Generation and Characterization of Recombinant Antibody-like ADP

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Generation and Characterization of Recombinant Antibody-Like ADP-Ribose Binding Proteins Bryan A. Gibson, Lesley B. Conrad, Dan Huang, and W. Lee Kraus Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00670 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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For Table of Contents Use Only

ADPR Macro

Fc

ADP-ribose Binding Reagents

ADPR ADPR ADPR ADPR Macro

Fc

WWE Substrate Protein

Fc

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Biochemistry

Antibody-Based Assays

ACS Paragon Plus Environment

Biochemistry

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Gibson, Conrad, and Kraus

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October 13, 2017

Title:

Generation and Characterization of Recombinant Antibody-Like ADP-Ribose Binding Proteins

Authors: Bryan A. Gibson1, 2, Lesley B. Conrad1, 2, 3, 5, Dan Huang1, 2, 4, 5, and W. Lee Kraus* 1, 2

1

2

3

4

5

Affiliations: The Laboratory of Signaling and Gene Expression, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX, 75390-8511. The Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-8511. The Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, 75390-9032. Department of Cardiovascular Diseases, Clinical Center for Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, P. R. China.

These authors contributed equally to this work.

*Address correspondence to: W. Lee Kraus, Ph.D. Cecil H. and Ida Green Center for Reproductive Biology Sciences The University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard Dallas, TX 75390-8511 Phone: 214-648-2388 Fax: 214-648-0383 E-mail: [email protected]

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Abstract ADP-ribosylation is an enzyme-catalyzed post-translational modification of proteins in which the ADP-ribose (ADPR) moiety of NAD+ is transferred to a specific amino acid in a substrate protein. The biological functions of ADP-ribosylation are numerous and diverse, ranging from normal physiology to pathological conditions. Biochemical and cellular studies of the diverse forms and functions of ADPR require immunological reagents that can be used for detection and enrichment. The lack of a complete set of tools that recognize all forms of ADPR [i.e., mono-, oligo-, and poly(ADP-ribose)] has hampered progress. Herein, we describe the generation and characterization of a set of recombinant antibody-like ADP-ribose binding proteins, in which naturally-occurring ADPR binding domains, including macrodomains and WWE domains, have been functionalized by fusion to the Fc region of rabbit immunoglobulin. These reagents, which collectively recognize all forms of ADPR with different specificities, are useful in a broad array of antibody-based assays, such as immunoblotting, immunofluorescent staining of cells, and immunoprecipitation. Observations from these assays suggest that the biology of ADPR is more diverse, rich, and complex than previously thought. The ARBD-Fc fusion proteins described herein will be useful tools for future exploration of the chemistry, biochemistry, and biology of ADP-ribose.

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Key Words: ADP-ribose binding domain; Immunoglobulin Fc region; Macrodomain; Mono(ADP-ribose), Oligo(ADP-ribose), Poly(ADP-ribose), PARPs; Recombinant protein; WWE domain.

Abbreviations: ADPR: ADP-ribose ARBD: ADP-ribose binding domain Fc: Immunoglobulin constant region IgG: Immunoglobulin G MAR: Mono(ADP-ribose) OAR: Oligo(ADP-ribose) PAR: Poly(ADP-ribose) PARP: Poly(ADP-ribose) polymerase

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Introduction ADP-ribosylation is an enzyme-catalyzed post-translational modification of proteins in which the ADP-ribose (ADPR) moiety of NAD+ is transferred to a specific amino acid in a substrate protein, with release of the nicotinamide moiety.1 ADP-ribosylation reactions are catalyzed by a variety of ADP-ribosyltransferase (ART) enzymes, including (1) bacterial toxins, such as Cholera toxin and Diphtheria toxin2,3, (2) Ecto-ADP-ribosyltransferases (ectoARTs), which are extracellular, membrane-bound, or secretory proteins that share homology in the catalytic domain with Clostridium C2 and C3 toxins (ARTCs)4, (3) members of the sirtuin family of enzymes in eukaryotes and prokaryotes5-7, and (4) members of the poly(ADP-ribose) polymerase (PARP) family of enzymes, which share homology in the catalytic domain with Diphtheria toxin (ARTDs).8-10 With the exception of some members of the PARP family, ARTs generally catalyze mono(ADP-ribosyl)ation reactions.2,4,8,11,12

Most bacterial ARTs transfer

ADP-ribose onto arginine residues, although asparagine and other amino acids are targeted as well.2,3 PARP family mono- and poly(ADP-ribosyl)transferase enzymes transfer ADP-ribose primarily onto glutamate, aspartate, serine, and lysine residues, generating mono-, oligo-, and poly(ADPR-ribose) (MAR, OAR, and PAR, respectively).8,11-15 The biological functions of ADP-ribosylation are numerous and diverse, ranging from normal physiology to pathological conditions, such as bacterial toxicity, aging, and cancer.16-18 Recent advances in PARP-directed therapeutics have shown promise for the treatment of cancers.19,20 While the cellular targets of bacterial toxins and the molecular effects of the ADPribosylation reactions that they catalyze have been well characterized2,3, the specific targets and effects of PARP-mediated ADP-ribosylation are less well understood.16 The few examples of the latter include: (1) NFAT (ADP-ribosylation increases DNA binding21), (2) C/EBPβ (ADP-

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Biochemistry

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ribosylation inhibits DNA binding and transcriptional activity22), (3) p53 (ADP-ribosylation inhibits nuclear export23), and (4) NELF-E (ADP-ribosylation inhibits RNA binding and NELFdependent promoter-proximal pausing by RNA polymerase II24). ADP-ribosylation of proteins can alter the biochemical activity of the ADP-ribosylated protein or create new interaction surfaces that drive protein-protein interactions.8 Interestingly,

Figure 1 A

has

devised

protein modules that specifically recognize and

Poly(ADP-ribose) WWE Domain H. sapiens RNF146

nature

Macrodomains H. sapiens macroH2A1.1 A. fulgidus AF1521

bind to various forms of ADP-ribose.25,26 These modules are found in a variety of proteins with diverse functions, including a

B

Mono(ADP-ribose) Macrodomains H. sapiens macroH2A1.1 A. fulgidus AF1521 H. sapiens PARP-14

number of PARP family members, and are

C IgG Monoclonal Antibody

likely to mediate many of the biological functions of ADP-ribosylation.8,25,26 well Fv Region Fc Region Replaced By ADP-ribose Binding Domain

characterized

ADP-ribose

Some binding

domains (ARBDs) include macrodomains and

D Affinity Tag(s)

