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Capillary electrophoresis separations of glycans labeled with 1-aminopyrene-3,6,8-trisulfonic acid were achieved with separation efficiencies ranging from 480000 to 640000 theoretical plates in a 60.2 cm, 25 μm inner diameter fused silica capillary. Under these separation conditions, the coefficient of variation in peak area is 10%, and if labeling efficiency is estimated at 100%, the limit of detection is 15 fM. The capillary electrophoresis method incorporated phospholipid additives to enhance the separation of glycans with slight differences in hydrodynamic volume. In addition, the phospholipid additives supported the integration of the lectin concanavalin A as well as the enzymes α1-2,3 mannosidase or β1-4 galactosidase to provide structural and compositional information about the glycans subject to separation. The use of in-capillary cleavage of terminal glycan residues with exoglycosidases offers a number of advantages over benchtop enzymatic sequencing, including reduced consumption of analyte, as well as enzyme. These methods were used to evaluate glycans derived from the glycoproteins α1-acid glycoprotein, fetuin, and ribonuclease B, as well as from glycoproteins collected from MCF7 cells. [ABSTRACT FROM AUTHOR]

Fireflies use bioluminescent signals to communicate with their mates. Luciferase has been thought to be the sole contributor to light color; however, populations of the Photinus pyralis firefly display variation in the color of their emitted signals yet have identical luciferase sequences. Here, we examined whether pigments could be present in the light organs of the twilight-active species P. pyralis and contribute to this variation. We detected patterns of expression that suggest ommochrome and pterin screening pigments are expressed in P. pyralis light organs and could filter light emitted by luciferase and play a role in signal tuning. There were no significant differences between the pigment gene expression of P. pyralis individuals with yellower and greener signals. Our study provides alternative mechanisms that could influence pigments in P. pyralis light organs that could also play a role in modifying signal color.

Streptococcus mutans is commonly associated with dental caries and the ability to form biofilms is essential for its pathogenicity. We recently identified the Pgf glycosylation machinery of S. mutans, responsible for the post-translational modification of the surface-associated adhesins Cnm and WapA. Since the four-gene pgf operon (pgfS-pgfM1-pgfE-pgfM2) is part of the S. mutans core genome, we hypothesized that the scope of the Pgf system goes beyond Cnm and WapA glycosylation. In silico analyses and tunicamycin sensitivity assays suggested a functional overlap between the Pgf machinery and the rhamnose-glucose polysaccharide synthesis pathway. Phenotypic characterization of pgf mutants (ΔpgfS, ΔpgfE, ΔpgfM1, ΔpgfM2, and Δpgf) revealed that the Pgf system is important for biofilm formation, surface charge, membrane stability, and survival in human saliva. Moreover, deletion of the entire pgf operon (Δpgf strain) resulted in significantly impaired colonization in a rat oral colonization model. Using Cnm as a model, we showed that Cnm is heavily modified with N-acetyl hexosamines but it becomes heavily phosphorylated with the inactivation of the PgfS glycosyltransferase, suggesting a crosstalk between these two post-translational modification mechanisms. Our results revealed that the Pgf machinery contributes to multiple aspects of S. mutans pathobiology that may go beyond Cnm and WapA glycosylation., (© 2023 John Wiley & Sons Ltd.)

Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N -acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed., Importance: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus . Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide., Competing Interests: The authors declare no conflict of interest.

