WO1996012797A9 - Production et utilisation de methyltransferases de l'homme et des plantes - Google Patents

Production et utilisation de methyltransferases de l'homme et des plantes

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Publication number
WO1996012797A9
WO1996012797A9 PCT/US1995/013691 US9513691W WO9612797A9 WO 1996012797 A9 WO1996012797 A9 WO 1996012797A9 US 9513691 W US9513691 W US 9513691W WO 9612797 A9 WO9612797 A9 WO 9612797A9
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methyltransferase
isoaspartyl
protein
aspartyl
human
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PCT/US1995/013691
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English (en)
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WO1996012797A1 (fr
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Priority to JP8514113A priority Critical patent/JPH10507919A/ja
Priority to EP95939576A priority patent/EP0796322A4/fr
Priority to AU41341/96A priority patent/AU4134196A/en
Publication of WO1996012797A1 publication Critical patent/WO1996012797A1/fr
Publication of WO1996012797A9 publication Critical patent/WO1996012797A9/fr

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  • the present invention relates to the production and use of methyltransferases. More specifically, the invention relates to the production, purification and use of recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase, an isolated polynucleotide coding for a plant L-isoaspartyl protein methyltransferass and a purified plant L-isoaspartyl protein methyltransferase.
  • Background of the Invention Proteins undergo spontaneous thermodynamically driven changes over time that can result in decreased functionality (Harding, JJ. (1985) Adv.Protein Che . 37:247-334).
  • the enzymic methylation reaction represents the first step of a process that results in the conversion of these altered aspartyl residues to normal L-aspartyl residues (McFadden, et al. (1987) ProcNatl. Acad.Sci.USA 84:2595-2599, Johnson, et al. (1987) J.BioLChem 262:5622-5629, Lowenson, et al.
  • Residue 22 can be either isoleucine (I) or leucine (L); residue 119 can either be isoleucine or valine (V); and residue 205 can either be lysine (K) or arginine (R) (Tsai and Clarke (1994) Biochem. Biophys. Res. Commun. 203:491497).
  • each isozyme described above can exist in at least eight forms (' 2*119* ⁇ 205- '22H 19 ⁇ 205' '22 ⁇ 119 ⁇ 205- '22 ⁇ 119 ⁇ 205' L 22 l 11 g 2 05- L 22' 119 R 205- L 22 V 119 205> L 22 V 119 R 205*-
  • FIGURE 1 shows construction of the human L-isoaspartyl/D-aspartyl methyltransferase expression plasmid, pDM2x of the present invention.
  • a 107bp Kpnl-Narl fragment is removed from plasmid pDM2 and replaced with a synthetic polylinker containing ribosome binding and initiator sites.
  • FIGURE 2 shows the novel polylinker fragment used in the present invention.
  • the polylinker contains multiple cloning sites [Kpn ⁇ , Kho ⁇ , Xba ⁇ , BamW ⁇ , Nhe ⁇ , Nco ⁇ ) and a strong prokaryotic ribosomal binding site (Shine, et al. (1974) Proc. Natl. Acad. Sci. USA 71:1342-1346).
  • FIGURE 3 shows the nucleotide and deduced amino acid sequence of the human L-isoaspartyl/D-aspartyl methyltransferase expression plasmid, pOM2x, of the present invention (SEQ ID N0:8).
  • FIGURE 4 shows the effect of isopropyl ff-D-thiogalactopyranoside (IPTG) concentration on the level of human L-isoaspartyl/D-aspartyl methyltransferase production in £. coli.
  • FIGURE 5 shows E. coli growth percent of total soluble protein obtained as human methyltransferase, and yield of human methyltransferase in the pDM2x expression system of the present invention as a function of time after IPTG induction.
  • IPTG isopropyl ff-D-thiogalactopyranoside
  • FIGURE 6 shows optimization of the protamine sulfate precipitation step according to the present invention.
  • FIGURE 7 shows optimization of ammonium sulfate precipitation fractionation of human L-isoaspartyl/D- aspartyl methyltransferase from the protamine sulfate clarified supernatant according to the present invention.
  • FIGURE 8 shows the anion exchange column purification step of the human L-isoaspartyl/D-aspartyl methyltransferase according to the present invention.
  • FIGURE 9 shows the pH dependence for concentrating purified human L-isoaspartyl/D-aspartyl methyltransferase according to the present invention.
  • FIGURE 10 shows electrospray mass spectral analysis of purified recombinant human methyltransferase of the present invention.
  • A A portion of the spectrum of material purified using dithiothreitol.
  • B A portion of the spectrum of material purified when 15mM 0-mercaptoethanol was substituted for 0.1 ⁇ M ditiothreitol in buffer A.
  • FIGURE 11 shows the ultraviolet absorbance spectrum of the purified human L-isoaspartyl/D-aspartyl methyltransferase of the present invention.
  • FIGURE 12 shows the purification of wheat germ L-isoaspartyl methyltransferase according to the present invention.
  • A DEAE-52 anion exchange cellulose treatment.
  • B Reverse ammonium sulfate gradient solubilization treatment.
  • C Sephacryl S-200 gel filtration.
  • FIGURE 13 shows the Polypeptide analysis of the purification of L-isoaspartyl methyltransferase from wheat germ according to the present invention.
  • FIGURE 14 shows the DNA sequencing strategy of the wheat germ methyltransferase cDNA insert employed in the present invention.
  • FIGURE 15 shows an alignment of the sequenced peptide fragments of L-isoaspartyl methyltransferase from wheat germ and its predicted amino acid sequence from pMBML
  • One particular objective of the present invention is to provide an isolated recombinant human L- isoaspartyl/D-aspartyl protein methyltransferase by expression of the cDNA encoding the enzyme.
  • this expression system permits an extremely efficient large-scale production of a pure recombinant enzyme.
  • the structure of the recombinant enzyme is different from that of the purified enzyme from human erythrocytes only at the N- ter inal alanine residue where the recombinant enzyme is not modified by the post-translational modification of acetylation.
  • this enzyme is suitable for use in human studies without the potential problem of antigenicity.
  • Other objectives of the present invention include the identification of cDNA encoding wheat L-isoaspartyl protein methyltransferase, and the provision of a purified plant L-isoaspartyl protein methyltransferase from wheat. High levels of methyltransferase are found in wheat, especially in seeds.
  • Still other objectives of the present invention are to provide a pharmaceutical preparation containing the enzyme to treat disorders resulting from protein degradation, and to provide an analytical tool for quality control of protein and peptide pharmaceuticals and for diagnosis of disease states associated with protein degradation.
  • Other objectives of the present invention will become apparent to one having ordinary skill in the art upon reference to the ensuing detailed description of the invention.
