Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides.

Daniel Nyberg Larsen,Peter Højrup,Jakub Z Kaczmarek,Grzegorz P Bereta,Christian Engelbrecht Mikkelsen,Mads Kierkegaard,Zuzanna Nowakowska,Jan Potempa

doi:10.1074/mcp.ra119.001700

Abstract

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.

Highlights

Citrullination is a deimination of arginine, which results in the loss of a single nitrogen and hydrogen along with the addition of an oxygen, resulting in a mass shift of 0.984 Da and loss of a single charge
In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional heptafluorobutyric acid (HFBA)-based (2D) separation system combined with LC-mass spectrometry (MS)
As the amount of sample was identical for all samples, the difference is likely caused by citrullination of the WT peptides resulting in a lower detection efficiency. This assumption was verified by measuring of the ratio between lysine terminated and arginine terminated peptides in the various samples as shown in Table 3 and 4. Taken together these results indicate that numerous P. gingivalis proteins were cleaved by RgpA/B, but almost all generated C-terminal arginines were modified by PPAD to citrulline

Summary

Introduction

Citrullination is a deimination of arginine, which results in the loss of a single nitrogen and hydrogen along with the addition of an oxygen, resulting in a mass shift of 0.984 Da and loss of a single charge. Citrullination is a post-translational modification that can only occur on arginine residues, either on the Nor C-terminal of the peptides or internally. Citrullination occurs in physiological and pathological conditions and is thought to play a range of different functions. The human peptidyl arginine deiminases (PAD) 1, 2, 3, 4, and 6 exert different roles as a result of expression in different cellular environments. The PAD6 function is not completely understood due to lack of substrate, but it is thought to have a role in reproduction[14]

Results

Discussion

Conclusion