Rapid progress in our understanding of glycosylation pathways in eukaryotes came with the application of genetic strategies to isolate mutants of mammalian cells and yeast with defects in glycan synthesis. This chapter reviews general methods used to isolate glycosylation mutants, the diversity of mutants that may be obtained, and the application of these mutants to address questions in glycobiology research. Many of the cell lines described in this chapter are available through the American Type Culture Collection. Glycosylation mutants of yeast are discussed in Chapter 21.

HISTORY

The success of bacterial genetics as an approach to defining genetic and biochemical pathways, together with the desire to understand the molecular basis of human genetic disorders and general metabolism, led to the development of somatic cell genetics in mammalian cells. The field began in the late 1950s with pioneering studies of cultured fibroblasts and the derivation of cell lines from Chinese hamster ovary tissue (CHO cells). Stable cell lines from animal tissues were obtained readily and propagated in vitro under partially defined growth conditions. Furthermore, mutants were isolated at a respectable frequency and their phenotypes remained stable over many generations. Studying various processes in cultured cell mutants circumvented the long generation times inherent in genetic studies of whole organisms and allowed systematic control of environmental factors, such as nutrients. Techniques already established for microbial organisms could now be applied to somatic cells.

Somatic cell genetic techniques were applied early on to glycobiology, yielding numerous mutants in glycoprotein biosynthesis and later in proteoglycan, glycosylphosphatidylinositol (GPI) anchor, and glycolipid biosynthesis. Similar strategies were used to obtain yeast glycosylation mutants (see Chapter 21). N-glycan synthesis in yeast and mammals is very similar in the early part of the pathway, including the formation of the mature 14-sugar dolicholglycan, transfer of the glycan to protein, and the trimming of three glucose residues and one mannose residue in the endoplasmic reticulum. The ability to isolate glycosylation mutants in culture made it possible to unravel pathways of glycan synthesis and degradation and to identify, isolate, and map structural and regulatory genes. Mutants often accumulate intermediates upstream of the block in a pathway and thereby reveal the chemical structure of substrates and the nature of reactions that constitute a metabolic pathway. Sequencing of mutant alleles reveals changes in specific amino acids that affect a glycosylating activity. In most cases, glycosylation mutations are loss-of-function mutations and they depress the activity of an enzyme in a pathway; but there are also gain-of-function mutations that activate a silent glycosylation gene, elevate the expression of an existing activity, or inactivate a negative regulatory factor (Figure 46.1). In nearly all situations, the mutations lead to the presence of altered glycans on cell-surface glycoconjugates and changes in biological responses that correlate glycan structure to function.

FIGURE 46.1

Alteration of cell-surface glycans by recessive and dominant glycosylation mutations. Symbol Key:

INDUCTION AND ISOLATION OF MUTANTS

In cell culture, mutations occur randomly at a low rate (<10⁻⁶ mutations per generation), and thus the likelihood of finding mutants is low. To increase this probability, mutations may be induced by treating cells with chemical (e.g., alkylating agents), physical (e.g., ionizing radiation), or biological (e.g., a virus) mutagens, thereby increasing the number of mutants in a population by several orders of magnitude. However, mammalian cells are diploid and cultured cells are often hyperdiploid or tetraploid. Because the ploidy state determines the number of gene copies per cell, it will affect the frequency of obtaining a mutant phenotype, particularly if the mutation is recessive, as expected for the majority of loss-of-function mutations. Surprisingly, however, the frequency of finding recessive mutants is often much higher than predicted. In CHO cells, many loci are functionally hemizygous (single copy), which means that a single hit generates a recessive mutant phenotype. The frequency of dominant mutations, which in most cases induce a gain-of-function phenotype, is usually independent of ploidy state.

