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Register a new userVariable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: implications for B-cell selection
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Added by
Caroline Gr\\\"onwall
Publication date
2018
fields
Biology
and research's language is
English
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Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly-mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decreased in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly also through non-antigen driven mechanisms.
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Glycosylation is a highly complex process to produce a diverse repertoire of cellular glycans that are attached to proteins and lipids. Glycans are involved in fundamental biological processes, including protein folding and clearance, cell proliferation and apoptosis, development, immune responses, and pathogenesis. One of the major types of glycans, N-linked glycans, is formed by sequential attachments of monosaccharides to proteins by a limited number of enzymes. Many of these enzymes can accept multiple N-linked glycans as substrates, thereby generating a large number of glycan intermediates and their intermingled pathways. Motivated by the quantitative methods developed in complex network research, we investigated the large-scale organization of such N-linked glycosylation pathways in mammalian cells. The N-linked glycosylation pathways are extremely modular, and are composed of cohesive topological modules that directly branch from a common upstream pathway of glycan synthesis. This unique structural property allows the glycan production between modules to be controlled by the upstream region. Although the enzymes act on multiple glycan substrates, indicating cross-talk between modules, the impact of the cross-talk on the module-specific enhancement of glycan synthesis may be confined within a moderate range by transcription-level control. The findings of the present study provide experimentally-testable predictions for glycosylation processes, and may be applicable to therapeutic glycoprotein engineering.
B cells develop high affinity receptors during the course of affinity maturation, a cyclic process of mutation and selection. At the end of affinity maturation, a number of cells sharing the same ancestor (i.e. in the same clonal family) are released from the germinal center, their amino acid frequency profile reflects the allowed and disallowed substitutions at each position. These clonal-family-specific frequency profiles, called substitution profiles, are useful for studying the course of affinity maturation as well as for antibody engineering purposes. However, most often only a single sequence is recovered from each clonal family in a sequencing experiment, making it impossible to construct a clonal-family-specific substitution profile. Given the public release of many high-quality large B cell receptor datasets, one may ask whether it is possible to use such data in a prediction model for clonal-family-specific substitution profiles. In this paper, we present the method Substitution Profiles Using Related Families (SPURF), a penalized tensor regression framework that integrates information from a rich assemblage of datasets to predict the clonal-family-specific substitution profile for any single input sequence. Using this framework, we show that substitution profiles from similar clonal families can be leveraged together with simulated substitution profiles and germline gene sequence information to improve prediction. We fit this model on a large public dataset and validate the robustness of our approach on an external dataset. Furthermore, we provide a command-line tool in an open-source software package (https://github.com/krdav/SPURF) implementing these ideas and providing easy prediction using our pre-fit models.
The wide spread of coronavirus disease 2019 (COVID-19) has declared a global health emergency. As one of the most important targets for antibody and drug developments, Spike RBD-ACE2 interface has received extensive attention. Here, using molecular dynamics simulations, we explicitly evaluated the binding energetic features of the RBD-ACE2 complex of both SARS-CoV and SARS-CoV-2 to find the key residues. Although the overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, the difference in binding affinity is as large as -16.35 kcal/mol. Energy decomposition analyses identified three binding patches in the SARS-CoV-2 RBD and eleven key residues (Phe486, Tyr505, Asn501, Tyr489, Gln493, Leu455 and etc) which are believed to be the main targets for drug development. The dominating forces are from van der Waals attractions and dehydration of these residues. It is also worth mention that we found seven mutational sites (Lys417, Leu455, Ala475, Gly476, Glu484, Gln498 and Val503) on SARS-CoV-2 which unexpectedly weakened the RBD-ACE2 binding. Very interestingly, the most repulsive residue at the RBD-ACE2 interface (E484), is found to be mutated in the latest UK variant, B1.1.7, cause complete virus neutralization escapes from highly neutralizing COVID-19 convalescent plasma. Our present results indicate that at least from the energetic point of view such E484 mutation may have beneficial effects on ACE2 binding. The present study provides a systematical understanding, from the energetic point of view, of the binding features of SARS-CoV-2 RBD with ACE2 acceptor. We hope that the present findings of three binding patches, key attracting residues and unexpected mutational sites can provide insights to the design of SARS-CoV-2 drugs and identification of cross-active antibodies.
Biochemistry and mechanics are closely coupled in cell adhesion. At sites of cell-matrix adhesion, mechanical force triggers signaling through the Rho-pathway, which leads to structural reinforcement and increased contractility in the actin cytoskeleton. The resulting force acts back to the sites of adhesion, resulting in a positive feedback loop for mature adhesion. Here we model this biochemical-mechanical feedback loop for the special case when the actin cytoskeleton is organized in stress fibers, which are contractile bundles of actin filaments. Activation of myosin II molecular motors through the Rho-pathway is described by a system of reaction-diffusion equations, which are coupled into a viscoelastic model for a contractile actin bundle. We find strong spatial gradients in the activation of contractility and in the corresponding deformation pattern of the stress fiber, in good agreement with experimental findings.
A recent experimental study found that the binding affinity between the cellular receptor human angiotensin converting enzyme 2 (ACE2) and receptor-binding domain (RBD) in spike (S) protein of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is more than 10-fold higher than that of the original severe acute respiratory syndrome coronavirus (SARS-CoV). However, main-chain structures of the SARS-CoV-2 RBD are almost the same with that of the SARS-CoV RBD. Understanding physical mechanism responsible for the outstanding affinity between the SARS-CoV-2 S and ACE2 is the urgent challenge for developing blockers, vaccines and therapeutic antibodies against the coronavirus disease 2019 (COVID-19) pandemic. Considering the mechanisms of hydrophobic interaction, hydration shell, surface tension, and the shielding effect of water molecules, this study reveals a hydrophobic-interaction-based mechanism by means of which SARS-CoV-2 S and ACE2 bind together in an aqueous environment. The hydrophobic interaction between the SARS-CoV-2 S and ACE2 protein is found to be significantly greater than that between SARS-CoV S and ACE2. At the docking site, the hydrophobic portions of the hydrophilic side chains of SARS-CoV-2 S are found to be involved in the hydrophobic interaction between SARS-CoV-2 S and ACE2. We propose a method to design live attenuated viruses by mutating several key amino acid residues of the spike protein to decrease the hydrophobic surface areas at the docking site. Mutation of a small amount of residues can greatly reduce the hydrophobic binding of the coronavirus to the receptor, which may be significant reduce infectivity and transmissibility of the virus.
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