Abstract
The present invention relates to a method for producing a site-specifically serine ADP-ribosylated protein or peptide being, comprising: (a) subjecting a protein or peptide comprising or consisting of an amino acid sequence comprising two or more serines, wherein at least one serine is phosphorylated and at least one serine is non-phosphorylated, to a serine ADP-ribosylation reaction. The present invention also relates to a site-specifically serine ADP-ribosylated protein or peptide produced by the method of the invention.
Claims
1. A method for producing a site-specifically serine ADP-ribosylated protein or peptide, comprising: (a) subjecting a protein or peptide comprising or consisting of an amino acid sequence comprising two or more serines, wherein at least one serine is phosphorylated and at least one serine is non-phosphorylated, to a serine ADP-ribosylation reaction.
2. The method of claim 1, further comprising (b) removing the phosphate group(s) from the at least one phosphorylated serine.
3. The method of claim 2, wherein the phosphate group(s) is/are removed by an enzyme, preferably a phosphatase, more preferably an enzyme selected from the group consisting of protein serine/threonine phosphatases, and most preferably selected from the group consisting of the lambda protein phosphatase and alkaline phosphatase, wherein the alkaline phosphatase is preferably selected from calf intestinal phosphatase (CIP), antarctic phosphatase (AnP), and shrimp alkaline phosphatase (SAP).
4. The method of any one of claims 1 to 3, wherein step (a) comprises the synthesis of the protein or peptide as defined in claim 1, wherein the synthesis preferably comprises a solid-phase peptide synthesis.
5. The method of any one of claims 1 to 4, wherein the serine ADP-ribosylation reaction is carried out in an ADP-ribosylation reaction mixture, said mixture comprising (i) a buffered solution, (ii) NAD.sup.+, (iii) PARP-1, PARP-2 or the PARP-1 variant E988Q, (iv) HPF1, (v) sonicated DNA, said sonicated DNA preferably comprising DNA fragments of 10 to 330 bp, and (vi) the protein or peptide as defined in claim 1, step (a).
6. The method of any one of claims 1 to 5, further comprising purifying the site-specifically serine ADP-ribosylated protein or peptide.
7. The method of claim 6, wherein the site-specifically serine ADP-ribosylated protein or peptide is purified by StageTip fractionation employing C8, C18, SCX, SAX or SDB-RPS chromatography media, cation or anion exchange chromatography, hydrophilic interaction chromatography, phosphopeptide enrichment, enrichment with an ADP-ribose-binding protein domain, boronate affinity chromatography, filtering the reaction with an ultrafiltration device, a spin column or a combination thereof.
8. The method of any one of claims 1 to 7, further comprising formulating the produced site-specifically serine ADP-ribosylated protein or peptide into a composition, preferably a pharmaceutical, diagnostic or cosmetic composition.
9. The method of any one of claims 1 to 8, wherein the protein or peptide further comprises at least one post-translational modification other than serine ADP-ribosylation and serine phosphorylation.
10. The method of claim 9, wherein the post-translational modification is selected from lipidation, N- or O-linked glycosylation, phosphorylation of an amino acid other than serine, acetylation, amidation, hydroxylation, mono- or di- or tri-methylation, ubiquitylation, SUMOylation, neddylation, butyrylation, propionylation, crotonylation, 2-hydroxyisobutyrylation, malonylation, succinylation, citrullination, pyrrolidone carboxylic acid and sulfation.
11. The method of any one of claims 1 to 10, wherein the method is carried out ex vivo or in vitro.
12. A site-specifically serine ADP-ribosylated protein or peptide produced by the method of any one of claims 1 to 11.
13. A site-specifically serine ADP-ribosylated protein or peptide comprising or consisting of (i) the amino acid sequence of any one of SEQ ID NOs 17 to 309, wherein within the amino acid sequence at least one serine is ADP-ribosylated and at least one serine is not ADP-ribosylated, and wherein the at least one serine not being ADP-ribosylated is optionally phosphorylated, or (ii) an amino acid sequence being at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% identical to the amino acid sequence of (i), provided that at least two, preferably all the serines within the amino acid sequence of (i) are conserved.
14. A binding molecule, preferably an antibody, specifically binding to the site-specifically serine ADP-ribosylated protein or peptide of claim 12 or 13.
15. A composition, preferably a pharmaceutical, diagnostic or cosmetic composition, comprising the site-specifically ADP-ribosylated protein or peptide of claim 12 or 13 or the binding molecule of claim 14.
Description
[0170] The Figures Show.
