Iozzo Renato V. Proteoglycan Protocols
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118 Plaas et al.
are extensively substituted with sulfate esters at C4 or C6 of the hexosamine residues, and
occasionally also on C2 of an adjacent hexuronic acid residue. All three polymer sequences
are cleaved at the
β
1,4-bonds by chondroitinase ABC (28), generating ∆-disaccharides
from the chain interior, and mono- or disaccharides from the chain terminal (see Fig. 2).
Each lyase product contains a free reducing end that can be stoichiometrically coupled to a
fluorescent tag, such as 2-aminoacridone, via reductive amination (19,26,29) (see Fig. 3).
The fluorotagged products can be resolved into discrete bands by electrophoresis on high
percentage polyacrylamide gels (26,30–33). Each product can be detected down to the
picomolar range by illuminating gels with ultraviolet light. Such images are electronically
recorded and further processed using computerized image analysis software.
2. Materials
2.1. Glycosaminoglycan Preparation from Tissues
and Proteoglycans
1. Proteinase K (fungal, > 20 U/mg, Gibco BRL (see Notes 1–3).
2. 100 mM ammonium acetate, pH 7.0, freshly prepared.
3. Absolute ethanol, precooled to –20°C.
4. Microcon centrifugal filter devices (YM-3) (Amicon).
5. Tabletop microcentrifuge.
2.2. Glycosaminoglycan Lyase Digestion
1. Chondroitinase ABC (Proteus vulgaris), (Seikagaku America), dissolved in water at
1.0 U/mL and stored in 10-µL aliquots at –80°C for up to 3 mo (see Notes 4–6).
2. 50 mM ammonium acetate, pH 7.0, freshly prepared.
3. Microcentrifuge tubes (1.5 mL capacity).
Fig. 1. Repeating disaccharide structures for hyaluronan, chondroitin sulfate, and dermatan
sulfate. The sulfation at C4 and C6 of the hexosamine and at C2 of the hexuronic acid in chon-
droitin and dermatan sulfate are indicated by R. Dermatan sulfate is characterized by a variable
proportion of
L-iduronate residues in the chain, while chondroitin sulfate contains only
D-glucuronate residues.
Analyses of Hyaluronan and Chondroitin/Dermatan Sulfate 119
Fig. 3. Schematic representation of saccharide fluorotagging by reductive amination with
2-aminoacridone (AMAC) and sodium cyanoborohydride. In this reaction a Schiff’s base is
formed between the aldol group of the reducing saccharide and the amino group of a fluorotag,
and this bond is then reduced to the stable secondary amine linkage to complete the formation
of the fluorotagged products (26,30).
Fig. 2. Schematic representation of chondroitinase ABC cleavage in the glycosaminoglycan
chain and the products generated from the interior and the nonreducing terminus.
120 Plaas et al.
2.3. Fluorescent Derivitization of Lyase Digestion Products
1. Glucose (Sigma Chemical), stock solution of 10 mmol/mL, prepared in water and stored
in 1-mL aliquots at –80°C and ∆-disaccharide standards (HPLC grade, Seikagaku
America), stock solution of 1 mg/mL dissolved in water and stored in 10-µL aliquots at
–20 °C (see Note 7).
2. Standard containing 1 nmol of glucose, and ∆-DiS (500–3000 pmol).
3. 2-aminoacridone (Molecular Probes).
4. Dimethylsulfoxide (99.7%) (Sigma-Aldrich Chemical).
5. Acetic acid (ACS grade) (Sigma-Aldrich Chemical).
6. Sodium cyanoborohydride (95 % w/v, in water) (Sigma-Aldrich Chemical) (see Note 8).
7. Glycerol (25% v/v, in water) (Sigma-Aldrich Chemical).
8. Oven or heating block at 37°C.
2.4. FACE Gel Electrophoresis of Fluorotagged
Lyase Digestion Products
1. Mono
TM
Composition gel cassettes (Glyko) stored at 4°C until use (see Notes 9 and 10).
2. Mono
TM
gel running buffer (Glyko) freshly prepared in water and cooled to 4°C.
3. Electrophoresis apparatus (Glyko).
4. High voltage power supply (Bio-Rad PowerPac 1000 or Pharmacia Biotech EPS 1000).
2.5. Gel Imaging
1. Light-impermeable tape (see Note 11).
2. UV transilluminator (300–360 nm) (see Note 12).
3. Gel documentation system (Glyko Strategene Eagle Eye, or cooled CCD camera).
2.6. Product Identification
1. Image analysis sofware (NIH Image Analysis Version 1.7; Gel-Pro Analyzer 3.0) (see
Notes 13–15).
