Cargando…

Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis

Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes ca...

Descripción completa

Detalles Bibliográficos
Autores principales: Caballé, Marc, Faijes, Magda, Planas, Antoni
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9692976/
https://www.ncbi.nlm.nih.gov/pubmed/36430454
http://dx.doi.org/10.3390/ijms232213975
_version_ 1784837412948541440
author Caballé, Marc
Faijes, Magda
Planas, Antoni
author_facet Caballé, Marc
Faijes, Magda
Planas, Antoni
author_sort Caballé, Marc
collection PubMed
description Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes capable of synthesizing this molecule under in vivo or in vitro conditions is a potential production strategy. Several GT4 enzymes from Bacteroides fragilis were considered as potential candidates in addition to the known BF9343_3149, but only this one showed glycolipid synthase activity. The enzyme was expressed as a SUMO fusion protein to produce soluble protein. It is a non-processive glycosyltransferase that prefers UDP-Gal over UDP-Glc as a donor substrate, and maximum activity was found at pH 7.3 and around 30–35 °C. It does not require metal cations for activity as other GT4 enzymes, but Zn(2+) inactivates the enzyme. The reaction occurs when the ceramide lipid acceptor is solubilized with BSA (100% conversion) but not when it is presented in mixed micelles, and anionic lipids do not increase activity, as in other membrane-associated glycolipid synthases. Further protein engineering to increase stability and activity can make feasible the enzymatic synthesis of α-GalCer for biomedical applications.
format Online
Article
Text
id pubmed-9692976
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-96929762022-11-26 Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis Caballé, Marc Faijes, Magda Planas, Antoni Int J Mol Sci Article Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes capable of synthesizing this molecule under in vivo or in vitro conditions is a potential production strategy. Several GT4 enzymes from Bacteroides fragilis were considered as potential candidates in addition to the known BF9343_3149, but only this one showed glycolipid synthase activity. The enzyme was expressed as a SUMO fusion protein to produce soluble protein. It is a non-processive glycosyltransferase that prefers UDP-Gal over UDP-Glc as a donor substrate, and maximum activity was found at pH 7.3 and around 30–35 °C. It does not require metal cations for activity as other GT4 enzymes, but Zn(2+) inactivates the enzyme. The reaction occurs when the ceramide lipid acceptor is solubilized with BSA (100% conversion) but not when it is presented in mixed micelles, and anionic lipids do not increase activity, as in other membrane-associated glycolipid synthases. Further protein engineering to increase stability and activity can make feasible the enzymatic synthesis of α-GalCer for biomedical applications. MDPI 2022-11-12 /pmc/articles/PMC9692976/ /pubmed/36430454 http://dx.doi.org/10.3390/ijms232213975 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Caballé, Marc
Faijes, Magda
Planas, Antoni
Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis
title Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis
title_full Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis
title_fullStr Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis
title_full_unstemmed Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis
title_short Characterization of a Glycolipid Synthase Producing α-Galactosylceramide in Bacteroides fragilis
title_sort characterization of a glycolipid synthase producing α-galactosylceramide in bacteroides fragilis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9692976/
https://www.ncbi.nlm.nih.gov/pubmed/36430454
http://dx.doi.org/10.3390/ijms232213975
work_keys_str_mv AT caballemarc characterizationofaglycolipidsynthaseproducingagalactosylceramideinbacteroidesfragilis
AT faijesmagda characterizationofaglycolipidsynthaseproducingagalactosylceramideinbacteroidesfragilis
AT planasantoni characterizationofaglycolipidsynthaseproducingagalactosylceramideinbacteroidesfragilis