  • one aspect of the present invention is an isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase obtained by expression of a polynucleotide having a sequence selected from the group consisting of SEQ ID N0:1 (isozyme I) and SEQ ID N0:2 (isozyme II), each including a total of 17 nucleotide sequences coding for eight amino acid sequences of isozyme I or II, i.e., I 2 2' 119 ⁇ 205' '22'119 R 205- '22 v 119 K 205'
  • Another aspect of the present invention is an isolated recombinant human L-isoaspartyl/D-aspartyl protein methyltransferase having an amino acid sequence selected from the group consisting of SEQ N0.3 (isozyme I) and SEQ ID N0.4 (isozyme II), each including eight amino acid sequences as above.
  • Another aspect of the present invention is an isolated polynucleotide having the coding sequence of the sequence indicated as SEQ ID N0:5, which codes for a plant L-isoaspartyl protein methyltransferase.
  • the 690 base pairs of this sequence beginning with the ATG codon at base numbers 117-199 and ending with the AGC codon prior to the TGA codon at positions 807-809 represent the coding sequence of this polynucleotide.
  • Another aspect of the present invention is an isolated recombinant plant L-isoaspartyl protein methyltransferase obtained by expression of a polynucleotide having the sequence indicated as SEQ ID N0:5.
  • Another aspect of the present invention is a purified plant L-isoaspartyl protein methyltransferase from wheat germ having the amino acid sequence indicated as SEQ ID NO: 6 or 7.
  • Another aspect of the present invention is a method of recombinantly producing human L-isoaspartyl/D- aspartyl protein methyltransferase, comprising: modifying a plasmid such as plasmid pDM2 (Genebank accession # S37495) that contains the full coding region of human L-isoaspartyl/D-aspartyl protein methyltransferase, using oligonucleotides, to provide multiple cloning sites, an efficient ribosome binding site, and a strong translational initiator region, said initiator region being designed to function in bacterial and/or eukaryotic expression system; transfecting the constructed vector into a host that contains an isopropyl j?-D-thiogalactopyranoside (IPTG)- inducible T7 polymerase gene; and inducing overexpression of the methyltransferase with IPTG, whereby the methyltransferase is produced.
  • IPTG isopropy
  • the enzyme is preferably obtained from an overexpressed human cDNA in £ coli such as BL2KDE3) grown in LB Broth. Further efficient and economical expression is achieved using a richer media, e.g., terrific broth (Sambrook, et al. (1989) "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory), which results in a higher final cell density and a longer exponential growth and methyltransferase production phase.
  • a richer media e.g., terrific broth (Sambrook, et al. (1989) "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory), which results in a higher final cell density and a longer exponential growth and methyltransferase production phase.
  • the final result can be a preparation where the human methyltransferase makes up about 10-30% of the total bacterial soluble protein.
  • Another aspect of the present invention is a method of purifying recombinantly produced human L- isoaspartyl/D-aspartyl protein methyltransferase present in a lysed bacterial extract in which the methyltransferase expression has been performed, comprising: adding a nucleotide precipitant such as protamine sulfate or polyethyleneimine to the extract to remove DNA present in the extract subsequent to removing the cellular debris; precipitating the methyltransferase with ammonium sulfate; removing the ammonium sulfate by dialysis; and purifying the methyltransferase from the dialysate using anion-exchange chromatography under novel conditions.
  • a nucleotide precipitant such as protamine sulfate or polyethyleneimine
  • Another aspect of the present invention is a method of purifying plant L-isoaspartyl protein methyltransferase from wheat, comprising: obtaining a crude cytos ⁇ i from raw wheat germ; fractionating the crude cytosol by DEAE-cellulose chromatography; adding ammonium sulfate to the pooled active fractions in the presence of a protein carrier; fractionating the resulting material by reverse ammonium sulfate gradient solubilization; and purifying the pooled active fractions by gel filtration chromatography, whereby the methyltransferase is purified as a monomeric 28,000 Da species.
  • Another aspect of the present invention is a method of treatment for a medical conditions associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue, comprising administering to the tissue an amount of methyltransferase, with or without S-adenosybnethionine, sufficient to convert said L-isoaspartyl/D-aspartyl residues to L-aspartyl residues in the tissue.
  • a method of diagnosis of disease states where L-isoaspartyl/D-aspartyl residues are accumulated comprising measuring the content of L- isoaspartyl/D-aspartyl residues accumulated in a disease associated protein, by using methyltransferase as a probe.
  • Another aspect of the invention is a method of determination of degradation of pharmaceutical polypeptides, comprising measuring the content of L-isoaspartyl and D-aspartyl residues in the polypeptides, by using methyltransferase as a probe.
  • Still another embodiment of the invention is a pharmaceutical preparation for treatment of a medical condition associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue, comprising human L-isoaspartyl/D-aspartyl protein methyltransferase, preferably with its substrate S-adenosylmethioni ⁇ e, and pharmaceutically acceptable carriers.
  • the methyltransferase can be used as a sensitive analytical probe of L-isoaspartyl and D-aspartyl residues in quality control of protein and peptide pharmaceuticals.
  • the uses in medical diagnostics for assaying for altered proteins and peptides in biological fluids such as the /.-amyloid product (Roher, A.E., et al. (1993) J.BioLChem. 268:3072-3083) is possible. Because the endogenous human methyltransferase is limited to the cytosol (Clarke, S.
  • the plasmid pBluescript SK(-) (pDM2: Genebank accession tt S37495) contains the entire coding region (SEQ ID N0:9) for the more acidic isozyme II of the human L-isoaspartyl/D-aspartyl methyltransferase (SEQ ID N0.9) (MacLaren, et al. (1992) Biochem. Biophys.Res.Commun. 185:277-283.).
  • This plasmid is obtained from a cDNA library derived from HUMAN brain tissue (Stratagene, La Jolla, CA) using a mouse cDNA as a probe.
  • a plasmid containing cDNA encoding isozyme I (SEQ ID N0:1) or isozyme II (SEQ ID N0.2) having sequences other than pDM2 (SEQ ID N0:9) can be used to produce isozyme I having the sequence indicated as SEQ ID N0:3 or isozyme II having the sequence indicated as SEQ ID N0:4 so that each isozyme can be produced in at least eight forms '22 l 119 R 205- '22 V 119 K 205- l 22 V 119 R 205- L 22 !
  • isozyme III with a -R C-terminus (226 residue long) can be produced in the same manner.
