Removal Of N-glycans Research Articles

Development of new NGLY1 assay systems - toward developing an early screening method for NGLY1 deficiency.

Cytosolic peptide: N-glycanase (PNGase/NGLY1 in mammals) is an amidase (EC:3.5.1.52) widely conserved in eukaryotes. It catalyzes the removal of N-glycans on glycoproteins, converting N-glycosylated Asn into Asp residues. This enzyme also plays a role in the quality control system for nascent glycoproteins. Since the identification of a patient with an autosomal recessive genetic disorder caused by NGLY1 gene dysfunction, known as NGLY1 deficiency or NGLY1 congenital disorder of deglycosylation (OMIM: 615273), in 2012, more than 100 cases have been reported worldwide. NGLY1 deficiency is characterized by a wide array of symptoms, such as global mental delay, intellectual disability, abnormal electroencephalography findings, seizure, movement disorder, hypolacrima or alacrima, and liver dysfunction. Unfortunately, no effective therapeutic treatments for this disease have been established. However, administration of adeno-associated virus 9 (AAV9) vector harboring human NGLY1 gene to an NGLY1-deficient rat model (Ngly1-/- rat) by intracerebroventricular injection was found to drastically improve motor function defects. This observation indicated that early therapeutic intervention could alleviate various symptoms originating from central nervous system dysfunction in this disease. Therefore, there is a keen interest in the development of facile diagnostic methods for NGLY1 deficiency. This review summarizes the history of assay development for PNGase/NGLY1 activity, as well as the recent progress in the development of novel plate-based assay systems for NGLY1, and also discusses future perspectives.

Human organic anion transporting polypeptide 1B3 (OATP1B3) is more heavily N-glycosylated than OATP1B1 in extracellular loops 2 and 5

Human organic anion transporting polypeptide 1B3 (OATP1B3) and 1B1 are two liver-specific and highly homologous uptake transporters, whose structures consist of 12 transmembrane domains. The present study showed that OATP1B3 is more heavily N-glycosylated than OATP1B1 in extracellular loop 2 (EL2) and EL5. OATP1B3 has six N-glycosylation sites, namely N134, N145, N151, N445, N503, and N516, which is twice of that of OATP1B1. Single removal of individual N-glycans seems to have minimal influence on the surface expression and function of OATP1B3. However, simultaneous removal of all N-glycans will lead to OATP1B3's large retention in the endoplasmic reticulum and cellular degradation and thus significantly disrupts its surface expression. While N-glycosylation plays a crucial role in the surface expression of OATP1B3, it also has some effect on the transport function of OATP1B3 per se, which is not due to a decrease of substrate binding affinity but due to a reduced transporter's turnover number. Taken together, N-glycosylation is essential for normal surface expression and function of OATP1B3. Its disruption by some liver diseases such as NASH might alter the pharmacokinetic/pharmacodynamic properties of OATP1B3's substrate drugs.

Sugar-mediated non-canonical ubiquitination impairs Nrf1/NFE2L1 activation

Proteasome is essential for cell survival, and proteasome inhibition induces proteasomal gene transcription via the activated endoplasmic-reticulum-associated transcription factor nuclear factor erythroid 2-like 1(Nrf1/NFE2L1). Nrf1 activation requires proteolytic cleavage by DDI2 and N-glycan removal by NGLY1. We previously showed that Nrf1 ubiquitination by SKP1-CUL1-F-box (SCF)FBS2/FBXO6, an N-glycan-recognizing E3 ubiquitin ligase, impairs its activation, although the molecular mechanism remained elusive. Here, we show that SCFFBS2 cooperates with the RING-between-RING (RBR)-type E3 ligase ARIH1 to ubiquitinate Nrf1 through oxyester bonds in human cells. Endo-β-N-acetylglucosaminidase (ENGASE) generates asparagine-linked N-acetyl glucosamine (N-GlcNAc) residues from N-glycans, and N-GlcNAc residues on Nrf1 served as acceptor sites for SCFFBS2-ARIH1-mediated ubiquitination. We reconstituted the polyubiquitination of N-GlcNAc and serine/threonine residues on glycopeptides and found that the RBR-specific E2 enzyme UBE2L3 is required for the assembly of atypical ubiquitin chains on Nrf1. The atypical ubiquitin chains inhibited DDI2-mediated activation. The present results identify an unconventional ubiquitination pathway that inhibits Nrf1 activation.

Involvement of the GH38 Family Exoglycosidase α-Mannosidase in Strawberry Fruit Ripening

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.

Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

Abstract LB058: N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models

Abstract Background: Current clinical application of trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate (ADC) has been severely limited by interstitial lung disease (ILD). Recent studies suggest a correlation between ILD and the uptake of T-DXd by alveolar macrophages (AMs), which are major innate immune cells in lung tissues. This underlying mechanism of AMs phagocytosing T-DXd was predicted to be the binding interaction between the crystallizable fragment (Fc) of trastuzumab and the Fcγ receptors on the cell surface of AMs, exerting effector functions. Since monoclonal antibodies typically undergo N-glycosylation in the Fc fragment, here we investigate the impacts of antibody N-glycan deglycosylation on T-DXd-induced ILD in preclinical models. Methods: N-glycan removal was performed on trastuzumab and T-DXd using PNGase F. Using immunofluorescence imaging combined with flow cytometry, we conducted a comparative analysis of the binding affinity and internalization efficiency of wild type and fully N-glycan deglycosylated HER2 antibodies. Next, we determined the in vitro cytotoxicity of WT and N-glycan deglycosylated T-DXd against both macrophages and HER2-positive cancer cells using CCK-8 assay. Furthermore, the deposition of N-glycan deglycosylated T-DXd in healthy lungs and their effects in ILD incidence and severity was determined in immunocompetent BALB/c mice carrying HER2-positive tumors. Results: First, we obtained complete N-glycan deglycosylated HER2 antibodies confirmed by SDS-PAGE and mass spectrometry. Next, we showed that N-glycan removal by PNGase F did not affect the Fab fragments of HER2 antibodies binding to the HER2 antigens on HER2-expressing cancer cells (OE19, SK-OV-3 and CT26-hHER2). Importantly, we did observe an approximately 30-50% reduction in the N-glycan deglycosylated HER2 antibodies binding with a panel of three macrophages (MH-S, RAW264.7, and PMA-induced THP-1) in comparison with WT antibodies, indicating a significant impact of N-glycan removal on innate immune cell-mediated effector functions. Moreover, in vitro cytotoxicity studies further support these observations that N-glycan deglycosylated T-DXd has significantly lower cytotoxicities against MH-S (p=0.046) and RAW264.7(p=0.020) in comparison with WT T-DXd. Correlating with our in vitro findings, in vivo biodistribution data showed a reduced lung deposition of HER2 antibodies and T-DXd after N-glycan removal. Immunofluorescence staining confirms the co-localization of HER2 antibodies with CD68-positive macrophages, indicating that AMs are the major contributor to the lung deposition of HER2 antibodies and T-DXd and N-glycan removal could effectively avoid ADC being phagocytosed by AMs. We also demonstrated chronic administration of N-glycan deglycosylated T-DXd could reduce ADC-associated ILD incidence and ameliorate ILD severity. Conclusions: Collectively, our study suggests that antibody N-glycan deglycosylation has important roles in regulating interactions between ADC and innate immune cells. This finding provides valuable clues for further optimizing ADC design and treatment strategies. Citation Format: Yu-Xuan Yang, Le-Tao Ma, Peng Guo. N-glycan deglycosylation ameliorates anti-HER2 antibody drug conjugate-induced interstitial pneumonia in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB058.

Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Site-specific N-glycosylation characterization and allergenicity analysis of globulin-1 S allele from wheat

N-glycans in many proteins are of great concern because of their strong association with food allergies. Triticum aestivum (bread wheat), a major food crop, is known as one of the “Big Eight” allergenic groups. However, little research has been done about N-glycans in wheat glycoproteins. In this study, a soluble wheat glycoprotein was purified from wheat and further identified as globulin-1 S allele (GSA). The wheat GSA displayed significant IgE-binding activity. Moreover, one N-glycosylation site and 6 kinds of N-glycans were identified by mass spectrometry, including 3 high mannose types and 3 complex types. Furthermore, the IgE-binding activity of wheat GSA is proved to be reduced by the removal of N-glycan, thermal treatment (temperatures > 80 °C), and strong acidic treatment (pH 3.0). These findings would provide a better understanding of the effects of N-glycosylation, thermal treatment, and acidic treatment on the molecular characteristics of GSA, and further provide new insights into the development of hypoallergenic wheat products.

Site-specific, covalent immobilization of PNGase F on magnetic particles mediated by microbial transglutaminase

In the workflow of global N-glycosylation analysis, endoglycosidase-mediated removal of glycans from glycoproteins is an essential and rate-limiting step. Peptide-N-glycosidase F (PNGase F) is the most appropriate and efficient endoglycosidase for the removal of N-glycans from glycoproteins prior to analysis. Due to the high demand for PNGase F in both basic and industrial research, convenient and efficient methods are urgently needed to generate PNGase F, preferably in the immobilized form to solid phases. However, there is no integrated approach to implement both efficient expression, and site-specific immobilization of PNGase F. Herein, efficient production of PNGase F with a glutamine tag in Escherichia coli and site-specific covalent immobilization of PNGase F with this special tag via microbial transglutaminase (MTG) is described. PNGase F was fused with a glutamine tag to facilitate the co-expression of proteins in the supernatant. The glutamine tag was covalently and site-specifically transformed to primary amine-containing magnetic particles, mediated by MTG, to immobilize PNGase F. Immobilized PNGase F could deglycosylate substrates with identical enzymatic performance to that of the soluble counterpart, and exhibit good reusability and thermal stability. Moreover, the immobilized PNGase F could also be applied to clinical samples, including serum and saliva.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV-Cell Interaction and Functional Activation of Endothelial Cells.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV–cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells’ interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton’s Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV–cell interactions and associated functions.

