Mediators of Galactose Sensitivity in UDP-Galactose 4′-Epimerase-impaired Mammalian Cells

Schulz, Jenny M.; Ross, Kerry L.; Malmström, Kerstin; Krieger, Monty; Fridovich‐Keil, Judith L.

doi:10.1074/jbc.m414045200

Cited by 31 publications

(46 citation statements)

References 37 publications

(51 reference statements)

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“…Prior studies have demonstrated that GALE-impaired yeast (Douglas and Hawthorne, 1964;Ross et al, 2004), mammalian tissue culture cells (Schulz et al, 2005) and patients (Openo et al, 2006) all accumulate elevated levels of gal-1P upon exposure to high levels of environmental galactose, ostensibly because, when GALE is deficient, GALT activity is compromised by an accumulation of product (UDP-gal) and a depletion of substrate (UDP-glc).…”

Section: Loss Of Gale Results In Abnormal Accumulation Of Gal-1pmentioning

confidence: 99%

“…GALE-deficient yeast are viable and apparently healthy, although they arrest growth upon exposure to even trace levels of environmental galactose (Douglas and Hawthorne, 1964;Ross et al, 2004). GALE-deficient Chinese hamster ovary (CHO) cells [ldlD cells (Krieger et al, 1989)] also demonstrate galactose-sensitive growth arrest, as well as defects in both N-and O-linked glycosylation (Kingsley et al, 1986;Schulz et al, 2005). Nonetheless, the relationship between yeast or tissue culture outcomes and the pathophysiology of epimerase deficiency galactosemia remains unclear.…”

Section: Introductionmentioning

confidence: 99%

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UDP-galactose 4′ epimerase (GALE) is essential for development ofDrosophila melanogaster

Sanders¹,

Sefton

Moberg

et al. 2010

Disease Models &Amp; Mechanisms

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SUMMARYUDP-galactose 4' epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

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Section: Loss Of Gale Results In Abnormal Accumulation Of Gal-1pmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

UDP-galactose 4′ epimerase (GALE) is essential for development ofDrosophila melanogaster

Sanders¹,

Sefton

Moberg

et al. 2010

Disease Models &Amp; Mechanisms

Self Cite

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“…This is because (i) human cells have salvage pathways for UDP-Galp biosynthesis (indeed, Gal-1-P and UDP-Galp accumulate in GalE null mammalian cells [103]) and (ii) although defects in human GalE lead to type III galactosemia (114), inhibition of human GalE over a period of days may be tolerable. In any case, three lines of evidence suggest that some selectivity against trypanosome GalE enzymes should be possible.…”

Section: Cruzi) Gdp-man Is Also the Precursor Of Gdp-fuc (See Below)mentioning

confidence: 99%

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

2007

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The cell surface glycoconjugates of trypanosomatid parasites are intimately involved in parasite survival, infectivity, and virulence in their insect vectors and mammalian hosts. Although there is a considerable body of work describing their structure, biosynthesis, and function, little is known about the sugar nucleotide pools that fuel their biosynthesis. In order to identify and quantify parasite sugar nucleotides, we developed an analytical method based on liquid chromatography-electrospray ionization-tandem mass spectrometry using multiple reaction monitoring. This method was applied to the bloodstream and procyclic forms of Trypanosoma brucei, the epimastigote form of T. cruzi, and the promastigote form of Leishmania major. The trypanosomatids are protozoan parasites that impose a major health burden on many countries in the developing world, causing a wide range of diseases over three continents. Like most eukaryotes, the cell surfaces and endosomal/lysosomal systems of these organisms are rich in glycoconjugates, some of which play essential roles in their survival, infectivity, or virulence. The glycoconjugate repertoires of the three trypanosomatids studied here, Trypanosoma brucei, T. cruzi, and Leishmania major, are fundamentally different, reflecting their disparate life cycles, modes of infection, and disease pathologies (see references 5,27,28,38,47,65, and 69 and references therein). The monosaccharides that make up these glycoconjugates vary between the three trypanosomatid species. For example, all three contain D-mannose (Man), D-Nacetylglucosamine (GlcNAc), D-glucosamine (GlcN), D-glucose (Glc), and D-galactopyranose (Galp), while only T. cruzi and L. major contain D-galactofuranose (Galf), only T. cruzi contains D-xylose (Xyl), L-rhamnopyranose (Rha), and L-fucose (Fuc), and only L. major contains D-arabinopyranose (Ara) ( Table 1). However, a unifying theme of their glycobiology is an abundance of cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and/or non-proteinlinked GPI structures.The protein-linked GPI glycans of the leishmania are the simplest in structure, consisting of the conserved Man␣1-2Man␣1-6Man␣1-4GlcN core. Those of T. cruzi are principally Man␣1-2Man␣1-2Man␣1-6Man␣1-4GlcN, and those of T. brucei are the most complex, with ␣-D-Galp and ␤-D-Galp side chains in the bloodstream form and sialylated poly-N-acetyllactosamine (poly-LacNAc) side chains in the procyclic form (27).The non-protein-linked GPI structures include the so-called glycoinositolphospholipids, or GIPLs, that are most abundant in the leishmania and T. cruzi species (58, 64, 90) but are also found in procyclic-form T. brucei (60,73,122) and bloodstream form T. brucei (39,63). Non-protein-linked GPIs also include the more exotic leishmania-specific lipophosphoglycan (LPG) structures (38,47,64,65). The leishmania LPGs contain characteristic phosphosaccharide repeats of Galp␤1-4Man␣1-P, which in L. major can have ␤-D-Galp and ␣-D-Arap side chains (66,67). These repeats are also found in the secr...

