Glycoproteomic analysis of the changes in protein N-glycosylation during neuronal differentiation in human-induced pluripotent stem cells and derived neuronal cells

Abstract

N-glycosylation of glycoproteins, a major post-translational modification, plays a crucial role in various biological phenomena. In central nervous systems, N-glycosylation is thought to be associated with differentiation and regeneration; however, the state and role of N-glycosylation in neuronal differentiation remain unclear. Here, we conducted sequential LC/MS/MS analyses of tryptic digest, enriched glycopeptides, and deglycosylated peptides of proteins derived from human-induced pluripotent stem cells (iPSCs) and iPSC-derived neuronal cells, which were used as a model of neuronal differentiation. We demonstrate that the production profiles of many glycoproteins and their glycoforms were altered during neuronal differentiation. Particularly, the levels of glycoproteins modified with an N-glycan, consisting of five N-acetylhexosamines, three hexoses, and a fucose (HN5H3F), increased in dopaminergic neuron-rich cells (DAs). The N-glycan was deduced to be a fucosylated and bisected biantennary glycan based on product ion spectra. Interestingly, the HN5H3F-modified proteins were predicted to be functionally involved in neural cell adhesion, axon guidance, and the semaphorin-plexin signaling pathway, and protein modifications were site-selective and DA-selective regardless of protein production levels. Our integrated method for glycoproteome analysis and resultant profiles of glycoproteins and their glycoforms provide valuable information for further understanding the role of N-glycosylation in neuronal differentiation and neural regeneration.

Glycan Modifications as Regulators of Stem Cell Fate

“…The HN5H3F-modified proteins were considered to be involved in neural cell adhesion, axon guidance, and the semaphorinplexin signaling pathway, and the observed protein modifications were protein, site, and differentiation selective regardless of protein production levels. Collectively, this data underscores the pivotal involvement of N-glycosylated proteins in the complex process of neuronal differentiation of iPSC [78]. Furthermore, an elevated abundance of sialic acid is evident in neuronal cell membranes when compared to other tissues [79].…”

Glycoproteome remodelling and granule-specificN-glycosylation accompany neutrophil granulopoiesis

“…In fact, maturation stage-specific glycosylation appears scarcely investigated with recently available and highly informative glycoproteomics approaches. Examples from the glycobiological literature include our own report on dynamic glycosylation underpinning myogenesis and muscle development 56 , and N -glycoproteome changes during neuronal 57 , and cardiomyocyte 58 differentiation reported by other groups.…”

Glycoproteome remodeling and organelle-specific N -glycosylation accompany neutrophil granulopoiesis

“…In fact, maturation stage–specific glycosylation appears scarcely investigated with recently available and highly informative glycoproteomics approaches as used herein. Examples from the glycobiological literature include our own report on dynamic glycosylation underpinning myogenesis and muscle development ( 51 ), and N -glycoproteome changes during neuronal ( 52 ), and cardiomyocyte ( 53 ) differentiation reported by other groups.…”