In vivo modification of the goat mammary gland glycosylation pathway.

Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.

Follicle-Stimulating Hormone Glycobiology.

FSH glycosylation varies in two functionally important aspects: microheterogeneity, resulting from oligosaccharide structure variation, and macroheterogeneity, arising from partial FSHß subunit glycosylation. Although advances in mass spectrometry permit extensive characterization of FSH glycan populations, microheterogeneity remains difficult to illustrate, and comparisons between different studies are challenging because no standard format exists for rendering oligosaccharide structures. FSH microheterogeneity is illustrated using a consistent glycan diagram format to illustrate the large array of structures associated with one hormone. This is extended to commercially available recombinant FSH preparations, which exhibit greatly reduced microheterogeneity at three of four glycosylation sites. Macroheterogeneity is demonstrated by electrophoretic mobility shifts due to the absence of FSHß glycans that can be assessed by Western blotting of immunopurified FSH. Initially, macroheterogeneity was hoped to matter more than microheterogeneity. However, it now appears that both forms of carbohydrate heterogeneity have to be taken into consideration. FSH glycosylation can reduce its apparent affinity for its cognate receptor by delaying initial interaction with the receptor and limiting access to all of the available binding sites. This is followed by impaired cellular signaling responses that may be related to reduced receptor occupancy or biased signaling. To resolve these alternatives, well-characterized FSH glycoform preparations are necessary.

N-glycan microheterogeneity regulates interactions of plasma proteins.

Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein-protein, or protein-small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin-hemoglobin (Hp-Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp-Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein-drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.

Structural Studies of Fucosylated N-Glycans by Ion Mobility Mass Spectrometry and Collision-Induced Fragmentation of Negative Ions.

There is considerable potential for the use of ion mobility mass spectrometry in structural glycobiology due in large part to the gas-phase separation attributes not typically observed by orthogonal methods. Here, we evaluate the capability of traveling wave ion mobility combined with negative ion collision-induced dissociation to provide structural information on N-linked glycans containing multiple fucose residues forming the Lewisx and Lewisy epitopes. These epitopes are involved in processes such as cell-cell recognition and are important as cancer biomarkers. Specific information that could be obtained from the intact N-glycans by negative ion CID included the general topology of the glycan such as the presence or absence of a bisecting GlcNAc residue and the branching pattern of the triantennary glycans. Information on the location of the fucose residues was also readily obtainable from ions specific to each antenna. Some isobaric fragment ions produced prior to ion mobility could subsequently be separated and, in some cases, provided additional valuable structural information that was missing from the CID spectra alone. Graphical abstract ᅟ.

Identification of Lewis and Blood Group Carbohydrate Epitopes by Ion Mobility-Tandem-Mass Spectrometry Fingerprinting.

Glycans have several elements that contribute to their structural complexity, involving a range of monosaccharide building blocks, configuration of linkages between residues and various degrees of branching on a given structure. Their analysis remains challenging and resolving minor isomeric variants can be difficult, in particular terminal fucosylated Lewis and blood group antigens present on N- and O-glycans. Accurately characterizing these isomeric structures by current techniques is not straightforward and typically requires a combination of methods and/or sample derivatization. Yet the ability to monitor the occurrence of these epitopes is important as structural changes are associated with several human diseases. The use of ion mobility-mass spectrometry (IM-MS), which separates ions in the gas phase based on their size, charge and shape, offers a new potential tool for glycan analysis and recent reports have demonstrated its potential for glycomics. Here we show that Lewis and blood group isomers, which have identical fragmentation spectra, exhibit very distinctive IM drift times and collision cross sections (CCS). We show that IM-MS/MS analysis can rapidly and accurately differentiate epitopes from parotid gland N-glycans and milk oligosaccharides based on fucosylated fragment ions with characteristic CCSs.

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity.

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.

Production, purification, and characterization of recombinant hFSH glycoforms for functional studies.

Previously, our laboratory demonstrated the existence of a ß-subunit glycosylation-deficient human FSH glycoform, hFSH(21). A third variant, hFSH(18), has recently been detected in FSH glycoforms isolated from purified pituitary hLH preparations. Human FSH(21) abundance in individual female pituitaries progressively decreased with increasing age. Hypo-glycosylated glycoform preparations are significantly more active than fully-glycosylated hFSH preparations. The purpose of this study was to produce, purify and chemically characterize both glycoform variants expressed by a mammalian cell line. Recombinant hFSH was expressed in a stable GH3 cell line and isolated from serum-free cell culture medium by sequential, hydrophobic and immunoaffinity chromatography. FSH glycoform fractions were separated by Superdex 75 gel-filtration. Western blot analysis revealed the presence of both hFSH(18) and hFSH(21) glycoforms in the low molecular weight fraction, however, their electrophoretic mobilities differed from those associated with the corresponding pituitary hFSH variants. Edman degradation of FSH(21/18)-derived ß-subunit before and after peptide-N-glycanase F digestion confirmed that it possessed a mixture of both mono-glycosylated FSHß subunits, as both Asn(7) and Asn(24) were partially glycosylated. FSH receptor-binding assays confirmed our previous observations that hFSH(21/18) exhibits greater receptor-binding affinity and occupies more FSH binding sites when compared to fully-glycosylated hFSH(24). Thus, the age-related reduction in hypo-glycosylated hFSH significantly reduces circulating levels of FSH biological activity that may further compromise reproductive function. Taken together, the ability to express and isolate recombinant hFSH glycoforms opens the way to study functional differences between them both in vivo and in vitro.

