Xiaomei Bai Brett Crawford and Jeffrey D Esko

1. Introduction

Mutant cell lines provide an excellent model for studying the structure, assembly and function of proteoglycans under the controlled conditions of tissue culture. Numerous proteoglycan-deficient strains have been isolated, mostly in Chinese hamster ovary cells, and in many cases the defects have been characterized both genetically and biochemically (see Table 1). Biochemical analysis of the mutants has confirmed that various enzyme activities detected in cell-free extracts using synthetic substrates actually play a role in proteoglycan assembly in vivo. The cell lines have allowed investigators to study how altering the composition of proteoglycans affects fundamental properties of cells, such as adhesion and signaling. Moreover, animal cell mutants provide the background for predicting the phenotype of organismal mutants defective in proteoglycan assembly.

Until recently, high-capacity selection methods for isolating glycosaminoglycan-deficient cell mutants have not been available. Instead, most mutants have been identified by indirect screening methods using a modified form of Lederberg-style replica plating, as originally devised for the isolation of microbial mutants (1). As applied to animal cells, replica plating involves the transfer of colonies growing on plastic culture dishes onto overlying disks of polyester cloth. The colonies on the replicas are then used to screen for mutants by metabolic labeling schemes or a biochemical assay. Mutants identified as lacking a particular activity are recovered from the original set of colonies present on the plate. Although the technique has moderate capacity compared to selection schemes (~105 colonies can be screened at one time), the frequency of mutations is sufficiently high after mutagenesis that a large collection of strains have been identified (see Table 1).

One limitation of replica plating is that it is not amenable to all types of cells, and mutations in some steps appear to be relatively rare. To circumvent this problem, direct selection techniques have been developed. One procedure employed repeated rounds

Table 1

Cell Mutants with Defined Defects in Glycosaminoglycan Biosynthesis

ComplementationGroup

Biochemical Defect

Phenotype pgsA (CHO) (28) pgsB (CHO) (29) pgsG (CHO) (20) pgsD (CHO) (30)

Xylosyltransferase

Galactosyltransferase I

Glucuronosyltransferase I

N-acetylglucosaminyl/ glucuronosyltransferase (EXT-1)

N-acetylglucosaminyl/ glucuronosyltransferase (EXT-1)

UDP-glucose/galactose (GlcNAc/GalNAc) 4-epimerase

Sulfate transporter

N-deacetylase/ N-sulfotransferase 1 (NDST-1)

CM-15 (COS cells) (36) N-deacetylase/N-sulfotransferase

(undefined locus)

2-O-sulfotransferase

Glycosaminoglycan-deficient Glycosaminoglycan-deficient Glycosaminoglycan-deficient Heparan sulfate-deficient

Heparan sulfate-deficient

Chondroitin sulfate-deficient when starved for GalNAc; GAG-deficient when starved for galactose

Normal glycosaminoglycans; deficient labeling with 35SO4

Undersulfated heparan sulfate

Undersulfated heparan sulfate

Deficient 2-O-sulfation of heparan sulfate of cell lysis mediated by guinea pig complement and an antibody against a carbohydrate domain of a surface proteoglycan (2). More recently, Tufaro and co-workers have exploited the dependence of Herpes simplex virus on cell surface proteoglycans to identify strains resistant to the viral cytopathic effect (3,4). One class of mutants isolated in this way lacks cell surface proteoglycans required for viral attachment and invasion. Since Herpes viruses in general depend on cell surface proteoglycans acting as co-receptors (5-9), these methods could be applicable to a variety of cell types.

Compared to replica plating, direct selection methods have high capacity since they can be applied to large populations of cells (~109). To expand this approach, it would be desirable to have a variety of agents targeted to specific glycosaminoglycan sequences that make up binding sites for ligands. In Asn-linked glycosylation, numerous mutants have been isolated by taking advantage of the specificity and cyto-toxicity of plant lectins (10). Plant lectins bind to oligosaccharides, often detecting subtle differences in anomeric linkage or in the stereochemistry of one or more sugars (11). Binding results in cell death or in the release of the cells from their substratum, depending on the nature of the lectin. Direct selections based on lectin resistance have yielded recessive, loss-of-function mutations in various glycosyltransferases (10,12), as well as dominant, gain-of-function mutants expressing novel activities (13). These cell lines have helped to unravel the branching pathway of Asn-linked glycosylation, the assembly and transport of lipid and nucleotide precursors, and the effects of altered glycosylation on glycoprotein function (10,14).

