Publications by authors named "Parastoo Azadi"

152 Publications

, a Pathogen from Patients with Chronic Granulomatous Disease, Produces a Penta-Acylated Hypostimulatory Glycero-D-talo-oct-2-ulosonic Acid-Lipid A Glycolipid (Ko-Lipid A).

Int J Mol Sci 2021 Mar 24;22(7). Epub 2021 Mar 24.

Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact () is hypostimulatory compared to , i.e., cytokine production in human blood requires 10-100 times more CFU/mL than . To better understand the pathogenicity of , we isolated its lipopolysaccharide (LPS) and characterized its lipid A. Unlike with typical , the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Man-(1→4)-β-GlcN3N-(1→6)-α-GlcN-(1⇿1)-α-GlcA tetra-saccharide substituted with five acyl chains: the amide-linked N-3' 14:0(3-OH), N-2' 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constituent of the core region of the LPS that is covalently attached to GlcpN3N of the lipid backbone may account for the acid resistance of LPS. In addition, the presence of Ko and only five acyl chains may explain the >10-fold lower proinflammatory potency of Ko-lipidA compared to lipid A, as measured by cytokine induction in human blood. These unusual structural properties of the Ko-lipid A glycolipid likely contribute to immune evasion during pathogenesis and resistance to antimicrobial peptides.
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http://dx.doi.org/10.3390/ijms22073303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036547PMC
March 2021

Glycoproteomic Sample Processing, LC-MS, and Data Analysis Using GlycReSoft.

Curr Protoc 2021 Mar;1(3):e84

Department of Biochemistry, Boston University, Boston, Massachusetts.

Identification of N- and O-glycosylation on specific sites of proteins, along with glycan structural information, is necessary to determine the roles glycoproteins play in normal and pathologic cellular functions. Because such glycosylation is macro- and micro-heterogeneous and alters the dissociation behavior of glycopeptides, specific sample preparation, mass spectrometry, and data analysis techniques are required. Advanced tandem mass spectrometry-based glycoproteomics coupled with powerful data mining algorithms are key elements for characterization of protein glycosylation. This article includes the detailed, streamlined sample preparation method for liquid chromatography-mass spectrometry data acquisition and subsequent bioinformatics-based data annotation using the publicly available GlycReSoft program for highly efficient identification and quantification of glycoprotein glycosylation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Characterization of glycans and site occupancy on purified glycoprotein Support Protocol 1: In-gel digestion of glycoproteins Support Protocol 2: Detection of glycoproteins from cells/tissue through glycopeptide enrichment Basic Protocol 2: Acquisition of glycopeptides through high-resolution nano-LC-MS/MS Basic Protocol 3: Identification and quantification of glycopeptides using GlycReSoft.
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http://dx.doi.org/10.1002/cpz1.84DOI Listing
March 2021

Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity.

PLoS Pathog 2021 Mar 18;17(3):e1009324. Epub 2021 Mar 18.

Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.
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http://dx.doi.org/10.1371/journal.ppat.1009324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009368PMC
March 2021

Engineered glycomaterial implants orchestrate large-scale functional repair of brain tissue chronically after severe traumatic brain injury.

Sci Adv 2021 Mar 5;7(10). Epub 2021 Mar 5.

Regenerative Bioscience Center, University of Georgia, Athens, GA 30602, USA.

Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain's poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. However, the efficacy of engineered CS (eCS) matrices in mediating structural and functional recovery chronically after sTBI has not been investigated. We report that neurotrophic factor functionalized acellular eCS matrices implanted into the rat M1 region acutely after sTBI significantly enhanced cellular repair and gross motor function recovery when compared to controls 20 weeks after sTBI. Animals subjected to M2 region injuries followed by eCS matrix implantations demonstrated the significant recovery of "reach-to-grasp" function. This was attributed to enhanced volumetric vascularization, activity-regulated cytoskeleton (Arc) protein expression, and perilesional sensorimotor connectivity. These findings indicate that eCS matrices implanted acutely after sTBI can support complex cellular, vascular, and neuronal circuit repair chronically after sTBI.
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http://dx.doi.org/10.1126/sciadv.abe0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935369PMC
March 2021

Comparative performance of bio-based coatings formulated with cellulose, chitin, and chitosan nanomaterials suitable for fruit preservation.

Carbohydr Polym 2021 May 10;259:117764. Epub 2021 Feb 10.

Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, United States.

