Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry

The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.     

Myrosinases TGG1 and TGG2 from Arabidopsis thaliana contain exclusively oligomannosidic N-glycans

In all eukaryotes N-glycosylation is the most prevalent protein modification of secretory and membrane proteins. Although the N-glycosylation capacity and the individual steps of the N-glycan processing pathway have been well studied in the model plant Arabidopsis thaliana, little attention has been paid to the characterization of the glycosylation status of individual proteins. We report here the structural analysis of all N-glycans present on the endogenous thioglucoside glucohydrolases (myrosinases) TGG1 and TGG2 from A. thaliana. All nine glycosylation sites of TGG1 and all four glycosylation sites of TGG2 are occupied by oligomannosidic structures with Man₅GlcNAc₂ as the major glycoform. Analysis of the oligomannosidic isomers from wild-type plants and mannose trimming deficient mutants by liquid chromatography with porous graphitic carbon and mass spectrometry revealed that the N-glycans from both myrosinases are processed by Golgi-located α-mannosidases.     

Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.     

Changes in antigen-specific IgG1 Fc N-glycosylation upon influenza and tetanus vaccination

Antibody effector functions have been shown to be influenced by the structure of the Fc N-glycans. Here we studied the changes in plasma or serum IgG Fc N-glycosylation upon vaccination of 10 Caucasian adults and 10 African children. Serum/plasma IgG was purified by affinity chromatography prior to and at two time points after vaccination. Fc N-glycosylation profiles of individual IgG subclasses were determined for both total IgG and affinity-purified anti-vaccine IgG using a recently developed fast nanoliquid chromatography-electrospray ionization MS (LC-ESI-MS) method. While vaccination had no effect on the glycosylation of total IgG, anti-vaccine IgG showed increased levels of galactosylation and sialylation upon active immunization. Interestingly, the number of sialic acids per galactose increased during the vaccination time course, suggesting a distinct regulation of galactosylation and sialylation. In addition we observed a decrease in the level of IgG1 bisecting N-acetylglucosamine whereas no significant changes were observed for the level of fucosylation. Our data indicate that dependent on the vaccination time point the infectious agent will encounter IgGs with different glycosylation profiles, which are expected to influence the antibody effector functions relevant in immunity.     

Profiling of isomer-specific IgG N-glycosylation in cohort of Chinese colorectal cancer patients

BackgroudGiven the increasing morbidity and mortality of colorectal cancer (CRC), it is urgent to develop a noninvasive screening strategy for early diagnosis of CRC. Altered IgG glycosylation is associated with CRC progression, whereas the association of IgG isomeric glycosylation with CRC were not investigated.MethodsMethylamidation of IgG N-glycans was conducted prior to PGC-based nanoLC-ESI-MS/MS analysis. Data processing was operated by a self-developed application based on MATLAB solution. Statistical analysis including K–S test, t-test, ROC curve and OPLS-DA were successively performed. Additionally, an independent set was utilized to validate the results.ResultsTotal 28 IgG glycans and 79 compositional isomers were identified, over half of which are firstly identified so far. Statistical analysis showed that CRC associates with increase in IgG agalactosylation, decrease in IgG sialylation and fucosylation of sialylated glycans. Additionally, it was found that three compositional isomers (H3N4F1-a, H3N4F1-b and H4N3S1F1-e) could distinguish CRC and early stages from controls with an accurate area under the receiver operating characteristic curve. Significantly, these results were validated in an independent set by multivariate statistical analysis.ConclusionsThis is the first comprehensively profiling of isomer-specific IgG N-glycosylation, which could differentiate normal controls from colorectal disease patients. The candidate IgG glyco-biomarkers provide important screening indicators for early diagnosis of CRC.General significanceColorectal cancer progression is strongly associated with isomer-specific IgG N-glycosylation.     

The non-classical N-glycan processing pathway of bovine brain ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is brain specific and not due to mannose-6-phosphorylation