  • Such plasmids include pBK phage vector (Strategene) and the pTrc99A expression plasmid (Pharmacia, Piscatway, NJ). £ coli is preferred for expression of human methyltransferase because characterization of isozyme II purified from human erythrocytes has shown that it is not post-translationally modified (Ingrosso, et al. (1989) J.Biol.Chem.
  • the £ coli expression system can produce the recombinant methyltransferase nearly identical to the human enzyme.
  • the pDM2 plasmid already contains a T7 promoter site ' in the proper position and orientation for transcription of the insert cDNA but does not contain a ribosomal binding site (MacLaren, et al. (1992) Biochem. Biophpys.Res.Commun. 185:277-283.).
  • the pDM2 plasmid is modified to give the overexpression vector, pDM2x, by replacing the region between the T7-promoter site and the start codon of the enzyme with a synthetic fragment containing a strong ribosomal binding site (Hine, et al. (1974) Proc.Natl.Acad.Sci. USA 71:1342-1346.) (Fig. 1).
  • the KpnINa ⁇ fragment from pDM2 is replaced with a synthetic linker containing multiple cloning sites, a eukaryotic initiator site, and a strong ribosomal binding site (Fig. 2).
  • pDM2 is a pBiuescript SK(-) (Stratagene) plasmid containing the human methyltransferase isozyme II cDNA inserted into its Eco ⁇ . ⁇ sites.
  • This replacement fragment has been engineered to also possess multiple cloning sites for the insertion the methyltransferase cDNA (Fig. 3) into different expression systems and organisms.
  • the £? ⁇ RI site at the 3'-end of the sequence shows 7 bases that are part of the pBiuescript SK(-) cloning plasmid.
  • the regions 5' from the Kpn ⁇ restriction site, including the T7-promoter site, are part of the pBiuescript SK(-) plasmid also.
  • the fragment has the sequence SEQ ID N0:8.
  • the nucleotide and translated amino acid sequence of the pDM2x expression plasmid from the T7-promoter site to the 3' Eco ⁇ U linker on the tail of the human cDNA insert is shown in Fig. 3.
  • the structure of the modified region of pDM2x and the overall plasmid structure have been confirmed by DNA sequence analysis and restriction endonuciease digest analysis, respectively. Any synthetic polylinker containing ribosome binding and initiator sites capable of being inserted into a methyltransferase-encoding vector are within the scope of the present invention.
  • the T7 RNA polymerase-driven expression plasmid is then transfected into £ coli strain BL2KDE3) (Studier, et al. (1986) J. Mol. Biol.
  • This strain of bacteria contains a phage T7 RNA polymerase gene in the chromosome under the control of the isopropyl /?-D-thiogalactopyranoside (IPTG)-inducible /acUV5 promoter.
  • IPTG isopropyl /?-D-thiogalactopyranoside
  • Other bacterial host strains containing an IPTG inducible T7 polymerase gene are also contemplated.
  • it is possible to use a dual plasmid system where the T7 polymerase is encoded behind a heat inducible promoter on plasmid pGP1 -2. This plasmid can be transformed into a variety of bacterial strains.
  • Methyltransferase Purification The initial batch purification steps used here to enrich the methyltransferase fraction in the lysed bacterial extract is a modification of the procedure used by Fu, et al. (Fu, et al. (1991) J.BioLChem. 266:14562-14572.). After lysis of the overexpressing bacteria by sonication, the cellular debris is removed by centrifugation. The nucleic acids remaining in the supernatant are removed by addition of a precipitant, protamine sulfate or polyethyleneimine, and centrifugation.
  • the amount of protamine sulfate required is optimized, and it is found that addition of 0.1 volumes of a 4% solution of protamine sulfate gives a good purification of methyltransferase in essentially quantitative yield.
  • the methyltransferase then is concentrated and purified further by precipitation with ammonium sulfate. Again, conditions for this step in preliminary experiments are optimized, and it is found that although the best purification occurs between 50 to 55% saturation, the best compromise between yield and purification occurs at 60% saturation and these conditions are used in the present large-scale purification.
  • the pelleted protein is then resuspended in a small volume of buffer A and dialyzed against buffer A to remove the ammonium sulfate. Then a rapid, one-step column procedure for purifying the methyltransferase from the dialysate by using
  • DEAE-cellulose anion exchange chromatography is performed. It is found that application of traditional methodologies where the enzyme was bound to a column in a noninteracting cationic buffer resulted in incomplete purification. However, the use of an interacting anionic buffer under nonequilibrium conditions was found to result in the isocratic elution of homogenous enzyme. If the chromatography is performed where the column is fully equilibrated before loading, then the methyltransferase is only slightly retarded and elutes close to the void volume along with small amounts of contaminating polypeptides (data not shown). Identification of L-lsoaspartyl Methyltransferase in Plants
  • Peptide-depe ⁇ dent L-isoaspartyl methyltransferase is found in the vegetative cells of the green alga C. reinhardtii, demonstrating its presence in at least one species in the Kingdom Protista.
  • methyltransferase activity is detected in both classes of the angiosperms, the monocots and the dicots. The level of activity in different tissues varies considerably. Of the species assayed, the highest specific activity of the methyltransferase is found in wheat embryos (germ). In contrast, almost no detectable L-isoaspartyl peptide-specific methyltransferase activity is found in the leaves of lettuce or the fruits of tomato.
  • Peptide-dependent L-isoaspartyl methyltransferase activity is highest in mature wheat seeds and the activity is significantly reduced following imbibition and germination.
  • Northern analysis shows that methyltransferase mRNA is expressed as a single 1200 nucleotide species only in seeds, and not in whole seedlings, leaves, or roots. The levels of the enzyme vary depending on stages of caryopsis development, and the highest level of methyltransferase mRNA is detected in stage IV seeds whose embryos have reached maximal size while no methyltransferase mRNA is detectable at stage II.
  • methyltransferase gene expression is increased approximately two fold over the effect of either agent alone.
  • the additive effect of a combined ABA-NaCI treatment suggests that the methyltransferase gene may be a salt-responsive gene in addition to an ABA-responsive gene.
  • the hormonal and environmental stress necessary for inducing expression of methyltransferase is preferably an ABA concentration of 10-100 ⁇ M, a salt concentration of 0.1-1 M, or a dehydration time of 5-24 hours (Although dehydration may be larger for other plants up to 7 days).
  • Active fractions are then saturated to 60-100% , preferably to 80%, with ammonium sulfate in the presence of a protein carrier such as Celite 545, poured into a column, and fractionated by reverse ammonium sulfate gradient solubilization at room temperature (Fig. 12B).
  • Active fractions containing approximately 20-50%, preferably 26-31 % saturated ammonium sulfate can be further purified preferably by using Sephacryl S-200 gel filtration chromatography, although other gel filtration materials are also contemplated.