Even with mutagenesis, the incidence of mutants with defects specifically in glycosylation genes is low, consistent with the observation that these genes represent only about 1% of the genome (see Chapter 7). Thus, selection or enrichment is needed in order to find rare mutants bearing a desired glycosylation phenotype (Table 46.1). Direct selection schemes based on resistance to cytotoxic plant lectins that bind to cell-surface glycans are especially useful for identifying mutants altered in N-glycans. Dozens of lectins are available, each with a different specificity for sugars in various arrangements (see Chapters 28 and 29). Mutants with a glycosylation defect become resistant to cytotoxic lectins by reducing the expression of a single sugar or a group of sugars on cell-surface glycans (Figures 46.1 and 46.2). Importantly, many mutants that are resistant to one or more lectins because of the loss of specific sugars become supersensitive to killing by a different group of lectins that recognize sugar residues exposed by the mutation. The latter group of lectins may be used to select for revertants in the original mutant population. Nontoxic lectins are also useful for selecting lectin-binding mutants. For example, lectins may interfere with cell adhesion, and mutant cells lacking a particular glycan may be selected because they continue to adhere in the presence of these lectins. Mutations that affect all stages of glycosylation reactions, including the generation and transport of nucleotide sugars, have been detected using lectins as selective agents (Table 46.2).

TABLE 46.1

Classes of glycosylation mutants obtained from different selections or screens

FIGURE 46.2

Selection of mutants with lectins or cytotoxic agents that bind to specific sugar residues. Symbol Key:

TABLE 46.2

Examples of mutants with defects in nucleotide sugar formation or transport

Virtually any agent that recognizes cell-surface glycans or a specific surface glycoprotein, GPI-anchored protein, or glycolipid can be used to isolate mutants with a glycosylation defect (Figure 46.2). Thus, glycosylation defects in GPI-anchor biosynthesis reduce expression of GPI-anchored proteins at the cell surface (see Chapter 11). Conjugation of glycan-binding proteins to a cytotoxin that has no receptor provides another agent to select mutants in systems where lectins are not available. For example, basic fibroblast growth factor (FGF-2)/saporin complexes have been used for the selection of mutants deficient in heparan sulfate (HS). Lectins, antibodies, or ligands that are fluorescently tagged also may be used to enrich for mutants that are either deficient in binding or have acquired a novel binding ability due to altered glycosylation or reduced expression of an antigen at the cell surface. Panning is a related technique that is based on lack of adhesion of cells to surfaces coated with a glycan-binding agent. For example, coating a plate with FGF-2 allows selection of mutant cells that fail to produce HS proteoglycans, because those mutants fail to adhere to a FGF-2-coated plate.

Radiation suicide is another direct selection method for obtaining glycosylation mutants. Incubation of cells with a radioactive sugar, sulfate, or other precursor of high radiospecific activity leads to labeled glycoproteins, glycolipids, or proteoglycans that, upon prolonged storage, will cause radiation damage and death to wild-type cells, whereas mutants with reduced incorporation of the label survive. Animal cells can also be replica-plated, much like microbial colonies, using porous cloth made of polyester or nylon as the replica (Figure 46.3). Colonies of cells on the disc can be used to measure incorporation of macromolecular precursors (e.g., radioactive sugars or sulfate) or to identify mutants that fail to bind to a lectin, an antibody, or a growth factor. An adaptation of this technique allows detection of mutants affecting a specific enzyme by direct assay for activity in colony lysates generated on the discs. Although this technique has great specificity, its limited capacity makes detection of rare mutants difficult, and mutagenesis prior to screening is usually a requirement.

FIGURE 46.3

Screening for mutants using animal cell replica plating. Animal cell colonies transferred to discs can be screened for defects in incorporation of radioactive precursors, binding to lectins and antibodies, or direct enzymatic assay. Mutants are depicted (more...)

