High-mannose Type N-glycans Research Articles

Antiadhesive nanosome elicits role of glycocalyx of tumor cell-derived exosomes in the organotropic cancer metastasis

Despite emerging importance of tumor cells-derived exosomes in cancer metastasis, the heterogeneity of exosome populations has largely hampered systemic characterization of their molecular composition, biogenesis, and functions. This study communicates a novel method for predicting and targeting pre-metastatic sites based on an exosome model “fluorescent cancer glyconanosomes” displaying N-glycans of cultured tumor cells. Glycoblotting by antiadhesive quantum dots provides a nice tool to shed light on the pivotal functions of the glycocalyx reconstructed from four cancer cell types without bias due to other compositions of exosomes. In vivo imaging revealed that circulation, clearance, and organotropic biodistribution of cancer glyconanosomes in mice depend strongly on cancer cell-type-specific N-glycosylation patterns, the compositions of key glycotypes, particularly dominant abundances of high mannose-type N-glycans and the position-specific sialylation. Notably, organ biodistribution of cancer glyconanosomes is reproducible artificially by mimicking cancer cell-type-specific N-glycosylation patterns, demonstrating that nanosomal glycoblotting method serves as promising tools for predicting and targeting pre-metastatic sites determined by the glycocalyx of extracellular vesicles disseminated from the primary cancer site.

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum.

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVNLec1. We found that Sf-RVN and Sf-RVNLec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVNLec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVNLec1 cells were Endo H-cleavable Man5GlcNAc2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.

Exposure of silver nanocolloids causes glycosylation disorders and embryonic deformities in medaka

Silver nanomaterials such as silver nanocolloids (SNC) contribute to environmental pollution and have adverse ecological effects on aquatic organisms. In particular, chemical exposure of fish during embryogenesis leads to deformities and puts the population at risk. Although glycans and glycosylation are known to be important for proper morphology in embryogenesis, little glycobiology-based research has examined morphological disorders caused by environmental pollutants. This study addressed the glycobiological effects of SNC exposure on medaka embryogenesis. After exposure of medaka embryos to SNC, deformities such as small heads and deformed eyes were observed. The expression of five glycan-related genes (alg2, gnsb, b4galt2, b3gat1a, and b3gat2) was significantly altered, with changes depending on the embryonic stage at exposure, with more severe deformities with exposure at earlier stages. In situ hybridization analyses indicated that the five genes were expressed mainly in the head region; exposure of SNC suppressed alg2 and gnsb and enhanced b4galt2 and b3gat1a expression relative to controls on day 7. Loss (siRNA)- and gain (RNA overexpression)-of-function experiments confirmed that alg2, gnsb, and b4galt2 are essential for embryogenesis. The effects of SNC exposure on glycan synthesis were estimated by glycan structure analysis. In the medaka embryo, high mannose–type glycans were dominant, and SNC exposure altered glycan synthesis. The alteration was more significant when exposure occurred at an early stage of medaka embryogenesis. Thus, SNC exposure causes embryonic deformities in medaka embryos through disordered glycosylation.

A Pradimicin-Based Staining Dye for Glycoprotein Detection.

Pradimicin A (PRM-A) and related compounds constitute an exceptional family of natural pigments that show Ca2+-dependent recognition of d-mannose (Man). Although these compounds hold great promise as research tools in glycobiology, their practical application has been severely limited by their inherent tendency to form water-insoluble aggregates. Here, we demonstrate that the 2-hydroxyethylamide derivative (PRM-EA) of PRM-A shows little aggregation in neutral aqueous media and retains binding specificity for Man. We also show that PRM-EA stains glycoproteins in dot blot assays, whereas PRM-A fails to do so, owing to severe aggregation. Significantly, PRM-EA is sensitive to glycoproteins carrying high mannose-type and hybrid-type N-linked glycans, but not to those carrying complex-type N-linked glycans. Such staining selectivity has never been observed in conventional dyes, suggesting that PRM-EA could serve as a unique staining agent for the selective detection of glycoproteins with terminal Man residues.

Purification, Characterization, and Gene Expression of Rice Endo-β-N-Acetylglucosaminidase, Endo-Os.

