Complex-type Oligosaccharides Research Articles

Engineering of a Biologically Active Glycosylated Glucagon-Like Peptide-1 Analogue.

Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.

Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Characterization of novel endo-β-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans

Endo-β-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

NMR assignments of the N-glycans of the Fc fragment of mouse immunoglobulin G2b glycoprotein.

The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each CH2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two CH2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.

Enzymatic Synthesis of a Fluorogenic Reporter Substrate and the Development of a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation.

We report a straightforward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of N-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequentially coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

Greening Capillary Electrophoresis, a promising sprout of Separation Science toward sustainability

As a result of miniaturization new avenues were open toward customizing, improving and rendering separation science more affordable and available to any laboratory worldwide. One of the best resolving liquid separation techniques that still benefits from miniaturization is capillary electrophoresis (CE), where analytes are separated by their hydrodynamic volume to charge ratio. The theory of CE was introduced almost one hundred years ago, but became popular in the 1970s, yielding by 2010 over 1000 papers produced yearly. This progress triggered sample preparation optimization efforts, which led to significant reduction of required chemicals for analysis and the decrease of overall sample processing times. Consequently, CE can be considered as a sustainable technique in the field of liquid phase separation science. In this paper a custom made, cheap capillary electrophoresis unit with LED induced fluorescent (LedIF) imaging detection was used to demonstrate applicability of modern electronics, consumer products, and 3D printing in generating scientific results, while keeping sustainability in mind. Samples were chosen according to the observed trends of the past decade, namely from biotherapeutics industry. Its golden standard, immunoglobulin G N-glycans were enzymatically digested and the released complex type oligosaccharides were labeled with charged fluorophore, according to one of the most advanced and optimized protocols. Results were compared to separation runs performed on a high quality commercially available instrument, used as the control. Results disclosed in this paper should not be subjected to direct quantitative comparison, but should be rather taken as a technical demonstration of the capabilities of current and future technology, which can be implemented and merged with existing solutions in a sustainable manner.

Differential expression of N-linked oligosaccharides in methotrexate-resistant primary central nervous system lymphoma cells

BackgroundOligosaccharides of glycoprotein, particularly negatively-charged sialylated N-glycans, on the surface of lymphomas play important roles in cell–cell interactions and bind immunoglobulin-like lectins, causing inflammatory responses and bioregulation. However, their characterizations have largely been unknown in central nervous system (CNS) lymphoma.MethodsHere, we investigated expression patterns of N-linked oligosaccharides of glycoproteins in cells derived from CNS lymphomas and clinical specimens.ResultsWe first generated methotrexate (MTX)-resistant cells derived from HKBML and TK as CNS lymphoma, and RAJI as non-CNS lymphoma and determined N-linked oligosaccharide structures in these cells and other non-CNS lymphoma-derived cells including A4/FUK, OYB, and HBL1. Major components of the total oligosaccharides were high-mannose type N-glycans, whose level increased in MTX-resistant HKBML and TK but decreased in MTX-resistant RAJI. We also detected sialylated biantennary galactosylated N-glycans with α1,6-fucosylation, A2G2F, and A2G2FB from HKBML, TK, and RAJI. Sialylated A4G4F was specifically isolated from RAJI. However, the ratios of these sialylated N-glycans slightly decreased against MTX-resistant compared to non-resistant cells. Interestingly, almost all complex-type oligosaccharides were α2,6-sialylated.DiscussionThis is the first study for the expression profile of N-oligosaccharides on MTX-resistant primary CNS lymphoma-derived cells HKBML and TK, and tumor tissues resected from patients with CNS lymphoma,ConclusionThese results propose a possibility that the differential expression of high-mannose types and sialylated A2G2F, A2G2FB, and A4G4F on the surface of CNS lymphomas may provide a hint for targets for diagnoses and treatments of the oligosaccharide type-specific lymphomas.

A Comparison of the Oligosaccharide Structures of Antithrombin Derived from Plasma and Recombinant Using POTELLIGENT® Technology

Human antithrombin (AT) has two isoforms of which the predominant α-form is glycosylated on all four possible glycosylation sites and the lower abundant β-isoform lacks the oligosaccharide on Asn135. The main oligosaccharide structure of human AT consists of biantennary complex-type oligosaccharides lacking a core fucose. Generally, Chinese hamster ovary (CHO) cells produce recombinant human AT (rhAT) with core-fucosylated oligosaccharides. However, rhAT lacking core-fucose oligosaccharides can be produced by POTELLIGENT® technology, which uses FUT8 knockout CHO cells in production. The rhAT has more variable glycan structures, such as tetra-antennary complex type, high-mannose type, and mannose 6-phosphate species as minor components compared to plasma-derived human AT (phAT). In addition, the site-specific glycan profile was different between two ATs. We evaluated the effect of these properties on efficacy and safety based on a comparison of rhAT made by that technology with phAT in terms of their respective oligosaccharide structures, site-specific oligosaccharide profiles, and the ratio of α- and β-forms. Although some structural differences were found between the rhAT and phAT, we concluded that these differences have no significant effect on the efficacy and safety of rhAT.

