Mechanism of Protein Aggregation Inhibition by Arginine: Blockage of Anionic Side Chains Favors Unproductive Encounter Complexes

Journal of the American Chemical Society, Год журнала: 2024, Номер 146(12), С. 8394 - 8406

Опубликована: Март 13, 2024

Aggregation refers to the assembly of proteins into nonphysiological higher order structures. While amyloid has been studied extensively, much less is known about amorphous aggregation, a process that interferes with protein expression and storage. Free arginine (Arg+) widely used aggregation inhibitor, but its mechanism remains elusive. Focusing on myoglobin (Mb), we recently applied atomistic molecular dynamics (MD) simulations for gaining detailed insights (Ng J. Phys. Chem. B 2021, 125, 13099). Building approach, current work first time demonstrates MD can directly elucidate inhibition mechanisms. Comparative without Arg+ reproduced experimental finding significantly decreased Mb propensity. Our data reveal that, Arg+, protein–protein encounter complexes readily form salt bridges hydrophobic contacts, culminating in firmly linked dimeric nuclei. promotes dissociation complexes. These "unproductive" are favored because binding D– E– lowers tendency these anionic residues interprotein bridges. Side chain blockage mediated largely by guanidinium group which binds carboxylates through H-bond-reinforced ionic contacts. revealed self-association dynamic quasi-infinite network, found no evidence this important inhibition. Instead, similar free ions. The computational strategy here should be suitable rational design inhibitors enhanced potency.

Язык: Английский

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Vaccine-Induced Immune Thrombotic Thrombocytopenia: Clinicopathologic Features and New Perspectives on Anti-PF4 Antibody-Mediated Disorders

Journal of Clinical Medicine, Год журнала: 2024, Номер 13(4), С. 1012 - 1012

Опубликована: Фев. 9, 2024

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet severe adverse complication first identified during the global vaccination effort against SARS-CoV-2 infection, predominantly observed following administration of ChAdOx1-S (Oxford-AstraZeneca) and Ad26.CoV2.S (Johnson & Johnson/Janssen) adenoviral vector-based vaccines. Unlike other anti-platelet factor 4 (PF4) antibody-mediated disorders, such as heparin-induced (HIT), VITT arises with development platelet-activating anti-PF4 antibodies 4-42 days post-vaccination, typically featuring thrombosis at unusual sites.

Язык: Английский

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A Handle on Mass Coincidence Errors in De Novo Sequencing of Antibodies by Bottom-up Proteomics

Journal of Proteome Research, Год журнала: 2024, Номер 23(8), С. 3552 - 3559

Опубликована: Июнь 27, 2024

Antibody sequences can be determined at 99% accuracy directly from the polypeptide product by using bottom-up proteomics techniques. Sequencing peptide level is limited isobaric residues leucine and isoleucine, incomplete fragmentation spectra in which order of two or more remains ambiguous due to lacking fragment ions for intermediate positions, combinations amino acids, potentially different lengths, example, GG = N GA Q. Here, we present several updates Stitch (v1.5), performs template-based assembly de novo peptides reconstruct antibody sequences. This version introduces a mass-based alignment algorithm that explicitly accounts mass coincidence errors. In addition, it incorporates postprocessing procedure assign I/L based on secondary fragments (satellite ions, i.e., w-ions). Moreover, evidence sequence assignments now evaluated with addition an integrated spectrum viewer. Lastly, input data wider selection sequencing algorithms are allowed, including Casanovo, PEAKS, Novor.Cloud, pNovo, MaxNovo, flat text FASTA. Combined, these changes make compatible larger range processing pipelines improve its tolerance peptide-level

Язык: Английский

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A handle on mass coincidence errors inde novosequencing of antibodies by bottom-up proteomics

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Фев. 22, 2024

Abstract Antibody sequences can be determined at 99% accuracy directly from the polypeptide product using bottom-up proteomics techniques. This circumvents need to isolate antibody-producing B-cell clone and enables reverse engineering of monoclonal antibodies lost hybridoma cell lines, as well secreted protein in bodily fluid. Sequencing peptide level is limited by common mass coincidences isobaric residues like leucine/isoleucine, but also incomplete fragmentation spectra which order two or more remains ambiguous due lacking fragment ions for intermediate positions. Likewise, different combinations amino acids, potentially length, coincide same ( e . g GG=N, GA=Q etc .). Here we present several updates Stitch (v1.5), performs template-based assembly de novo reads reconstruct antibody sequences. version introduces a mass-based alignment algorithm that explicitly accounts coincidence errors. In addition, it incorporates postprocessing procedure assign I/L based on secondary fragments (satellite ions, i e. w- ions). Moreover, evidence sequence assignments now evaluated with addition an integrated spectrum viewer. allows input data wider selection sequencing algorithms, including Casanovo, PEAKS, Novor.Cloud, pNovo, MaxNovo, flat text FASTA. Combined, these changes make compatible larger range processing pipelines improve its tolerance peptide-level

Язык: Английский

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Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

Analytical Chemistry, Год журнала: 2024, Номер 96(16), С. 6209 - 6217

Опубликована: Апрель 12, 2024

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from patient's blood. Intact mass measurements indicate that significant fraction these represent limited number clones. MS analysis large antibody fragments (the light chain and the Fc/2 Fd heavy chain) confirms monoclonal nature this component repertoire reveals presence mature complex biantennary

Язык: Английский

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Do-It-Yourself De Novo Antibody Sequencing Workflow that Achieves Complete Accuracy of the Variable Regions

Journal of Proteome Research, Год журнала: 2025, Номер unknown

Опубликована: Май 5, 2025

Antibodies are widely used as research tools or therapeutic agents. Knowing the sequences of variable regions an antibody─both heavy chain and light chain─is a prerequisite for production recombinant antibodies. Mass spectrometry-based de novo sequencing is frequently used, sometimes only approach to gaining this information. Here, we describe workflow that enables accurate sequence determination monoclonal antibodies based on mass spectrometry data freely available software tools. This workflow, which developed using homemade anti-FLAG antibody reference sample, achieved 100% accuracy with clear distinction between leucine (L) isoleucine (I). Using successfully decoded anti-HA antibody, had no prior knowledge its sequence. Based result, generated demonstrated it has same specificity, sensitivity, affinity commercial antibody.

