Total cell N-glycosylation is altered during neuronal differentiation of iPSC to NSC and is disturbed by trisomy 21

ABSTRACT Down syndrome (DS) is a genetic condition caused by trisomy 21 (T21) and characterized by a range of neurological symptoms including intellectual disability, early onset of neurodegeneration and dementia, some of which can be attributed to accelerated aging. N-glycosylation is a protein modification that plays a critical role in numerous biological processes and its dysregulation is associated with a wide range of diseases, in some even before the onset of symptoms. N-glycosylation of total plasma proteins, as well as specific plasma proteins, such as immunoglobulin G, has been shown to change in DS, displaying an accelerated aging phenotype consistent with the various symptoms of premature aging that occur in DS. However, little is known about how T21 affects the N-glycosylation of other cellular proteins. To better understand how T21 affects N-glycosylation during neural differentiation, we characterized and compared the total released N-glycans of induced pluripotent stem cells (iPSCs) and their neural stem cell (NSC) derivatives. We analyzed six different isogenic clones all derived from a single individual with mosaic DS and thus all sharing the same genetic background; however, three had a normal disomic karyotype (D21), while the other three had an additional copy of chromosome 21 (T21). We characterized the total cell N-glycosylation profiles using ultra high performance liquid chromatography (UHPLC) and subsequent tandem mass spectrometry analysis to determine proposed glycan structures. Our results revealed both qualitative and quantitative differences in the composition of N-glycomes between iPSCs and NSCs, with NSCs showing a higher amount of complex N-glycans and a lower amount of mannosidic N-glycans when compared to iPSCs. Moreover, we found differences in N-glycosylation patterns between D21 and T21 cells. Notably, T21 cells exhibited a significant increase in the amount of pseudohybrid N-glycans. Trisomy 21 also caused a significant decrease in the abundance of a hybrid monoantennary fucosylated glycan (H6N3F1). Our findings define the released N-glycan profile of total cells for both D21 and T21 iPSCs and NSCs and suggest that the presence of a third copy of chromosome 21 impacts N-glycosylation patterns already in the stem cell state.