N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase

Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS enzymes are N-glycosylated, but the biological functions of their glycans have remained elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structural stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. We expressed the enzyme in Chinese hamster ovary Lec1 cells, which produce high-mannose type N-glycans similar to the TS N-glycosylation pattern in vivo. Our MALDI-TOF mass spectrometry data revealed that up to eight putative N-glycosylation sites were glycosylated. In addition, we determined that N-glycan removal via endoglycosidase Hf treatment of TconTS1 led to a decrease in substrate affinity relative to the untreated enzyme but had no impact on the conversion rate. Furthermore, we observed no changes in secondary structure elements of hypoglycosylated TconTS1 in CD experiments. Finally, our molecular dynamics simulations provided evidence for interactions between monosaccharide units of the highly flexible N-glycans and some conserved amino acids located at the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and enzyme–substrate complex stability. The here-observed modulation of catalytic activity via N-glycans represents a so-far-unknown structure–function relationship potentially inherent in several members of the TS enzyme family.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2.

ABSTRACTHuman angiotensin I-converting enzyme 2 (hACE2) is a type I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for the attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment, or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 was localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV or SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation does not play a role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell-to-cell fusion mediated by SARS-CoV S protein but not that mediated by SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which may be the result of low cell surface expression of the deglycosylated ACE2 receptor.IMPORTANCE Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction. In addition, we found that the removal of N-glycans of ACE2 impaired its ability to support an efficient transduction of SARS-CoV and SARS-CoV-2 S pseudotyped viruses. Our data suggest that the role of deglycosylation of ACE2 on reducing infection is likely due to a reduced expression of the viral receptor on the cell surface. These findings offer insight into the glycan structure and function of ACE2 and potentially suggest that future antiviral therapies against coronaviruses and other coronavirus-related illnesses involving inhibition of ACE2 recruitment to the cell membrane could be developed.

Determining The Role of Surface Glycans in The Pharmacokinetics of Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication and are thereby expected to be promising carriers for drug delivery. Understanding the factors that affect sEV pharmacokinetics is crucial for its application as a drug delivery carrier. In this study, the role of sEV surface glycans was investigated by evaluating the effects of enzymatic deglycosylation treatment on sEV pharmacokinetics. First, control glycoprotein fetuin was used to optimize the glycosidase treatment conditions. B16-BL6-derived sEVs labeled with fusion proteins comprising Gag protein and Gaussia luciferase (gLuc) (Gag-gLuc) were then treated with glycosidases, Peptide-N-Glycosidase F or O-glycosidase, which cleaves N- and O-glycans, respectively. Glycosidase-treated sEVs showed physicochemical characteristics comparable to those of the untreated sEVs. However, removal of N-glycans from B16-BL6 sEVs enhanced cellular uptake by the peritoneal macrophages, while the removal of O-glycans had minimal impact, as evaluated by flow cytometry. To determine the effect of surface glycans on the sEV pharmacokinetics, Gag-gLuc labeled B16-BL6 sEVs treated with or without glycosidases were then intravenously administered to mice. Glycosidase-treated sEVs showed almost identical clearance from the blood circulation as that of the untreated sEVs. These results suggest minimal impact of surface glycans on sEV pharmacokinetics, despites its effect on cellular uptake.

Non-productive binding of cellobiohydrolase i investigated by surface plasmon resonance spectroscopy

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers.Graphic abstract

Mouse IgG3 binding to macrophage-like cells is prevented by deglycosylation of the antibody or by Accutase treatment of the cells

The binding of mouse IgG3 to Fcγ receptors (FcγR) and the existence of a mouse IgG3-specific receptor have been discussed for 40 years. Recently, integrin beta-1 (ITGB1) was proposed to be a part of an IgG3 receptor involved in the phagocytosis of IgG3-coated pathogens. We investigated the interaction of mouse IgG3 with macrophage-like J774A.1 and P388D1 cells. The existence of an IgG3-specific receptor was verified using flow cytometry and a rosetting assay, in which erythrocytes clustered around the macrophage-like cells coated with an erythrocyte-specific IgG3. Our findings confirmed that receptors binding antigen-free IgG3 are present on J774A.1 and P388D1 cells. We demonstrated for the first time that the removal of N-glycans from IgG3 completely abolished its binding to the cells. Moreover, we discovered that the cells treated with Accutase did not bind IgG3, indicating that IgG3-specific receptors are substrates of this enzyme. The results of antibody-mediated blocking of putative IgG3 receptors suggested that apart from previously proposed ITGB1, FcγRII, FcγRIII, also additional, still unknown, receptor is involved in IgG3 binding. These findings indicate that there is a complex network of glycan-dependent interactions between mouse IgG3 and the surface of effector immune cells.