Inflammatory Stress Causes N-Glycan Processing Deficiency in Ocular Autoimmune Disease

High levels of proinflammatory cytokines have been associated with a loss of tissue function in ocular autoimmune diseases, but the basis for this relationship remains poorly understood. Here we investigate a new role for tumor necrosis factor α in promoting N-glycan-processing deficiency at the surface of the eye through inhibition of N-acetylglucosaminyltransferase expression in the Golgi. Using mass spectrometry, complex-type biantennary oligosaccharides were identified as major N-glycan structures in differentiated human corneal epithelial cells. Remarkably, significant differences were detected between the efficacies of cytokines in regulating the expression of glycogenes involved in the biosynthesis of N-glycans. Tumor necrosis factor α but not IL-1β had a profound effect in suppressing the expression of enzymes involved in the Golgi branching pathway, including N-acetylglucosaminyltransferases 1 and 2, which are required for the formation of biantennary structures. This decrease in gene expression was correlated with a reduction in enzymatic activity and impaired N-glycan branching. Moreover, patients with ocular mucous membrane pemphigoid were characterized by marginal N-acetylglucosaminyltransferase expression and decreased N-glycan branching in the conjunctiva. Together, these data indicate that proinflammatory cytokines differentially influence the expression of N-glycan-processing enzymes in the Golgi and set the stage for future studies to explore the pathophysiology of ocular autoimmune diseases.

Spontaneously Cleavable Glycosylated Linker Capable of Extended Release of Its Conjugated Peptide.

Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling. This tunability will allow optimization towards the intended use of the peptides to be released. Furthermore, because glycosylation is a reliable method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are expected to permit the preparation of antibodies in aqueous buffers even in the case of sparingly soluble antigen peptides.

PrPc: The Normal Prion

PrPc is a normal cell‐surface glycoprotein that is conformationally characterized by two alpha helices and two complex‐type N‐linked oligosaccharide chains. This protein is unique in its propensity to misfold into a neurodegenerative disease‐causing proteinaceous infectious particle, known as a prion. PrPc can undergo conversion into PrPSc through spontaneous misfolding, a genetic mutation of the human PRNP gene, or exposure to a prion from an external source. When this happens, its composition shifts from an alpha‐helical, soluble protein low in beta pleated sheets to an insoluble, protease‐resistant particle with a high percentage of beta sheets. This resulting misfolded form, known formally as PrPSc, is pathogenic and aggregates in neural tissue by recruiting and converting more PrPc to PrPSc, ultimately causing neuronal cell dysfunction followed by cell death. The prion's form of “self‐replication” without utilizing genetic material is particularly fascinating, as it appears to defy the central dogma of biology and differs from all other pathogenic particle infection mechanisms. It is notable that some prions have evolved to act as transmissible infectious agents, similar to viruses, despite the absence of genetic material. This distinguishes some prion diseases, such as chronic wasting disease (CWD) and scrapie, from their inherited disease counterparts. The biosynthesis pathway of PrPc is similar to other cellular proteins, with replication occurring on the ribosomes on the endoplasmic reticulum. However, whereas malfunctioning proteins are normally tagged by ubiquitin and then degraded in a proteasome, the ubiquitin‐proteasome complex is inhibited during prion formation, allowing PrPSc to form and survive degradation, leading to its buildup in cytosol and subsequent neurotoxicity. Human and animal prion diseases have been classified into three broad categories‐‐genetic, sporadic, and acquired‐‐based on properties of the PrP proteins and prion infection morphology. Physical symptoms of prion infection include deviant behavior, dementia, ataxia, insomnia, paraplegia, and paresthesias. Commonly discussed prion‐induced diseases (differentiated via host type and slight conformational discrepancies between misfolded proteins) include scrapie, kuru, CJD, bovine spongiform encephalopathy (mad cow disease), and CWD. Here, we present current research on prions, with a focus on CWD, which was first found in Colorado and affects deer and other cervids.The Olathe North High School Team 1 MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine the structure‐function relationships and histology of PrPc, or the normal cellular prion.Support or Funding InformationThis project is a joint venture between the Milwaukee School of Engineering and the Medical Professions Academy.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Site-specific N-glycosylation analysis of soluble Fc\u03b3 receptor IIIb in human serum

