Acid Glycan Research Articles

Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells.

Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.

Polysaccharide Extracts Derived from Defloration Waste of Fruit Pitaya Regulates Gut Microbiota in a Mice Model

Flower thinning is often used during the planting of fruit trees to improve fruit quality and promote large fruit. Flower buds become an agricultural by-product of the planting process. Pitaya (Hylocereus undatus) is a popular fruit in many tropical regions, which is widely cultivated in Southeast Asian countries. Probiotics such as Lactobacillus plantarum have been shown to exhibit an anti-obesity effect by regulating gut microbiota. This study investigated the effect of polysaccharides from pitaya flower buds (PFW) extracted with water on the regulation of gut microbiota and body weight control in mice fed with a high-fat diet. The effects of PFW on the growth of L. plantarum were analyzed and the propagation of L. plantarum was promoted in an aqueous solution containing PFW. In an in vivo study, mice were fed with a high-fat diet supplemented with PFW for 12 weeks; PFW treatment effectively controlled body weight and reduced short bowel syndrome of mice induced by the high-fat diet. Gut microbiota sequencing revealed that Lachnospiraceae and Lactobacillaceae were the main bacteria targeted by PFW. Moreover, transcript analysis demonstrated that PFW alleviated obesity through amino acid metabolism, carbohydrate metabolism, and glycan metabolism. Overall, PFW is a valuable food supplement that can regulate gut microbiota and may have potential to ameliorate the physiological damage caused by a high-fat diet.

High sensitivity acidic N-glycan profiling with MS-enhancing derivatization and mixed mode chromatography

Acidic N-linked glycan content is often associated with a protein drug’s stability, efficacy and immune response. It has often been a challenge to analyze these types of glycans, including those that are differentiated by the incorporation of N-acetyl (NANA) and N-glycolyl neuraminic acid (NGNA) residues. In this study, a strategy for rapid N-glycan profiling by mixed mode chromatography is proposed as a complement to established HILIC methodologies. Hybrid silica chromatographic surfaces are used to improve recoveries during a column’s initial use and to eliminate the need for column conditioning. In addition, the loss of labeled acidic glycans, especially phosphorylated glycan species, during SPE purification is addressed through the use of a citrate containing eluent. Yields for both singly and doubly phosphorylated glycan species are markedly improved. Combined with a mixed mode anion exchange reversed phase separation, these advances afford a class separation of glycans derivatized with labels designed to enhance positive ion mode MS detection. These labeled glycan species are separated according to their charge and with an added level of resolution imparted by the reversed phase retention mechanism. The separation technique itself can be accomplished with a low ionic strength gradient running from 0 to 22 mM ammonium formate such that high sensitivity detection can be achieved by both fluorescence and mass spectrometry. Using analytical scale chromatography, features in an N-glycan profile were easily interrogated to well below a 0.1% relative abundance. As such, it became possible to characterize N-glycans from recombinant beta glucuronidase and to quickly identify a number of unique phosphorylated glycan species.

Targeting cell surface glycans with lectin-coated fluorescent nanodiamonds.

Glycosylation is arguably the most important functional post-translational modification in brain cells and abnormal cell surface glycan expression has been associated with neurological diseases and brain cancers. In this study we developed a novel method for uptake of fluorescent nanodiamonds (FND), carbon-based nanoparticles with low toxicity and easily modifiable surfaces, into brain cell subtypes by targeting their glycan receptors with carbohydrate-binding lectins. Lectins facilitated uptake of 120 nm FND with nitrogen-vacancy centers in three types of brain cells – U87-MG astrocytes, PC12 neurons and BV-2 microglia cells. The nanodiamond/lectin complexes used in this study target glycans that have been described to be altered in brain diseases including sialic acid glycans via wheat (Triticum aestivum) germ agglutinin (WGA), high mannose glycans via tomato (Lycopersicon esculentum) lectin (TL) and core fucosylated glycans via Aleuria aurantia lectin (AAL). The lectin conjugated nanodiamonds were taken up differently by the various brain cell types with fucose binding AAL/FNDs taken up preferentially by glioblastoma phenotype astrocyte cells (U87-MG), sialic acid binding WGA/FNDs by neuronal phenotype cells (PC12) and high mannose binding TL/FNDs by microglial cells (BV-2). With increasing recognition of glycans having a role in many diseases, the lectin bioconjugated nanodiamonds developed here are well suited for further investigation into theranostic applications.

Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling.

All cells are decorated with a highly dense and complex structure of glycan chains, which are mostly attached to proteins and lipids. In this context, sialic acids are a family of nine-carbon acidic monosaccharides typically found at the terminal position of glycan chains, modulating several physiological and pathological processes. Sialic acids have many structural and modulatory roles due to their negative charge and hydrophilicity. In addition, the recognition of sialic acid glycans by mammalian cell lectins, such as siglecs, has been described as an important immunological checkpoint. Furthermore, sialic acid glycans also play a pivotal role in host–pathogen interactions. Various pathogen receptors exposed on the surface of viruses and bacteria are responsible for the binding to sialic acid sugars located on the surface of host cells, becoming a critical point of contact in the infection process. Understanding the molecular mechanism of sialic acid glycans recognition by sialic acid-binding proteins, present on the surface of pathogens or human cells, is essential to realize the biological mechanism of these events and paves the way for the rational development of strategies to modulate sialic acid-protein interactions in diseases. In this perspective, nuclear magnetic resonance (NMR) spectroscopy, assisted with molecular modeling protocols, is a versatile and powerful technique to investigate the structural and dynamic aspects of glycoconjugates and their interactions in solution at the atomic level. NMR provides the corresponding ligand and protein epitopes, essential for designing and developing potential glycan-based therapies. In this review, we critically discuss the current state of knowledge about the structural features behind the molecular recognition of sialic acid glycans by different receptors, naturally present on human cells or pathogens, disclosed by NMR spectroscopy and molecular modeling protocols.

Comprehensive analysis of lectin-glycan interactions reveals determinants of lectin specificity.

Lectin-glycan interactions facilitate inter- and intracellular communication in many processes including protein trafficking, host-pathogen recognition, and tumorigenesis promotion. Specific recognition of glycans by lectins is also the basis for a wide range of applications in areas including glycobiology research, cancer screening, and antiviral therapeutics. To provide a better understanding of the determinants of lectin-glycan interaction specificity and support such applications, this study comprehensively investigates specificity-conferring features of all available lectin-glycan complex structures. Systematic characterization, comparison, and predictive modeling of a set of 221 complementary physicochemical and geometric features representing these interactions highlighted specificity-conferring features with potential mechanistic insight. Univariable comparative analyses with weighted Wilcoxon-Mann-Whitney tests revealed strong statistical associations between binding site features and specificity that are conserved across unrelated lectin binding sites. Multivariable modeling with random forests demonstrated the utility of these features for predicting the identity of bound glycans based on generalized patterns learned from non-homologous lectins. These analyses revealed global determinants of lectin specificity, such as sialic acid glycan recognition in deep, concave binding sites enriched for positively charged residues, in contrast to high mannose glycan recognition in fairly shallow but well-defined pockets enriched for non-polar residues. Focused fine specificity analysis of hemagglutinin interactions with human-like and avian-like glycans uncovered features representing both known and novel mutations related to shifts in influenza tropism from avian to human tissues. As the approach presented here relies on co-crystallized lectin-glycan pairs for studying specificity, it is limited in its inferences by the quantity, quality, and diversity of the structural data available. Regardless, the systematic characterization of lectin binding sites presented here provides a novel approach to studying lectin specificity and is a step towards confidently predicting new lectin-glycan interactions.