  • the L-isoaspartyl methyltransferase elutes in a highly purified state in a fraction nearly corresponding to the total volume of the column.
  • This step is unique in that the methyltransferase is not fractionated on the basis of its size. Rather it is suggested that the methyltransferase associates with the Sephacryl S-200 resin through hydrophobic interactions due to a solvent effect created by the relatively high concentration of ammonium sulfate in the fractions (Belew, et al. (1978) J. Chromatogr. 147, 205-212). In the absence of ammonium sulfate, the methyltransferase elutes from the Sephacryl S-200 column in a position consistent with a monomeric molecular weight, along with numerous contaminating polypeptides. Thus, the successful isolation of a highly purified enzyme preparation from the gel filtration column is attributed to this unusual absorption phenomenon.
  • This polypeptide corresponds to the L-isoaspartyl methyltransferase as assessed by renaturing individual gel slices in the presence of Triton X-100 as described by Clarke (Clarke (1981) Biochim. Biophys. Acta 670, 195-202).
  • the purity of this preparation can be as high as 80-100%, estimated from densitometry of the Coomassie-stained gel (Fig. 13).
  • the DNA sequence of the 952-bp cDNA insert in the plasmid pMBMI is determined using the sequencing strategy shown in Fig. 14.
  • the DNA sequence of the methyltransferase cDNA and its deduced amino acid sequence are indicated as SEQ ID N0s:5 and 6, respectively.
  • the calculated molecular weight of the 230 amino acid polypeptide deduced for the 690-bp open reading frame is 24,710.
  • purified methyltransferase migrated as a 28,000 Da polypeptide as determined by SDS-pol ⁇ acrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS-pol ⁇ acrylamide gel electrophoresis
  • the methyltransferase cDNA insert is inserted into well-known prokaryotic expression vectors as described for the human enzyme and used to transform competent £ coli, followed by induction of the T7 polymerase gene with IPTG.
  • the expression of the plant enzyme can be done in exactly the same manner as the human enzyme.
  • the bacterial expression system is designed to express cDNA sequences regardless of their phylogenetic origin.
  • the expressed recombinant protein is purified as described above.
  • Methyltransferase catalyzes the S-adenosylmethionine-dependent methylation of atypical L-isoaspartyl and D-aspartyl residues in peptides and proteins. This reaction can not only be used as an analytical tool to detect the presence of these altered residues in aged and stressed proteins, but can also initiate a non-enzymatic pathway that can result in the conversion of these residues to normal L-aspartyl residues.
  • the methyltransferase enzymes of the present invention can be used in connection with the determination of L-isoaspartate and D-aspartyl residues in peptides as disclosed in U.S. Patent No. 5,273,886 to Aswad, incorporated herein by the previous reference thereto above. Briefly, this method involves breaking the polypeptide into fragments using a proteolytic enzyme and then quantitatively methylating the isoaspartyl residues in the fragments using a methyltransferase enzyme. The total amount of methyl groups incorporated into the fragments is an indication of the amount of isoaspartyl residues in the polypeptide.
  • the amount of isoaspartyl residues in the polypeptide can be used as an indication of the amount of damage to proteins, such as those used in therapeutic applications.
  • the structure of the recombinant enzyme is different from that of the purified enzyme from human erythrocytes at the N-terminal alanine residue, and, as determined by electrospray mass spectroscopy, the recombinant enzyme does not contain covalent post-translational modifications. Thus this enzyme is suitable for use in human studies without the potential problem of a ⁇ tige ⁇ icity. Because the endogenous human methyltransferase is limited to the cytosol (Clarke, S.
  • treatment for a medical condition associated with an increase in L-isoaspartyl/D-aspartyl residues of polypeptides in a tissue can be performed.
  • medical conditions include those resulting from crosslinki ⁇ g of matrix proteins and degradation of flexibility of skin tissues such as cataracts, Alzheimer's disease and the like.
  • the human or plant enzymes can be used, and the dosage of the enzyme is such that the concentration of the enzyme in the preparation is in the range of 0.440 ⁇ M.
  • the enzyme can be formulated simply in the form of an ointment with S-adenosylmethionine and a pharmaceutically acceptable carrier.
  • a typical ointment can contain the enzyme in an amount of 0.001-10% by weight and S-adenosylmethionine in an amount of 0.00004-0.4% by weight.
  • human enzyme is preferably used in an amount such that the concentration of the enzyme in the extracellular space is in the range of 0.4-40 ⁇ M.
  • the enzyme can be provided as an injectable solution typically containing the enzyme in an amount of 0.001 -10% by weight and S- adenosylmethio ⁇ ine in an amount of 0.00004-0.4% by weight in a pharmaceutically acceptable carrier.
  • injectable solution typically containing the enzyme in an amount of 0.001 -10% by weight and S- adenosylmethio ⁇ ine in an amount of 0.00004-0.4% by weight in a pharmaceutically acceptable carrier.
  • L-isoaspartyl and D-aspartyl residues can accumulate in the amyloid protein of Alzheimer's disease. Since a fraction of /9-amyloid protein is found in the cerebrospinal fluid (CSF), it may also be possible to treat Alzheimer's disease by injecting the enzyme into the CSF.
  • CSF cerebrospinal fluid
  • Another medical condition is degradation of flexibility in a vascular system
  • human enzyme is preferably used in an amount such that the concentration of the enzyme in an erythrocyte, endothelial tissue, coronary artery tissue, immune cells, receptors of all cells or lungs is in the range of 0.440 ⁇ M.
  • the enzyme can be provided as an injectable intravenous solution typically containing the enzyme in an amount of 0.001-10% by weight and S-adenosylmethionine in an amount of 0.00004 0.4% by weight in a pharmaceutically acceptable carrier.
  • the solution can be administered by means of a catheter or direct injection.
  • human enzyme is preferably used in an amount such that the concentration of the enzyme in egg or sperm cells is in the range of 0.440 ⁇ M.
  • the enzyme can be provided as an injectable solution typically containing the enzyme in an amount of 0.001 -10% by weight and S-adenosylmethionine in an amount of 0.00004-0.4% by weight with a pharmaceutically acceptable carrier.
  • L-isoaspartyl and D-aspartyl residues are accurately recognized by methyltransferase, it is possible to determine the presence of these damaged residues in pharmaceutical polypeptides so that the purity and shelf-life of such protein products can be verified.
  • These assays are performed by incubating the pharmaceutical preparation with S-adenosyl [ C-methyl ⁇ ethionine in the presence of the purified methyltransferase and determining the radioactivity transferred to the pharmaceutical. This is done by incubating the reaction products with an alkaline solution to release bound methyl esters as radioactive methanol, which is then collected in scintillation fluid as described (Lowenson, et al. (1991) J. Biol. Chem. 266:19396-19406).
