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Article

Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities

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Université de Paris, INSERM U1149, 16 rue Henri Huchard, 75018 Paris, France
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Université de Paris, CICB-Paris, CNRS UMR8601, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints-Pères, 75006 Paris, France
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Mass Spectrometry Laboratory, Institut Jacques Monod, UMR 7592, Université de Paris, CNRS, F-75205 Paris, France
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School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
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Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France
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Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques (SABNP), Univ Evry, INSERM U1204, Université Paris-Saclay, 91025 Evry, France
*
Author to whom correspondence should be addressed.
Present address: Division of Anatomy, Biochemistry and Genetics, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, An-Najah National University, P.O. Box 7, Nablus, Palestine.
Molecules 2019, 24(11), 2135; https://doi.org/10.3390/molecules24112135
Received: 15 May 2019 / Revised: 4 June 2019 / Accepted: 5 June 2019 / Published: 6 June 2019
Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities. View Full-Text
Keywords: protein N-glycosylation; lipid-linked oligosaccharide; congenital disorders of glycosylation; endoplasmic reticulum; peptidoglycan biosynthesis protein N-glycosylation; lipid-linked oligosaccharide; congenital disorders of glycosylation; endoplasmic reticulum; peptidoglycan biosynthesis
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MDPI and ACS Style

Massarweh, A.; Bosco, M.; Chantret, I.; Léger, T.; Jamal, L.; Roper, D.I.; Dowson, C.G.; Busca, P.; Bouhss, A.; Gravier-Pelletier, C.; Moore, S.E.H. Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities. Molecules 2019, 24, 2135. https://doi.org/10.3390/molecules24112135

AMA Style

Massarweh A, Bosco M, Chantret I, Léger T, Jamal L, Roper DI, Dowson CG, Busca P, Bouhss A, Gravier-Pelletier C, Moore SEH. Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities. Molecules. 2019; 24(11):2135. https://doi.org/10.3390/molecules24112135

Chicago/Turabian Style

Massarweh, Ahmad, Michael Bosco, Isabelle Chantret, Thibaut Léger, Layla Jamal, David I. Roper, Christopher G. Dowson, Patricia Busca, Ahmed Bouhss, Christine Gravier-Pelletier, and Stuart E.H. Moore. 2019. "Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities" Molecules 24, no. 11: 2135. https://doi.org/10.3390/molecules24112135

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