PMP-HPLC Fingerprint of Partial Acid Hydrolysate of Lycium Barbarum Polysaccharides Based on the Chemometric Methods

In this paper, the fingerprints of partial acid hydrolysates of Lycium barbarum polysaccharides (LBPs) were constructed, combined with a variety of chemometric methods, to evaluate the differences of LBPs from different origins and identify the authenticity of commercial samples. Sixteen batches of Lycium barbarum polysaccharides (LBPs) from different areas were obtained by the method of water extraction and alcohol precipitation, which were partially hydrolyzed by trifluoroacetic acid. The PMP pre-column derivatization-HPLC method was applied to analyse the monosaccharides and oligosaccharides in the partial hydrolysates of LBPs. The similarity of samples of different origins was calculated by the “similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(TCM)”software. And the fingerprints were further evaluated by chemometric methods including cluster analysis (HCA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The results showed that the partial acid hydrolysis products of LBPs contained seven monosaccharides, galacturonic acid glycans of DP 2-4 and three kinds of disaccharides. The similarities of 16 batches of LBPs of different origins were high ranging from 0.911 to 0.997. HCA, PCA and PLS-DA all divided LBPs from different origins into three categories, and the classification results had certain correlation with the origins. The three iconic components that influenced the differences between origins were screened out by PLS-DA, which were glucose (Glc), arabinose (Ara) and galactose (Gal), respectively. The established fingerprint in this paper could be used for the quality control of LBPs.

Glyconectin Cell Adhesion Epitope, β-d-GlcpNAc3S-(1→3)-α-l-Fucp, Is Involved in Blastulation of Lytechinus pictus Sea Urchin Embryos

Glycans, as the most peripheral cell surface components, are the primary candidates to mediate the initial steps of cell recognition and adhesion via glycan–glycan binding. This molecular mechanism was quantitatively demonstrated by biochemical and biophysical measurements at the cellular and molecular level for the glyconectin 1 β-d-GlcpNAc3S-(1→3)-α-l-Fucp glycan structure (GN1). The use of adhesion blocking monoclonal antibody Block 2 that specifically recognize this epitope showed that, besides Porifera, human colon carcinoma also express this structure in the apical glycocalyx. Here we report that Block 2 selectively immune-precipitate a Mr 580 × 103 (g580) acidic non-glycosaminoglycan glycan from the total protein-free glycans of Lytechinus pictus sea urchin hatched blastula embryos. Immuno-fluorescence confocal light microscopy and immunogold electron microscopy localized the GN1 structure in the apical lamina glycocalyx attachments of ectodermal cells microvilli, and in the Golgi complex. Biochemical and immune-chemical analyses showed that the g580 glycan is carrying about 200 copies of the GN1 epitope. This highly polyvalent g580 glycan is one of the major components of the glycocalyx structure, maximally expressed at hatched blastula and gastrula. The involvement of g580 GN1 epitope in hatched blastula cell adhesion was demonstrated by: (1) enhancement of cell aggregation by g580 and sponge g200 glycans, (2) inhibition of cell reaggregation by Block 2, (3) dissociation of microvilli from the apical lamina matrix by the loss of its gel-like structure resulting in a change of the blastula embryonal form and consequent inhibition of gastrulation at saturating concentration of Block 2, and (4) aggregation of beads coated with the immune-purified g580 protein-free glycan. These results, together with the previous atomic force microscopy measurements of GN1 binding strength, indicated that this highly polyvalent and calcium ion dependent glycan–glycan binding can provide the force of 40 nanonewtons per single ectodermal cell association of microvilli with the apical lamina, and conservation of glycocalyx gel-like structure. This force can hold the weight of 160,000 cells in sea water, thus it is sufficient to establish, maintain and preserve blastula form after hatching, and prior to the complete formation of further stabilizing basal lamina.

Vibrio parahaemolyticus alters the community composition and function of intestinal microbiota in Pacific white shrimp, Penaeus vannamei

