Iozzo Renato V. Proteoglycan Protocols

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308 Chen et al.

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Isolation of Mutants 309

309

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Selection of Glycosaminoglycan-Deficient Mutants

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 iden-

tified 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 cul-

ture 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 com-

pared to selection schemes (~10

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

310 Bai et al.

of cell lysis mediated by guinea pig complement and an antibody against a carbohy-

drate 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 (~10

). 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

Table 1

Cell Mutants with Defined Defects in Glycosaminoglycan Biosynthesis

ComplementationGroup Biochemical Defect Phenotype

pgsA (CHO) (28) Xylosyltransferase Glycosaminoglycan-deficient

pgsB (CHO) (29) Galactosyltransferase I Glycosaminoglycan-deficient

pgsG (CHO) (20) Glucuronosyltransferase I Glycosaminoglycan-deficient

pgsD (CHO) (30) N-acetylglucosaminyl/ Heparan sulfate-deficient

glucuronosyltransferase (EXT-1)

Gro2C (mouse L-cells) N-acetylglucosaminyl/ Heparan sulfate-deficient

(3,31) glucuronosyltransferase (EXT-1)

ldlD (CHO) (32,33) UDP-glucose/galactose Chondroitin sulfate-deficient

(GlcNAc/GalNAc) 4-epimerase when starved for GalNAc;

GAG-deficient when starved

for galactose

pgsC (CHO) (34) Sulfate transporter Normal glycosaminoglycans;

deficient labeling with

pgsE (CHO) (35) N-deacetylase/ Undersulfated heparan sulfate

N-sulfotransferase 1 (NDST-1)

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

(undefined locus)

pgsF (CHO) (26) 2-O-sulfotransferase Deficient 2-O-sulfation

of heparan sulfate

Isolation of Mutants 311

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 iden-

tified. 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. Combin-

ing 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 µg/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. Recom-

binant 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

312 Bai et al.

many cells 5- to 17-µm-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 × 10

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 µg/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 tempera-

ture 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 10

, 10

, and 10

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 (~10

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 10

- to

-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 ces-

sation 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 contain-

ing 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

or [6-

H]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.).

Isolation of Mutants 313

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

incubator.

5. Transfer the disk to growth medium containing 10 µCi of H

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

, the colonies can be screened by blotting methods

using a suitably labeled GAG-binding protein (e.g.,

125

I-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

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

314 Bai et al.

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 (~10

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

or other radioactive metabolic precursors as a global screen for GAG defi-

ciency. 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.

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Xylosides 317

317

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Xyloside Priming of Glycosaminoglycan Biosynthesis

and Inhibition of Proteoglycan Assembly

Timothy A. Fritz and Jeffrey D. Esko

1. Introduction

A powerful approach for studying the relationship of proteoglycan (PG) structure

to function employs inhibitors to block glycosaminoglycan (GAG) biosynthesis.

Although true enzyme-based, active site-directed inhibitors of the glycosyltransferases

and sulfotransferases have not yet been described, decoys consisting of β-D-xylose

linked to hydrophobic aglycones have been available for some time (1). As shown

over 25 years ago (2), xylosides block PG assembly by serving as alternate substrates,

thereby diverting GAG assembly from xylosylated proteoglycan core proteins onto

the exogenous xyloside primer. This method of derailing PG biosynthesis has been

used to explore PG function in cells, tissues, and animals. The priming of oligosaccha-

rides on xylosides has also been used to define the nature of mutations in cell lines

deficient in PG biosynthesis (3–5), to co-localize glycosyltransferases in Golgi

subcompartments (6–8), and as a model for glycoside primers that affect other kinds

of glycoconjugates (9–12).

Beta-D-xylosides consist of xylose in beta linkage to an aglycone (see Fig. 1). The

aglycones typically consist of a hydrophobic compound which aids in carrying the

polar sugar moiety across cell membranes into the Golgi apparatus, where GAG bio-

synthesis takes place. While all β-D-xylosides prime chondroitin/dermatan sulfate,

studies have shown that their ability to prime heparan sulfate depends on the structure

of the aglycone (13,14). Xylosides that prime heparan sulfate require an aglycone

containing fused rings that assume a more or less planar configuration. Priming of

heparan sulfate also depends on concentration, and usually requires a higher dose than

is needed for priming chondroitin sulfate. This structural specificity is thought to arise

by recognition of the aglycone by the α-GlcNAc transferase that initiates the forma-

tion of the repeating disaccharide units of heparan sulfate (15,16).