Mapping the Expressed Glycome and Glycosyltransferases of Zebrafish Liver Cells as a Relevant Model System for Glycosylation Studies

Abstract

The emergence of zebrafish as a model organism for human diseases was accompanied by the development of cellular model systems that extended the possibilities for in vitro manipulation and in vivo studies after cell implantation. The exploitation of zebrafish cell systems is, however, still hampered by the lack of genomic and biochemical data. Here, we lay a path toward the efficient use of ZFL, a zebrafish liver-derived cell system, as a platform for studying glycosylation. To achieve this, we established the glycomic profile of ZFL by a combination of mass spectrometry and NMR. We demonstrated that glycoproteins were substituted by highly sialylated multiantennary N-glycans, some of them comprising the unusual zebrafish epitope Galβ1-4[Neu5Ac(α2,3)]Galβ1-4[Fuc(α1,3)]GlcNAc, and core 1 multisialylated O-glycans. Similarly, these analyses established that glycolipids were dominated by sialylated gangliosides. In parallel, analyzing the expression patterns of all putative sialyl- and fucosyltransferases, we directly correlated the identified structures to the set of enzymes involved in ZFL glycome. Finally, we demonstrated that this cell system was amenable to metabolic labeling using functionalized monosaccharides that permit in vivo imaging of glycosylation processes. Altogether, glycomics, genomics, and functional studies established ZFL as a relevant cellular model for the study of glycosylation.

Integrative View of α2,3-Sialyltransferases (ST3Gal) Molecular and Functional Evolution in Deuterostomes: Significance of Lineage-Specific Losses

“…In the Actinopterygii branch, the spotted gar Lepisosteus oculatus shows eight α2,3-sialyltransferase-related sequences, whereas teleost genomes ( D . rerio and Gasterosteus aculeatus ) show up to 12 β-galactoside α2,3-sialyltransferase-related sequences expressed in the zebrafish liver cell line model ZFL ( Vanbeselaere et al 2012 ). Finally, six α2,3-sialyltransferase-related sequences were identified in the lamprey Petromyzon marinus .…”

Mass spectrometry for protein sialoglycosylation

“…Permethylation is currently the most popular derivatization method for sialylated O ‐glycans, which has been used for the O ‐glycosylation profiling of recombinant glycoproteins, mucins, and proteins from serum or cells . Excluding permethylation, the derivatization methods commonly used in sialylated N ‐glycan analysis have not been widely employed for sialylated O ‐glycans.…”

“…Permethylation is currently the most popular derivatization method for sialylated O-glycans, which has been used for the Oglycosylation profiling of recombinant glycoproteins, mucins, and proteins from serum or cells. [262][263][264][265][266] Excluding permethylation, the mode. 270 The analyte-ILM mixture shows homogeneity well on the target plate, which may be useful for automated and quantitative analysis using MALDI-MS.…”

Advancement of Sialyltransferase Inhibitors: Therapeutic Challenges and Opportunities

“…Zebrafish are an emerging live whole animal model for early pharmacological assessment of safety and toxicity in drug development, and have been used for examining the expression and evolution of ST8Sia III and its link to neuronal migration . Mapping of the glycome and GTs of zebrafish liver cells provides a useful tool for future glycosylation studies in this model system …”