Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer with limited treatment options. Mannose, a common monosaccharide taken up by cells through the same transporters as glucose, has been shown to induce growth retardation and enhance cell death in response to chemotherapy in several cancers, including PDAC. However, the molecular targets and mechanisms underlying mannose's action against PDAC are not well understood. In this study, we used an integrative approach of network pharmacology, bioinformatics analysis, and experimental verification to investigate the pharmacological targets and mechanisms of mannose against PDAC. Our results showed that the protein Src is a key target of mannose in PDAC. Additionally, computational analysis revealed that mannose is a highly soluble compound that meets Lipinski's rule of five and that the expression of its target molecules is correlated with survival rates and prognosis in PDAC patients. Finally, we validated our findings through in vitro and in vivo experiments. In conclusion, our study provides evidence that mannose plays a critical role in inhibiting PDAC growth by targeting Src, suggesting that it may be a promising therapeutic candidate for PDAC.

The Protein-O-mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the post-translational O-mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in M. tuberculosis, severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its O-mannosyltransferase activity in bacteria, in the non-pathogenic Mycobacterium smegmatis strain. Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily.

Chronic obstructive pulmonary disease (COPD) kills millions of people annually and patients suffering from exacerbations of this disorder display high morbidity and mortality. The clinical course of COPD is associated with dysbiosis and infections, but the underlying mechanisms are poorly understood. Glycosylation of proteins play roles in regulating interactions between microbes and immune cells, and knowledge on airway glycans therefore contribute to the understanding of infections. Furthermore, glycans have biomarker potential for identifying smokers with enhanced risk for developing COPD as well as COPD subgroups. Here, we characterized the N-glycosylation in the lower airways of healthy never-smokers (HNS, n = 5) and long-term smokers (LTS) with (LTS+, n = 4) and without COPD (LTS-, n = 8). Using mass spectrometry, we identified 57 highly confident N-glycan structures whereof 38 oligomannose, complex, and paucimannose type glycans were common to BAL samples from HNS, LTS-, and LTS+ groups. Hybrid type N-glycans were identified only in the LTS+ group. Qualitatively and quantitatively, HNS had lower inter-individual variation between samples compared to LTS- or LTS+. Cluster analysis of BAL N-glycosylation distinguished LTS from HNS. Correlation analysis with clinical parameters revealed that complex N-glycans were associated with health and absence of smoking whereas oligomannose N-glycans were associated with smoking and disease. The N-glycan profile from monocyte-derived macrophages differed from the BAL N-glycan profiles. In conclusion, long-term smokers display substantial alterations of N-glycosylation in the bronchoalveolar space, and the hybrid N-glycans identified only in long-term smokers with COPD deserve to be further studied as potential biomarkers.

While glycans underlie many biological processes, such as protein folding, cell adhesion and cell-cell recognition, deep evolution of glycosylation machinery remains an understudied topic. N-linked glycosylation is a conserved process in which mannosidases are key trimming enzymes. One of them is the glycoprotein endo-α-1,2-mannosidase which participates in the initial trimming of mannose moieties from an N-linked glycan inside the cis-Golgi. It is unique as the only endo-acting mannosidase found in this organelle. Relatively little is known about its origins and evolutionary history; so far it was reported to occur only in vertebrates. In this work, a taxon-rich bioinformatic survey to unravel the evolutionary history of this enzyme, including all major eukaryotic clades and a wide representation of animals, is presented. The endomannosidase was found to be more widely distributed in animals and other eukaryotes. The protein motif changes in context of the canonical animal enzyme were tracked. Additionally, the data show the two canonical vertebrate endomannosidase genes, MANEA and MANEAL, arose at the second round of the two vertebrate genome duplications and one more vertebrate paralog, CMANEAL, is uncovered. Finally, a framework where N-glycosylation co-evolved with complex multicellularity is described. A better understanding of the evolution of core glycosylation pathways is pivotal to understanding biology of eukaryotes in general, and the Golgi apparatus in particular. This systematic analysis of the endomannosidase evolution is one step towards this goal.

