Proximity Ligation Assay Allows the Detection, Localization, and Quantification of Protein Arginine Methylation in Fixed Tissue

Summary

Proximity ligation assay is a very useful technique to localize and quantify arginine methylation of a given protein when the modified arginine residue is unknown and/or if no specific antibody is available.

Abstract

Arginine methylation is emerging as a key post-translational modification involved in a large range of biological processes. Its study in tissue is often limited by the lack of a specific antibody recognizing the target arginine residue. Proximity ligation assay (PLA) was originally developed to study protein/protein interactions. Here, we describe in detail a PLA protocol dedicated to the detection of arginine methylation that we applied to the glucocorticoid receptor (GR). Having previously shown that PRMT5 dimethylates GRs in cells, we used PLA with a pan symmetrical dimethyl antibody and an anti-GR antibody to measure GR methylation in breast tumors. We demonstrate that PLA offers a unique approach to measure arginine methylation of a target protein, even when the site of methylation has not been identified. This technique could be extended to other post-translational modifications where effective pan antibodies are available. Hence, we detail the PLA technology used to detect arginine methylation in fixed tissue using GR as an example.

Introduction

Arginine methylation by protein arginine methyltransferases (PRMTs) is an abundant post-translational modification (PTM) involved in numerous biological processes. PRMTs catalyze the transfer of methyl groups from the S-adenosyl methionine to arginine residues. The PRMT family comprises nine members classified according to the type of methylation they perform. All members perform monomethylation (MMA). Type 1 (PRMT1, 2, 3, 4, 6, and 8) PRMTs catalyze asymmetrical dimethylation (ADMA), whereas type 2 (PRMT5 and 9) catalyze symmetrical dimethylation (SDMA), and type 3 (PRMT7) only generate MMA¹. By methylating numerous substrates, the different P....

Protocol

Written informed consent was obtained from each patient. The study protocol was approved by the institutional ethics committee of the Cancer Research Center of Lyon.

1. Choice of the antibodies

1. Use primary antibodies validated by IHC or immunofluorescence (IF).
   ​NOTE: The primary antibodies selected for the study are crucial to the success of PLA and more particularly in fixed tissue. Using an antibody validated by IHC or IF will increase the success rate of the experiment. Optimization and specificity of antibodies are required before conducting experiments, ideally by performing control experiment....

Results

Using the procedure described above, it is possible to detect and quantify the methylation of a protein of interest. Here, we show the example of the methylation of GR by PRMT5. The antibodies and the experimental conditions for PLA were previously applied to cells¹⁰. Briefly, primary antibodies targeting GR and SDMA are recognized by proximity probes conjugated with complementary oligonucleotides. Then, the hybridization of a circular DNA probe occurs when the proteins are in close proximity. Sub.......

Discussion

Arginine methylation, like other PTMs, contributes to the fine regulation of protein functions. However, its impact is underestimated due to the difficulty in assessing these modifications, primarily because of a lack of tools. This is particularly true when studying methylation in vivo, where the only way to measure arginine methylation is to possess specific antibodies recognizing the methylated residue of the protein of interest. This clearly constitutes a limitation as the methylated arginine residue must be.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesion slides TOMO 90°, x100	VWR	631-1239
anti-GR antibody (mouse)	Santa Cruz	sc393232
anti-GR antibody (mouse)	santa cruz	sc393232
anti-PRMT5 antibody (rabbit)	Merck	07-405
anti-SDMA antibody (rabbit)	CST	13222
Automate d'inclusion	Leica	ASP 6025	Paraffin infiltration and block preparation
Autostainer XL	Leica	ST5010	Autostainer
Cassettes Q path macrostar III  x1500	VWR	720-2233
CC1	Roche	5279801001
Citrate Buffer pH 6 10x, 100 mL	MMF	F/T0050
Dako antibody diluent	Dako Agilent	S202230-2	antibody diluent
Discovery ChromoMap Diaminobenzidine (DAB) kit	Roche	760-159	Diaminobenzidine (DAB) kit
Discovery Wash	Roche	7311079001
Duolink insitu  PLA probe anti-mouse minus	Sigma-Aldrich	DUO92004	PLA  kit (probe anti-rabbit minus)
Duolink insitu detection reagents brightfield	Sigma-Aldrich	DUO92012	PLA  kit (in situ detection reagents)
Duolink insitu PLA probe anti-rabbit plus	Sigma-Aldrich	DUO92002	PLA  kit (probe anti-rabbit plus)
Duolink insitu wash buffer brightfield	Sigma-Aldrich	DUO82047	PLA  kit (in situ wash buffer)
Ethanol 96% VOL TECHNISOLV, 5 L	VWR	83804.360
Ethanol absolute ≥99.8%, AnalaR NORMAPUR ACS,  5 L	VWR	20821.365
EZ Prep 10x	Roche	5279771001
Formol, ready to use, 5 L	MMF	F/40877-36	Formalin
Fully automated glass coverslipper	Leica	CV5030	automated coverslipper
Glass coverslips 24 x 40	Dutscher	100037
Hematoxylin	Ventana	760-2021
IHC instrument	Roche	DISCOVERY XT	Automation of IHC
LCS	Roche	5264839001
Microtome	Thermo Scientific	Microm HM340E	Cutting of the tissues including in blocks
Mounting Medium Pertex	Histolab	00801-FR
PAP Pen for immunostaining	Sigma-Aldrich	Z672548-1EA
Paraffin Wax tek III, 4 x 2, 5 kg	Sakura	4511
Pasteur Disposable Pipettes	Fisher Scientific	12583237
PBS Buffer 10x, 100 mL	MMF	F/T0020
Reaction Buffer 10x	Roche	5353955001
Ribo Wash 10x	Roche	5266262001
RiboCC1	Roche	5266297001
Secondary antibody anti-mouse	Abcam	ab133469
Secondary antibody OmniMap anti-rabbit HRP	Roche	760-4311
Tissue Embedding center	MMF	EC 350
Xylene (mixture of isomers) ≥98.5%, AnalaR NORMAPUR ACS, 5 L	VWR	28975.360
Zeiss Axio Imager M2 microscope			upright bright-field microscope