CA2266760A1 - Methylation of arginine and related amino acids - Google Patents
Methylation of arginine and related amino acids Download PDFInfo
- Publication number
- CA2266760A1 CA2266760A1 CA002266760A CA2266760A CA2266760A1 CA 2266760 A1 CA2266760 A1 CA 2266760A1 CA 002266760 A CA002266760 A CA 002266760A CA 2266760 A CA2266760 A CA 2266760A CA 2266760 A1 CA2266760 A1 CA 2266760A1
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- slm
- sam68
- wasp
- hnrnp
- methylated
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- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 102000009076 src-Family Kinases Human genes 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
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- 241000701447 unidentified baculovirus Species 0.000 description 1
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- 229910000166 zirconium phosphate Inorganic materials 0.000 description 1
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Abstract
The subject invention provides for methods of assaying arginine methyltransferase activity by means of measuring the methylation of SLM-1, SLM-2. Sam68, hnRNP K, WASP
or derivatives thereof. Measurement of SLM-1, SLM-2, Sam68, hnRNP K, or WASP methylation may be used to determine whether or not a cell is cancerous. The methylation of WASP protein may also be used as a diagnostic tool for individuals with the Wiskott-Aldrich Syndrome.
or derivatives thereof. Measurement of SLM-1, SLM-2, Sam68, hnRNP K, or WASP methylation may be used to determine whether or not a cell is cancerous. The methylation of WASP protein may also be used as a diagnostic tool for individuals with the Wiskott-Aldrich Syndrome.
Description
METHYLATION OF ARGININE AND RELATED AMINO ACIDS
FIELD OF THE INVENTION
This invention relates generally to the measurement of the methylation of arginine and related amino acids, and more particularly to the methylation of SLM-1, SLM-2, Sam68, hnRNP K and WASP in particular.
BACKGROUND OF THE INVENTION
Proteins that provide an intracellular link from the extracellular milieu are called signal transduction proteins.
These proteins contain numerous post-translation modifications including tyrosine phosphorylation (Pawson, 1995) and arginine methylation (Gary and Clarke, 1998). The tyrosine kinases and the arginine methyltransferases that modify the signal proteins are often found associated with growth factor receptors and are involved in transferring the information to the inside of the cell. It is known that many growth factor receptors and soluble tyrosine kinases are oncogenes and can transform cells.
Thus substrates of tyrosine kinases or post-translational modifications in response to growth factor receptors can serve as an indication of whether or not a cell is cancerous. In addition to being post-translationally modified, many proteins contain proline motifs and phosphotyrosine residues that serve as specific binding sites for SH3 and SH2 domain containing proteins (Pawson, 1995).
Post-translation modification includes N-methylation of the side chain of the guanidine arginine residues. Related amino acids, such as lysine and histidine, can also be subject of methylation.
Three mammalian protein arginine methyltransferases have been cloned including PRMT1 (Scott et al., 1998 and Lin et al., 1996), PRMT2 (Scott et al., 1998) and PRMT3 (Tang et al., 1998). From the EST database, it has been predicted that others will be identified (Tang et al., 1998). Arginine methylation is thought to be an irreversible post-translational modification. Protein arginine N-methyltransferase enzymes catalyze the formation of asymmetric 1V~,IV~-dimethylarginine residues in proteins by transferring methyl groups from S-adenosylmethionine to the guanidino nitrogen atoms of arginine. These enzymes generally methylate arginine in the context of RGG or arginine-glycine rich domains.
Known substrates for PRMTs include hnRNP proteins including hnRNP A1 and myelin basic protein. PRMT1 interacts with several proteins including the cytoplasmic domain of the interferon alpha receptor 1 (Abramovich et al, 1997) and the immediate early gene products including TIS21 and BTG1 interact with PRMT1 (Lin et al., 1996). These findings suggest that PRMT1 activity may be regulated by cell surface receptors and may be involved in signal transduction aspects of downstream of the interferons. PRMT1 is predominantly nuclear and methylates arginines that contain glycines at their C-terminus in repeats such as arginine-glycine or arginine-glycine-glycine and so forth (Scott et al., 1998 and Lin et al., 1996). PRMT2 has not been shown to have any arginine methyltransferase activity, perhaps because the appropriate substrate has not been identified (Scott et al., 1998). Interestingly, PRMT2 contains an SH3 domain in its sequence and thus has the potential to link arginine methyltransferase to signalling proteins. It is well known that SH3 domains bind proline rich repeats that are frequently found in cytoskeletal and signalling proteins (Pawson, 1995). PRMT3 is predominantly cytoplasmic and also favours the methylation of arginine amino acids that contain a C-terminal glycine (Tang et al., 1998).
SUMMARY OF THE INVENTION
The subject invention provides for methods of assaying methylation of arginine and related amino acids, more particularly methods of assaying arginine methyltransferase activity by means of measuring the methylation of suitable substrates, such as SLM-1, SLM-2, Sam68, hnRNP K or WASP
proteins. The invention is described with respect to the specific methyltransferase substrates SLM-1, SLM-2, Sam68, hnRNP K and WASP, however, it will be appreciated that other substrates can be methylated in a similar fashion, and accordingly the invention has application to such substrates although it is described herein with reference to SLM-1, SLM-2, Sam68, hnRNP K and WASP.
The subject invention also provides for a method of measuring the percentage of methylated substrate relative to total substrate in a cell. The methylation of substrate such as SLM-1, SLM-2, Sam68, hnRNP K or WASP may be used to determine whether or not a cell is transformed or has the potential to become cancerous. The methylation of WASP may also be used to diagnose individuals with Wiskott-Aldrich syndrome. Another aspect of the invention is to provide for antibodies that may be used to detect polypeptides that contain methylated arginines.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
This invention relates to the measurement of methylation of arginine and related amino acids such as lysine and histidine. The invention is described with respect to arginine, however, it will be appreciated that related amino acids, such as lysine and histidine, can be methylated in a similar fashion, and accordingly the invention has similar application to lysine and histidine although it is described herein with reference to arginine.
By arginine methylation or by methylated arginines, it is meant that arginine residues in a peptide contain one or more methyl group. By methylation, it is meant 1V~-monomethlyarginine (MMA) , N~,1V~-dimethylarginine residues (asymmetric, DMA) and N~,N'~-dimethylarginine (symmetric, DMA').
Antibodies specific for arginine methylation would be able to recognize MMA, DMA and/or DMA' in a polypeptide and/or oligopeptide.
Those skilled in the art will appreciate that there must exist a protein module that binds methylated arginine residues much like SH2 domains bind phosphotyrosine residues.
As hereinbefore mentioned the invention relates to a method for assaying a medium for the presence of a substance that affects a methylarginine specific antibody or methylarginine binding domain to a methylarginine ligand regulatory system comprising a specific methylarginine binding antibody or protein module or a subdomain thereof, and a methylated ligand which is capable of interacting with said specific methylarginine binding antibody or protein module or a subdomain thereof to form a specific methylarginine binding antibody or protein module- methylated ligand complex, said specific methylarginine binding antibody or protein module or subdomain and/or said methylated ligand being present in a known concentration, and incubating with a substance which is suspected of affecting a specific methylarginine binding antibody or protein module-methylated ligand regulatory system, under conditions which permit the formation of said specific methylarginine binding antibody or protein module methylated ligand complex, and assaying for said specific methylarginine binding antibody or protein module methylated ligand complex, free specific methylarginine binding antibody or protein module or subdomains thereof, or non-complexed methylated ligand.
