Abstract
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 enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−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. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were 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 border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.
Highlights
N-glycosylation is one of the most vital, widespread, and complex co- and post-translational protein modifications [1]
Knock out mice of Mgat1 and Mgat2 resulted in death around embryonic day 9 [3], and during early post-natal development [4], respectively. These studies revealed the importance of hybrid and more importantly complex type N-glycans for development of mammals, and that oligomannose type N-glycans are inadequate for embryonic development
Current knowledge on the cellular roles of hybrid type N-glycans is quite limiting since glycosylation mutant cell lines for the Mgat2 gene have not been established until now
Summary
N-glycosylation is one of the most vital, widespread, and complex co- and post-translational protein modifications [1]. Changes in N-glycan structures at the cell surface alter the spatial arrangement of a major cell–cell adhesion molecule (E-cadherin) [7,8], and voltage gated potassium channels (Kv3.1b and a) [7,9,10] in the plasma membrane. We employed the clustered regularly interspaced short palindromic repeats/caspase (CRISPR/Cas9) technique, a recently established technology [17,18], to knock out Mgat in Pro cells This glycosylation mutant CHO cell line is referred to as the K16 cell line and it is the first established glycosylation mutant cell line that predominantly expresses hybrid type N-glycans. Results of this study demonstrate the importance of oligomannose, hybrid, and complex types of N-glycans at the cell surface and their effects on cellular architecture and properties, and health and disease of mammals
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