Ecto-nucleotide phosphodiesterase/pyrophosphatase 6 (eNPP6) is a glycosylphosphatidylinositol (GPI)-anchored alkaline lysophospholipase C which is predominantly expressed in brain myelin and kidney. Due to shedding of the GPI-anchor eNPP6 occurs also as a soluble isoform (s-eNPP6). eNPP 6 consists of two identical monomers of 55 kDa joined by a disulfide bridge, and possesses four N-glycans in each monomer. In brain s-eNPP6 the N-glycans are mainly hybrid and high mannose type structures, reminiscent of processed mannose-6-phosphorylated glycans. Here we completed characterization of the site-specific glycan structures of bovine brain s-eNPP6, and determined the endo H-sensitivity glycan profiles of s-eNPP6 from bovine liver and kidney. Whereas in brain s-eNPP6 all of the N-glycans were endo H-sensitive, in liver and kidney only one of the glycosylation sites was occupied by an endo H-sensitive glycan, likely N406, which is located within the cleft formed by the dimer interface. Thus, the non-classical glycan processing pathway of brain eNPP 6 is not due to mannose-6-phosphorylation, suggesting that there is an alternative Golgi glycan-processing pathway of eNPP6 in brain. The resulting brain-specific expression of accessible hybrid and oligomannosidic glycans may be physiologically important within the cell–cell communication system of the brain.     

Glycomics and proteomics analyses of mouse uterine luminal fluid revealed a predominance of Lewis Y and X epitopes on specific protein carriers

Sperm motility and maturation are known to be affected by a host of factors encountered en route in both male and female genital tracts prior to fertilization. Using a concerted proteomics and glycomics approach with advanced mass spectrometry-based glycan sequencing capability, we show in this work that 24p3, an abundant mouse uterine luminal fluid (ULF) glycoprotein also called lipocalin 2 (Lcn2), is highly fucosylated in the context of carrying multiple Lewis X and Y epitopes on complex type N-glycans at its single glycosylation site. The predominance of Lewis X/Y along with Neu5Acalpha2-6 sialylation was found to be a salient feature of the ULF glycome, and several other protein carriers were additionally identified including the highly abundant lactotransferrin, which is N-glycosylated at two sites, both with a similar range of highly fucosylated N-glycans. A comparative glycomics analysis of the male genital tract fluids revealed that there is a gradient of glycomic complexity from the cauda to caput regions of the epididymis, varying from high mannose to sialylated complex type N-glycans but mostly devoid of fucosylation. The seminal vesicle fluid glycome, on the other hand, carries equally abundant multimeric Lewis X structures but is distinctively lacking in additional fucosylation of the terminal galactose to give the Lewis Y epitope typifying the glycome of female ULF. One-dimensional shotgun proteomics analysis identified over 40 proteins in the latter, many of which are reported for the first time, and a majority are notably involved in immune defense and antigen processing. Further sperm binding and motility assays suggest that the Lewis X/Y epitopes do contribute to the sperm motility-enhancing activity of 24p3, whereas lactotransferrin is largely inactive in this context despite being similarly glycosylated. These findings underline the importance of glycoproteomics in delineating both the specific glycan structures and their carriers in assigning glycobiological functions.     

Rapid high yield production of different glycoforms of Ebola virus monoclonal antibody

Background

Fc-glycosylation of monoclonal antibodies (mAbs) has profound implications on the Fc-mediated effector functions. Alteration of this glycosylation may affect the efficiency of an antibody. However, difficulties in the production of mAbs with homogeneous N-glycosylation profiles in sufficient amounts hamper investigations of the potential biological impact of different glycan residues.

Methodology/Principal Findings

Here we set out to evaluate a transient plant viral based production system for the rapid generation of different glycoforms of a monoclonal antibody. Ebola virus mAb h-13F6 was generated using magnICON expression system in Nicotiana benthamiana, a plant species developed for commercial scale production of therapeutic proteins. h-13F6 was co-expressed with a series of modified mammalian enzymes involved in the processing of complex N-glycans. Using wild type (WT) plants and the glycosylation mutant ΔXTFT that synthesizes human like biantennary N-glycans with terminal N-acetylglucosamine on each branch (GnGn structures) as expression hosts we demonstrate the generation of h-13F6 complex N-glycans with (i) bisected structures, (ii) core α1,6 fucosylation and (iii) β1,4 galactosylated oligosaccharides. In addition we emphasize the significance of precise sub Golgi localization of enzymes for engineering of IgG Fc-glycosylation.

Conclusion

The method described here allows the efficient generation of a series of different human-like glycoforms at large homogeneity of virtually any antibody within one week after cDNA delivery to plants. This accelerates follow up functional studies and thus may contribute to study the biological role of N-glycan residues on Fcs and maximizing the clinical efficacy of therapeutic antibodies.     

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Background

Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses.

Results

The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Le^x and H type 2 antennae in sialylated complex-type N-glycans.

Conclusion

The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.     