In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-β-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-β-GlcNAc-ases and preliminarily reported the gene information of rice endo-β-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-β-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-β-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manβ1 unit; this substrate specificity was almost the same as that of other plant endo-β-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

Microbiome signatures of progression toward celiac disease onset in at-risk children in a longitudinal prospective cohort study

Other than exposure to gluten and genetic compatibility, the gut microbiome has been suggested to be involved in celiac disease (CD) pathogenesis by mediating interactions between gluten/environmental factors and the host immune system. However, to establish disease progression markers, it is essential to assess alterations in the gut microbiota before disease onset. Here, a prospective metagenomic analysis of the gut microbiota of infants at risk of CD was done to track shifts in the microbiota before CD development. We performed cross-sectional and longitudinal analyses of gut microbiota, functional pathways, and metabolites, starting from 18 mo before CD onset, in 10 infants who developed CD and 10 matched nonaffected infants. Cross-sectional analysis at CD onset identified altered abundance of six microbial strains and several metabolites between cases and controls but no change in microbial species or pathway abundance. Conversely, results of longitudinal analysis revealed several microbial species/strains/pathways/metabolites occurring in increased abundance and detected before CD onset. These had previously been linked to autoimmune and inflammatory conditions (e.g., Dialister invisus, Parabacteroides sp., Lachnospiraceae, tryptophan metabolism, and metabolites serine and threonine). Others occurred in decreased abundance before CD onset and are known to have anti-inflammatory effects (e.g., Streptococcus thermophilus, Faecalibacterium prausnitzii, and Clostridium clostridioforme). Additionally, we uncovered previously unreported microbes/pathways/metabolites (e.g., Porphyromonas sp., high mannose-type N-glycan biosynthesis, and serine) that point to CD-specific biomarkers. Our study establishes a road map for prospective longitudinal study designs to better understand the role of gut microbiota in disease pathogenesis and therapeutic targets to reestablish tolerance and/or prevent autoimmunity.

Man-Specific Lectins from Plants, Fungi, Algae and Cyanobacteria, as Potential Blockers for SARS-CoV, MERS-CoV and SARS-CoV-2 (COVID-19) Coronaviruses: Biomedical Perspectives.

Betacoronaviruses, responsible for the “Severe Acute Respiratory Syndrome” (SARS) and the “Middle East Respiratory Syndrome” (MERS), use the spikes protruding from the virion envelope to attach and subsequently infect the host cells. The coronavirus spike (S) proteins contain receptor binding domains (RBD), allowing the specific recognition of either the dipeptidyl peptidase CD23 (MERS-CoV) or the angiotensin-converting enzyme ACE2 (SARS-Cov, SARS-CoV-2) host cell receptors. The heavily glycosylated S protein includes both complex and high-mannose type N-glycans that are well exposed at the surface of the spikes. A detailed analysis of the carbohydrate-binding specificity of mannose-binding lectins from plants, algae, fungi, and bacteria, revealed that, depending on their origin, they preferentially recognize either complex type N-glycans, or high-mannose type N-glycans. Since both complex and high-mannose glycans substantially decorate the S proteins, mannose-specific lectins are potentially useful glycan probes for targeting the SARS-CoV, MERS-CoV, and SARS-CoV-2 virions. Mannose-binding legume lectins, like pea lectin, and monocot mannose-binding lectins, like snowdrop lectin or the algal lectin griffithsin, which specifically recognize complex N-glycans and high-mannose glycans, respectively, are particularly adapted for targeting coronaviruses. The biomedical prospects of targeting coronaviruses with mannose-specific lectins are wide-ranging including detection, immobilization, prevention, and control of coronavirus infection.