  • diagnosis of disease states in which L-isoaspartyl and D-aspartyl residues accumulate may be performed by measuring the content of L-isoaspartyl and D-aspartyl residues accumulated in a disease associated protein, by using methyltransferase as a probe. Since a fraction of /9-amyloid protein is found in the cerebrospinal fluid (CSF), it is possible to develop a diagnostic test for Alzheimer's disease by measuring the content of L- isoaspartyl and D-aspartyl residues in samples of CSF. It has not hitherto been possible to obtain an accurate diagnosis of this disease which incapacitates millions of Americans. The diagnostic test can be accomplished using the same assay described above for protein pharmaceutical quality control.
  • EXPERIMENT 1 NUCLEOTIDE SEQUENCE OF HUMAN METHYLTRANSFERASE CODING REGION cDNA Library Synthesis and Clone Screening
  • a cDNA library constructed from the temporal cortex of the brain of a 2-year-old female human was purchased from Stratagene (#935205).
  • the cDNA was synthesized from oligo-dT isolated mRNA, and packaged into the EcoW sites of the lambda ZAP bacteriophage vector (Stratagene).
  • the library was propagated in £ coli BB4 and 22 plates containing 5 x 10 5 plaques each (1.1 x 10 7 plaques total) were screened using a radiolabeled 769 bp Haelll fragment from the coding region of a 1580 bp murine methyltransferase cDNA (Romanik, et al. (1992) Gene, 118:217-222).
  • the fragment was labelled with [ ⁇ - 32 P]-dCTP to a specific activity of 10 9 cpm/ ⁇ g with the PRIME-IT random priming kit (Stratagene). Standard plaque lift and Southern blot procedures (Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory) produced three positive signals. The clones for these plaques were isolated by subsequent screenings. The clones were repackaged into plasmids in XLI-Blue cells via in vivo excision according to the ⁇ ZAP protocol. Successful excision was denoted by ampicillin resistance.
  • the cells containing the insert-carrying plasmids of interest were grown in LB/Ampicillin medium, and their plasmids isolated and purified using Qiagen plasmid isolation columns.
  • Nucleotide Sequence Determination and Analysis The nucleotide sequences of the clones were determined on both strands by the dideox ⁇ chain-terminating method (Sanger, et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467) using the Sequenase 2.0 kit (USB), M13 and T7 universal primers, and synthesized 22mer primers. The sequence data were analyzed with DNAStar programs on a Macintosh computer.
  • the pDM2 plasmid was modified to give the overexpression vector, pDM2x, by replacing the region between the T7-promoter site and the start codon of the enzyme with a synthetic fragment containing a strong ribosomal binding site, as previously described (Figs. 1-3). Bacterial Growth
  • £ coli strain DH5 ⁇ (Gibco-BRL, Gaithersburg, MD) was used for cloning and propagation of plasmid constructs. Transformation of £ coli was accomplished by the one-step method described by Chung, et al. [Proc.Natl.Acad.Sci. USA 11989) 86:2172-519). For protein expression, £ coli strain BL2KDE3) (Studier, et al. J.Mo/.Bio/.i ⁇ 9S6) 189:113-130) was transformed with the pDM2x expression plasmid.
  • the concentration of active methyltransferase was determined by measuring base-labile methyl ester formation on the methyl-acceptor ovalbumin using a vapor diffusion assay (Gilbert, et al. Biochemistry (1988) 27:5227-5233). Final concentrations in a 50 ⁇ L reaction mixture were 10 ⁇ M S-adenosyl-L
  • Fig. 4 shows that only very low amounts (0.03 mM) of IPTG were required to induce expression of the methyltransferase.
  • a 1 liter solution of LB broth at 37° C containing ampicillin at 100 ⁇ g/ml was inoculated with BL2KOE3) £ coli harboring the pDM2x methyltransferase expression plasmid and grown to an optical density of 0.2 at 600 nm.
  • the culture was divided into 4 equal volumes (250 ml each) and brought to final concentrations of 0, 0.03 M, 0.50 mM, 04 8.00 mM isopropyl 9-D-thiogalactop ⁇ ranoside. Vigorous shaking at 37°C was continued and samples were collected over 3 hours. Each sample was placed on ice and the cells were pelleted by low speed centrifugation. The pelleted cells were resuspended in buffer A, sonicated, and assayed for methyltransferase activity. The concentration of soluble protein was determined by the modified Lowry assay. A specific activity of 10,000 pmoles/min/mg was used to calculate methyltransferase concentration. Importantly, the methyltransferase was found to comprise up to 20% of soluble total protein in fully active form and, as determined by SDS-PAGE, no methyltransferase polypeptide was found in the insoluble fraction where inclusion bodies are usually found.
  • Buffer A was used throughout all the preparations and contained 5 mM sodium phosphate, pH 8.0, 5 M EDTA, 25 ⁇ M phenylmethanesulfonyl fluoride (PMSF), 0.1 mM dithiothreitol (DTT), and 10% v/v glycerol.
  • the cell pellet (14 g) was suspended in 200 ml of buffer A at 4°C.
  • the bacterial cells were lysed by sonication for 2 min on power level 5 at continuous output using a Branson W-350 sonifier with microtip probe.
  • the sonication was done in a 500 ml beaker cooled in an ice-water slurry bath to dissipate heat-buildup and in a manner ensuring that thorough mixing took place.
  • These sonication settings were optimized in preliminary experiments where samples were lysed at various power levels for various times and assayed for enzyme content by methyltransferase activity. Cell disruption began at power level 3, while activity of the enzyme began to decrease at level 7, presumably due to overheating.
  • the lysed cells were centrifuged at 13,000 g for 15 min and the supernatant saved. 200 ml of buffer A was added to the pelleted debris and this material was again sonicated, pelleted, and the supernatant saved. The supernatants of the two extracts were combined to give a final volume of 390 ml.
  • Nucleic acids were precipitated by slow addition of protamine sulfate (0.1 volumes (39 ml) of a 4% w/v protamine sulfate solution (Grade X, Sigma)) at 4°C with mixing to the combined supernatant fraction. After mixing for 30 min, the solution was centrifuged at 13,000 g for 15 min and the supernatant saved. The methyltransferase was concentrated and further purified by slowly adding solid ammonium sulfate (Ultrapure, ICN) to 60% saturation (167 g) at 4°C, mixing for 30 min, and centrifuging at 13,000 g for 15 min (Scopes, R.K.
  • protamine sulfate 0.1 volumes (39 ml) of a 4% w/v protamine sulfate solution (Grade X, Sigma)
  • the methyltransferase was concentrated and further purified by slowly adding solid ammonium sulfate (Ultrapure,
  • the final purification step used DEAE-cellulose chromatography under nonequilibrium conditions at room temperature.
  • a 14.7 cm high x 2.5 cm I.D. DE-52 (Whatman) column was equilibrated at a flow rate of 2 ml/min with buffer A as determined by measuring the pH of the eluate.
  • the column was washed with buffer A with NaCI added to a final concentration of 1 M at a flow rate of 2 ml/min for 1 h, and finally washed with buffer A in the absence of NaCI for 6 hours.
  • 3 ml of the dialyzed ammonium sulfate preparation was then loaded onto the column and washed at 2 ml/min with buffer A for 2 hours. Material bound to the column at this point was eluted by washing with buffer A with 1 M NaCI. 2 minute (4 ml) fractions were collected.
  • the initial batch purification steps used here to enrich the methyltransferase fraction in the lysed bacterial extract is a modification of the procedure used by Fu, et al. (Fu, et al. (1991) J.BioLChem. 266:14562-14572). After lysis of the overexpressing bacteria by sonication, the cellular debris was removed by centrifugation. The nucleic acids remaining in the supernatant were removed by addition of a precipitant, protamine sulfate, and centrifugation.
  • Fig. 6, Table 1 various volumes of a 4% w/v protamine sulfate in buffer A were pipetted into 1.0 ml of centrifugation cleared sonicate supernatant and vortexed. Nucleic acids and other debris were precipitated at 13,000 g for 15 min. Supernatants were assayed for total protein and methyltransferase as described in Fig. 4.
  • methyltransferase then was concentrated and purified further by precipitation with ammonium sulfate. Again, conditions for this step in preliminary experiments were optimized. The amount of total protein and methyltransferase precipitated at various percentages of ammonium sulfate saturation is shown in Fig. 7. In Fig. 7, solid ammonium sulfate was added to 1.0 ml samples to give the indicated percent saturation and mixed on a rocking platform for 30 min at 4°C. Protein was pelleted by 13,000 g for 15 min. Pelleted protein was resuspended in an original volume of buffer A and assayed for total protein and methyltransferase as described in Fig. 4.
  • Polypeptides were visualized by rapid silver staining (Blum, et al. (1987) Electrophoresis 8:93-99.). Fractions were analyzed on two mini gels: 1 ⁇ L Sonicate and Load, 10 ⁇ L of 10-21 on gel #1 and; 15 ⁇ L of LMWS/100, 10 ⁇ L fractions 22-25, 27,29,31,33,35, I ⁇ L of fractions 76-80 on gel #2. Under these conditions, the methyltransferase elutes as a narrow spike of enzyme centered on fraction 18 and as a broader peak from fractions 21 to 40. The methyltransferase in these two peaks appears to be identical.
  • Both peaks are characterized by predominant 25 kDa polypeptides in SDS gel analysis (Fig. 9).
  • Fig. 9 1 ml fractions of purified enzyme were dialyzed against 20 mM sodium citrate at the pH values indicated and the fractions were concentrated by ultrafiltration using Centricon-10 micro concentrators (Amicon, Beverly, MA).
  • the first peak shows a low level of contamination (less than 5% for fraction 18) by other polypeptides while the second, broader peak shows no other protein contamination.
  • the specific activity of the methyltransferase is identical in both peaks, and their weight by mass spectroscopy is also identical (see below).
  • Table 1 summarizes the purification steps involved in obtaining large amounts of homogenous methyltransferase. It shows that the largest loss in yield occurs during the ammonium sulfate precipitation step; the reason for this is not understood.
  • the column chromatography step using only one-sixth of the total preparation (Table 1) was performed. Repeated cycles of DEAE-cellulose chromatography can thus be used to readily generate additional homogenous enzyme.
  • the purified recombinant enzyme is stable at room temperature for up to 2 months and repeated freeze-thaw cycles have no effect on activity. Furthermore, it was found that the methyltransferase can be heated for up to 30 min at 50° C with no loss of activity.
  • N-terminal sequencing of the purified methyltransferase was performed by automated Edman degradation analysis.
  • the experimentally-determined sequence of the first twenty residues was exactly that predicted from the cDNA with the removal of the initiator methionine as found in the human enzyme (Ingrosso, et al.(1989) J.BioLChem. 264:20131-20139) (Table 2). It was found no evidence for a blocked amino terminus, such as the acet ⁇ lated alanine residue present in the human enzyme (Ingrosso, et al.(1989) J.BioLChem. 264:20131-20139).
  • the molecular weight of the purified enzyme was measured at 24,551 ⁇ 3 Da by electrospray mass spectroscopy (Fig. 10 (A)).
  • Fig. 10 (A) a portion of the spectrum of material purified was described above using dithiothreitol as the reductant in buffer A is shown. No other peaks were discerned at other molecular weights. This average value matched exactly the predicted value of 24,551 Da of the unmodified product encoded by the cDNA sequence.
  • Fresh carrots, yellow corn, Romaine lettuce, green peas, white potatoes, spinach, cherry tomatoes, and alfalfa were purchased at a local distributor. Alfalfa seeds and raw wheat germ were from Rainbow Acres, Inc. (Los Angeles, CA), while soybean seeds were from Arrowhead Mills, Inc. (Hereford, TX). Winter wheat ⁇ Triticium aestt ' vum cultivar Augusta) seeds were provided by Dr. Robert Forsberg of the University of Wisconsin (Madison, Wl). Danver Half Long carrot seeds, Golden Jubilee corn seeds, Romaine lettuce seeds, sugar snap pea seeds, New Zealand spinach seeds, and Bonny Best tomato seeds were from the Chas. H. Lilly Co. (Portland, OR).
  • Crude cytosol was extracted from the plant tissues by homogenization using a mortar and pestle.
  • liquid nitrogen was poured over plant tissue (typically, 20 g of fresh tissue or 5 g of seeds) until the tissue was completely frozen.
  • 3 g of hydrated PVPP polyvinyl-polypyrrolidone
  • 3 g of hydrated PVPP polyvinyl-polypyrrolidone
  • Extraction buffer (20 L of 100 M HEPES, pH 7.5, 10 mM 2-mercaptoethanol, 1 ⁇ m leupeptin, 1 mM PMSF, 10 mM sodium hydrosulfite, and 10 mM sodium metabisulfite at 4°C) was added to the mortar, and the slurry was ground further.
  • the resulting crude homogenate was pressed through four layers of cheesecloth and then centrifuged at 2200 g for 30 min at 4°C to remove the insoluble PVPP and undisrupted plant material.