Regardless of the technique used to isolate mutants, the resulting strains must be cloned and carefully characterized for stability and the molecular basis of mutation. Additional genetic analyses include somatic cell hybridization for dominance/recessive testing and assigning mutants to different genetic complementation groups. Biochemical analysis involves the characterization of glycan structures produced by mutant cells (see Chapter 47), the quantitation and analysis of intermediates, and assays for activities thought to be missing or acquired based on the properties of the mutant. Identifying the molecular basis of mutation requires isolation of a complementing cDNA that reverts the mutant phenotype and determining whether the mutation arose from defective transcription, translation, or stability of the gene product or from a missense or nonsense mutation in the coding region of the gene.

DERIVATION OF CELL LINES FROM MICE OR HUMANS WITH A GLYCOSYLATION MUTATION

Transgenic mice that overexpress a glycosylation gene or mutant mice that lack a glycosylation activity due, in most cases, to targeted gene inactivation (see Chapter 38) are a source of mutant cells that may be analyzed in culture and used for glycobiology research. Cells may be derived from mutant or transgenic embryos, grown as primary cultures, or immortalized by viral transformation. By crossing mutant mice with the Immortomouse, which carries a temperature-sensitive SV40 T antigen in every cell, immortalized mutant cell strains can be derived from essentially any cell type. For mutations that cause embryos to die during gestation, mutant embryonic stem (ES) cells can be derived from blastocysts, provided the mutation is not cell-lethal. The resulting mutant ES cell lines can be used to investigate functions for specific glycans during differentiation in embryoid cell culture or in vivo in mouse chimeras. A chimera is obtained by injecting wild-type or mutant ES cells into the inner cell mass of a mouse blastocyst. If the ES cells survive, the resulting mouse is a mosaic of cells derived from the ES cells and cells derived from the blastocyst. Mutant ES cells may not contribute equally well to all tissues. For example, ES cells lacking GlcNAc transferase I (GlcNAcT-I) are unable to make complex or hybrid N-glycans (see Chapter 8), but they differentiate normally into many cell types in cultured embryoid bodies. However, following introduction into blastocysts, mutant ES cells lacking GlcNAcT-I do not contribute to the organized layer of bronchial epithelium in chimeric embryos.

Similarly, cell lines can be obtained from other organisms, although with greater difficulty compared to mice. For example, human fibroblasts and lymphocytes are easy to obtain but difficult to convert to immortal cell lines. Studies of fibroblasts from patients with defects in glycosylation have led to the elucidation of the underlying defect (see Chapter 42). Many lines bearing defects in lysosomal degradation are available as well.

MUTANTS SELECTED FOR RESISTANCE TO PLANT LECTINS

Selection schemes based on isolating rare mutants resistant to cytotoxic plant lectins have yielded a large number of glycosylation mutants affected in diverse aspects of glycan synthesis. Table 46.2 lists examples of mutant strains altered in nucleotide sugar formation or transport into the Golgi. As might be expected, some defects are pleiotropic. For example, the UDP-Gal transporter defect in the Lec8 mutant affects transfer of galactose to O-linked and N-linked structures on glycoproteins as well as to glycosaminoglycans (GAGs) and glycolipids. The ldlD mutant is particularly interesting in this regard, because it lacks the epimerase responsible for converting UDP-Glc to UDP-Gal and UDP-GlcNAc to UDP-GalNAc (Figure 46.4). Because there are salvage pathways for importing galactose and N-acetylgalactosamine into cells (see Chapter 4), the composition of different classes of glycans can be controlled in ldlD cells by nutritional supplementation with either galactose or N-acetylgalactosamine. Alternatively, supplementing cells with increased serum glycoproteins such as fetuin will also bypass the defect because galactose and N-acetylgalactosamine are salvaged following lysosomal degradation. Most of the mutants in Table 46.2 lack a glycosylation activity or fail to make a precursor. Two CHO cell mutants, ldlB and ldlC (not shown in the table) carry mutations in the conserved oligomeric Golgi (COG) complex used for trafficking glycan biosynthetic enzymes between the endoplasmic reticulum and Golgi. SAP mutants of CHO and D33W25-1 cells have dominant mutations that activate a latent enzyme, in this case, a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc. Such gain-of-function mutants provide access to gene products that may normally be expressed only in a few, very specialized cells in the body. Therefore, dominant mutants are important in glycosylation gene discovery, in identifying mechanisms of glycosylation gene regulation, and for defining pathways of glycan biosynthesis.

