Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases.

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Citrullination, which is catalyzed by protein arginine deiminases (PADs 1–4 and 6), is a post-translational modification (PTM) that effectively neutralizes the positive charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homologue of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at R26, and this PTM leads to the increased transcription of more than 200 genes under the control of the estrogen receptor. Given that our understanding of PAD2 biology remains incomplete, we initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Herein, we report that the substrate specificity and calcium dependence of PAD2 are similar to those of PADs 1, 3, and 4. However, unlike those isozymes, PAD2 appears to use a substrate-assisted mechanism of catalysis in which the positively charged substrate guanidinium depresses the pKa of the nucleophilic cysteine. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isozyme-specific inhibitors.

Binding of pertussis toxin (PTx) was examined by glycan microarray; 53 positive hits fell into four general groups. One group represents sialylated bi-antennary compounds with an N-glycan core terminating in α2-6 linked sialic acid. The second group consists of multi-antennary compounds with a canonical N-glycan core, but lacking terminal sialic acids, which represents a departure from previous understanding of PTx binding to N-glycans. The third group consists of Neu5Acα2-3(Lactose or N-acetyllactosamine) that lack the branched mannose core found in Nglycans, thus their presentation is more similar to O-linked glycans and glycolipids. The fourth group of compounds consists of Neu5Acα2-8Neu5Acα2-8Neu5Ac, which is seen in the c series gangliosides and some N-glycans. Quantitative analysis by SPR of the relative affinities of PTx for terminal Neu5Acα2-3 versus Neu5Acα2-6, as well as the affinities for the trisaccharide Neu5Acα2-8Neu5Acα2-8Neu5Ac versus disaccharide, revealed identical global affinities, even though the amount of bound glycan varied by 4-to 5-fold. These studies suggest that the conformational space occupied by a glycan can play an important role in binding, independent of affinity. Characterization of N-terminal and C-terminal binding sites on the S2 and S3 subunits by mutational analysis revealed that binding to all sialylated compounds was mediated by the Cterminal binding sites, and binding to non-sialylated N-linked glycans is mediated by the Nterminal sites present on both the S2 and S3 subunits. A detailed understanding of the glycans recognized by pertussis toxin is essential to understand which cells are targeted in clinical disease.Vaccination has greatly reduced whooping cough (pertussis) morbidity and mortality; alarmingly however, the number of cases has been increasing in the US since a historic low in 1976 (1,2). Pertussis toxin (PTx) is often considered the major virulence factor of B pertussis, as PTx mutants are avirulent in mouse models, and consequently PTx is included as a component in all acellular pertussis vaccines (3). PTx alone is responsible for the systemic manifestations of lymphocytosis and hyperinsulinemia, and is the chief candidate for defense against innate and adaptive immune systems past the initial colonization (4-7).PTx is a member of the AB 5 family of bacterial toxins, which includes cholera toxin from Vibrio cholerae, heat-labile toxin from Escherichia coli, and Shiga toxin from Shigella † This work was supported by National Institutes of Health Grants R01 AI 064893 (A.A.W.) , and S5 in the ratio 1:1:2:1 (8). The A subunit, named S1 in PTx, is an ADP-ribosyltransferase which targets the α-subunit of some GTP-binding proteins (9). The B-pentamer is required for cell-targeting and cytosolic entry of S1 into mammalian cells, but also has activities independent of S1, such as antigen-independent T cell activation and mitogenicity (8,(10)(11)(12)(13)(14)(15). The fact that the binding (B) portion of the toxin has activity independent of the enzymatic acti...