Gly-Ser Linker

ADPR Binding Domain

Fc Domain

WWE domains.25,26 Macrodomains recognize

Bacterial Expression Vector

MAR, as well as the terminal ADPR moieties

Modular Specificity

in OAR and PAR, allowing them to bind to all three forms of ADPR (i.e., MAR, OAR, and PAR) (Figure 1, A and B).27-29 In contrast, WWE domains recognize the iso-ADPR linkages joining ADPR monomers, restricting their binding to OAR and PAR (Figure 1, A and B).30-32 In addition to the biology that they provide, these and other types of ARBDs can be functionalized to serve as useful research tools for the molecular recognition of various forms of ADPR.22,24,29,33-36

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Biochemistry


October 13, 2017

Although recent developments in the mass spectrometry-based identification of ADPRmodified amino acids have enhanced the study of specific ADP-ribosylation events on target proteins37, the lack of a complete set of immunological tools that recognize the diverse forms of ADPR has hampered progress. Anti-ADPR polyclonal antibodies have been reported, but the specificity and utility of these antibodies has not been assessed broadly and, like other polyclonal antibodies, they are difficult to produce in a constant supply.38-44 Instead, the PARP field has relied on the anti-PAR monoclonal antibody 10H, which is thought to bind to PAR with a lower limit for the length of polymers detected of 10 to 20 ADPR units.45,46 Although useful, this antibody has left the field blind to mono- and oligo(ADP-ribosyl)ation, as well as their biological importance. Herein, we describe the generation and characterization of a set of recombinant antibody-like ADP-ribose binding proteins, in which natural ARBDs have been functionalized with the Fc region of rabbit immunoglobulin. These reagents are useful in a broad array of antibody-based assays, such as immunoblotting, immunofluorescent staining of cells, and immunoprecipitation.

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Biochemistry

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October 13, 2017

Materials and Methods

Antibodies and ADPR Binding Reagents Alexa Fluor 488- and HRP-conjugated goat anti-rabbit IgG antibodies (A-11008 and 31346, respectively) and HRP-conjugated goat anti-mouse IgG antibody (31430) were purchased from Thermo Fisher Scientific. The anti-poly(ADP-ribose) mouse monoclonal antibody 10H was purchased from Millipore (MAB3192).

The custom anti-PARP-1 rabbit polyclonal

antiserum, which was generated by using an antigen comprising the amino-terminal half of PARP-147, is now available from Active Motif (39559).

The ARBD-Fc fusion proteins

described herein are now available through Millipore (MABE1016, RRID AB_2665466; MABE1031, RRID AB_2665467: MABE1075, RRID AB_2665468; MABE1076, RRID: AB_2665468).

Construction of Plasmid Vectors for the Expression of ADPR Binding Domain-Fc Fusion Proteins in Bacteria WWE(RNF-146)-Fc. DNA encoding the WWE(RNF146)-Fc reagent, comprising (1) the WWE domain from H. sapiens RNF14631,32,48,49 (UniProt ID Q9NTX7), which we refer to as WWE(RNF146), (2) a flexible linker, and (3) the constant or “Fc” region of rabbit immunoglobulin, was synthesized as three overlapping gene blocks by Integrated DNA Technologies (IDT). These three fragments were assembled and amplified into a single double stranded DNA fragment using PCR, and were subsequently cloned into the pET19b vector (Novagen) between the NdeI and BamHI restriction endonuclease sites. Macro(AF1521)-Fc and Macro(mH2A1.1)-Fc. DNA encoding the macrodomain from

7


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October 13, 2017

A. fulgidus AF152127,50 (UniProt ID O28751) or H. sapiens macroH2A1.128,29 (UniProt ID A0A0D2UG83) [i.e., Macro(AF1521) and Macro(mH2A1.1), respectively] were amplified using PCR from plasmid DNA (kindly provided by from M. Bycroft and Y. Yu, respectively). The WWE cassette from the WWE(RNF146)-Fc/pET19b plasmid (described above) was excised using the NdeI and SalI sites (the latter is within the linker region) and replaced with PCR amplified DNA encoding Macro(AF1521) and Macro(mH2A1.1), which were also digested using NdeI and SalI. Macro3X(PARP14)-Fc and Macro2/3(PARP14)-Fc. DNA encoding either the triple macrodomain

cassette

[i.e.

Macro3X(PARP14)]

or

macrodomains

2

and

3

[i.e.

Macro2/3(PARP14)] from H. sapiens PARP-1451 (UniProt ID Q460N5) was amplified from cDNA prepared as previously described.24

The WWE cassette from the WWE(RNF146)-

Fc/pET19b plasmid (described above) was excised using the NdeI and SalI sites, and replaced with PCR amplified DNA encoding Macro3X(PARP14) or Macro2/3(PARP14), which were also digested using NdeI and SalI.

Expression and Purification of the Antibody-like ADPR Binding Reagents in Bacteria Expression. The ADPR binding reagents were expressed in bacteria using the pET19bbased vectors described above. Due to expression issues with the Macro3X(PARP14)-Fc, a different protocol was used. E. coli strain BL21(DE3) Rosetta2 pLysS was made competent using a CaCl2 protocol and transformed with the pET19b-based plasmids encoding one of the ADPR binding reagents described above.

For WWE(RNF146)-Fc, Macro(AF1521)-Fc,

Macro(mH2A1.1)-Fc, and Macro2/3(PARP14)-Fc, the transformed bacteria were grown in LB containing ampicillin and chloramphenicol at 37°C until reaching an OD595

8


nm

of 0.4-0.6.

