Abstract
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 ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewisx and sialyl-Lewisx 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.
Highlights
The LEC12 N-glycan profile (Fig. 9B) demonstrates both an increase in the relative abundance of fucosylation events, and an increase in the number of fucose residues attached to a single N-glycan, with up to seven such residues observed on a single structure
Fucosylation of the polyLacNAc extensions was confirmed by GC-MS linkage analysis that demonstrated a significant increase in the levels of terminal fucose 3,4-linked GlcNAc when compared with the wild type
It is apparent that CHO cells can make very large N-glycans with up to 26 LacNAc units
Summary
GC-MS linkage analysis (see supplemental Table S3) demonstrates an increase in both terminal fucose and 3,4-linked GlcNAc when compared with the sCHO wild type data, further corroborating the FucT gain-of-function. The most abundant complex structures in the N-glycan profile of the LEC11 cells were fucosylated, mono- or di-sialylated glycans, bearing between 2 and 16 LacNAc units (NeuAc0 –2Fuc1– 4Hex5–19HexNAc4 –18).
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