Structural plasticity of the Semliki Forest virus glycome upon interspecies transmission.

Cross-species viral transmission subjects parent and progeny alphaviruses to differential post-translational processing of viral envelope glycoproteins. Alphavirus biogenesis has been extensively studied, and the Semliki Forest virus E1 and E2 glycoproteins have been shown to exhibit differing degrees of processing of N-linked glycans. However the composition of these glycans, including that arising from different host cells, has not been determined. Here we determined the chemical composition of the glycans from the prototypic alphavirus, Semliki Forest virus, propagated in both arthropod and rodent cell lines, by using ion-mobility mass spectrometry and collision-induced dissociation analysis. We observe that both the membrane-proximal E1 fusion glycoprotein and the protruding E2 attachment glycoprotein display heterogeneous glycosylation that contains N-linked glycans exhibiting both limited and extensive processing. However, E1 contained predominantly highly processed glycans dependent on the host cell, with rodent and mosquito-derived E1 exhibiting complex-type and paucimannose-type glycosylation, respectively. In contrast, the protruding E2 attachment glycoprotein primarily contained conserved under-processed oligomannose-type structures when produced in both rodent and mosquito cell lines. It is likely that glycan processing of E2 is structurally restricted by steric-hindrance imposed by local viral protein structure. This contrasts E1, which presents glycans characteristic of the host cell and is accessible to enzymes. We integrated our findings with previous cryo-electron microscopy and crystallographic analyses to produce a detailed model of the glycosylated mature virion surface. Taken together, these data reveal the degree to which virally encoded protein structure and cellular processing enzymes shape the virion glycome during interspecies transmission of Semliki Forest virus.

Hypo-glycosylated human follicle-stimulating hormone (hFSH(21/18)) is much more active in vitro than fully-glycosylated hFSH (hFSH(24)).

Hypo-glycosylated hFSH(21/18) (possesses FSHß(21) and FSHß(18)bands) was isolated from hLH preparations by immunoaffinity chromatography followed by gel filtration. Fully-glycosylated hFSH(24) was prepared by combining the fully-glycosylated FSHß(24) variant with hCGα and isolating the heterodimer. The hFSH(21/18) glycoform preparation was significantly smaller than the hFSH(24) preparation and possessed 60% oligomannose glycans, which is unusual for hFSH. Hypo-glycosylated hFSH(21/18) was 9- to 26-fold more active than fully-glycosylated hFSH(24) in FSH radioligand assays. Significantly greater binding of (125)I-hFSH(21/18) tracer than hFSH(24) tracer was observed in all competitive binding assays. In addition, higher binding of hFSH(21/18) was noted in association and saturation binding assays, in which twice as much hFSH(21/18) was bound as hFSH(24). This suggests that more ligand binding sites are available to hFSH(21/18) in FSHR than to hFSH(24). Hypo-glycosylated hFSH(21/18) also bound rat FSHRs more rapidly, exhibiting almost no lag in binding, whereas hFSH(24) specific binding proceeded very slowly for almost the first hour of incubation.

Increase in sialylation and branching in the mouse serum N-glycome correlates with inflammation and ovarian tumour progression.

Ovarian cancer is the most lethal gynaecological cancer and is often diagnosed in late stage, often as the result of the unavailability of sufficiently sensitive biomarkers for early detection, tumour progression and tumour-associated inflammation. Glycosylation is the most common posttranslational modification of proteins; it is altered in cancer and therefore is a potential source of biomarkers. We investigated the quantitative and qualitative effects of anti-inflammatory (acetylsalicylic acid) and pro-inflammatory (thioglycolate and chlorite-oxidized oxyamylose) drugs on glycosylation in mouse cancer serum. A significant increase in sialylation and branching of glycans in mice treated with an inflammation-inducing compound was observed. Moreover, the increases in sialylation correlated with increased tumour sizes. Increases in sialylation and branching were consistent with increased expression of sialyltransferases and the branching enzyme MGAT5. Because the sialyltransferases are highly conserved among species, the described changes in the ovarian cancer mouse model are relevant to humans and serum N-glycome analysis for monitoring disease treatment and progression might be a useful biomarker.