Sustainable nanomaterials (SNMs) from wood, sugarcane and crab shell were prepared and used to coat selected fruits. The properties of SNMs and selected fruits were characterized and strawberry was used as an example to test antifungal activity and freshness preservation of the SNMs. The SNMs with their nano-structured morphology form strong shear-thinning dispersions for easy spraying on fruit surfaces. The fruit surface free energy was influenced by its surface morphology, predominant surface wax components, and cutin monomers. The antifungal activity of SNMs was influenced by their surface functional groups and particle size (crystals vs fibers). The coblend of wood nanocrystals (WCNCs) and chitosan nanofiber (CSNFs) exhibited the best antifungal property, which was comparable with the performance of the fungicide thiabendazole (80 mg L). The weight loss and color change of the WCNC/CSNF coated strawberries decreased by nearly half compared with the control samples, showing coating effectiveness on preserving fruit freshness.
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http://dx.doi.org/10.1016/j.carbpol.2021.117764DOI Listing
May 2021

FUT4 and FUT6 Are Arabinofuranose-Specific Fucosyltransferases.

Front Plant Sci 2021 9;12:589518. Epub 2021 Feb 9.

The Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States.

The bulk of plant biomass is comprised of plant cell walls, which are complex polymeric networks, composed of diverse polysaccharides, proteins, polyphenolics, and hydroxyproline-rich glycoproteins (HRGPs). Glycosyltransferases (GTs) work together to synthesize the saccharide components of the plant cell wall. The fucosyltransferases (FUTs), FUT4, and FUT6, are members of the plant-specific GT family 37 (GT37). FUT4 and FUT6 transfer fucose (Fuc) onto arabinose (Ara) residues of arabinogalactan (AG) proteins (AGPs) and have been postulated to be non-redundant AGP-specific FUTs. FUT4 and FUT6 were recombinantly expressed in mammalian HEK293 cells and purified for biochemical analysis. We report an updated understanding on the specificities of FUT4 and FUT6 that are involved in the synthesis of wall localized AGPs. Our findings suggest that they are selective enzymes that can utilize various arabinogalactan (AG)-like and non-AG-like oligosaccharide acceptors, and only require a free, terminal arabinofuranose. We also report with GUS promoter-reporter gene studies that and gene expression is sub-localized in different parts of developing roots.
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http://dx.doi.org/10.3389/fpls.2021.589518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900004PMC
February 2021

Structural elucidation and immuno-stimulatory activity of a novel polysaccharide containing glucuronic acid from the fungus Echinodontium tinctorium.

Carbohydr Polym 2021 Apr 27;258:117700. Epub 2021 Jan 27.

Chemistry and Biochemistry Program, University of Northern British Columbia, Prince George, British Columbia, V2N 4Z9, Canada. Electronic address:

An immuno-stimulatory polysaccharide (EtISPFa) was purified from water extract of the fungus Echinodontium tinctorium. EtISPFa has an estimated weight average molecular weight (M) of 1354 kDa and is composed of glucose (66.2 %), glucuronic acid (10.1 %), mannose (6.7 %), galactose (6.4 %), xylose (5.6 %), rhamnose (3.1 %), fucose (1.8 %), and arabinose (0.2 %). It has multiple glycosidic linkages, with 3-Glcp (19.8 %), 4-GlcpA (10.8 %), 6-Glcp (10.7 %), and 3,6-Glcp (8.7 %) being the most prominent. NMR analysis showed that EtISPFa has a backbone containing mostly of 3-substituted β-glucopyranose with 4-substituted glucopyranosyluronic acid. Short side chains consisting of an average of two β-glycopyranose residues, connected through 1→6 linkages, are attached to the 6-position of about every 4th or 5th backbone glucose residue. EtISPFa is a novel glucuronic acid-containing β-glucan capable of significantly inducing the production of cytokines IL-17, IL-16, MIP-2, G-CSF,GM-CSF, LIF, MIP-1α, MIP-1β, and RANTES in vitro. EtISPFa should be further explored for its immuno-stimulatory activity in vivo.
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http://dx.doi.org/10.1016/j.carbpol.2021.117700DOI Listing
April 2021

High expression of tissue -linked glycans is associated with a malignant phenotype of cholangiocarcinoma.

J Int Med Res 2021 Feb;49(2):300060520976864

Liver Fluke and Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.

Objective: This study aimed to investigate the expression of -linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue.

Methods: Sixty patients with cholangiocarcinoma were included in the study. Permethylated -linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1.

Results: -linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight -linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II-IV), lymphatic invasion, and vascular invasion.

Conclusion: Our study shows expression of -linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.
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http://dx.doi.org/10.1177/0300060520976864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869157PMC
February 2021

Binding of FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity.

Front Microbiol 2020 11;11:531596. Epub 2021 Jan 11.

Division of Infectious Diseases, Department of Medicine, Medical College of Georgia, Augusta University, Augusta, GA, United States.

CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.
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http://dx.doi.org/10.3389/fmicb.2020.531596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829508PMC
January 2021

Glucuronidation of Methylated Quercetin Derivatives: Chemical and Biochemical Approaches.

J Agric Food Chem 2020 Dec 8;68(50):14790-14807. Epub 2020 Dec 8.

BioDiscovery Institute and Department of Biological Sciences, University of North Texas, Denton 76203, Texas, United States.

