Glycosyltransferase Genes Research Articles

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

The released polysaccharide inhibits cell aggregation and biofilm formation in the cyanobacterium Synechocystis sp. PCC 6803

ABSTRACT Cyanobacteria secrete exopolysaccharide (EPS), which can be intimately associated with the cell surfaces (known as capsular polysaccharide, CPS), or released into the surrounding environment (released polysaccharide, RPS). The aim of this study was to explore the role of EPS in cell aggregation and biofilm formation in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Three mutants (Δsll5043, Δslr1063 and Δslr1076) were obtained through disrupting three putative glycosyltransferase genes (sll5043, slr1063 and slr1076) separately. Decreased contents and altered monosaccharide composition of both RPS and CPS were observed in the three mutants compared with the wild type (WT). RPS from the WT (WT-RPS) had high emulsification activity, while RPSs from the mutants had no emulsification activity. Unlike the WT, the three mutants formed cell aggregates and biofilms. Compared with the WT, the three mutants possessed lower cell hydrophobicity and less negative cell surface charge, which could result from the altered production of CPS. 40 μg ml–1 WT-RPS significantly decreased cell hydrophobicity of the three mutants, and significantly inhibited cell aggregation and biofilm formation, while the RPS from each mutant itself had no effect on cell hydrophobicity of the mutant, and did not affect cell aggregation and biofilm formation. The inhibitory effect of WT-RPS on cell aggregation and biofilm formation was thus suggested to be related to its ability to reduce cell hydrophobicity. The difference in cell aggregation and biofilm formation between the WT and the three mutants could be related to differences in cell hydrophobicity, cell surface charge and physicochemical properties of RPS. Our results in this study indicated that both CPS and RPS affected cell aggregation and biofilm formation in Synechocystis sp. strain PCC 6803, and WT-RPS inhibited cell aggregation and biofilm formation.

Elongating maize root: zone-specific combinations of polysaccharides from type I and type II primary cell walls

The dynamics of cell wall polysaccharides may modulate the cell wall mechanics and thus control the expansion growth of plant cells. The unique composition of type II primary cell wall characteristic of grasses suggests that they employ specific mechanisms for cell enlargement. We characterized the transcriptomes in five zones along maize root, clustered the expression of genes for numerous glycosyltransferases and performed extensive immunohistochemical analysis to relate the changes in cell wall polysaccharides to critical stages of cell development in Poaceae. Specific patterns of cell wall formation differentiate the initiation, realization and cessation of elongation growth. Cell walls of meristem and early elongation zone represent a mixture of type I and type II specific polysaccharides. Xyloglucans and homogalacturonans are synthesized there actively together with mixed-linkage glucans and glucuronoarabinoxylans. Rhamnogalacturonans-I with the side-chains of branched 1,4-galactan and arabinan persisted in cell walls throughout the development. Thus, the machinery to generate the type I primary cell wall constituents is completely established and operates. The expression of glycosyltransferases responsible for mixed-linkage glucan and glucuronoarabinoxylan synthesis peaks at active or late elongation. These findings widen the number of jigsaw pieces which should be put together to solve the puzzle of grass cell growth.

Human Natural Antibodies to Mammalian Carbohydrate Antigens as Unsung Heroes Protecting against Past, Present, and Future Viral Infections.