N-linked glycan structures of the human Fcγ receptors produced in NS0 cells.

Immune recognition of nonself is coordinated through complex mechanisms involving both innate and adaptive responses. Circulating antibodies communicate with effector cells of the innate immune system through surface receptors known as Fcγ receptors (FcγRs). The FcγRs are single-pass transmembrane glycoproteins responsible for regulating innate effector responses toward antigenic material. Although immunoglobulin G (IgG) antibodies bind to a range of receptors, including complement receptors and C-type lectins, we have focused on the Fcγ receptors. A total of five functional FcγRs are broadly classified into three families (FcγRI, FcγRII, and FcγRIII) and together aid in controlling both inflammatory and anti-inflammatory responses of the innate immune system. Due to the continued success of monoclonal antibodies in treating cancer and autoimmune disorders, research is typically directed toward improving the interaction of antibodies with the FcγRs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies using recombinant forms of the FcγRs are often used to quantitate changes in binding affinity, a key indicator of a likely biological outcome. However, analysis of the FcγRs themselves is imperative as recombinant FcγRs differ greatly from those observed in humans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG-FcγR interaction. Here, we present data for the N-linked glycans present on FcγRs produced in NS0 cells, namely, FcγRIa, FcγRIIa, FcγRIIB, FcγRIIIa, and FcγRIIIb. Importantly, these FcγRs demonstrate typical murine glycosylation in the form of α-galactose epitopes, N-glycolylneuraminic acid, and other key glycan properties that are generally expressed in murine cell lines and therefore are not typically observed in humans.

Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development.

Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (∼12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.

5-AZA-2'-deoxycytidine induced demethylation influences N-glycosylation of secreted glycoproteins in ovarian cancer.

Glycosylation is the most common posttranslational modification of proteins and is highly reflective of changes in the environment of a cell. Epigenetic modifications to the genome are stably transmitted to daughter cells without the requirement for genetic sequence alterations. Aberrant regulation of both epigenetic programming and glycosylation patterning are integral aspects of carcinogenesis. The objective of this study was to determine the interplay between these two complex cellular processes. We demonstrate that global DNA methylation changes in ovarian cancer epithelial cells (OVCAR3) resulted in significant alterations in the glycosylation of secreted glycoproteins. These changes included a reduction in core fucosylation, increased branching and increased sialylation. We further show that the change in core fucose levels was mirrored by altered expression of GMDS and FX, key enzymes in fucose biosynthesis. Alterations in the expression of key glycosyltransferase enzymes such as MGAT5 reflect the changes seen in the branching and sialylation of secreted glycans. Overall, our results highlight that modifications to the epigenetic machinery have a profound effect on the glycan structures generated by cells, which may be a key step in understanding metastasis and drug resistance during cancer progression.

Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines.

Recombinant human erythropoietin has been used to treat anemia associated with chronic renal disease. This paper provides a comprehensive comparative analysis of Dynepo and three other commercial erythropoiesis stimulating agents, Eprex, NeoRecormon and Aranesp. We found significant differences in the type, levels and amount of O-acetylation of sialic acids. Sialic acids and O-acetylation present provide protection from clearance from circulation. Aranesp had up to six O-acetyl groups attached to the sialic acids. Eprex and NeoRecormon had only minor amounts of O-acetylation while Dynepo had none. Dynepo had no Neu5Gc, which is potentially immunogenic for humans. Dynepo contained the least amount of disialylated and Aranesp the highest amount of tetrasialylated glycans. NeoRecormon and Eprex contained more trisialylated, but less tetrasialylated glycans than Dynepo and Aranesp. Dynepo had the highest amount of tetraantennary glycans and the lowest amounts of triantennary glycans with a ß1-6-GlcNAc linkage. All the samples contained poly-N-acetyl-lactosamine repeats with Dynepo having the least. The major N-acetyl-lactosamine extensions in Dynepo and Aranesp were on biantennary glycans, whereas in NeoRecomon and Eprex they were on triantennary glycans. The sLe(x) epitope was only detected in Dynepo.

Glycosylation of liver acute-phase proteins in pancreatic cancer and chronic pancreatitis.