Botanical supplements derived from grapes are functional in animal model systems for the amelioration of neurological conditions, including cognitive impairment. Rats fed with grape extracts accumulate 3'--methyl-quercetin-3--β-d-glucuronide () in their brains, suggesting as a potential therapeutic agent. To develop methods for the synthesis of and the related 4'--methyl-quercetin-7--β-d-glucuronide (), 3--methyl-quercetin-3'--β-d-glucuronide (), and 4'--methyl-quercetin-3'--β-d-glucuronide (), which are not found in the brain, we have evaluated both enzymatic semisynthesis and full chemical synthetic approaches. Biocatalysis by mammalian UDP-glucuronosyltransferases generated multiple glucuronidated products from 4'--methylquercetin, and is not cost-effective. Chemical synthetic methods, on the other hand, provided good results; , , and were obtained in six steps at 12, 18, and 30% overall yield, respectively, while was synthesized in five steps at 34% overall yield. A mechanistic study on the unexpected regioselectivity observed in the quercetin glucuronide synthetic steps is also presented.
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http://dx.doi.org/10.1021/acs.jafc.0c04500DOI Listing
December 2020

Unravelling Chemical Composition of Agave Spines: News from Lem.

Plants (Basel) 2020 Nov 25;9(12). Epub 2020 Nov 25.

CONACYT-Research Fellow Colegio de Postgraduados Campus Campeche, Carretera Haltunchén-Edzná km 17.5, Sihochac, Campeche 24450, Mexico.

Spines are key plant modifications developed to deal against herbivores; however, its physical structure and chemical composition have been little explored in plant species. Here, we took advantage of high-throughput chromatography to characterize chemical composition of Lem. spines, a species traditionally used for fiber extraction. Analyses of structural carbohydrate showed that spines have lower cellulose content than leaf fibers (52 and 72%, respectively) but contain more than 2-fold the hemicellulose and 1.5-fold pectin. Xylose and galacturonic acid were enriched in spines compared to fibers. The total lignin content in spines was 1.5-fold higher than those found in fibers, with elevated levels of syringyl (S) and guaiacyl (G) subunits but similar S/G ratios within tissues. Metabolomic profiling based on accurate mass spectrometry revealed the presence of phenolic compounds including quercetin, kaempferol, (+)-catechin, and (-)-epicatechin in spines, which were also detected in situ in spines tissues and could be implicated in the color of these plants' structures. Abundance of (+)-catechins could also explain proanthocyanidins found in spines. Agave spines may become a plant model to obtain more insights about cellulose and lignin interactions and condensed tannin deposition, which is valuable knowledge for the bioenergy industry and development of naturally dyed fibers, respectively.
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http://dx.doi.org/10.3390/plants9121642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759909PMC
November 2020

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

PLoS One 2020 10;15(11):e0241249. Epub 2020 Nov 10.

Department of Surgery, Schulze Diabetes Institute, University of Minnesota, Minneapolis, Minnesota, United States of America.

Objectives: Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection.

Methods: N- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS).

Results: A total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets.

Conclusions: These results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241249PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654812PMC
December 2020

Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2.

Glycobiology 2020 Oct 29. Epub 2020 Oct 29.

Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602.

The emergence of the COVID-19 pandemic caused by SARS-CoV-2 has created the need for development of new therapeutic strategies. Understanding the mode of viral attachment, entry and replication has become a key aspect of such interventions. The coronavirus surface features a trimeric spike (S) protein that is essential for viral attachment, entry and membrane fusion. The S protein of SARS-CoV-2 binds to human angiotensin converting enzyme 2 (hACE2) for entry. Herein, we describe glycomic and glycoproteomic analysis of hACE2 expressed in HEK293 cells. We observed high glycan occupancy (73.2 to 100%) at all seven possible N-glycosylation sites and surprisingly detected one novel O-glycosylation site. To deduce the detailed structure of glycan epitopes on hACE2 that may be involved in viral binding, we have characterized the terminal sialic acid linkages, the presence of bisecting GlcNAc, and the pattern of N-glycan fucosylation. We have conducted extensive manual interpretation of each glycopeptide and glycan spectrum, in addition to using bioinformatics tools to validate the hACE2 glycosylation. Our elucidation of the site-specific glycosylation and its terminal orientations on the hACE2 receptor, along with the modeling of hACE2 glycosylation sites can aid in understanding the intriguing virus-receptor interactions and assist in the development of novel therapeutics to prevent viral entry. The relevance of studying the role of ACE2 is further increased due to some recent reports about the varying ACE2 dependent complications with regard to age, sex, race, and pre-existing conditions of COVID-19 patients.
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http://dx.doi.org/10.1093/glycob/cwaa101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665489PMC
October 2020

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility.

Sci Adv 2020 Sep 16;6(38). Epub 2020 Sep 16.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA 02139, USA.

Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.
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http://dx.doi.org/10.1126/sciadv.aba4015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494350PMC
September 2020

Mass Spectrometric Method for the Unambiguous Profiling of Cellular Dynamic Glycosylation.

