Transportadores de nucleótidos de azúcar : mecanismo de transporte y relevancia funcional

En eucariontes, la mitad de las proteínas celulares son secretadas o permanecen unidas a membrana. Ambos tipos de proteínas son sintetizadas en ribosomas unidos a membrana y translocados al lumen del Retículo Endoplasmático. Desde allí, migran vía vesículas al aparato de Golgi y luego al destino final ya sea dentro o fuera de la célula. En el lumen del aparato de Golgi, un gran porcentaje de estas proteínas son glicosiladas por glicosiltransferasas que usan nucleótidos de azúcar como sustratos. Estos derivados nucleotídicos deben ser transportados desde el citosol, donde la mayoría son sintetizados, hacia el lumen del aparato Golgi. El transporte al lumen de esta organela es mediado por transportadores específicos, llamados transportadores de nucleótidos de azúcar. C.elegans es un organismo bien caracterizado tanto desde el punto de vista genético como en cuanto al desarrollo, para el cual sólo se han descrito 2 de 18 putativos transportadores de nucleótidos de azúcar. En esta tesis, se caracterizó un transportador de nucleótidos de azúcar de C.elegans codificado por el gen C03H5.2 que transloca UDP-Nacetilglucosamina y UDP-N-acetilgalactosamina. Se obtuvo evidencia que los dos sustratos son transportados en forma independiente y simultánea, un mecanismo nuevo que es diferente del transporte competitivo no cooperativo previamente descrito para otro transportador de nucleótido de azúcar. También hemos obtenido evidencia de redundancia funcional entre C03H5.2 y otro transportador de nucleótidos de azúcar de C.elegans, SRF-3. Esto sugiere un mecanismo que asegura una adecuada biosíntesis de glicoconjugados en organismos multicelulares que a su vez, es necesario para un correcto desarrollo tisular. El Síndrome de Adhesión Leucocitaria II es un síndrome humano causado por mutaciones en el transportador de GDP-fucosa. Hemos establecido y realizado los experimentos que muestran que las mutantes en este transportador tienen actividad parcial. Entonces, no es necesario invocar otras proteínas para explicar la actividad de transporte residual observada en vesículas del aparato de Golgi de células de pacientes.In eukaryotes, half of cellular proteins are secreted or membrane bound. Both groups of proteins are synthesized on membrane bound polysomes and translocated into the lumen of the endoplasmic reticulum and thereafter migrate via vesicles to the Golgi apparatus. In the lumen of the Golgi apparatus, most of these proteins become glycosylated by substrates which are nucleotide-sugars. These nucleotide derivatives must be transported from the cytosol, the site where most are synthesized, into the lumen of the above organelle. Transport into the lumen of the Golgi apparatus is mediated by specific transporters, named nucleotide sugar transporters. C.elegans is a genetically and developmentally well characterized organism for which only 2 out of 18 putative nucleotide sugar transporter-like proteins have been described. Here we have characterized a nucleotide sugar transporter of C. elegans encoded by the gene CO3H5.2 which translocates UDP-N-acetylglucosamine and UDP- N-acetylgalactosamine. Evidence was obtained that the two substrates are translocated via an independent and simultaneous manner, a novel mechanism that is different from the previously described multisubstrate nucleotide sugar transporters which are competititve and non cooperative. We also obtained evidence for functional redundancy between C03H5.2 and SRF-3, another nucleotide sugar transporter from C.elegans. This suggests a mechanism to ensure adequate glycoconjugate biosynthesis in multicellular organisms which in turn may be necessary for proper tissue development. Leukocyte Adhesion Deficiency II is a human syndrome caused by mutations in the GDP-fucose transporter. We have designed and performed experiments that show that mutants in this transporter have parcial activity. Thus it is not necessary to invoke other proteins to explain the residual transport activity observed in Golgi vesicles of patient cells.Fil: Caffaro, Carolina E.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Inhibition of Golgi Apparatus Glycosylation Causes Endoplasmic Reticulum Stress and Decreased Protein Synthesis*

Nucleotide sugar transporters of the Golgi apparatus play an essential role in the glycosylation of proteins, lipids, and proteoglycans. Down-regulation of expression of the transporters for CMP-sialic acid, GDP-fucose, or both unexpectedly resulted in accumulation of glycoconjugates in the Golgi apparatus rather than in the plasma membrane. Pulse-chase experiments with radiolabeled sugars and amino acids showed decreased synthesis and secretion of both nonglycoproteins and glycoproteins. Further studies revealed that the above silencing induced endoplasmic reticulum stress and inhibited protein translation initiation. Together these results suggest that global inhibition of Golgi apparatus glycosylation may lead to important secondary metabolic changes, unrelated to glycosylation

A Nucleotide Sugar Transporter Involved in Glycosylation of the <i>Toxoplasma</i> Tissue Cyst Wall Is Required for Efficient Persistence of Bradyzoites

<div><p><i>Toxoplasma gondii</i> is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host <i>via</i> encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a <i>Toxoplasma</i> nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. <i>Toxoplasma</i> strains deleted for the <i>TgNST1</i> gene (Δ<i>nst1</i>) form cyst-like structures <i>in vitro</i> but no longer interact with lectins, suggesting that Δ<i>nst1</i> strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. <i>In vivo</i> infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δ<i>nst1</i> parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of <i>Toxoplasma</i> tissue cysts.</p></div

Deletion of <i>TgNST1</i> affects GRA2 mobility in a SDS-PAGE.

