Addition Of Galactose Research Articles

Unique Glycans in Synaptic Glycoproteins in Mouse Brain.

The synapse is an essential connection between neuronal cells in which the membrane and secreted glycoproteins regulate neurotransmission. The post-translational modifications of glycoproteins with carbohydrates, although essential for their functions as well as their specific localization, are not well understood. Oddly, whereas galactose addition to glycoproteins is required for neuronal functions, galactosylation is severely restricted for Asn-linked on N-glycans in the brain, and genetic evidence highlights the important roles of galactose in brain functions and development. To explore this novel glycosylation, we exploited an orthogonal technology in which a biotinylated sialic acid derivative (CMP-biotin-Sia) is transferred to terminally galactosylated proteins by a recombinant sialyltransferase (rST6Gal1). This approach allowed us to identify the carrier proteins as well as their localization on brain sections. Immunohistochemical analysis of the biotinylated glycoproteins in brain sections demonstrates that they are largely positioned in the pre- and postsynaptic membranes. Consistent with this positioning, glycoproteomic analyses of the labeled glycoproteins identified a number of them that are involved in synaptic function, cell adhesion, and extracellular matrix interactions. The discovery of these galactosylated N-glycoproteins and their relative confinement to synapses provide novel insights into the unusual and specific nature of protein glycosylation in the brain.

Mutual Relationships of Nanoconfined Hexoses: Impacts on Hydrodynamic Radius and Anomeric Ratios.

Although all hexose sugars share the same chemical formula, C6H12O6, subtle differences in their stereochemical structures lead to their various biological roles. Due to their prominent role in metabolism, hexose sugars are commonly found in nanoconfined environments. The complexity of authentic nanoconfined biological environments makes it challenging to study how confinement affects their behavior. Here, we present a study using a common model system, AOT reverse micelles, to study hexose sugars in nanoconfinement. We examine how reverse micelles affect the hexoses, how the hexoses affect reverse micelle formation, and the differences between specific hexoses: glucose, mannose, and galactose. We find that addition of glucose, mannose or galactose to reverse micelles that already contain water leaves their size smaller or nearly unchanged. Introducing aqueous hexose solution yields reverse micelles smaller than those prepared with the same volume of water. We use 1H NMR to show how the nanoconfined environment impacts hexose sugars' anomeric ratios. Nanoconfined mannose and galactose display smaller changes in their anomeric ratios compared to glucose. These conclusions may provide insights about the biological roles of each hexose when studied under a more authentic nanoconfined system.

Abstract PR-13: Disruption of sulfatide metabolism by targeting UGT8 is an actionable metabolic vulnerability for pancreatic cancer early interception

Abstract The existence of asymptomatic precursor lesions that are known to predate invasive pancreatic ductal adenocarcinoma (PDAC) by years provides a compelling rationale for cancer interception efforts. One such precursor is intraductal papillary mucinous neoplasm (IPMN). Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) imaging of metabolites in tissue sections from resected patient IPMN cases (N=23) and from a KrasG12D/GnasR201C mouse model of IPMN, we have made a discovery of long chain hydroxylated sulfatide species as being selectively enriched in IPMN. Spatial transcriptomics analysis of serial sections from the same IPMN tissues confirmed cognate transcripts, including ceramide galactosyltransferase (CGT, also known as UGT8) and galactose-3-O-sulfotransferase (CST, also known as Gal3St1) enzymes that, respectively, catalyze the addition of galactose from UDP-galactose to ceramide and the O-sulfation of the galactose residue on galactosylceramide (GalCer), to also be highly enriched in IPMN. Functional studies using CRISPR/cas9 UGT8 knockout Kras;Gnas IPMN cells demonstrated that loss of UGT8, the key enzyme that produces the sulfatide precursor GalCer, results in attenuation of sulfatide biosynthesis and impaired mitochondrial function, as evidenced by reduced mitochondrial respiration and ATP production. These changes were met with concomitant decreases in proliferation and invasiveness, and increased susceptibility to intrinsic apoptosis. Targeting of sulfatide metabolism using a selective small molecule inhibitor of UGT8 suppressed sulfatide biosynthesis, induced ceramide-mediated compensatory mitophagy, and subsequent apoptosis in Kras;Gnas IPMN cells. In vivo, UGT8 inhibition suppressed tumor growth in an IPMN allograft model. In conclusion, our work identifies enhanced sulfatide metabolism as an early metabolic alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia and represents an actionable vulnerability for PDAC early interception. Citation Format: Riccardo Ballarò, Yihui Chen, Fredrik I Thege, Rongzhang Dou, Jimin Min, Michele Yip-Schneider, Jianjun Zhang, Ranran Wu, Ehsan Irajizad, Yuki Makino, Kimal I Rajapakshe, Mark W Hurd, Ricardo A León-Letelier, Edwin Ostrin, Jody Vykoukal, Jennifer B Dennison, Kim-Anh Do, Samir Hanash, Robert A Wolff, Paola A Guerrero, Michael P Kim, C Max Schmidt, Anirban Maitra, Johannes F Fahrmann. Disruption of sulfatide metabolism by targeting UGT8 is an actionable metabolic vulnerability for pancreatic cancer early interception [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-13.