Purified derivatives of SLM-1, SLM-2, Sam68, hnRNP K and WASP
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives may be purified from a variety of cells. By "purified" it is meant, when referring to a peptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, e.g., polypeptides, polynucleic acids, and the like of the same type. The term "purified" as used herein preferably means at least 95% by weight, more preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 can be present).
The term "pure" as used herein preferably has the same numerical limits as "purified" immediately above. The term "isolated" as used herein refers to polypeptide or polynucleotide molecules separated not only from other peptides, or from DNAs or RNAs, respectively, that are present in the natural source of the macromolecule but also from other macromolecules and preferably refers to a macromolecule found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a solution of the same.
FIELD OF THE INVENTION
This invention relates generally to the measurement of the methylation of arginine and related amino acids, and more particularly to the methylation of SLM-1, SLM-2, Sam68, hnRNP K and WASP in particular.
BACKGROUND OF THE INVENTION
Proteins that provide an intracellular link from the extracellular milieu are called signal transduction proteins.
These proteins contain numerous post-translation modifications including tyrosine phosphorylation (Pawson, 1995) and arginine methylation (Gary and Clarke, 1998). The tyrosine kinases and the arginine methyltransferases that modify the signal proteins are often found associated with growth factor receptors and are involved in transferring the information to the inside of the cell. It is known that many growth factor receptors and soluble tyrosine kinases are oncogenes and can transform cells.
Thus substrates of tyrosine kinases or post-translational modifications in response to growth factor receptors can serve as an indication of whether or not a cell is cancerous. In addition to being post-translationally modified, many proteins contain proline motifs and phosphotyrosine residues that serve as specific binding sites for SH3 and SH2 domain containing proteins (Pawson, 1995).
Post-translation modification includes N-methylation of the side chain of the guanidine arginine residues. Related amino acids, such as lysine and histidine, can also be subject of methylation.
Three mammalian protein arginine methyltransferases have been cloned including PRMT1 (Scott et al., 1998 and Lin et al., 1996), PRMT2 (Scott et al., 1998) and PRMT3 (Tang et al., 1998). From the EST database, it has been predicted that others will be identified (Tang et al., 1998). Arginine methylation is thought to be an irreversible post-translational modification. Protein arginine N-methyltransferase enzymes catalyze the formation of asymmetric 1V~,IV~-dimethylarginine residues in proteins by transferring methyl groups from S-adenosylmethionine to the guanidino nitrogen atoms of arginine. These enzymes generally methylate arginine in the context of RGG or arginine-glycine rich domains.
Known substrates for PRMTs include hnRNP proteins including hnRNP A1 and myelin basic protein. PRMT1 interacts with several proteins including the cytoplasmic domain of the interferon alpha receptor 1 (Abramovich et al, 1997) and the immediate early gene products including TIS21 and BTG1 interact with PRMT1 (Lin et al., 1996). These findings suggest that PRMT1 activity may be regulated by cell surface receptors and may be involved in signal transduction aspects of downstream of the interferons. PRMT1 is predominantly nuclear and methylates arginines that contain glycines at their C-terminus in repeats such as arginine-glycine or arginine-glycine-glycine and so forth (Scott et al., 1998 and Lin et al., 1996). PRMT2 has not been shown to have any arginine methyltransferase activity, perhaps because the appropriate substrate has not been identified (Scott et al., 1998). Interestingly, PRMT2 contains an SH3 domain in its sequence and thus has the potential to link arginine methyltransferase to signalling proteins. It is well known that SH3 domains bind proline rich repeats that are frequently found in cytoskeletal and signalling proteins (Pawson, 1995). PRMT3 is predominantly cytoplasmic and also favours the methylation of arginine amino acids that contain a C-terminal glycine (Tang et al., 1998).
SUMMARY OF THE INVENTION
The subject invention provides for methods of assaying methylation of arginine and related amino acids, more particularly methods of assaying arginine methyltransferase activity by means of measuring the methylation of suitable substrates, such as SLM-1, SLM-2, Sam68, hnRNP K or WASP
proteins. The invention is described with respect to the specific methyltransferase substrates SLM-1, SLM-2, Sam68, hnRNP K and WASP, however, it will be appreciated that other substrates can be methylated in a similar fashion, and accordingly the invention has application to such substrates although it is described herein with reference to SLM-1, SLM-2, Sam68, hnRNP K and WASP.
The subject invention also provides for a method of measuring the percentage of methylated substrate relative to total substrate in a cell. The methylation of substrate such as SLM-1, SLM-2, Sam68, hnRNP K or WASP may be used to determine whether or not a cell is transformed or has the potential to become cancerous. The methylation of WASP may also be used to diagnose individuals with Wiskott-Aldrich syndrome. Another aspect of the invention is to provide for antibodies that may be used to detect polypeptides that contain methylated arginines.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
This invention relates to the measurement of methylation of arginine and related amino acids such as lysine and histidine. The invention is described with respect to arginine, however, it will be appreciated that related amino acids, such as lysine and histidine, can be methylated in a similar fashion, and accordingly the invention has similar application to lysine and histidine although it is described herein with reference to arginine.
By arginine methylation or by methylated arginines, it is meant that arginine residues in a peptide contain one or more methyl group. By methylation, it is meant 1V~-monomethlyarginine (MMA) , N~,1V~-dimethylarginine residues (asymmetric, DMA) and N~,N'~-dimethylarginine (symmetric, DMA').
Antibodies specific for arginine methylation would be able to recognize MMA, DMA and/or DMA' in a polypeptide and/or oligopeptide.
Those skilled in the art will appreciate that there must exist a protein module that binds methylated arginine residues much like SH2 domains bind phosphotyrosine residues.
As hereinbefore mentioned the invention relates to a method for assaying a medium for the presence of a substance that affects a methylarginine specific antibody or methylarginine binding domain to a methylarginine ligand regulatory system comprising a specific methylarginine binding antibody or protein module or a subdomain thereof, and a methylated ligand which is capable of interacting with said specific methylarginine binding antibody or protein module or a subdomain thereof to form a specific methylarginine binding antibody or protein module- methylated ligand complex, said specific methylarginine binding antibody or protein module or subdomain and/or said methylated ligand being present in a known concentration, and incubating with a substance which is suspected of affecting a specific methylarginine binding antibody or protein module-methylated ligand regulatory system, under conditions which permit the formation of said specific methylarginine binding antibody or protein module methylated ligand complex, and assaying for said specific methylarginine binding antibody or protein module methylated ligand complex, free specific methylarginine binding antibody or protein module or subdomains thereof, or non-complexed methylated ligand.
Purified derivatives of SLM-1, SLM-2, Sam68, hnRNP K and WASP
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives may be purified from a variety of cells. By "purified" it is meant, when referring to a peptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, e.g., polypeptides, polynucleic acids, and the like of the same type. The term "purified" as used herein preferably means at least 95% by weight, more preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 can be present).
The term "pure" as used herein preferably has the same numerical limits as "purified" immediately above. The term "isolated" as used herein refers to polypeptide or polynucleotide molecules separated not only from other peptides, or from DNAs or RNAs, respectively, that are present in the natural source of the macromolecule but also from other macromolecules and preferably refers to a macromolecule found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a solution of the same.
5 "Isolated" and "purified" do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure substances or as solutions.