Identification and quantification of N-linked oligosaccharides released from glycoproteins: an inter-laboratory study

As characterization of glycosylation is required for the licensing of recombinant glycoprotein therapeutics, technique comparability must be assessed. Eleven UK laboratories (seven industrial, two regulatory or government, two academic) participated in an inter-laboratory study to analyze N-glycans present in four mixtures prepared by PNGase F cleavage of commercial glycoproteins: human alpha1-acid glycoprotein (H alpha1), bovine alpha1-acid glycoprotein (B alpha1), bovine pancreatic ribonuclease B (RNaseB), and human serum immunoglobulin G (hIgG). Participants applied their routine glycan mapping methodology using predominantly chromatography and mass spectrometry to identify and quantify components. Data interpretation focused on the relative amounts of different glycan structures present, the degree of sialylation, antennary and the galactosylation profiles, fucosylation and bisecting GlcNAc content, and the number of glycan components identified. All laboratories found high levels of sialylation for H alpha1 and B alpha1 (Z-numbers 271 +/- 24 and 224 +/- 18, respectively), but varying ratios of di-, tri-, and tetra-antennary chains. The Z-score for hIgG glycans had high variability as values obtained from mass spectrometric and chromatographic methods clustered separately. The proportion of the major penta-mannosyl chain from RNaseB was between 29 and 62%. Proportions of fucosylated and bisected GlcNAc chains from hIgG were between 58 and 96% and 9 and 23%, respectively. Mass spectrometric approaches consistently identified more glycan species, especially when both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) were present. These data highlight the need for well-characterized reference standards to support method validation and regulatory guidance on selection of approaches. Pharmacopoeial specifications must acknowledge method variability.     

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.     

N-glycans modulate Kv1.5 gating but have no effect on Kv1.4 gating

Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K⁺ (K_v) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family K_v channel isoform, K_v1.5, but have no effect on gating of a second Shaker isoform, K_v1.4. Each isoform contains one potential N-glycosylation site located along the S1-S2 linker; immunoblot analyses verified that K_v1.4 and K_v1.5 were N-glycosylated. The conductance-voltage (G-V) relationships and channel activation rates for two glycosylation-site deficient K_v1.5 mutants, K_v1.5^N290Q and K_v1.5^S292A, and for wild-type K_v1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing ∼ 18 mV compared to wild-type K_v1.5 activation. External divalent cation screening experiments suggested that K_v1.5 sialic acids contribute to an external surface potential that modulates K_v1.5 activation. Channel availability was unaffected by changes in K_v1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate K_v1.5 activation. The sialic acids fully account for effects of N-glycans on K_v1.5 gating. Conversely, K_v1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for K_v1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous K_v1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated.     

Comparison of the Fc glycosylation of fetal and maternal immunoglobulin G

Human immunoglobulin G (IgG) molecules are composed of two Fab portions and one Fc portion. The glycans attached to the Fc portions of IgG are known to modulate its biological activity as they influence interaction with both complement and various cellular Fc receptors. IgG glycosylation changes significantly with pregnancy, showing a vast increase in galactosylation and sialylation and a concomitant decrease in the incidence of bisecting GlcNAc. Maternal IgGs are actively transported to the fetus by the neonatal Fc receptor (FcRn) expressed in syncytiotrophoblasts in the placenta, providing the fetus and newborn with immunological protection. Two earlier reports described significant differences in total glycosylation between fetal and maternal IgG, suggesting a possible glycosylation-selective transport via the placenta. These results might suggest an alternative maternal transport pathway, since FcRn binding to IgG does not depend on Fc-glycosylation. These early studies were performed by releasing N-glycans from total IgG. Here, we chose for an alternative approach analyzing IgG Fc glycosylation at the glycopeptide level in an Fc-specific manner, providing glycosylation profiles for IgG1 and IgG4 as well as combined Fc glycosylation profiles of IgG2 and 3. The analysis of ten pairs of fetal and maternal IgG samples revealed largely comparable Fc glycosylation for all the analyzed subclasses. Average levels of galactosylation, sialylation, bisecting GlcNAc and fucosylation were very similar for the fetal and maternal IgGs. Our data suggest that the placental IgG transport is not Fc glycosylation selective.     

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.     

