Types Of Post-translational Modifications Research Articles

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications.

Post-translational modifications (PTMs) endow proteins with new properties to respond to environmental changes or growth needs. With the development of advanced proteomics techniques, hundreds of distinct types of PTMs have been observed in a wide range of proteins from bacteria, archaea, and eukarya. To identify the roles of these PTMs, scientists have applied various approaches. However, high dynamics, low stoichiometry, and crosstalk between PTMs make it almost impossible to obtain homogeneously modified proteins for characterization of the site-specific effect of individual PTM on target proteins. To solve this problem, the genetic code expansion (GCE) strategy has been introduced into the field of PTM studies. Instead of modifying proteins after translation, GCE incorporates modified amino acids into proteins during translation, thus generating site-specifically modified proteins at target positions. In this review, we summarize the development of GCE systems for orthogonal translation for site-specific installation of PTMs.

Spatiotemporal and direct capturing global substrates of lysine-modifying enzymes in living cells

Protein-modifying enzymes regulate the dynamics of myriad post-translational modification (PTM) substrates. Precise characterization of enzyme-substrate associations is essential for the molecular basis of cellular function and phenotype. Methods for direct capturing global substrates of protein-modifying enzymes in living cells are with many challenges, and yet largely unexplored. Here, we report a strategy to directly capture substrates of lysine-modifying enzymes via PTM-acceptor residue crosslinking in living cells, enabling global profiling of substrates of PTM-enzymes and validation of PTM-sites in a straightforward manner. By integrating enzymatic PTM-mechanisms, and genetically encoding residue-selective photo-crosslinker into PTM-enzymes, our strategy expands the substrate profiles of both bacterial and mammalian lysine acylation enzymes, including bacterial lysine acylases PatZ, YiaC, LplA, TmcA, and YjaB, as well as mammalian acyltransferases GCN5 and Tip60, leading to discovery of distinct yet functionally important substrates and acylation sites. The concept of direct capturing substrates of PTM-enzymes via residue crosslinking may extend to the other types of amino acid residues beyond lysine, which has the potential to facilitate the investigation of diverse types of PTMs and substrate-enzyme interactive proteomics.

N-glycosylation as a eukaryotic protective mechanism against protein aggregation.

The tendency for proteins to form aggregates is an inherent part of every proteome and arises from the self-assembly of short protein segments called aggregation-prone regions (APRs). While posttranslational modifications (PTMs) have been implicated in modulating protein aggregation, their direct role in APRs remains poorly understood. In this study, we used a combination of proteome-wide computational analyses and biophysical techniques to investigate the potential involvement of PTMs in aggregation regulation. Our findings reveal that while most PTM types are disfavored near APRs, N-glycosylation is enriched and evolutionarily selected, especially in proteins prone to misfolding. Experimentally, we show that N-glycosylation inhibits the aggregation of peptides in vitro through steric hindrance. Moreover, mining existing proteomics data, we find that the loss of N-glycans at the flanks of APRs leads to specific protein aggregation in Neuro2a cells. Our findings indicate that, among its many molecular functions, N-glycosylation directly prevents protein aggregation in higher eukaryotes.

Characterization of Diagenetiforms in an Expanded Proteome of the Extinct Moa (Dinornithidae): Identifying Biological, Diagenetic, Experimental Artifact, and Mislabeled Modifications in Degraded Tissues

Proteomic analyses of extinct moa (Dinornithidae; ~800–1000 years) bone tissue previously revealed preserved collagens (I, II, and V), as well as several biological post-translational modifications (PTMs) and diagenetic peptide sequence alterations. The diagenetiforms detected in that study provided a baseline of PTM preservation in degraded tissues, identifying sequence alterations that could be accounted for in bioinformatic data searches (e.g., carboxymethyllysine). Subsequently, an improved extraction and sample preparation methodology, coupled with higher resolution mass spectrometry analyses, identified a wealth of previously unidentified non-collagenous proteins (NCPs) from the specimen. Here, in-depth analyses of the PTMs preserved in the expanded data set provide a detailed look at the types of PTMs (i.e., biological, diagenetic, and potential experimental artifacts) that occur in degraded tissues, the proteins they occur on, and the amino acids they modify. In total, 10 biological PTMs (e.g., ubiquitylation) and 18 diagenetic PTMs, including two advanced glycation end products (e.g., dihydroxy methylglyoxal adduction) and 12 types of oxidative damage (e.g., pyrrolidone formation from proline), were detected. In addition, peptides displaying diagenetic backbone cleavage (hydrolysis) were frequently observed to possess unidentified, variable mass shifts at their broken terminus, which search software would attempt to erroneously identify as different PTMs. The modifications characterized in the bones of this specimen, both in collagens and in NCPs, provide insight into patterns of preservation and degradation that paleoproteomic studies can utilize when searching and interpreting data sets from fossil tissue.