Biochemistry

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Recombinant protein expression was induced by the addition of 1 mM IPTG for 2 hours at 37°C. For Macro3X(PARP14)-Fc, the transformed bacteria were grown in LB containing ampicillin and chloramphenicol at 37°C until reaching an OD595 nm of 0.2. The culture was cooled to 18°C and grown to an OD595 nm of 90 percent pure (Figure 2, top). In contrast,

25 –

Lane

ARBD-Fc Fusion

Mol. Wt (kDa)

1)

WWE(RNF146)-Fc

(~39 kDa)

2)

Macro(AF1521)-Fc

(~50 kDa)

3)

Macro(mH2A1.1)-Fc

(~51 kDa)

4)

Macro3X(PARP14)-Fc

(~99 kDa)

most preparations of Macro3X(PARP14)-Fc had two major contaminating bands (Figure 2, top),

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October 13, 2017

which did not affect the utility of the protein in ADPR detection (see below). The purified proteins have expected specificities for ADPR as follows: (1) WWE(RNF146)-Fc: OAR and PAR; (2) Macro(AF1521)-Fc: MAR, OAR, and PAR; (3) Macro(mH2A1.1)-Fc: MAR, OAR, and PAR; and (4) Macro3X(PARP14)-Fc: MAR.27-32 Macro3X(PARP14)-Fc had a lower yield compared to the other reagents, thus we developed a modified version of the PARP14 macrodomain-based reagent to produce larger amounts. In the modified construct, we included macrodomains 2 and 3 of PARP14 only, excluding macrodomain 1, yielding Macro2/3(PARP14)-Fc. In this regard, a previous study indicated that macrodomains 2 and 3 are the critical ADPR readers in PARP-14.51 We expressed Macro2/3(PARP14)-Fc in E. coli and purified it as described above. The yields were improved and the ratio of full-length protein to lower molecular weight contaminants was increased compared

to

Macro3X(PARP14)-Fc

(Figure

S1A).

As

described

below,

both

Macro3X(PARP14)-Fc and Macro2/3(PARP14)-Fc behaved similarly in all assays and were used interchangeably as indicated.

Generation of Protein-linked Mono-, Oligo-, and Poly(ADP-ribose) Standards To test the ability of the ARBD-Fc fusion proteins to bind to and recognize specific forms of ADPR, we required a source of MAR, OAR, and PAR. To generate different forms of ADPR, we used purified recombinant PARP-1 (which can automodify with OAR and PAR) and PARP-3 (which can automodify with MAR) in biochemical reactions with NAD+. To stimulate the catalytic activity of PARP-1 and PARP-3, we added sonicated salmon sperm DNA. We controlled the extent of automodification of PARP-1 by altering the concentration of NAD+ in the reactions and the time of incubation (i.e., 3 µM NAD+ for 30 min. for OAR and 250 µM

19


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Biochemistry


October 13, 2017

NAD+ for 5 min. for PAR) (Figure 3A). Reactions lacking NAD+ were used as a control. For an initial confirmation that the PARP-1 reactions yielded the expected products, we performed an immunoblot analysis of the

Figure 3 reactions with the 10H antiPAR

monoclonal

A

B

antibody,

1

1) PARP-3 2) PARP-3 + 250 µM NAD+ 3) PARP-1 4) PARP-1 + 3 µM NAD+ 5) PARP-1 + 250 µM NAD+

which recognizes PAR, but not

Yield 1) 2) 3) 4) 5)

MAR or OAR (Figure 3B). As expected, the reaction in which

None Mono(ADP-ribose) None Oligo(ADP-ribose) Poly(ADP-ribose)

D

PARylated PARP-1 (Figure 3B, lane

5),

while

the

1

3

4

4

WWE(RNF146)-Fc 1

5

MW (kDa)

MW (kDa)

250 150 100 75

250 150 100 75

50

50

37

37

2

3

4

5

Poly Oligo

E

Macro(mH2A1.1)-Fc

5

1

2

3

4

F

Macro3X(PARP14)-Fc 1

5

MW (kDa)

MW (kDa)

250 150 100 75

250 150 100 75

250 150 100 75

50

50

50

37

37

37

2

3

4

5

Poly Oligo Mono

G Mono

no signal.

3

MW (kDa)

other

reactions and controls showed

2

2

C

Mono

Macro(AF1521)-Fc

PAR was generated showed a signal at the expected size of

Anti-PAR (10H)

Reaction Conditions Used to Generate ADPR

Oligo

The PAR chains

Poly Anti-PAR (10H)

generated in this way are on WWE(RNF146)-Fc

average about 35 ADPR units Macro(AF1521)-Fc

in length (Figure S2, right). In Macro(mH2A1.1)-Fc

contrast, the OAR chains are on Macro3X(PARP14)-Fc

average about 10 ADPR units in length (Figure S2, left).

Immunoblotting and Dot Blotting with Recombinant ARBD-Fc Fusion Proteins

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To explore in more detail the specificity of the various ARBD-Fc fusion proteins for different forms of ADPR, we performed immunoblot analyses of automodification reactions containing PARP-3-MAR, PARP-1-OAR, and PARP-1-PAR, as well as control reactions lacking NAD+ (Figure 3, C-F, Figure S1B). The results with most of the reagents were as expected, based on the previously reported specificity of the ARBDs27-32, with one exception (Table 1). We observed that (1) the WWE domain from H. sapiens RNF146 recognized OAR and PAR, but not MAR (Figure 3C); (2) the macrodomain from A. fulgidus AF1521 recognized MAR, OAR, and PAR (Figure 3D); (3) the macrodomain from H. sapiens macroH2A1.1 recognized MAR and PAR, but not OAR (Figure 3E); and (4) the macrodomains from H. sapiens PARP-14 recognized MAR, but not OAR or PAR (Figure 3F). Similar results were obtained in dot blot assays using the same sources of MAR, OAR, and PAR (Figure 3G). The recognition of MAR and PAR, but not OAR, by the macroH2A1.1 macrodomain was paradoxical, because we expected macrodomains to recognize any terminal ADPR moiety (i.e., a single protein-linked ADPR moiety or the terminal ADPR moiety of a protein-linked OAR or PAR chain) (Figure 1, A and B). This observation may provide information about the nature of H. sapiens macroH2A1.1 or OAR (discussed in more detail below).

Testing the ARBD-Fc Fusion

Proteins

in

Cell-based Assays Antibodies useful

tools

are for

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biochemical and molecular assays performed using cells, such as immunoblotting of cellular extracts, immunofluorescent staining of intact cells, and immunoprecipitations. With this in mind, we tested the performance of the ARBD-Fc fusion proteins in a series of cell-based immunological assays. Figure 4

Immunoblotting. First, we tested the

ADPR-binding

immunoblotting

reagents

assays

using

A

MCF-7

in

nuclear

MW (kDa) 250 150 100 75

MW (kDa) 250 150 100 75

50

50

37

37

25

25

15

15

extracts prepared from HeLa and MCF-7 cells under basal growth conditions (Figure 4A).