FIGURE 46.4

Pleiotropic effects of mutations in UDP-Glc/UDP-GlcNAc-4-epimerase. ldlD mutant CHO cells may be rescued by salvage reactions that generate the UDP-Gal and UDP-GalNAc necessary for the synthesis of many classes of glycans. Symbol Key:

Some lectin-resistant mutants are defective in the formation of dolichol-P-oligosaccharides or in the processing reactions that remove glucose and mannose residues after transfer of the glycan chain from the dolichol intermediate to glycoproteins (see Chapter 8). The latter mutants revealed the identity and importance of α-mannosidases in the formation of N-linked glycans in cultured cells. However, when the α-mannosidase II gene was ablated in mice, no effect was seen in certain tissues because another previously unknown α-mannosidase allowed N-glycans to be synthesized. This finding emphasizes a limitation of somatic cell mutants—the investigation is restricted to the cell line in which the gene is mutated. Because many glycosyltransferases appear to be developmentally regulated in a tissue-specific manner, studying mutants of a single cell type might preclude the discovery of alternate pathways.

Other examples of defects in N-linked glycan synthesis are given in Table 46.3. Note that some mutations affect the kinetic properties of an enzyme (e.g., Lec1A) or its subcellular localization (e.g., Lec4A). Sequencing mutant alleles provides leads for further site-directed mutagenesis of the gene in order to define important functional domains of the protein required for catalysis or compartmentalization.

TABLE 46.3

Examples of mutants altered in late N-glycan synthesis

Another class of lectin-resistant mutants consists of strains with a gain-of-function dominant phenotype due to the increased expression of a glycosyltransferase that is normally silent or expressed at very low levels. The activation of these glycosyltransferase genes may reflect a mutation in a regulatory region of the gene or in a trans-acting factor. The generation of gain-of-function mutants provides the opportunity to detect and analyze the effects of novel genes, which in many cases, were not previously known to exist. For example, LEC14 and LEC18 mutations activate previously unknown transferases that add branching N-acetylglucosamine residues to the core of N-glycans (Table 46.4).

TABLE 46.4

Dominant gain-of-function mutants expressing a new activity

MUTANTS IN GPI-ANCHOR BIOSYNTHESIS

Lectins that selectively bind to GPI anchors have not been described, but there are bacterial toxins that bind these glycans and they may be used to select GPI-anchor mutants. Originally, however, many GPI mutants were isolated by strategies that took advantage of antibodies to a GPI-anchored protein (e.g., Thy-1 on T-cell lymphoma cells). Cells expressing Thy-1 on their surface were incubated with an antibody to Thy-1 and serum-containing complement components, which lysed cells expressing the Thy-1 antigen. Loss of GPI-anchor biosynthesis reduced the expression of Thy-1 on the surface and conferred resistance to the cytolytic effect. Other mutants have been obtained by sorting cells that do not bind to a fluorescent antibody. The mutants obtained to date fall into more than 20 genetic complementation groups, each having a different lesion in GPI-anchor biosynthesis (Table 46.5; see Chapter 11). These mutants reveal the complexity of GPI-anchor biosynthesis: Multiple gene products are involved in forming the N-acetylglucosamine linkage to PI, the first committed intermediate in the pathway; Dol-P-Man is utilized as the donor of mannose; at least three enzymes are involved in the attachment of ethanolamine phosphate residues; and five genes are required for the transfer of the GPI anchor to protein. The available strains demonstrate the importance of genetic approaches for identifying genes that might not be obvious from measuring biosynthetic reactions in vitro.