Comparative Analysis of Post-Translational Modifications of Human Serum Albumin Derived from Human Plasma and Recombinant Sources in China.

The variations in sequence, three-dimensional structure, and post-translational modifications (PTMs) of human serum albumin (HSA) are crucial for its physiological functions. This study aims to analyze and compare the disparities in PTMs between HSA derived from human plasma and genetically recombinant sources for clinical treatments in China. Six distinct PTMs, namely acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation, were identified using pan-specific antibodies via Western blot analysis. The samples, comprising human plasma-derived HSA (pHSA) from six different manufacturers and recombinant HSA (rHSA) expressed in yeast and Oryza sativa, underwent detection for various types of PTMs. Additionally, a 4D label-free quantitative proteomic analysis was performed to identify N-glycosylation and the aforementioned PTMs in both pHSA and rHSA samples. This analysis aimed to discern disparities in modification sites and levels. Through Western blot analysis, all six pHSA and two rHSA samples displayed positive bands for albumin (66.5 kDa) across the six PTMs. Subsequent analysis using 4D label-free quantitative proteomics revealed 25 (29) acetylated, 30 (32) succinylated, 41 (50) malonylated, 15 (23) phosphorylated, 36 (30) beta-hydroxybutyrylated, and 27 (34) lactylated modification sites in pHSA and rHSA samples, with no N-glycosylation modification sites detected. The analysis identified 1 acetylation (ALB_K160), 2 beta-hydroxybutyrylation (ALB_K569, ALB_K426), and 3 crotonylation (ALB_K264, ALB_K581, ALB_K560) specific modification sites in pHSA, as well as 3 crotonylation (ALB_K560, ALB_K562, ALB_K75), 1 succinylation (ALB_K490), and 23 phosphorylation specific modification sites in rHSA. In pHSA (rHSA), 2 (6) acetylation, 10 (12) succinylation, 0 (9) crotonylation, 1 (9) phosphorylation, 6 (0) beta-hydroxybutyrylation, and 0 (7) lactylation specific modification sites were found. Moreover, in the shared modification sites between pHSA and rHSA, pHSA exhibited up-regulation of amberylation (16:1) and beta-hydroxybutyrylation (12:2) in more sites, and up-regulation of acetylation (7:11), crotonylation (2:11), phosphorylation (1:8), and lactylation (1:14) in fewer sites compared to rHSA. In clinical practice, both pHSA and rHSA utilized in China commonly display acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation. Notably, there exist distinctions in the site characteristics and modification levels of these alterations between pHSA and rHSA. Further experimental inquiries are imperative to delve into the implications of these disparities in PTMs on the biological functionality, effectiveness, and safety of pHSA and rHSA.

Demystifying PTM Identification Using MODplus: Best Practices and Pitfalls.

Tandem mass spectrometry (MS/MS) facilitates the rapid identification of posttranslational modifications (PTMs), which play a pivotal role in regulating numerous biological processes. This chapter explores recent advancements that expand the types of detectable PTMs and enhance the speed of the PTM searches. We also delve into computational challenges associated with searching for a multitude of PTMs simultaneously. The latter section introduces an automated procedure to identify an extensive range of PTMs using MODplus, a free PTM analysis software tool. We guide the reader through the preparation of the modification search, the determination of optional search parameters, the execution of the search, and the analysis of results, exemplified by a case study using specific MS/MS dataset.

Lipidation alters the phase-separation of resilin-like polypeptides.