Unfortunately, plant lectins that bind to glycosaminoglycans have not yet been identified. Monoclonal antibodies could potentially fulfill this role, but few antibodies with defined sequence specificity have been described or they do not fix complement (15-17). To circumvent this problem, chimeric toxins consisting of a GAG-binding protein coupled to a cytotoxin have been made. The prototype GAG-dependent cyto-toxin consists of basic fibroblast growth factor (FGF-2) fused to a ribosome-inactivat-ing protein, saporin (SAP) (18). Application of this chimeric toxin to cultured cells results in cell death, mediated through high-affinity FGF signaling receptors and/or low-affinity heparan sulfate co-receptors (19,20). CHO cells express very few high-affinity FGF receptors, and therefore the cytotoxic activity depends critically on the expression of cell surface heparan sulfate chains. Although the use of chimeric toxins targeted to glycosaminoglycans is a relatively new concept, novel groups of mutants have already been identified (20). Thus, the technique should have broad impact since a variety of cytotoxins can be made with different specificities dependent on the GAG-binding moiety of the chimera (21).

The following technical description takes advantage of the cytotoxin FGF-Saporin (see Note 1). The latter can be produced by chemical cross-linking of saporin to FGF-2 (19,22-24) or by expression of a recombinant chimera in Escherichia coli (18,25). The generation and use of other chimeras should follow the same principles. Combining this technique with replica plating provides both the capacity and biochemical specificity needed to identify desirable mutant cell lines.

2. Materials

2.1. Selection

1. Culture medium appropriate for specific cell line under study. For CHO cells, use Ham's F12 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL of penicillin, and 100 ^g/mL of streptomycin.

2. FGF-SAP. The original preparation was obtained from Selective Genetics, Inc. (San Diego, CA), but the company no longer produces the material for commercial distribution. Recombinant material can be generated using published procedures for expression and purification of the chimera in E. coli (18,25) (see Note 2).

3. 10% trichloroacetic acid (TCA). Caution: TCA is caustic and will cause severe burns. Coomassie brilliant blue G (0.05% ) in methanol/water/acetic acid, 45/45/10 (v/v). The destaining solution consists of methanol/water/acetic acid, 45/45/10 (v/v).

2.2. Replica Plating

1. Polyester cloth disks. Polyester cloth of different pore sizes can be obtained from Tetko, Inc. (Elmsford, NY). The optimal pore size should be determined empirically, but for many cells 5- to 17-^m-pore-diameter cloth works well (PeCap 7-5, PeCap 7-17). The disks should be prepared as described (1).

2. Sterile 4-mm-diameter glass beads (Pyrex, Fisher) prepared as described (1).

3. Sterile Whatman #1 filter paper cut to fit the culture dish prepared as described (1).

3. Methods

3.1. Determine the Dose-Response Curve for the Cytotoxin

1. Plate wild-type cells in a 96-well dish at a density of ~1 x 103 cells/well in 0.2 mL of growth medium supplemented with serum and antibiotics. Add 0.05, 0.1, 0.2, 0.4, 0.6, 0.8. 1.0, 2.0, 4.0, and 8.0 ^g/mL FGF-SAP to individual wells.

2. After 4-5 d, decant the spent medium and rinse the wells with buffered saline to remove dead cells. Fix the remaining attached cells with 10% TCA for 15 min at room temperature and then rinse the plate 3 times with water. Stain the cells with Coomassie blue and remove excess dye with destaining solution.

3. Examine the staining intensity in each well and select the concentration of cytotoxin that completely inhibited cell growth.

3.2. Selection of Resistant Mutants

1. Plate cells in growth medium containing the optimal concentration of cytotoxin. A pilot experiment is necessary to determine the incidence of mutants in the population. Seed multiple 100-mm-diameter plates at 102, 103, 104, and 105 cells. If cells are plated at too high a density, the incidence of resistant strains may be too great to pick individual clones easily. Plating the cells at too low of a density wastes the cytotoxin and other materials (see Note 3).

2. With direct selection, it may not be necessary to treat cells with a mutagen to find the desired mutants, since the capacity of the technique is high (~109 cells). However, if resistant mutants are not observed, then a mutagen should be employed. The details of mutagen-treatment have been described elsewhere (1). Typically one can expect a 102- to 104-fold increase in the incidence of toxin-resistant mutants after chemical mutagenesis, but the increased likelihood of finding mutants should be weighed against the enhanced probability of inducing DNA damage and multiple mutations (see Note 4).

3. Since saporin is a ribosome-inactivating agent (RNA N-glycosidase), an immediate cessation of growth does not occur, and detachment of dead cells from the plate takes a few days. After 4 d, remove dead cells and the spent medium, and add fresh medium containing cytotoxin. After ~10 d in culture, visible colonies arise that can be picked with a glass cloning cylinder or a Pasteur pipet (1). To assure their purity and stability, these clones should be repurified by serial dilution in microtiter plates in the presence of selection medium.

