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A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories., Graphical Abstract Highlights A broad-based interlaboratory study of the glycosylation of a reference antibody: NISTmAb. 103 reports were received from 76 diverse laboratories worldwide. Analysis involved two samples, the NISTmAb and an enzymatically modified sample, enabling within-lab separation of random and systematic errors using the “Youden two-sample” method. Consensus values were derived and similar performance across all experimental methods was noted., Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

African sleeping sickness is caused by Trypanosoma brucei, a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized the N-glycome of tsetse saliva glycoproteins. Tsetse salivary N-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that the N-glycan profiles of T. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type N-glycans. In overlay assays, these sugars were differentially recognized by the mannose receptor and DC-SIGN C-type lectins. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivary N-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host., Author summary In addition to helping the ingestion of a bloodmeal, the saliva of vector insects can modulate vertebrate immune responses. However, most research has focused on the salivary proteins, while the sugars (glycans) that modify them remain unexplored. Here we studied N-glycosylation, a common post-translational modification where sugar structures are attached to specific sites of a protein. Insect salivary N-glycans may affect how the saliva is recognized by the host, possibly playing a role during pathogen transmission. In this manuscript, we present the first detailed structural characterization of the salivary N-glycans in the tsetse fly Glossina morsitans, vector of African trypanosomiasis. We found that tsetse fly glycoproteins are mainly modified by simple N-glycans with short mannose modifications, which are recognised by mammalian C-type lectins (mannose receptor and DC-SIGN). Furthermore, we show that salivary glycoproteins bind to the surface of the trypanosomes that are transmitted to the vertebrate host; this opens up interesting questions as to the role of these glycoproteins in the successful establishment of infection by this parasite. Overall, our work represents a novel contribution towards the salivary N-glycome of an important insect vector, and towards the understanding of vector saliva and its complex effects in the vertebrate host.

Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy, which has been produced on several platforms. In this study, a novel single-chain anti-VEGF monoclonal antibody (scVEGFmAb) was produced in the goat mammary gland by adenoviral transduction. scVEGFmAb was purified by affinity chromatography. N-glycans were analyzed by exoglycosidase digestion and hydrophilic interaction ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The biological activity of scVEGFmAb was assessed by scratch and mouse aortic ring assays. scVEGFmAb was produced at 0.61 g/L in the goat milk, and its purification rendered 95 % purity. N-glycans attached to scVEGFmAb backbone were mainly neutral biantennary core fucosylated with Galβ1,4GlcNAc motif, and charged structures were capped with Neu5Ac and Neu5Gc. The chimeric molecule significantly prevented cell migration and suppressed microvessel sprouting. These results demonstrated for the first time the feasibility of producing an anti-VEGF therapeutic antibody in the milk of non-transgenic goats with the potential to counteract tumor angiogenesis.

During Leishmania transmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host’s immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile of N-glycans from the salivary glycoproteins of Lutzomyia longipalpis, vector of visceral leishmaniasis in the Americas. In silico predictions suggest half of Lu. longipalpis salivary proteins may be N-glycosylated. SDS-PAGE coupled to LC–MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity of N-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC–MS/MS, MALDI-TOF–MS, and exoglycosidase treatments. We found that the N-glycan composition of Lu. longipalpis saliva mostly consists of oligomannose sugars, with Man5GlcNAc2 being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.

African sleeping sickness is caused byTrypanosoma brucei,a parasite transmitted by the bite of a tsetse fly. Trypanosome infection induces a severe transcriptional downregulation of tsetse genes encoding for salivary proteins, which reduces its anti-hemostatic and anti-clotting properties. To better understand trypanosome transmission and the possible role of glycans in insect bloodfeeding, we characterized theN-glycome of tsetse saliva glycoproteins. Tsetse salivaryN-glycans were enzymatically released, tagged with either 2-aminobenzamide (2-AB) or procainamide, and analyzed by HILIC-UHPLC-FLR coupled online with positive-ion ESI-LC-MS/MS. We found that theN-glycan profiles ofT. brucei-infected and naïve tsetse salivary glycoproteins are almost identical, consisting mainly (>50%) of highly processed Man3GlcNAc2 in addition to several other paucimannose, high mannose, and few hybrid-type glycans. In overlay assays, these sugars were differentially recognized by the C-type lectins mannose receptor and DC-SIGN. We also show that salivary glycoproteins bind strongly to the surface of transmissible metacyclic trypanosomes. We suggest that although the repertoire of tsetse salivaryN-glycans does not change during a trypanosome infection, the interactions with mannosylated glycoproteins may influence parasite transmission into the vertebrate host.

