Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3

The nucleotide-sugar transporters (NSTs) are integral membrane proteins of the Golgi apparatus \nand the Endoplasmic Reticulum (ER), that controls the flux of nucleotide-sugars from the cytosol to \nthe lumen, where they serve as substrate for specific glycosyltransferases during the synthesis of \n...

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Detalles Bibliográficos
Autor principal: Favarolo, María Belen
Otros Autores: Baumeister, Ralph
Formato: Tesis de maestría acceptedVersion
Lenguaje:Inglés
Publicado: Facultad de Farmacia y Bioquímica 2016
Materias:
Glicosilación
Transporte
Estructura-función
Transporter
Structure
Function
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2811
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2811.dir/2811.PDF
Aporte de:
Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_2811
record_format dspace
spelling I28-R145-HWA_28112022-03-07 The nucleotide-sugar transporters (NSTs) are integral membrane proteins of the Golgi apparatus \nand the Endoplasmic Reticulum (ER), that controls the flux of nucleotide-sugars from the cytosol to \nthe lumen, where they serve as substrate for specific glycosyltransferases during the synthesis of \ndiverse glycoconjugates (glycolipids, glycoproteins and proteoglycans). The NSTs are members of \nSLC35 family, a group of highly conserved hydrophobic proteins with multiple transmembrane \ndomains with a size of 30-40kDa. Mutations in these proteins, cause development anomalies in \neukaryotic organisms, from protozoa to mammals, indicating the physiological relevance of these \ntransporters. \nFor the nucleotide-sugar transport process an antiporter mechanism, nucleotide sugar/ nucleoside \nmonophosphate, have been proposed. Nevertheless the molecular basis of ligand recognition and \ntransport is unknown. Like other membrane transport proteins the progress in structure-function \nstudies are limited due to the complexity in the transport activity characterization assays and the \ndifficulty to obtain large amounts of purified protein. In this context the general aim of this work \nwas to determine the transport mechanism of NST at a molecular level, using the murine SLC35A3 \ntransporter, specific for UDP-GlcNAc as a model. Based on bioinformatics tools, GFP-Fusion \ntechnology, a complementation activity assay and cysteine scanning mutagenesis we developed an \napproach to identify and characterize residues critical for the UDP-GlcNAc transport activity by \nSLC35A3. We functionally characterized the MmSLC35A3 transporter as a UDP-GlcNAc \ntransporter based on bioinformatic tools, phenotypic correction and Cysteine Scanning Mutagenesis \nassays. Two critical residues for the MmSLC35A3 functioning, Glu47 and Lys50, localized in the \nsecond transmembrane domain were identified. On the other hand, based on the approach \ndeveloped by Newstead et al (2007), we were able to optimize the MmSLC35A3 expression and \npurification, obtaining a yield of 3.9 mg of protein per L of culture, estimating that this approach \nwill leads to a function-structure studies on MmSLC35A3 pure. This last approach will be based on the labeling of the cysteine residues on the cysteine unique mutants with fluorescents probes. The \nobjective of this label is to know which residues are exposed to the cytosolic or the luminal space Fil: Favarolo, María Belen. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Baumeister, Ralph Facultad de Farmacia y Bioquímica Bredeston, Luis M. Favarolo, María Belen 2016-11-23 application/pdf Adamo, Ana Abdian, Patricia Rühe, Jürgen Glicosilación Transporte Estructura-función Transporter Structure Function eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3 Estudios de estructura-función sobre el transportador de UDP-GlcNAc, SLC35A3 info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2811 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2811.dir/2811.PDF
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Inglés
orig_language_str_mv eng
topic Glicosilación
Transporte
Estructura-función
Transporter
Structure
Function
Ciencias de la vida
spellingShingle Glicosilación
Transporte
Estructura-función
Transporter
Structure
Function
Ciencias de la vida
Favarolo, María Belen
Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
topic_facet Glicosilación
Transporte
Estructura-función
Transporter
Structure
Function
Ciencias de la vida
description The nucleotide-sugar transporters (NSTs) are integral membrane proteins of the Golgi apparatus \nand the Endoplasmic Reticulum (ER), that controls the flux of nucleotide-sugars from the cytosol to \nthe lumen, where they serve as substrate for specific glycosyltransferases during the synthesis of \ndiverse glycoconjugates (glycolipids, glycoproteins and proteoglycans). The NSTs are members of \nSLC35 family, a group of highly conserved hydrophobic proteins with multiple transmembrane \ndomains with a size of 30-40kDa. Mutations in these proteins, cause development anomalies in \neukaryotic organisms, from protozoa to mammals, indicating the physiological relevance of these \ntransporters. \nFor the nucleotide-sugar transport process an antiporter mechanism, nucleotide sugar/ nucleoside \nmonophosphate, have been proposed. Nevertheless the molecular basis of ligand recognition and \ntransport is unknown. Like other membrane transport proteins the progress in structure-function \nstudies are limited due to the complexity in the transport activity characterization assays and the \ndifficulty to obtain large amounts of purified protein. In this context the general aim of this work \nwas to determine the transport mechanism of NST at a molecular level, using the murine SLC35A3 \ntransporter, specific for UDP-GlcNAc as a model. Based on bioinformatics tools, GFP-Fusion \ntechnology, a complementation activity assay and cysteine scanning mutagenesis we developed an \napproach to identify and characterize residues critical for the UDP-GlcNAc transport activity by \nSLC35A3. We functionally characterized the MmSLC35A3 transporter as a UDP-GlcNAc \ntransporter based on bioinformatic tools, phenotypic correction and Cysteine Scanning Mutagenesis \nassays. Two critical residues for the MmSLC35A3 functioning, Glu47 and Lys50, localized in the \nsecond transmembrane domain were identified. On the other hand, based on the approach \ndeveloped by Newstead et al (2007), we were able to optimize the MmSLC35A3 expression and \npurification, obtaining a yield of 3.9 mg of protein per L of culture, estimating that this approach \nwill leads to a function-structure studies on MmSLC35A3 pure. This last approach will be based on the labeling of the cysteine residues on the cysteine unique mutants with fluorescents probes. The \nobjective of this label is to know which residues are exposed to the cytosolic or the luminal space
author2 Baumeister, Ralph
author_facet Baumeister, Ralph
Favarolo, María Belen
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Favarolo, María Belen
author_sort Favarolo, María Belen
title Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
title_short Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
title_full Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
title_fullStr Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
title_full_unstemmed Study of the structure and the function of the UDP-GlcNAc transporter, SLC35A3
title_sort study of the structure and the function of the udp-glcnac transporter, slc35a3
publisher Facultad de Farmacia y Bioquímica
publishDate 2016
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_2811
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_2811.dir/2811.PDF
work_keys_str_mv AT favarolomariabelen studyofthestructureandthefunctionoftheudpglcnactransporterslc35a3
AT favarolomariabelen estudiosdeestructurafuncionsobreeltransportadordeudpglcnacslc35a3
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