Insect cells as hosts for the expression of recombinant glycoproteins

Baculovirus-mediated expression in insect cells has become well-established for the production of recombinant glycoproteins. Its frequent use arises from the relative ease and speed with which a heterologous protein can be expressed on the laboratory scale and the high chance of obtaining a biologically active protein. In addition to Spodoptera frugiperda Sf9 cells, which are probably the most widely used insect cell line, other mainly lepidopteran cell lines are exploited for protein expression. Recombinant baculovirus is the usual vector for the expression of foreign genes but stable transfection of - especially dipteran - insect cells presents an interesting alternative. Insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. For large scale culture, conditions have been developed which meet the special requirements of insect cells.With regard to protein folding and post-translational processing, insect cells are second only to mammalian cell lines. Evidence is presented that many processing events known in mammalian systems do also occur in insects. In this review, emphasis is laid, however, on protein glycosylation, particularly N-glycosylation, which in insects differs in many respects from that in mammals. For instance, truncated oligosaccharides containing just three or even only two mannose residues and sometimes fucose have been found on expressed proteins.These small structures can be explained by post-synthetic trimming reactions. Indeed, cell lines having a low level of N-acetyl--glucosaminidase, e.g. Estigmene acrea cells, produce N-glycans with non-reducing terminal N-acetylglucosamine residues. The Trichoplusia ni cell line TN-5B1-4 was even found to produce small amounts of galactose terminated N-glycans. However, there appears to be no significant sialylation of N-glycans in insect cells. Insect cells expressed glycoproteins may, though, be 1,3-fucosylated on the reducing-terminal GlcNAc residue. This type of fucosylation renders the N-glycans on one hand resistant to hydrolysis with PNGase F and on the other immunogenic. Even in the absence of 1,3-fucosylation, the truncated N-glycans of glycoproteins produced in insect cells constitute a barrier to their use as therapeutics. Attempts and strategies to mammalianise the N-glycosylation capacity of insect cells are discussed.     

Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures

As we pursue the means to improve yields to meet growing therapy demands, it is important to examine the impact of process control on glycosylation patterns to ensure product efficacy and consistency. In this study, we describe a dynamic on-line fed-batch strategy based on low glutamine/glucose concentrations and its impact on cellular metabolism and, more importantly, the productivity and N-glycosylation quality of a model recombinant glycoprotein, interferon gamma (IFN-gamma). We found that low glutamine fed-batch strategy enabled up to 10-fold improvement in IFN-gamma yields, which can be attributed to reduced specific productivity of ammonia and lactate. Furthermore, the low glutamine concentration (0.3 mM) used in this fed-batch strategy could maintain both the N-glycosylation macro- and microheterogeneity of IFN-gamma. However, very low glutamine (<0.1 mM) or glucose (<0.70 mM) concentrations can lead to decreased sialylation and increased presence of minor glycan species consisting of hybrid and high-mannose types. This shows that glycan chain extension and sialylation can be affected by nutrient limitation. In addition to nutrient limitation, we also found that N-glycosylation quality can be detrimentally affected by low culture viability. IFN-gamma purified at low culture viability had both lower sialylation as well as glycans of lower molecular masses, which can be attributed to extensive degradation by intracellular glycosidases released by cytolysis. Therefore, in order to maintain good N-glycosylation quality, there is a need to consider both culture viability and nutrient control setpoint in a nutrient-limiting fed-batch culture strategy. A greater understanding of these major factors that affect N-glycosylation quality would surely facilitate future development of effective process controls.     

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non–small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.     

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1–4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.     

Glycans in sera of amyotrophic lateral sclerosis patients and their role in killing neuronal cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by degeneration of upper and lower motor neurons. To date, glycosylation patterns of glycoproteins in fluids of ALS patients have not been described. Moreover, the aberrant glycosylation related to the pathogenesis of other neurodegenerative diseases encouraged us to explore the glycome of ALS patient sera. We found high levels of sialylated glycans and low levels of core fucosylated glycans in serum-derived N-glycans of patients with ALS, compared to healthy volunteer sera. Based on these results, we analyzed the IgG Fc N(297)-glycans, as IgG are major serum glycoproteins affected by sialylation or core fucosylation and are found in the motor cortex of ALS patients. The analyses revealed a distinct glycan, A2BG2, in IgG derived from ALS patient sera (ALS-IgG). This glycan increases the affinity of IgG to CD16 on effector cells, consequently enhancing Antibody-Dependent Cellular Cytotoxicity (ADCC). Therefore, we explore whether the Fc-N(297)-glycans of IgG may be involved in ALS disease. Immunostaining of brain and spinal cord tissues revealed over-expression of CD16 and co-localization of intact ALS-IgG with CD16 and in brain with activated microglia of G93A-SOD1 mice. Intact ALS-IgG enhanced effector cell activation and ADCC reaction in comparison to sugar-depleted or control IgG. ALS-IgG were localized in the synapse between brain microglia and neurons of G93A-SOD1 mice, manifesting a promising in vivo ADCC reaction. Therefore, glycans of ALS-IgG may serve as a biomarker for the disease and may be involved in neuronal damage.