Glycosylation Mapping Research Articles

Mapping and functional analysis of S1 subunit glycosylation of avian infectious bronchitis virus

Glycosylation of the S1 subunit is a critical post-translational modification in coronavirus antigen proteins and plays a key role in vaccine development. The biological role of highly mutable glycosylation sites in virus evolution, however, remains underexplored. In this study, we identified nine glycosylation sites with high mutation rates on the S1 subunit of Avian Infectious Bronchitis Virus (IBV) through evolutionary analysis. Functional analysis of mutant strains revealed that specific mutations, particularly at position 76 (N → V), significantly enhance the strain's protective efficacy and limit viral replication. These findings provide important insights into the role of glycosylation in viral evolution and offer valuable guidance for the development of more effective IBV vaccines.

Monitoring multiple quality attributes of a complex Fc-fusion protein during cell culture production processes by mD-LC-MS peptide mapping

Fc-fusion proteins represent a successful class of biopharmaceutical products. They are considered highly heterogeneous products due to the common degradation of amino acids that occurs during their production in upstream and downstream processes (e.g., oxidation and deamidation) and, above all, their complex glycosylation profile. Multi-dimensional liquid chromatography-mass spectrometry (mD-LC-MS) has recently gained much interest for process analytical technology, enabling the integration of this analytical technology in production and purification environments. In this study, an online mD-LC-MS/MS peptide mapping method was developed for monitoring multiple quality attributes, including the N-glycosylation state of a complex Fc-fusion protein, which is made by combining two heavily glycosylated cytokines with an Fc domain. This fully automated workflow includes sample purification, reduction, digestion, peptide mapping, and subsequent mass spectrometric analysis. Two immobilized enzyme cartridges based on trypsin and Lys-C protease were employed to generate a detailed glycosylation mapping, as trypsin allowed the identification of only one of four glycosylation sites, while Lys-C was more informative for two other sites. Site-specific glycosylation information such as antennarity, sialyation, and core fucosylation state was also determined. In addition to glycans, other post-translational modifications could be monitored simultaneously during the cell culture production processes by the mD-LC-MS/MS approach. In summary, the generated data demonstrate the applicability of mD-LC-MS for the monitoring and trending of multiple attributes for complex antibody formats over production processes in an automated and fast manner, compared to the complex and time-consuming traditional offline assays.

Efficient HCD-pd-EThcD approach for N-glycan mapping of therapeutic antibodies at intact glycopeptide level

N-glycosylation is a critical quality attribute for monoclonal antibody (mAb)-based therapeutics due to its significant impact on drug efficacy and safety. Extensive glycosylation mapping is therefore necessary for mAb drug development and quality control. We utilized a higher-energy dissociation product ions-triggered electron-transfer/higher-energy collision dissociation (HCD-pd-EThcD) approach to mapping N-glycosylation in therapeutic mAbs. Due to the improved duty cycle and targeted ability, HCD-pd-EThcD could provide extensive N-glycan identifications as well as higher quality spectra than EThcD mode. On average, ten types of N-glycan were uncovered in two different lots of trastuzumab, demonstrating a significant increment in N-glycan species compared to only four types identified by EThcD. After integrating pre-enrichment of glycopeptides, up to 16 N-glycans were recognized. Significantly, this strategy facilitated the identification of glycopeptides containing fucosylated and sialylated glycans, meanwhile enabled the recognition of different N-glycan classes (high mannose, hybrid, and complex). Further application in the glycosylation analysis of adalimumab and bevacizumab resulted in 19 and 8 N-glycans species, providing a more comprehensive insight into their glycosylation modification status. We demonstrated the benefits of an integrated strategy in characterizing various N-glycans of mAb therapeutics and offer an alternative approach for their quality control at the intact glycopeptides level.

