Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells

Abstract

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis.

Keywords: CHO cells; antibodies; engineer glycans; glycan binding; glycosylation mutants; lectins; mammalian cells.

Figure 1. Glycans expressed by CHO Cells and altered in the Lec1 CHO mutant

The CHO cells from which the glycosylation mutants described in Table 1 were derived express the major classes of glycans depicted in the figure (North et al., 2010) using a broadly accepted symbol nomenclature (Varki et al., 2009). Minor glycans found on particular glycoproteins at very low levels such that they were not readily detected by mass spectrometry, include O-fucose, O-glucose, O-xylose and O-GlcNAc glycans at particular consensus sites on EGF repeats in extracellular domains of certain membrane proteins such as Notch receptors (Rana and Haltiwanger, 2011), and O-mannose glycans on alpha-dystroglycan and a few other proteins (Aguilan et al., 2009). The synthesis of N-glycans requires the participation of many glycosyltransferases, nucleotide sugar synthases and transporters, as well as factors that regulate the integrity of the secretory pathway. Thus, activating or inactivating mutations in a large number of genes that alter glycosylation will be reflected in altered N-glycan synthesis and structure. The lectins used in the assays described here all bind to N-glycans. Lectin-resistant Lec1 CHO mutants lack the glycosyltransferase MGAT1 and are blocked in N-glycan synthesis so that the substrate of MGAT1 replaces all complex N-glycans (dashed line). Complex N-glycans are not synthesized in Lec1 CHO cells but no other glycans are altered.

Figure 2. Lectin resistance test

Resistance to a panel of increasing concentrations of plant lectins for CHO cells (Pro^-5) versus Lec1 mutant cells derived from Pro^-5. Lec1 cells lack MGAT1 activity. When the first well of each row was confluent, all wells were scored by inverted light microscopy for their degree of confluence, and the cells were then stained using Methylene Blue. Lec1 cells are more resistant than CHO cells to L-PHA, WGA, RIC and LCA, but more sensitive than CHO cells to Con A.

Figure 3. Lectin and antibody binding test

Comparison of lectin and antibody binding to CHO cells and LEC11 CHO cells with an activated Fut6 gene that encodes α(1,3)fucosyltransferase VI (Zhang et al., 1999). The lectins are Pisum sativum (PSA) that recognizes the Fuc in the complex N-glycan core GlcNAc and Aleuria aurentia lectin (AAL) that binds to any terminal fucose residue. The antibodies are anti-SSEA-1, which binds to the glycan epitope shown, also the Lewis X (LeX) blood group, and anti-CSLEX-1, which binds to sialylated LeX (SLeX). This research was originally published in The Journal of Biological Chemistry. North, S. J., Huang, H-H., Sundaram, S., Jang-Lee, J., Etienne, A. T., Trollope, A., Al-Chalabi, S., Dell, A., Stanley P. and Haslam, S. M. (2010) Glycomics profiling of Chinese hamster ovary (CHO) cell glycosylation mutants reveals N-glycans of a novel size and complexity. J. Biol. Chem., 285, 5759–5775 © the American Society for Biochemistry and Molecular Biology. Permission from The Journal of Biological Chemistry is automatically given and referred to as they request. See Fig. 3 legend.