Functional cloning and characterization of a UDP- glucuronic acid decarboxylase: The pathogenic fungus
            <i>Cryptococcus neoformans</i>
            elucidates UDP-xylose synthesis

Bar‐Peled, Maor; Griffith, Cara L.; Doering, Tamara L.

doi:10.1073/pnas.211229198

Cited by 137 publications

(132 citation statements)

References 33 publications

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“…Using serial analysis of gene expression or SAGE, Steen and collaborators 50 showed that C. neoformans cells present in the cerebral spinal fluid of rabbit with cryptococcal meningitis are actively engaged in protein synthesis, protein degradation, stress response, small molecule transport and signaling but none of the genes recently described involved in the capsule biosynthesis pathway [51][52][53] has been identified thus far. It may be that none are triggered in this early machinery and/or that the pathogenesis of cryptococcal meningitis in the rabbit differs from that of disseminated cryptococcosis in the mouse or in humans (one major difference is that rabbits have higher body temperature than both mice or humans).…”

Section: Discussionmentioning

confidence: 99%

Capsule Structure Changes Associated with Cryptococcus neoformans Crossing of the Blood-Brain Barrier

Charlier

Chrétien

Baudrimont

et al. 2005

The American Journal of Pathology

187

188

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Cryptococcus neoformans is a yeast responsible for disseminated meningoencephalitis in patients with cellular immune defects. The major virulence factor is the polysaccharide capsule. We took advantage of a relevant murine model of disseminated meningoencephalitis to study the early events associated with blood-brain barrier (BBB) crossing. Mice were sacrificed at 1, 6, 24, and 48 hours post-intravenous inoculation, and classical histology, electron microscopy, and double immunofluorescence were used to study tissues and yeasts. Crossing of the BBB occurred early after inoculation, did not involve the choroid plexus but instead occurred at the level of the cortical capillaries, and caused early and severe damage to the structure of the microvessels. Seeding of the leptomeninges was not the primary event but occurred secondary to leakage of cortical pseudocysts. Organ invasion was associated with changes in cryptococcal capsule structure and cell size, which differed in terms of magnitude and kinetics, depending on both the organs involved, and potentially, on the bed structure of the local capillary. The rapid changes in capsule structure could contribute to inability of the host immune response to control cryptococcal infection in extrapulmonary spaces.

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Section: Discussionmentioning

confidence: 99%

Capsule Structure Changes Associated with Cryptococcus neoformans Crossing of the Blood-Brain Barrier

Charlier

Chrétien

Baudrimont

et al. 2005

The American Journal of Pathology

187

188

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“…In brief, a pET-28a plasmid carrying the decarboxylase (UXS1) cDNA (a kind gift of Dr. Tamara Doering) was used to transform E. coli strain BL21(DE3)pLysS. After induction using isopropyl-␤-D-thiogalactopyranoside, the cells were harvested and lysed as described (24). To generate UDP-xylose, 10 l of soluble bacterial protein extract was incubated overnight with 250 nmol of UDP-glucuronic acid (UDP-GlcA) and 125 nmol of NAD ϩ in Tris-Cl, pH 7.4, buffer in a total volume of 50 l at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…As acceptor, a peptide (DDDSIEGSGGR) corresponding to residues 55-65 of Drosophila syndecan (25) was used, which contains the features of an acidic region and a Ser-Gly sequence found around chondroitin and heparan sulfate attachment sites (31,32), as well as an arginine residue which apparently aids detection by MALDI-TOF mass spectrometry (33). For most experiments, the UDP-Xyl donor was synthesized, without subsequent purification, using recombinant Cryptococcus UDP-GlcA decarboxylase (24).…”

Section: Identification and Analysis Of The Drosophila Oxt Gene Andmentioning

confidence: 99%

Functional Characterization of Drosophila melanogaster Peptide O-Xylosyltransferase, the Key Enzyme for Proteoglycan Chain Initiation and Member of the Core 2/I N-Acetylglucosaminyltransferase Family

