Sialic Acid Structures Research Articles

Host glycosylation of immunoglobulins impairs the immune response to acute Lyme disease

Lyme disease is caused by the bacteria Borreliella burgdorferi sensu lato (Bb) transmitted to humans from the bite of an infected Ixodes tick. Current diagnostics for Lyme disease are insensitive at the early disease stage and they cannot differentiate between active infections and people with a recent history of antibiotic-treated Lyme disease. Machine learning technology was utilized to improve the prediction of acute Lyme disease and identify sialic acid and galactose sugar structures (N-glycans) on immunoglobulins associated specifically at time points during acute Lyme disease time. A plate-based approach was developed to analyze sialylated N-glycans associated with anti-Bb immunoglobulins. This multiplexed approach quantitates the abundance of Bb-specific IgG and the associated sialic acid, yielding an accuracy of 90% in a powered study. It was demonstrated that immunoglobulin sialic acid levels increase during acute Lyme disease and following antibiotic therapy and a 3-month convalescence, the sialic acid level returned to that found in healthy control subjects (p<0.001). Furthermore, the abundance of sialic acid on Bb-specific IgG during acute Lyme disease impaired the host's ability to combat Lyme disease via lymphocytic receptor FcγRIIIa signaling. After enzymatically removing the sialic acid present on Bb-specific antibodies, the induction of cytotoxicity from acute Lyme disease patient antigen-specific IgG was significantly improved. Taken together, Bb-specific immunoglobulins contain increased sialylation which impairs the host immune response during acute Lyme disease. Furthermore, this Bb-specific immunoglobulin sialyation found in acute Lyme disease begins to resolve following antibiotic therapy and convalescence. Funding for this study was provided by the Coulter-Drexel Translational Research Partnership Program as well as from a Faculty Development Award from the Drexel University College of Medicine Institute for Molecular Medicine and Infectious Disease and the Department of Microbiology and Immunology.

Sialic acids in infection and their potential use in detection and protection against pathogens.

In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.

Insights into the Structure, Metabolism, Biological Functions and Molecular Mechanisms of Sialic Acid: A Review.

Sialic acid (SA) is a kind of functional monosaccharide which exists widely in edible bird's nest (EBN), milk, meat, mucous membrane surface, etc. SA is an important functional component in promoting brain development, anti-oxidation, anti-inflammation, anti-virus, anti-tumor and immune regulation. The intestinal mucosa covers the microbial community that has a significant impact on health. In the gut, SA can also regulate gut microbiota and metabolites, participating in different biological functions. The structure, source and physiological functions of SA were reviewed in this paper. The biological functions of SA through regulating key signaling pathways and target genes were discussed. In summary, SA can modulate gut microbiota and metabolites, which affect gene expressions and exert its biological activities. It is helpful to provide scientific reference for the further investigation of SA in the functional foods.

Development of a Synthetic Strategy for Gangliosides Using the Unique Chemical Structure of Sialic Acid

Gangliosides are the components of lipid rafts involved in several biological events such as signal transduction. The synthesis of structurally well-defined gangliosides is necessary in order to elucidate the formation mechanisms and functions of lipid rafts at the molecular level. However, previous methods to achieve stereocontrol in glycosidic bond formation of sialic acid have shown that it is substrate-dependent. Therefore, the chemical synthesis of gangliosides and their glycans remains a challenging task in carbohydrate chemistry. This review presents the development of a 1,5-lactam synthetic strategy for gangliosides.

Development of a Synthetic Strategy for Gangliosides Using the Unique Chemical Structure of Sialic Acid

ガングリオシドは、シグナル伝達などの生命現象の足場となる脂質ラフトの構成分子である。脂質ラフトの形成機構とその機能を分子レベルで解明するには、化学合成による均一なガングリオシド分子の供給が必要不可欠である。しかしながら、従来のシアル酸のグリコシド結合形成の立体制御法は反応基質に依存するため、ガングリオシドおよびその糖鎖の化学的構築は未だ困難である。本総説では、ガングリオシド糖鎖構築に有用な1,5-ラクタム合成戦略の開発とガングリオシド合成への展開について紹介する。