[0171] FIG. 1. A. Schematic representation of the products of an ADP-ribosylation reaction when using a substrate that comprises at least two target serine. To note, the state-of-the-art methodology to enzymatically generate ADP-ribosylated substrates with more than one target serine cannot ensure site-specificity, creating a mixture of different ADPr species (same amino acid sequence, but modification on different residues). B. Schematic representation of a synthetic peptide corresponding to the amino acids 494-524 of human PARP-1. To note, this amino acid sequence comprises three target serines (underlined), which means that state-of-the-art enzymatic ADP-ribosylation reactions will produce a mixture of different ADPr species. C. Analysis of the ADP-ribosylation of four different variants of synthetic peptides corresponding to the amino acids 494-524 of human PARP-1 (2 μg). In vitro ADP-ribosylation assays were performed as previously described (Bonfiglio et al, Mol Cell, 2017) with 5 μM NAD.sup.+ and 63 nM (0.05 μCi/μl).sup.32P-NAD.sup.+, Activated DNA, recombinant PARP-1 (0.1 μM) in the presence or absence of recombinant HPF1 WT (1 μM) for 20 minutes at 25° C. Samples were resolved by SDS-PAGE and analysed by autoradiography. D. Analysis of the ADP-ribosylation of two different variants of synthetic peptides corresponding to the amino acids 494-524 of human PARP-1. In vitro ADP-ribosylation assays were performed as described in panel C, in the presence or absence of recombinant HPF1 WT or Y238A/R239A mutant (1 μM). Samples were resolved by SDS-PAGE and analysed by autoradiography. FIG. 1D is taken from in Bonfiglio et al., 2017, loc. lit. E. Representative chromatogram illustrating the non-selective Ser-ADPr on substrates with more than one target serine. Biotinylated PARP-1 (494-511) was subjected to incomplete in vitro enzymatic ADPr and analysed by LC-MS. As depicted, the presence of two target serine (Ser499 and S507) results in non-selective reactions generating heterogenous mixtures of modified species that differ in the positions at which ADP-ribose is attached. For simplicity, the chromatogram shows only the m/z ranges in which mono- and di-ADP-ribosylated PARP-1 peptides were detected (m/z=624.63-624.97 and m/z=759.91-760.13).
[0172] FIG. 2. A. Schematic representation of the protein or peptide with naturally-occurring amino acid sequence that is intended to be site-specifically ADP-ribosylated. To note, the amino acid sequence of the protein or peptide to be modified comprises at least two target serines. B. Schematic workflow for the means and methods for production of site-specific serine ADP-ribosylated protein or peptide with naturally-occurring amino acid sequences with two or more serines. The strategy consists of (i) providing a protein or peptide comprising or consisting of a naturally-occurring amino acid sequence comprising two or more serines, wherein at least one serine is phosphorylated and at least one serine is non-phosphorylated; (ii) subjecting such protein or protein to a serine ADP-ribosylation reaction; (iii) removing phosphorylation from the phosphorylated serine residues of (ii), thereby obtaining a site-specific Ser-ADPr protein or peptide with naturally-occurring amino acid sequence.
[0173] FIG. 3. A. Production of a site-specific ADP-ribosylated form of a PARP-1 peptide corresponding to the human amino acid sequence (494-514). 2 μg of Biotinylated PARP-1 (494-517) S507ph peptide (lane 2) were subjected to phosphatase treatment (400U of Lambda Phosphatase for 1 h at 30° C.) alone (lane 4), or to in vitro ADP-ribosylation (lane 6, 2 mM NAD.sup.+, activated DNA, recombinant PARP-1 (0.1 μM) and recombinant HPF1 WT (1 μM) for 6 h at 25° C.) followed by phosphatase treatment (lane 8, 400U of Lambda Phosphatase for 1 h at 30° C.). 