<p>Total parasite extracts from parental, control, <i>Δnst1</i> and <i>Δnst1:NST1</i> strains were separated by SDS-PAGE and proteins transferred to PVDF membrane. The mobility of GRA2 protein was analyzed using a mouse anti-GRA2 antibody (upper panel). The blot was stripped and reprobed with antibodies to SAG1 as a loading control (lower panel).</p

<i>Toxoplasma</i> TgNST1 is a nucleotide-sugar transporter that transports UDP-GlcNAc and UDP-GalNac.

<p>A) Analysis of protein expression by western blot. Total extracts from <i>Saccharomyces cerevisiae</i> strain PRY225 and the same strain transformed with the p426 vector encoding the <i>Toxoplasma</i> NST1HA protein (TgNST1) were resolved by SDS-PAGE followed by transfer to PVDF membrane. Detection of the recombinant protein was done using an anti-HA antibody. B) 1 mg of membrane vesicles from yeast transformed with an empty vector or the vector encoding the TgNST1HA protein were incubated with radioactive and unlabeled UDP-GlcNAc or UDP-GalNAc for 4 min. Final concentration of nucleotide-sugar is 2 µM per sample. Vesicles were separated by centrifugation and radioactivity within vesicles was measured in the pellet after acid precipitation. Transport activity is calculated as the difference of radioactive solutes in the vesicle pellet after incubation at 30°C vs 0°C and expressed as pmol of nucleotide sugar/mg protein/min.</p

Design and verification of the strain deficient in NST1 and the corresponding complemented strain.

<p>A) Workflow of the generation of the wild type, Δ<i>nst1</i> and <i>Δnst1:NST1</i> strains. 5′- and 3′-FR indicates the 5′- and 3′-flanking regions of the TgNST1 gene used for the homologous recombination required for deletion of the gene. Primers used for PCR analysis are numbered. B) The engineered strains of panel A were analyzed by PCR using primers that specifically amplify the 5′- or 3′-flanking regions (primers 1+2 and 3+4, respectively), the NST1 coding region (primers 5+6), the HPT cassette (primers 7+8), or the 5′- to 3′- flanking regions (primers 9+10).</p

TgNST1 activity is not required for virulence during the acute stage of infection in mice.

<p>Mice were infected i.p. with 500 WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> tachyzoites. A) At the indicated days post-infection (pi), animals were injected i.p. with 200 µL of d-luciferin in PBS (150 mg substrate/kg body weight), anesthetized with isoflurane and imaged using the Xenogen IVIS200 charge-couple. Mice were imaged ventrally for 5 min. B) Infected animals shown in panel A were weighed at the indicated days post-infection and loss of weight was plotted as the percentage of the initial weight over time. Weights for animals infected with WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> parasites are shown with green, red and blue lines, respectively.</p

<i>TgNST1</i> deletion interferes with recognition by the DBA and WGA lectins.

<p>WT, <i>Δnst1</i>, or <i>Δnst1:NST1</i> parasites were grown under bradyzoite-inducing conditions for 4 days <i>in vitro</i>, fixed in formaldehyde, permeabilized and stained using the FITC-conjugated lectins DBA (A) or WGA (B). Antibodies against the bradyzoite-specific antigen SRS9 and Alexa 647-conjugated goat anti-rabbit antisera (pseudocolor red) were use to identify bradyzoites within cysts. Scale bar represents 10 µm. C) Mice infected with the WT, <i>Δnst1</i> and <i>Δnst1:NST1</i> strains were sacrificed 3 weeks post-infection, perfused with heparine/saline and brains were drop-fixed in paraformaldehyde, sucrose-embedded and cryosectioned for immunohistochemistry. 40 µm sections were stained using the FITC-DBA lectin and antibody against the bradyzoite-protein SRS9, as in parts A and B. 1 µm slice images were taken using a Zeiss confocal microscope and a single slice is shown. Scale bar represents 10 µm.</p

Mice infected with the Δ<i>nst1</i> strain show reduced numbers of cysts in the brain.

<p>Mice were infected i.p. with 500 WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> tachyzoites. 3 weeks post-infection, animals were sacrificed by perfusion and half the brain was homogenized for cyst enumeration using antibodies against the bradyzoite-specific antigen SRS9 to facilitate detection of cysts. The figure shows representative results from two independent experiments. Horizontal bars indicate the mean for each group. Asterisks indicate statistically significant differences (p = 0.05) between indicated groups.</p

Δ<i>nst1</i> parasites are not defective in tropism to the brain.

<p>Mice were infected i.p. with 500 WT, Δ<i>nst1</i> or Δ<i>nst1:NST1</i> tachyzoites.12 days post-infection, animals were sacrificed and the posterior left quarter of the brain was used to quantify the number of parasites present by real-time quantitative PCR detection of <i>Toxoplasma</i>'s B1 gene normalized to the detection levels of mouse actin (ACT1).</p