Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity.
Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z approximately 13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Gal beta1-4GlcNAc)(n) units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis(x) and sialyl-Lewis(x) determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.
North
Simon J
SJ
Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, United Kingdom.
Huang
Hung-Hsiang
HH
Sundaram
Subha
S
Jang-Lee
Jihye
J
Etienne
A Tony
AT
Trollope
Alana
A
Chalabi
Sara
S
Dell
Anne
A
Stanley
Pamela
P
Haslam
Stuart M
SM
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011134:Polysaccharides