTC AA GTGCCAGCCCTCGCGGGTGCA CCACCGCAGCGTCAAGGTGGC CAAGGTGGAGT AT GTCAGAAAGAAGCCCAAGTTG AAAGAAGTGCAGGTGCGGCTG GAGGAGCACCTGGAGTGCGCG TGCGCGGCCTCGAGCGCGGGC CCGGAGCACCGCGAGGAGGAG GCAGGGACCCTCCTGCCCGCG CCTGGTGGCGTCCATCCTCAG GGTTGCCTCAG AGCCCACGATGGCTGCCAGAG C
  • a DNA sequence encoding an acid sequence can be used when the PDGF-A isoform of the present invention is produced by a genetic engineering method.
  • Such a DNA sequence encodes the amino acid sequence of the above formula [I] or an amino acid sequence identical to a part of the amino acid sequence, and comprises the PDGF-A isoform of the present invention. Any DNA sequence may be used as long as it can express the form.
  • PDGF PDGF of the present invention.
  • DNA sequence of the portion coding for the propeptide in such DNA examples include those represented by the following formula [VI].
  • the polypeptide of the formula [IV] consisting of the 20 amino acids described above as a specific example of the signal peptide in the precursor protein of the PDGF-A isoform of the present invention.
  • the DNA containing the DNA sequence encoding the polypeptide may be any that encodes the polypeptide and can express the PDGF-A isoform precursor protein of the present invention. Any type of DNA may be used.
  • the DNA sequence of the portion encoding such a signal peptide in DNA is represented by the following formula [H]. Is exemplified.
  • DNA containing a DNA sequence encoding the PDGF-A isoform as described above can be prepared from cDNA, the triester method, the phosphophyte method, or the like. It can be prepared using a usual method such as a method of chemically synthesizing using the method.
  • the method for preparing a DNA containing the DNA sequence encoding the PDGF-A isoform of the present invention will be described with reference to the method for preparing from cDNA as an example.
  • Preparation, 2 PDGF — can be carried out by appropriately combining conventional methods such as selection of a cDNA clone containing a DNA sequence that encodes the A-isoform. You.
  • Preparation of the DNA library can be performed according to a conventional method. For example, total RNA is extracted from the fetal blood vessel of an animal according to a conventional method, and the extracted RNA is passed through an oligo (dT) cellulose column to purify poly (A) RNA. Obtained m Let the RNA be type II, and let the double-stranded DNA be, for example,
  • Selection of cDNA clones containing the DNA sequence encoding the PDGF-A IT form from the DNA library can be performed by plaque hybridization using a phage vector. Method (Maniates et al., Molecular cloning, Cold Spring Harbor Laboratory,
  • the probe used for screening is a known PDGF-A Synthetic oligonucleotides of 40- to 60-mer prepared with reference to the nucleotide sequence of the isoform can be used.
  • a clone to be hybridized is selected using the probe, the recombinant phage DNA (or recombinant plasmid DNA) is prepared according to a conventional method, and the inserted cD
  • the nucleotide sequence of the NA fragment was determined by the dideoxythiene terminator method (Sager, F. et al., Proc. Natl. Acad. Sci. USA, 74, 5463 (19777)) or the Determined by the Girno coat method (Methods in Enzymology, _6_5_, 497 (1980)).
  • the thus obtained cDNA sequence DNA encoding PDGF-A isoform or open reading frame of PDGF-A isoform precursor protein After the DNA is introduced into an appropriate expression vector, host cells (such as Escherichia coli, yeast, and Bacillus subtilis) are transformed using the obtained recombinant plasmid. The transformant is cultured to express the PDGF-A isoform of the present invention or its precursor protein.
  • the obtained PDGF-A isoform or its precursor protein of the present invention can be isolated and purified by a conventional method for isolating and purifying a physiologically active substance. It can be.
  • RNA was extracted from the aorta extirpated from a 4 week gestational heron embryo kept at 70 ° C according to the thermal phenol method.
  • the extracted RNA was prepared according to the method of Maniatis et al.
  • the recovered DNA was used as a 50 a1 reaction solution (67 mM potassium phosphate buffer (pH 7.4), 6.7 mM magnesium chloride, 1 mM 2_mercaptoethanol, 3 3 / Md NTP, 2 units of Klenow fragment (manufactured by Boehringer Mannheim)]] at 37 ° C for 1 hour to blunt the cDNA fragment. Terminated. 50 blunt-ended cDNA fragments
  • the cDNA fragment was Ec0RI terminated.
  • Ec0RI-terminated cDNA fragment was treated with EcoRI and alkaline phosphatase; IZap DNA was mixed, and the reaction mixture was mixed at 50 / zl [66 mM tris-HCl Buffer (H7.6), 6.6 mM magnesium chloride, 10 mM DTT, 0.1 mM ATP, 100 units of T4 DNA ligase] at 16 ° C for 12 hours I let it.
  • This reaction solution was subjected to ADNA Invitrono II according to the method of Williams et al. (Genetic Engineering, 2, PI enum Press (1989)). Invitrono, using a packaging kit (in vitro Packaging kit (manufactured by Aramasham)). Escherichia coli Y1088 (ATCC No. 37195) was transduced to prepare a cDNA library of 5 ⁇ 105 or more.
  • a plaque hybridizer probed with a 50-mer oligonucleotide synthesized from PDGF-A Screening was carried out using the molecular method (Molecular Cloning, described above). As a result, 2 plaques were pro portion and hives re Dizu in 2 X 1 0 5 plaques. Recombinant phage DNA was prepared from the probe and hybridized clones according to the plate lysate method (Molecular cloning, described above).