Based on factual scientific health claims, prebiotics have gained significant importance in ever-growing food and pharmaceutical industries. The diverse nature of distinct prebiotics influences the host differently in distinguishable patterns. Functional oligosaccharides are either plant-derived or commercially prepared. Raffinose, stachyose, and verbascose are the 3 types of raffinose family oligosaccharides (RFOs) that have been extensively used as medicine, cosmetic, and food additives. These dietary fiber fractions avert the adhesion and colonization by enteric pathogens and add nutrition metabolites for a healthy immune system. Enrichment of RFOs in healthy foods should be promoted as these oligosaccharides augment gut microecology by enhancing the health conferring microbes i.e. bifidobacteria and lactobacilli. RFOs influence the host's multiorgan systems due to their physiological and physicochemical properties. For example, the fermented microbial products of such carbohydrates affect neurological processes, including memory, mood, and behavior in humans. Raffinose-type sugar uptake is thought to be a ubiquitous property of bifidobacteria. This review paper summarizes the source of RFOs and their metabolizing entities, highlighting bifidobacterial carbohydrate utilization and health benefits.

Trehalose is a major circulatory sugar in the haemolymph of insects. It provides instant energy and protection against stress. Trehalose metabolism is associated with insect growth and development. The architecture and spatio-temporal expression dynamics of trehalose metabolism and transport genes are key for regulation. These genes are controlled by various transcription factors, largely linked to nutrition, insect development, and metamorphosis. Also, trehalose levels are affected by substrate affinities and modifications of enzymes involved in the pathway. A feedback mechanism involving the precursors and products can regulate trehalose metabolism. Further, the neuroendocrine system controls trehalose levels under normal and stressed conditions by producing different hormones. Hypotrehalosemic hormones work under surplus energy conditions to activate haemolymph trehalose uptake and degradation. In contrast, hypertrehalosemic hormones stimulate trehalose production in the fat body and its transport to the haemolymph. However, trehalose metabolism regulation in insects needs to be studied in detail. This review discusses aspects of trehalose synthesis, transport, and degradation dynamics in developmental transition and stress response. Unraveling the epigenetic factors, transcriptional control and chemical or genetic modulators can provide further insights into the intricate regulation of trehalose in a development- and tissue-specific manner. This molecular information about effectors and regulators of trehalose metabolism can be applied in developing diverse biotechnological applications.

Early detection is urgently needed to improve the patient's pancreatic ductal adenocarcinoma (PDAC) survival. Previously, we identified a novel tumor-associated glycan, H-type3, which is expressed on PDAC cells and is detected by rBC2LCN (recombinant N-terminal domain of BC2L-C identified from Burkholderia cenocepacia) lectin. Here, we identified that SERPINA3 is an rBC2LCN-reactive glycoprotein (BC2-S3) secreted from PDAC cells into the blood in patients with PDAC by liquid chromatography-tandem mass spectrometry analysis and lectin blotting. In immune staining, BC2-S3 was detected specifically in the tumor but not in normal tissues of PDAC. Lectin-ELISA was then developed to measure the serum level of BC2-S3 in healthy control (HC, n = 99) and patients with PDAC (n = 88). BC2-S3 exhibited higher in patients with PDAC than in those with HC. BC2-S3 showed similar diagnostic performance in all stages of PDAC (stages IA-IV, true positive rate = 76.1%, true negative rate = 81.8%) to CA19-9 (72.7%, 75.8%). Remarkably, BC2-S3 showed a significantly higher detection rate (89.7%) for early stage PDAC (IA-IIA) than CA19-9 (62.1%, P = 0.029). The combination of BC2-S3 and CA19-9 further improved the diagnostic ability for all stages of PDAC (81.8%, 87.9%). In conclusion, BC2-S3 is a glycobiomarker candidate for PDAC.

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D 1H, 13C-HSQC, and 1H,1H-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that ~½ of the population was 2,6-di-O-acetylated, ~¼ was 2-O-acetylated, whereas ~¼ did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: →2)-β-d-Manp-(1→3)-β-d-Manp2Ac6Ac-(1→4)-β-d-GlcpA-(1→3)-α-d-GlcpNAc-(1→, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840T differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue.