Human natural antibodies to mammalian carbohydrate antigens (MCA) bind to carbohydrate-antigens synthesized in other mammalian species and protect against zoonotic virus infections. Three such anti-MCA antibodies are: (1) anti-Gal, also produced in Old-World monkeys and apes, binds to α-gal epitopes synthesized in non-primate mammals, lemurs, and New-World monkeys; (2) anti-Neu5Gc binds to Neu5Gc (N-glycolyl-neuraminic acid) synthesized in apes, Old-World monkeys, and many non-primate mammals; and (3) anti-Forssman binds to Forssman-antigen synthesized in various mammals. Anti-viral protection by anti-MCA antibodies is feasible because carbohydrate chains of virus envelopes are synthesized by host glycosylation machinery and thus are similar to those of their mammalian hosts. Analysis of MCA glycosyltransferase genes suggests that anti-Gal appeared in ancestral Old-World primates following catastrophic selection processes in which parental populations synthesizing α-gal epitopes were eliminated in enveloped virus epidemics. However, few mutated offspring in which the α1,3galactosyltransferase gene was accidentally inactivated produced natural anti-Gal that destroyed viruses presenting α-gal epitopes, thereby preventing extinction of mutated offspring. Similarly, few mutated hominin offspring that ceased to synthesize Neu5Gc produced anti-Neu5Gc, which destroyed viruses presenting Neu5Gc synthesized in parental hominin populations. A present-day example for few humans having mutations that prevent synthesis of a common carbohydrate antigen (produced in >99.99% of humans) is blood-group Bombay individuals with mutations inactivating H-transferase; thus, they cannot synthesize blood-group O (H-antigen) but produce anti-H antibody. Anti-MCA antibodies prevented past extinctions mediated by enveloped virus epidemics, presently protect against zoonotic-viruses, and may protect in future epidemics. Travelers to regions with endemic zoonotic viruses may benefit from vaccinations elevating protective anti-MCA antibody titers.

Analysis of 1321 Eubacterium rectale genomes from metagenomes uncovers complex phylogeographic population structure and subspecies functional adaptations

BackgroundEubacterium rectale is one of the most prevalent human gut bacteria, but its diversity and population genetics are not well understood because large-scale whole-genome investigations of this microbe have not been carried out.ResultsHere, we leverage metagenomic assembly followed by a reference-based binning strategy to screen over 6500 gut metagenomes spanning geography and lifestyle and reconstruct over 1300 E. rectale high-quality genomes from metagenomes. We extend previous results of biogeographic stratification, identifying a new subspecies predominantly found in African individuals and showing that closely related non-human primates do not harbor E. rectale. Comparison of pairwise genetic and geographic distances between subspecies suggests that isolation by distance and co-dispersal with human populations might have contributed to shaping the contemporary population structure of E. rectale. We confirm that a relatively recently diverged E. rectale subspecies specific to Europe consistently lacks motility operons and that it is immotile in vitro, probably due to ancestral genetic loss. The same subspecies exhibits expansion of its carbohydrate metabolism gene repertoire including the acquisition of a genomic island strongly enriched in glycosyltransferase genes involved in exopolysaccharide synthesis.ConclusionsOur study provides new insights into the population structure and ecology of E. rectale and shows that shotgun metagenomes can enable population genomics studies of microbiota members at a resolution and scale previously attainable only by extensive isolate sequencing.

Aminoglycosides are efficient reagents to induce readthrough of premature termination codon in mutant B4GALNT1 genes found in families of hereditary spastic paraplegia.

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1, in which gradual neurological disorders emerged with aging.

Production and characterization of low molecular weight heparosan in Bacillus megaterium using Escherichia coli K5 glycosyltransferases

Low molecular weight heparosan is an un-sulfated polysaccharide primarily used as a precursor for heparin synthesis that has recently been used in drug delivery applications. Heparosan synthesis from recombinant bacterial systems provides a safer alternative to naturally producing pathogenic bacterial systems. In this study, we engineered a functional heparosan synthesis pathway in Bacillus megaterium by the expression of E. coli K5 kfiC and kfiA glycosyltransferase genes. Upregulation of individual UDP-sugar precursor pathway genes enhanced the heparosan production, indicating that UDP-precursor sugar concentrations were limiting the biosynthesis. The engineered B. megaterium yielded a maximum heparosan concentration of 394 mg/L in batch bioreactor. The heparosan titer was further increased to 1.32 g/L in fed-batch fermentation. Nuclear magnetic resonance analysis revealed that the chemical structure of B. megaterium derived heparosan was identical to E. coli K5 heparosan. The heparosan molecular weight varied from 31 to 60 kDa, indicating its potential as a precursor for chemoenzymatic heparin biosynthesis. This study provides an efficient process to produce heparosan from non-pathogenic B. megaterium.

The wclY gene of Escherichia coli serotype O117 encodes an α1,4-glucosyltransferase with strict acceptor specificity but broad donor specificity.

The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcβ1-3-L-Rhaα1-4-D-Glcα1-4-D-Galβ1-3-D-GalNAcα1-4]n. A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6-tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1,4-Glc-transferase that transfers Glc to the Gal moiety of Galβ1-3GalNAcα-OPO3-PO3-phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1,4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.

MP16-11 HISTOLOGIC AND ENZYMATIC BACKGROUND FOR BIOSYNTHESIS OF ABERRANT GLYCOSYLATED PROSTATE-SPECIFIC ANTIGEN

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-11 HISTOLOGIC AND ENZYMATIC BACKGROUND FOR BIOSYNTHESIS OF ABERRANT GLYCOSYLATED PROSTATE-SPECIFIC ANTIGEN Tohru Yoneyama*, Yuki Tobisawa, Tomokazu Ishikawa, Tomonori Kaneko, Takatoshi Kaya, Shingo Hatakeyama, Hayato Yamamoto, Mihoko Sutoh Yoneyama, Takahiro Yoneyama, Yasuhiro Hashimoto, and Chikara Ohyama Tohru Yoneyama*Tohru Yoneyama* More articles by this author , Yuki TobisawaYuki Tobisawa More articles by this author , Tomokazu IshikawaTomokazu Ishikawa More articles by this author , Tomonori KanekoTomonori Kaneko More articles by this author , Takatoshi KayaTakatoshi Kaya More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Hayato YamamotoHayato Yamamoto More articles by this author , Mihoko Sutoh YoneyamaMihoko Sutoh Yoneyama More articles by this author , Takahiro YoneyamaTakahiro Yoneyama More articles by this author , Yasuhiro HashimotoYasuhiro Hashimoto More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.011AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Although we previously reported that the prostate cancer (PC) diagnostic performance of serum aberrant Siaα2,3Gal-glycosylated prostate-specific antigen (S2,3PSA) test or LacdiNAc-glycosylated PSA (LDN-PSA) test much superior to conventional PSA test (Ishikawa et al., IJMS, 2017; Yoneyama et al., Cancer Sci. 2019), histopathologic and enzymatic background for biosynthesis of aberrant-glycosylated PSA in prostate tissue has still unknown. In this study, we investigate the origin of aberrant-glycosylated PSA in prostate tissues. METHODS: Total RNA and protein were extracted from benign prostate gland and each Gleason pattern of PC tissue that was macro-dissected from FFPE prostate section in 102 patients who underwent radical prostatectomy in Hirosaki University. The expression level of sialylated- and LDN- synthesis-related glycosyltransferase genes were evaluated by droplet digital PCR, and also %S2,3PSA or LDN-PSA/total PSA levels were analyzed by automated immunoassay systems. RESULTS: The expression level of Siaα2,6Gal- synthesis-related α2,6sialyltransferase (ST6GAL1) was significantly decreased in Gleason 4 & 5 tissue compare to Gleason 3 and benign prostate gland. Siaα2,3Gal- synthesis-related α2,3sialyltransferases (ST3GAL3,4,6) was not vary from tumor malignancy. The %S2,3PSA level was also significantly increased in Gleason 4 & 5 tissues suggesting that upregulation of the Siaα2,3Gal- ratio on PSA regulated by ST6GAL1 expression. The expression level of LDN- synthesis-related β1,4-N-acetylgalactosyltransferase 4 in prostate tissue was higher than that of β4GALNT3, and β4GALNT4 expression increased in Gleason 4 & 5 tissues compared to benign prostate gland, suggesting that LDN- synthesis in prostate tissues mainly regulated by β4GALNT4. LDN-PSA/total PSA level was also significantly increased in Gleason 4 & 5 tissues compared to benign prostate gland. Thus, LDN-PSA was mainly synthesized in cancer tissues and increased in high grade Gleason 4 & 5 tissues. CONCLUSIONS: Aberrant glycosylation (Siaα2,3Gal- or LDN-) related glycosyltransferase expression significantly increased in PC tissue with higher Gleason pattern and aberrant glycosylated PSA mainly secreted from PC tissue. Source of Funding: none © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e218-e219 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tohru Yoneyama* More articles by this author Yuki Tobisawa More articles by this author Tomokazu Ishikawa More articles by this author Tomonori Kaneko More articles by this author Takatoshi Kaya More articles by this author Shingo Hatakeyama More articles by this author Hayato Yamamoto More articles by this author Mihoko Sutoh Yoneyama More articles by this author Takahiro Yoneyama More articles by this author Yasuhiro Hashimoto More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement PDF downloadLoading ...

Multi‐omics Studies Reveal Altered Hippocampal N‐Glycosylation in High Fat Diet‐Induced Obese Mice

BackgroundObesity is a major public health issue afflicting over 93 million people in the US and is projected to continue increasing. Impairments of cognitive abilities such as memory loss and mood disorders such as depression and anxiety are debilitating conditions associated with obesity. N‐glycosylation, an essential posttranslational modification of proteins is required for hippocampal long‐term potentiation and memory consolidation. However, whether altered N‐glycosylation is a potential mechanism involved in impaired hippocampal functioning in obesity remains unknown.AimOur goal was to develop a multi‐omics platform including glycomics, proteomics, and transcriptomics to determine the impact of high‐fat‐diet (HFD) consumption on hippocampal N‐glycosylation and expression of cell surface receptors in the context of diet‐induced obesity.HypothesisWe hypothesize that in HFD‐induced obese mice abnormal hippocampal N‐glycosylation occurs, affecting cell surface expression and signaling of key receptors.MethodsC57BL/6J male mice were fed a HFD (45% fat) for 8 weeks to induce obesity or a low‐fat diet (LFD, 10% fat) as the lean control group. Mice were euthanized, and brain tissues collected. Hippocampi were surgically dissected and processed for glycomics, proteomics, glycoproteomics, and RT‐qPCR analysis. Hippocampal cell membrane fractions (MF) were obtained, and N‐glycans released and analyzed by nano‐Liquid Chromatography‐Mass Spectrometry (nanoLC‐MS). Peptides and glycopeptides derived from MF were analyzed by nano‐LC‐FusionOrbitrap MS. Expression level of glycosyltransferase genes were obtained by qPCR.ResultsThe N‐glycome of LFD‐fed mice was dominated by highly branched fucosylated and sialylated N‐glycans (61%) while in HFD‐fed obese mice these N‐glycans were significantly decreased (51%). Correspondingly, transcript analysis of the glycosyltransferases showed that MGAT2 gene, which encodes the enzyme responsible for initiating the synthesis of complex branched N‐glycans, was significantly decreased by 50% in hippocampi of HFD‐fed obese mice compared to lean controls. Our findings show a decrease in complex branched glycans on the hippocampal cell surface of HFD‐obese mice that may be due to the downregulation of the MGAT2 gene. Proteomics and glycoproteomic analysis revealed site‐specific occupancy of over hundreds of hippocampal cell surface receptors.ConclusionsOur results suggest that chronic consumption of HFD and induced obesity results in abnormal hippocampal cell surface N‐glycosylation, and a possible enzyme associated with the altered glycosylation phenotype was identified. Aberrant N‐glycosylation may contribute to altered neuronal or glial cell function. This approach may be useful to identify novel targets for the development of new treatments for obesity‐associated mood and cognitive deficits.Support or Funding InformationFunding from National Institutes of Health, NIDDK (award 5R21DK118379) to M.B.

Enhancing A82846B production by artificial attB-assisted overexpression of orf10\u2013orf11 genes in Kibdelosporangium aridum SIPI-3927

A82846B, producing by Kibdelosporangium aridum, is an important precursor of the semi-synthetic glycopeptide antibiotic Oritavancin. K. aridum produces three components A82846A, B and C, so it is essential to increase A82846B titer and reduce A82846A and C titers by overexpressing halogenase and glycosyltransferase genes. Firstly, we constructed the genetically engineered strain SIPI-3927-attB harboring artificial attB site via homologous recombination. Secondly, two strains SIPI-3927-C1 and C2 were also constructed by integrating halogenase genes vcm8 and orf10 into artificial attB sites of SIPI-3927-attB, respectively. Meantime, three strains SIPI-3927-C3, C4 and C5 containing overexpressing glycosyltransferase A, B and C genes were obtained, respectively. Through fermentation analyses, the results showed that SIPI-3927-C1 and C2 could increase A82846B ratios, in which SIPI-3927-C1 showed a better performance. Moreover, the titer of SIPI-3927-C3 was highest in those of three strains. Finally, the strain SIPI-3927-C6 was constructed by integrating both orf10-encoded halogenase and orf11-encoded glycosyltransferase A, of which the yield and ratio of A82846B in shake-flask fermentation reached 1200 mg/L and 73.6%, respectively. Besides, the yield and ratio of A82846B in SIPI-3927-C6 grew up to 2520 mg/L and 86.5% in the 5-L fermenter culture, respectively. In conclusion, overexpressing orf10 gene can increase A82846B ratio,while overexpressing orf11 gene can increase A82846B titer as well. The artificial attB site is effective for inserting new genes.

GCNT1-Mediated O-Glycosylation of the Sialomucin CD43 Is a Sensitive Indicator of Notch Signaling in Activated T Cells.

Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.

Novel Molecular Mechanisms of Gangliosides in the Nervous System Elucidated by Genetic Engineering.

Acidic glycosphingolipids, i.e., gangliosides, are predominantly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they are considered to be involved in the development and function of nervous systems. Recent studies involving genetic engineering of glycosyltransferase genes have revealed novel aspects of the roles of gangliosides in the regulation of nervous tissues. In this review, novel findings regarding ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice are summarized. In particular, the roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems are reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in studies of congenital neurological disorders due to genetic mutations of ganglioside synthase genes and also in the techniques for the analysis of ganglioside functions are introduced.

Transcriptome Analysis Reveals Biosynthesis of Important Bioactive Constituents and Mechanism of Stem Formation of Dendrobium huoshanense

The stem of Dendrobium huoshanense C.Z. Tang and S.J. Cheng was widely used as a medicinal herb in health care products due to its broad pharmacological activities. However, the molecular regulation mechanism of stem development and biosynthetic pathways of important bioactive substances are still unclear in D. huoshanense. In this study, the bioactive compounds in leaves, stems and roots, and the identification of candidate genes involved in stem formation and biosynthesis of active compounds via transcriptome sequence were analyzed. The accumulation of total polysaccharides and flavonoids were varied significantly in different tissues. A comparative transcriptomic analysis revealed several differentially expressed genes (DEGs) involved in polysaccharides biosynthesis (103 genes), including fructose and mannose related genes (29 genes) and glycosyltransferase genes (74 genes), and flavonoids biosynthesis (15 genes). Some candidate genes that participated in photoperiod regulation (27 genes), starch and sucrose metabolism (46 genes), and hormone-induced activation of signaling pathways (38 genes) may be involved in stem formation. In sum, this study provides a foundation for investigating the molecular processes in the biosynthesis of active compounds and stem development. The transcriptome data presented here provides an important resource for the future studies of the molecular genetics and functional genomics in D. huoshanense and optimized control of the active compounds produced by D. huoshanense.

A mutation map for human glycoside hydrolase genes.

Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs.

Defining the human kidney N-glycome in normal and cancer tissues using MALDI imaging mass spectrometry.

Clear‐cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late‐stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well‐described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N‐glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin‐fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N‐linked glycan‐based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N‐glycans with bisecting N‐acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor‐specific N‐glycans were detected with tri‐ and tetra‐antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N‐glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N‐glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers.

Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose

BackgroundThe exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains.ResultsIn this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant−) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA.ConclusionsScreening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

Glycosphingolipid Biosynthesis Pathway in the Spinal Cord and Dorsal Root Ganglia During Inflammatory Pain: Early and Late Changes in Expression Patterns of Glycosyltransferase Genes