ACS Chem Biol 2020 10 4;15(10):2692-2701. Epub 2020 Sep 4.

Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States.

Various biological processes at the cellular level are regulated by glycosylation which is a highly microheterogeneous post-translational modification (PTM) on proteins and lipids. The dynamic nature of glycosylation can be studied through metabolic incorporation of non-natural sugars into glycan epitopes and their detection using bio-orthogonal probes. However, this approach possesses a significant drawback due to nonspecific background reactions and ambiguity of non-natural sugar metabolism. Here, we report a probe-free strategy for their direct detection by glycoproteomics and glycomics using mass spectrometry (MS). The method dramatically simplifies the detection of non-natural functional group bearing monosaccharides installed through promiscuous sialic acid, -acetyl-d-galactosamine (GalNAc) and -acetyl-d-glucosamine (GlcNAc) biosynthetic pathways. Multistage enrichment of glycoproteins by cellular fractionation, subsequent ZIC-HILIC (zwitterionic-hydrophilic interaction chromatography) based glycopeptide enrichment, and a spectral enrichment algorithm for the MS data processing enabled direct detection of non-natural monosaccharides that are incorporated at low abundance on the N/O-glycopeptides along with their natural counterparts. Our approach allowed the detection of both natural and non-natural sugar bearing glycopeptides, N- and O-glycopeptides, differentiation of non-natural monosaccharide types on the glycans and also their incorporation efficiency through quantitation. Through this, we could deduce interconversion of monosaccharides during their processing through glycan salvage pathway and subsequent incorporation into glycan chains. The study of glycosylation dynamics through this method can be conducted in high throughput, as few sample processing steps are involved, enabling understanding of glycosylation dynamics under various external stimuli and thereby could bolster the use of metabolic glycan engineering in glycosylation functional studies.
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http://dx.doi.org/10.1021/acschembio.0c00453DOI Listing
October 2020

Evaluating the Utility of Permethylated Polysaccharide Solution NMR Data for Characterization of Insoluble Plant Cell Wall Polysaccharides.

Anal Chem 2020 10 2;92(19):13221-13228. Epub 2020 Sep 2.

Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States.

Plant cell wall polysaccharide analysis encompasses the utilization of a variety of analytical tools, including gas and liquid chromatography, mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. These methods provide complementary data, which enable confident structural proposals of the many complex polysaccharide structures that exist in the complex matrices of plant cell walls. However, cell walls contain fractions of varying solubilities, and a few techniques are available that can analyze all fractions simultaneously. We have discovered that permethylation affords the complete dissolution of both soluble and insoluble polysaccharide fractions of plant cell walls in organic solvents such as chloroform or acetonitrile, which can then be analyzed by a number of analytical techniques including MS and NMR. In this work, NMR structure analysis of 10 permethylated polysaccharide standards was undertaken to generate chemical shift data providing insights into spectral changes that result from permethylation of polysaccharide residues. This information is of especial relevance to the structure analysis of insoluble polysaccharide materials that otherwise are not easily investigated by solution-state NMR methodologies. The preassigned NMR chemical shift data is shown to be vital for NMR structure analysis of minor polysaccharide components of plant cell walls that are particularly difficult to assign by NMR correlation data alone. With the assigned chemical shift data, we analyzed the permethylated samples of destarched, alcohol-insoluble residues of switchgrass and poplar by two-dimensional NMR spectral profiling. Thus, we identified, in addition to the major polysaccharide components, two minor polysaccharides, namely, <5% 3-linked arabinoxylan (switchgrass) and <2% glucomannan (poplar). In particular, the position of the arabinose residue in the arabinoxylan of the switchgrass sample was confidently assigned based on chemical shift values, which are highly sensitive to local chemical environments. Furthermore, the high resolution afforded by the H NMR spectra of the permethylated switchgrass and poplar samples allowed facile relative quantitative analysis of their polysaccharide composition, utilizing only a few milligrams of the cell wall material. The concepts herein developed will thus facilitate NMR structure analysis of insoluble plant cell wall polysaccharides, more so of minor cell wall components that are especially challenging to analyze with current methods.
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http://dx.doi.org/10.1021/acs.analchem.0c02379DOI Listing
October 2020

Structural signatures in EPR3 define a unique class of plant carbohydrate receptors.

Nat Commun 2020 07 30;11(1):3797. Epub 2020 Jul 30.

Department of Molecular Biology and Genetics, Aarhus University, 8000, Aarhus C, Denmark.