Thrombospondin 1 (THBS1) is a secreted extracellular matrix glycoprotein that regulates a variety of cellular and physiological processes. THBS1's diverse functions are attributed to interactions between the modular domains of THBS1 with an array of proteins found in the extracellular matrix. THBS1's three Thrombospondin type 1 repeats (TSRs) are modified with O-linked glucose-fucose disaccharide and C-mannose. It is unknown whether these modifications impact trafficking and/or function of THBS1 in vivo. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) and is sequentially extended to the disaccharide by β3glucosyltransferase (B3GLCT). The C-mannose is added by one or more of four C-mannosyltransferases. O-fucosylation by POFUT2/B3GLCT in the endoplasmic reticulum has been proposed to play a role in quality control by locking TSR domains into their three-dimensional fold, allowing for proper secretion of many O-fucosylated substrates. Prior studies showed the siRNA knockdown of POFUT2 in HEK293T cells blocked secretion of TSRs 1-3 from THBS1. Here we demonstrated that secretion of THBS1 TSRs 1-3 was not reduced by CRISPR-Cas9-mediated knockout of POFUT2 in HEK293T cells and demonstrated that knockout of Pofut2 or B3glct in mice did not reduce the trafficking of endogenous THBS1 to secretory granules of platelets, a major source of THBS1. Additionally, we demonstrated that all three TSRs from platelet THBS1 were highly C-mannosylated, which has been shown to stabilize TSRs in vitro. Combined, these results suggested that POFUT2 substrates with TSRs that are also modified by C-mannose may be less susceptible to trafficking defects resulting from the loss of the glucose-fucose disaccharide.

In Parkinson's disease, there is an accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. In neural cells, cytokine-induced SYN aggregation is modulated by heparan sulfate (HS) derived from glypican-1 (GPC1) by amyloid precursor protein (APP) and nitric oxide (NO)-dependent cleavage. We have explored possible interplay between APP, GPC1, and SYN in undifferentiated and differentiated neural progenitor cells (NPCs) by modulating APP and GPC1 processing. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies recognizing APP degradation products, HS released from GPC1, and SYN aggregates (filamentous SYN [SYNfil]). Suppression of HS release from GPC1 by inhibition of β-secretase or by NO deprivation resulted in no or slight increase in SYNfil aggregation. Stimulation of HS release by ascorbate did not further increase SYNfil staining. Interleukin-6 (IL-6) induced increased APP and GPC1 processing and SYNfil formation, which was reduced when β-secretase was inhibited and when HS release was impeded by NO deprivation. Ascorbate restored APP and GPC1 processing but did not affect SYNfil formation. Ascorbate-dependent differentiation of NPC resulted in the expression of tyrosine hydroxylase (TH) which colocalized with SYNfil. Suppression of APP processing by inhibition of β-secretase greatly disturbed the differentiation process. IL-6 induced coclustering of APP-degradation products, TH, HS, and SYNfil, which could be reversed by stimulation of HS release from GPC1 by excess ascorbate. We suggest that continuous release of HS from GPC1 moderates SYN aggregation and supports differentiation of NPC to dopaminergic neurons.

DC-SIGN and Galectin-3 are two different lectins and have been reported to participate in regulation of several virus infections. WHO has pointed that H5N1 and H7N9 avian influenza viruses (AIVs) play continuous threats to global health. AIV hemagglutinin (HA) protein-a highly glycosylated protein-mediates influenza infection and was proposed to have DC-SIGN and Gal3 interactive domains. This study aims to address the individual and collaborative roles of DC-SIGN and Gal3 toward AIVs infection. Firstly, A549 cells with DC-SIGN expression or Gal3-knockdown, via lentiviral vector-mediated CD209 gene expression or LGALS-3 gene knockdown, respectively were generated. Quantitative reverse transcription PCR (qRT-PCR) results indicated that DC-SIGN expression and Gal3 knockdown in A549 cells significantly promoted and ameliorated HA or NP gene expression, respectively after H5N1 and H7N9-reverse genetics (RG) virus postinfections (P < 0.05). Similar results observed in immunoblotting, indicating that DC-SIGN expression significantly facilitated H5N1-RG and H7N9-RG infections (P < 0.05), whereas Gal3 knockdown significantly reduced both viral infections (P < 0.05). Furthermore, we found that DC-SIGN and Gal3 co-expression significantly enhanced infectivity of both H5N1-RG and H7N9-RG viruses (P < 0.01) and higher regulatory capabilities by DC-SIGN and Gal3 in H5N1-RG than H7N9-RG were noted. The promoting effect mainly relied on exogenous Gal3 and DC-SIGN directly interacting with the HA protein of H5N1 or H7N9 AIVs, subsequently enhancing virus infection. This study sheds light on two different lectins individually and collaboratively regulating H5N1 and H7N9 AIVs infection and suggests that inhibitors against DC-SIGN and Gal3 interacting with HA could be utilized as alternative antiviral strategies.