Angiotensin(1-7)-Stearic Acid Conjugate: Synthesis and Characterization

The novel coronavirus, SARS-CoV-2, broken out as the COVID-19 epidemic, is transported into the cytoplasm by angiotensin-converting enzyme-2 (ACE2), a key protein of the renin-angiotensin-system (RAS). ACE2 is a protective protein that reduces angiotensin (Ang) II, the bioactive component of RAS, by converting it to its potent antagonist, Ang-(1-7) peptide, in order to provide a pathophysiological response to stimuli. Although ACE-2 is upregulated especially in pulmonary endothelial cells and alveolar epithelial cells, downregulation of ACE-2 in the lung owing to loss of key regulatory factors explains the enzyme-dependent lethality of SARS-CoV-2. The N-terminal domain (NTD) of S1, one of the protein subunits of coronaviruses, is known to recognize acetylated sialic acids on glycosylated cell surface receptors. In this study, the stearic acid-peptide conjugate mimicking the sialic acid structure was synthesized, which will be able to balance uncontrolled inflammatory response and excessive cytokine production, and depending on these to suppress pneumonia and acute respiratory distress syndrome (ARDS), against SARS-CoV-2. It was expected that fatty acid acylation would greatly enhance cellular internalization and cytosolic distribution of the peptide through the cell membrane. Thus, we synthesized fatty acyl derivative of the N-Ac-Gly4-Ang (1-7) peptide. The peptide was synthesized using Fmoc/tBu solid-phase peptide chemistry and characterized by FT-IR, Zetasizer, and LC-ESI-MS. This study provided more detailed insights into understanding and meeting the basic structural requirements for optimal cellular delivery and formulation of the stearyl Ang (1-7)-peptide conjugate.

Sialylation of CD44 by ST8sia6 is required for recognition by the inhibitory receptor Siglec-7

Abstract Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human. Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.

Altered IgG glycosylation at COVID-19 diagnosis predicts disease severity.

The nature of the immune responses associated with COVID‐19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID‐19. We have analyzed IgG Fc N‐glycan traits of 82 SARS‐CoV‐2+ unvaccinated patients, at diagnosis, by nano‐LC‐ESI‐MS. We determined the impact of IgG Fc glyco‐variations in the induction of NK cells activation, further evaluating the association between IgG Fc N‐glycans and disease severity/prognosis. We found that SARS‐CoV‐2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID‐19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID‐19 patients appears to induce NK cells activation associated with increased release of IFN‐γ and TNF‐α, which indicates the presence of pro‐inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.

Human apolipoprotein E isoforms are differentially sialylated and the sialic acid moiety in ApoE2 attenuates ApoE2-Aβ interaction and Aβ fibrillation

The APOE genotype is the most prominent genetic risk factor for the development of late-onset Alzheimer’'s disease (LOAD); however, the underlying mechanisms remain unclear. In the present study, we found that the sialylation profiles of ApoE protein in the human brain are significantly different among the three isoforms, with ApoE2 exhibiting the most abundant sialic acid modification whereas ApoE4 had the least. We further observed that the sialic acid moiety in ApoE2 significantly affected the interaction between ApoE2 and Aβ peptides. The removal of sialic acid in ApoE2 increased the ApoE2 binding affinity for the Aβ17–24 region of Aβ and promoted Aβ fibrillation. These findings provide a plausible explanation for the well-documented differential roles of ApoE isoforms in Aβ pathogenesis. Specifically, compared to the other two isotypes, the higher expression of sialic acid in ApoE2 may contribute to the less potent interaction between ApoE2 and Aβ and ultimately the slower rate of brain Aβ deposition, a mechanism thought to underlie ApoE2-mediated decreased risk for AD. Future studies are warranted to determine whether the differential sialylation in ApoE isoforms may also contribute to some of their other distinct properties, such as their divergent preferences in associations with lipids and lipoproteins, as well as their potential impact on neuroinflammation through modulation of microglial Siglec activity. Overall, our findings lead to the insight that the sialic acid structure is an important posttranslational modification (PTM) that alters ApoE protein functions with relevance for AD.