DuringLeishmaniatransmission sand flies inoculate parasites and saliva into the skin of vertebrates. Saliva has anti-haemostatic and anti-inflammatory activities that evolved to facilitate bloodfeeding, but also modulate the host’s immune responses. Sand fly salivary proteins have been extensively studied, but the nature and biological roles of protein-linked glycans remain overlooked. Here, we characterised the profile ofN-glycans from the salivary glycoproteins ofLutzomyia longipalpis, vector of visceral leishmaniasis in the Americas.In silicopredictions suggest half ofLu. longipalpissalivary proteins may beN-glycosylated. SDS-PAGE coupled to LC-MS analysis of sand fly saliva, before and after enzymatic deglycosylation, revealed several candidate glycoproteins. To determine the diversity ofN-glycan structures in sand fly saliva, enzymatically released sugars were fluorescently tagged and analysed by HPLC, combined with highly sensitive LC-MS/MS, MALDI-TOF-MS, and exoglycosidase treatments. We found that theN-glycan composition ofLu. longipalpissaliva mostly consists of oligomannose sugars, with Man5GlcNAc2being the most abundant, and a few hybrid-type species. Interestingly, some glycans appear modified with a group of 144 Da, whose identity has yet to be confirmed. Our work presents the first detailed structural analysis of sand fly salivary glycans.

The study of protein N-glycosylation is essential in biological and biopharmaceutical research as N-glycans have been reported to regulate a wide range of physiological and pathological processes. Monitoring glycosylation in diagnosis, prognosis, as well as biopharmaceutical development and quality control are important research areas. A number of techniques for the analysis of protein N-glycosylation are currently available. Here we examine three methodologies routinely used for the release of N-glycans, in the effort to establish and standardize glycoproteomics technologies for quantitative glycan analysis from cultured cell lines. N-glycans from human gamma immunoglobulins (IgG), plasma and a pool of four cancer cell lines were released following three approaches and the performance of each method was evaluated.

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (FIT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for FIT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.

The study of protein O-glycosylation is receiving increasing attention in biological, medical, and biopharmaceutical research. Improved techniques are required to allow reproducible and quantitative analysis of O-glycans. An established approach for O-glycan analysis relies on their chemical release in high yield by hydrazinolysis, followed by fluorescent labeling at the reducing terminus and high-performance liquid chromatography (HPLC) profiling. However, an unwanted degradation known as "peeling" often compromises hydrazinolysis for O-glycan analysis. Here we addressed this problem using low-molarity solutions of ethylenediaminetetraacetic acid (EDTA) in hydrazine for O-glycan release. O-linked glycans from a range of different glycoproteins were analyzed, including bovine fetuin, bovine submaxillary gland mucin, and serum immunoglobulin A (IgA). The data for the O-glycans released by hydrazine with anhydrous EDTA or disodium salt dihydrate EDTA show high yields of the native O-glycans compared with the peeled product, resulting in a markedly increased robustness of the O-glycan profiling method. The presented method for O-glycan release demonstrates significant reduction in peeling and reduces the number of sample handling steps prior to release. (C) 2014 Elsevier Inc. All rights reserved.

Colorectal cancer (CRC) affects both women and men living in societies with a high sedentary lifestyle. Amongstthe phenotypic changes exhibited by tumor cells, a widerange of glycosylationshas been reported for colon cancer-derived cell lines and CRC tissues. These aberrant modifications affect different aspectsof glycosylation, including an increase in core fucosylation and GlcNAc branching on N-glycans, alteration of O-glycans, upregulated sialylation and O-GlcNAcylation. Although O-GlcNAcylation and complex glycosylations differ in many aspects, sparse evidences report on the interference of O-GlcNAcylation with complex glycosylation. Nevertheless, this relationship is still a matter of debate. Combining different approaches onthree human colon cell lines (HT29, HCT116 and CCD841CoN), we herein report that silencing O-GlcNAc transferase (OGT, the sole enzyme driving O-GlcNAcylation), only slightly affectsoverall N-and O-glycosylation patterns. Interestingly, silencingof OGT in HT29 cells upregulates E-cadherin (a major actor of epithelial-to-mesenchymal transition) and changes its glycosylation.On the other hand, OGT silencing perturbs biosynthesis ofglycosphingolipids resulting ina decreaseingangliosidesandan increase in globosides.Together, these results provide novel insights regarding the selective regulation of complex glycosylations by O-GlcNAcylation in colon cancer cells. 19;21-22