HeLa

Nuclear Extracts

All of the reagents yielded robust B

WWE(RNF146)-Fc

signals in the assays, and each reagent

– –

– +

+ –

+ +

Macro(mH2A1.1)-Fc H2O2 PARPi

– –

produced a unique pattern of detection that

MW (kDa) 250 150 100 75

MW (kDa) 250 150 100 75

differed from the other reagents and

50

50

37

37

25

25

15

15

10

10

between the cell types (Figure 4A). We also tested the reagents in whole cell extracts from Hela cells treated with 2 mM hydrogen

peroxide

(H2O2;

a

DNA

damaging agent that activates the catalytic activity of PARP-1 and possibly other

Macro(AF1521)-Fc – –

– +

+ –

+ +

– +

+ –

+ +

H2O2 PARPi

Macro2/3(PARP14)-Fc H2O2 PARPi

– –

MW (kDa) 250 150 100 75

MW (kDa) 250 150 100 75

50

50

37

37

25

25

15

15

10

10

– +

+ –

+ +

H2O2 PARPi

Whole Cell Extracts

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Biochemistry

nuclear PARPs), 20 µM PJ34 (PARPi; a broad-spectrum inhibitor of nuclear PARPs54,55), or both, H2O2 and PJ34 combined (Figure 4B). These treatments allowed us to increase (i.e., H2O2) or decrease (i.e., PJ34) the levels of ADP-ribosylation in predictable ways to determine the effect on detection by the reagents. The signals from all four ARBD-Fc reagents were dramatically

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Biochemistry


October 13, 2017

increased in response to treatment with H2O2, an effect that was blocked by co-treatment with PJ34 (Figure 4B). Interestingly, we observed inhibition by PJ34 of the H2O2- stimulated signal obtained using the PARP-14-based reagent , a PARPi that is thought to target primarily the nuclear polyenzymes PARPs 1 and 254 (Figure 4B, lower right). This may be due to the detection of initial protein-proximal PARP-1- and PARP-2-mediated mono(ADP-ribosyl)ation events, or perhaps PARP-3 mono(ADP-ribosyl)ation events that are modulated in some way by PARPs 1 and 2. To test the specificity of the signals detected in the immunoblotting assays with extracts from H2O2-treated HeLa cells, we used three different approaches. First, we blocked the ADPRbinding reagents using free ADPR prior to incubation

with

the

membrane

for

immunoblotting. This eliminated the signal

Figure 5 A

WWE (RNF146)-Fc + ADP-Ribose –

Macro (AF1521)-Fc – +

Macro (mH2A1.1)-Fc –

+

Blocking During Blotting

for all three MAR-binding reagents, but did not affect the signal with WWE(RNF146)-

B

WWE (RNF146)-Fc +

Macro (AF1521)-Fc –

+

Macro (mH2A1.1)-Fc – +

Treatment of Membrane

hydroxylamine (NH2OH), which cleaves glutamate

and

Macro2/3 (PARP14)-Fc – + MW

(kDa) 250 150 100 75 50 37

aspartate

residues56, prior to immunoblotting.

MW (kDa) 250 150

25

NH2OH –

from

+

37

Second, we treated the membranes with

ADPR

–

50

Fc, which binds to the linkage between ADPR units in OAR and PAR (Figure 5A).

Macro2/3 (PARP14)-Fc

100 75

25

β-actin

43

This C

eliminated the signal for all four of the ADPR-binding reagents (Figure 5B), but had

WWE (RNF146)-Fc + ARH3 –

Treatment of Extract

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no appreciable effect on signals detected by

Macro (AF1521)-Fc – +

Macro (mH2A1.1)-Fc – +

Macro2/3 (PARP14)-Fc – + MW

(kDa) 250 150 100 75 50 37 25

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Biochemistry


October 13, 2017

an acetyl-lysine antibody or a dimethyl-lysine antibody (Figure S3).

The results with

hydroxylamine treatment suggest that the bulk of the signal in H2O2-treated HeLa cells is from ADP-ribosylated glutamate or aspartate residues. Finally, we incubated the extracts with purified ARH3 (Figure S4), a glycohydrolase that cleaves PAR chains, as well as the proximal ADPR units from proteins

57-59

reagents (Figure 5C).

. Again, this eliminated the signal for all four of the ADPR-binding A previous report suggested that ARH3 is capable of cleaving the

proximal ADPR from Ser residues, but not from Glu, Arg, or Lys residues.57 However, given that HeLa cells are known to have a complex ADP-ribosylated Asp and Glu proteome24, and the fact that hydroxylamine also eliminated the signal, our results suggest that ARH3 is capable of cleaving ADPR from Asp and Glu under the conditions used here. Collectively, our results indicate that the immunoblotting signals that we detect with the ADPR-binding reagents are dependent on the presence and recognition of ADPR. Immunofluorescent staining.

Next, we tested the ADPR-binding reagents in

immunofluorescent cell staining assays using HeLa cells treated with 2 mM H2O2 or 20 µM PJ34 (Figure 6). The cells were stained using a conventional immunofluorescent staining protocol using the reagents with methanol fixation and co-staining for DNA using TO-PRO-3. Staining for ADPR was evident in the basal (vehicle-treated) condition with WWE(RNF146)-Fc, Macro(mH2A1.1)-Fc, and Macro3X(PARP14)-Fc, but not Macro(AF1521)-Fc (Figure 6; see the enlarged images in Figure 6B). In all cases where staining was observed in the basal condition, it was reduced by treatment with PJ34, and the background signal with all four reagents was low (Figure 6). The staining in the basal condition was primarily nuclear, although cytoplasmic ‘speckles’ were evident with WWE(RNF146)-Fc and perinuclear cytoplasmic staining was evident with Macro3X(PARP14)-Fc (Figure 6B). Interestingly, staining in the basal condition

24


Biochemistry


October 13, 2017

was excluded from the nucleoli in many cells with

WWE(RNF146)-Fc

A

and

Merge

With all four reagents,

treatment with H2O2 increased the amount of

Vehicle H2O2 PARPi

and

Macro(mH2A1.1)-Fc in the basal condition

B

immunofluorescent

results cell

from staining

+ – –

– + –

– – +

the

Macro(AF1521)-Fc

Macro3X(PARP14)-Fc

WWE(RNF146)-Fc

Macro(mH2A1.1)-Fc

Nucleolar Exclusion

10 µM

Merge

the

ADPR

was reduced by H2O2 treatment (Figure 6B). Together,

– – +

Merge

particular, the exclusion of nucleolar staining WWE(RNF146)-Fc

– + –

DNA

degree (Figure 6B; see also Figure 4B). In

with

+ – –

ADPR

staining, as well as pattern of staining to some

observed

50 µM

DNA

Macro(mH2A1.1)-Fc (Figure 6B; see the yellow arrows).