Biology exploits biomacromolecular phase separation to form condensates, known as membraneless organelles. Despite significant advancements in deciphering sequence determinants for phase separation, modulating these features in vivo remains challenging. A promising approach inspired by biology is to use post-translational modifications (PTMs)-to modulate the amino acid physicochemistry instead of altering protein sequences-to control the formation and characteristics of condensates. However, despite the identification of more than 300 types of PTMs, the detailed understanding of how they influence the formation and material properties of protein condensates remains incomplete. In this study, we investigated how modification with myristoyl lipid alters the formation and characteristics of the resilin-like polypeptide (RLP) condensates, a prototypical disordered protein with upper critical solution temperature (UCST) phase behaviour. Using turbidimetry, dynamic light scattering, confocal and electron microscopy, we demonstrated that lipidation-in synergy with the sequence of the lipidation site-significantly influences RLPs' thermodynamic propensity for phase separation and their condensate properties. Molecular simulations suggested these effects result from an expanded hydrophobic region created by the interaction between the lipid and lipidation site rather than changes in peptide rigidity. These findings emphasize the role of "sequence context" in modifying the properties of PTMs, suggesting that variations in lipidation sequences could be strategically used to fine-tune the effect of these motifs. Our study advances understanding of lipidation's impact on UCST phase behaviour, relevant to proteins critical in biological processes and diseases, and opens avenues for designing lipidated resilins for biomedical applications like heat-mediated drug elution.

The SysteMHC Atlas v2.0, an updated resource for mass spectrometry-based immunopeptidomics.

The SysteMHC Atlas v1.0 was the first public repository dedicated to mass spectrometry-based immunopeptidomics. Here we introduce a newly released version of the SysteMHC Atlas v2.0 (https://systemhc.sjtu.edu.cn), a comprehensive collection of 7190 MS files from 303 allotypes. We extended and optimized a computational pipeline that allows the identification of MHC-bound peptides carrying on unexpected post-translational modifications (PTMs), thereby resulting in 471K modified peptides identified over 60 distinct PTM types. In total, we identified approximately 1.0 million and 1.1 million unique peptides for MHC class I and class II immunopeptidomes, respectively, indicating a 6.8-fold increase and a 28-fold increase to those in v1.0. The SysteMHC Atlas v2.0 introduces several new features, including the inclusion of non-UniProt peptides, and the incorporation of several novel computational tools for FDR estimation, binding affinity prediction and motif deconvolution. Additionally, we enhanced the user interface, upgraded website framework, and provided external links to other resources related. Finally, we built and provided various spectral libraries as community resources for data mining and future immunopeptidomic and proteomic analysis. We believe that the SysteMHC Atlas v2.0 is a unique resource to provide key insights to the immunology and proteomics community and will accelerate the development of vaccines and immunotherapies.

RMTLysPTM: recognizing multiple types of lysine PTM sites by deep analysis on sequences.

Post-translational modification (PTM) occurs after a protein is translated from ribonucleic acid. It is an important living creature life phenomenon because it is implicated in almost all cellular processes. Identification of PTM sites from a given protein sequence is a hot topic in bioinformatics. Lots of computational methods have been proposed, and they provide good performance. However, most previous methods can only tackle one PTM type. Few methods consider multiple PTM types. In this study, a multi-label classification model, named RMTLysPTM, was developed to recognize four types of lysine (K) PTM sites, including acetylation, crotonylation, methylation and succinylation. The surrounding sites of a lysine site were selected to constitute a peptide segment, representing the lysine at the center. Deep analysis was conducted to count the distribution of 2-residues with fixed location across the four types of lysine PTM sites. By aggregating the distribution information of 2-residues in one peptide segment, the peptide segment was encoded by informative features. Furthermore, a prediction engine that can precisely capture the traits of the above representations was designed to recognize the types of lysine PTM sites. The cross-validation results on two datasets (Qiu and CPLM training datasets) suggested that the model had extremely high performance and RMTLysPTM had strong generalization ability by testing it on protein Q16778 and CPLM testing datasets. The model was found to be generally superior to all previous models and those using popular methods and features. A web server was set up for RMTLysPTM, and it can be accessed at http://119.3.127.138/.

