Site-specific incorporation of citrulline into proteins in mammalian cells

Abstract

Citrullination is a post-translational modification (PTM) of arginine that is crucial for several physiological processes, including gene regulation and neutrophil extracellular trap formation. Despite recent advances, studies of protein citrullination remain challenging due to the difficulty of accessing proteins homogeneously citrullinated at a specific site. Herein, we report a technology that enables the site-specific incorporation of citrulline (Cit) into proteins in mammalian cells. This approach exploits an engineered E. coli-derived leucyl tRNA synthetase-tRNA pair that incorporates a photocaged-citrulline (SM60) into proteins in response to a nonsense codon. Subsequently, SM60 is readily converted to Cit with light in vitro and in living cells. To demonstrate the utility of the method, we biochemically characterize the effect of incorporating Cit at two known autocitrullination sites in Protein Arginine Deiminase 4 (PAD4, R372 and R374) and show that the R372Cit and R374Cit mutants are 181- and 9-fold less active than the wild-type enzyme. This technology possesses the potential to decipher the biology of citrullination.

Fig. 1. SM60, a photocaged-citrulline and its conversion to citrulline with 365 nm UV light.

a Conversion of peptidyl-arginine to peptidyl-citrulline by the PADs. b Chemical structures of SM60 and SM70. c Schematic representation of the incorporation of SM60 into proteins by an engineered LeuRS-tRNA^Leu pair and subsequent conversion to citrulline. d Decaging of SM60 to citrulline with 365 nm UV irradiation. Left and right panels indicate the HPLC and ion chromatograms, showing the disappearance of SM60 and the formation of citrulline (Cit), respectively, with increasing UV exposure. Quantitative analyses of decaging and Cit formation are shown in the insets. n = 2 independent experiments, data are presented as mean value ± SD. Assay mixture: 1 mM SM60, 2 mM DTT, phosphate-buffered saline pH 7.4. Source data are provided as a Source Data file.

Fig. 2. Site-specific incorporation of SM60 in EGFP and subsequent conversion to citrulline in HEK293T cells.

a EGFP-39-TAG reporter expression by EcLeuRS-tRNA_CUA^EcLeu pair in HEK293T cells in the presence of SM60 indicated by the fluorescence of EGFP. b Quantification of EGFP-39-TAG reporter expression efficiency in the presence of an increasing concentration of SM60. n = 3 independent experiments, data are presented as mean value ± SD. The solid circles represent data points for biologically independent replicates. Source data are provided as a Source Data file. c Coomassie stain of purified EGFP containing SM60. Full gel is given in Supplementary Fig. 7. d Deconvoluted mass spectrum of EGFP before and after 365 nm UV irradiation (1 min), indicating the presence of SM60 and citrulline, respectively, at position 39. Non-deconvoluted spectra are given in Supplementary Figs. 8 and 9.

Fig. 3. Autocitrullination of PAD4, incorporation of citrulline in PAD4 and implications thereof.

a Chemical structure of Rh-PG and the fluorescence labeling of autocitrullinated PAD4. The bands at 0 min in the presence of calcium and at all the time points in the absence of calcium correspond to the basal levels of autocitrullination during the expression and purification of PAD4 from E. coli. n = 2 independent experiments, data are presented as mean value ± SD. b Expression of R372Cit PAD4 in the presence of engineered release factor, eRF1-E55D and SM60 in HEK293T cells, indicating the essential role of eRF1-E55D for efficient TAG suppression. c Coomassie stains for WT, R372Cit and R374Cit PAD4, indicating their purity. d Michaelis–Menten kinetics for WT, R372Cit, and R374Cit PAD4. n = 2 independent experiments, data are presented as mean value ± SD. e Western blot analysis of histone H3 citrullination in live HEK293T cells by WT and R374Cit PAD4. The normalization procedure is given in the “Methods” section. n = 3 independent experiments, data are presented as mean value ± SD. The samples derive from the same experiment and the blots were processed in parallel (see Supplementary Fig. 20c for a detailed explanation). f Thermal shift profiles of WT and R374Cit PAD4. Western blot images are given in Supplementary Fig. 17. The table indicates the melting temperatures (T_m). n = 2 independent experiments, data are presented as mean value ± SD. The full gels corresponding to panels (a), (b), and (e) are given in Supplementary Fig. 20. Source data for panels (a) and (d–f) are provided as a Source Data file.

Fig. 4. Autocitrullination sites in PAD4.

a Heat map representing the time-dependent change in peptides containing arginine or citrulline at the indicated positions (autocitrullination sites). Ca²⁺-untreated samples are negative controls. b Time-dependent autocitrullination at the major sites. R218 site could not be shown because of disorder in that region (PDB: 1WDA). The increase in autocitrullination at these sites follows the same color code as in panel (a).