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.

LEC10 Chinese hamster ovary (CHO) cells are gain-of-function mutants that express N-acetylglucosaminyltransferase III (GlcNAc-TIII), the glycosyltransferase that adds the bisecting GlcNAc to complex N-glycans. LEC10 cells are useful for glycosylation engineering of recombinant glycoproteins, including antibody therapeutics, for defining lectin recognition specificities and for determining biological functions of the bisecting GlcNAc. We show that three CHO mutants, LEC10, LEC10A, and LEC10B, arose due to transcriptional activation of the quiescent CHO Mgat3 gene. They each express Mgat3 gene transcripts of approximately 4.7 kb at different levels (LEC10B > LEC10 > LEC10A). Southern analyses gave a single band in LEC10, LEC10A, and parent CHO DNA with four restriction enzymes but an additional band with three of them in LEC10B genomic DNA, indicative of a duplication event in LEC10B. The deduced amino acid sequence of the Mgat3 gene expressed in each CHO mutant and in parent CHO genomic DNA is identical. However, 5' UTR sequences differ with LEC10 and LEC10B containing a 5' UTR segment of the Atf4 gene downstream of the Mgat3 gene in human and mouse. Somatic cell hybrid analysis indicated that the LEC10B Mgat3 gene was induced by a cis mechanism. LEC10B glycoproteins bound more erythroagglutinin lectin (E-PHA) than LEC10 glycoproteins and MALDI-TOF MS revealed a broad spectrum of complex, bisected N-glycans expressed by the LEC10B mutant. LEC10B is therefore the cell line of choice for producing recombinant glycoproteins carrying bisected N-glycans and for investigating biological functions of the bisecting GlcNAc.

The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt−/− retina, and Notch target gene expression was decreased in Eogt−/−endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development.DOI: http://dx.doi.org/10.7554/eLife.24419.001

In living color: Many mammalian glycans associated with signaling receptors contain terminal or penultimate N‐acetyllactosamine. A highly specific method for labeling this disaccharide on cell‐surface glycoproteins of live cultured cells and zebrafish embryos is reported. The two‐step chemoenzymatic approach involves in situ fucosylation followed by a bioorthogonal click reaction (see scheme; α(1,3)FucT=α(1,3)‐fucosyltransferase).

Abstract. Two CHO glycosylation mutants that were previously shown to lack N-linked carbohydrates with GleNAe/31,6Manotl,6 branches, and to belong to the same genetic eomplementation group, are shown here to differ in the activity of N-

Inactivating mutations of Large reduce the functional glycosylation of alpha-dystroglycan (alpha-DG) and lead to muscular dystrophy in mouse and humans. The N-terminal domain of Large is most similar to UDP-glucose glucosyltransferases (UGGT), and the C-terminal domain is related to the human i blood group transferase beta1,3GlcNAcT-1. The amino acids at conserved motifs DQD+1 and DQD+3 in the UGGT domain are necessary for mammalian UGGT activity. When the corresponding residues were mutated to Ala in mouse Large, alpha-DG was not functionally glycosylated. A similar result was obtained when a DXD motif in the beta1,3GlcNAcT-1 domain was mutated to AIA. Therefore, the first putative glycosyltransferase domain of Large has properties of a UGGT and the second of a typical glycosyltransferase. Co-transfection of Large mutants affected in the different glycosyltransferase domains did not lead to complementation. While Large mutants were more localized to the endoplasmic reticulum than wild-type Large or revertants, all mutants were in the Golgi, and only very low levels of Golgi-localized Large were necessary to generate functional alpha-DG. When Large was overexpressed in ldlD.Lec1 mutant Chinese hamster ovary (CHO) cells which synthesize few, if any, mucin O-GalNAc glycans and no complex N-glycans, functional alpha-DG was produced, presumably by modifying O-mannose glycans. To investigate mucin O-GalNAc glycans as substrates of Large, a new CHO mutant Lec15.Lec1 that lacked O-mannose and complex N-glycans was isolated and characterized. Following transfection with Large, Lec15.Lec1 cells also generated functionally glycosylated alpha-DG. Thus, Large may act on the O-mannose, complex N-glycans and mucin O-GalNAc glycans of alpha-DG.

In living color: Many mammalian glycans associated with signaling receptors contain terminal or penultimate N‐acetyllactosamine. A highly specific method for labeling this disaccharide on cell‐surface glycoproteins of live cultured cells and zebrafish embryos is reported. The two‐step chemoenzymatic approach involves in situ fucosylation followed by a bioorthogonal click reaction (see scheme; α(1,3)FucT=α(1,3)‐fucosyltransferase).

Six ␤4-galactosyltransferase (␤4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat ؊ 2 line of Chinese hamster ovary cells (Gat ؊ 2 CHO). Two independent mutants termed Pro ؊ 5Lec20 and Gat ؊ 2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary Nglycans synthesized by Pro ؊ 5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in ␤4GalT-1 transcripts in Pro ؊ 5Lec20 cells. The Gat ؊ 2Lec20 mutant expressed ␤4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the ␤4GalT-1 gene coding region. Northern analysis with probes from the remaining five ␤4GalT genes revealed that Gat ؊ 2 CHO and Gat ؊ 2Lec20 cells express all six ␤4GalT genes. Unexpectedly, the ␤4GalT-6 gene is not expressed in either Pro ؊ 5 or Pro ؊ 5Lec20 cells. Thus, in addition to a deficiency in ␤4GalT-1, Pro ؊ 5Lec20 cells lack ␤4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the ␤4GalT-1 ؊ (Gat ؊ 2Lec20) and ␤4GalT-1 ؊ /␤4GalT-6 ؊ (Pro ؊ 5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, ␤4GalT-1 is a key enzyme in the galactosylation of Nglycans, but is not involved in glycolipid synthesis in CHO cells. ␤4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different ␤4GalTs. They may be used in glycosylation engineering and in investigating roles for ␤4GalT-1 and ␤4GalT-6 in generating specific glycan ligands.