Engineered ChymotrypsiN for Mass Spectrometry-Based Detection of Protein Glycosylation.

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.

Beyond Autoantibodies: Biologic Roles of Human Autoreactive B Cells in Rheumatoid Arthritis Revealed by RNA-Sequencing.

OBJECTIVE: To obtain the comprehensive transcriptome profile of human citrulline-specific B cells from patients with rheumatoid arthritis (RA). METHODS: Citrulline- and hemagglutinin-specific B cells were sorted by flow cytometry using peptide-streptavidin conjugates from the peripheral blood of RA patients and healthy individuals. The transcriptome profile of the sorted cells was obtained by RNA-sequencing, and expression of key protein molecules was evaluated by aptamer-based SOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. RESULTS: Citrulline-specific B cells, in comparison to citrulline-negative B cells, from patients with RA differentially expressed the interleukin-15 receptor α (IL-15Rα) gene as well as genes related to protein citrullination and cyclic AMP signaling. In analyses of an independent cohort of cyclic citrullinated peptide-seropositive RA patients, the expression of IL-15Rα protein was enriched in citrulline-specific B cells from the patients' peripheral blood, and surprisingly, all B cells from RA patients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production of AREG directly led to increased migration and proliferation of fibroblast-like synoviocytes, and, in combination with anti-citrullinated protein antibodies, led to the increased differentiation of osteoclasts. CONCLUSION: To the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing several US Food and Drug Administration-approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.