Biology subjects

Dominguez-Vega, E.

Publications and source records attributed to Dominguez-Vega, E..

5 recordsLinked to original sources

bioRxiv2026

MuSK antibodies differently affect the MuSK signaling cascade depending on valency and epitope specificity

Muscle-specific kinase (MuSK) is a pivotal player in forming and maintaining healthy neuromuscular junctions (NMJ). In MuSK myasthenia gravis (MG), autoantibodies targeting MuSK disrupt its function, impairing neuromuscular transmission and causing fatigable skeletal muscle weakness. MuSK autoantibodies predominantly belong to the IgG4 subclass, which bind in a monovalent fashion due to Fab-arm exchange, although autoantibodies of other subclasses also exist. Polyclonal autoreactive IgG from patients may therefore harbor a variety of monovalent and bivalent MuSK antibodies with potentially distinct effects on MuSK signaling. To further unravel the pathomechanisms underlying MuSK MG, we have investigated how MuSK antibody-binding affects MuSK functioning with a diverse panel of (patient-derived) monoclonal MuSK antibodies. Our findings reveal that the valency of antibody-binding influences binding kinetics to MuSK, inhibition of agrin-induced MuSK activation, Dok7 binding to MuSK and NMJ gene expression. Monovalent binding to the frizzled domain of MuSK did not inhibit agrin-induced MuSK activation, while monovalent binding to the Ig-like domain 1 does. Moreover, the kinetics of Dok7 degradation induced by bivalent MuSK antibodies appear to depend on binding-epitope of MuSK. Surprisingly, none of the clones tested (both bivalent and monovalent) increased MuSK internalization. Taken together, the cumulative pathogenic effect of polyclonal MuSK antibodies in individual MuSK MG patients thus likely depends on autoantibody titer, affinity and the unique composition of MuSK autoantibodies varying in epitope and valency. This research enriches our understanding of the intricate interactions between antibodies and MuSK in MuSK MG and offers potential insights into novel therapeutic strategies using MuSK antibodies.

immunology↗

bioRxiv2025

Increasing Applicability of Automated mD-LC-MS Peptide Mapping for Biopharmaceuticals via Streamlined In-Solution Digestion

Since the introduction of the first multidimensional liquid chromatography mass spectrometry (mD-LC-MS) approach for antibody peak characterization, multiple developments have been reported including integration of various chromatographic approaches and precise fractionation, providing high quality and reliable peptide data in a drastically decreased total analysis time. Most of these platforms rely on the use of immobilized enzyme reactors (IMERs) for digestion, limiting the applicability to few enzymes such as trypsin. Recently, the introduction of in-solution enzymatic digestion in mD-LC-MS systems has been proposed as an alternative to IMERs. Here, we make use of current innovations in 2D-LC commercial systems such as active solvent modulation valves, which permits direct mixing of the fractionated peaks with the endoprotease and reducing agent in an online manner, to integrate in-solution digestion in a straightforward manner in mD-LC-MS peak characterization platforms. Following antibody digestion, the generated peptides are automatically trapped, separated and detected by mass spectrometry. Efficient reduction and tryptic digestion was obtained using short incubation times (15 min). The approach was further expanded to alternative endoproteases such as chymotrypsin and thermolysine with other digestion specificities and allow an easy exchange between enzymes with similar buffer and digestion conditions. As a proof-of-principle that strategy was applied to achieve peptide maps from ion exchanged separated mAb peaks showing good digestion efficiency and high sequence coverages.

biochemistry↗

bioRxiv2024

Fc proteoforms of ACPA IgG discriminate autoimmune responses in plasma and synovial fluid of rheumatoid arthritis patients and associate with disease activity

Autoantibodies and their post-translational modifications (PTMs) are insightful markers of autoimmune diseases providing diagnostic and prognostic clues, thereby informing clinical decisions. However, current autoantibody analyses focus mostly on IgG1 glycosylation representing only a subpopulation of the actual IgG proteome. Here, by taking rheumatoid arthritis (RA) as prototypic autoimmune disease, we sought to circumvent these shortcomings and illuminate the importance of (auto)antibody proteoforms employing a novel comprehensive mass spectrometry (MS)-based analytical workflow. Profiling of anti-citrullinated protein antibodies (ACPA) IgG and total IgG in paired samples of plasma and synovial fluid revealed a clear distinction of autoantibodies from total IgG and between biofluids. This discrimination relied on comprehensive subclass-specific PTM profiles including previously neglected features such as IgG3 CH3 domain glycosylation, allotype ratios, and non-glycosylated IgG. Intriguingly, specific proteoforms were found to correlate with markers of inflammation and disease accentuating the need of such approaches in clinical investigations and calling for further mechanistic studies to comprehend the role of autoantibody proteoforms in defining autoimmune responses.

immunology↗

bioRxiv2021

Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

As the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

biochemistry↗

bioRxiv2020

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis

Human IgG4 usually displays anti-inflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 antibodies naturally engage in a stochastic process termed Fab-arm exchange in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis (MG). In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a novel pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/296293v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@45c221org.highwire.dtl.DTLVardef@1cc31cdorg.highwire.dtl.DTLVardef@d9b09borg.highwire.dtl.DTLVardef@1d9b681_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