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 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.
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA. hallma@ecu.edu.
M Kristen
MK
Hall
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA. weidnerd@ecu.edu.
Douglas A
DA
Weidner
Department of Biology, East Carolina University, 1000 E. 5th Street, Greenville, NC 27858, USA. zhuy@ecu.edu.
Yong
Y
Zhu
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA. dayals13@students.ecu.edu.
Sahil
S
Dayal
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA. whitmana13@students.ecu.edu.
Austin A
AA
Whitman
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Greenville, NC 27834, USA. schwalber@ecu.edu.
Ruth A
RA
Schwalbe
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D006224:Cricetinae; D003412:Cricetulus; D055786:Gene Knockout Techniques; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D008358:Mannose / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
D015820:Cadherins; D037102:Lectins; D008565:Membrane Proteins; D011134:Polysaccharides; D000217:Acyltransferases; C021802:2-acylglycerol O-acyltransferase; D008358:Mannose
N-glycan; cadherin; cell motility; cell surface glycan; cell–cell adhesion; glycobiology; lateral heterogeneity of proteins in membranes; transmembrane glycoprotein