3. Methods
3.1. Glycosaminoglycan Preparation from Tissues
and Proteoglycans
1. Typically, 1 mg of tissue or 50 µg of purified proteoglycans are suspended in 300 µl of ammo-
nium acetate buffer, pH 7.0, and digested at 60°C for 18–24 h with 50 µg of proteinase K.
2. The enzyme is inactivated by heating the samples at 100°C for 5 min, and undigested tissue
debris is removed by centrifugation in a tabletop microcentrifuge (10,000g, 10 min at
room temperature).
3. The clarified supernatants are cooled on ice, and glycosaminoglycan/peptides are precipi-
tated by addition of 1.0 mL of ice-cold ethanol (to give a final concentration of 75% v/v)
followed by storage at –20° C for 4 h (see Notes 1 and 2).
4. Precipitates are pelleted in a tabletop microcentrifuge at 10,000g for 15 min at 4°C.
5. Pellets are redissolved in water and portions are taken for estimation of total glycosami-
noglycans by hexuronic acid content (34) or dimethymethylene blue dye binding (35)
measurements (see Notes 3, 4 and 7).
3.2. Glycosaminoglycan Lyase Digestion
1. Glycosaminoglycan portions, 1–5 µg, are aliquoted into microcentrifuge tubes and dried
in a vacuum concentrator.
Analyses of Hyaluronan and Chondroitin/Dermatan Sulfate 121
2. They are resuspended in 100 µL of ammonium acetate buffer, pH 7.3, and digested at
37°C for 8–16 h with 5 mU of chondroitinase ABC (see Notes 4–6).
3. The digests are cooled on ice, and enzyme protein or undigested glycosaminoglycans are
precipitated by addition of 900 µL of ice-cold ethanol at –20°C for 2 h.
4. Precipitates are removed in a microcentrifuge (10,000g, 20 min., at 4°C) and the ∆-disac-
charides are recovered in the supernatant, which is dried by Speed Vac lyophilization.
3.3. Fluorescent Derivitization of Lyase Digestion Products
1. A 0.1 M solution of AMAC is prepared by dissolving 25 mg of the reagent in 1 mL of
dimethylsulfoxide/acetic acid (85/15, v/v).
2. Then 5 µL of the freshly prepared reagent is added to the dried lyase products and the
standards, dried in the bottom of the tube. Samples are vortex mixed carefully and spun
birefly in the tabletop centrifuge to recover the mixture into the bottom of the tube. Samples
are maintained at room temperature for 5–15 min, during which a 1 M solution of
cyanoborohydride in water is prepared and a 5-µL aliquot immediately added to the AMAC-
disaccharide mixture (see Notes 7 and 8).
3. Fluorotagging is carried out for 16 h at 37°C.
4. Samples are cooled to room temperature, and then mixed with 30 µL of 25% glycerol. These
should be analyzed immediately by gel electrophoresis, or stored stably for up to 6 months
at –80°C.
3.4. FACE Gel Electrophoresis of Fluorotagged Lyase Digestion
Products
1. The electrophoresis tank is filled with precooled running buffer and set into a large con-
tainer on ice to maintain tank and buffer temperatures at 4°C before and during the run.
2. The precast Mono composition gels are removed from the package, the loading wells
extensively washed with the Mono gel running buffer, and the gel cassette placed into the
electrophoresis tank.
3. A 4- to 6-µL portion (representing 10–15% of the total) of the standard mixture and the
fluorotagged samples is loaded per well, and electrophoresis is done at 500 V, with a
starting current of ~25 mA per gel and a final current of ~10 mA per gel (see Note 9).
4. Electrophoresis is terminated when the salt front reaches the bottom of the gel, which is
usually achieved after ~80 min (see Note 10).
3.5. Gel Imaging and Product Quantitation (see Figs. 4–6)
1. After completion of the electrophoresis, one gel cassette at a time is removed from the
tank. The outside of the glass plates as well as the loading wells are extensively washed
with distilled water to remove running buffer and excess unreacted AMAC reagent (see
Note 11).
2. The sample wells and the stacking gel are covered with light impermeable tape, and the gel
cassette is placed on the transilluminator light box (see Note 12).
3. The gels are viewed and the images captured and stored in TIFF file formats.
4. Using standard image analysis software, an average pixel density per picomole of the
∆-disaccharide standards (based on the glucose standard) is determined. From that,
∆-disaccharide contents and composition in experimental samples are calculated
(see Notes 9, 13–15).
5. A typical FACE gel of the chondroitinase ABC digestion products obtained from gly-
cosaminoglycans prepared from fetal and adult bovine cartilages and adult human cornea
122 Plaas et al.
Fig. 4. Quantitation of a fluorotagged saccharide after FACE separation using pixel density
measurements. (A) The fluorescent image and the inverted image used for determination of pixel
density values of various concentrations of fluorotagged galactose. (B) Double log plot of
pixel density per band vs. picomoles of saccharide per band, with near linearity between
~20–200 pmol of saccharide.