TABLE 46.5

Mutants defective in GPI-anchor biosynthesis

MUTANTS IN PROTEOGLYCAN ASSEMBLY

A large collection of mutants defective in glycosaminoglycan (GAG)/proteoglycan biosynthesis has been isolated (Table 46.6). Many of these mutants were obtained by replica plating methods using sulfate incorporation to monitor GAG production in colonies (see Figure 46.3). Mutants in the early steps of GAG biosynthesis (complementation groups A, B, and G) lack both chondroitin sulfate (CS) and heparan sulfate (HS) chains, and enzymatic assays showed that they lack enzymes responsible for the assembly of the core protein linkage tetrasaccharide shared by both these GAG species (see Chapter 16). Another class of mutants (group D) is defective only in HS biosynthesis. This mutation defines a bifunctional enzyme (EXT1) that catalyzes the alternating addition of N-acetylglucosamine and glucuronic acid residues to growing HS chains. Some of the mutant alleles depress both enzyme activities, whereas others only affect the glucuronic acid transfer activity. Thus, the mutants define different functional domains of the protein, which have been mapped by sequencing various mutant alleles. Mutants in the GlcNAc N-deacetylase/N-sulfotransferase (Ndst1) (another bifunctional enzyme) have only a partial deficiency in N-sulfation of HS chains. Further analysis of the mutant showed that more than one isozyme is present in CHO cells and that the defect affects only one locus. Thus, the mutants revealed early on that the assembly of HS is much more complex than had been appreciated on the basis of known structures, enzymatic reactions measured in cell extracts, or intermediates observed in pulse-labeling experiments.

TABLE 46.6

Mutants defective in proteoglycan assembly

MUTANTS DEFECTIVE IN GLYCOLIPID OR O-GLYCAN SYNTHESIS

Glycolipid and O-glycan structures are often relatively simple in cultured cells. For example, CHO cells synthesize mainly ganglioside GM3 and lactosylceramide with a small amount of glucosylceramide. O-glycans initiated with N-acetylgalactosamine contain up to only four sugars in glycoproteins from CHO cells. All of these structures are affected in the mutants described in Table 46.2 in which CMP-sialic acid, UDP-Gal, UDP-GalNAc, or GDP-fucose syntheses are reduced or altered. Similarly, a defective sialyltransferase or galactosyltransferase may cause these structures to be truncated, depending on its acceptor specificity. A mutant of B16 melanoma cells that is defective in cerebroside glucosyltransferase lacks all glycolipids because this enzyme catalyzes the first committed step in the synthesis pathway (see Chapter 10). However, cultured cell mutants defective in protein O-GalNAc transferases or protein O-fucosyltransferase have not been isolated. This is most likely due to lack of application of the selection technologies described above, in part because of the paucity of cytotoxic lectins or toxins that bind to O-glycans and glycolipids or because of redundancy of enzymes in the system (see Chapters 9 and 10). Mice lacking specific glycolipid synthetic enzymes and glycosyltransferases that transfer N-acetylgalactosamine or fucose to protein have been generated and provide a source of mutant cells that may be studied in culture. Interestingly, cells lacking the O-GlcNAc transferase that acts in the cytoplasm to transfer N-acetylglucosamine to protein have not been obtained, and mouse mutants defective in this transferase become arrested in development at the two-cell-stage embryo, demonstrating that O-GlcNAc addition is essential for cell viability.

USES OF SOMATIC CELL GLYCOSYLATION MUTANTS

Glycosylation mutants of cultured cells have been used to address many questions in glycobiology and for glycosylation engineering of recombinant glycoproteins. Because mutant selections are broad and often not intentionally biased, they generate mutants defective in both known and novel reactions. Thus, glycosylation mutants play an important role in research to define the pathways and regulation of glycosylation in mammals. In this regard, they are more useful tools than mutant mice because cells in culture are viable in the absence of glycolipids, GPI anchors, proteoglycans, O-GalNAc and O-fucose glycans, and complex or hybrid N-glycans.