Fc-receptors for immunoglobulin G (FcγRs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of FcγRs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous FcγRIIIa and FcγRIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant FcγRIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of FcγRIIIb purified from human serum based on mass spectrometric analysis. Our data indicate a distinct and common tendency of the glycoforms exhibited at each N-glycosylation site between the native and the previously reported recombinant FcγRIII glycoproteins. Among the six N-glycosylation sites of serum soluble FcγRIIIb, Asn45 was shown to be exclusively occupied by high-mannose-type oligosaccharides, whereas the remaining sites were solely modified by the complex-type oligosaccharides with sialic acid and fucose residues. The results of our endogenous FcγRIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.

鶏卵由来の二分枝糖鎖を用いるヒト型三分枝糖鎖の半合成

鶏卵由来の二分枝糖鎖を用いるヒト型三分枝糖鎖の半合成

Semisynthesis of Complex-type Triantennary Oligosaccharides from a Biantennary Oligosaccharide Isolated from Hen Egg Yolk

Semisynthesis of Complex-type Triantennary Oligosaccharides from a Biantennary Oligosaccharide Isolated from Hen Egg Yolk

Semisynthesis of Complex-Type Biantennary Oligosaccharides Containing Lactosamine Repeating Units from a Biantennary Oligosaccharide Isolated from a Natural Source.

Poly-N-acetyllactosamine (poly-LacNAc) structures on glycoproteins play important roles in essential biological events such as cell-cell adhesion. Here, we report a new strategy for the semisynthesis of LacNAc-extended complex-type biantennary oligosaccharides. We found an efficient isopropylidenation reaction that selectively protects the terminal Gal-3,4-OH of a biantennary complex-type nonasaccharide isolated from a natural source. This finding enabled the conversion of the nonasaccharide into the two types of oligosaccharides containing di-LacNAc units at one or two antennae via ten-step chemical sequences.

Improved therapeutic efficacy of mammalian expressed-recombinant interferon gamma against ovarian cancer cells

Human interferon gamma (hIFNγ) affects tumour cells and modulates immune responses, showing promise as an anti-cancer biotherapeutic. This study investigated the effect of glycosylation and expression system of recombinant hIFNγ in ovarian carcinoma cell lines, PEO1 and SKOV3. The efficacy of E. coli- and mammalian-expressed hIFNγ (hIFNγ-CHO and HEK293, glycosylated/de-glycosylated) on cytostasis, cell death (MTT, and Guava-ViaCount® flow-cytometry) and apoptotic signalling (Western blot of Cdk2, histone H3, procaspase-3, FADD, cleaved PARP, and caspase-3) was examined. Hydrophilic Interaction Liquid Chromatography determined the structure of N-linked glycans present in HEK293-expressed hIFNγ (hIFNγ-HEK). PEO1 was more sensitive to hIFNγ than SKOV3, but responses were dose-dependent and expression platform/glycosylation status-independent, whereas SKOV3 responded to mammalian-expressed hIFNγ in a dose-independent manner, only. Complex-type oligosaccharides dominated the N-glycosylation pattern of hIFNγ-HEK with some terminal sialylation and core fucosylation. Cleaved PARP and cleaved caspase-3 were not detected in either cell line, but FADD was expressed in SKOV3 with levels increased following treatment. In conclusion, hIFNγ did not induce apoptosis in either cell line. Mammalian- expressed hIFNγ increased cell death in the drug-resistant SKOV3. The presence of FADD in SKOV3, which may inhibit apoptosis through activation of NF-κB, could serve as a novel therapeutic target.

High molecular weight components containing N-linked oligosaccharides of Ascaris suum extract inhibit the dendritic cells activation through DC-SIGN and MR