Automated and Faster Affinity Capture Method for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates.

Traditionally the biotransformation of antibody drug conjugates (ADCs) has been evaluated by affinity capture on streptavidin magnetic beads coated with a biotinylated capture reagent. To reduce the complexity of the analyte, the affinity captured ADCs are digested with enzymes ("on-bead" or after elution), and/or interchain disulfides are reduced to generate LC and HC fragments prior to mass spectrometry analysis. The "on-bead" enzymatic digestion with IdeS and PNGase F is not efficient and requires longer incubation times to achieve complete Fc and N-glycan removal. This results in a prolonged sample preparation time (7-18 h) and is not suitable for labile ADCs due to the possibility of assay-induced artifacts. To address these challenges, we developed an affinity capture method, where the ADCs are first captured onto streptavidin cartridges coated with a biotinylated generic capture reagent, followed by a 15 min "on-cartridge" digestion with IdeS or PNGase F. The ADCs are then eluted and directly analyzed by LC-HRMS. This method was successfully applied for the biotransformation assessment of site-specific ADCs with payload conjugated on the Fab or Fc. The reduced complexity of the analyte (Fc and N-glycan removal) combined with HRMS enabled sensitive and accurate identification of minor mass change catabolites and changes in the DAR distribution. This automated cartridge-based affinity capture method is fast with a total sample preparation time of less than 4 h (hands-on time of less than 1 h) and can be utilized for any human mAb/ADC independent of isotype (IgG1, IgG2, and IgG4).

Effect of deglycosylation on immunoreactivity and in vitro pepsin digestibility of major cashew (Anacardium occidentale L.) allergen, Ana o 1.

Major cashew allergen, Ana o 1, was purified in its native form from cashew seeds and subjected to enzymatic deglycosylation using PNGase F to assess the potential role of N-glycans in immunoreactivity. Western and dot blotting with pooled human plasma containing anticashew IgE revealed that deglycosylation increased IgE-binding of Ana o 1. Removal of N-glycans may have exposed previously masked Ana o 1 epitopes. Purified glycosylated and deglycosylated Ana o 1 were also subjected to in vitro pepsin digestion at pH 3.0 for 2 hr. Both glycosylated and deglycosylated Ana o 1 remained stable and reactive with IgE antibodies following digestion. PRACTICAL APPLICATION: Understanding the role of glycosylation in Ana o 1 immunoreactivity may provide insight into the potential development of hypoallergenic cashews/cashew products for sensitive individuals in the future.

Venom Profiling of the Insular Species Bothrops alcatraz: Characterization of Proteome, Glycoproteome, and N-Terminome Using Terminal Amine Isotopic Labeling of Substrates.

Bothrops alcatraz, a species endemic to Alcatrazes Islands, is regarded as critically endangered due to its small area of occurrence and the declining quality of its habitat. We recently reported the identification of N-glycans attached to toxins of Bothrops species, showing similar compositions in venoms of the B. jararaca complex (B. jararaca, B. insularis, and B. alcatraz). Here, we characterized B. alcatraz venom using electrophoretic, proteomic, and glycoproteomic approaches. Electrophoresis showed that B. alcatraz venom differs from B. jararaca and B. insularis; however, N-glycan removal revealed similarities between them, indicating that the occupation of N-glycosylation sites contributes to interspecies variability in the B. jararaca complex. Metalloproteinase was the major toxin class identified in the B. alcatraz venom proteome followed by serine proteinase and C-type lectin, and overall, the adult B. alcatraz venom resembles that of B. jararaca juvenile specimens. The comparative glycoproteomic analysis of B. alcatraz venom with B. jararaca and B. insularis indicated that there may be differences in the utilization of N-glycosylation motifs among their different toxin classes. Furthermore, we prospected for the first time the N-terminome of a snake venom using the terminal amine isotopic labeling of substrates (TAILS) approach and report the presence of ∼30% of N-termini corresponding to truncated toxin forms and ∼37% N-terminal sequences blocked by pyroglutamic acid in B. alcatraz venom. These findings underscore a low correlation between venom gland transcriptomes and proteomes and support the view that post-translational processes play a major role in shaping venom phenotypes.