Glycosylation mutants make glycans with truncated or altered structures and thus provide an opportunity to study functional roles for cell-surface glycans in the context of a living cell. Important insights have been gained into specific sugars required for viral, bacterial, or parasite adhesion and infection and for leukocyte cell adhesion and motility. In addition, functional roles for glycans in the intracellular sorting and secretion of glycoproteins, in growth factor binding and activation, and in receptor functions have been identified using glycosylation mutants. For example, a panel of CHO glycosylation mutants was used in a coculture assay to show that ligand-induced Notch signaling is reduced when GDP-fucose levels are low, but this is not affected by reductions in sialic acid or Gal. Similarly, one of the first demonstrations for coreceptor functions for HS employed mutant CHO cells defective in HS synthesis and engineered to express the FGF receptor.

Although glycosylation is in many cases dispensable for survival of isolated cells in a culture dish, it is often crucial in vivo. Gene ablation studies in mice have identified several instances in which an intact glycosylation pathway is essential for embryogenesis. Examples include mutants that lack complex and hybrid N-glycans and proteoglycan mutants defective in HS, whereas the corresponding mutants in CHO cells do not cause an obvious phenotype. Thus, one theme that emerges from the study of mutants is that glycosylation is critical in the context of a multicellular organism but dispensable in isolated cells. This conclusion has been driven home in recent years by the discovery of human genetic diseases termed congenital disorders of glycosylation, which arise from mutations in genes involved in glycosylation (see Chapter 42).

CHO cells have become the cells of choice for the biotechnology industry in the production of recombinant therapeutic glycoproteins and in glycosylation engineering (see Chapter 51). For example, CHO cells with a Lec1 mutation have been used to produce the lysosomal enzyme glucocerebrosidase for the treatment of patients with Gaucher’s disease, who lack this enzyme. Glucocerebrosidase from cells lacking GlcNAcT-I have only oligomannosyl N-glycans and are thereby efficiently targeted to the mannose receptor on reticuloendothelial cells and ultimately to lysosomes. LEC11 CHO cells have been used to generate recombinant soluble complement receptor carrying sialyl Lewis^x, which targets the molecule to damaged endothelium where it is most effective. In a third example, CHO cells with multiple mutations that simplify N- and O-glycans are being used by X-ray crystallographers to produce homogeneous preparations of membrane glycoproteins with highly truncated N- and O-glycans that do not inhibit their crystallization.

Somatic cell genetics arose from the desire to manipulate the genome of cultured cells in vitro. Today, the availability of genomic sequences from multiple organisms has shifted the emphasis in genetics toward the generation of mutant organisms using the techniques of transgenesis, homologous recombination for gene replacement, or conditional gene inactivation. However, the study of somatic cell mutants still plays an important role in glycobiology research because it provides a less-expensive and faster method for studying the effects of deleting or newly expressing particular glycosylation gene products in a cell. Gain-of-function mutants may of course be generated by transfection of cDNAs encoding glycosylation genes, and reduced expression of any gene can be achieved by the use of RNA interference (RNAi) or antisense cDNA strategies. Although extremely valuable, the latter approaches generally target only known genes, whereas cell-based genetics makes it possible to discover new genes by screening for phenotypic changes directly related to glycosylation changes. Additionally, cells and mutants with well-characterized glycosylation pathways are ideal hosts for investigating the activity encoded by a putative glycosylation gene identified in genome sequence databases. These mutant cells also provide a platform to test the severity of human mutations in a complementation test: The normal human gene rescues defective glycosylation when transfected into the mutant cell, but the same gene with pathological mutations does not. Thus, somatic cell mutants provide access to novel genes involved in glycosylation, which in turn guide strategies for sophisticated gene-manipulation experiments in animals. By combining the two approaches, the biological function of a particular glycosyltransferase, sugar residue, or lectin can be defined. Coupled with powerful new mass spectrometry techniques for determining glycan structures from small samples of tissue or cells, glycosylation mutants of cells and animals provide complementary material for structure/function analyses and identifying mechanistic bases of glycan functions in mammals.

FURTHER READING

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