Precision control of ammonium release in Azotobacter vinelandii.

The capture and reduction of atmospheric dinitrogen gas to ammonium can be accomplished through the enzyme nitrogenase in a process known as biological nitrogen fixation (BNF), by a class of microbes known as diazotrophs. The diazotroph Azotobacter vinelandii is a model organism for the study of aerobic nitrogen fixation, and in recent years has been promoted as a potential producer of biofertilizers. Prior reports have demonstrated the potential to partially deregulate BNF in A. vinelandii, resulting in accumulation and extracellular release of ammonium. In many cases, deregulation requires the introduction of transgenic genes or elements to yield the desired phenotype, and the long-term stability of these strains has been reported to be somewhat problematic. In this work, we constructed two strains of A. vinelandii where regulation can be precisely controlled without the addition of any foreign genes or genetic markers. Regulation is maintained through native promoters found in A. vinelandii that can be induced through the addition of extraneous galactose. These strains result in varied degrees of regulation of BNF, and as a result, the release of extracellular ammonium is controlled in a precise, and galactose concentration-dependent manner. In addition, these strains yield high biomass levels, similar to the wild-type A. vinelandii strain and are further able to produce high percentages of the bioplastic polyhydroxybutyrate.

Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host’s immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.Graphical abstractIllustration of GnT-I activity. (a) Transfer of GlcNAc to Man5-PA, creating Man5GlcNAc3-PA. (b) Transfer of GlcNAc to MM-PA, creating MGn-PA. (c) Transfer of GlcNAc to GnM-PA, creating GnGn-PA. Blue squares represent N-acetylglucosamine, green cycles depict mannose. Graphic illustration of N-glycans were created using bioRENDER.

Mapping of the gene network that regulates glycan clock of ageing.

Glycans are an essential structural component of immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation), a trait which was shown to be associated with ageing. We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in the in vitro IgG expression system. Upregulation of three genes, EEF1A1, MANBA and TNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in this in vitro expression system.

Differential metabolites screening in yak (Bos grunniens) seminal plasma after cryopreservation and the evaluation of the effect of galactose on post-thaw sperm motility

