Biology subjects

Kaltashov, I. A.

Publications and source records attributed to Kaltashov, I. A..

5 recordsLinked to original sources

bioRxiv2023

Reverse engineering of a pathogenic antibody reveals the molecular mechanism of vaccine-induced immune thrombotic thrombocytopenia

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which - vaccine-induced immune thrombotic thrombocytopenia (VITT) - is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patients blood indicates that its major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the CDR regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies, such as their ability to activate platelets in a PF4-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at Fc{gamma}RIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising polyspecificity not only sheds light on VITT etiology, but also opens up a range of opportunities to manage this pathology. Significance StatementVaccine-induced immune thrombotic thrombocytopenia (VITT) is a dangerous side effect of adenoviral-vectored vaccines that is linked to the emergence of autoantibodies recognizing platelet factor 4 (PF4). We have engineered a recombinant VITT antibody by sequencing a VITT patient-derived anti-PF4 monoclonal antibody that causes platelet activation and triggers thrombosis. This antibody was used to characterize architecture of the pathogenic immune complexes with a combination of biophysical and computational approaches, revealing the molecular mechanism of VITT. The results of this work demonstrate the critical role of electrostatics in PF4 recognition by the pathogenic antibody and the polyspecificity of the latter. Availability of the engineered VITT antibody will be invaluable for future studies aiming at understanding the general mechanistic features of autoimmune pathologies.

immunology↗

bioRxiv2023

Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT)

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but extremely dangerous side effect that has been reported for several adenoviral (Ad)-vectored COVID-19 vaccines. VITT pathology had been linked to production of antibodies that recognize platelet factor 4 (PF4), an endogenous chemokine. In this work we characterize anti-PF4 antibodies obtained from a VITT patients blood. Intact-mass MS measurements indicate that a significant fraction of this ensemble is comprised of antibodies representing a limited number of clones. MS analysis of large antibody fragments (the light chain, as well as the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire, and reveals the presence of a fully mature complex biantennary N-glycan within its Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS analysis were used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to IgG2 subclass and verify that the light chain belongs to the {lambda}-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site (absent in the germline sequence) is a result of a single mutation giving rise to an NDT motif in the antibody sequence. Peptide mapping also provides a wealth of information on lower-abundance proteolytic fragments derived from the polyclonal component of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1 through IgG4) and both types of the light chain ({lambda} and {kappa}). The structural information reported in this work will be indispensable for understanding the molecular mechanism of VITT pathogenesis.

biochemistry↗

bioRxiv2023

Molecular architecture and platelet-activating properties of small immune complexes assembled on intact heparin and their possible involvement in heparin-induced thrombocytopenia

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

biochemistry↗

bioRxiv2021

The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community - the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of structural heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native mass spectrometry (MS) as a means of characterizing its interactions with both the host cell-surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.

biochemistry↗

bioRxiv2020

The utility of native MS for understanding the mechanism of action of repurposed therapeutics in COVID-19: heparin as a disruptor of the SARS-CoV-2 interaction with its host cell receptor.

The emergence and rapid proliferation of the novel coronavirus (SARS-CoV-2) resulted in a global pandemic, with over six million cases and nearly four hundred thousand deaths reported world-wide by the end of May 2020. A rush to find the cures prompted re-evaluation of a range of existing therapeutics vis-a-vis their potential role in treating COVID-19, placing a premium on analytical tools capable of supporting such efforts. Native mass spectrometry (MS) has long been a tool of choice in supporting the mechanistic studies of drug/therapeutic target interactions, but its applications remain limited in the cases that involve systems with a high level of structural heterogeneity. Both SARS-CoV-2 spike protein (S-protein), a critical element of the viral entry to the host cell, and ACE2, its docking site on the host cell surface, are extensively glycosylated, making them challenging targets for native MS. However, supplementing native MS with a gas-phase ion manipulation technique (limited charge reduction) allows meaningful information to be obtained on the non-covalent complexes formed by ACE2 and the receptor-binding domain (RBD) of the S-protein. Using this technique in combination with molecular modeling also allows the role of heparin in destabilizing the ACE2/RBD association to be studied, providing critical information for understanding the molecular mechanism of its interference with the virus docking to the host cell receptor. Both short (pentasaccharide) and relatively long (eicosasaccharide) heparin oligomers form 1:1 complexes with RBD, indicating the presence of a single binding site. This association alters the protein conformation (to maximize the contiguous patch of the positive charge on the RBD surface), resulting in a notable decrease of its ability to associate with ACE2. The destabilizing effect of heparin is more pronounced in the case of the longer chains due to the electrostatic repulsion between the low-pI ACE2 and the heparin segments not accommodated on the RBD surface. In addition to providing important mechanistic information on attenuation of the ACE2/RBD association by heparin, the study demonstrates the yet untapped potential of native MS coupled to gas-phase ion chemistry as a means of facilitating rational repurposing of the existing medicines for treating COVID-19. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/142794v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@ce764corg.highwire.dtl.DTLVardef@b8909corg.highwire.dtl.DTLVardef@11e13fcorg.highwire.dtl.DTLVardef@1b2383a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