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“…After 2 min incubation, 100 l of 500 mM KH 2 PO 4 was added to the reaction mixture to stop the reaction. The final product, UDP-GlcNAc or UDP-N-acetyl-D-galactosamine (UDP-GalNAc), was measured and quantified mainly according to the method described by Schulz et al (24). In this method, the elution positions for UDP-GlcNAc and UDPGalNAc showed individual peaks.…”

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confidence: 99%

Identification of Novel Acetyltransferase Activity on the Thermostable Protein ST0452 from Sulfolobus tokodaii Strain 7

2010

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and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-D-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.The nucleotide-sugar molecule is used as a substrate for the construction of the polymer structure of carbohydrates and as a starting material for the biosynthesis of modified-sugar-nucleotide conjugates; for example, TDP-glucose is the starting material for the construction of TDP-rhamnose (25). Several sugar-1-phosphate nucleotidylyltransferases (sugar-1-P NTases) involved in some biosynthetic reactions, such as glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase), N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase), and mannose-1-phosphate guanylyltransferase, have been identified from many bacteria, including pathogens, as well as from eukaryotes (5,10,11,17,25,27,29). However, only limited information on the archaeal enzyme with sugar-1-P NTase activity has been obtained.Our group has previously reported the first thermostable enzyme with sugar-1-P NTase activity from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7 (31). The sugar-1-P NTase activity of the ST0452 protein, which was primarily identified by sequence similarity with the Escherichia coli RmlA protein, indicated the utilization of Glc-1-P and GlcNAc-1-P as a sugar-1-P substrate and all deoxynucleoside triphosphates (dNTPs) and UTP as nucleoside triphosphate (NTP) substrates, but glucosamine-1-phosphate (GlcN-1-P) was not utilized as a substrate. It was also reported that the GlcNAc-1-P UTase activity of the ST0452 protein was improved by the introduction of site-directed mutagenesis (30).The identification of GlcNAc-1-P UTase activity on the ST0452 protein suggests that the protein may have activities and features similar to those of the bacterial GlmU enzyme (19). However, the ST0452 protein exhibits only a low level of sequence identity (less than 20% identity) with E. coli GlmU (data not shown). The phylogenetic analysis of the ST0452 protein and related proteins indicates that the ST0452 protein and homologous proteins form a group independent of other related enzymes identified as RmlA or GlmU (Fig. 1). In spite of the low level of sequence similarity of the ST0452 protein with the bacterial GlmU, GlcNAc-1-P UTase activity was detected on the protein. Twe...

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Mediators of Galactose Sensitivity in UDP-Galactose 4′-Epimerase-impaired Mammalian Cells

Cited by 31 publications

References 37 publications

UDP-galactose 4′ epimerase (GALE) is essential for development ofDrosophila melanogaster

UDP-galactose 4′ epimerase (GALE) is essential for development ofDrosophila melanogaster

Sugar Nucleotide Pools of Trypanosoma brucei , Trypanosoma cruzi , and Leishmania major

Identification of Novel Acetyltransferase Activity on the Thermostable Protein ST0452 from Sulfolobus tokodaii Strain 7

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