2 μg of a PARP-1 (494-514) S507A peptide were used as a control (lanes 1, 3, 5 and 7). Using this system along the lines described herein, a single species site-specific ADP-ribosylated peptide PARP-1 at Ser-499 was obtained. (Lane 1) PARP-1 (494-514) S507A; (Lane 2) PARP-1 (494-514) S507ph; (Lane 3) PARP-1 (494-514) S507A treated with phosphatase; (Lane 4) PARP-1 (494-514) S507ph treated with phosphatase; (Lane 5) PARP-1 (494-514) S507A subjected to in vitro ADPr; (Lane 6) PARP-1 (494-514) S507ph subjected to in vitro ADPr; (Lane 7) PARP-1 (494-514) S507A subjected to in vitro ADPr followed by phosphatase treatment; and (Lane 8) PARP-1 (494-514) S507ph subjected to in vitro ADPr followed by phosphatase treatment. B. Deconvoluted MS spectra displaying the different peptide species described in FIG. 2B. Unmodified biotinylated PARP-1 (494-511) peptide with two target serine (Ser499 and Ser507) was synthesized with phosphorylation in Ser507 and subjected to enzymatic HPF1/PARP-1 ADPr, generating an intermediate species comprising Ser499-ADPr and Ser507-phosphorylation. After ADPr reaction, phosphate from Ser507 was removed by Lambda phosphatase, producing biotinylated PARP-1 (494-511) Ser499-ADPr peptide. As depicted, chemically synthesized peptide containing S507ph is shifted 79.96 Da with respect to the unmodified counterpart (mass of phosphorylation=79.96 Da). Next, in vitro reaction produces a shift of 541.06 Da (mass of ADPr=541.06 Da) and finally, treatment with lambda phosphatase produces a negative shift of 79.96 Da, indicating the removal of phosphorylation. The final product is pure PARP-1 (494-511) Ser499 ADP-ribosylated peptide. C. Production of a site-specific ADP-ribosylated form of a Histone H2B peptide corresponding to the human amino acid sequence (1-32). 2 μg of Biotinylated Histone H2B (1-32) S14ph peptide (lane 2) were subjected to phosphatase treatment (400U of Lambda Phosphatase for 1 h at 30° C.) alone (lane 1), or to in vitro ADP-ribosylation (lane 3, 2 mM NAD.sup.+, activated DNA, recombinant PARP-1 (0.1 μM) and recombinant HPF1 WT (1 μM) for 6 h at 25° C.) followed by phosphatase treatment (lane 4, 400U of Lambda Phosphatase for 1 h at 30° C.). Using this system along the lines described herein, a single species site-specific ADP-ribosylated peptide Histone H2B at Ser-6 was obtained. (Lane 1) Histone H2B (1-32); (Lane 2) Histone H2B (1-32) S14ph; (Lane 3) Histone H2B (1-32) S14ph subjected to in vitro ADPr; (Lane 4) Histone H2B (1-32) S14ph subjected to in vitro ADPr followed by phosphatase treatment.
[0174] FIG. 4. A. A peptide with more than one target serine residue is still ADP-ribosylated after mutation of the main target serine residue. Representative stained gels (Imperial™ Protein Stain) illustrating the non-selective Ser-ADPr on substrates with more than one target serine. Histone H2A peptides in which the major target serine has been substituted by alanine (Histone H2A (1-19) S1A) are still ADP-ribosylated due to the presence of other target serines. Biotinylated Histone H2A (1-19) peptide (Lane 1) was reacted with 2 mM NAD.sup.+ and 0.12 μM PARP-1 in the absence (Lane 2) or presence (Lane 3) of 1.5 μM HPF1 for 75 minutes at RT. Similarly, Biotinylated Histone H2A (1-19) S1A peptide (Lane 4) was reacted with 2 mM NAD.sup.+ and 0.12 μM PARP-1 in the absence (Lane 5) or presence (Lane 6) of 1.5 μM HPF1 for 75 minutes at RT. B. 2 μg of Biotinylated H2A (1-19) S18, 19ph peptide (lane 2) was subjected to phosphatase treatment (400U of Lambda Phosphatase for 1 h at 30° C.) alone (lane 4), or to in vitro ADP-ribosylation (lane 5, 2 mM NAD.sup.+, Activated DNA, recombinant PARP-1 (0.1 μM) and recombinant HPF1 WT (1 μM) for 6 h at 25° C.) followed by phosphatase treatment (lane 6, 400U of Lambda Phosphatase for 1 h at 30° C.). 2 μg of a H2A (1-19) peptide were used as a control (lanes 1 and 3). Following the strategy described in this application, a single species site-specific ADP-ribosylated peptide H2A at Ser-1 was obtained. (Lane 1) H2A (1-19) (Lane 2) H2A (1-19) S18, 19ph (Lane 3) H2A (1-19) treated with phosphatase (Lane 4) H2A (1-19) S18, 19ph treated with phosphatase (Lane 5) H2A (1-19) S18, 19ph subjected to in vitro ADPr (Lane 6) H2A (1-19) S18, 19ph subjected to in vitro ADPr followed by phosphatase treatment.
[0175] The examples illustrate the invention.