Glycosphingolipids (GSLs) are abundant, ceramide-containing lipids in the nervous system that play key functional roles in pain and inflammation. We measured gene expression (Ugcg, St3gal5, St8sia1, B4galNT1, Ugt8a, and Gal3st1) of glycosyltransferases involved in GSL synthesis in murine dorsal root ganglion (DRG) and spinal cord after complete Freund’s adjuvant (CFA)-induced unilateral hind-paw inflammation (1 day vs. 15 days). Chronic inflammation (15 days) sensitized both ipsilateral and contralateral paws to pain. One day of induced unilateral hind-paw inflammation (1d-IUHI) increased Ugcg, St8sia1, B4galnt1, and Gal3st1 expression in ipsilateral cord, suggesting that sulfatide and b-series gangliosides were also elevated. In addition, 1d-IUHI increased Ugcg, st3gal5 and Gal3st1 expression in contralateral cord, suggesting that sulfatide and a-/b-series gangliosides were elevated. By contrast, 1d-IUHI decreased Ugcg, St3gal5, and St8sia1 expression bilaterally in the DRG, suggesting that b-series gangliosides were depressed. Since intrathecal injection of b-series ganglioside induced mechanical allodynia in naïve mice, it seems reasonable that b-series gangliosides synthesized from upregulated St8sia1 in the ipsilateral spinal cord are involved in mechanical allodynia. By contrast, chronic inflammation led to a decrease of Ugcg, St3gal5, B4galnt1, and Gal3st1 expression in spinal cord bilaterally and an increase of St8sia1 expression in the ipsilateral DRG, suggesting that a-/b-series gangliosides in the spinal cord decreased and b-series gangliosides in ipsilateral DRG increased. These changes in glycosyltransferase gene expression in the DRG and the spinal cord may contribute to the modification of pain sensitivity in both inflamed and non-inflamed tissues and the transition from early to chronic inflammatory pain.

Genetic determinants of genus-level glycan diversity in a bacterial protein glycosylation system.

The human pathogens N. gonorrhoeae and N. meningitidis display robust intra- and interstrain glycan diversity associated with their O-linked protein glycosylation (pgl) systems. In an effort to better understand the evolution and function of protein glycosylation operating there, we aimed to determine if other human-restricted, Neisseria species similarly glycosylate proteins and if so, to assess the levels of glycoform diversity. Comparative genomics revealed the conservation of a subset of genes minimally required for O-linked protein glycosylation glycan and established those pgl genes as core genome constituents of the genus. In conjunction with mass spectrometric–based glycan phenotyping, we found that extant glycoform repertoires in N. gonorrhoeae, N. meningitidis and the closely related species N. polysaccharea and N. lactamica reflect the functional replacement of a progenitor glycan biosynthetic pathway. This replacement involved loss of pgl gene components of the primordial pathway coincident with the acquisition of two exogenous glycosyltransferase genes. Critical to this discovery was the identification of a ubiquitous but previously unrecognized glycosyltransferase gene (pglP) that has uniquely undergone parallel but independent pseudogenization in N. gonorrhoeae and N. meningitidis. We suggest that the pseudogenization events are driven by processes of compositional epistasis leading to gene decay. Additionally, we documented instances where inter-species recombination influences pgl gene status and creates discordant genetic interactions due ostensibly to the multi-locus nature of pgl gene networks. In summary, these findings provide a novel perspective on the evolution of protein glycosylation systems and identify phylogenetically informative, genetic differences associated with Neisseria species.

Transcriptome analysis of genes related to fruit texture in watermelon

Changes in texture are the most significant changes in fruit development and maturity. The formation of texture is a complex physiological and biochemical process that is regulated by many factors.This study measured the cell wall material content of Plant Introduction 186490 (PI186490) and ‘sanbai’ during different fruit development. The pectin, cellulose and hemicellulose contents of PI186490 were more than 2 times higher than those of ‘sanbai’ in the same period. Therefore, pectin, cellulose and hemicellulose may play key roles in the fruit texture difference of watermelon. At the same time, 85 candidate genes related to watermelon fruit texture were screened by using RNA-seq technology. Analysis of these genes revealed that such genes as pectinesterase genes, pectate lyase genes, glycosyltransferase genes, cellulose synthase genes, polygalacturonase genes and xyloglucan genes were differentially expressed in two varieties. It is possible to regulate the changes in the texture of watermelon pulp by affecting the content of the cell wall material components. In addition, some proteins, phytohormone-related genes, transcription factors and peroxidases may also be associated with watermelon fruit texture. These findings contribute to the body of knowledge regarding watermelon quality and provide a theoretical basis for the improvement of watermelon fruit texture.