Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Cell Biology, CONGENITAL DISORDERS, OLIGOSACCHARIDES, DEGRADATION, Click chemistry, Congenital disorders of glycosylation, Endoplasmic reticulum, Golgi apparatus, Metabolic labeling, N-Glycosylation, Cells, Cultured, Click Chemistry, Congenital Disorders of Glycosylation, Endoplasmic Reticulum, Fibroblasts, Fluorescent Dyes, Glycoproteins, Glycosylation, Golgi Apparatus, Humans, Mannose, Microscopy, Fluorescence, Protein Processing, Post-Translational, Sialic Acids, Staining and Labeling, 0601 Biochemistry and Cell Biology, Developmental Biology, 3101 Biochemistry and cell biology

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.