Suitable cell sources for the production of purified SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include cells naturally producing SLM-1, SLM-2, Sam68, hnRNP K, or WASP including most mammalian cells, cells not naturally encoding an expressible SLM-1, SLM-2, Sam68, hnRNP K, or WASP
gene but genetically modified to do so, and cells naturally producing SLM-1, SLM-2 and Sam68 but genetically modified so as to produce elevated levels of SLM-1, SLM-2, Sam68, hnRNP K, or WASP. Preferred cell sources for SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivative molecules are selected so that at least 5%, preferably at least 50% and more preferably at least 90% of the SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivative molecules are methylated. Purification methods for SLM-l, SLM-2, Sam68, hnRNP K, WASP and their derivatives that depend on affinity reagents specific for methylated arginines, necessarily employ SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives isolated from cells that methylate SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives that have been produced in cells that lack arginine methyltransferase activity but have been methylated in vitro with enzymes with arginine methyltransferase activity.
It will be appreciated that an important advantage of the subject invention is to apply recombinant DNA techniques so as to provide for cellular lysates that contain SLM-1, SLM-2, Sam68, hnRNP K, or WASP in significantly higher (at least 2-fold, and preferably at least 10-fold) concentrations than found in naturally occurring cells or cell lines that have not been modified by exogenous SLM-1, SLM-2, or Sam68 encoding nucleic acid sequences. Since SLM-1, SLM-2, Sam68, hnRNP K, and WASP derivatives are not naturally produced, it is apparent that cells from which SLM-1, SLM-2, Sam68, hnRNP K, and WASP
derivatives can be isolated do not naturally encode SLM-1, SLM-2, Sam68, hnRNP K, or WASP derivatives but are genetically modified to do so.
Cells from which SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives may be isolated include both prokaryotic and eukaryotic cells. Preferred cellular sources for the isolation of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include mammalian cells possessing high levels of arginine methyltransferase activity. Of particular interest are mammalian cells transfected with arginine methyltransferases such as PRMT1, PRMT2, and PRMT3. Other mammalian cell sources of interest for the purification of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include mammalian cells stimulated by growth factors that bind to growth factor receptors that stimulate arginine methyltransferase activity. Another preferred source for preparations from which to purify SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives is insect cells, preferably grown in tissue culture, and genetically modified by baculovirus expression vectors or the like to express SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives and an arginine methyltransferase. A particularly preferred source of SLM-1, SLM-2, Sam68, hnRNP K, or WASP derivatives is the SF9 cell line. Another source would be to produce recombinant SLM-1, SLM-2, Sam68, hnRNP K, or WASP in bacteria or yeast as a fusion protein with tags such as histidine repeats or glutathione S-transferase proteins. It will be appreciated that purified SLM-1, SLM-2, Sam68, hnRNP K, or WASP can be methylated in vitro to yield purified methylated SLM-1, SLM-2, Sam68, knRNP K, or WASP.
Purification of SLM-1, SLM-2, Sam68, hnRNP K, or WASP
Derivatives Affinity purification of SLM-1, SLM-2, Sam68, hnRNP K, or WASP and their derivatives may employ various immobilized reagents specific for SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivatives, i.e., affinity reagents. The affinity purification may be performed in batches or employ chromatography columns. The affinity reagents may be immobilized to a variety of inert matrices prepared in bead form. Suitable immobilization matrices include cross-linked agarose beads, Sepharose, cross-linked polyacrylamide beads, Sephacryl, and the like. When the affinity reagents used are antibodies, a preferred immobilizing matrix is protein A
sepharose. Affinity reagents of interest include antibodies, purified SH3 domains, purified SH2 domains, and homopolymeric RNA. Purification of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP can be performed by using methylated arginine specific antibodies. Purification of non-methylated SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivatives may also be achieved through the use of SLM-l, SLM-2, Sam68, hnRNP K, or WASP
specific antibodies as affinity reagents.
In addition to production of purified SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives by purification of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives produced in cells, purified SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives may be produced by organic chemical reactions performed in vitro. Automated equipment for the direct synthesis of the polypeptides disclosed herein is commercially available. Such equipment provides convenient access to peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques. The use of such commercially available polypeptide synthesis machines and the like are a preferred method of synthesizing oligopeptides SLM-1, SLM-2, Sam68, hnRNP K or WASP derivatives having about 5-25 amino acids. It will be appreciated by those skilled in the art that oligopeptides can be synthesized containing arginines that are methylated.
Other methods for synthesis of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include the in vitro transcription of DNA sequences encoding SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives coupled with the in vitro translation of the RNA transcripts thus produced. In vitro transcription systems are well known in the art. In vitro transcription systems typically involve the creation of nucleotide sequences in which the coding sequence of interest is located downstream from a strong promoter, such as a promoter specific for SP-6 or T7 RNA polymerases, followed by the addition of an RNA polymerase specific for the promoter, and substrates required for the reaction. Similarly, in vitro translation systems are well known in the art and may be used to produce SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivative polypeptides from a variety of transcripts produced by in vitro transcription systems.
Uses for methylarginine specific antibodies The invention provides for antibodies capable of recognizing polypeptides containing methylated arginines. By the term "antibodies," it is intended both polyclonal and monoclonal antibodies with natural immunoglobulin sequences, synthetic antibody derivatives, and the like; antibodies may be modified so as to be joined to any of a variety of labels, fluorescent, radioactive, enzymatic, biotin/avidin or the like.
Synthetic antibody derivatives include natural immunoglobulin sequences that have been mutated and selected for altered binding specificity, various immunoglobulin gene derived polypeptides, typically single chain, produced by genetically modified bacteria, antibodies modified so as to contain modified constant regions and the like; a review of such synthetic antibody derivatives based on the principles of antibody formation is provided in Harlow and Lane, Antibodies, Coldspring Harbor Laboratory, Coldspring Harbor Press, A
Laboratory Manual, 1988.
Antibodies specific for methylated arginines may be generated by using proteins or peptides that contain one or more methylated arginines. By induction of antibodies it is intended not only the stimulation of an immune response by injection into animals, but analogous steps in the production of synthetic antibodies such as the screening of recombinant immunoglobulin libraries (Orlandi et al., 1989 or Huse et al., 1989), or the in vitro stimulation of lymphocyte populations.
Of particular interest is the development of antibody preparations, monoclonal antibodies, specific for methylated arginine containing polypeptides, i.e., monospecific antibodies.
Short oligopeptides, i.e., containing about 20 amino acids or less, are of particular interest for both the induction and the screening of mono-specific antibodies specific for epitopes of interest. In general, oligopeptides for use in the induction of epitope specific monospecific antibodies will have an amino acid sequence corresponding to at least a portion of the epitope of interest.
It is also of interest to produce antibody preparations that are capable of specifically binding to the methylated forms of SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Uses for such methylation state detecting antibodies include the measurement of the degree of methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell.
Assays The subject invention provides methods and reagents for performing assays capable of measuring the amount of arginine methyltransferase activity present in a cell and the fraction of proteins that contain methylated arginines.
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives may be used as substrates for the detection and quantification of arginine methyltransferase activity from a variety of cellular sources. It is desirable to measure arginine methyltransferase activity for several reasons. Of particular interest is the measurement of arginine methyltransferase activity produced by arginine methyltransferase encoded by oncogenes and proto-oncogenes.
Thus assays for arginine methyltransferase may be employed to determine whether a cell is cancerous or has cancer potential.
Also of interest is the measurement of arginine methyltransferase activity attributable to the stimulation membrane bound ligand receptors associated with arginine methyltransferase activity, since the extent of methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be used to measure 5 the extent to which ligands are binding to receptors.