The Sigma-1 receptor is an ER-localized type II membrane protein

The Sigma-1 receptor (S1R) is a transmembrane protein with important roles in cellular homeostasis in normal physiology and in disease. Especially in neurodegenerative diseases, S1R activation has been shown to provide neuroprotection by modulating calcium signaling, mitochondrial function and reducing endoplasmic reticulum (ER) stress. S1R missense mutations are one of the causes of the neurodegenerative Amyotrophic Lateral Sclerosis and distal hereditary motor neuronopathies. Although the S1R has been studied intensively, basic aspects remain controversial, such as S1R topology and whether it reaches the plasma membrane. To address these questions, we have undertaken several approaches. C-terminal tagging with a small biotin-acceptor peptide and BirA biotinylation in cells suggested a type II membrane orientation (cytosolic N-terminus). However, N-terminal tagging gave an equal probability for both possible orientations. This might explain conflicting reports in the literature, as tags may affect the protein topology. Therefore, we studied untagged S1R using a protease protection assay and a glycosylation mapping approach, introducing N-glycosylation sites. Both methods provided unambiguous results showing that the S1R is a type II membrane protein with a short cytosolic N-terminal tail. Assessments of glycan processing, surface fluorescence-activated cell sorting, and cell surface biotinylation indicated ER retention, with insignificant exit to the plasma membrane, in the absence or presence of S1R agonists or of ER stress. These findings may have important implications for S1R-based therapeutic approaches.

Novel Insight Into Glycosaminoglycan Biosynthesis Based on Gene Expression Profiles.

Glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate, except for hyaluronan that is a free polysaccharide, are covalently attached to core proteins to form proteoglycans. More than 50 gene products are involved in the biosynthesis of GAGs. We recently developed a comprehensive glycosylation mapping tool, GlycoMaple, for visualization and estimation of glycan structures based on gene expression profiles. Using this tool, the expression levels of GAG biosynthetic genes were analyzed in various human tissues as well as tumor tissues. In brain and pancreatic tumors, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be upregulated. In breast cancerous tissues, the pathways for biosynthesis of chondroitin and dermatan sulfate were predicted to be up- and down-regulated, respectively, which are consistent with biochemical findings published in the literature. In addition, the expression levels of the chondroitin sulfate-proteoglycan versican and the dermatan sulfate-proteoglycan decorin were up- and down-regulated, respectively. These findings may provide new insight into GAG profiles in various human diseases including cancerous tumors as well as neurodegenerative disease using GlycoMaple analysis.

Comparative Glycosylation Mapping of Recombinant and Plasma-Derived Human Factor VIII Reveals Key Biological Differences

Human coagulation factor VIII (FVIII) is a key co-factor in the coagulation pathway. Its functional deficiency is directly linked to Hemophilia A. Both human plasma-derived (pdFVIII) and recombinant FVIII (rFVIII) have been developed as effective reagents for treating the bleeding episodes. However, it remains debatable if these two pharmaceutical products are equivalent in clinical efficiency and safety profile despite of their identical amino acid sequences. Several studies have indicated that rFVIII comes with higher risk of inhibitor antibody development. In this report, we systemically analyzed the site-specific pattern of pdFVIII and rFVIII in glycosylation. A total of 61 N-glycan structures or isomers and 31 O-glycan compositions were identified. The likely immunogenic epitope, N-glycolylneuraminic acid (Neu5Gc), was absent in either recombinant or plasma-derived FVIII. However, potentially immunogenic glycopeptides, including Alpha-Gal epitope, has been identified in rFVIII. The glycosylation site localization between rFVIII and pdFVIII show significant divergence. The specific differences in glycosylation pattern of pdFVIII and rFVIII may be one of the important biological basis leading to their immunological disparity in human patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Global mapping of glycosylation pathways in human-derived cells

Glycans are one of the fundamental classes of macromolecules and are involved in a broad range of biological phenomena. A large variety of glycan structures can be synthesized depending on tissue or cell types and environmental changes. Here, we developed a comprehensive glycosylation mapping tool, termed GlycoMaple, to visualize and estimate glycan structures based on gene expression. We informatically selected 950 genes involved in glycosylation and its regulation. Expression profiles of these genes were mapped onto global glycan metabolic pathways to predict glycan structures, which were confirmed using glycomic analyses. Based on the predictions of N-glycan processing, we constructed 40 knockout HEK293 cell lines and analyzed the effects of gene knockout on glycan structures. Finally, the glycan structures of 64 cell lines, 37 tissues, and primary colon tumor tissues were estimated and compared using publicly available databases. Our systematic approach can accelerate glycan analyses and engineering in mammalian cells.

Site-Specific Glycosylation Mapping of Fc Gamma Receptor IIIb from Neutrophils of Individual Healthy Donors.