Fig. 5. FACE analyses of ∆-disaccharide standards. Two separate preparations of 1 nmol each of
∆-di-HA, ∆-di-0S, ∆-di-6S, ∆-di-4S, and ∆-di-E (based on the quantity of disaccharide obtained
from the supplier) and 1 nmol of glucose were fluorotagged and 10% portions of each reaction
mixture (panel A, L1 and L2) were analyzed by FACE (see Subheading 3.5. in text for details).
Analyses of Hyaluronan and Chondroitin/Dermatan Sulfate 123
is shown in Fig. 6. Two images representing different exposure times were used to visu-
alize and quantitate the abundant ∆-disaccharides and minor products, respectively. The
results are summarized in Table 1.
4. Notes
1. The separation of glycosaminoglycans from other protease products, such as collagen
peptides, is usually required, since these can interfere with the lyase digestion and the
recovery of products. Unsulfated chondroitin or short chains of hyaluronan and chon-
droitin/dermatan sulfate, however, are poorly recovered after ethanol precipitation. Alter-
natively, buffer salts and protein products can be removed by centrifugation (15 min at
9,500g at room temperature) of digests in MicroCon 3 devices. The glycosaminoglycan
Fig. 6. FACE analyses of chondroitinase ABC-digested tissue glycosaminoglycans. 10 µg
of glycosaminoglycans prepared from fetal bovine (FB) or adult bovine (AB) cartilages and
from human cornea (HC) were digested with chondroitinase ABC, fluorotagged, and analyzed
by FACE as outlined in the experimental procedure. The left-hand panel shows a short-exposure
image that was used for quantitation of the major disaccharide products (∆-di-0S, ∆-di-6S,
and ∆-Di-4S). The right-hand panel shows a longer exposure that was used to visualize and
quantitate minor products, such as ∆-di-HA, and the nonreducing terminal monosaccharide
galNAc4S (indicated by white arrows). Nonspecific fluorescent bands from the reagents them-
selves are indicated by (*). A standard curve for product quantitation by pixel densities (B)
was generated from the standard ∆-disaccharides and glucose shown in lane S (ranging from
25–270 pmol) and at a 1:4 dilution in lane S'.
124 Plaas et al.
peptides are retained on the filters, quantitatively recovered in water, and dried for further
processing.
2. It should be noted that glucose is present in all tissues and biological fluids at ~0.8 mg/mL
or less, which is solubilized with the protease digestion, but can be effectively removed
from the glycosaminoglycans using the ethanol precipitation or MicroCon filtration proce-
dure (see Subheading 3.1.). Conversely, fluorotagging and FACE analyses of a portion
of the digests prior to the glycosaminoglycan purification step will provide a simple, yet
accurate and sensitive, quantitation of the tissue or fluid glucose.
3. The recoveries of glycosaminoglycans as well as the completion of the lyase digestions can
be determined by analyzing known amounts (1–5 µg) of commercially available stan-
dards, such as chondroitin, chondroitin sulfates (from Seikagaku America or Sigma-
Aldrich Chemical) or hyaluronan (from Pharmacia or Sigma-Aldrich Chemical).
4. Chondroitinase digestions may not proceed to completion if the glycosaminoglycans are
insufficiently dissolved before digestion. This can be improved by subjecting the gly-
cosaminoglycan/water suspensions to several short cycles of agitation using a Vortex mixer
and 10–15 min incubation at 37°C before the addition of the lyase. Attention should also
be paid to maintaining the pH at 7–8 during the digestion, and it can be monitored by
inclusion of 10 µL of 0.05% phenol red (26,33) in the digestion buffer.
5. Additional chondroitin lyases are commercially available. These include chondroitinase
ACII (Arthrobacter aurescens), chondroitinase B (Flavobacterium heparinum), and
proteinase-free ABC (Proteus vulgaris). It should be noted that each enzyme has dis-
tinct substrate specificity (9,10) and cleavage mode (36–39) that will result in a mixture
of ∆-di- and oligosaccharides products from the chondroitin/dermatan sulfate chain inte-
rior. Conditions for optimal resolution of products generated by these enzymes by FACE
will need to be established.
6. Chondroitinase ABC also digests hyaluronan, by cleavage at
β
1,4 linkages between
glcNAc and glcA, and ∆-di-HA product are therefore commonly present in lyase digests
of total tissue glycosaminoglycans. Cleavage of hyaluronan, however, takes place at a
significantly lower rate than that of dermatan and chondroitin sulfates. For the most accu-
rate determination of tissue hyaluronan contents, samples should be digested sequentially
with hyaluranidase SD (Seikagaku America) and chondroitinases ABC and ACII (26).
