School of BioSciences - Research Publications
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ItemProfiling Cell Wall Monosaccharides and Nucleotide-Sugars from Plants.(Wiley-Blackwell, 2019-06)The cell wall is an intricate mesh largely composed of polysaccharides that vary in structure and abundance. Apart from cellulose biosynthesis, the assembly of matrix polysaccharides such as pectin and hemicellulose occur in the Golgi apparatus before being transported via vesicles to the cell wall. Matrix polysaccharides are biosynthesized from activated precursors or nucleotide sugars. The composition and assembly of the cell wall is an important aspect in plant development and plant biomass utilization. The application of anion-exchange chromatography to determine the monosaccharide composition of the insoluble matrix polysaccharides enables a complete profile of all major sugars in the cell wall from a single run. While porous carbon graphite chromatography and tandem mass spectrometry delivers a sensitive and robust nucleotide sugar profile from plant extracts. Here we describe detailed methodology to quantify nucleotide sugars within the cell and profile the non-cellulosic monosaccharide composition of the cell wall.
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ItemGDP-L-fucose transport in plants: The missing piece(TAYLOR & FRANCIS INC, 2017)
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ItemUDP-Glucuronic Acid Transport Is Required for Virulence of Cryptococcus neoformans(AMER SOC MICROBIOLOGY, 2018-01-30)Glycans play diverse biological roles, ranging from structural and regulatory functions to mediating cellular interactions. For pathogens, they are also often required for virulence and survival in the host. In Cryptococcus neoformans, an opportunistic pathogen of humans, the acidic monosaccharide glucuronic acid (GlcA) is a critical component of multiple essential glycoconjugates. One of these glycoconjugates is the polysaccharide capsule, a major virulence factor that enables this yeast to modulate the host immune response and resist antimicrobial defenses. This allows cryptococci to colonize the lung and brain, leading to hundreds of thousands of deaths each year worldwide. Synthesis of most glycans, including capsule polysaccharides, occurs in the secretory pathway. However, the activated precursors for this process, nucleotide sugars, are made primarily in the cytosol. This topological problem is resolved by the action of nucleotide sugar transporters (NSTs). We discovered that Uut1 is the sole UDP-GlcA transporter in C. neoformans and is unique among NSTs for its narrow substrate range and high affinity for UDP-GlcA. Mutant cells with UUT1 deleted lack capsule polysaccharides and are highly sensitive to environmental stress. As a result, the deletion mutant is internalized and cleared by phagocytes more readily than wild-type cells are and is completely avirulent in mice. These findings expand our understanding of the requirements for capsule synthesis and cryptococcal virulence and elucidate a critical protein family.IMPORTANCECryptococcus neoformans causes lethal meningitis in almost two hundred thousand immunocompromised patients each year. Much of this fungal pathogen's ability to resist host defenses and cause disease is mediated by carbohydrate structures, including a complex polysaccharide capsule around the cell. Like most eukaryotic glycoconjugates, capsule polysaccharides are made within the secretory pathway, although their precursors are generated in the cytosol. Specific transporters are therefore required to convey these raw materials to the site of synthesis. One precursor of particular interest is UDP-glucuronic acid, which donates glucuronic acid to growing capsule polysaccharides. We discovered a highly specific, high-affinity transporter for this molecule. Deletion of the gene encoding this unusual protein abolishes capsule synthesis, alters stress resistance, and eliminates fungal virulence. In this work, we have identified a novel transporter, elucidated capsule synthesis and thereby aspects of fungal pathogenesis, and opened directions for potential antifungal therapy.
