Abstract
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.
Highlights
In mammals, protein N-glycosylation is essential for life [1,2], and, in the human population, 1
The solid-phase extraction extraction (SPE) extraction of reaction mixtures using Vm-beads facilitated the rapid analysis of lipid II
Vm-beads facilitated the rapid analysis of lipid and its hydrolysis products in reaction mixtures containing liver microsomal proteins
Summary
Protein N-glycosylation is essential for life [1,2], and, in the human population, 1. N-glycosylation is essential for lifeof[1,2], in the human population, in of genes required for (CDG). This process underlie a group rare inherited metabolic diseases congenitalmutations disorders glycosylation [3,4]. N-glycosylation is the endoplasmic reticulum (ER) by the sequential addition ofshown monosaccharides to dolichyl-phosphate initiated in the endoplasmic reticulum (ER) by the sequential addition of monosaccharides to (DP) to yield mature Glc Man GlcNAc2 -PP-dolichol (DLO) [5,6]. N-glycan biosynthesis, andthe theproduction production of oligosaccharylphosphates in congenital disorders of glycosylation. Dolichyl phosphate (left) is glycosylated on the cytoplasmic in congenital disorders of glycosylation. Dolichyl phosphate (left) is glycosylated on the cytoplasmic face of the ER to yield Man5GlcNAc2-PP-dolichol which is flipped into the ER and further elongated face of thetoER to yield Man GlcNAc2 -PP-dolichol which is flipped into the ER and further elongated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