Receptor-mediated perception of surface-exposed carbohydrates like lipo- and exo-polysaccharides (EPS) is important for non-self recognition and responses to microbial associated molecular patterns in mammals and plants. In legumes, EPS are monitored and can either block or promote symbiosis with rhizobia depending on their molecular composition. To establish a deeper understanding of receptors involved in EPS recognition, we determined the structure of the Lotus japonicus (Lotus) exopolysaccharide receptor 3 (EPR3) ectodomain. EPR3 forms a compact structure built of three putative carbohydrate-binding modules (M1, M2 and LysM3). M1 and M2 have unique βαββ and βαβ folds that have not previously been observed in carbohydrate binding proteins, while LysM3 has a canonical βααβ fold. We demonstrate that this configuration is a structural signature for a ubiquitous class of receptors in the plant kingdom. We show that EPR3 is promiscuous, suggesting that plants can monitor complex microbial communities though this class of receptors.
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http://dx.doi.org/10.1038/s41467-020-17568-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392887PMC
July 2020

Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria.

Nat Chem Biol 2020 10 27;16(10):1062-1070. Epub 2020 Jul 27.

Department of Microbiology, Cornell University, Ithaca, NY, USA.

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.
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http://dx.doi.org/10.1038/s41589-020-0595-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857696PMC
October 2020

Ganglioside GM2: a potential biomarker for cholangiocarcinoma.

J Int Med Res 2020 Jul;48(7):300060520903216

Liver Fluke and Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.

Objective: To investigate the expression of glycosphingolipids in serum and tissue from patients with cholangiocarcinoma compared with healthy controls.

Methods: Nanospray ionization-linear ion trap mass spectrometry (NSI-MS) was used to demonstrate the comparative structural glycomics of glycosphingolipids in serum from patients with cholangiocarcinoma (n=15), compared with healthy controls (n = 15). GM2 expression in cholangiocarcinoma tissues (n = 60) was evaluated by immunohistochemistry.

Results: Eleven glycosphingolipids were detected by NSI-MS: CMH (ceramide monohexose), Lac-Cer (galactose (Gal)β1-4 glucose (Glc)β1-1'-ceramide), Gb3 (Galα1-4Galβ1-4Glcβ1-1'-ceramide), Gb4/Lc4 (N-acetylgalactosamine (GalNAc)β1-3Galα1-4Galβ1-4Glcβ1-1'-ceramide/Galβ1-4 N-acetylglucosamine (GlcNAc)β1-3Galβ1-4Glcβ1-1'-ceramide), GM3 (N-acetylneuraminic acid (NeuAc)2-3Galβ1-4Glcβ1-1'-ceramide), GM2 (GalNAcβ1-4[NeuAc2-3]Galβ1-4Glcβ1-1'-ceramide), GM1 (Galβ1-3GalNAcβ1-4[NeuAc2-3]Galβ1-4Glcβ1-1'-ceramide), hFA (hydroxylated fatty acid)-CMH, hFA-Lac-Cer, hFA-Gb3, and hFA-GM3. Lac-Cer was the most abundant structure among the lactosides and globosides (normal, 24.40% ± 0.11%; tumor, 24.61% ± 2.10%), while GM3 predominated among the gangliosides (normal, 29.14% ± 1.31%; tumor, 30.53% ± 4.04%). The two glycosphingolipids that significantly differed between healthy controls and patients with cholangiocarcinoma were Gb3 and GM2. High expression of GM2 was associated with vascular invasion in tissue from patients with cholangiocarcinoma.

Conclusions: Altered expression of glycosphingolipids in tissue and serum from patients with cholangiocarcinoma may contribute to tumor growth and progression. The ganglioside GM2, which significantly increased in the serum of patients with cholangiocarcinoma, represents a promising target as a biomarker for cholangiocarcinoma.
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http://dx.doi.org/10.1177/0300060520903216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375732PMC
July 2020

Carbohydrate Structure Analysis: Methods and Applications.

SLAS Technol 2020 Aug;25(4):305-306

Complex Carbohydrate Research Center at the University of Georgia, Athens, GA, USA.

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http://dx.doi.org/10.1177/2472630320937003DOI Listing
August 2020

An atypical lipoteichoic acid from elicits a broadly cross-reactive and protective immune response.

J Biol Chem 2020 07 18;295(28):9513-9530. Epub 2020 May 18.

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

is a leading cause of food-poisoning and causes avian necrotic enteritis, posing a significant problem to both the poultry industry and human health. No effective vaccine against is currently available. Using an antiserum screen of mutants generated from a transposon-mutant library, here we identified an immunoreactive antigen that was lost in a putative glycosyltransferase mutant, suggesting that this antigen is likely a glycoconjugate. Following injection of formalin-fixed whole cells of HN13 (a laboratory strain) and JGS4143 (chicken isolate) intramuscularly into chickens, the HN13-derived antiserum was cross-reactive in immunoblots with all tested 32 field isolates, whereas only 5 of 32 isolates were recognized by JGS4143-derived antiserum. The immunoreactive antigens from both HN13 and JGS4143 were isolated, and structural analysis by MALDI-TOF-MS, GC-MS, and 2D NMR revealed that both were atypical lipoteichoic acids (LTAs) with poly-(β1→4)-ManNAc backbones substituted with phosphoethanolamine. However, although the ManNAc residues in JGS4143 LTA were phosphoethanolamine-modified, a few of these residues were instead modified with phosphoglycerol in the HN13 LTA. The JGS4143 LTA also had a terminal ribose and ManNAc instead of ManN in the core region, suggesting that these differences may contribute to the broadly cross-reactive response elicited by HN13. In a passive-protection chicken experiment, oral challenge with JGS4143 lead to 22% survival, whereas co-gavage with JGS4143 and α-HN13 antiserum resulted in 89% survival. This serum also induced bacterial killing in opsonophagocytosis assays, suggesting that HN13 LTA is an attractive target for future vaccine-development studies.
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http://dx.doi.org/10.1074/jbc.RA119.009978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363129PMC
July 2020

Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy.