Sperm survival and activity depend on the provision of energy and nutrients from seminal plasma (SP). This study aimed to investigate the variations of metabolites within SP before and after freezing and subsequently explore the potential regulatory mechanisms affecting yak sperm cryodamage due to changes in metabolites in the SP. Untargeted metabolomics analysis was performed to screen for differential metabolites, followed by KEGG analysis to identify enriched signaling pathways. The combinatorial analysis of metabolomics and sperm proteomics revealed the influence of key SP metabolites on sperm proteins. Subsequently, the relevant differentially expressed proteins were verified by Western blot analysis. Finally, the mechanism underlying the positive effect of galactose on sperm motility was determined by assessing the change in ATP content in sperm before and after freezing and thawing. The data showed that a total of 425 and 269 metabolites were identified in the positive and negative ion modes, respectively. Freezing and thawing resulted in the up-regulation of 70 metabolites and the down-regulation of 29 metabolites in SP. The primary impact of freezing and thawing was observed in carbohydrate metabolism, including pyruvate metabolism, pentose phosphate pathway, galactose metabolism, the TCA cycle, and butanoate metabolism. In the combined analysis and Western blot results, a significant positive correlation was observed between galactose and Aldo-keto reductase family 1 member B1 (AKR1B1) (P < 0.05), which has the ability to convert galactose into galactol. Furthermore, the addition of galactose to thawed semen improved sperm motility by increasing AKR1B1 protein in sperm and was associated with the content of ATP. These data identify differential metabolites between fresh and frozen-thawed SP and suggest that galactose is a valuable additive for cryopreserved sperm, providing a theoretical basis for further exploration of the refrigerant formula for yak sperm cryopreservation.

In-Depth Mass Spectrometry Analysis Reveals the Plasma Proteomic and N-Glycoproteomic Impact of an Amish-Enriched Cardioprotective Variant in B4GALT1

B4GALT1 encodes β-1,4-galactosyltransferase 1, an enzyme that plays a major role in glycan synthesis in the Golgi apparatus by catalyzing the addition of terminal galactose. Studies increasingly suggest that B4GALT1 may be involved in the regulation of lipid metabolism pathways. Recently, we discovered a single-site missense variant Asn352Ser (N352S) in the functional domain of B4GALT1 in an Amish population, which decreases the level of LDL-cholesterol (LDL-c) as well as the protein levels of ApoB, fibrinogen, and IgG in the blood. To systematically evaluate the effects of this missense variant on protein glycosylation, expression, and secretion, we developed a nano-LC-MS/MS-based platform combined with TMT-labeling for in-depth quantitative proteomic and glycoproteomic analyses in the plasma of individuals homozygous for the B4GALT1 missense variant N352S versus non-carriers (n = 5 per genotype). A total of 488 secreted proteins in the plasma were identified and quantified, 34 of which showed significant fold changes in protein levels between N352S homozygotes and non-carriers. We determined N-glycosylation profiles from 370 glycosylation sites in 151 glycoproteins and identified ten proteins most significantly associated with decreased galactosylation and sialyation in B4GALT1 N352S homozygotes. These results further support that B4GALT1 N352S alters the glycosylation profiles of a variety of critical target proteins, thus governing the functions of these proteins in multiple pathways, such as those involved in lipid metabolism, coagulation, and the immune response.

Galactosyl, alkyl, and acidic groups modify uptake and subcellular deposition of pyropheophorbide-a by epithelial tumor cells and determine photosensitizing efficacy

Photosensitizers currently used for photodynamic therapy of cancer show enhanced accumulation in tumor tissue but lack cancer cell specificity. To augment cellular uptake, the targeting of pyropheophorbide-a to carbohydrate-binding components of cancer cells was explored. Galactose was attached to pyropheophorbide-a at positions 172 and 20. Since the modification at position 172 removed a carboxylic group, which is relevant for cell specificity, this study evaluated the relative contribution of galactosyl and carboxyl groups at either position 172 or 20, with or without a (hexyloxy)ethyl at position 3, to cellular uptake by human epithelial cancer cells. The subcellular deposition was monitored using fluorescence microscopy and the photoreaction was quantified using biomarkers. The results demonstrated that any galactose addition suppresses transmembrane diffusion and promotes endocytosis and lysosomal accumulation. An anionic group at position 172 or 20 enhances lysosomal retention. Neutralization of the carboxylic group at position 172 facilitates transfer to mitochondria/endoplasmatic reticulum and promotes tumor cell-specific retention. Replacing (hexyloxy)ethyl with an ethyl group at position 3 increased both cellular uptake and egress but did not alter subcellular localization. These findings suggest that specific neutral galactosylated pheophorbides may provide an enhanced therapeutic effect for those tumor types that do not retain unmodified pyropheophorbide. However, the galactose conjugates also serve as substrates for preferential uptake by liver cells resulting in hepatic sequestration, reduced systemic distribution, and lower accumulation in tumor tissue.