EXAMPLE 1—PRODUCTION OF A SITE-SPECIFIC ADP-RIBOSYLATED FORM OF A PARP-1 PEPTIDE CORRESPONDING TO THE HUMAN AMINO ACID SEQUENCE (494-514)
[0176] Unlike the commonly-used approach of replacing the undesired target serine with another residue, the methodology described in the present application does not require the replacement of serine residue(s) and the consequent use of a non-naturally-occurring sequence to prepare a protein or peptide being site-specifically serine ADP-ribosylated (FIG. 2). By these means, a site-specific -Ser499-ADP-ribosylated peptide corresponding to the naturally-occurring human amino acid sequence (494-514) of PARP-1 (FIGS. 3A and B) was obtained. Importantly, the PARP-1 Ser499-ADPr site is the main endogenous ADPr site upon DNA damage. A standard approach to prevent ADPr of Ser507 would be replacing this residue with alanine (FIG. 3A). However, the final ADP-ribosylated product will not carry the natural sequence, which might represent a drawback for downstream applications (e.g. generation of antibodies). To overcome this limitation, a novel strategy was designed that consists of obtaining by standard chemical synthesis, a phosphorylated Ser507 peptide that prevents ADPr of Ser507. After subjecting this peptide to ADPr reactions, the unmodified Ser-499 becomes ADP-ribosylated. As the resulting peptide is both phosphorylated and ADP-ribosylated, the phosphate is afterwards removed from the peptide without affecting ADP-ribosylation (e.g. a second enzymatic reaction with a recombinant commercially-available phosphatase). The resulting peptide is ADP-ribosylated on a specific serine and comprises a naturally-occurring sequence (FIGS. 2 and 3A and B). By the above-described means also a Histone H2B (1-32) which is site-specifically ADPr at Ser-6 was obtained (FIG. 3C).
EXAMPLE 2—PRODUCTION OF A SITE-SPECIFIC ADP-RIBOSYLATED FORM OF A H2A PEPTIDE CORRESPONDING TO THE HUMAN AMINO ACID SEQUENCE (1-19)
[0177] When a protein or peptide comprising at least two target serines is subjected to an ADP-ribosylation reaction, a mixture of different ADP-ribosylated species (same amino acid sequence, but modification on different residues) is generated (FIGS. 1A and 4). For example, the peptide corresponding to the human amino acid sequence (1-19) of H2A contains, in addition to the Ser-1, two additional Ser residues (Ser-18 and Ser-19) that have never been described in the literature as targets of ADPr. However, when mutating the only reported target serine (Ser-1), the peptide is still ADP-ribosylated (FIG. 4A, lane 6), demonstrating the limitations of using a peptide with at least two target serine to obtain site-specific ADP-ribosylated species. The methodology described in the present application solves this problem by preventing the ADP-ribosylation of undesired Ser residues and therefore allowing the preparation of a protein or peptide being site-specifically serine ADP-ribosylated. The novel strategy consists of using a phosphorylated Ser peptide (Ser18 and Ser19 of this example, FIG. 4B) that prevents ADPr of those residues. Similarly to what has been described in Example 1, after subjecting the phosphorylated peptide to ADPr reactions, the unmodified Ser-1 becomes ADP-ribosylated and to remove the phosphates of Ser18 and Ser19, the peptide is subjected to a phosphate treatment (FIG. 4B).
EXAMPLE 3—EXPERIMENTAL CONDITIONS UNDER WHICH CONSIDERABLE AMOUNTS OF PURE PARP-1 SER-499 MONO-ADP-RIBOSYLATED PEPTIDE WERE OBTAINED
[0178] Given that some important applications require considerable amounts of serine ADP-ribosylated peptides (e.g. generation of antibodies), the inventors scaled up the reactions and under the following tested conditions have been able to produce ˜500 μg of pure PARP-1 Ser-499 mono-ADP-ribosylated peptide:
Solvent: Water
[0179] Buffer: 50 mM Tris-HCl, pH=7.5;
Salts: 50 mM NaCl and 1 mM MgCl.SUB.2.;
NAD.SUP.+.: 2 mM;
PARP-1: 100 nM;
HPF1: 1.5 μM;
[0180] Substrate peptide: 700 μg (Biotinylated PARP-1 (494-517) S507ph peptide)
[0181] The reaction mix was incubated for 360 minutes at RT, adding 2 mM NAD.sup.+ every 120 minutes and stopped by adding 1 μM Olaparib. Afterwards, 1 mM MnCl.sub.2, 1×PMP buffer (New England Biolabs), 8000 U of Lambda Protein Phosphatase (Lambda PP, New England Biolabs) and 1 μM PARG were added to the reaction mix and it was incubated for 300 minutes at 30° C. Afterwards, the serine mono-ADP-ribosylated peptides were separated from the other constituents of the reaction mix by using reverse chromatography (C18 cartridge). Pure mono-Ser-499 ADP-ribosylated Biotinylated PARP-1 (494-517) peptide was eluted with 30% Acetonitrile.