Язык: Английский

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Vaccine-Induced Immune Thrombotic Thrombocytopenia—Clinicopathologic Features and New Perspectives on Anti-PF4 Antibody-Mediated Disorders

Опубликована: Дек. 20, 2023

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but severe adverse event that was first observed during the global vaccination campaign against SARS-CoV-2 infection, specifically in those receiving adenoviral vector-based vaccines for Coronavirus disease 2019 (COVID-19). VITT develops 4 to 42 days post-vaccination and characterized by development of platelet-activating anti-platelet factor (PF4) antibodies, leading thrombosis at unusual sites. The rise awareness subsequent prompt recognition paramount reducing mortality. Moreover, as campaigns around world continue, better understanding not only has important clinical implications also crucial future vaccine development. In this review, we summarize features, pathophysiology, incidence rates well highlight other anti-PF4 antibody-mediated disorders growing significance.

Язык: Английский

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Characterization of Large Immune Complexes with Size Exclusion Chromatography and Native Mass Spectrometry Supplemented with Gas Phase Ion Chemistry

Analytical Chemistry, Год журнала: 2024, Номер unknown

Опубликована: Фев. 6, 2024

Large immune complexes formed by the cross-linking of antibodies with polyvalent antigens play critical roles in modulating cell-mediated immunity. While both size and shape are important determinants Fc receptor-mediated signaling responsible for phagocytosis, degranulation, and, some instances, autoimmune pathologies, their characterization remains extremely challenging due to large structural heterogeneity. We use native mass spectrometry (MS) supplemented limited charge reduction gas phase determine stoichiometry a bivalent (homodimeric) antigen, 163 kDa aminopeptidase P2 (APP2), monoclonal antibody (mAb) APP2. The observed (APP2·mAb)n populate wide range stoichiometries (n = 1–4) largest detected species exceeding 1 MDa, although gas-phase dissociation products also evident spectra. frequently considering nuisance that complicates interpretation MS data, provides an additional dimension complex quaternary structure. APP2/mAb associations identical composition but slightly different elution times exclusion chromatography exhibit notable differences spontaneous fragmentation profiles. latter indicates presence extended linear cyclized configurations. unique ability distinguish between such isomeric structures will be invaluable variety applications where biological effects determined assemble receptor clusters certain density on cell surfaces, as platelet activation clustering low-affinity receptors FcγRIIa surface.

Язык: Английский

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Recombinant Anti-PF4 Antibodies Derived from Patients with Vaccine-Induced Immune Thrombocytopenia and Thrombosis (VITT) Facilitate Research and Laboratory Diagnosis of VITT

Vaccines, Год журнала: 2024, Номер 13(1), С. 3 - 3

Опубликована: Дек. 24, 2024

Background/Objectives: Adenoviral vector-based vaccines against COVID-19 rarely cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a severe adverse reaction caused by IgG antibodies platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become scarce resource. Recombinant (rAbs) derived from VITT characteristic amino acid sequences of anti-PF4 an alternative to pathophysiology. Methods: Amino the variable region immunoglobulin light heavy chain patients were obtained mass spectrometry sequencing rAbs synthetized reverse-engineering. Six different produced: CR23003, CR23004, CR23005 (from vaccinated with Jcovden, Johnson & Johnson-Janssen (Beerse, Belgium)), CR22046, CR22050 CR22066 two Vaxzevria, AstraZeneca (Cambridge, UK)). These further characterized using anti-PF4/heparin ELISAs, rapid anti-PF4/polyanion chemiluminescence assays, PF4-induced activation assay (PIPA) their capacity induce procoagulant platelets. Results: bound PF4 alone, not PF4/polyanion complexes in assays. Chemiluminescence assays both IgG/heparin ELISA showed concentration-dependent binding all six rAbs, however, reactivities among them. PIPA similar, pattern. varied reactivity majority tested able Conclusions: The reflect VITT-typical capacities ability activate Therefore, these offer attractive new option

Язык: Английский

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Molecular architecture and platelet-activating properties of small immune complexes assembled on heparin and platelet factor 4

Communications Biology, Год журнала: 2024, Номер 7(1)

Опубликована: Март 11, 2024

Abstract Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading a reduction in circulating platelets with increased risk of thrombosis. It precipitated by polymerized immune complexes consisting pathogenic antibodies that recognize small chemokine platelet factor 4 (PF4) bound heparin. Characterization these extremely challenging due the enormous structural heterogeneity such macromolecular assemblies and their constituents. Native mass spectrometry demonstrates up three PF4 tetramers can be assembled on chain, consistent molecular modeling studies showing facile polyanion wrapping along polycationic belt surface. Although accommodate maximum only two antibodies, resulting are capable activation despite modest size. Taken together, provide further insight into mechanisms HIT other disorders where anti-PF4 play central role.

Язык: Английский

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