Integrated Structural Analysis of N-Glycans and Free Oligosaccharides Allows for a Quantitative Evaluation of ER Stress

Endoplasmic reticulum (ER) stress has been reported in a variety of diseases. Although ER stress can be detected using specific markers, it is still difficult to quantitatively evaluate the degree of stress and to identify the cause of the stress. The ER is the primary site for folding of secretory or transmembrane proteins as well as the site where glycosylation is initiated. This study therefore postulates that tracing the biosynthetic pathway of asparagine-linked glycans (N-glycans) would be a reporter for reflecting the state of the ER and serve as a quantitative descriptor of ER stress. Glycoblotting-assisted mass spectrometric analysis of the HeLa cell line enabled quantitative determination of the changes in the structures of N-glycans and degraded free oligosaccharides (fOSs) in response to tunicamycin- or thapsigargin-induced ER stress. The integrated analysis of neutral and sialylated N-glycans and fOSs showed the potential to elucidate the cause of ER stress, which cannot be readily done by protein markers alone. Changes in the total amount of glycans, increase in the ratio of high-mannose type N-glycans, increase in fOSs, and changes in the ratio of sialylated N-glycans in response to ER stress were shown to be potential descriptors of ER stress. Additionally, drastic clearance of accumulated N-glycans was observed in thapsigargin-treated cells, which may suggest the observation of ER stress-mediated autophagy or ER-phagy in terms of glycomics. Quantitative analysis of N-glycoforms composed of N-glycans and fOSs provides the dynamic indicators reflecting the ER status and the promising strategies for quantitative evaluation of ER stress.

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Mevo lectin specificity toward high-mannose structures with terminal αMan(1,2)αMan residues and its implication to inhibition of the entry of Mycobacterium tuberculosis into macrophages.

Mannose-binding lectins can specifically recognize and bind complex glycan structures on pathogens and have potential as antiviral and antibacterial agents. We previously reported the structure of a lectin from an archaeal species, Mevo lectin, which has specificity toward terminal α1,2 linked manno-oligosaccharides. Mycobacterium tuberculosis expresses mannosylated structures including lipoarabinomannan (ManLAM) on its surface and exploits C-type lectins to gain entry into the host cells. ManLAM structure has mannose capping with terminal αMan(1,2)αMan residues and is important for recognition by innate immune cells. Here, we aim to address the specificity of Mevo lectin toward high-mannose type glycans with terminal αMan(1,2)αMan residues and its effect on M. tuberculosis internalization by macrophages. Isothermal titration calorimetry studies demonstrated that Mevo lectin shows preferential binding toward manno-oligosaccharides with terminal αMan(1,2)αMan structures and showed a strong affinity for ManLAM, whereas it binds weakly to Mycobacterium smegmatis lipoarabinomannan, which displays relatively fewer and shorter mannosyl caps. Crystal structure of Mevo lectin complexed with a Man7D1 revealed the multivalent cross-linking interaction, which explains avidity-based high-affinity for these ligands when compared to previously studied manno-oligosaccharides lacking the specific termini. Functional studies suggest that M. tuberculosis internalization by the macrophage was impaired by binding of Mevo lectin to ManLAM present on the surface of M. tuberculosis. Selectivity shown by Mevo lectin toward glycans with terminal αMan(1,2)αMan structures, and its ability to compromise the internalization of M. tuberculosis in vitro, underscore the potential utility of Mevo lectin as a research tool to study host-pathogen interactions.

Biochemical properties of a native β-1,4-mannanase from Aspergillus aculeatus QH1 and partial characterization of its N-glycosylation

N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal β-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from Aspergillus aculeatus QH1, displaying a full length of 1302 bp with an open reading frame of 1134 bp encoding for a GH5 subfamily 7 β-1, 4-mannanase, namely AacMan5_7A. The enzyme was purified and exhibited an optimal activity at pH 4.6 and 60 °C, hydrolyzing glucomannan and galactomannan, but not yeast mannan. AacMan5_7A is an N-glycosylated protein decorated with a high-mannose type glycan. Further through UPLC-ESI-MS/MS analysis, one of the four predicted N-glycosylation sites at N255 position was experimentally verified. The present study expands the information of N-glycosylation in fungal CAZymes, providing scientific bases for enhancing the production of fungal enzymes and their applications in food, feed, and plant biomass conversions.

Purification and molecular characterization of a truncated-type Ara h 1, a major peanut allergen: oligomer structure, antigenicity, and glycoform.