  • the resulting supernatant was centrifuged further at 172200xg for 50 min at 4°C and then filtered through two layers of Miracloth (Calbiochem, San Diego,
  • Methyltransferase activity was identified using a vapor-phase diffusion assay that quantitates the number of radiolabeled methyl groups transferred for S-adenosyl-L-l ⁇ t ⁇ /- 14 C) methionine to a peptide substrate by quantitating the release of [ 14 C] methanol resulting from the hydrolysis of base-labile methyl esters.
  • VYP-(L-isoAsp)-HA SEQ ID N0:15
  • KASA-(L-isoAsp)-LAKY SEQ ID N0:16
  • AA L-isoAsp)-F-NH 2
  • VYG D-Asp
  • PA PA
  • KASA-(D-Asp)-LAKY SEQ ID N0:18
  • Endogenous cytosolic oligopeptides are also potential methyl acceptors; therefore, parallel experiments were conducted in the presence and absence of the peptide substrate (Table 3).
  • Peptide dependent L-isoaspartyl methyltransferase was found in the vegetative cells of the green alga C. reinhardt ⁇ , demonstrating its presence in at least one species in the Kingdom Protista.
  • methyltransferase activity was detected in both classes of the angiosper s, the monocots and the dicots. The level of activity in different tissues varied considerably. Of the species assayed, the highest specific activity of the methyltransferase was found in wheat embryos.
  • the specific activity of the enzyme in plant seeds (0.66 • 14.0 pmol/min/mg) is comparable to the levels found in £ coli (1-2.5 pmol/min/mg; Fu, et al., 1991) and human erythrocytes (1.9 • 9.4 pmol/min/mg; Ota, et al., 1988; Gilbert, et al. 1988).
  • Methylation assays were performed in triplicate.
  • the present purification strategy was based on the partial purification of the protein carboxyl
  • the loaded column was washed isocratically with 1 L of buffer followed by a 6-L gradient of 25-200 mM NaCI in the above buffer.
  • the protein profile and the NaCI gradient were monitored by measuring absorbance at 280 nm and conductivity, respectively, in the corresponding fractions. Every fifth fraction was assayed for L-isoaspartyl methyltransferase using VYP-(L-isoAsp)-HA (SEQ ID N0:15) as the peptide substrate.
  • VYP-(L-isoAsp)-HA SEQ ID N0:15
  • One peak of methyltransferase activity was pooled (fractions 80-110,600 mL, see brackets) and further purified by reverse ammonium sulfate gradient solubilization as described by King (Biochemistry 11, 367-371, 1972).
  • the pH of the DE-52 pooled material was adjusted to 8.38 with 20 mL of 1 M Tris-HCl, pH 7.97. 15.62 g of Celite 545 (Baker Analyzed Reagent, 11 g of Celite/1 g of protein) was then added with stirring to 80% saturation (56.1 g of ammonium sulf ate/100 mL initial volume) in a 30-mi ⁇ period at room temperature, and then stirring was continued for an additional 45 min.
  • This Celite mixture containing precipitated cytosolic proteins was poured into a 3 cm diameter x 19 cm column and packed with the aid of a peristalic pump at room temperature. The column was washed isocratically with 150 mL
  • Fig. 13 active fractions containing methyltransferase from each purification step were analyzed by SDS-PAGE using the buffer system described by Laemmli (Laemmli (1970) Nature 227, 600-685). Protein fractions were mixed in a ratio of 2:1 (v/v) with sample buffer [180 M Tris-HCl (pH 6.8), 6.0% (w/v) SDS, 2.1 M 2-mercaptoethanol, 35.5% (v/v) glycerol, and 0.004% (w/v) bromophenol blue]
  • this polypeptide corresponds to the L- isoaspartyl methyltransferase by renaturing individual gel slices in the presence of Triton X-100 as described by Clarke (Clarke (1981) Biochim.Biophys.Acta 670, 195-202). The purity of this preparation was estimated to be 86% from densitometry of the Coomassie-stained gel. The remaining minor polypeptide contaminants could be removed by an additional chromatography step. Dialyzed methyltransferase was 0 loaded onto a Mono Q anion exchange column and eluted with a linear gradient of 0-1 M sodium acetate.
  • Methyltransferase purified through the Mono Q step [12,500 pmol/min/mg at pH 7.5 with the VYP- (isoAsp)-HA peptide as the substrate] was used to study the specificity of the wheat germ enzyme. Like 5 the £ coli and human erythrocyte methyltransferases, the wheat germ enzyme efficiently methylates L- isoaspartyl residues in synthetic peptides.
  • Homogeneous methyltransferase suitable for sequence analysis was obtained by reverse-phase 0 HPLC (high-performance liquid chromatography) of the enzyme purified through the Sephacryl S-200 step.
  • EXPERIMENT 7 cDNA CODING FOR WHEAT METHYLTRANSFERASE Synthetic Oligonucleotide Probes
  • Oligonucleotide probes were synthesized using .-cyanoethyl N.N-diisopropylphosphora idite chemistry in a Gene Assembler Plus DNA synthesizer (Pharmacia LKB Biotechnology).
  • oligonucleotides were synthesized on the basis of the partial amino acid sequence data (see above) and then used to amplify a region of the methyltransferase cDNA from a wheat cDNA library constructed with poly (A) + RNA isolated from 48-h-etiolated wheat seedlings (Hatfield, et al.(1990)
  • An 850-bp PCR product was amplified using a 384-fold degenerate oligonucleotide, MB3, representing the nucleic acid sequence at the 5'-region of the methyltransferase cDNA (corresponding to the peptide YLKQYGV) and a primer encoding the T7 promoter of the pBiuescript vector.
  • the identity of the 850-bp PCR product was verified by Southern hybridization using a 64-fold degenerate oligonucleotide, MB1, representing the nucleic acid sequence in the middle region of the methyltransferase cDNA (corresponding to the peptide GIEHIPE).
  • the 850-bp PCR product was used as a template in a PCR reaction with the MB3 primer and a 288-fold degenerate oligonucleotide, MB4, representing the nucleic acid sequence at the 3'-region of the methyltransferase cDNA (corresponding to the peptide LQVIDK).
  • PCR amplification produced a 600-bp fragment containing only the methyltransferase cDNA sequence, as determined by PCR dideoxy chain-termination sequencing.