Macro(mH2A1.1)-Fc

WWE(RNF146)-Fc ADPR

stained

Figure 6

assays

Vehicle H2O2

+ –

– +

+ –

– +

ADPR

demonstrate that (1) ARBD-Fc fusion proteins work well for detecting ADPR in this assay and (2) cells produce different forms of ADPR

Merge

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 53

in different subcellular compartments and

Macro(AF1521)-Fc

under different conditions. Immunoprecipitation.

Finally,

we

tested

the

ADPR-binding

Macro3X(PARP14)-Fc

reagents

in

immunoprecipitation assays using nuclear extracts from 293T cells treated with H2O2 (Figure 7). Many immunological assays are based on molecular interactions with the Fc region of the immunoglobulin

(antibody)

that

lends

specificity

to

the

assay.

For

example,

immunoprecipitation is typically performed using bacterial immunoglobulin binding proteins,

25


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October 13, 2017

such as Staphylococcus sp. Protein A or Protein G, which bind to the Fc region. As a first step in exploring the utility of the ARBD-Fc fusion proteins in immunoprecipitation assays, we performed a simple Protein A binding assay to determine if the ARBD-FC fusions could be recovered quantitatively from binding reactions. We incubated equivalent amounts of IgG heavy chain or one of the four ARBD-Fc fusion proteins with Protein A-agarose resin. After washing the resin, we eluted the IgG or ARBD-Fc fusion proteins and analyzed the eluate by SDS-PAGE (Figure 7A). We observed efficient binding in all cases, and the extent of binding for the ARBD-Fc fusion proteins was as good, if not better, than what was observed with IgG heavy chain (Figure 7A). After

determining

that

the

ARBD-Fc fusion proteins are bound

Figure 7 A

Protein A-Agarose Binding

efficiently by Protein A, we assayed (1) IgG (Heavy Chain)

B MW (kDa) 250

2% Input

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Biochemistry

Immunoprecipitation 1

2

3

4

5

Without H2O2

150

their

ability

to

immunoprecipitate

(2) Macro(AF1521)-Fc (3) WWE(RNF146)-Fc

100 75

With H2O2

250

automodified PARP-1 from nuclear extracts prepared from 293T cells with

(4) Macro(mH2A1.1)-Fc

150

(5) Macro2/3(PARP14)-Fc

100

Oligo/Poly Mono

75

WB: PARP-1

or without H2O2 treatment (Figure 7B). Automodified PARP-1 from these cells contains a mixture of MAR, OAR, and PAR modifications (Figure 7B). We observed that all of the reagents were able to immunoprecipitate automodified PARP-1 from the nuclear extract, and the patterns of recognition were largely as expected (Figure 7B). For example, WWE(RNF146)-Fc immunoprecipitated OAR- and PAR-modified PARP-1 efficiently (Figure 7B).

The WWE

domain that it contains binds the iso-ADPR linkage between ADPR monomers in PAR (Figure 1A), thus providing many binding sites per each molecule of automodified PARP-1. In contrast, Macro(AF1521)-Fc immunoprecipitated OAR- and PAR-modified PARP-1 less efficiently

26


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Page 28 of 53

October 13, 2017

(Figure 7B). The macrodomain that it contains binds the terminal ADPR unit in PAR (Figure 1A), thus providing a limited number of binding sites per each molecule of automodified PARP1.

Macro(mH2A1.1)-Fc and Macro2/3(PARP14)-Fc both immunoprecipitated MARylated

PARP-1 efficiently (Figure 7B).

The extent to which ARBD interactions with MAR are

modulated by the linkage to the modified amino acid, or by nearby amino acids in the modified protein, remains an open question. Unexpectedly, Macro(mH2A1.1)-Fc, which recognized both MAR and PAR in the biochemical assays (Figure 3), was unable to immunoprecipitate OAR- and PAR-modified PARP-1 (Figure 7B).

Taken together, these results demonstrate that this

collection of ARBD-Fc fusion proteins can be used to immunoprecipitate MAR-, OAR-, and PAR-modified proteins.

27


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Discussion Biochemical and cellular studies of the diverse forms and functions of ADPR require immunological reagents that can be used for detection and enrichment. Prior to this report, the state-of-the-art has been the anti-PAR monoclonal antibody 10H, which is thought to bind to PAR with a lower limit for the length of polymers detected of around 10 ADPR units.45,46 Thus, 10H cannot detect MAR or OAR, limiting its utility and leaving the field blind to mono- and oligo(ADP-ribosyl)ation events. Herein, we have described the generation and characterization of recombinant antibody-like ADP-ribose binding proteins, in which natural ARBDs have been functionalized with the Fc region of rabbit immunoglobulin. Specific recognition of the diverse forms of ADPR by these reagents comes from the particular ARBD included in each fusion protein (e.g., various macrodomains and a WWE domain) (Table 1). Importantly, the set of ARBD-Fc fusion proteins that we have generated includes those that can recognize MAR and OAR, as well as one that can recognize all forms of ADPR (Table 1). As we have demonstrated herein and in recently published studies22,24, these reagents are useful in a broad array of antibody-based assays, such as immunoblotting, immunofluorescent staining of cells, and immunoprecipitation.

Functionalization of Naturally-Occurring ARBDs as Research Tools Our ARBD-Fc fusion proteins represent one example of the use of naturally-occurring ARBDs as tools for exploring the chemistry, biochemistry, and biology of ADP-ribose. Other examples include: (1) ARBD-GFP (green fluorescent protein) fusion proteins, which allow realtime tracking of localized MARylation and PARylation events in cells.29,34,35 and (2) ARBD-

28


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GST (glutathione-S-transferase) fusion proteins, which can be used for enrichment in biochemical and molecular assays. With respect to the latter, the Af1521 macrodomain, which recognizes MAR and the terminal ADP-ribose of PAR, has been fused with a GST tag and used to enrich for ADP-ribosylated targets in genomic and proteomic screens.33,36 Similarly, the RNF146 WWE domain has been fused with a GST tag and used to determine the genomic localization of PAR.33