Donor‐Reactivity‐Controlled Sialylation Reactions

AbstractAlthough tremendous efforts have been made for the efficient preparation of sialosides, controlling the stereochemical outcome of sialylation reaction still remains one of the most challenging tasks due to the unique chemical structure of sialic acid. We developed a new strategy to statistically analyze the stereoselectivity of sialylation reactions on six types of p‐tolyl thiosialosides in NIS/TfOH system using Relative Reactivity Value (RRV) as the indicator. Analysis of the reaction mechanism showed the formation of the relatively stable glycosyl bromide and glycosyl chloride intermediates from halide‐ and triflate‐containing promotors in the absence of an acceptor. We found that the α/β‐stereoselectivity, yields, and intermediate changes were associated with their donor reactivity. These findings enable to tailor the most suitable building blocks for stereo‐controlled sialylation reactions.

Characterization of Gangliosides in Three Sea Urchin Species by HILIC-ESI-MS/MS.

Sea urchin gangliosides (SU-GLSs) are well acknowledged for their nerve regeneration activity and neuroprotective property. The present study sought to characterize and semi-quantitate different SU-GLS subclasses in three sea urchin species, including Strongylocentrotus nudus, Hemicentrotus pulcherrimus, and Glyptocidaris crenularis. A total of 14 SU-GLS subclasses were identified by a hydrophilic interaction liquid chromatography-Q-Exactive tandem mass spectrometry method. Three sialic acid (Sia) structures, including Neu5Ac, Neu5Gc, and KDN, were identified in SU-GLSs, of which Neu5Ac and Neu5Gc had their corresponding sulfated forms. The linkage among Sias was determined to be 2-8. Additionally, KDN2-6Glc1-1Cer, KDN2-8Neu5Gc2-6Glc1-1Cer, and KDN2-8Neu5Gc2-8Neu5Gc2-6Glc-1Cer were speculated to be novel SU-GLS structures. Furthermore, the total SU-GLS content was 2.0-7.3 mg/g in the three sea urchin species. These results will provide useful data for developing a SU-GLS database of aquatic products. Besides, this study will provide a theoretical basis to explore the nutritional values of seafood products further.

The influences of cycle stage and pregnancy upon cell glycosylation in the endometrium of the mare

From Day 6.5–7 post-conception until its loss around Day 22, the equine embryo is enclosed in a mucinous capsule that prevents direct intercellular interaction between the trophectoderm and uterine epithelium. The embryo is, however, bathed in glycoprotein-rich secretions. In this study, lectin histochemistry was used to characterise the distribution and glycan composition of uterine glycoproteins destined for secretion, and to ascertain the local effect of an embryo on glycosylation in the endometrium. Endometrial biopsies were taken from mares in estrus, on Days 5, 8, 12 and 15 of diestrus, and on Days 12 and 15 of pregnancy and processed for lectin histochemistry. During estrus, lumenal epithelial cells were as truncated pyramids and mainly non-ciliated with glycosylated granules in the cytoplasm. Occasional ciliated cells contained few granules. Five days post-ovulation, non-ciliated cells of the lumenal epithelium were taller, and had accumulated many highly glycosylated apical granules. By Days 12 and 15 post-ovulation these cells were more cuboidal and some showed fewer secretory granules. In marked contrast, by Days 12 and 15 of pregnancy, the ciliated cells were distended, with numerous granules but non-ciliated cells had only a few in the apical cytoplasm. Glycosylation changed dramatically in pregnancy in the luminal and superficial gland epithelium, with fewer fucosylated termini, more N-acetyl galactosamine residues, together with an overall reduction in sialic acid and several other sugar structures. Glycosylation in ciliated cells on Days 12 and 15 of pregnancy showed a striking similarity to that of the blastocyst capsule. The data strongly suggests that glycoprotein production by luminal epithelial cells is influenced by the presence of a conceptus. We speculate that, as well as providing nourishment for the developing embryo, epithelial secretory glycoproteins may contribute components to the capsule, which develops only partially in embryos cultured in vitro.

Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo.

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo.