Heparan sulfate (HS) is a sulfated polysaccharide with a wide range of biological activities. There is an increasing interest in the development of structurally homogeneous HS oligosaccharides as therapeutics. However, the factors influencing the pharmacokinetic properties of HS-based therapeutics remain unknown. Here, we report the pharmacokinetic properties of a panel of dodecasaccharides (12-mers) with varying sulfation patterns in healthy mice and uncover the pharmacokinetic properties of an octadecasaccharide (18-mer) in acutely injured mice. In the 12-mer panel, 1 12-mer, known as dekaparin, is anticoagulant, and 3 12-mers are nonanticoagulant. The concentrations of 12-mers in plasma and urine were determined by the disaccharide analysis using liquid chromatography coupled with tandem mass spectrometry. We observed a striking difference between anticoagulant and nonanticoagulant oligosaccharides in the 12-mer panel, showing that anticoagulant dekaparin had a 4.6-fold to 8.6-fold slower clearance and 4.4-fold to 8-fold higher plasma exposure compared to nonanticoagulant 12-mers. We also observed that the clearance of HS oligosaccharides is impacted by disease. Using an antiinflammatory 18-mer, we discovered that the clearance of 18-mer is reduced 2.8-fold in a liver failure mouse model compared to healthy mice. Our results suggest that oligosaccharides are rapidly cleared renally if they have low interaction with circulating proteins. We observed that the clearance rate of oligosaccharides is inversely associated with the degree of binding to target proteins, which can vary in response to pathophysiological conditions. Our findings uncover a contributing factor for the plasma and renal clearance of oligosaccharides which will aid the development of HS-based therapeutics.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

The O157:H7 strain of Escherichia coli is responsible for frequent outbreaks of hemorrhagic colitis worldwide. Its lipopolysaccharide is a virulence factor and contains an O antigen having repeating units with the tetrasaccharide structure [2-D-PerNAcα1-3-L-Fucα1-4-D-Glcβ1-3-D-GalNAcα1-]n. Genes encoding glycosyltransferases WbdN, WbdO, and WbdP are responsible for the biosynthesis of this repeating unit. We have previously characterized the second enzyme in the pathway, WbdN, which transfers Glc in β1-3 linkage to GalNAcα-O-PO3-PO3-(CH2)11-O-Ph (GalNAc-PP-PhU). In this work, Fuc-transferase WbdO from E. coli O157:H7 expressed in BL21 bacteria was characterized using the product of WbdN as the acceptor substrate. We showed that WbdO is specific for GDP-β-L-Fuc as the donor substrate. Compounds that contained terminal Glc or Glcβ1-3GalNAc structures but lacked the diphosphate group did not serve as acceptor substrates. The structure of the WbdO product was identified by mass spectrometry and Nuclear magnetic resonance (NMR) as L-Fucα1-4-D-Glcβ1-3-D-GalNAc PP-PhU. WbdO is an unusual bivalent metal ion-dependent Fuc-transferase classified as an inverting GT2 family enzyme that has 2 conserved sequences near the N-terminus. The Asp37 residue within the 36VDGGSTD42 sequence was found to be essential for catalysis. Mutation of Asp68 to Ala within the conserved 67YDAMNK72 sequence resulted in a 3-fold increase in activity. These studies show that WbdOO157 is a highly specific Fuc-transferase with little homology to other characterized Fuc-transferases.

During our biochemical characterization of select bacterial phosphatases belonging to the haloacid dehalogenase superfamily of hydrolases, we discovered a strong bias of Salmonella YidA for glucose-1-phosphate (Glc-1-P) over galactose-1-phosphate (Gal-1-P). We sought to exploit this ability of YidA to discriminate these two sugar-phosphate epimers in a simple coupled assay that could be a substitute for current cumbersome alternatives. To this end, we focused on Gal-1-P uridylyltransferase (GalT) that is defective in individuals with classical galactosemia, an inborn disorder. GalT catalyzes the conversion of Gal-1-P and UDP-glucose to Glc-1-P and UDP-galactose. When recombinant YidA was coupled to GalT, the final orthophosphate product (generated from selective hydrolysis of Glc-1-P by YidA) could be easily measured using the inexpensive malachite green reagent. When this new YidA-based colorimetric assay was benchmarked using a recombinant Duarte GalT variant, it yielded kcat/Km values that are ~2.5-fold higher than the standard coupled assay that employs phosphoglucomutase and glucose-6-phosphate dehydrogenase. Although the simpler design of our new GalT coupled assay might find appeal in diagnostics, a testable expectation, we spotlight the GalT example to showcase the untapped potential of sugar-phosphate phosphatases with distinctive substrate-recognition properties for measuring the activity of various metabolic enzymes (e.g. trehalose-6-phosphate synthase, N-acetyl-glucosamine-6-phosphate deacetylase, phosphofructokinase).