Advanced glycation end products (AGEs), formed via the Maillard reaction (MR) during processing of foods, have been implicated in inflammatory and degenerative diseases in human beings. Cellular damage is primarily caused by AGE binding with the receptor for AGEs (RAGE) on cell membranes. An isoform of RAGE, soluble RAGE (sRAGE), acts as a decoy receptor binding circulating AGEs preventing cellular activation. Pet food manufacturing involves processing methods similar to human food processing that may increase dietary AGEs (dAGEs). We hypothesized that diet, plasma and urine AGEs, and serum sRAGE concentrations would differ between thermally processed diets. This study examined the association of four differently processed diets: ultra-processed canned wet food (WF); ultra-processed dry food (DF); moderately processed air-dried food (ADF) and minimally processed mildly cooked food (MF) on total plasma levels of the AGEs, carboxymethyllysine (CML), carboxyethyllysine (CEL), methylglyoxal hydroimidazolone-1, glyoxal hydroimidazolone-1, argpyrimidine, urine CML, CEL and lysinoalanine, and serum sRAGE concentration. Ultra-high-performance liquid chromatography-tandem mass spectrometry was used to measure AGEs. sRAGE concentration was measured using a commercial canine-specific enzyme-linked immunosorbent assay kit. Total dAGEs (mg/100 kcal as fed) were higher in WF than in other diets. Plasma total AGEs (nM/50 μL) were significantly higher with WF, with no difference found between DF, ADF, and MF; however, ADF was significantly higher than MF. Urine CML (nmol AGEs/mmol creatinine) was significantly higher with DF than with WF and MF. There were no significant differences in total urine AGEs or serum sRAGE concentration between diets. In conclusion, different methods of processing pet foods are associated with varied quantities of AGEs influencing total plasma AGE concentration in healthy dogs. Serum sRAGE concentration did not vary across diets but differences in total AGE/sRAGE ratio were observed between MF and WF and, ADF and DF., (© 2024 The Authors. Journal of Animal Physiology and Animal Nutrition published by Wiley‐VCH GmbH.)

Heparan sulfate (HS) is a linear polysaccharide that plays a key role in cellular signaling networks. HS functions are regulated by its 6-O-sulfation, which is catalyzed by three HS 6-O-sulfotransferases (HS6STs). Notably, HS6ST2 is mainly expressed in the brain and HS6ST2 mutations are linked to brain disorders, but the underlying mechanisms remain poorly understood. To determine the role of Hs6st2 in the brain, we carried out a series of molecular and behavioral assessments on Hs6st2 knockout mice. We first carried out strong anion exchange-high performance liquid chromatography and found that knockout of Hs6st2 moderately decreases HS 6-O-sulfation levels in the brain. We then assessed body weights and found that Hs6st2 knockout mice exhibit increased body weight, which is associated with abnormal metabolic pathways. We also performed behavioral tests and found that Hs6st2 knockout mice showed memory deficits, which recapitulate patient clinical symptoms. To determine the molecular mechanisms underlying the memory deficits, we used RNA sequencing to examine transcriptomes in two memory-related brain regions, the hippocampus and cerebral cortex. We found that knockout of Hs6st2 impairs transcriptome in the hippocampus, but only mildly in the cerebral cortex. Furthermore, the transcriptome changes in the hippocampus are enriched in dendrite and synapse pathways. We also found that knockout of Hs6st2 decreases HS levels and impairs dendritic spines in hippocampal CA1 pyramidal neurons. Taken together, our study provides novel molecular and behavioral insights into the role of Hs6st2 in the brain, which facilitates a better understanding of HS6ST2 and HS-linked brain disorders., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Importance: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species., Competing Interests: The authors declare no conflict of interest.

Glycosidic linkages in oligosaccharides play essential roles in determining their chemical properties and biological activities. MS n has been widely used to infer glycosidic linkages but requires a substantial amount of starting material, which limits its application. In addition, there is a lack of rigorous research on what MS n protocols are proper for characterizing glycosidic linkages. In this work, to deliver high-quality experimental data and analysis results, we propose a machine learning-based framework to establish appropriate MS n protocols and build effective data analysis methods. We demonstrate the proof-of-principle by applying our approach to elucidate sialic acid linkages (α2'-3' and α2'-6') in a set of sialyllactose standards and NIST sialic acid-containing N-glycans as well as identify several protocol configurations for producing high-quality experimental data. Our companion data analysis method achieves nearly 100% accuracy in classifying α2'-3' vs α2'-6' using MS 5 , MS 4 , MS 3 , or even MS 2 spectra alone. The ability to determine glycosidic linkages using MS 2 or MS 3 is significant as it requires substantially less sample, enabling linkage analysis for quantity-limited natural glycans and synthesized materials, as well as shortens the overall experimental time. MS 2 is also more amenable than MS 3/4/5 to automation when coupled to direct infusion or LC-MS. Additionally, our method can predict the ratio of α2'-3' and α2'-6' in a mixture with 8.6% RMSE (root-mean-square error) across data sets using MS 5 spectra. We anticipate that our framework will be generally applicable to analysis of other glycosidic linkages.