  • the inserted Ec0RI fragment was obtained from Plasmidec Yuichi pBluescript SK (-) (Nucleic Acide Res., 12, 70-35 to 7056 (1994)): Stratagene was subcloned and structural analysis was performed by digestion with various restriction enzymes. As a result of the analysis, the nucleotide sequence of the inserted cDNA fragment of two clones, P1 and P3, having different restriction enzyme cleavage maps was determined by the dideoxythiamine terminus method (Sanger, F., et al. , Pro Nat 1. Acad.
  • P1 has a total length of 1 In Kb, a signal peptide consisting of a 5 'untranslated region and 20 amino acids, followed by a propane peptide consisting of 68 amino acids and a PDGF-A isoform It encodes a PDGF-A isoform precursor protein consisting of (see Figure 1).
  • This precursor protein of PDGF-A is cleaved by peptide enzyme between arginine, the 68th amino acid, and the next threonine, resulting in 110 amino acids. It was thought to be secreted as PDGF-A, which is composed of amino acids (see Fig. 2).
  • This PDGF-A isoform is derived from human endothelial cell-derived PDGF-A isoform (Tong B. D et al, Nature, 328, 619-621, (19) 87))) and 90% homology, and the carboxyl termini of both were identical. Therefore, this PDGF-A isoform is considered to belong to the group of PDGF-A1.
  • P 3 has a total length of 2 Kb
  • P3 As shown in (PDGF-A3), the coding at the 5 'end was initiated from the middle of the PDGF-A precursor protein.
  • P3 also encodes the 60 bp and 204 bp DNA sequences encoding the same signal peptide and propeptide as P1 at 5 ′. It has been clarified that it has the terminal side.
  • P3 is between the 5 'end and the 386th base, that is, from the N-terminal of the final mature PDGF-A to the 107th amino acid Is the nucleotide sequence and amino acid sequence Both were completely consistent with those of P 1.
  • the amino acid sequence between the 387th base and the 497th base of P3 is a specific sequence not present in P1, and the 498th base is not present in P1.
  • the amino acid sequences of P1 and P3 were identical.
  • the PDGF-A encoded by P1 becomes a carboxyl-terminus with the sequence of aspartic acid mono-arginine after the 108th amino acid, whereas P3 After diverging from the common part with P1, PDGF-A becomes a carboxyl terminus with 38 amino acid sequences (see Fig. 3).
  • P1 and P3 are generated by selective splicing from the same PDGF-A gene, and the nucleotide sequence specific to P3 is PDGF-A It may be coded by an independent exon in the gene (see Fig. 4).
  • the PDGF-A3 force coded by this P3 is the PDGF-A isoform of the present invention.
  • P D G F A has been used to date as a human immunoglobulin (Nature, 320, 695-699 (19986)), endothelial cells
  • PDGF-A 3 is the previously reported PDGF — It was completely different from the Aisoform (A1 or A2) in the structure of the carboxyl terminus. PDGF-A2 is also expressed in egg cells and shows high homology with the human glioma PDGF-A2 (see Fig. 5). This is probably because the peculiar structure of the carboxyl terminus of PDGF-A3 derived from the egret blood vessel is not due to the species difference but to the origin of the blood vessel.
  • PDGF-A has potent growth factor activity on mesenchymal cells and is thought to play an important role in the development of atherosclerosis.
  • the growth factor activity of PDGF-A depends on the structure of the carboxyl terminus, and the shorter PDGF-A1 is much less active than the longer PDGF-A2 This is known (Nature, 328,
  • PDGF-A3 also exhibits unique growth factor activity and expression patterns, and is useful as a reagent for studying the development of atherosclerosis.
  • PDGF-A isoform precursor protein has a different amino acid sequence from previously reported human PDGF-A precursor protein. Two amino acids, alanine-proline, have been inserted. For this reason, the precursor protein of the PDGF-A isoform derived from blood vessels of the present invention is useful for preparing a specific antibody against the precursor protein, and the precursor protein is used as the precursor protein.
  • the DNA to be coded is the genetic engineering technique of the present invention.

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Abstract

Un cADN qui code le PDGF-A3, une isoforme nouvelle de la chaîne A de facteur de croissance dérivé de plaquettes, a à présent été reproduit à partir de l'aorte d'un f÷tus de lapin. Le PDGF-A3 a une séquence d'acide aminé différente de celle de la chaîne A de facteur de croissance dérivé de plaquettes connue à l'extrémité C de celle-ci, et est supposé présenter une activité de facteur de croissance spécifique contre les cellules de mésenchyme.
PCT/JP1991/000381 1990-03-26 1991-03-25 Isoforme de chaine a de facteur de croissance derive de plaquettes (pdgf) Ceased WO1991014777A1 (fr)

Applications Claiming Priority (2)

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JP2/76314 1990-03-26
JP7631490 1990-03-26

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JPN CIRC. J., Vol. 54, 1990, NAKAHARA K. et al., "Characterization of Two Types of Vascular PDGF-A Chain Isoform Complementary DNA Clones", p. 713-714. *
NATURE, Vol.320, 1986, BETSHOLCZ C. et al., "cDNA Sequence and Chromosomal Localization of Human Platelet-Derived Growth Factor A - Chain and its Expression in Tumor Cell Lines", p. 695-699. *

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