Abstract 6301: Post-translational modifications regulate sensitivity to Polatuzumab Vedotin in DLBCL

Abstract Diffuse Large B-Cell Lymphoma (DLBCL) is an aggressive malignancy of which one-third of diagnosed patients ultimately relapse and die. New treatment paradigms for relapsed-refractory patients are needed. Most DLBCL tumors are reliant on signaling through their cell surface B-cell receptor (BCR) mediated by ITAMs on CD79A and CD79B to drive anti-apoptotic NF-kB and PI3 kinase signaling. In order to understand how surface BCR is regulated, we performed whole genome CRISPR screens in 9 DLBCL cell lines in which we sorted for high and low surface CD79B or added the CD79B targeted antibody-drug conjugate Polatuzumab-Vedotin (POLA-V). Strikingly, independent of genetic or cell of origin subtype, sgRNAs encoding the entire N-linked glycosylation pathway and specific sugar modification by addition of galactose and sialic acid were found to be enriched in high CD79B fractions and depleted in POLA-V treated samples compared to control. Further testing showed that removing sialic acid by treating with a small molecule inhibitor of sialic acid, genetic perturbation of golgi sugar transporters SLC35A1 and SLC35A2, or the a2,6 sialyltransferase ST6GAL1 resulted in more surface CD79B antibody binding with no significant change in CD79B protein abundance. The specificity of the effect of sialic acid loss on POLA antibody binding was confirmed by removing cell surface sialic acid with exogenous sialidase and observing increased binding of POLA, but not other therapeutic antibodies like Rituximab or CD19 FMC-63. Loss of SLC35A1, SLC35A2, and ST6GAL1 synergized with POLA-V but not the drug conjugate MMAE alone, showing a BCR dependent mechanism of sialic acid loss on POLA-V sensitization. In a further cell of origin subtype analysis of the germinal center B-cell (GCB) DLBCL cell lines, loss of the E3 ligase KLHL6 was found to synergize with POLA-V. Importantly, KLHL6 is heavily mutated across all germinal center derived malignancies, is more highly expressed in germinal center B-cells than other members of the B-cell lineage, and plays an unknown role in the development of autoimmune diseases. Further work will give evidence for the target of KLHL6 mediated ubiquitination and will show how this gene can drive lymphoma and autoimmune disease development in the germinal center. Citation Format: Sean R. Corcoran, Louis M. Staudt, Daniel Hodson. Post-translational modifications regulate sensitivity to Polatuzumab Vedotin in DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6301.

Synthesis of O-Linked Glycoconjugates in the Nervous System.

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids are glycosylated by distinct glycosylation enzymes as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent careful analyses by MS have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport, metabolic stress responses, cell-cell interactions and so on. Discoveries of human diseases associated with glycosylation enzyme deficits have proved modification of lipids and proteins with carbohydrates play critical roles in human health and disease in the nervous systems.

Phosphatase control of cytokine-mediated overproduction of galactose-deficient IgA1, the main autoantigen in IgA nephropathy