Oligo- and polysacharides, proteoglycans

After clearing up the confusion in the previously used nomenclature, the paper discusses the family of small-leucin-rich glycoproteins. After mentioning the family and its role in the organisation of the ground substance and in the regulation of inflammation, one unique molecule of proteoglycans is presented. This molecule is the inter-α-trypsin inhibitor. It has turned out that this peculiar molecule originally contains glycosaminoglycan beside the neutral carbohydrates. After summarizing the numerous connections of the proteoglycan molecule, the "self-sacrificing" nature of the molecule is presented. It is noted that the functions of the ovaries are also controlled by this molecule. The unique structure and fundamental function of the CD44 is also analysed. The CD44 is a transmembrane proteoglycan receptor with diverse functions and actions. The organisation has intramolecular, transmembrane sections, a stalk and one globular part and contains glycosaminoglycan and oligosacharids, too. The construction contains sialic acid residues at end positions of the molecular chains, which hinder it from joining other sialic acid structures in terminal positions. Therefore, this molecule has to be separated to open up a way for the sequencing neutral molecule (e.g., hyaluronan) to be attached. CD44 is the most important, but not the only receptor of hyaluronan. It can form connections with a lot of constructions, e.g., blood cells, the human endothelial, adventitial and mesenchymal. Human extracellular matrix proteinases can change the receptor's structures. Both the molecular and the supramolecular complexes boast unique features. The giant hyaluronan chains can connect to hyaluronan-binding proteoglycans - particularly hyaladherins to hyaluronectins - to create supramolecular connections and networks. The molecular mass of the fragments of hyaluronan is in the range of some kDs, while the whole molecule weighs up to several million Ds. The fragments are independent masses and can form attachments by themselves. The fragments have important inflammatory effects. Previously unidentified proteoglycan fragments have also been identified. Five of the ten identified fragments are secreted after synthetizing and stored in the granula of human cells. These strange characteristics are connected to the features of chromogranins/secretogranins. Nevertheless, this is another system of the organism, the functional, molecular structures of which are synthetized in neuroendocrine cells. Orv Hetil. 2018; 159(16): 620-627.

Sialidases: Therapeutic and Antiatherogenic Potential.

This review focuses on the biological role and clinical relevance of relatively poor studied enzymes known as sialidases. We describe structure and function of sialic acid, in particular as a component of gangliosides and plasma lipoproteins. Several types of sialidases are known in mammals, of which trans-sialidase is of special interest, since it is capable of removing sialic acid from low density lipoprotein (LDL) particles and transferring it to different acceptors in blood plasma. Desialylation of LDL, in turn, endows it a capacity to accumulate in the smooth muscle cells of human aortic intima, and therefore is important for atherogenesis. Moreover, sialidases appear to be involved in a variety of pathological processes, including viral infections and cancer, which makes these enzymes an attractive therapeutic target.

Metabolic Modulation of Cell Surface Sialoform of Macrophages

Macrophages and their precursors, monocytes, mediate the innate immune system through the initiation and regulation of inflammation and contribute to the adaptive immunity via variety mechanisms. Macrophage cell surface expresses a dense layer of glycans often terminated with sialic acids (SAs). SAs are a family of 9-carbon containing acidic monosaccharides and terminate cell surface glycans of either glycoproteins or glycolipids. The C-5-amino derivative represents the well-known neuraminic acid, and its amino functional group can be either acetylated (N-acetylneuraminic acid, Neu5Ac) or glycolylated (N-glycolylneuraminic acid, Neu5Gc). The most abundant SA is Neu5Ac. Notably, humans make Neu5Ac but are incapable of synthesizing Neu5Gc. The NeuGc deficiency in humans is attributed to a truncation in the gene encoding cytidine monophosphate sialic acid hydroxylase (CMAH). In this study, we investigated Neu5Gc incorporation of both THP-1 monocytes and THP-1 macrophages and to see its impact on macrophage functions. In addition, we designed a Neu5Gly as a Neu5Gc analog and investigated its metabolic inhibition of Neu5Gc incorporation into THP-1 monocytes and THP-1 macrophages. As a result, there is no inhibition of the Neu5Gc incorporation found when cells were treated with both Neu5Gc and Neu5Gly for both THP-1 monocytes and THP-1 macrophages, while Neu5Gly incorporation were found for both THP-1 monocytes and THP-1 macrophages. The ability to alter cell surface sialic acid structures will provide insights into structure-function relationships and the importance of individual structures in glycan-mediated processes. Further, the ability to remodel cell surface glycan architectures is expected to offer a novel approach to manipulate cellular physiology and phenotypic outcomes. Support or Funding InformationThis work was supported by Faculty Research Development Fund and the research fund from the Center for Gene Regulation in Health and Disease (GRHD) at Cleveland State University supported by Ohio Department of Development (ODOD). The authors acknowledge the National Science Foundation Major Research Instrumentation Grants for supporting NMR and QTrap 5500 mass spectrometer instrument requisition at Cleveland State University Figure 1Open in figure viewerPowerPoint Cell surface sialosides Figure 2Open in figure viewerPowerPoint Metabolic modulation of cell-surface sialic acids of monocytes and macrophages Scheme 1Open in figure viewerPowerPoint Synthesis of Zwitterionic Sialic Acid - Neu5Gly