Functioning and mechanisms of PTMs in renal diseases

Post-translational modifications (PTMs) are crucial epigenetic mechanisms that regulate various cellular biological processes. The use of mass spectrometry (MS)-proteomics has led to the discovery of numerous novel types of protein PTMs, such as acetylation, crotonylation, 2-hydroxyisobutyrylation, β-hydroxybutyrylation, protein propionylation and butyrylation, succinylation, malonylation, lactylation, and histone methylation. In this review, we specifically highlight the molecular mechanisms and roles of various histone and some non-histone PTMs in renal diseases, including diabetic kidney disease. PTMs exhibit diverse effects on renal diseases, which can be either protective or detrimental, depending on the specific type of protein PTMs and their respective targets. Different PTMs activate various signaling pathways in diverse renal pathological conditions, which could provide novel insights for studying epigenetic mechanisms and developing potential therapeutic strategies for renal diseases.

Structural impairment of p53 C-terminal due to the effect of phosphorylation and acetylation: a study on the interdependence of PTM

The C-terminal of tumor suppressor protein p53 is intrinsically disordered while unbound. This particular segment often shows structural plasticity when bound to other binding partners. The disordered component undergoes a disordered to ordered transition upon recognition. Post-translational modifications (PTMs), namely phosphorylation and acetylation, significantly alter the structural motifs of the segment. Among the various types of PTMs, phosphorylation, and acetylation of p53 at both N- and C- terminals lead to stabilization and activation. It has been noted experimentally that phosphorylation often regulates (enhances or reduces) the acetylation at specific sites. The phosphorylation of Thr377 and Ser378 reduces the acetylation of Lys373 and Lys382. Mutations of Thr377 and Ser378 to neutral Ala enhance and phospho mimic Asp reduce the acetylation of Lys373 and Lys382. Simulations of several single-point and pair-wise mutated systems have been generated to compare how the presence or absence of phosphorylation favors or disfavors the acetylation by thermodynamic and conformational analysis. We are using implicit solvent replica exchange molecular dynamics simulations to get 200 ns well-converged conformational ensembles of each system. Different sets of systems having both single and double PTMs are simulated. The results admit the appreciable change in the secondary structural level upon specific PTM. Also, the residual structure of the unbound p53 with single-point PTM varies significantly with pair-wise modifications. These observations further shed light on the relationship between the interdependencies of the specific PTM sites and the secondary structural levels. Communicated by Ramaswamy H. Sarma

Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation.

The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses. Posttranslational modifications (PTMs) of NLRP3, including ubiquitination and phosphorylation, control inflammasome activation and determine the intensity of inflammation. However, the role of other PTMs in controlling NLRP3 inflammasome activation remains unclear. This study found that TLR priming induced NLRP3 ISGylation (a type of PTM in which ISG15 covalently binds to the target protein) to stabilize the NLRP3 protein. Viral infection, represented by SARS-COV-2 infection, and type I IFNs induced expression of ISG15 and the predominant E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice). HERCs promoted NLRP3 ISGylation and inhibited K48-linked ubiquitination and proteasomal degradation, resulting in the enhancement of NLRP3 inflammasome activation. Concordantly, Herc6 deficiency ameliorated NLRP3-dependent inflammation as well as hyperinflammation caused by viral infection. The results illustrate the mechanism by which type I IFNs responses control inflammasome activation and viral infection-induced aberrant NLRP3 activation. This work identifies ISGylation as a PTM of NLRP3, revealing a priming target that modulates NLRP3-dependent immunopathology.

New Strategies for Probing the Biological Functions of Protein Post-translational Modifications in Mammalian Cells with Genetic Code Expansion.