Nature is rich with examples of highly specialized biological materials produced by organisms for functions, including defense, hunting, and protection. Along these lines, velvet worms (Onychophora) expel a protein-based slime used for hunting and defense that upon shearing and dehydration forms fibers as stiff as thermoplastics. These fibers can dissolve back into their precursor proteins in water, after which they can be drawn into new fibers, providing biological inspiration to design recyclable materials. Elevated phosphorus content in velvet worm slime was previously observed and putatively ascribed to protein phosphorylation. Here, we show instead that phosphorus is primarily present as phosphonate moieties in the slime of distantly related velvet worm species. Using high-resolution nuclear magnetic resonance (NMR), natural abundance dynamic nuclear polarization (DNP), and mass spectrometry (MS), we demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with glycans linked to large slime proteins, while transcriptomic analyses confirm the expression of 2-AEP synthesizing enzymes in slime glands. The evolutionary conservation of this rare protein modification suggests an essential functional role of phosphonates in velvet worm slime and should stimulate further study of the function of this unusual chemical modification in nature.

Streptococcus mutans is a key pathogen associated with dental caries and is often implicated in infective endocarditis. This organism forms robust biofilms on tooth surfaces and can use collagen-binding proteins (CBPs) to efficiently colonize collagenous substrates, including dentin and heart valves. One of the best characterized CBPs of S. mutans is Cnm, which contributes to adhesion and invasion of oral epithelial and heart endothelial cells. These virulence properties were subsequently linked to post-translational modification (PTM) of the Cnm threonine-rich repeat region by the Pgf glycosylation machinery, which consists of 4 enzymes: PgfS, PgfM1, PgfE, and PgfM2. Inactivation of the S. mutans pgf genes leads to decreased collagen binding, reduced invasion of human coronary artery endothelial cells, and attenuated virulence in the Galleria mellonella invertebrate model. The present study aimed to better understand Cnm glycosylation and characterize the predicted 4-epimerase, PgfE. Using a truncated Cnm variant containing only 2 threonine-rich repeats, mass spectrometric analysis revealed extensive glycosylation with HexNAc2. Compositional analysis, complemented with lectin blotting, identified the HexNAc2 moieties as GlcNAc and GalNAc. Comparison of PgfE with the other S. mutans 4-epimerase GalE through structural modeling, nuclear magnetic resonance, and capillary electrophoresis demonstrated that GalE is a UDP-Glc-4-epimerase, while PgfE is a GlcNAc-4-epimerase. While PgfE exclusively participates in protein O-glycosylation, we found that GalE affects galactose metabolism and cell division. This study further emphasizes the importance of O-linked protein glycosylation and carbohydrate metabolism in S. mutans and identifies the PTM modifications of the key CBP, Cnm., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The compositional and structural analysis of GAGs is challenging due to their heterogenous structures. Strong anion exchange (SAX) HPLC can aid in the compositional analysis of GAGs and can separate complex mixtures based on charge and degree of sulfation. Herein we describe the digestion and release of GAGs from tissue, and the compositional analysis using SAX-HPLC., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells 1,2 . Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections 3,4 . Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase 5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD22 6,7 , which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Offspring of obese mothers suffer higher risks of type 2 diabetes due to increased adiposity and decreased β cell function. To date, the sex-differences in offspring islet insulin secretion during early life has not been evaluated extensively, particularly prior to weaning at postnatal day 21 (P21). To determine the role of maternal obesity on offspring islet insulin secretion, C57BL/6J female dams were fed chow or western diet from 4 weeks prior to mating to induce maternal obesity. First, offspring of chow-fed and obese dams were evaluated on postnatal day 21 (P21) prior to weaning for body composition, glucose and insulin tolerance, and islet phasic insulin-secretion. Compared to same-sex controls, both male and female P21 offspring born to obese dams (MatOb) had higher body adiposity and exhibited sex-specific differences in glucose tolerance and insulin secretion. The male MatOb offspring developed the highest extent of glucose intolerance and lowest glucose-induced insulin secretion. In contrast, P21 female offspring of obese dams had unimpaired insulin secretion. Using SAX-HPLC, we found that male MatOb had a decrease in pancreatic heparan sulfate glycosaminoglycan, which is a macromolecule critical for islet health. Notably, 8-weeks-old offspring of obese dams continued to exhibit a similar pattern of sex-differences in glucose intolerance and decreased islet insulin secretion. Overall, our study suggests that maternal obesity induces sex-specific changes to pancreatic HSG in offspring and a lasting effect on offspring insulin secretion, leading to the sex-differences in glucose intolerance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Casasnovas, Damron, Jarrell, Orr, Bone, Archer-Hartmann, Azadi and Kua.)