SLAS Technol 2020 08 4;25(4):367-379. Epub 2020 May 4.

Complex Carbohydrate Research Center, The University of Georgia, Athens, GA, USA.

Glycoproteins play key roles in various molecular and cellular functions and are among the most difficult to analyze biomolecules on account of their microheterogeneity, non-template-driven synthesis, and low abundances. The stability, serum half-life, immunogenicity, and biological activity of therapeutic glycoproteins, including antibodies, vaccines, and biomarkers, are regulated by their glycosylation profile. Thus, there is increasing demand for the qualitative and quantitative characterization and validation of glycosylation on glycoproteins. One of the most important derivatization processes for the structural characterization of released glycans by mass spectrometry (MS) is permethylation. We have recently developed a permethylation strategy in microscale that allows facile permethylation of glycans and permits the processing of large sample sets in nanogram amounts through high-throughput sample handling. Here, we are reporting the wide potential of micropermethylation-based high-throughput structural analysis of glycans from various sources, including human plasma, mammalian cells, and purified glycoproteins, through an automated tandem electrospray ionization-mass spectrometry (ESI-MSn) platform. The glycans released from the plasma, cells, and glycoproteins are permethylated in microscale in a 96-well plate or microcentrifuge tube and isolated by a C18 tip-based cleanup through a shorter and simple process. We have developed a workflow to accomplish an in-depth automated structural characterization MS program for permethylated N/O-glycans through an automated high-throughput multistage tandem MS acquisition. We have demonstrated the utility of this workflow using the examples of sialic acid linkages and bisecting GlcNAc (-acetylglucosamine) on the glycans. This approach can automate the high-throughput screening of glycosylation on large sample sets of glycoproteins, including clinical glycan biomarkers and glycoprotein therapeutics.
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http://dx.doi.org/10.1177/2472630320912929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931992PMC
August 2020

Deducing the N- and O-glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2.

Glycobiology 2020 12;30(12):981-988

Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Rd, Athens, GA 30602, USA.

The current emergence of the novel coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands the development of new therapeutic strategies to prevent rapid progress of mortalities. The coronavirus spike (S) protein, which facilitates viral attachment, entry and membrane fusion is heavily glycosylated and plays a critical role in the elicitation of the host immune response. The spike protein is comprised of two protein subunits (S1 and S2), which together possess 22 potential N-glycosylation sites. Herein, we report the glycosylation mapping on spike protein subunits S1 and S2 expressed on human cells through high-resolution mass spectrometry. We have characterized the quantitative N-glycosylation profile on spike protein and interestingly, observed unexpected O-glycosylation modifications on the receptor-binding domain of spike protein subunit S1. Even though O-glycosylation has been predicted on the spike protein of SARS-CoV-2, this is the first report of experimental data for both the site of O-glycosylation and identity of the O-glycans attached on the subunit S1. Our data on the N- and O-glycosylation are strengthened by extensive manual interpretation of each glycopeptide spectra in addition to using bioinformatics tools to confirm the complexity of glycosylation in the spike protein. The elucidation of the glycan repertoire on the spike protein provides insights into the viral binding studies and more importantly, propels research toward the development of a suitable vaccine candidate.
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http://dx.doi.org/10.1093/glycob/cwaa042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239183PMC
December 2020

Molecular Mechanism of Polysaccharide Acetylation by the Arabidopsis Xylan -acetyltransferase XOAT1.

Plant Cell 2020 07 30;32(7):2367-2382. Epub 2020 Apr 30.

Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602

Xylans are a major component of plant cell walls. -Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis () xylan -acetyltransferase 1 (XOAT1), a member of the plant-specific Trichome Birefringence Like (TBL) family. Detailed characterization of XOAT1-catalyzed reactions by real-time NMR confirms that it exclusively catalyzes the 2--acetylation of xylan, followed by nonenzymatic acetyl migration to the -3 position, resulting in products that are monoacetylated at both -2 and -3 positions. In addition, we report the crystal structure of the catalytic domain of XOAT1, which adopts a unique conformation that bears some similarities to the α/β/α topology of members of the GDSL-like lipase/acylhydrolase family. Finally, we use a combination of biochemical analyses, mutagenesis, and molecular simulations to show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate, Ac-Ser-216, by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally uses an Arg residue in the formation of an oxyanion hole.
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http://dx.doi.org/10.1105/tpc.20.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346548PMC
July 2020

Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles.