PURPOSE: Glycosylation of acute-phase proteins (APP), which is partially regulated by cytokines, may be distinct in disease and provide useful tumour markers. Thus, we have examined the glycosylation of major serum APP in pancreatic cancer (PaC), chronic pancreatitis (CP) and control patients. EXPERIMENTAL DESIGN: Using a specific anti-sialyl Lewis X antibody and N-glycan sequencing, we have determined glycosylation changes on α-1-acid glycoprotein (AGP), haptoglobin (HPT), fetuin (FET), α-1-antitrypsin (AT) and transferrin (TRF). RESULTS: Increased levels of sialyl Lewis X (SLe(x) ) were detected on AGP in advanced PaC and CP and on HPT, FET, AT and TRF in CP. An increase in N-glycan branching was detected on AGP and HPT in the advanced stage of PaC and CP and on FET and TRF in the CP. A core fucosylated structure was increased on AGP and HPT only in the advanced PaC patients. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in APP SLe(x) and branching are probably associated with an inflammatory response because they were detected in both advanced PaC and CP patients and these conditions give rise to inflammation. On the contrary, the increase in APP core fucosylation could be cancer associated and the presence of this glycoform may give an advantage to the tumour.

Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens.

The envelope spike of HIV is one of the most highly N-glycosylated structures found in nature. However, despite extensive research revealing essential functional roles in infection and immune evasion, the chemical structures of the glycans on the native viral envelope glycoprotein gp120--as opposed to recombinantly generated gp120--have not been described. Here, we report on the identity of the N-linked glycans from primary isolates of HIV-1 (clades A, B, and C) and from the simian immunodeficiency virus. MS analysis reveals a remarkably simple and highly conserved virus-specific glycan profile almost entirely devoid of medial Golgi-mediated processing. In stark contrast to recombinant gp120, which shows extensive exposure to cellular glycosylation enzymes (>70% complex type glycans), the native envelope shows barely detectable processing beyond the biosynthetic intermediate Man5GlcNAc2 (<2% complex type glycans). This oligomannose (Man5-9GlcNAc2) profile is conserved across primary isolates and geographically divergent clades but is not reflected in the current generation of gp120 antigens used for vaccine trials. In the context of vaccine design, we also note that Manalpha1-->2Man-terminating glycans (Man6-9GlcNAc2) of the type recognized by the broadly neutralizing anti-HIV antibody 2G12 are 3-fold more abundant on the native envelope than on the recombinant monomer and are also found on isolates not neutralized by 2G12. The Manalpha1-->2Man residues of gp120 therefore provide a vaccine target that is physically larger and antigenically more conserved than the 2G12 epitope itself. This study revises and extends our understanding of the glycan shield of HIV with implications for AIDS vaccine design.

The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat.

Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man(8)GlcNAc(2) isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.

Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly.

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.

Identification of N-linked carbohydrates from severe acute respiratory syndrome (SARS) spike glycoprotein.

N-glycans were released from the SARS coronavirus (SARS-CoV) spike glycoprotein produced in Vero E6 cells and their structures were determined by a combination of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, negative ion electrospray collision-induced dissociation time-of-flight mass spectrometry and normal-phase high-performance liquid chromatography with exoglycosidase digestion. Major glycans were high-mannose (Man(5-9)GlcNAc(2)), hybrid and bi-, tri- and tetra-antennary complex with and without bisecting GlcNAc and core fucose. Complex glycans with fewer than the full complement of galactose residues were present and sialylation was negligible. Treatment with the glucosidase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) inhibited N-glycan processing as evidenced by the appearance of glycans of composition Glc(3)Man(7-9)GlcNAc(2). However, some complex glycans remained suggesting the presence of an alpha-endomannosidase. Our data in tissue culture indicate that inhibition of N-glycan processing may be considered as a therapeutic strategy against SARS CoV infections.

Identification of N-glycans from Ebola virus glycoproteins by matrix-assisted laser desorption/ionisation time-of-flight and negative ion electrospray tandem mass spectrometry.

The larger fragment of the transmembrane glycoprotein (GP1) and the soluble glycoprotein (sGP) of Ebola virus were expressed in human embryonic kidney cells and the secreted products were purified from the supernatant for carbohydrate analysis. The N-glycans were released with PNGase F from within sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) gels. Identification of the glycans was made with normal-phase high-performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionisation mass spectrometry, negative ion electrospray ionisation fragmentation mass spectrometry and exoglycosidase digestion. Most glycans were complex bi-, tri- and tetra-antennary compounds with reduced amounts of galactose. No bisected compounds were detected. Triantennary glycans were branched on the 6-antenna; fucose was attached to the core GlcNAc residue. Sialylated glycans were present on sGP but were largely absent from GP1, the larger fragment of the transmembrane glycoprotein. Consistent with this was the generally higher level of processing of carbohydrates found on sGP as evidenced by a higher percentage of galactose and lower levels of high-mannose glycans than were found on GP1. These results confirm and expand previous findings on partial characterisation of the Ebola virus transmembrane glycoprotein. They represent the first detailed data on carbohydrate structures of the Ebola virus sGP.