Glycans of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. The spike protein has been the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Analytical reports have described the glycan heterogeneity of the spike protein. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, experimental data on the role of discrete glycoforms on the spike protein pathobiology remains scarce. Building an understanding of their roles in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor-binding affinity revealed opposite regulatory effects of the RBD glycans with and without sialylation, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor-binding affinity. In combination with molecular dynamics simulation, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic glycan remodeling offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology.

Glucosylceramide synthase (UGCG) is a key enzyme in the biosynthesis of glycosphingolipids and its activity is related to the resistance to anticancer drugs and is involved in the derangement of metabolism in various diseases. Moreover, UGCG acts as a major controller of the balanced levels of individual brain sphingolipids that may trigger neurodegeneration in Gaucher disease and in Parkinson disease associated to pathogenic variants in the glucocerebrosidase-encoding gene GBA. We have developed an effective method for determining UGCG activity in vitro using deuterated ceramide as an acceptor, and quantitation of the formed deuterated glucosylceramide by liquid chromatography coupled with tandem mass spectrometry. The method enabled us to determine the kinetic parameters of UGGC and the effect of the inhibitor GZ667161 on the enzyme activity expressed in model cells, as well as to measure UGCG specific activity in human fibroblasts using a simple crude cell homogenate. This novel approach may be useful in determining the actual UGCG activity levels in patient cells and tissues of animal models of diseases, and to study novel drugs targeting glycosphingolipid metabolism.

We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5 and 28 days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50 percent of the protein content of every 0.5 d colostrum sample, which reduced to ≤3 percent in mature milk. In parallel, we analyzed IgG captured from the bovine milk samples to characterize its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation; data are available via ProteomeXchange with identifier PXD037755. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf's immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.

This report describes the isolation and characterization of two new antibodies, R-6C (IgM) and R-13E (IgM), which were generated in C57BL/6 mice (Mus musculus) using the Tic (JCRB1331) human induced pluripotent cell (hiPSC) line as an antigen, and their comparisons with two existing antibodies, R-10G (IgG1) and R-17F (IgG1). Their epitopes were studied by western blotting after various glycosidase digestions, binding analyses using enzyme-linked immunosorbent assays (ELISAs) and microarrays with various synthetic oligosaccharides. The minimum epitope structures identified were: Siaα2-3Galβ1-3GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S)β1 (R-6C), Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1 (R-13E), Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S)β1 (R-10G), and Fucα1-2Galβ1-3GlcNAβ1-3Galβ1-4Glc (lacto-N-fucopentaose I) (R-17F). Most glycoprotein epitopes are expressed as O-glycans. The common feature of these epitopes is the presence of an N-acetyllactosamine type 1 structure (Galβ1-3GlcNAc) at their nonreducing termini, followed by a type 2 structure (Galβ1-4GlcNAc); this arrangement comprises a type 1-type 2 motif. This motif is also shared by TRA-1-60, a traditional onco-fetal antigen. In contrast, the R-10G epitope has a type 2-type 2 motif. Among these antibodies, R-17F and R-13E exhibit cytotoxic activity toward hiPSCs. R-17F and R-13E exhibit extremely high similarity in the amino acid sequences in their complementarity-determining regions (CDRs), which is consistent with their highly similar glycan recognition. These antibodies are excellent tools for investigating the biological functions of glycoconjugates in hiPSCs/hESCs; they could be useful for the selection, isolation and selective killing of such undifferentiated pluripotent stem cells.

Altered and aberrant glycosylation signatures have been linked to being a hallmark in a variety of human disorders including cancers. Cancer stem cells, capable of self-renewal and differentiation, have recently been credited with a unique notion of disease genesis and implicated as the cause for initiation and recurrence of the disease in a new regime of neoplastic transformations hypothesis. Many biomarkers relating to diagnostic and prognostic intents have been discovered using the ubiquitous and abundant surface glycan patterns on cancer stem cells. Various technological advancements have been developed to identify and determine concerns with glycosylation structure. However, the nature and purpose of the glycan moiety on these glycosylation pattern have not yet been thoroughly investigated. This review, thus, summarises the process of glycosylation in CSCs, variations in glycosylation patterns in various stem cells, aberrant glycosylation patterns in cancer, the role of glycosylation in tumour cell adhesion, cell-matrix interactions, and signalling, as well as cancer detection and treatment. The function of carbohydrates as prospective serum biomarkers, some clinically authorized biomarkers, and potential novel biomarkers relating to cancer disease diagnosis and prognosis are also discussed in the review.