Soluble Siglec-5 associates to PSGL-1 and displays anti-inflammatory activity.

Interactions between endothelial selectins and the leukocyte counter-receptor PSGL1 mediates leukocyte recruitment to inflammation sites. PSGL1 is highly sialylated, making it a potential ligand for Siglec-5, a leukocyte-receptor that recognizes sialic acid structures. Binding assays using soluble Siglec-5 variants (sSiglec-5/C4BP and sSiglec-5/Fc) revealed a dose- and calcium-dependent binding to PSGL1. Pre-treatment of PSGL1 with sialidase reduced Siglec-5 binding by 79 ± 4%. In confocal immune-fluorescence assays, we observed that 50% of Peripheral Blood Mononuclear Cells (PBMCs) simultaneously express PSGL1 and Siglec-5. Duolink-proximity ligation analysis demonstrated that PSGL1 and Siglec-5 are in close proximity (<40 nm) in 31 ± 4% of PBMCs. In vitro perfusion assays revealed that leukocyte-rolling over E- and P-selectin was inhibited by sSiglec-5/Fc or sSiglec-5/C4BP, while adhesion onto VCAM1 was unaffected. When applied to healthy mice (0.8 mg/kg), sSiglec-5/C4BP significantly reduced the number of rolling leukocytes under basal conditions (10.9 ± 3.7 versus 23.5 ± 9.3 leukocytes/field/min for sSiglec-5/C4BP-treated and control mice, respectively; p = 0.0093). Moreover, leukocyte recruitment was inhibited over a 5-h observation period in an in vivo model of TNFalpha-induced inflammation following injection sSiglec-5/C4BP (0.8 mg/kg). Our data identify PSGL1 as a ligand for Siglec-5, and soluble Siglec-5 variants appear efficient in blocking PSGL1-mediated leukocyte rolling and the inflammatory response in general.

Lipooligosaccharide locus classes and putative virulence genes among chicken and human Campylobacter jejuni isolates.

Background Campylobacter cause morbidity and considerable economic loss due to hospitalization and post infectious sequelae such as reactive arthritis, Guillain Barré- and Miller Fischer syndromes. Such sequelae have been linked to C. jejuni harboring sialic acid structures in their lipooligosaccharide (LOS) layer of the cell wall. Poultry is an important source of human Campylobacter infections but little is known about the prevalence of sialylated C. jejuni isolates and the extent of transmission of such isolates to humans.ResultsGenotypes of C. jejuni isolates from enteritis patients were compared with those of broiler chicken with pulsed-field gel electrophoresis (PFGE), to study the patterns of LOS biosynthesis genes and other virulence associated genes and to what extent these occur among Campylobacter genotypes found both in humans and chickens. Chicken and human isolates generally had similar distributions of the putative virulence genes and LOS locus classes studied. However, there were significant differences regarding LOS locus class of PFGE types that were overlapping between chicken and human isolates and those that were distinct to each source.ConclusionsThe study highlights the prevalence of virulence associated genes among Campylobacter isolates from humans and chickens and suggests possible patterns of transmission between the two species.

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN® GS-/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc.

Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y.

Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed N-glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput N-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases.