Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

Glycosylation modulates growth, maintenance, and stress signaling processes.Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore,expanding our understanding of N-glycans in altering biological processes is of utmost interest.Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9)technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line,K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabledus to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-establishedPro´5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively.Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Celldissociation and migration assays demonstrated the greatest strength of cell--cell adhesion and fastestmigratory rates for oligomannose N-glycans, and these properties decreased as oligomannose typewere converted to hybrid type, and further decreased upon conversion to complex type. Next,we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin,a cell--cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell--cell borderwhen N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans wereprocessed to hybrid and then to complex. Thus, we provide evidence that all three general types ofN-glycans impact plasma membrane architecture and cellular properti