The emergence of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race and pre-existing conditions of COVID-19 patients., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development., (© The Author(s) 2020. Published by Oxford University Press.)

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods., (© 2020 De Leoz et al.)

The original version of this Article contained errors in Figures 1, 3 and 4. In panels b and d of Figure 1, the labels 'Sdc4 -/- ' were inadvertently replaced by 'Sdc4 +/+ '. In panels c and d of Figure 3, the labels 'Sdc4 -/- ' were replaced by 'Sdc2 -/- '. In panel f of Figure 3, the labels 'FGF2' were replaced by 'VEGFA 165 '. In panel e of Figure 6, a 'Sdc2 -/- ' label was inadvertently included. This has now been corrected in the PDF and HTML versions of the Article.

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA 165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA 165 binding sites and formation of a ternary Sdc2-VEGFA 165 -VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA 165 -VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.

Synapses are fundamental units of communication in the brain. The prototypical synapse-organizing complex neurexin-neuroligin mediates synapse development and function and is central to a shared genetic risk pathway in autism and schizophrenia. Neurexin's role in synapse development is thought to be mediated purely by its protein domains, but we reveal a requirement for a rare glycan modification. Mice lacking heparan sulfate (HS) on neurexin-1 show reduced survival, as well as structural and functional deficits at central synapses. HS directly binds postsynaptic partners neuroligins and LRRTMs, revealing a dual binding mode involving intrinsic glycan and protein domains for canonical synapse-organizing complexes. Neurexin HS chains also bind novel ligands, potentially expanding the neurexin interactome to hundreds of HS-binding proteins. Because HS structure is heterogeneous, our findings indicate an additional dimension to neurexin diversity, provide a molecular basis for fine-tuning synaptic function, and open therapeutic directions targeting glycan-binding motifs critical for brain development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases. The data support a pathogenic mechanism where TGF-ß signals enhance the proteolytic processing of pro-Ctsk by modulating the expression of chondroitin 4-sulfate (C4-S). In MLII, elevated C4-S corresponds with TGF-ß-mediated increases in chst11 expression. Inhibiting chst11 impairs the proteolytic activation of Ctsk and alleviates the MLII phenotypes. These findings uncover a regulatory loop between TGF-ß signaling and Ctsk activation that is altered in the context of lysosomal disease. This work highlights the power of ABPs to identify mechanisms underlying pathogenic development in living animals., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

N-Glycans of human proteins possess both α2,6- and α2,3-linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3-linkage due to the absence of α2,6-sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)-producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC-Sambucus nigra (SNA) lectin that preferentially binds α2,6-linked SA. The presence of α2,6-linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2-fold compared to the control. For host cell engineering, the CHOZN(®) GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single-cell clones were derived from the enriched population and selected based on FITC-SNA staining and St6gal1 expression. Two clones ("ST6GAL1 OE Clone 31 and 32") were confirmed for the presence of α2,6-linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6-linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human-like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of "bio-better" protein therapeutics and cell culture vaccine production., (© 2015 American Institute of Chemical Engineers.)

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M)) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P)) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P) bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P) (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M) (21%). The cytoprotective effect of the asialo-rhuEPO(P) was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

For the rapid production of influenza vaccine antigens in unlimited quantities, a transition from conventional egg-based production to cell-based and recombinant systems is required. The need for higher-yield, lower-cost, and faster production processes is critical to provide adequate supplies of influenza vaccine to counter global pandemic threats. In this study, recombinant hemagglutinin proteins of influenza virus were expressed in the microalga Schizochytrium sp., an established, fermentable organism grown in large scale for the manufacture of polyunsaturated fatty acids for animal and human health applications. Schizochytrium was capable of exporting the full-length membrane-bound proteins in a secreted form suitable for vaccine formulation. One recombinant hemagglutinin (rHA) protein derived from A/Puerto Rico/8/34 (H1N1) influenza virus was evaluated as a vaccine in a murine challenge model. Protective immunity from lethal challenge with homologous virus was elicited by a single dose of 1.7, 5 or 15 µg rHA with or without adjuvant at survival rates between 80-100%. Full protection (100%) was established at all dose levels with or without adjuvant when mice were given a second vaccination. These data demonstrate the potential of Schizochytrium sp. as a platform for the production of recombinant antigens useful for vaccination against influenza.