Arainine methyltransferase assays Arginine methyltransferase assays of interest measure the rate of methylation of SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives by arginine methyltransferase in a cell, 10 rather than simply measuring the amount of methylated SLM-l, SLM-2, Sam68, hnRNP K, or WASP present in a cell. Thus arginine methyltransferase assays of interest employ a method for distinguishing arginine methylation events that take place during an assay from arginine methylation events that occur before an assay. Arginine methyltransferase assays may employ the step of adding a methyl group, preferably S-adenosyl methionine and the like, to an assay mixture containing suitable buffers and salts. Methyl sources may be radioactively labelled on the terminal carbon or hydrogens so as to provide for the detection of arginine methyltransferase activity.
Arginine methyltransferase assays employing radioactive labels may or may not employ the step of addition of SLM-l, SLM-2, Sam68, hnRNP K, WASP or their derivatives, because arginine methyltransferase substrates initially present in the cell, or SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivatives added externally, and subsequently methylated by a radioactive methyl source, may subsequently be isolated by addition of SLM-1, SLM-2, Sam68, hnRNP K, or WASP-specific antibodies, followed by the step of radiometric quantitation.
Generally it will be preferable to add SLM-1, SLM-2, Sam68, hnRNP K, or WASP, preferably produced by recombinant means, to the assay mixture. After the arginine methyltransferase reaction has been allowed to progress, the amount of radioactive label incorporated into SLM-1, SLM-2, Sam68, hnRNP K, or WASP is measured by radiometric means. In order to measure the amount of labelling, the unincorporated label must be removed prior to radiometric measurement. This removal can be achieved through a variety of means including immunoprecipitation of SLM-1, SLM-2, Sam68, hnRNP K or WASP
with anti-SLM-1, SLM-2, Sam68, hnRNP K or WASP antibodies.
An important advantage of the subject invention is that the polypeptides provided for permit the detection and quantification of arginine methyltransferase activity without requiring the addition of radioactively labelled methyl groups.
Methods for measuring arginine methyltransferase activity without the addition of radioactively labelled methyl groups include assays involving the use of (1) SLM-1, SLM-2, Sam68, hnRNP K or WASP derivatives that contain epitopes not present on SLM-1, SLM-2, Sam68, hnRNP K, or WASP, (2) antibodies specific for that epitope, and (3) anti-methylated-arginine antibodies or protein domains that specifically bind to arginine methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Such assays involve the addition of the SLM-l, SLM-2, Sam68, hnRNP K, or WASP derivatives to the assay mixture, followed by the immunoprecipitation or immunobilization of the SLM-1, SLM-2, Sam68, hnRNP K, or WASP derivative by means of the epitope specific antibody so as to separate the SLM-1, SLM-2, Sam68, hnRNP K or WASP derivative from other cellular proteins containing methylated arginines (including endogenous SLM-1, SLM-2, Sam68, hnRNP K, and WASP).
In addition to providing methods and reagents for use in the detection of arginine methyltransferase activity present in a cell, the subject invention provides methods and reagents for determining what fraction of the SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell is methylated as well as determining the absolute amount of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in a cell. By cell, it is intended not only individual cells, but multiple cells. Arginine methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be detected by a variety of means. If the methyl donor in the assay contains a radioactive label, then arginine methyltransferase activity may be detected by separating the labelled SLM-1, SLM-2, Sam68, hnRNP K, or WASP from the unincorporated label and quantitating the amount of label incorporated into the SLM-l, SLM-2, Sam68, hnRNP K, or WASP substrate. When non-radioactively labelled methyl donors are used in assays, methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be detected by means of generally known immunoassays in which the immunospecific reagent employed is specific for methylated arginines.
The subject invention provides for methods and reagents for performing assays capable of determining what fraction of SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell is methylated. Such assay may employ well known immunoassay technology such as ELISA, RIA, western blotting, and the like.
The use of SLM-1, SLM-2, Sam68, hnRNP K, or WASP specific antibodies (as well as SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives) as provided for by the subject invention may be used in connection with the preciously described well-established immunoassay technology, in order to provide for assays capable of detecting the extent of SLM-1, SLM-2, Sam68, hnRNP K, or WASP methylation in a cell. In general, such assays will employ two types of SLM-1, SLM-2, Sam68, hnRNP K, or WASP specific antibodies (or similar binding reagent) in an immobilized phase, namely (1) antibodies capable of binding SLM-l, SLM-2, Sam68, hnRNP K, or WASP in both methylated and non-methylated form or antibodies capable of binding only the non-methylated form of SLM-1, SLM-2, Sam68, hnRNP K, or WASP, and (2) antibodies capable of binding the methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP. By employing two types of specific binding reagent, it is possible to determine the relative quantities of the methylated and unmethylated forms of SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in a sample. The binding of SLM-1, SLM-2, Sam68, hnRNP K, or WASP (methylated and non-methylated) to an immobilized antibody phase may be detected by the addition of a third antibody, preferably labelled, and having an affinity for exposed epitopes on the antibody bound SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Comparison of binding of the labelled antibody to SLM-1, SLM-2, Sam68, hnRNP K, or WASP bound to the 2 different types of immobilized antibody may be used to determine the fraction of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP present among the total SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in the sample.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
Example 1 Identifying protein arginine methyltransferase substrates.
HeLa cells (0.5 x 106) were transfected with 5 ug of each plasmid expressing myc epitope tagged mouse SLM-1 (Di Fruscio et al., 1999), mouse SLM-2 (Di Fruscio et al., 1999), mouse Sam68 (Wong et al., 1992), brine shrimp GRP33 (Cruz-Alvarez and Pellicer, 1987), human hnRNP K (Michael et al., 1997) and mouse Qkl (Ebersole et al., 1996) as described previously (Richard et al., 1995). Twelve hours after transfection the cells were lysed in 500 ul lysis buffer (1% Triton X-100, 20 mM Tris-HC1 pH 7.4, 150 mM NaCl, 50 mM
NaF, 100 uM sodium vanadate, 0.01% phenylmethanesulfonyl fluoride, 1 ug/ml aprotinin, and 1 ug/ml leupeptin) on ice for 15 minutes. The debris was removed by centrifugation and the supernatant was divided equally into two fractions. Half was immunoprecipitated with control immunoglobulin G and the other half was immunoprecipitated with the anti-myc antibody 9E10 (American Tissue Culture Collection) for 1 hour on ice. Then 20 ul of 50% slurry of protein A Sepharose was added and incubated with end-over-end mixing for 30 minutes. The immunoprecipitated proteins were washed extensively and 2 ug of purified recombinant GST-PRMT1 and 2 ul of [3H-methyl]-adenosyl-1-methionine was added in 25 mM Tris-HCl pH 7.4, 1 mM EDTA for 30 minutes at room temperature. PRMT1 is a protein arginine methyltransferase that uses the methyl groups from S-adenosyl-methionine to transfer them to the guandino nitrogens of arginine (Gary and Clarke, 1998). The reaction was stopped by adding 20 ul of 2X Laemmli buffer (Laemmli, 1970) and loaded on an SDS 10%-polyacrylamide gel.
Suitable cell sources for the production of purified SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include cells naturally producing SLM-1, SLM-2, Sam68, hnRNP K, or WASP including most mammalian cells, cells not naturally encoding an expressible SLM-1, SLM-2, Sam68, hnRNP K, or WASP
gene but genetically modified to do so, and cells naturally producing SLM-1, SLM-2 and Sam68 but genetically modified so as to produce elevated levels of SLM-1, SLM-2, Sam68, hnRNP K, or WASP. Preferred cell sources for SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivative molecules are selected so that at least 5%, preferably at least 50% and more preferably at least 90% of the SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivative molecules are methylated. Purification methods for SLM-l, SLM-2, Sam68, hnRNP K, WASP and their derivatives that depend on affinity reagents specific for methylated arginines, necessarily employ SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives isolated from cells that methylate SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives that have been produced in cells that lack arginine methyltransferase activity but have been methylated in vitro with enzymes with arginine methyltransferase activity.
It will be appreciated that an important advantage of the subject invention is to apply recombinant DNA techniques so as to provide for cellular lysates that contain SLM-1, SLM-2, Sam68, hnRNP K, or WASP in significantly higher (at least 2-fold, and preferably at least 10-fold) concentrations than found in naturally occurring cells or cell lines that have not been modified by exogenous SLM-1, SLM-2, or Sam68 encoding nucleic acid sequences. Since SLM-1, SLM-2, Sam68, hnRNP K, and WASP derivatives are not naturally produced, it is apparent that cells from which SLM-1, SLM-2, Sam68, hnRNP K, and WASP
derivatives can be isolated do not naturally encode SLM-1, SLM-2, Sam68, hnRNP K, or WASP derivatives but are genetically modified to do so.
Cells from which SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives may be isolated include both prokaryotic and eukaryotic cells. Preferred cellular sources for the isolation of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include mammalian cells possessing high levels of arginine methyltransferase activity. Of particular interest are mammalian cells transfected with arginine methyltransferases such as PRMT1, PRMT2, and PRMT3. Other mammalian cell sources of interest for the purification of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include mammalian cells stimulated by growth factors that bind to growth factor receptors that stimulate arginine methyltransferase activity. Another preferred source for preparations from which to purify SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives is insect cells, preferably grown in tissue culture, and genetically modified by baculovirus expression vectors or the like to express SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives and an arginine methyltransferase. A particularly preferred source of SLM-1, SLM-2, Sam68, hnRNP K, or WASP derivatives is the SF9 cell line. Another source would be to produce recombinant SLM-1, SLM-2, Sam68, hnRNP K, or WASP in bacteria or yeast as a fusion protein with tags such as histidine repeats or glutathione S-transferase proteins. It will be appreciated that purified SLM-1, SLM-2, Sam68, hnRNP K, or WASP can be methylated in vitro to yield purified methylated SLM-1, SLM-2, Sam68, knRNP K, or WASP.
Purification of SLM-1, SLM-2, Sam68, hnRNP K, or WASP
Derivatives Affinity purification of SLM-1, SLM-2, Sam68, hnRNP K, or WASP and their derivatives may employ various immobilized reagents specific for SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivatives, i.e., affinity reagents. The affinity purification may be performed in batches or employ chromatography columns. The affinity reagents may be immobilized to a variety of inert matrices prepared in bead form. Suitable immobilization matrices include cross-linked agarose beads, Sepharose, cross-linked polyacrylamide beads, Sephacryl, and the like. When the affinity reagents used are antibodies, a preferred immobilizing matrix is protein A
sepharose. Affinity reagents of interest include antibodies, purified SH3 domains, purified SH2 domains, and homopolymeric RNA. Purification of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP can be performed by using methylated arginine specific antibodies. Purification of non-methylated SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivatives may also be achieved through the use of SLM-l, SLM-2, Sam68, hnRNP K, or WASP
specific antibodies as affinity reagents.
In addition to production of purified SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives by purification of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives produced in cells, purified SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives may be produced by organic chemical reactions performed in vitro. Automated equipment for the direct synthesis of the polypeptides disclosed herein is commercially available. Such equipment provides convenient access to peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques. The use of such commercially available polypeptide synthesis machines and the like are a preferred method of synthesizing oligopeptides SLM-1, SLM-2, Sam68, hnRNP K or WASP derivatives having about 5-25 amino acids. It will be appreciated by those skilled in the art that oligopeptides can be synthesized containing arginines that are methylated.
Other methods for synthesis of SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives include the in vitro transcription of DNA sequences encoding SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives coupled with the in vitro translation of the RNA transcripts thus produced. In vitro transcription systems are well known in the art. In vitro transcription systems typically involve the creation of nucleotide sequences in which the coding sequence of interest is located downstream from a strong promoter, such as a promoter specific for SP-6 or T7 RNA polymerases, followed by the addition of an RNA polymerase specific for the promoter, and substrates required for the reaction. Similarly, in vitro translation systems are well known in the art and may be used to produce SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivative polypeptides from a variety of transcripts produced by in vitro transcription systems.
Uses for methylarginine specific antibodies The invention provides for antibodies capable of recognizing polypeptides containing methylated arginines. By the term "antibodies," it is intended both polyclonal and monoclonal antibodies with natural immunoglobulin sequences, synthetic antibody derivatives, and the like; antibodies may be modified so as to be joined to any of a variety of labels, fluorescent, radioactive, enzymatic, biotin/avidin or the like.
Synthetic antibody derivatives include natural immunoglobulin sequences that have been mutated and selected for altered binding specificity, various immunoglobulin gene derived polypeptides, typically single chain, produced by genetically modified bacteria, antibodies modified so as to contain modified constant regions and the like; a review of such synthetic antibody derivatives based on the principles of antibody formation is provided in Harlow and Lane, Antibodies, Coldspring Harbor Laboratory, Coldspring Harbor Press, A
Laboratory Manual, 1988.
Antibodies specific for methylated arginines may be generated by using proteins or peptides that contain one or more methylated arginines. By induction of antibodies it is intended not only the stimulation of an immune response by injection into animals, but analogous steps in the production of synthetic antibodies such as the screening of recombinant immunoglobulin libraries (Orlandi et al., 1989 or Huse et al., 1989), or the in vitro stimulation of lymphocyte populations.
Of particular interest is the development of antibody preparations, monoclonal antibodies, specific for methylated arginine containing polypeptides, i.e., monospecific antibodies.
Short oligopeptides, i.e., containing about 20 amino acids or less, are of particular interest for both the induction and the screening of mono-specific antibodies specific for epitopes of interest. In general, oligopeptides for use in the induction of epitope specific monospecific antibodies will have an amino acid sequence corresponding to at least a portion of the epitope of interest.
It is also of interest to produce antibody preparations that are capable of specifically binding to the methylated forms of SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Uses for such methylation state detecting antibodies include the measurement of the degree of methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell.
Assays The subject invention provides methods and reagents for performing assays capable of measuring the amount of arginine methyltransferase activity present in a cell and the fraction of proteins that contain methylated arginines.
SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives may be used as substrates for the detection and quantification of arginine methyltransferase activity from a variety of cellular sources. It is desirable to measure arginine methyltransferase activity for several reasons. Of particular interest is the measurement of arginine methyltransferase activity produced by arginine methyltransferase encoded by oncogenes and proto-oncogenes.
Thus assays for arginine methyltransferase may be employed to determine whether a cell is cancerous or has cancer potential.
Also of interest is the measurement of arginine methyltransferase activity attributable to the stimulation membrane bound ligand receptors associated with arginine methyltransferase activity, since the extent of methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be used to measure 5 the extent to which ligands are binding to receptors.
Arainine methyltransferase assays Arginine methyltransferase assays of interest measure the rate of methylation of SLM-1, SLM-2, Sam68, hnRNP K, WASP
and their derivatives by arginine methyltransferase in a cell, 10 rather than simply measuring the amount of methylated SLM-l, SLM-2, Sam68, hnRNP K, or WASP present in a cell. Thus arginine methyltransferase assays of interest employ a method for distinguishing arginine methylation events that take place during an assay from arginine methylation events that occur before an assay. Arginine methyltransferase assays may employ the step of adding a methyl group, preferably S-adenosyl methionine and the like, to an assay mixture containing suitable buffers and salts. Methyl sources may be radioactively labelled on the terminal carbon or hydrogens so as to provide for the detection of arginine methyltransferase activity.
Arginine methyltransferase assays employing radioactive labels may or may not employ the step of addition of SLM-l, SLM-2, Sam68, hnRNP K, WASP or their derivatives, because arginine methyltransferase substrates initially present in the cell, or SLM-1, SLM-2, Sam68, hnRNP K, WASP or their derivatives added externally, and subsequently methylated by a radioactive methyl source, may subsequently be isolated by addition of SLM-1, SLM-2, Sam68, hnRNP K, or WASP-specific antibodies, followed by the step of radiometric quantitation.
Generally it will be preferable to add SLM-1, SLM-2, Sam68, hnRNP K, or WASP, preferably produced by recombinant means, to the assay mixture. After the arginine methyltransferase reaction has been allowed to progress, the amount of radioactive label incorporated into SLM-1, SLM-2, Sam68, hnRNP K, or WASP is measured by radiometric means. In order to measure the amount of labelling, the unincorporated label must be removed prior to radiometric measurement. This removal can be achieved through a variety of means including immunoprecipitation of SLM-1, SLM-2, Sam68, hnRNP K or WASP
with anti-SLM-1, SLM-2, Sam68, hnRNP K or WASP antibodies.
An important advantage of the subject invention is that the polypeptides provided for permit the detection and quantification of arginine methyltransferase activity without requiring the addition of radioactively labelled methyl groups.
Methods for measuring arginine methyltransferase activity without the addition of radioactively labelled methyl groups include assays involving the use of (1) SLM-1, SLM-2, Sam68, hnRNP K or WASP derivatives that contain epitopes not present on SLM-1, SLM-2, Sam68, hnRNP K, or WASP, (2) antibodies specific for that epitope, and (3) anti-methylated-arginine antibodies or protein domains that specifically bind to arginine methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Such assays involve the addition of the SLM-l, SLM-2, Sam68, hnRNP K, or WASP derivatives to the assay mixture, followed by the immunoprecipitation or immunobilization of the SLM-1, SLM-2, Sam68, hnRNP K, or WASP derivative by means of the epitope specific antibody so as to separate the SLM-1, SLM-2, Sam68, hnRNP K or WASP derivative from other cellular proteins containing methylated arginines (including endogenous SLM-1, SLM-2, Sam68, hnRNP K, and WASP).
In addition to providing methods and reagents for use in the detection of arginine methyltransferase activity present in a cell, the subject invention provides methods and reagents for determining what fraction of the SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell is methylated as well as determining the absolute amount of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in a cell. By cell, it is intended not only individual cells, but multiple cells. Arginine methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be detected by a variety of means. If the methyl donor in the assay contains a radioactive label, then arginine methyltransferase activity may be detected by separating the labelled SLM-1, SLM-2, Sam68, hnRNP K, or WASP from the unincorporated label and quantitating the amount of label incorporated into the SLM-l, SLM-2, Sam68, hnRNP K, or WASP substrate. When non-radioactively labelled methyl donors are used in assays, methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP may be detected by means of generally known immunoassays in which the immunospecific reagent employed is specific for methylated arginines.
The subject invention provides for methods and reagents for performing assays capable of determining what fraction of SLM-1, SLM-2, Sam68, hnRNP K, or WASP in a cell is methylated. Such assay may employ well known immunoassay technology such as ELISA, RIA, western blotting, and the like.
The use of SLM-1, SLM-2, Sam68, hnRNP K, or WASP specific antibodies (as well as SLM-1, SLM-2, Sam68, hnRNP K, WASP and their derivatives) as provided for by the subject invention may be used in connection with the preciously described well-established immunoassay technology, in order to provide for assays capable of detecting the extent of SLM-1, SLM-2, Sam68, hnRNP K, or WASP methylation in a cell. In general, such assays will employ two types of SLM-1, SLM-2, Sam68, hnRNP K, or WASP specific antibodies (or similar binding reagent) in an immobilized phase, namely (1) antibodies capable of binding SLM-l, SLM-2, Sam68, hnRNP K, or WASP in both methylated and non-methylated form or antibodies capable of binding only the non-methylated form of SLM-1, SLM-2, Sam68, hnRNP K, or WASP, and (2) antibodies capable of binding the methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP. By employing two types of specific binding reagent, it is possible to determine the relative quantities of the methylated and unmethylated forms of SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in a sample. The binding of SLM-1, SLM-2, Sam68, hnRNP K, or WASP (methylated and non-methylated) to an immobilized antibody phase may be detected by the addition of a third antibody, preferably labelled, and having an affinity for exposed epitopes on the antibody bound SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
Comparison of binding of the labelled antibody to SLM-1, SLM-2, Sam68, hnRNP K, or WASP bound to the 2 different types of immobilized antibody may be used to determine the fraction of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP present among the total SLM-1, SLM-2, Sam68, hnRNP K, or WASP present in the sample.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
Example 1 Identifying protein arginine methyltransferase substrates.
HeLa cells (0.5 x 106) were transfected with 5 ug of each plasmid expressing myc epitope tagged mouse SLM-1 (Di Fruscio et al., 1999), mouse SLM-2 (Di Fruscio et al., 1999), mouse Sam68 (Wong et al., 1992), brine shrimp GRP33 (Cruz-Alvarez and Pellicer, 1987), human hnRNP K (Michael et al., 1997) and mouse Qkl (Ebersole et al., 1996) as described previously (Richard et al., 1995). Twelve hours after transfection the cells were lysed in 500 ul lysis buffer (1% Triton X-100, 20 mM Tris-HC1 pH 7.4, 150 mM NaCl, 50 mM
NaF, 100 uM sodium vanadate, 0.01% phenylmethanesulfonyl fluoride, 1 ug/ml aprotinin, and 1 ug/ml leupeptin) on ice for 15 minutes. The debris was removed by centrifugation and the supernatant was divided equally into two fractions. Half was immunoprecipitated with control immunoglobulin G and the other half was immunoprecipitated with the anti-myc antibody 9E10 (American Tissue Culture Collection) for 1 hour on ice. Then 20 ul of 50% slurry of protein A Sepharose was added and incubated with end-over-end mixing for 30 minutes. The immunoprecipitated proteins were washed extensively and 2 ug of purified recombinant GST-PRMT1 and 2 ul of [3H-methyl]-adenosyl-1-methionine was added in 25 mM Tris-HCl pH 7.4, 1 mM EDTA for 30 minutes at room temperature. PRMT1 is a protein arginine methyltransferase that uses the methyl groups from S-adenosyl-methionine to transfer them to the guandino nitrogens of arginine (Gary and Clarke, 1998). The reaction was stopped by adding 20 ul of 2X Laemmli buffer (Laemmli, 1970) and loaded on an SDS 10%-polyacrylamide gel.
The gel was then soaked in 3H Enhance (ICN) for 30 minutes and then the gel was equilibrated with water for 1 hour, dried and exposed to film. It was observed that in myc immunoprecipitates Sam68, SLM-1, SLM-2, GRP33, and hnRNP K but not Qk1 were labelled with PRMT1. These data demonstrate that SLM-1, SLM-2, Sam68, GRP33 and hnRNP K are substrates for methyltransferase in vitro and may also be substrates for methyltransferase in vivo. Type I methyltransferases are known to methylate arginines that contain a C-terminal glycine (Gary and Clarke, 1998), and indeed the methylated proteins contain numerous RG repeats.
Example 2 Preparation of antibodies which recognize dimethylarainine residues.
Peptides were synthesized that correspond to the arginine-glycine repeats of Sam68, hnRNP K and Wiskott-Aldrich syndrome protein (WASP). These peptides were synthesized unmethylated or with the arginines containing asymmetric dimethylarginines. The peptides for Sam68 were:
Sam68 P3 (GVSVRGRGAAPPPPPVPRGRGVGP) Sam68 P3* (GVSVR*GR*GAAPPPPPVPR*GR*GVGP) wherein R* denotes dimethylarginine Sam68 P4 (TRGATVTRGVPPPPTVRGAPTPR) Sam68 P4* (TR*GATVTR*GVPPPPTVR*GAPTPR) wherein R* denotes dimethylarginine P3* and P4* were coupled by the amino terminus to KLH and injected in rabbits to generate rabbit polyclonal antibodies.
The goal of generating these antibodies is to have antibodies that would recognize all dimethylated arginines in polypeptides or at the very least recognize the dimethylated arginines in the context of the peptide generated. Thus, in the former situation the antibody may recognize any protein or peptide that contains dimethylated arginines. In the latter situation, the antibody would recognize only dimethylated Sam68 and no other protein. After analyzing the antisera obtained from the P3* injected rabbit and the P4* injected rabbit both situations were obtained. The anti-sera from the rabbit injected with P4*
only recognized P4* and had some cross-reactivity to the unmethylated Sam68 P4 peptides. This antibody did not recognize P3 methylated or unmethylated. The anti-sera from 5 the rabbit injected with P3*, interestingly, recognized many peptides that contained dimethylated arginines. This anti-sera did not recognize the unmethylated counterpart of these peptides. These analyses were performed by using the ELISA.
Other peptides that were used for these analyses include:
10 Sam68 PO (RLTPSRPSPLPHRPRGGGGGPRGG) Sam68 PO* (RLTPSRPSPLPHRPR*GGGGGPR*GG) wherein R* denotes dimethylarginine WASP P1 biotin-RQEPLPPPPPPSRGGNQLPR
WASP P1* biotin-RQEPLPPPPPPSR(*)GGNQLPR
Example 2 Preparation of antibodies which recognize dimethylarainine residues.
Peptides were synthesized that correspond to the arginine-glycine repeats of Sam68, hnRNP K and Wiskott-Aldrich syndrome protein (WASP). These peptides were synthesized unmethylated or with the arginines containing asymmetric dimethylarginines. The peptides for Sam68 were:
Sam68 P3 (GVSVRGRGAAPPPPPVPRGRGVGP) Sam68 P3* (GVSVR*GR*GAAPPPPPVPR*GR*GVGP) wherein R* denotes dimethylarginine Sam68 P4 (TRGATVTRGVPPPPTVRGAPTPR) Sam68 P4* (TR*GATVTR*GVPPPPTVR*GAPTPR) wherein R* denotes dimethylarginine P3* and P4* were coupled by the amino terminus to KLH and injected in rabbits to generate rabbit polyclonal antibodies.
The goal of generating these antibodies is to have antibodies that would recognize all dimethylated arginines in polypeptides or at the very least recognize the dimethylated arginines in the context of the peptide generated. Thus, in the former situation the antibody may recognize any protein or peptide that contains dimethylated arginines. In the latter situation, the antibody would recognize only dimethylated Sam68 and no other protein. After analyzing the antisera obtained from the P3* injected rabbit and the P4* injected rabbit both situations were obtained. The anti-sera from the rabbit injected with P4*
only recognized P4* and had some cross-reactivity to the unmethylated Sam68 P4 peptides. This antibody did not recognize P3 methylated or unmethylated. The anti-sera from 5 the rabbit injected with P3*, interestingly, recognized many peptides that contained dimethylated arginines. This anti-sera did not recognize the unmethylated counterpart of these peptides. These analyses were performed by using the ELISA.
Other peptides that were used for these analyses include:
10 Sam68 PO (RLTPSRPSPLPHRPRGGGGGPRGG) Sam68 PO* (RLTPSRPSPLPHRPR*GGGGGPR*GG) wherein R* denotes dimethylarginine WASP P1 biotin-RQEPLPPPPPPSRGGNQLPR
WASP P1* biotin-RQEPLPPPPPPSR(*)GGNQLPR
15 wherein R* denotes dimethylarginine WASP P2 biotin-APPPPTPRGPPPPGRGGPPPPPP
WASP P2* biotin-APPPPTPR(*)GPPPPGR(*)GGPPPPPP
wherein R* denotes dimethylarginine hnRNPK P1 biotin-SPRRGPPPPPPGRGGRGGSR
hnRNPK P1* biotin-SPRR(*)GPPPPPPGR(*)GGR(*)GGSR
wherein R* denotes dimethylarginine hnRNPK P2 biotin-RARNLPLPPPPPPRGGDL
hnRNPK P2* biotin-RARNLPLPPPPPPR(*)GGDL
wherein R* denotes dimethylarginine These studies demonstrate that antibodies can be generated that will recognize dimethylarginine residues in polypeptides. The presence of dimethylated arginines in a medium can be monitored and examined by using the medium to disrupt the interaction between a peptide containing dimethylated arginines and a methylarginine specific antibody. As such it is possible to examine whether a given medium contains polypeptides with dimethylated arginines.
Example 3 Effect of methylation on binding domains.
The Sam68 P3 peptide binds the SH3 domains of p59f~"' and PLC~y-1 (Richard et al., 1995). HeLa cells were lysed and incubated with the SH3 domains of p59fs'I' and PLCy-1 covalently coupled to beads in the absence or presence of unmethylated or methylated peptides. It was observed that P3 or P4 peptides described in Example 2 competed out the binding of Sam68 to the SH3 domains, but that the methylated version of those peptides P3* or P4* could not compete out the binding of Sam68 to the SH3 domain of p59f~ and PLCy-1. These data suggest that methylated peptides cannot bind certain SH3 domain such as those of p59f}"' and PLCy-1. Thus, these data suggest that most likely a novel protein module exists that has the ability to bind dimethylated arginine residues, much like phosphotyrosine polypeptides associate with SH2 and PTB domain containing proteins (Pawson, 1995).
WASP P2* biotin-APPPPTPR(*)GPPPPGR(*)GGPPPPPP
wherein R* denotes dimethylarginine hnRNPK P1 biotin-SPRRGPPPPPPGRGGRGGSR
hnRNPK P1* biotin-SPRR(*)GPPPPPPGR(*)GGR(*)GGSR
wherein R* denotes dimethylarginine hnRNPK P2 biotin-RARNLPLPPPPPPRGGDL
hnRNPK P2* biotin-RARNLPLPPPPPPR(*)GGDL
wherein R* denotes dimethylarginine These studies demonstrate that antibodies can be generated that will recognize dimethylarginine residues in polypeptides. The presence of dimethylated arginines in a medium can be monitored and examined by using the medium to disrupt the interaction between a peptide containing dimethylated arginines and a methylarginine specific antibody. As such it is possible to examine whether a given medium contains polypeptides with dimethylated arginines.
Example 3 Effect of methylation on binding domains.