4. Examine the glycosaminoglycan composition of the resistant colonies by radiolabel-ing the cells with 35SO4 or [6-3H]glucosamine following established procedures (Chapters 1,9,30).

3.3. Selection and Replica Plating

In some cases it may be desirable to couple selection with replica plating in order to subsort the mutants into different biochemical groups. The details of replica plating have been described previously (1) and are presented here in abbreviated form (Fig. 1; Subheadings 3.1. and 3.2.).

125|-bFGF

125|-bFGF

Incorporation

Fig. 1. Selection and replica plating of animal cells for glycosaminoglycan-deficient mutants.

Incorporation

Fig. 1. Selection and replica plating of animal cells for glycosaminoglycan-deficient mutants.

1. From the pilot experiments described above, choose the appropriate number of cells to seed in 100-mm-diameter tissue culture plates so that selection with the cytotoxic agent will result in ~100 resistant colonies/dish. Under these conditions only 5-10 plates are needed to screen 500-1000 resistant colonies.

2. After 4 d, remove the dead cells by replacing the growth medium. Float two layers of polyester cloth in each dish and add glass beads as a ballast to hold the disk against the bottom of the plate (1).

3. Change the medium on day 8. After 12 d, remove the media, decant the glass beads, and remove the top disk with a pair of tweezers. Gently remove the bottom disk and place it into fresh (bacterial) dish with 5 mL of medium.

4. Gently rinse the master dish with growth medium and overlay it with Whatman #1 filter paper and fresh glass beads. This step prevents satellite colonies from growing while screening of the replica disks is underway. The master dish should be placed at 33°C in a CO2 incubator.

5. Transfer the disk to growth medium containing 10 ^Ci of H235SO4 in order to radiolabel newly made proteoglycans. Precipitate radioactive proteoglycans on the disks with 10% TCA, and wash the disk three times by dipping them in 2% TCA followed by one dip in water. Stain the disk with Coomassie blue to visualize the colonies. Dry the disk and image the colonies on film or a phosphorimager. As an alternative to measuring proteoglycan biosynthesis with 35SO4, the colonies can be screened by blotting methods using a suitably labeled GAG-binding protein (e.g., 125I-FGF) (20).

6. Compare the autoradiographic image to the Coomassie blue staining, and circle the colonies that are present on the disk but lack an autoradiographic halo.

7. To recover the colony, remove the glass beads and Whatman paper from the master dish. Rinse the plate once with saline solution, orient the stained disk in the plate, and circle the desired colony on the bottom of the plate. Pick the colony with glass cloning cylinders or a Pasteur pipet (1).

4. Notes

1. Saporin is a type I ribosome-inactivating inhibitor that enzymatically depurinates riboso-mal RNA. By itself, the toxin is inactive against cells due to limited uptake, but its fusion to a ligand for a cell surface receptor greatly enhances its potency. Toxicity presumably requires uptake of the cytotoxin by endocytosis and delivery to the cytoplasm, but the mechanism underlying escape from the endocytic pathway is unknown. Because of the complex mechanism underlying its action, one might expect to derive resistant mutants with altered endocytic properties or ribosomal structure. For unknown reasons, all of the CHO cell mutants derived to date have defects in proteoglycan assembly, suggesting that these alternate modes of resistance may be relatively rare events.

2. Optimizing the concentration of the cytotoxin is essential for each cell line. FGF-SAP binds to high-affinity, tyrosine kinase receptors as well as low-affinity heparan sulfate co-receptors, and evidence suggests that the killing effect may be a combination of both receptor subtypes (19). Thus, resistance could be due to loss of either class of receptors, depending on their relative number and the concentration of ligand that is used.

3. There are two key characteristics of an ideal cell line for selection. First, the cells must give rise to colonies from single cells at high efficiency. Second, it is favorable if the cell line is hypodiploid or aneuploid, since having multiple copies of the targeted gene will drastically reduce the probability of isolating the desired mutants. In CHO cells, many loci appear to be functionally and/or physically hemizygous, thus increasing the likelihood of finding mutants. However, the selection strategy may work even in diploid lines as well since its capacity is very high (~ 109 cells).

4. The combination of replica plating with direct selection provides a powerful method for identifying mutants in proteoglycan biosynthesis. One advantage of the combined procedure is that it provides the capacity to find rare mutants and the selectivity to find strains with specific biochemical characteristics. For example, the method described here can be used with 35SO4 or other radioactive metabolic precursors as a global screen for GAG deficiency. Alternatively, a radioactive or fluorescently labeled ligand or antibody can be used to pick out defects affecting a specific binding sequence (26,27). Colonies can also be screened by direct enzymatic assay to find variants in a specific biochemical step (1). Thus, the combination of selection and screening provides the order of magnitude and specificity required for identifying even rare mutants in proteoglycan biosynthesis.

References

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