Limited N-Glycan Processing Impacts Chaperone Expression Patterns, Cell Growth and Cell Invasiveness in Neuroblastoma

SIMPLE SUMMARY: The attachment of sugar residues to proteins is known as glycosylation. Glycosylation is a stepwise process which is significantly altered during cancer and may thus serve as both a therapeutic and/or diagnostic target. However, our understanding of how and which glycan structures contribute to aggressive cancer behaviors is lacking. In this study, we examined how specific N-glycans contribute to aggressive neuroblastoma behavior. Here, we use genetic editing to direct N-glycan expression in neuroblastoma cells, thus enabling us to assign specific N-glycan structures to cellular properties of neuroblastoma. In addition, we also examine how altering the process of N-glycosylation influences the expression of endoplasmic reticulum (ER) folding and transport proteins. It is critical to understand how N-glycosylation processing may influence ER folding and transport protein expression. This study demonstrates that alterations of N-glycosylation influence neuroblastoma progression by diminishing or enhancing cellular properties associated with cancer progression, along with influencing the expression pattern of ER folding and transport proteins. ABSTRACT: Enhanced N-glycan branching is associated with cancer, but recent investigations supported the involvement of less processed N-glycans. Herein, we investigated how changes in N-glycosylation influence cellular properties in neuroblastoma (NB) using rat N-glycan mutant cell lines, NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3), as well as the parental cell line NB_1. The two earlier mutant cells have compromised N-acetylglucosaminyltransferase-I (GnT-I) and GnT-II activities. Lectin blotting showed that NB_1(-Mgat3) cells had decreased activity of GnT-III compared to NB_1. ESI-MS profiles identified N-glycan structures in NB cells, supporting genetic edits. NB_1(-Mgat1) had the most oligomannose N-glycans and the greatest cell invasiveness, while NB_1(-Mgat2) had the fewest and least cell invasiveness. The proliferation rate of NB_1 was slightly slower than NB_1(-Mgat3), but faster than NB_1(-Mgat1) and NB_1(-Mgat2). Faster proliferation rates were due to the faster progression of those cells through the G1 phase of the cell cycle. Further higher levels of oligomannose with 6–9 Man residues indicated faster proliferating cells. Human NB cells with higher oligomannose N-glycans were more invasive and had slower proliferation rates. Both rat and human NB cells revealed modified levels of ER chaperones. Thus, our results support a role of oligomannose N-glycans in NB progression; furthermore, perturbations in the N-glycosylation pathway can impact chaperone systems.