Wilson

2002

Journal of Biological Chemistry

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Chondroitin and heparan sulfates are essential players in animal development and are synthesized by a series of glycosyltransferases, the first of which is UDP-␣-D-xylose:proteoglycan core protein ␤-D-xylosyltransferase (EC 2.4.2.26). In the present study, a Drosophila melanogaster gene (CG17771), previously designated as a homologue of core 2 and I ␤1,6-N-acetylglucosaminyltransferases, was shown to encode an active peptide O-xylosyltransferase. A novel coupled assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated transfer of xylose to the peptide DDDSIEGSGGR. Analysis of sequences of various peptide O-xylosyltransferase and ␤1,6-N-acetylglucosaminyltransferase sequences indicates that they are members of a large multifunctional protein family with a range of roles in ␤-glycosylation of either peptide or glycan substrates. Because in contrast to mammals, there is only one fly peptide O-xylosyltransferase gene, it is anticipated that, given the key roles of proteoglycans, the hereby designated oxt gene is essential for viability.

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“…The aqueous phases were combined and analyzed by HPLC using a Phenosphere-ODS2 column (250 3 4.6 mm; Phenomenex, Torrance, CA) or a Spheresorb-ODS2 column (250 3 4.6 mm; Waters, Milford, MA) eluted at 1 mL min ÿ1 with 0.5 M KH 2 PO 4 (A) for 15 min, followed by a gradient to 80% A and 20% methanol over 14 min at a flow rate of 0.7 mL min ÿ1 . Peaks eluted from the ODS2 column were injected onto a Phenosphere-SAX ion exchange column (250 3 4.6 mm; Phenomenex) and eluted at 1 mL min ÿ1 with a linear 2-to 600-mM ammonium-formate gradient formed over 25 min (Bar-Peled et al, 2001). Nucleotides and nucleotide-sugars were detected by UV absorbance using a Waters photodiode array detector.…”

Section: Enzyme Assaysmentioning

confidence: 99%

A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis

et al. 2004

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L-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-b-L-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-b-L-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 308C. The apparent K m for NADPH is 90 mM and 16.9 mM for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-b-L-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.L-Rhamnose is a component of the plant cell wall pectic polysaccharides rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II; Ridley et al., 2001) and is also present in diverse secondary metabolites including anthocyanins, flavonoids, and triterpenoids (Das et al., 1987;Bar-Peled et al., 1991;van Setten et al., 1995;Shinozaki et al., 1996;Markham et al., 2000), in certain types of plant glycoproteins (Haruko and Haruko, 1999), and in arabinogalactan proteins (Pellerin et al., 1995). The specific enzymes that attach rhamnose to each molecule are known as rhamnosyltransferases (RhaTs). To date, only a small number of RhaTs have been studied, and those were involved in flavonoid rhamnosylation. The characterized RhaTs utilize UDP-b-L-rhamnose (UDP-b-L-Rha) as the donor substrate (Kamsteeg et al., 1978;Feingold, 1982;Bar-Peled et al., 1991), although in mung bean (Vigna radiata), both dTDP-b-L-rhamnose (dTDP-b-L-Rha) and UDP-b-L-Rha were reported to act as sugar donors for the rhamnosylation of flavonoids (Barber and Neufeld, 1961).We are studying the enzymes involved in the synthesis of the nucleotide-rhamnose as part of our effort to understand the synthesis of pectic polysaccharides. To date, the rhamnosylation of plant polysaccharides and glycoproteins has not been studied. Thus, the identity of the activated form(s) of rhamnose needed for the synthesis of these macromolecules is not known with certainty. The enzymes required for the synthesis of the activated form(s) of rhamnose in plants have also not been purified.In contrast, much more is known about the synthesis of rhamnose in...

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Functional cloning and characterization of a UDP- glucuronic acid decarboxylase: The pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis

Cited by 137 publications

References 33 publications

Capsule Structure Changes Associated with Cryptococcus neoformans Crossing of the Blood-Brain Barrier

Capsule Structure Changes Associated with Cryptococcus neoformans Crossing of the Blood-Brain Barrier

Functional Characterization of Drosophila melanogaster Peptide O-Xylosyltransferase, the Key Enzyme for Proteoglycan Chain Initiation and Member of the Core 2/I N-Acetylglucosaminyltransferase Family

A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis

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