Peanut allergies are among the most severe food allergies, and several allergenic proteins referred to as Ara h 1-Ara h 17 have been identified from peanut seeds. The molecular characterization of Ara h 1 (63kDa), a glycosylated allergen, has almost been completed, and the occurrence of two homologous genes (clone 41B and clone P17) has been identified. In this study, we found a new variant of Ara h 1 i.e. 54kDa, in which the N-terminal amino acid sequence was EGREGEQ-, indicating that the N-terminal domain of 63kDa Ara h 1had been removed. This new isoform was obtained from the run-through fraction of hydrophobic interaction chromatography while 63kDa Ara h 1 was tightly bound to the hydrophobic resins, suggesting that the removal of the N-terminal domain resulted in extreme hydrophilic properties. We found that 63kDa Ara h 1 occurs as higher order homo-oligomeric conformations such as decamer or nonamer, while 54kDa Ara h 1 occurs exclusively as a homotrimer, indicating that the N-terminal domain of the 63kDa molecule may be involved in higher order oligomerization. When antisera from peanut-allergic patients were treated with both the Ara h 1 molecules, the immunoglobulin E (IgE) antibodies in these sera reacted with each Ara h 1 molecule, suggesting that the C-terminal as well as the N-terminal domains of Ara h 1 contribute significantly to the epitope formations of this peanut glycoallergen. Furthermore, the glycoform analyses of N-glycans linked to 63kDa and 54kDa Ara h 1 subunits revealed that both typical high-mannose type and β-xylosylated type N-glycans are linked to the molecules. The cross-reactivity of IgE against Ara h 1 in the serum of one peanut allergy patient was completely lost by de-N-glycosylation, indicating the N-glycan of Ara h 1 was the sole epitope for the Ara h 1- specific IgE in the patient.

Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei

BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L−1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-d-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-β-d-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Advances in enrichment of phosphorylated peptides and glycopeptides by smart polymer-based materials

翻译后修饰是蛋白质组学研究的前沿和重点,它不仅调节着蛋白质的折叠、状态、活性、定位以及蛋白质间的相互作用,也能帮助科学家更全面地了解生物体的生命过程,为疾病的预测、诊断和治疗提供更加强大的支撑和依据。翻译后修饰产物(例如磷酸化肽和糖肽)丰度很低,且存在着强烈的背景干扰,很难直接用质谱进行分析,因此迫切需要开发高效的富集材料和技术来选择性富集翻译后修饰产物。近年来,智能聚合物基材料通过外部物理、化学或生物刺激可逆地改变其结构和功能,实现对磷酸化肽和糖肽高度可控的吸附和脱附,进而衍生开发出一系列新颖的富集方法,极大地吸引研究者们的兴趣。一方面,智能聚合物基材料的响应变化包括材料疏水性的增加或减少、形状和形貌的改变、表面电荷的重新分布以及亲和配体的暴露或隐藏等特性。这些特性使得目标物和智能聚合物基材料之间的亲和力可以通过简单改变外部条件(如温度、pH值、溶剂极性和生物分子等)实现更可控和更智能的精细调节。另一方面,智能聚合物基材料为集成功能模块提供了便捷的可扩展平台,例如特定的识别组件,显著提高了目标物质的分离选择性。智能聚合物基材料在分离方面展现出巨大的潜力,这为蛋白质翻译后修饰产物的分析和研究带来了希望。围绕上述主题,该文依据Web of Science近20年来近50篇代表性文献,概述了智能聚合物基材料在磷酸化肽和糖肽分离及富集中的发展方向。

High-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues are unique to porcine islets.

ObjectivesIslet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection.MethodsN- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS).ResultsA total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets.ConclusionsThese results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors.

High-mannose type N-glycan binding specificity of a novel lectin from the red alga (Betaphycus gelatinus)