Fc gamma receptors (FcγRs) translate antigen recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and FcγR impacts their interaction dramatically. Regarding FcγR glycosylation profiling, major analytical challenges are associated with the presence of multiple glycosylation sites in close proximity and large structural heterogeneity. To address these challenges, we developed a straightforward and comprehensive analytical methodology to map FcγRIIIb glycosylation in primary human cells. After neutrophil isolation and immunoprecipitation, glycopeptides containing a single site each were generated by a dual-protease in-gel digestion. The complex mixture was resolved by liquid chromatography–tandem mass spectrometry (LC-MS/MS) providing information on the level of individual donors. In contrast to recently published alternatives for FcγRIIIb, we assessed its site-specific glycosylation in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying FcγRIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous FcγRIIIa on natural killer cells. This method will allow assessment of differences in FcγRIII glycosylation between individuals, cell types, subcellular locations, and pathophysiological conditions.

Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2.

The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.

Development of Immobilized Enzyme Reactors for the characterization of the glycosylation heterogeneity of a protein

The mapping of post-translational modifications (PTMs) of proteins can be addressed by bottom-up proteomics strategy using proteases to achieve the enzymatic digestion of the biomolecule. Glycosylation is one of the most challenging PTM to characterize due to its large structural heterogeneity. In this work, two Immobilized Enzyme Reactors (IMERs) based on trypsin and pepsin protease were used for the first time to fasten and improve the reliability of the specific mapping of the N-glycosylation heterogeneity of glycoproteins. The performance of the supports was evaluated with the digestion of human Chorionic Gonadotropin hormone (hCG), a glycoprotein characterized by four N- and four O-glycosylation sites, prior to the analysis of the digests by nanoliquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Firstly, the repeatability of the nanoLC-MS/MS was evaluated and a method to control the identification of the identified glycans was developed to validate them regarding the retention time of glycopeptides in reversed phase nanoLC separation. The repeatability of the digestion with trypsin-based IMER was evaluated on the same hCG batch and on three independent batches with common located glycans up to 75%. Then, the performance of the IMER digestions was compared to in-solution digestions to evaluate the qualitative mapping of the glycosylation. It has given rise to 42 out of 45 common glycans between both digestions modes. For the first time, the complementarity of trypsin and pepsin was illustrated for the glycosylation mapping as trypsin led to identifications on 2 out of 4 glycosylation site while pepsin was informative on the 4 glycosylation site. The potential of IMERs for the study of the glycosylation of a protein was illustrated with the comparison of two hCG-based drugs, Ovitrelle® and Pregnyl®.

Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase

Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.

Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex

The oligosaccharyltransferase complex, localized in the endoplasmic reticulum (ER) of eukaryotic cells, is responsible for the N-linked glycosylation of numerous protein substrates. The membrane protein STT3 is a highly conserved part of the oligosaccharyltransferase and likely contains the active site of the complex. However, understanding the catalytic determinants of this system has been challenging, in part because of a discrepancy in the structural topology of the bacterial versus eukaryotic proteins and incomplete information about the mechanism of membrane integration. Here, we use a glycosylation mapping approach to investigate these questions. We measured the membrane integration efficiency of the mouse STT3-A and yeast Stt3p transmembrane domains (TMDs) and report a refined topology of the N-terminal half of the mouse STT3-A. Our results show that most of the STT3 TMDs are well inserted into the ER membrane on their own or in the presence of the natural flanking residues. However, for the mouse STT3-A hydrophobic domains 4 and 6 and yeast Stt3p domains 2, 3a, 3c, and 6 we measured reduced insertion efficiency into the ER membrane. Furthermore, we mapped the first half of the STT3-A protein, finding two extra hydrophobic domains between the third and the fourth TMD. This result indicates that the eukaryotic STT3 has 13 transmembrane domains, consistent with the structure of the bacterial homolog of STT3 and setting the stage for future combined efforts to interrogate this fascinating system.

Membrane insertion and topology of the translocon-associated protein (TRAP) gamma subunit

Translocon-associated protein (TRAP) complex is intimately associated with the ER translocon for the insertion or translocation of newly synthesised proteins in eukaryotic cells. The TRAP complex is comprised of three single-spanning and one multiple-spanning subunits. We have investigated the membrane insertion and topology of the multiple-spanning TRAP-γ subunit by glycosylation mapping and green fluorescent protein fusions both in vitro and in cell cultures. Results demonstrate that TRAP-γ has four transmembrane (TM) segments, an Nt/Ct cytosolic orientation and that the less hydrophobic TM segment inserts efficiently into the membrane only in the cellular context of full-length protein.

Protein Arrays I: Antibody Arrays.