IgA nephropathy (IgAN) is an autoimmune disease characterized by the deposition of galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes in the kidneys. Elevated serum levels of Gd-IgA1, the main autoantigen in IgAN, are associated with mucosal infections and poor renal outcome in IgAN patients, but little is known about the activation of IgA1-secreting cells overproducing this autoantigen. We found that in peripheral blood mononuclear cells (PBMCs), cytokine stimulation elevated Gd-IgA1 production in B cells from IgAN patients but not in those from healthy controls (p < 0.01). These results were replicated in immortalized B cells derived from PBMCs of IgAN patients and healthy controls. Using single-cell transcriptomics, we identified subsets of IgA1-secreting cells from IgAN patients, but not from healthy controls, with decreased expression of C1GALT1 in response to cytokine stimulation. The C1GALT1-encoded glycosyltransferase is responsible for addition of galactose to IgA1 O-glycans, and its reduced activity is associated with elevated serum levels of Gd-IgA1. These newly identified subsets of IgA1-secreting cells with reduced C1GALT1 expression exhibited reduced expression of several genes related to cytokine-mediated signaling, including those encoding phosphatases, such as SOCS1. siRNA knock-down of SOCS1, and the related SOCS3, increased Gd-IgA1 production in cells derived from PBMCs of healthy controls, indicating a role of these regulators in abnormal cytokine signaling and Gd-IgA1 overproduction. These results revealed that specific subsets of IgA1-secreting cells may be responsible for autoantigen production in IgAN due to abnormal regulation of cytokine-mediated signaling, a process that may occur in inflammatory responses in IgAN patients.

Yeast-Mycelial Dimorphism in Pichia pastoris SMD1168 Is Triggered by Nutritional and Environmental Factors

This study reports, for the first time, morphological transition from yeast-like to filamentous form, normally associated with pathogenicity/increased protein secretion, in Pichia pastoris SMD1168 strain. The response was recorded in response to nutritional and environmental cues. The factors affecting this switch were extracellular pH (under nitrogen starvation conditions), carbon and nitrogen source under nitrogen- and carbon-limiting conditions respectively. Under nitrogen-limiting conditions, addition of fructose and sucrose in the culture medium induced filamentous morphology in a segregated form whereas addition of galactose led to a mixture of yeast and the filamentous form of the cells. Under carbon-limiting conditions, isoleucine and proline forced a filamentous form whereas glycine, valine, alanine and phenylalanine promoted yeast-like morphology. Similar dimorphic shift was also displayed by a recombinant methanol slow utilizing (Muts) strain (SMD-GCSF Muts) producing human granulocyte colony-stimulating factor in response to change in the initial inoculum level. Analysis of the extracellular metabolome by GC-MS indicated that several amino acids (leucine, proline, tyrosine), carboxylic acids (phenylacetic-, propanoic acid), alcohols and butylamine were present at different levels in the culture broth of the two morphological forms. High accumulation of proline and butylamine was seen in the extracellular culture filtrate of the filamentous form of the yeast. Presence of quorum-sensing molecules (phenylethyl alcohol, dodecanol) suggested complex network of pathways involved in this morphological transition.

Optimization of the Conditions for Cultivation of Yersinia pseudotuberculosis in the Process of Obtaining Cell Mass

Objective of the study was to optimize the conditions of submerged recurrent cultivation and the composition of the nutrient medium for obtaining the cell mass of Yersinia pseudotuberculosis strains, used as an adsorbent in the preparation of diagnostic plague immunoglobulins. Materials and methods. We utilized Y. pseudotuberculosis adsorbent strains 6; 31; 68; 69; and 70 belonging to V, I, III, IV, and V serotypes, respectively, received from the State Collection of Pathogenic Bacteria at the premises of the RusRAPI “Microbe”. The cultivation process was carried out on an incubator-shaker, laboratory and pilot fermenters with variation of the process parameters, different options for feeding and nutrient media. Results and discussion. In the course of work, the optimal parameters of recurring submerged cultivation have been established. It was found that the highest biomass yield is provided by a combination of a nutrient medium – a carbon substrate in the form of a broth, based on an enzymatic fibrin hydrolysate, with the addition of galactose as a substrate feeding. Thereat, the morphology and immunochemical properties of microbial cells obtained through modified preparation process do not differ from those produced in the control run. The optimization of the parameters for Y. pseudotuberculosis cell mass cultivation with subsequent upscaling has been performed.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