The red alga, Betaphycus gelatinus is one of carrageenan sources in the world. The lectin from the red alga B. gelatinus was isolated by a combination of aqueous ethanol extraction, ethanol precipitation, ion-exchange chromatography and gel filtration chromatography and was designated as BGL after the specific name of alga. Lectin gave a single band with molecular mass of about 19,000 Da in both non-reducing and reducing SDS-PAGE conditions, therefore lectin exists in monomer form. The hemagglutination activities of BGL were stable over a wide range of pH from 3 to 10, temperature up 60 oC and not affected by either the presence of EDTA or addition of divalent cations, indicating that lectin requires no metal for biological activity. The hemagglutination activities of BGL were not inhibited by monosaccharides and glycoproteins, D-glucose, D-mannose, D-galactose, D-xylose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetyl-neuraminic acid, N-acety-D-mannosamine, transferrin and fetuin, but strongly inhibited by glycoproteins bearing high-mannose type N-glycan, such as yeast mannan and porcine thyroglobulin. Lectin BGL is specific for N-glycans and may recognize terminal (α1–3) or (α1–6)-linked mannose residues in structure Man(α1-6)[Man(α1-3)]Man(α1-6)[Man(α1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc of N-glycans. High-mannose type N-glycan binding specificities of this lectin highly resemble with those of the anti-cancer, anti-virus and anti-bacteria lectins from the red algae, carrageenophytes, including Eucheuma serra (ESA-2), Eucheuma denticulatum (EDA-2), Kappaphycus striatum (KSA-2), Kappaphycus alvarezii (KAA-1 and KAA-2) and Solieria filiformis (SfL1 and SfL2). The red alga B. gelatinus could promise to be a good source of valuable lectins for application in biochemistry and biomedicine.

Man-Specific, GalNAc/T/Tn-Specific and Neu5Ac-Specific Seaweed Lectins as Glycan Probes for the SARS-CoV-2 (COVID-19) Coronavirus.

Seaweed lectins, especially high-mannose-specific lectins from red algae, have been identified as potential antiviral agents that are capable of blocking the replication of various enveloped viruses like influenza virus, herpes virus, and HIV-1 in vitro. Their antiviral activity depends on the recognition of glycoprotein receptors on the surface of sensitive host cells—in particular, hemagglutinin for influenza virus or gp120 for HIV-1, which in turn triggers fusion events, allowing the entry of the viral genome into the cells and its subsequent replication. The diversity of glycans present on the S-glycoproteins forming the spikes covering the SARS-CoV-2 envelope, essentially complex type N-glycans and high-mannose type N-glycans, suggests that high-mannose-specific seaweed lectins are particularly well adapted as glycan probes for coronaviruses. This review presents a detailed study of the carbohydrate-binding specificity of high-mannose-specific seaweed lectins, demonstrating their potential to be used as specific glycan probes for coronaviruses, as well as the biomedical interest for both the detection and immobilization of SARS-CoV-2 to avoid shedding of the virus into the environment. The use of these seaweed lectins as replication blockers for SARS-CoV-2 is also discussed.

In Vitro N-Glycan Mannosyl-Phosphorylation of a Therapeutic Enzyme by Using Recombinant Mnn14 Produced from Pichia pastoris.

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

Expression of a Large Single-Chain 13F6 Antibody with Binding Activity against Ebola Virus-Like Particles in a Plant System.

Pathogenic animal and human viruses present a growing and persistent threat to humans worldwide. Ebola virus (EBOV) causes zoonosis in humans. Here, two structurally different anti-Ebola 13F6 antibodies, recognizing the heavily glycosylated mucin-like domain (MLD) of the glycoprotein (GP), were expressed in transgenic Nicotiana tabacum plants and designed as inexpensive and effective diagnostic antibodies against Ebola virus disease (EVD). The first was anti-EBOV 13F6 full size antibody with heavy chain (HC) and light chain (LC) (monoclonal antibody, mAb 13F6-FULL), while the second was a large single-chain (LSC) antibody (mAb 13F6-LSC). mAb 13F6-LSC was constructed by linking the 13F6 LC variable region (VL) with the HC of mAb 13F6-FULL using a peptide linker and extended to the C-terminus using the endoplasmic reticulum (ER) retention motif KDEL. Agrobacterium-mediated plant transformation was employed to express the antibodies in N. tabacum. PCR, RT-PCR, and immunoblot analyses confirmed the gene insertion, transcription, and protein expression of these antibodies, respectively. The antibodies tagged with the KDEL motif displayed high-mannose type N-glycan structures and efficient binding to EBOV-like particles (VLPs). Thus, various forms of anti-EBOV plant-derived mAbs 13F6-FULL and LSC with efficient binding affinity to EBOV VLP can be produced in the plant system.