Protein post-translational modification (PTM) is a major mechanism for functional diversification of the human genome and plays a crucial role in almost every aspect of cellular processes, and the dysregulation of the protein PTM network has been associated with a variety of human diseases. Using high-resolution mass spectrometry, protein PTMs can be efficiently discovered and profiled under various biological and physiological conditions. However, it is often challenging to address the biological function of PTMs with biochemical and mutagenesis-based approaches. Specifically, this field lacks methods that allow gain-of-function studies of protein PTMs to understand their functional consequences in living cells. In this context, the genetic code expansion (GCE) strategy has made tremendous progress in the direct installation of PTMs and their analogs in the form of noncanonical amino acids (ncAAs) for gain-of-function investigations.In addition to studying the biological functions of known protein PTMs, the discovery of new protein PTMs is even more challenging due to the lack of chemical information for designing specific enrichment methods. Genetically encoded ncAAs in the proteome can be used as specific baits to enrich and subsequently identify new PTMs by mass spectrometry.In this Account, we discuss recent developments in the investigation of the biological functions of protein PTMs and the discovery of protein PTMs using new GCE strategies. First, we leveraged a chimeric design to construct several broadly orthogonal translation systems (OTSs). These broad OTSs can be engineered to efficiently incorporate different ncAAs in both E. coli and mammalian cells. With these broad OTSs, we accomplish the following: (1) We develop a computer-aided strategy for the design and genetic incorporation of length-tunable lipidation mimics. These lipidation mimics can fully recapitulate the biochemical properties of natural lipidation in membrane association for probing its biological functions on signaling proteins and in albumin binding for designing long-acting protein drugs. (2) We demonstrate that the binding affinity between histone methylations and their corresponding readers can be substantially increased with genetically encoded electron-rich Trp derivatives. These engineered affinity-enhanced readers can be applied to enrich, image, and profile the interactome of chromatin methylations. (3) We report the identification and verification of a novel type of protein PTM, aminoacylated lysine ubiquitination, using genetically encoded PTM ncAAs as chemical probes. This approach provides a general strategy for the identification of unknown PTMs by increasing the abundance of PTM bait probes.

Citrullination and the protein code: crosstalk between post-translational modifications in cancer.

Post-translational modifications (PTMs) of proteins are central to epigenetic regulation and cellular signalling, playing an important role in the pathogenesis and progression of numerous diseases. Growing evidence indicates that protein arginine citrullination, catalysed by peptidylarginine deiminases (PADs), is involved in many aspects of molecular and cell biology and is emerging as a potential druggable target in multiple diseases including cancer. However, we are only just beginning to understand the molecular activities of PADs, and their underlying mechanistic details in vivo under both physiological and pathological conditions. Many questions still remain regarding the dynamic cellular functions of citrullination and its interplay with other types of PTMs. This review, therefore, discusses the known functions of PADs with a focus on cancer biology, highlighting the cross-talk between citrullination and other types of PTMs, and how this interplay regulates downstream biological events. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Citrullination of matrisomal proteins in health and diseases.

Proteins once translated are subjected to post-translational modifications (PTMs) that can critically modify their characteristics. Citrullination is a unique type of PTM that is catalysed by peptidylarginine deiminase (PAD) enzymes, which regulate a multitude of physiological functions such as apoptosis, gene expression and immune response by altering the structure and function of cellular proteins. However, emerging data have unravelled compelling evidence to support that PAD-mediated citrullination is not exclusive to cellular proteins; rather citrullination of extracellular matrix (ECM) proteins also plays a major contributing role in various physiological/pathological conditions. Here, we discuss putative mechanisms for citrullination-induced alterations in the function of ECM proteins. Further, we put emphasis on influential roles of ECM citrullination in various pathological scenarios to underscore the clinical potential of its manipulation in human diseases. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

One-step production of fully biotinylated and glycosylated human Fc gamma receptors.

Initiating and regulating humoral immunity, Fc gamma receptors (FcγRs) have been identified both as therapeutics and as drug targets, and thus production of biologically active FcγRs is highly demanded for biopharmaceutical development. Focusing on low-affinity FcγRs IIA (131H/R allotypes), IIB, and IIIA (176F/V), this study used human 293-F cells to achieve correct post-translational modifications (PTMs) including biotinylation, N-glycosylation, and disulfides. Approaches involving co-expression of FcγR-AviTag and Escherichia coli biotin ligase BirA, endoplasmic reticulum retention, stable and transient transfections, and optimization of transgene ratio were investigated. Protein electrophoresis under reducing and non-reducing conditions, enzymatic deglycosylation, streptavidin pull-down assays, and binding kinetic analysis collectively indicated that the produced FcγR ectodomains were fully biotinylated, N-glycosylated, had formed disulfide bond, and exhibited expected binding affinities toward IgG1 trastuzumab and its Fc mutants. A clear trade-off between production yield and PTM quality was also observed. Achieving multiple types of PTMs completely by one-step cell culture should have applications for the production of a variety of complex proteins of biomedical importance.