Helminths, as well as their secretory/excretory products, induce a tolerogenic immune microenvironment. High molecular weight components (PI) from Ascaris suum extract down-modulate the immune response against ovalbumin (OVA). The PI exerts direct effect on dendritic cells (DCs) independent of TLR 2, 4 and MyD88 molecule and, thus, decreases the T lymphocytes response. Here, we studied the glycoconjugates in PI and the role of C-type lectin receptors (CLRs), DC-SIGN and MR, in the modulation of DCs activity. Our data showed the presence of glycoconjugates with high mannose- and complex-type N-linked oligosaccharide chains and phosphorylcholine residues on PI. In addition, these N-linked glycoconjugates inhibited the DCs maturation induced by LPS. The binding and internalization of PI-Alexa were decreased on DCs previously incubated with mannan, anti-DC-SIGN and/or anti-MR antibodies. In agreement with this, the incubation of DCs with mannan, anti-DC-SIGN and/or anti-MR antibodies abolished the down-modulatory effect of PI on these cells. It was also observed that the blockage of CLRs, DC-SIGN and MR on DCs reverted the inhibitory effect of PI in in vitro T cells proliferation. Therefore, our data show the involvement of DC-SIGN and MR in the recognition and consequent modulatory effect of N-glycosylated components of PI on DCs.

Development of N- and O-linked oligosaccharide engineeredSaccharomyces cerevisiae strain.

Yeast cells have been engineered for the production of glycoproteins as biopharmaceuticals with humanized N-linked oligosaccharides. The suppression of yeast-specific O-mannosylation is important to reduce immune response and to improve heterologous protein productivity in the production of biopharmaceuticals. However, so far, there are few reports of the engineering of both N-linked and O-linked oligosaccharides in yeast cells. In the present study, we describe the generation of a Saccharomyces cerevisiae strain capable of producing a glycoprotein with humanized Man5GlcNAc2 N-linked oligosaccharides, an intermediate of mammalian hybrid- and complex-type oligosaccharides, while suppressing O-mannosylation. First, a yeast strain that produces a glycoprotein with Man5GlcNAc2 was isolated by introducing msdS encoding α-1,2-mannosidase into a strain synthesizing Man8GlcNAc2 N-linked oligosaccharides. Next, to suppress O-mannosylation, an O-mannosyltransferase-deficient strain was generated by disrupting PMT1 and PMT2 Although the relative amount of O-linked oligosaccharides in the disruptant was reduced to approximately 40% of that in wild type cells, this strain exhibited growth defects and decreased protein productivity. To overcome the growth defects, we applied a mutagenesis technique that is based on the disparity theory of evolution. Finally, to improve protein productivity of the growth-recovered strain, vacuolar proteases PEP4 and PRB1 were further disrupted. Thus, by combining genetic engineering and disparity mutagenesis, we generated an Saccharomyces cerevisiae strain whose N- and O-linked oligosaccharide synthetic pathways were engineered to effectively produce the heterologous protein.

Evaluation of the effect of post-translational modification toward protein structure: Chemical synthesis of glycosyl crambins having either a high mannose-type or a complex-type oligosaccharide.

Glycoproteins are assembled and folded in the endoplasmic reticulum (ER) and transported to the Golgi for further processing of their oligosaccharides. During these processes, two types of oligosaccharides are used: that is, high mannose-type oligosaccharide in the ER and complex-type oligosaccharide in the Golgi. We were interested to know how two different types of oligosaccharides could influence the folding pathway or the final three-dimensional structure of the glycoproteins. For this purpose, we synthesized a new glycosyl crambin having complex-type oligosaccharide and evaluated the folding process, the final protein structure analyzed by NMR, and compared the CD spectra with previously synthesized glycosyl crambin bearing high mannose-type oligosaccharides. From our analysis, we found that the two different oligosaccharides do not influence the folding pathway in vitro and the final structure of the small glycoproteins. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 446-452, 2016.

Semisynthesis of Intact Complex-Type Triantennary Oligosaccharides from a Biantennary Oligosaccharide Isolated from a Natural Source by Selective Chemical and Enzymatic Glycosylation.

Attachment of oligosaccharides to proteins is a major post-translational modification. Chemical syntheses of oligosaccharides have contributed to clarifying the functions of these oligosaccharides. However, syntheses of oligosaccharide-linked proteins are still challenging because of their inherent complicated structures, including diverse di- to tetra-antennary forms. We report a highly efficient strategy to access the representative two types of triantennary oligosaccharides through only 9- or 10-step chemical conversions from a biantennary oligosaccharide, which can be isolated in exceptionally homogeneous form from egg yolk. Four benzylidene acetals were successfully introduced to the terminal two galactosides and two core mannosides of the biantennary asialononasaccharide bearing 24 hydroxy groups, followed by protection of the remaining hydroxy groups with acetyl groups. Selective removal of one of the benzylidene acetals gave two types of suitably protected glycosyl acceptors. Glycosylation toward the individual acceptors with protected Gal-β-1,4-GlcN thioglycoside and subsequent deprotection steps successfully yielded two types of complex-type triantennary oligosaccharides.