SUMMARYGalactose toxicity (Gal‐Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP‐galactose (UDP‐Gal) and UDP‐glucose is catalyzed by the enzymes galactokinase, UDP‐sugar pyrophosphorylase (USP) and UDP‐galactose 4‐epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose‐mediated toxicity in plants. In this study, we investigated the role of galactose‐containing metabolites like galactose‐1‐phosphate (Gal‐1P) and UDP‐Gal in Gal‐Tox. Recently published data from mouse models suggest that a reduction of the Gal‐1P level via an mRNA‐based therapy helps to overcome Gal‐Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal‐1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L−1 Gal). Nucleotide sugar analysis via high‐performance liquid chromatography‐mass spectrometry revealed highly elevated UDP‐Gal levels in roots of seedlings grown on 1.5 mmol L−1 galactose versus 1.5 mmol L−1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal‐feeding conditions, indicating that glycoproteins are a major target for elevated UDP‐Gal levels in plants.

Glucose-Induced Biofilm Formation in Bacillus thuringiensis KPWP1 is Associated with Increased Cell Surface Hydrophobicity and Increased Production of Exopolymeric Substances.

Bacillus thuringiensis is an agriculturally and medically important bacteria as it produces insecticidal Cry proteins and can form biofilm on different plant surfaces. Previous studies reported that the ubiquitous carbon source glucose could induce restricted motility and fractal pattern formation in the growing colonies of pH, salt and arsenate tolerant Bacillus thuringiensis KPWP1. As bacteria are evolved with the ability to exhibit multicellular behavior and biofilm formation under limiting conditions for survival, the present study was focused on exploring the effect of glucose in biofilm formation by Bacillus thuringiensis KPWP1. A significant rise in biofilm loads was observed with increased glucose concentrations in growth media. Compared to control, six times more biofilm load was marked in presence of 2% of glucose. Interestingly, it was observed that the effect was glucose specific and also not due to any change in the sugar-induced physicochemical property of the growth media as the addition of galactose or arabinose could not induce any significant increase in KPWP1 biofilm load. Scanning electron-, confocal laser scanning-microscopic studies and biochemical tests revealed that increased concentrations of glucose could induce increased production of exopolymeric substances, increased number of densely-packed micro-colonies in KPWP1 biofilm and increased hydrophobicity and adherence properties in KPWP1cells.

Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome.

Complex dietary carbohydrate structures including β(1–4) galacto-oligosaccharides (GOS) are resistant to digestion in the upper gastrointestinal (GI) tract and arrive intact to the colon where they benefit the host by selectively stimulating microbial growth. Studies have reported the beneficial impact of GOS (alone or in combination with other prebiotics) by serving as metabolic substrates for modulating the assembly of the infant gut microbiome while reducing GI infections. N-Acetyl-D-lactosamine (LacNAc, Galβ1,4GlcNAc) is found in breast milk as a free disaccharide. This compound is also found as a component of human milk oligosaccharides (HMOs), which have repeating and variably branched lactose and/or LacNAc units, often attached to sialic acid and fucose monosaccharides. Human glycosyl-hydrolases do not degrade most HMOs, indicating that these structures have evolved as natural prebiotics to drive the proper assembly of the infant healthy gut microbiota. Here, we sought to develop a novel enzymatic method for generating LacNAc-enriched GOS, which we refer to as humanized GOS (hGOS). We showed that the membrane-bound β-hexosyl transferase (rBHT) from Hamamotoa (Sporobolomyces) singularis was able to generate GOS and hGOS from lactose and N-Acetyl-glucosamine (GlcNAc). The enzyme catalyzed the regio-selective, repeated addition of galactose from lactose to GlcNAc forming the β-galactosyl linkage at the 4-position of the GlcNAc and at the 1-position of D-galactose generating, in addition to GOS, LacNAc, and Galactosyl-LacNAc trisaccharides which were produced by two sequential transgalactosylations. Humanized GOS is chemically distinct from HMOs, and its effects in vivo have yet to be determined. Thus, we evaluated its safety and demonstrated the prebiotic's ability to modulate the gut microbiome in 6-week-old C57BL/6J mice. Longitudinal analysis of gut microbiome composition of stool samples collected from mice fed a diet containing hGOS for 5 weeks showed a transient reduction in alpha diversity. Differences in microbiome community composition mostly within the Firmicutes phylum were observed between hGOS and GOS, compared to control-fed animals. In sum, our study demonstrated the biological synthesis of hGOS, and signaled its safety and ability to modulate the gut microbiome in vivo, promoting the growth of beneficial microorganisms, including Bifidobacterium and Akkermansia.

Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation.

Monoclonal antibodies are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute that has ramifications for patient safety and physiological efficacy-one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-F-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from Days 4 to 7 in a batch process used for IgG1 production from Chinese hamster ovary cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by ∼30%. The supplements caused significant changes in gene expression by Day 4 of the cultures where 2FP addition significantly reduced fucosyltransferase 8 and nucleotide sugar transporter gene expression (by ∼2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1-6 genes (by 1.5-3-fold). These gene expression data alongside glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes.

β1,4-Galactosyltransferase V Modulates Breast Cancer Stem Cells through Wnt/β-catenin Signaling Pathway.

PurposeBreast cancer stem cells (BCSCs) contribute to the initiation, development, and recurrence of breast carcinomas. β1,4-Galactosyltransferase V (B4GalT5), which catalyzes the addition of galactose to GlcNAcβ1-4Man of N-glycans, is involved in embryogenesis. However, its role in the modulation of BCSCs remains unknown.Materials and MethodsThe relationship between B4GalT5 and breast cancer stemness was investigated by online clinical databases and immunohistochemistry analysis. Mammosphere formation, fluorescence-activated cell sorting (FACS), and in-vivo assays were used to evaluate B4GalT5 expression in BCSCs and its effect on BCSCs. B4GalT5 regulation of Wnt/β-catenin signaling was examined by immunofluorescence and Ricinus communis agglutinin I pull-down assays. Cell surface biotinylation and FACS assays were performed to assess the association of cell surface B4GalT5 and BCSCs.ResultsB4GalT5, but not other B4GalTs, was highly correlated with BCSC markers and poor prognosis. B4GalT5 significantly increased the stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and promoted the production of CD44+CD24–/low cells and the formation of mammospheres. Furthermore, B4GalT5 overexpression resulted in dramatic tumor growth in vivo. Mechanistically, B4GalT5 modified and protected Frizzled-1 from degradation via the lysosomal pathway, promoting Wnt/β-catenin signaling which was hyperactivated in BCSCs. B4GalT5, located on the surface of a small subset of breast carcinoma cells, was not responsible for the stemness of BCSCs.ConclusionB4GalT5 modulates the stemness of breast cancer through glycosylation modification to stabilize Frizzled-1 and activate Wnt/β-catenin signaling independent of its cell surface location. Our studies highlight a previously unknown role of B4GalT5 in regulating the stemness of breast cancer and provide a potential drug target for anticancer drug development.

A photoelectrochemical biosensor based on the direct electron transfer to galactose oxidase

A novel photoelectrochemical biosensor for quantitative detection of galactose was successfully developed by immobilizing galactose oxidase on TiO2 nanorod arrays modified F-doped tin oxide (FTO) electrode. Specifically, the direct electron transfer to galactose oxidase was achieved. TiO2 nanorod arrays directly grew on FTO electrode from peroxotitanium complex precursor under low-temperature and normal-pressure conditions. The large surface area and good uniformity of TiO2 nanorod arrays facilitated direct electron transfer between the electrode surface and galactose oxidase. The photocurrent spectra of as-prepared biosensor showed an obvious photocurrent response under UV light irradiation. The generated photocurrent of the stable platform was significantly enhanced after the addition of galactose in solution and the photocurrent intensity possessed linear relationship with the galactose concentration. Moreover, CaCl2, uric acid and ascorbic acid had no interference with the detection of galactose on the working electrode. This sensor can be reused and measure the concentration of galactose in lactose-free milk.