7. For quantitative fluorotagging of larger amounts (>5 µg) of ∆-disaccharide products
increased volumes of both AMAC and cyanoborohydride solutions (up to 40 µL each)
should be used. Reaction is carried out as described under Subheading 3.3. and glycerol
added to a final concentration of 20% (v/v).
Table 1
Chonodroitin/Dermatan Sulfate Contents
and Disaccharide Compositional Analyses of Cartilage and Cornea
Glycosaminoglycan contents CS/CS disaccharide composition
(mg/mg dry wt tissue) (% of total ∆-dis)
Tissue CS/DS HA
b
∆-di-0S: ∆-di-4S: ∆-di-6S
Fetal cartilage (FB
a
) 130 1.8 19 54 27
Adult cartilage (AB
b
) 105 1.4 15 36 44
Adult cornea (HC) 5.75 0.11 63 31 6
a
See Fig. 6a.
b
For accurate hyaluronan quantitation, see Subheading 3.5., item 4.
Analyses of Hyaluronan and Chondroitin/Dermatan Sulfate 125
8. Completion of the reductive amination procedure also requires that the pH of the
fluorotagging reaction mixture is maintained between 3.5 and 7.0. As a weak buffer-
ing in this pH range is provided by acetic acid and cyanoborohydride, only freshly
prepared reagents should be used, to minimize decomposition of reducing agent or
evaporation of acetic acid prior to fluorotagging.
9. Samples can be diluted before electrophoresis using a freshly prepared solution of
dimethylsulfoxide (42%), acetic acid (7.5%), glycerol (20%) and 0.5 M cyano-
borohydride.
10. Electrophoresis times should be monitored to adjust for variable rates of sample
migration on different gel batches obtained from the supplier. For this, gels are briefly
removed from the electrophoresis tank at 20- to 30-min intervals during the electro-
phoresis, and the migration of saccharides visualized with a hand-held UV light
source.
11. Removal of excess AMAC reagent from the loading wells must be performed care-
fully, to avoid spillover of reagent onto the glass or into the gel. Its intense fluores-
cence otherwise masks the weaker fluorescent signals of individual saccharide bands.
12. If the transilluminator light source has a wavelength range of ~300 nm, the glass
plates covering the gel may absorb incoming light and possibly emitted fluorescence.
Fluorescence of lyase products can be intensified relative to the gel background by
carefully removing one glass plate and placing the exposed gel surface directly on the
transilluminator, with the remaining glass plate facing image-capture system.
13. Different image-capturing equipment and image analysis software can be used to
quantitate lyase products. It is important, however, to calibrate each system for a
near-linear range of product quantitation using pixel density measurements. A typical
example of such a calibration is given in Fig 4. Various concentrations (12.5–400
pmol) of fluoratagged galactose were electrophoresed, the bands were visualized by
illumination at 300 nm (panel A), and the image captured by a Strategene Eagle Eye
II gel documentation system. The pixel densities of the bands were determined by
NIH Image Analysis Software, version 1.57. The double-log plot of pixel density vs
saccharide concentration shows linearity between ~20–200 pmole (panel B). A simi-
lar quantitation range is obtained when images are captured with a cooled CCD cam-
era and pixel densities determined by Gel-Pro Image analyses software (26,33).
14. A sample containing a range of concentrations (20–300 pmol) of ∆-disaccharide
standards is routinely included in one lane of each FACE gel, to generate a standard
curve of pixel densities per picomole of saccharide for product identification and
quantitation in experimental samples. It is recommended, however, that the nominal
amount of the individual ∆-disaccharides as provided by the supplier, be validated first.
This can be performed by FACE analyses, as illustrated in Fig. 5. Portions of ∆-disac-
charide standards (1 nmol, based the amount [200 µg] of standard per vial from the
supplier) and glucose (1 nmol, prepared from a stock solution of 1 mg/mL) are
fluorotagged and electrophoresed (panel A). The pixel density values obtained for
each ∆-disaccharide standard deviated by ~10–30 % from values for the glucose stan-
dard. In all subsequent runs (for example, see Fig. 6), the absolute amounts of each
∆-disaccharide standard can then be adjusted relative to the glucose standard.
15. Composition or identity of separated products should be verified by treatment of lyase
products with mercuric acetate, sulfatases, and exoglycosidases (33). As these are
generally carried out before the fluorotagging, the buffer conditions, salt removal,
and product recoveries should be optimized for each treatment.
126 Plaas et al.
Acknowledgment:
We would like to thank Dr. A. Calabro for his insightful comments during the prepa-
ration of the chapter and help in assembling Figs. 1–3.
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