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ItemThree UDP-xylose transporters participate in xylan biosynthesis by conveying cytosolic UDP-xylose into the Golgi lumen in Arabidopsis(OXFORD UNIV PRESS, 2018-02-20)UDP-xylose (UDP-Xyl) is synthesized by UDP-glucuronic acid decarboxylases, also termed UDP-Xyl synthases (UXSs). The Arabidopsis genome encodes six UXSs, which fall into two groups based upon their subcellular location: the Golgi lumen and the cytosol. The latter group appears to play an important role in xylan biosynthesis. Cytosolic UDP-Xyl is transported into the Golgi lumen by three UDP-Xyl transporters (UXT1, 2, and 3). However, while single mutants affected in the UDP-Xyl transporter 1 (UXT1) showed a substantial reduction in cell wall xylose content, a double mutant affected in UXT2 and UXT3 had no obvious effect on cell wall xylose deposition. This prompted us to further investigate redundancy among the members of the UXT family. Multiple uxt mutants were generated, including a triple mutant, which exhibited collapsed vessels and reduced cell wall thickness in interfascicular fiber cells. Monosaccharide composition, molecular weight, nuclear magnetic resonance, and immunolabeling studies demonstrated that both xylan biosynthesis (content) and fine structure were significantly affected in the uxt triple mutant, leading to phenotypes resembling those of the irx mutants. Pollination was also impaired in the uxt triple mutant, likely due to reduced filament growth and anther dehiscence caused by alterations in the composition of the cell walls. Moreover, analysis of the nucleotide sugar composition of the uxt mutants indicated that nucleotide sugar interconversion is influenced by the cytosolic UDP-Xyl pool within the cell. Taken together, our results underpin the physiological roles of the UXT family in xylan biosynthesis and provide novel insights into the nucleotide sugar metabolism and trafficking in plants.
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ItemA hypomorphic allele of SLC35D1 results in Schneckenbecken-like dysplasia(OXFORD UNIV PRESS, 2019-11-01)We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a 'Schneckenbecken-like dysplasia.' The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.
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ItemAbsolute Quantitation of In Vitro Expressed Plant Membrane Proteins by Targeted Proteomics (MRM) for the Determination of Kinetic Parameters.(Humana Press, 2018)The purification of a functional soluble protein from biological or in vitro expression systems can be problematic and the enrichment of a functional membrane protein for biochemical analyses can be a serious technical challenge. Recently we have been characterizing plant endomembrane nucleotide sugar transporters using a yeast expression system. However, rather than enriching these in vitro expressed proteins to homogeneity, we have been conducting biochemical characterization of these transport proteins in yeast microsomal fractions. While this approach has enabled us to estimate a variety of kinetic parameters, the accurate determination of the turnover number of an enzyme-substrate complex (k cat) requires that the catalytic site concentration (amount of protein) in the total reaction volume is known. As a result, we have been employing targeted proteomics (multiple reaction monitoring) with peptide standards and a triple quadrupole mass spectrometer to estimate the absolute amount of protein in a mixed protein microsomal fraction. The following method details the steps required to define the absolute quantitation of an in vitro expressed membrane protein to define complete kinetic parameters.
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ItemEnrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings(Humana Press, 2017)The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.
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ItemProteomic characterization of Golgi membranes enriched from Arabidopsis suspension cell cultures(Springer, 2016)The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.
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ItemXylose donor transport is critical for fungal virulence(PUBLIC LIBRARY SCIENCE, 2018-01)Cryptococcus neoformans, an AIDS-defining opportunistic pathogen, is the leading cause of fungal meningitis worldwide and is responsible for hundreds of thousands of deaths annually. Cryptococcal glycans are required for fungal survival in the host and for pathogenesis. Most glycans are made in the secretory pathway, although the activated precursors for their synthesis, nucleotide sugars, are made primarily in the cytosol. Nucleotide sugar transporters are membrane proteins that solve this topological problem, by exchanging nucleotide sugars for the corresponding nucleoside phosphates. The major virulence factor of C. neoformans is an anti-phagocytic polysaccharide capsule that is displayed on the cell surface; capsule polysaccharides are also shed from the cell and impede the host immune response. Xylose, a neutral monosaccharide that is absent from model yeast, is a significant capsule component. Here we show that Uxt1 and Uxt2 are both transporters specific for the xylose donor, UDP-xylose, although they exhibit distinct subcellular localization, expression patterns, and kinetic parameters. Both proteins also transport the galactofuranose donor, UDP-galactofuranose. We further show that Uxt1 and Uxt2 are required for xylose incorporation into capsule and protein; they are also necessary for C. neoformans to cause disease in mice, although surprisingly not for fungal viability in the context of infection. These findings provide a starting point for deciphering the substrate specificity of an important class of transporters, elucidate a synthetic pathway that may be productively targeted for therapy, and contribute to our understanding of fundamental glycobiology.
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ItemThe Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development(NATURE PORTFOLIO, 2016-07)Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.