Homeostasis of the intestinal microbiota is crucial to the health of the host, and pathogen invasion often results in dynamic changes in the composition, structure, and functions of gut microbes. Vibrio parahaemolyticus is an important opportunistic pathogen of Pacific white shrimp (Penaeus vannamei) often leading to high mortality. The V. parahaemolyticus strain that causes acute hepatopancreatic necrosis disease (AHPND) expresses the virulence genes, pirA and pirB. However, some non-AHPND strains of V. parahaemolyticus that do not carry pir genes can still cause disease in shrimp resulting in huge economic losses. The TJA114 strain of V. parahaemolyticus, which was isolated from diseased shrimp, showed strong pathogenicity toward P. vannamei and was found to lack the pir gene. This study investigated the alteration in bacterial community composition and function of intestinal microbiota in P. vannamei following infection with V. parahaemolyticus TJA114. We used 16S rRNA high-throughput sequencing and liquid chromatography with tandem mass spectrometry technologies to identify the altered bacterial taxa involved in disease in experimentally infected shrimp. These included the potentially pathogenic Oceanospirillum, Lewinella, and Saprospira and the potentially beneficial Agarivorans, Ferrimonas, and Shewanella. The alteration in composition of these bacterial taxa was closely associated with some differentially expressed metabolites identified by shrimp fecal metabolome analysis. These metabolites were mainly involved in level 3 KEGG pathways including lysine, arginine, primary and secondary bile acid biosynthesis, and purine, pyrimidine, glycerophospholipid, glycan, proline, and histidine metabolism. The findings of this study provide novel insights into the pathogenesis of vibriosis in P. vannamei caused by infection with the non-AHPND V. parahaemolyticus.

Gut bacteria communities differ between Gynaephora species endemic to different altitudes of the Tibetan Plateau

Abstract Knowledge about the impact of different ecological environments on the composition and function of gut bacteria of Gynaephora species is largely unknown. Here, we characterized the gut bacterial communities of the two Gynaephora species (Gynaephora menyuanensis and G. alpherakii) inhabiting different altitudes of the Tibetan Plateau (TP) by 16S rRNA and metagenomic sequencing. We found that G. menyuanensis, which inhabits a lower altitude, had a higher richness in gut bacteria than that of G. alpherakii, which inhabits a higher altitude, whereas G. alpherakii exhibited more unique gut bacteria. The gut bacteria of G. menyuanensis and G. alpherakii were dominated by Proteobacteria and Firmicutes, respectively. Wolbachia and Pseudomonas were significantly more abundant in G. menyuanensis, while Carnobacterium, Enterococcus and Bacillus were more abundant in G. alpherakii. Metagenomic analysis showed that genes involved in metabolism were most enriched in both Gynaephora species. Functional differences were found in four KEGG pathways (metabolism, genetic information processing, environmental information processing, and cellular processes) between the two Gynaephora species. For the metabolism pathway, genes involved in global and overview maps, energy metabolism and metabolism of cofactors and vitamins were more enriched in G. menyuanensis, whereas in G. alpherakii, there was enrichment for genes involved in carbohydrate metabolism, amino acid metabolism and glycan biosynthesis and metabolism. These results indicated that the two Gynaephora species occurring at different altitudes had evident compositional and functional differences in gut bacteria, likely associated with ecological adaptation. Overall, investigation of the composition and function of Gynaephora species opens a new horizon for understanding the gut bacteria, which may provide valuable information about probiotics and control of Gynaephora as a pest.

Sequence and N-glycan diversity analysis of immunoglobulin G from buffalo milk using RP-UHPLC MS/MS.

Immunoglobulin G is the abundant antibody present in the colostrum and milk of major dairy animals. In the present study, buffalo milk IgG was characterized for its amino acid sequence and glycan diversity using reverse phase liquid chromatography coupled to ESI-Q-TOF MS in tandem mode. Amino acid sequence analysis of heavy chain constant region revealed the presence of two IgG subtypes namely IgG1 and IgG3, with IgG1 being the abundant. The complete light chain constant region sequence was also determined. N-glycan sequence analysis at a highly conserved site Asn-Ser-Thr revealed the presence of mainly biantennary complex type with core fucosylation (34%), bisecting GlcNAc (19%) and sialylation with both Neu5Ac and Neu5Gc (14%). The observed glycan diversity in buffalo milk IgG is in part comparable with bovine colostrum as well as human, bovine, goat serum counterparts.

Trafficking of JC virus-like particles across the blood-brain barrier.

Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood–brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.

The Nontypeable Haemophilus influenzae Major Adhesin Hia Is a Dual-Function Lectin That Binds to Human-Specific Respiratory Tract Sialic Acid Glycan Receptors.