While our ARBD-Fc fusion proteins are not the first examples of

functionalization of natural ADRBs for use as research tools, they are the first examples to include all of the key features of a monoclonal antibody. These include (1) monospecificity (i.e., monoclonality), (2) binding to protein A and G, (3) binding to Ig-directed secondary antibodies, and (4) renewable production. The Ig Fc region is the target of many antibody-directed tools, reagents, and methods, underlying the utility of the ARBD-Fc fusion proteins in a wide range of immunological assays. Recent reports have indicated that some macrodomains possess intrinsic ADPribosylhydrolase activity.60-65 In theory, such an activity might limit the utility of macrodomains as reagents for the detection of ADPR. In this regard, we note that only one of the three macrodomains that we tested herein, namely the macrodomain from AF1521, has ADPribosylhydrolase activity, while the macrodomains from PARP-14 and macroH2A1.1 do not.61,63 Furthermore, the ADP-ribosylhydrolase activity reported for macrodomains, like AF1521, is generally weak.60-65 or is undetectable under the conditions used in the assays described herein. In our hands, we have not found this to be a significant issue with the Macro(AF1521)-Fc reagent. If it becomes an issue for future macrodomain-based reagents, the activity can be blocked by site-specific mutation of the macrodomain, without loss of ADPR binding.60-65

29


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Biochemistry


October 13, 2017

Using ARBD-Fc Fusion Proteins to Uncover the Nature of Different Forms of ADPR While not a major focus of this work describing the generation and characterization of a set ARBD-Fc fusion proteins, our work has revealed some interesting aspects of the chemistry, biochemistry, and biology of ADP-ribose. For example, our results showing a dramatic loss of signal upon hydroxylamine treatment when using the ARBD-Fc fusion proteins in immunoblotting assays with cell extracts (Figure 5B) suggest that most of the ADP-ribosylation in HeLa cells occurs on glutamate and aspartate residues (the chemistry of ADP-ribosylation is such that hydroxylamine is only expected to cleave ADPR from these residues56). Results from previous studies have suggested that ADP-ribosylation can occur on a variety of residues (e.g., Glu, Asp, Lys, Ser, Arg) and that the proportion of the amino acids that are modified may vary by cell or tissue type.13,36,56

This approach using hydroxylamine treatment coupled with

immunoblotting may be a useful way to get an initial assessment of the relative levels of glutamate and aspartate ADP-ribosylation in biological samples prior to more detailed analyses. Furthermore, from previous studies, we know that macrodomains recognize MAR, as well as the terminal ADPR moieties in OAR and PAR, allowing them to bind to all three forms of ADPR (i.e., MAR, OAR, and PAR) (Figure 1, A and B).27-29 In contrast, WWE domains recognize the iso-ADPR linkages joining ADPR monomers, restricting their binding to OAR and PAR (Figure 1, A and B).30-32 The fact that the different macrodomains that we tested exhibited different specificities for MAR, OAR, and PAR (Table 1) suggest that they can recognize additional and distinct features of these molecules. The binding of MAR and PAR, but not OAR, by the macroH2A1.1 macrodomain is perplexing (Figure 3, E and G; Table 1), but it might suggest that binding by this macrodomain could be inhibited by branch points near the terminal ends of ADPR chains in OAR. Determining if this is indeed the case will require further tests.

30


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The binding of MAR, but not OAR or PAR, by the PARP-14 macrodomains (Figs. 3, F and G; Table 1), may indicate that (1) additional interactions occur between the PARP-14 macrodomains and ADP-ribosylated proteins or amino acids, or (2) the binding of the PARP-14 macrodomains to the terminal ADP-ribose units of poly(ADP-ribose) chains is inhibited by the penultimate ADP-ribose units.

The extent to which ARBD interactions with MAR are

modulated by the linkage to the modified amino acid or nearby amino acids in the modified protein remains an open question. Collectively, such observations suggest that the biology of MAR, OAR, and PAR is more diverse, rich, and complex than previously thought. In this regard, the immunofluorescent cell staining assays shown in Figure 6 shown a diverse array of staining patterns with the different ARBD-Fc fusion proteins, suggesting different biologies for ADPR in different subcellular compartments. The ARBD-Fc fusion proteins described herein will be useful tools for future exploration of the chemistry, biochemistry, and biology of ADPribose.

31


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October 13, 2017

Supporting Information The following supporting Information accompanies this manuscript: (1) Supporting materials and methods. (2) Supporting figures and legends: Expression, purification, and characterization of a PARP14 macrodomain 2/3 construct (Figure S1); Deproteinization and analysis of poly(ADP-ribose) synthesized in vitro (Figure S2); Treatment of the membrane with hydroxylamine does not have an appreciable effect on the signals for acetyl-lysine or dimethyl-lysine in immunoblotting assays (Figure S3). (3) Supporting references.

Funding This work was supported by a grant from the National Institutes of Health/National Institute of Diabetes, Digestive, and Kidney Disorders (DK069710) and the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K.

Acknowledgments The authors would like to thank (1) Dr. Mark Bycroft (Medical Research Council UK, Laboratory of Molecular Biology) for providing the AF1521 macrodomain cDNA, (2) Yonghao Yu (UT Southwestern Medical Center, Dallas) for providing the macroH2A1.1 macrodomain cDNA, (3) Rosemary Plagens for assistance with production of the ARBD-Fc fusion proteins, and (4) members of the Kraus lab for providing critical feedback on this work and the manuscript.

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October 13, 2017

Author Contributions W.L.K. conceived of the ARBD-Fc fusion proteins. B.A.G. designed and generated the fusion protein expression constructs, expressed and purified the fusion proteins, and conducted the biochemical assays. L.B.C. performed the ADPR detection assays (immunoblotting and immunofluorescence) in HeLa and MCF-7 cells.

B.A.G., L.B.C., and D.H. designed and

executed the experiments, and analyzed the data, with input from W.L.K. W.L.K. secured funding and provided intellectual support. B.A.G, L.B.C., and W.L.K. prepared the figures and wrote the paper.

Disclosures W.L.K. is a founder and consultant for Ribon Therapeutics, Inc. W.L.K. and B.A.G. hold the patent on the ARBD-Fc fusion proteins described herein (United States Patent No. 9,599,606).

UT Southwestern Medical Center has licensed the fusion proteins to EMD

Millipore, which markets them for research purposes.

33


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October 13, 2017

Table 1. Summary of the selectivity of the 10H anti-PAR monoclonal antibody, as well as four ARBD-Fc fusion proteins, for mono-, oligo-, and poly(ADP-ribose). Mono (MAR)

Oligo (OAR)a

Poly (PAR)b

Anti-PAR (10H)

–

–

+

WWE(RNF146)-Fc

–

+

+

Macro(AF1521)-Fc

+

+

+

Macro(mH2A1.1)-Fc

+

–

+

Macro3X(PARP14)-Fc

+

–

–

Macro2/3(PARP14)-Fc

+

–

–

Detection Reagent

a

The OAR chains generated for use in the assays described in this study had an average size of about 10 ADPR units in length (Figure S2, left).

b

The PAR chains generated for use in the assays described in this study had an average size of about 35 ADPR units in length (Figure S2, right).

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Page 36 of 53


October 13, 2017

Figure Legends

Figure 1. Design of antibody-like ADP-ribose binding reagents. (A and B) The chemical structure of (A) a poly(ADP-ribosyl)ated amino acid or (B) a mono(ADP-ribosyl)ated amino acid, with the amino acids shown in heteroatomic colors and the ADP-ribose units colored in grey (proximal to the amino acid) and black. The blue box with a dashed line highlights the chemical moiety in the ADP-ribose modification recognized by the WWE domain (the iso-ADP-ribose linkage between two ADP-ribose monomers). The green boxes with dashed lines highlight the chemical moiety in the ADP-ribose modification recognized by the macrodomains. The WWE and macrodomains used in this study are indicated (species and protein). (C) Ribbon diagram depicting the X-ray crystal structure of a monoclonal IgG antibody (PDBID:1IGY), with the variable fragment (Fv) in green and the homodimerized Fc fragment in purple and pink. (D) Schematic diagram of the plasmid constructs used to express the ADP-ribose binding domain-Fc (ARBD-Fc) fusion proteins in bacteria.

The constructs contain DNA segments

encoding: (1) 10xHis and/or Strep[II] tags, (2) an ADP-ribose binding domain (green), (3) a flexible glycine and serine linker, and (4) rabbit IgG constant fragment (Fc) (pink).

Figure 2. Expression and purification of ADP-ribose binding domain-Fc (ARBD-Fc) fusion proteins. ARBD-Fc fusion proteins were expressed in E. coli and purified using Ni-NTA affinity chromatography. (Top) The purified proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. The arrows indicate protein bands with the expected molecular weight

35


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Biochemistry


October 13, 2017

of the ARBD-Fc fusion proteins. The asterisks indicate breakdown products or contaminating E. coli proteins, which do not alter the functionality of the ARBD-Fc fusion protein. (Bottom) List of four ARBD-Fc fusion proteins and their expected molecular weights in kilodaltons (kDa).

Figure 3. Immunoblot and dot blot analyses of mono-, oligo-, and poly(ADP-ribosyl)ated PARP proteins using ARBD-Fc fusion proteins. (A) Purified recombinant PARP-1 and PARP-3 were incubated with or without NAD+ under different reaction conditions to promote auto(ADP-ribosyl)ation. The yield was mono(ADPribosyl)ated PARP-3 (red), oligo(ADP-ribosyl)ated PARP-1 (green), or poly(ADP-ribosyl)ated PARP-1 (blue). (B through F) Immunoblot analyses. The mono, oligo, and poly(ADP-ribosyl)ated PARP proteins were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the 10H anti-PAR monoclonal antibody, as well as four ARBD-Fc fusion proteins, as labeled. Each lane contained approximately the same number of terminal ADPribose units, to our best approximation.

The molecular weights in kilodaltons (kDa) are

indicated. (G) Dot blot analyses. Purified recombinant PARP-1 and PARP-3 were incubated with or without NAD+ under different reaction conditions to promote auto(ADP-ribosyl)ation. The yield was mono(ADP-ribosyl)ated PARP-3 (red), oligo(ADP-ribosyl)ated PARP-1 (green), or poly(ADP-ribosyl)ated PARP-1 (blue). Serial dilutions of the auto(ADP-ribosyl)ated PARP proteins were applied to nitrocellulose membranes for dot blotting using the 10H anti-PAR monoclonal antibody, as well as four ARBD-Fc fusion proteins, as labeled. Each spot contained approximately the same number of terminal ADP-ribose units, to our best approximation.

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Figure 4. Immunoblot blot analysis of ADP-ribosylation in HeLa and MCF-7 cells using ARBD-Fc fusion proteins. (A) Nuclear extracts were prepared from HeLa cells (left) and MCF-7 cells (right) maintained under standard culture conditions. Aliquots of the nuclear extracts containing equal total protein levels were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the ARBD-Fc fusion proteins as indicated above each lane. (B) Whole cell extracts were prepared from HeLa cells grown in culture following treatment with or without 2 mM H2O2 and 20 µM PARP inhibitor PJ34 (PARPi), as indicated. Aliquots of the whole cell extracts containing equal total protein levels were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the ARBD-Fc fusion proteins as indicated.

Figure 5. Blocking or removing ADP-ribose eliminates detection by ARBD-Fc fusion proteins in immunoblotting assays using cell extracts. Whole cell extracts were prepared from HeLa cells treated with 2 mM H2O2. Aliquots of the extracts containing equal total protein levels were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting using the ARBD-Fc fusion proteins as indicated above each lane. (A) Immunoblotting after blocking the ADPR-binding reagents with 10 mM free ADPR prior to incubation with the membrane (B) Immunoblotting after treating the membranes with 1 M hydroxylamine, which cleaves ADPR from glutamate and aspartate residues56, prior to immunoblotting.

37


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October 13, 2017

(C) Immunoblotting after incubating the extracts with 1 µM purified ARH3, a glycohydrolase that cleaves PAR chains as well as the proximal ADPR units from proteins57-59, prior to immunoblotting.

Figure 6. Immunofluorescent staining of ADP-ribosylation in HeLa cells using ARBD-Fc fusion proteins. HeLa cells grown in culture on glass cover slips were treated with vehicle, H2O2, or PJ34 (PARPi), as indicated. Following treatment, the cells were fixed with methanol, stained with TO-PRO-3 (a DNA stain), and immunostained for ADP-ribose using the ARBD-Fc fusion proteins, as indicated. The cover slips were affixed to glass slides, and the cells were imaged for fluorescence by laser scanning confocal microscopy. (A) Fluorescence images for all conditions and all ADPR-Fc fusion proteins, as indicated. The ADPR (green), DNA (red), or merged images are indicated. Scale bar = 50 µm (same for all images). (B) Nine-fold magnification of selected conditions from (A). Exclusion of staining from the nucleoli is indicated by the yellow arrows. Scale bar = 10 µm (same for all images).

