title: Assessing ER and Golgi N-glycosylation process using metabolic labeling in mammalian cultured cells.
abstract: Modifications of N-glycosylation in disease states are common and illustrate the crucial requirement of glycosylation in human biology. Mainly based on glycan permethylation and the use of mass spectrometry analysis, we can easily understand that many different methods to analyze the N-glycome have seen the day. While extremely powerful, these methods are mainly used to analyze qualitative variations of N-glycosylation of human serum proteins and do not necessarily reflect the glycosylation status of derived mammalian cultured cells. This chapter summarizes two methods that we are routinely using in our laboratory to assess the ER and Golgi N-glycosylation process. The proposed methodology allows pinpointing ER as well as Golgi glycosylation deficiencies in mammalian cultured cells. The first approach is based on direct metabolic labeling of cultured mammalian cells with [2-(3)H] mannose followed by sequential extraction and HPLC analysis of the purified oligosaccharides. The second one is based on the copper-catalyzed azide alkyne cycloaddition (CuAAC) strategy. We propose the use of alkyne-tagged sialic acid (SialNAl) to visualize the Golgi glycosylation efficiency. Their metabolic incorporation into newly synthesized glycoproteins can then be chemoselectively coupled to complementary azide-functionalized fluorophores, and visualized by using confocal laser scanning microscopy. To summarize, we present here a detailed description of our know-how in the field of ER and Golgi N-glycosylation.
authors.affiliation: Center for Human Genetics, KU Leuven University, Herestraat 49, Leuven, Belgium.
authors.forename: Romain
authors.initials: R
authors.lastname: Péanne
authors.forename: Jorick
authors.initials: J
authors.lastname: Vanbeselaere
authors.forename: Dorothée
authors.initials: D
authors.lastname: Vicogne
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Mir
authors.forename: Christophe
authors.initials: C
authors.lastname: Biot
authors.forename: Gert
authors.initials: G
authors.lastname: Matthijs
authors.forename: Yann
authors.initials: Y
authors.lastname: Guérardel
authors.forename: François
authors.initials: F
authors.lastname: Foulquier
mesh_terms: D002478:Cells, Cultured; D057930:Click Chemistry; D018981:Congenital Disorders of Glycosylation / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011499:Protein Processing, Post-Translational; D012794:Sialic Acids / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D012794:Sialic Acids; D008358:Mannose
keywords: Click chemistry; Congenital disorders of glycosylation; Endoplasmic reticulum; Golgi apparatus; Metabolic labeling; N-Glycosylation