NTHi is a human-adapted pathogen that colonizes the human respiratory tract. Strains of NTHi express multiple adhesins; however, there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1 and HMW2 (HMW1/2). Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein that has a critical role in colonization of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2-3- and 2-6-linked sialic acid glycans found in the human respiratory tract. Here, we show that the high-affinity binding domain of Hia, binding domain 1 (BD1), is responsible for binding to α2-6-sialyllactosamine (2-6 SLN) glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower-affinity binding activity for 2-3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and HMW2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of this major NTHi adhesin and putative vaccine candidate will direct and inform development of future vaccines and therapeutic strategies for this important pathogen.IMPORTANCE Host-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work, we show that the major NTHi adhesin Hia preferentially binds to Neu5Ac-α2-6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for microbes that colonize the human airway.

De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs.

A number of studies have shown that glycosylation of proteins plays diverse functions in the lives of organisms, has crucial biological and physiological roles in pathogen–host interactions, and is involved in a large number of biological events in the immune system, and in virus and bacteria recognition. The large amount of scientific interest in glycoproteins of molluscan hemocyanins is due not only to their complex quaternary structures, but also to the great diversity of their oligosaccharide structures with a high carbohydrate content (2–9%). This great variety is due to their specific monosaccharide composition and different side chain composition. The determination of glycans and glycopeptides was performed with the most commonly used methods for the analysis of biomolecules, including peptides and proteins, including Matrix Assisted Laser Desorption/Ionisation–Time of Flight (MALDI-TOF-TOF), Liquid Chromatography - Electrospray Ionization-Mass Spectrometry (LC/ESI-MS), Liquid Chromatography (LC-Q-trap-MS/MS) or Nano- Electrospray Ionization-Mass Spectrometry (nano-ESI-MS) and others. The molluscan hemocyanins have complex carbohydrate structures with predominant N-linked glycans. Of interest are identified structures with methylated hexoses and xyloses arranged at different positions in the carbohydrate moieties of molluscan hemocyanins. Novel acidic glycan structures with specific glycosylation positions, e.g., hemocyanins that enable a deeper insight into the glycosylation process, were observed in Rapana venosa, Helix lucorum, and Haliotis tuberculata. Recent studies demonstrate that glycosylation plays a crucial physiological role in the immunostimulatory and therapeutic effect of glycoproteins. The remarkable diversity of hemocyanin glycan content is an important feature of their immune function and provides a new concept in the antibody–antigen interaction through clustered carbohydrate epitopes.

Comprehensive microbiome and metabolome analyses reveal the physiological mechanism of chlorotic Areca leaves

As an important economic crop in tropical areas, Areca catechu L. affects the livelihood of millions of farmers. The Areca yellow leaf phenomenon (AYLP) leads to severe crop losses and plant death. To better understand the relationship of microbes and chlorotic Areca leaves, microbial community structure as well as its correlation with differential metabolites was investigated by high-throughput sequencing and metabolomic approaches. High-throughput sequencing of the internal transcribed spacer 1 and 16S rRNA gene revealed that fungal diversity was dominated by Ascomycota and the bacterial community consisted of Proteobacteria as well as Actinobacteria. The microbiota structure on chlorotic Areca leaves exhibited significant changes based on non-metric multidimensional scaling analysis, which were attributed to 477 bacterial genera and 183 fungal genera. According to the results of the Kruskal-Wallis test, several potential pathogens were enriched on chlorotic Areca leaves. Further analysis based on metabolic pathways predicted by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States revealed the metabolism of half-yellow leaves and yellow leaves microbiota were significantly elevated in amino acid metabolism, carbohydrate metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, partial xenobiotics biodegradation and metabolism. Furthermore, 22 significantly variable metabolites in Areca leaves were identified by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry and statistical analysis. Moreover, we further investigated the correlation between the predominant microbes and differential metabolites. Taken together, the association between AYLP and microbiome of Areca leaves was explored from the microecological perspective by omics techniques, and these findings provide new insights into possible prevention, monitoring and control of AYLP in the future.

N-glycomic profiling of colorectal cancer according to tumor stage and location.

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.

Biosynthesis and Export of Bacterial Glycolipids.

Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.

Transcriptional analysis reveals physiological response to acute acidification stress of barramundi Lates calcarifer (Bloch) in coastal areas.