One of the most widely used methods for glycan analysis is fluorescent labeling of released glycans followed by hydrophilic interaction chromatography-(ultra-)high-performance liquid chromatography [HILIC-(U)HPLC]. Here, we compare the data obtained by (U)HPLC-fluorescence (FLR) coupled to electrospray ionization-mass spectrometry (ESI-MS) for procainamide and 2-aminobenzamide (2-AB)-labeled N-glycans released from human immunoglobulin G (IgG). Fluorescence profiles from procainamide show comparable chromatographic separation to those obtained for 2-AB but gave higher fluorescence intensity as well as significantly improved ESI efficiency (up to 30 times that of 2-AB). Thus, labeling with procainamide increases the ability to identify minor glycan species that may have significant biological activity.

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

The study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as biological fluid. Saliva is rich in glycoproteins which are secreted from the bloodstream or produced by salivary glands. Mucins, which are highly O-glycosylated proteins, are particularly abundant in human saliva. Their glycosylation is associated with blood group and secretor status, and represents a reservoir of potential disease biomarkers. This study aims to analyse and compare O-glycans released from whole human mouth saliva collected 3 times a day from a healthy individual over a 5 days period. O-linked glycans were released by hydrazinolysis, labelled with procainamide and analysed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FLR) coupled to electrospray ionization mass spectrometry (ESI-MS/MS). The sample preparation method showed excellent reproducibility and can therefore be used for biomarker discovery. Our data demonstrates that the O-glycosylation in human saliva changes significantly during the day. These changes may be related to changes in the salivary concentrations of specific proteins.

Immunoglobulin A (IgA) is a glycoprotein of which altered glycosylation has been associated with several pathologies. Conventional methods for IgA N- and O-glycosylation analysis are tedious, thus limiting such analyses to small sample sizes. Here we present a high-throughput strategy for the simultaneous analysis of serum-derived IgA1 N- and O-glycopeptides using matrix-assisted laser/desorption ionisation Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry (MS). Six non-fucosylated diantennary complex type glycoforms were detected on the Asn144-containing glycopeptide. Thirteen distinct glycoforms were identified for the Asn340-containing tailpiece glycopeptide, mainly of the diantennary complex type, and low amounts of triantennary glycoforms. Simultaneously with these N-glycopeptides, 53 compositional glycoforms of the hinge region O-glycopeptide were profiled in a single high resolution MALDI-FTICR spectrum. Since many pregnancy associated changes have been recognized for immunoglobulin G, we sought to demonstrate the clinical applicability of this method in a cohort of 29 pregnant women, from whom samples were collected at three time points during pregnancy and three time points after delivery. Pregnancy associated changes of N-glycan bisection were different for IgA1 as compared to IgG-Fc described earlier. We foresee further applications of the developed method for larger patient cohorts to study IgA N- and O-glycosylation changes in pathologies.

Tannerella forsythia, a Gram-negative member of the Bacteroidetes has evolved to harvest and utilize sialic acid. The most common sialic acid in humans is a mono-N-acetylated version termed Neu5Ac (5-N-acetyl-neuraminic acid). Many bacteria are known to access sialic acid using sialidase enzymes. However, in humans a high proportion of sialic acid contains a second acetyl group attached via an O-group, i.e. chiefly O-acetylated Neu5,9Ac2 or Neu5,4Ac2. This diacetylated sialic acid is not cleaved efficiently by many sialidases and in order to access diacetylated sialic acid, some organisms produce sialate-O-acetylesterases that catalyse the removal of the second acetyl group. In the present study, we performed bioinformatic and biochemical characterization of a putative sialate-O-acetylesterase from T. forsythia (NanS), which contains two putative SGNH-hydrolase domains related to sialate-O-acetylesterases from a range of organisms. Purification of recombinant NanS revealed an esterase that has activity against Neu5,9Ac2 and its glycolyl form Neu5Gc,9Ac. Importantly, the enzyme did not remove acetyl groups positioned at the 4-O position (Neu5,4Ac2). In addition NanS can act upon complex N-glycans released from a glycoprotein [erythropoietin (EPO)], bovine submaxillary mucin and oral epithelial cell-bound glycans. When incubated with its cognate sialidase, NanS increased sialic acid release from mucin and oral epithelial cell surfaces, implying that this esterase improves sialic acid harvesting for this pathogen and potentially other members of the oral microbiome. In summary, we have characterized a novel sialate-O-acetylesterase that contributes to the sialobiology of this important human pathogen and has potential applications in the analysis of sialic acid diacetylation of biologics in the pharmaceutical industry.

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets.

The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method.