  • the DNA sequence of the 952-bp cDNA insert in the plasmid pMBM1 was determined using the sequencing strategy shown in Fig. 14. Referring to Fig. 14, both strands of the pMBM1 clone containing the wheat methyltransferase cDNA insert (a 952-bp £coRI fragment) were sequenced by dideoxy chain- termination sequencing. Oligonucleotides were synthesized using the sequence of a PCR product containing 600 bp of the wheat methyltransferase cDNA and then used as primers to sequence the 952-bp fragment as shown in the sequencing strategy in this figure. The DNA sequence of the methyltransferase cDNA and ⁇ 27- its deduced amino acid sequence are indicated as SEQ ID NOs:5 and 6, respectively. Referring to SEQ ID NOs:5 and 6, respectively. Referring to SEQ ID NOs:5 and 6, respectively. Referring to SEQ ID NOs:5 and 6, respectively. Referring to SEQ ID NOs:5 and 6,
  • the sequence of the coding strand of the 944-bp insert of the plasmid pMBMI is shown without the terminal £ oRI linkers (GGAATTCC) that were added to the cDNA library.
  • the 690-bp methyltransferase cDNA initiates at the ATG codon at position 118 and terminates at the TGA codon at position 808.
  • the calculated molecular weight of the 230 amino acid polypeptide deduced for the 690-bp open reading frame is 24,710.
  • purified methyltransferase migrated as a 28,000-Da polypeptide as determined by SDS-PAGE (Fig. 13).
  • EXPERIMENT 8 COMPARISON OF SEQUENCED PEPTIDE FRAGMENTS AND PREDICTED SEQUENCE
  • Fig. 15 discrepancies at 12 sites between the predicted amino acid sequence of the wheat cDNA and the sequence of the peptide fragments of the wheat germ L-isoaspartyl methyltransferase were found (Fig. 15).
  • T trypsin
  • V S. aureus V8 protease
  • peptides were recovered by reverse-phase HPLC and sequenced by automated Edma ⁇ degradation. Peptide sequences of fragments numbered in the order of elution are shown by lines in comparison to the deduced cDNA sequence. The presence of a space indicates that unambiguous identification of the amino acid residue could not be made in this cycle. Additional residues identified in a particular cycle are indicated in parentheses above.
  • EXPERIMENT 9 SKIN TREATMENT USING METHYLTRANSFERASE Purified methyltransferase (either isolated from plant sources or as a recombinant human enzyme produced in bacteria) and S-adenosyl-L-methionine are mixed to final concentrations of 0.01 % and 0.00004% respectively in a water-based cream containing glycerin and mineral oil.
  • a skin or hair cream is formulated using water, glycerin, cetearyl alcohol, palm oil glyceride, Ceteareth-20, mineral oil, petrolatum, sorbitol, avocado oil, DMSO (or other carrier molecules), steric acid, alantoin, squalane, methylparaben, Sodium Carbomer 941, recombinant human L-isoaspartyl/D-aspartyl methyltransferase (or plant methyltransferase), propylparaben, S-adenosyl-L-methionine, Quaternium-15, fragrance, FD&C Yellow
  • a skin mist (“Methyl ist”) is formulated using water, glycerin, cetearyl alcohol, DMSO (or other carrier molecules), citric acid, recombinant human L-isoaspartyl/D-aspartyl methyltransferase (or plant methyltransferase), S-adenosyl-L-methionine, Quaternium-15, and fragrance.
  • a transdermal skin patch is formulated using the same ingredients as the above skin mist. These preparations can be directly applied to the skin.
  • the methyltransferase skin cream (a), mist (b) and patch (c) are used by the following methods respectively: (a) applying a dab (0.5ml) of a viscous formulation to a central spot and spreading over an area of about 100 cm 2 by rubbing with small circular motions directly by hand with or without latex gloves andlor with or without applicants such as towels and tissues; (b) using a less viscous formulation ("MethylMist”) and spraying on with a spray bottle or atomizer in which the residue can be wiped into the skin as described above for the skin cream; and
  • An injectable or topical preparation for treatment of the eye (e.g., for preventing cataracts).
  • the injectable preparation can be packaged in carrier liposomes or microporous structures. These preparations can be directly injected to the eye, brain or blood stream.
  • a sample of cerebrospinal fluid (0.01 ml) diluted in 0.2 M sodium citrate buffer, pH 6.0 is mixed with 0.03 ml of a mixture of S-adenosyl-[ 14 C-methylR-methionine (100 cpm/pmol) and purified methyltransferase (from plant sources or human recombinant) in the same buffer.
  • the latter mixture contains 400 pmol of radiolabelled S-adenosylmethionine and 10 micrograms of purified methyltransferase.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • GGCAGCATCA AAATGMRNCC TCTGATGGGG GTGATATACG TGCCTTTAAC AGATAAAGAA 660
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • GGCAGCATCA AAATGMRNCC TCTGATGGGG GTGATATACG TGCCTTTAAC AGATAAAGAA 660
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
  • GTAATACGAC TCACTATAGG GCGAATTGGG TACCTCGAGT CTAGAGGATC CTTTGTTTAA 60 CTTTAAGAAG GAAAGCTAGC CATG GCC TGG AAA TCC GGC GGC GCC AGC CAC 111
  • GGC GGA AAC CAA ATG TTG GAG CAG TAT GAC AAG CTA CAA GAT GGC AGC 687 Gly Gly Asn Gin Met Leu Glu Gin Tyr Asp Lys Leu Gin Asp Gly Ser
  • ATC AAA ATG AAG CCT CTG ATG GGG GTG ATA TAC GTG CCT TTA ACA GAT 735 He Lys Met Lys Pro Leu Met Gly Val He Tyr Val Pro Leu Thr Asp 205 210 215
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE (ix) FEATURE:

Abstract

On obtient une L-isoaspartyle/D-aspartyle protéine méthyltransférase recombinante isolée de l'homme par surexpression d'ADNc codant pour l'isozyme II dans une souche de E.coli et on obtient un clone d'ADNc de l'enzyme du blé, ainsi qu'une enzyme purifiée à partir du blé. Ces enzymes sont utiles dans le traitement d'états pathologiques et dans le diagnostic de maladies associées à une augmentation de restes de L-isoaspartyle/D-aspartyle de polypeptides dans un tissu.
PCT/US1995/013691 1994-10-19 1995-10-19 Production et utilisation de methyltransferases de l'homme et des plantes WO1996012797A1 (fr)

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EP95939576A EP0796322A4 (fr) 1994-10-19 1995-10-19 Production et utilisation de methyltransferases de l'homme et des plantes
AU41341/96A AU4134196A (en) 1994-10-19 1995-10-19 Production and use of human and plant methyltransferases

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FR2862661B1 (fr) * 2003-11-26 2007-10-05 Agronomique Inst Nat Rech Utilisation de la l-isoaspartyl methyltransferase comme marqueur de longevite des semences.
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