To understand the physiological response of estuarine fish to acidification, barramundi (Lates calcarifer) juveniles were exposed to acidified seawater in experimental conditions. The molecular response of barramundi to acidification stress was assessed by RNA-seq analysis. A total of 2188 genes were identified as differential expression genes. The gene ontology classification system and Kyoto Encyclopedia of Genes and Genomes database analysis showed that acidification caused differential expressions of genes and pathways in the gills of barramundi. Acidification had a great influence on the signal transduction pathway in cell process. Furthermore, we detected that numerous unigenes involved in the pathways associated with lipid metabolism, carbohydrate metabolism, amino acid metabolism, glycan biosynthesis and metabolism specific and non-specific immunity were changed. This study indicates that the physiological responses in barramundi especially the immune system and energy allocation correspond to the variation of environmental pH. This study reveals the necessity for assessment of the potential of estuarine fishes to cope with acidification of the environment and the need to develop strategies for fish conservation in coastal areas.

Bioinformatics studies on a function of the SARS-CoV-2 spike glycoprotein as the binding of host sialic acid glycans

SARS-CoV and SARS-CoV-2 do not appear to have functions of a hemagglutinin and neuraminidase. This is a mystery, because sugar binding activities appear essential to many other viruses including influenza and even most other coronaviruses in order to bind to and escape from the glycans (sugars, oligosaccharides or polysaccharides) characteristic of cell surfaces and saliva and mucin. The S1 N terminal Domains (S1-NTD) of the spike protein, largely responsible for the bulk of the characteristic knobs at the end of the spikes of SARS-CoV and SARS-CoV-2, are here predicted to be “hiding” sites for recognizing and binding glycans containing sialic acid. This may be important for infection and the ability of the virus to locate ACE2 as its known main host cell surface receptor, and if so it becomes a pharmaceutical target. It might even open up the possibility of an alternative receptor to ACE2. The prediction method developed, which uses amino acid residue sequence alone to predict domains or proteins that bind to sialic acids, is naïve, and will be advanced in future work. Nonetheless, it was surprising that such a very simple approach was so useful, and it can easily be reproduced in a very few lines of computer program to help make quick comparisons between SARS-CoV-2 sequences and to consider the effects of viral mutations.

Abstract A95: A genome-wide CRISPR screen identifies novel ligands for the Siglec family of glyco-immune checkpoint receptors

Abstract Cancer cells are frequently characterized by extensive remodeling of their cell surface glycocalyx, the complex pattern of sugar modifications that decorate cell surface proteins. In particular, cancer cells often overexpress an unusually high density of sialic acid glycans on their plasma membrane. This modification shields tumors from immune detection through engagement of inhibitory Siglec (Sialic acid-binding immunoglobulin-type lectin) receptors, which are widely expressed in innate immune cells. Blockade of these Siglec-based immune checkpoints has shown the potential to increase immune activity against highly sialylated cancer cells. The specific glycosylated ligands that serve as counter-receptors for Siglecs, however, are not well characterized. Here we describe a strategy for better defining these immune checkpoints using a CRISPR-based genetic screening approach. A library of gRNAs was screened using FACS to identify genes whose ablation significantly reduced cell-surface binding of recombinantly expressed, fluorescently labeled Siglecs. We thereby generated a list of genes with putative roles in driving the cell-surface expression of sialic acid-containing Siglec ligands. Our screen correctly identified known genes involved in sialic acid biosynthesis, validating the specificity of this approach. Additionally, this screen revealed that knockdown of a single heavily O-glycosylated protein plasma membrane protein dramatically reduced binding of Siglec7, the Siglec receptor expressed specifically in NK cells. Follow-up work has shown that Siglec7 binds to this ligand with high affinity and that genetic blockade of this interaction potentiates NK cell killing activity in vitro. This study has thus defined a novel and specific glyco-immune interaction with potential significance in driving tumor escape from innate immune surveillance. Citation Format: Simon Wisnovsky, Carolyn Bertozzi. A genome-wide CRISPR screen identifies novel ligands for the Siglec family of glyco-immune checkpoint receptors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A95.