Int J Biol Macromol 2020 Aug 26;157:158-169. Epub 2020 Apr 26.

Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA. Electronic address:

Plant-based expression system has many potential advantages to produce biopharmaceuticals, but plants cannot be directly used to express human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit plant-based expression system for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco in order to generate capacities for β1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human β1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) was co-expressed to produce rhuEPO bearing β1,4-galactose-extended N-glycan chains as well as compare their β1,4-galactosylation efficiencies. Five mammalian genes encoding enzymes/transporter for sialic acid biosynthesis, transport and transfer were co-expressed to build sialylation capacity in plants. The human MGAT3 was co-expressed to produce N-glycan chains with bisecting GlcNAc. Our results demonstrated that the above transgenes were incorporated into tobacco genome and transcribed. ST/GalT was found to be more efficient than GalT for β1,4-galactosylation. Furthermore, co-expressing MGAT3 generated N-glycans likely bearing bisected GlcNAc. However, our current efforts did not result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of β1,4-galactose-extended N-glycan chains for tissue protective purposes.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.04.199DOI Listing
August 2020

Slc35a1 deficiency causes thrombocytopenia due to impaired megakaryocytopoiesis and excessive platelet clearance in the liver.

Haematologica 2021 03 1;106(3):759-769. Epub 2021 Mar 1.

Jiangsu Institute of Hematology, NHC Key Laboratory of Thrombosis and Hemostasis, The First Affiliated Hospital of Soochow University, Suzhou, China

Sialic acid is a common terminal residue of glycans on proteins and acidic sphingolipids such as gangliosides and has important biological functions. The sialylation process is controlled by more than 20 different sialyltransferases, many of which exhibit overlapping functions. Thus, it is difficult to determine the overall biological function of sialylation by targeted deletion of individual sialyltransferases. To address this issue, we established a mouse line with the Slc35a1 gene flanked by loxP sites. Slc35a1 encodes the cytidine-5’-monophosphate (CMP)-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation. Here we report our study regarding the role of sialylation on megakaryocytes and platelets using a mouse line with significantly reduced sialylation in megakaryocytes and platelets (Plt Slc35a1– /–). The major phenotype of Plt Slc35a1–/– mice was thrombocytopenia. The number of bone marrow megakaryocytes in Plt Slc35a1–/– mice was reduced, and megakaryocyte maturation was also impaired. In addition, an increased number of desialylated platelets was cleared by Küpffer cells in the liver of Plt Slc35a1–/– mice. This study provides new insights into the role of sialylation in platelet homeostasis and the mechanisms of thrombocytopenia in diseases associated with platelet desialylation, such as immune thrombocytopenia and a rare congenital disorder of glycosylation (CDG), SLC35A1-CDG, which is caused by SLC35A1 mutations.
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http://dx.doi.org/10.3324/haematol.2019.225987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927894PMC
March 2021

Binding of Phage-Encoded FlaGrab to Motile Flagella Inhibits Growth, Downregulates Energy Metabolism, and Requires Specific Flagellar Glycans.

Front Microbiol 2020 20;11:397. Epub 2020 Mar 20.

Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada.

Many bacterial pathogens display glycosylated surface structures that contribute to virulence, and targeting these structures is a viable strategy for pathogen control. The foodborne pathogen expresses a vast diversity of flagellar glycans, and flagellar glycosylation is essential for its virulence. Little is known about why encodes such a diverse set of flagellar glycans, but it has been hypothesized that evolutionary pressure from bacteriophages (phages) may have contributed to this diversity. However, interactions between phages and host flagellar glycans have not been characterized in detail. Previously, we observed that Gp047 (now renamed FlaGrab), a conserved phage protein, binds to flagella displaying the nine-carbon monosaccharide 7-acetamidino-pseudaminic acid, and that this binding partially inhibits cell growth. However, the mechanism of this growth inhibition, as well as how might resist this activity, are not well-understood. Here we use RNA-Seq to show that FlaGrab exposure leads 11168 cells to downregulate expression of energy metabolism genes, and that FlaGrab-induced growth inhibition is dependent on motile flagella. Our results are consistent with a model whereby FlaGrab binding transmits a signal through flagella that leads to retarded cell growth. To evaluate mechanisms of FlaGrab resistance in , we characterized the flagellar glycans and flagellar glycosylation loci of two strains naturally resistant to FlaGrab binding. Our results point toward flagellar glycan diversity as the mechanism of resistance to FlaGrab. Overall, we have further characterized the interaction between this phage-encoded flagellar glycan-binding protein and , both in terms of mechanism of action and mechanism of resistance. Our results suggest that encodes as-yet unidentified mechanisms for generating flagellar glycan diversity, and point to phage proteins as exciting lenses through which to study bacterial surface glycans.
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http://dx.doi.org/10.3389/fmicb.2020.00397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099621PMC
March 2020

New strategies for profiling and characterization of human milk oligosaccharides.