Antibody arrays represent one of the very early protein array systems where antibodies are used to capture and detect target proteins in a high-throughput platform. The development of high-quality antibodies, nanomaterial-based novel detection probes, as well as innovative imaging technologies and computational tools has tremendously improved the sensitivity, specificity, and robustness of antibody arrays during the past decade. In this protocol we will incorporate the most updated innovations and developments of antibody arrays into the step-by-step experimental procedures. This includes antibody printing, sample preparation, array detection, as well as imaging and data analysis. Antibody array could be used for cytokine profiling or mapping of phosphorylation, glycosylation, or other post-translational modifications of target proteins.

Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)

The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout–Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.

Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation

While early structural models of helix-bundle integral membrane proteins posited that the transmembrane α-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.

The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane

Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an Nlumen–Ccytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane.

An Accessible Protocol for Solid-Phase Extraction of N-Linked Glycopeptides through Reductive Amination by Amine-Functionalized Magnetic Nanoparticles

In light of the significance of glycosylation for wealthy biological events, it is important to prefractionate glycoproteins/glycopeptides from complex biological samples. Herein, we reported a novel protocol of solid-phase extraction of glycopeptides through a reductive amination reaction by employing the easily accessible 3-aminopropyltriethoxysilane (APTES)-functionalized magnetic nanoparticles. The amino groups from APTES, which were assembled onto the surface of the nanoparticles through a one-step silanization reaction, could conjugate with the aldehydes from oxidized glycopeptides and, therefore, completed the extraction. To the best of our knowledge, this is the first example of applying the reductive amination reaction into the isolation of glycopeptides. Due to the elimination of the desalting step, the detection limit of glycopeptides was improved by 2 orders of magnitude, compared to the traditional hydrazide chemistry-based solid phase extraction, while the extraction time was shortened to 4 h, suggesting the high sensitivity, specificity, and efficiency for the extraction of N-linked glycopeptides by this method. In the meantime, high selectivity toward glycoproteins was also observed in the separation of Ribonuclease B from the mixtures contaminated with bovine serum albumin. What's more, this technique required significantly less sample volume, as demonstrated in the successful mapping of glycosylation of human colorectal cancer serum with the sample volume as little as 5 μL. Because of all these attractive features, we believe that the innovative protocol proposed here will shed new light on the research of glycosylation profiling.

Sweet-Heart — An integrated suite of enabling computational tools for automated MS2/MS3 sequencing and identification of glycopeptides

High efficiency identification of intact glycopeptides from a shotgun glycoproteomic LC-MS(2) dataset remains problematic. The prevalent mode of identifying the de-N-glycosylated peptides is littered with false positives and addresses only the issue of site occupancy. Here, we present Sweet-Heart, a computational tool set developed to tackle the heart of the problems in MS(2) sequencing of glycopeptide. It accepts low resolution and low accuracy ion trap MS(2) data, filters for glycopeptides, couples knowledge-based de novo interpretation of glycosylation-dependent fragmentation pattern with protein database search, and uses machine-learning algorithm to score the computed glyco and peptide combinations. Higher ranking candidates are then compiled into a list of MS(2)/MS(3) entries to drive subsequent rounds of targeted MS(3) sequencing of putative peptide backbone, allowing its validation by database search in a fully automated fashion. With additional fishing out of all related glycoforms and final data integration, the platform proves to be sufficiently sensitive and selective, conducive to novel glycosylation discovery, and robust enough to discriminate, among others, N-glycolyl neuraminic acid/fucose from N-acetyl neuraminic acid/hexose. A critical appraisal of its computing performance shows that Sweet-Heart allows high sensitivity comprehensive mapping of site-specific glycosylation for isolated glycoproteins and facilitates analysis of glycoproteomic data. The biological relevance of protein site-specific glycosylation cannot be meaningfully addressed without first defining its pattern by direct analysis of glycopeptides. Sweet-Heart is a novel suite of computational tools allowing for automated analysis of mass spectrometry-based glycopeptide sequencing data. It is developed to accept ion trap MS2/MS3 data and uses a machine learning algorithm to score and rank the candidate peptide core and glycosyl substituent combinations. By eliminating the need for manual, labor-intensive, and subjective data interpretation, it facilitates high throughput shotgun glycoproteomic data analysis and is conducive to identification of unanticipated glycosylation, as demonstrated here with a recombinant EGFR.

Human Vitamin K Epoxide Reductase and Its Bacterial Homologue Have Different Membrane Topologies and Reaction Mechanisms

Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.