The Sam68 P3 peptide binds the SH3 domains of p59f~"' and PLC~y-1 (Richard et al., 1995). HeLa cells were lysed and incubated with the SH3 domains of p59fs'I' and PLCy-1 covalently coupled to beads in the absence or presence of unmethylated or methylated peptides. It was observed that P3 or P4 peptides described in Example 2 competed out the binding of Sam68 to the SH3 domains, but that the methylated version of those peptides P3* or P4* could not compete out the binding of Sam68 to the SH3 domain of p59f~ and PLCy-1. These data suggest that methylated peptides cannot bind certain SH3 domain such as those of p59f}"' and PLCy-1. Thus, these data suggest that most likely a novel protein module exists that has the ability to bind dimethylated arginine residues, much like phosphotyrosine polypeptides associate with SH2 and PTB domain containing proteins (Pawson, 1995).
References Abramovich et al, 1997 EMBO J. 16:260-266 Cruz-Alvarez, M. and Pellicer, A. 1987. Cloning of a full-length complementary cDNA for as Artemia salina glycine-rich protein. J. Biol. Chem., 262:13377-13380.
Di Fruscio, M., Chen, T. and Richard, S. 1999. Two novel Sam68-like mammalian proteins SLM-1 and SLM-2: SLM-1 is a Src substrate during mitosis, Proc. Natl. Acad. Sci. USA, 96:2710-2715.
Ebersole, T.A., Chen, Q., Justice, M.J. and Artzt, K. 1996. The quaking gene unites signal transduction and RNA binding in the developing nervous system. Nature Genetics, 12:260-265.
Gary, J.D. and Clarke, S. 1998. RNA and protein interactions modulated by protein arginine methylation. Prog. Nuc. Acid Res.
Mol. Biol. 61:65-131.
Huse et al., 1989 Science 256: 1275-1281 Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685.
Lin et al., 1996 JBC 271:15034-44 Michael W. M., Eder, P.S. and Dreyfuss, G. 1997. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. EMBO J., 16:3587-3598.
Orlandi et al., 1989 PNAS USA 86:3833-3837 Pawson, T. 1995. Protein modules and signalling networks.
Nature, 373:573-580.
Di Fruscio, M., Chen, T. and Richard, S. 1999. Two novel Sam68-like mammalian proteins SLM-1 and SLM-2: SLM-1 is a Src substrate during mitosis, Proc. Natl. Acad. Sci. USA, 96:2710-2715.
Ebersole, T.A., Chen, Q., Justice, M.J. and Artzt, K. 1996. The quaking gene unites signal transduction and RNA binding in the developing nervous system. Nature Genetics, 12:260-265.
Gary, J.D. and Clarke, S. 1998. RNA and protein interactions modulated by protein arginine methylation. Prog. Nuc. Acid Res.
Mol. Biol. 61:65-131.
Huse et al., 1989 Science 256: 1275-1281 Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685.
Lin et al., 1996 JBC 271:15034-44 Michael W. M., Eder, P.S. and Dreyfuss, G. 1997. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. EMBO J., 16:3587-3598.
Orlandi et al., 1989 PNAS USA 86:3833-3837 Pawson, T. 1995. Protein modules and signalling networks.
Nature, 373:573-580.
Richard, S. Yu, D., Blumer, K.J., Hausladen, D., Olszowy, M.W., Connelly, P.A. and Shaw, A. S. 1995. Association of p62, a multi-functional SH2- and SH3-binding protein, with src-family tyrosine Kinases, Grb2, and phospholipase Cg-1. Mol. Cell.
Biol., 15:186-197.
Scott et al., 1998 Genomics 48:330-340 Tang et al., 1998 JBC 273: 16935-45 Wong, G., Muller, O., Clark, R., Conroy, L., Moran, M.F., Polakis, P. and McCormick, F. 1992. Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell, 69:551-558.
Biol., 15:186-197.
Scott et al., 1998 Genomics 48:330-340 Tang et al., 1998 JBC 273: 16935-45 Wong, G., Muller, O., Clark, R., Conroy, L., Moran, M.F., Polakis, P. and McCormick, F. 1992. Molecular cloning and nucleic acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell, 69:551-558.
Sequences Figure 1. Amino acid sequence of SLM-1 - ID NO: 1.
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: Mus musculus Figure 2. Amino acid sequence of SLM-2 - ID N0: 2.
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: Mus musculus Figure 3. Amino acid sequence of Sam68 - ID NO: 3.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 443 amino acids 10 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
15 (vi) ORIGINAL SOURCE: brain library (A) ORGANISM: Mus musculus Figure 4. Amino acid sequence of hnRNPK - ID NO: 4.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: human Figure 5. Amino acid sequence of WASP - ID NO: 5.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 520 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: mouse Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly;
H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln;
R, Arg; S,Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: Mus musculus Figure 2. Amino acid sequence of SLM-2 - ID N0: 2.
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 346 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: Mus musculus Figure 3. Amino acid sequence of Sam68 - ID NO: 3.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 443 amino acids 10 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
15 (vi) ORIGINAL SOURCE: brain library (A) ORGANISM: Mus musculus Figure 4. Amino acid sequence of hnRNPK - ID NO: 4.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: human Figure 5. Amino acid sequence of WASP - ID NO: 5.
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 520 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: full-length ORF
(vi) ORIGINAL SOURCE: brain library (A) ORGANISM: mouse Abbreviations for the amino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly;
H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln;
R, Arg; S,Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Claims (6)
1. A method of assaying arginine methyltransferase activity which comprises measuring methylation of SLM-1, SLM-2, Sam68, hnRNP K, or WASP.
2. A method for assaying a medium for the presence of a substance that affects an interaction between an arginine methyl-specific antibody and a methylated arginine ligand comprising incubating a methylarginine binding antibody or protein module or a subdomain thereof and a methylated ligand which is capable of interacting with said specific methylarginine binding antibody or protein module or subdomain thereof to form a specific methylarginine binding antibody or protein module- methylated ligand complex, with a substance which is suspected of affecting a specific methylarginine binding antibody or protein module-methylated ligand regulatory system, under conditions which permit the formation of said specific methylarginine binding antibody or protein module methylated ligand complex, and assaying for said specific methylarginine binding antibody or protein module methylated ligand complex, free specific methylarginine binding antibody or protein module or subdomains thereof, or non-complexed methylated ligand.
3. A method as claimed in 2 wherein the substance is from an individual with or suspected of having the Wiskott-Aldrich Syndrome.
4. An isolated antibody that specifically recognizes arginine methylated amino acids.
5. Use of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP peptides as inhibitors of SH3 domain.
6. Use of methylated SLM-1, SLM-2, Sam68, hnRNP K, or WASP peptides to suppress the immune system.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008164517A (en) * | 2006-12-28 | 2008-07-17 | Sentan Seimei Kagaku Kenkyusho:Kk | Immunological assay method of methylated heterogeneous-nuclear-ribonucleoprotein and its use |
CN104677998A (en) * | 2013-11-29 | 2015-06-03 | 沈阳药科大学 | Novel biomarker for evaluating lung cancer disease through plasma |
-
1999
- 1999-04-08 CA CA002266760A patent/CA2266760A1/en not_active Abandoned
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008164517A (en) * | 2006-12-28 | 2008-07-17 | Sentan Seimei Kagaku Kenkyusho:Kk | Immunological assay method of methylated heterogeneous-nuclear-ribonucleoprotein and its use |
CN104677998A (en) * | 2013-11-29 | 2015-06-03 | 沈阳药科大学 | Novel biomarker for evaluating lung cancer disease through plasma |
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