Glycobiology 2020 Sep;30(10):774-786

Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602, USA.

Human breast milk is an incredibly rich and complex biofluid composed of proteins, lipids and complex carbohydrates, including a diverse repertoire of free human milk oligosaccharides (HMOs). Strikingly, HMOs are not digested by the infant but function as prebiotics for bacterial strains associated with numerous benefits. Considering the broad variety of beneficial effects of HMOs, and the vast number of factors that affect breast milk composition, the analysis of HMO diversity and complexity is of utmost relevance. Using human milk samples from a cohort of Bangladeshi mothers participating in a study on malnutrition and stunting in children, we have characterized breast milk oligosaccharide composition by means of permethylation followed by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) analysis. This approach identified over 100 different glycoforms and showed a wide diversity of milk composition, with a predominance of fucosylated and sialylated HMOs over nonmodified HMOs. We observed that these samples contain on average 80 HMOs, with the highest permethylated masses detected being >5000 mass units. Here we report an easily implemented method developed for the separation, characterization and relative quantitation of large arrays of HMOs, including higher molecular weight sialylated HMOs. Our ultimate goal is to create a simple, high-throughput method, which can be used for full characterization of sialylated and/or fucosylated HMOs. These results demonstrate how current analytical techniques can be applied to characterize human milk composition, providing new tools to help the scientific community shed new light on the impact of HMOs during infant development.
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http://dx.doi.org/10.1093/glycob/cwaa028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526734PMC
September 2020

Species-Specific Recognition of Mediated by UV-Inducible Pili and S-Layer Glycosylation Patterns.

mBio 2020 03 10;11(2). Epub 2020 Mar 10.

Molecular Biology of Archaea, Institute of Biology II-Microbiology, University of Freiburg, Freiburg, Germany

The UV-inducible pili system of (Ups) mediates the formation of species-specific cellular aggregates. Within these aggregates, cells exchange DNA to repair DNA double-strand breaks via homologous recombination. Substitution of the pilin subunits UpsA and UpsB with their homologs from showed that these subunits facilitate species-specific aggregation. A region of low conservation within the UpsA homologs is primarily important for this specificity. Aggregation assays in the presence of different sugars showed the importance of -glycosylation in the recognition process. In addition, the -glycan decorating the S-layer of is different from the one of Therefore, each species seems to have developed a unique UpsA binding pocket and unique glycan composition to ensure aggregation and, consequently, also DNA exchange with cells from only the same species, which is essential for DNA repair by homologous recombination. Type IV pili can be found on the cell surface of many archaea and bacteria where they play important roles in different processes. The UV-inducible pili system of (Ups) pili from the crenarchaeal species are essential in establishing species-specific mating partners, thereby assisting in genome stability. With this work, we show that different species have specific regions in their Ups pili subunits, which allow them to interact only with cells from the same species. Additionally, different species have unique surface-layer glycosylation patterns. We propose that the unique features of each species allow the recognition of specific mating partners. This knowledge for the first time gives insights into the molecular basis of archaeal self-recognition.
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http://dx.doi.org/10.1128/mBio.03014-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064770PMC
March 2020

Biocompatibility and structural characterization of glycosaminoglycans isolated from heads of silver-banded whiting (Sillago argentifasciata Martin & Montalban 1935).

Int J Biol Macromol 2020 May 16;151:663-676. Epub 2020 Feb 16.

Department of Biomolecular Sciences, Pharmacognosy Division, Research Institute of Pharmaceutical Sciences School of Pharmacy, University of Mississippi, Oxford, MS 38677-1848, USA. Electronic address:

Glycosaminoglycans (GAGs) were extracted from heads of silver-banded whiting (SBW) fish and subjected to preliminary biocompatibility testing per ISO 10993: intracutaneous irritation, maximization sensitization, systemic toxicity, and cytotoxicity. When the GAG solution was injected intradermally, the observed irritation was within ISO limits and comparable to a marketed control. There was no evidence of sensitization, systemic toxicity, or cellular toxicity on the test organisms treated with the GAG mixture from SBW fish heads. Fractionation by size-exclusion chromatography has shown three distinct fractions: F1 as low molecular weight hyaluronic acid (190 kDa), F2 (82 kDa) and F3 (64 kDa), both as chondroitin sulfates. Structural characterization by 1D and 2D nuclear magnetic resonance spectroscopy and disaccharide analysis have shown sulfation ratios at positions C4:C6 of the F2 and F3 fractions respectively as 70:20% and 50:30%, and the balance of non-sulfated and 4,6-di-sulfated units. The preliminary results here suggest that GAG-based extracts from SBW fish heads are suitable alternative products to be used in soft tissue augmentation, although further long-term biocompatibility studies are still required.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.02.160DOI Listing
May 2020