Figure 7. Immunoprecipitation of ADP-ribosylated PARP-1 from 293T cells using ARBDFc fusion proteins. (A) Protein A-agarose binding assay with ADPR-Fc fusion proteins. The ADPR-Fc fusion proteins indicated were incubated with protein A-agarose.

The efficiency of binding was

assessed by SDS-PAGE analyses of the eluted material, with subsequent staining using Coomassie brilliant blue.

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(B) 293T cells were treated with vehicle or H2O2, as indicated. Nuclear extracts prepared from the treated cells were subjected to immunoprecipitation using the indicated ADPR-Fc fusion proteins (the lane numbers above the gel correspond to the number labels for the ADPR-Fc fusion proteins shown in panel A). The immunoprecipitated material was separated by SDSPAGE, transferred to nitrocellulose, and subjected to immunoblotting using an anti-PARP-1 antibody. The molecular weights in kilodaltons (kDa) are indicated.

39


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October 13, 2017

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Page 47 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

A

Poly(ADP-ribose) WWE Domain H. sapiens RNF146

B

Mono(ADP-ribose) Macrodomains H. sapiens macroH2A1.1 A. fulgidus AF1521 H. sapiens PARP-14

Macrodomains H. sapiens macroH2A1.1 A. fulgidus AF1521

C IgG Monoclonal Antibody

Fv Region Fc Region Replaced By ADP-ribose Binding Domain

D Affinity Tag(s)

Gly-Ser Linker

ADPR Binding Domain

Fc Domain

Bacterial Expression Vector

Modular Specificity


Figure 1 – Gibson et al. (2017)

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

MW (kDa) 150 –

1

2

3

Page 48 of 53

MW (kDa)

4

250 –

100 –

150 –

75 –

100 – 75 –

50 –

* *

50 –

37 –

37 – 25 –

Lane

ARBD-Fc Fusion

Mol. Wt (kDa)

1)

WWE(RNF146)-Fc

(~39 kDa)

2)

Macro(AF1521)-Fc

(~50 kDa)

3)

Macro(mH2A1.1)-Fc

(~51 kDa)

4)

Macro3X(PARP14)-Fc

(~99 kDa)



Page 49 of 53

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

A

Reaction Conditions Used to Generate ADPR 1) PARP-3 2) PARP-3 + 250 μM NAD+ 3) PARP-1 4) PARP-1 + 3 μM NAD+ 5) PARP-1 + 250 μM NAD+

B

1

Yield 1) None 2) Mono(ADP-ribose) 3) None 4) Oligo(ADP-ribose) 5) Poly(ADP-ribose)

D

Macro(AF1521)-Fc 1

2

3

4

Anti-PAR (10H) 2

3

4

C

WWE(RNF146)-Fc 1

5

MW (kDa)

MW (kDa)

250 150 100 75

250 150 100 75

50

50

37

37

E

Macro(mH2A1.1)-Fc 1

5

2

3

4

F

2

3

4

5

Poly Oligo Mono

Macro3X(PARP14)-Fc 1

5

MW (kDa)

MW (kDa)

MW (kDa)

250 150 100 75

250 150 100 75

250 150 100 75

50

50

50

37

37

37

2

3

4

5

Poly Oligo Mono

G Mono

Oligo

Poly Anti-PAR (10H)

WWE(RNF146)-Fc

Macro(AF1521)-Fc

Macro(mH2A1.1)-Fc

Macro3X(PARP14)-Fc



Biochemistry

A

MCF-7

HeLa

MW (kDa) 250 150 100 75

MW (kDa) 250 150 100 75

50

50

37

37

25

25

15

15

B

WWE(RNF146)-Fc

MW (kDa) 250 150 100 75

– –

– +

+ –

+ +

Macro(mH2A1.1)-Fc H2O2 PARPi

MW (kDa) 250 150 100 75 50

37

37

25

25

15

15

10

10

Macro(AF1521)-Fc

MW (kDa) 250 150 100 75

– +

+ –

+ +

– –

– +

+ –

+ +

H2O2 PARPi

Macro2/3(PARP14)-Fc H2O2 PARPi

MW (kDa) 250 150 100 75

50

50

37

37

25

25

15

15

10

10

– –

– +


+ –

+ +

H2O2 PARPi

Whole Cell Extracts

50

– –

Nuclear Extracts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 53


Page 51 of 53

A

WWE (RNF146)-Fc –

+

–

+


+

Macro2/3 (PARP14)-Fc –

+

Blocking During Blotting

ADP-Ribose

Macro (AF1521)-Fc

MW (kDa) 250 150 100 75 50 37 25

B

WWE (RNF146)-Fc –

+

–

+


+


+

Treatment of Membrane

NH2OH

Macro (AF1521)-Fc

MW (kDa) 250 150 100 75 50 37 25

β-actin

C

43

WWE (RNF146)-Fc ARH3

–

+

Macro (AF1521)-Fc –

+


+

Treatment of Extract

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry


+

MW (kDa) 250 150 100 75 50 37 25



Biochemistry

A

Macro(mH2A1.1)-Fc

ADPR

WWE(RNF146)-Fc

Merge

DNA

50 µM

+ – –

– + –

– – +

+ – –

– + –

– – +

Merge

DNA

ADPR

Vehicle H2O2 PARPi

Macro3X(PARP14)-Fc

WWE(RNF146)-Fc

Macro(mH2A1.1)-Fc

Nucleolar Exclusion

10 µM

Merge

ADPR

B

Macro(AF1521)-Fc

+ –

– +

+ –

– +

ADPR

Vehicle H2O2

Merge

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 52 of 53

Macro(AF1521)-Fc

Macro3X(PARP14)-Fc



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

A

Protein A-Agarose Binding

(1) IgG (Heavy Chain)

B MW (kDa) 250

2% Input

Page 53 of 53

Immunoprecipitation 1

2

3

4

5

Without H2O2

150

(2) Macro(AF1521)-Fc (3) WWE(RNF146)-Fc

100 75

With H2O2

250

(4) Macro(mH2A1.1)-Fc

150

(5) Macro2/3(PARP14)-Fc

100

Oligo/Poly Mono

75

WB: PARP-1



Generation and Characterization of Recombinant Antibody-like ADP

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