Identification and application of species-specific peptide biomarkers from soft-shelled turtles (Pelodiscus sinensis) using post-translational modification detection-based peptidomics analysis.

The soft-shelled turtle is a commercially aquatic species in Asian countries, which serves as an important source of collagen with high nutritional and medicinal value, so it is of great significance to distinguish soft-shelled turtle derived collagen from others or adulterations. In this work, peptidomics analysis based on post-translational modification (PTM) assay was used to discover specific peptide biomarkers of soft-shelled turtle gelatin (STG). In total eight specific sequences and 74 peptides with different PTM types were screened out, and seven peptides with good signal responses and STG specificity were selected and validated as STG-specific peptide biomarkers. These peptide biomarkers could be used for distinguishing STG from other animal gelatins, and applied for ensuring the quality of collagens or gelatins from soft-shelled turtle with authenticity and traceability.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Y-mtPTM: Yeast mitochondrial posttranslational modification database.

One powerful strategy of how to increase the complexity of cellular proteomes is through posttranslational modifications (PTMs) of proteins. Currently, there are ∼400 types of PTMs, the different combinations of which yield a large variety of protein isoforms with distinct biochemical properties. Although mitochondrial proteins undergoing PTMs were identified nearly 6 decades ago, studies on the roles and extent of PTMs on mitochondrial functions lagged behind the other cellular compartments. The application of mass spectrometry for the characterization of the mitochondrial proteome as well as for the detection of various PTMs resulted in the identification of thousands of amino acid positions that can be modified by different chemical groups. However, the data on mitochondrial PTMs are scattered in several data sets, and the available databases do not contain a complete list of modified residues. To integrate information on PTMs of the mitochondrial proteome of the yeast Saccharomyces cerevisiae, we built the yeast mitochondrial posttranslational modification (y-mtPTM) database (http://compbio.fmph.uniba.sk/y-mtptm/). It lists nearly 20,000 positions on mitochondrial proteins affected by ∼20 various PTMs, with phosphorylated, succinylated, acetylated, and ubiquitylated sites being the most abundant. A simple search of a protein of interest reveals the modified amino acid residues, their position within the primary sequence as well as on its 3D structure, and links to the source reference(s). The database will serve yeast mitochondrial researchers as a comprehensive platform to investigate the functional significance of the PTMs of mitochondrial proteins.

A cellular platform for the evaluation of the effect of O-glycan modification on B cell receptor signaling

Abstract The importance of N-glycosylation, a ubiquitous post-translational modification (PTM) of cell-surface and secreted proteins in vertebrates, in the regulation of B cell receptor (BCR) signaling has been well documented. On the other hand, the importance of O-glycosylation, another type of ubiquitous PTM, in B cell function is getting recognized only recently. A simplified system to study the effects of O-glycan modifications in BCR signaling would facilitate the efforts to understand the mechanism of B cell regulation by O-glycans. To this end, we reconstituted BCR components and CD45 with orthogonal self-labeling protein tags in the mouse myeloma J558L cell line, which is deficient in the components of BCR and several co-receptors. The enzymes that modify O-glycans were further introduced, and the cells were subjected to imaging-based quantitative analysis of the distance between BCR and CD45, as well as the evaluation of BCR signaling by Western blotting. Gene expression profiles among the cells were also compared by RNA sequencing. Our study revealed that core 2 branching of O-glycans has minimal effect on the distance between BCR and CD45, whereas the addition of an α2–8-linked sialic acid by ST8Sia-VI reduces the distance between them, facilitating the downstream BCR signaling. Our system may be adapted for the functional evaluation of other PTMs in BCR signaling. Supported by a grant from the Ministry of Science and Technology (MOST)/National Science and Technology Council (NSTC) of Taiwan (MOST 109-2311-B-001-009-MY3)