Sulfation of Chondroitin Sulfate in Human Articular Cartilage
THE EFFECT OF AGE, TOPOGRAPHICAL POSITION, AND ZONE OF CARTILAGE
ON TISSUE COMPOSITION*
Michael T.
Bayliss
§,
David
Osborne¶,
Sandra
Woodhouse¶, and
Catherine
Davidson
From the
Royal Veterinary College, Royal College
Street, London NW1 OTU, ¶ Lilly Research Centre Ltd., Windlesham,
Surrey GU20 6PH, and the
Wellcome/CRC Institute, University of
Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom
 |
ABSTRACT |
The chondroitin ABC lyase digestion
products of normal human femoral condyle articular cartilage and of
purified aggrecan were analyzed for their mono- and nonsulfated
disaccharide composition. Changes in the total tissue chemistry were
most pronounced during the period from birth to 20 years of age, when
the -[GlcA
,3GalNAc6]- disaccharide content increased from
approximately 50% to 85% of the total disaccharide content and there
was a concomitant decrease in the content of the 4-sulfated
disaccharide. In general, the disaccharide content of the deeper layers
of immature cartilage were richer in the 4-sulfated residue than the
upper regions of the tissue. As the tissue aged and decreased in
thickness, the disaccharide composition became more evenly 6-sulfated.
The newly synthesized chondroitin sulfate chains had a similar
composition to the endogenous chains and also underwent the same age
and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used
to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the
sulfation pattern at this position did not necessarily reflect the
internal disaccharide composition of the chains. In summary, the
sulfation pattern of chondroitin sulfate disaccharides from human
normal articular cartilage varies with the age of the specimen, the
position (topography) on the joint surface, and the zone of cartilage
analyzed. Furthermore, these changes in composition are a consequence
of both extracellular, post-translational processing of the core
protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.
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INTRODUCTION |
Proteoglycans are major components of the extracellular matrix of
articular cartilage and provide the tissue with many of its
characteristic physicochemical properties, including its ability to
generate an osmotic swelling pressure, which enables it to withstand a
wide range of compressive loads (1). Proteoglycans are also known to
directly influence chondrocyte activity, either through cell-matrix
interactions or by binding specific growth factors in the extracellular
matrix, thereby modifying their temporal and spatial effects. Many of
these biological interactions are properties of the glycosaminoglycan
chains, which are covalently attached to the protein cores of
individual proteoglycan molecules. In articular cartilage, the
glycosaminoglycans are mainly chondroitin and keratan sulfate
chains, as well as a small proportion of dermatan sulfate chains, and
they exert their action by virtue of the high electronegative charge
and spatial arrangement of their constituent sulfate groups.
Aggrecan, the major type of proteoglycan found in articular cartilage,
consists of a protein core to which are attached many chondroitin
sulfate chains that are predominantly 4- or 6-sulfated (2). Aging of
human articular cartilage is accompanied by many changes in the
structure of aggrecan and the multimolecular aggregate that it forms
with hyaluronan and link protein. These molecular changes are a
consequence of biosynthetic and catabolic events regulated by many
cellular and extracellular events. The extent to which these mechanisms
are expressed within articular cartilage is, therefore, not uniform,
and factors such as species, site (which joint), zone (through the
tissue depth), and region (topographical distribution) all dictate
specific qualitative and quantitative changes in proteoglycan. However,
it is the age of the individual that appears to have the most profound
effect on the composition of cartilage (3-5). Joint diseases occur
most frequently in the older age group, and it is important that the
changes associated with joint pathology are identified and
distinguished from normal, age-related events. Although age-related
changes in the proportion of 4- and 6-sulfated disaccharides have been
described in human articular cartilage, these have been carried out on
a relatively small number of specimens from a mixture of joints and no
attempt has been made to separate changes coincident with maturation
from those associated with subsequent aging (4, 6-8). Moreover, the
topographical and zonal variations in sulfation pattern have not been
defined, and there are no data concerning the relative contribution of
the biosynthetic and degradative pathways.
The need for a better understanding of the sulfation pattern of human
aggrecan is prompted by two published observations. The first of these
concerns the generally accepted view that osteoarthritic chondrocytes
synthesize proteoglycans with an "immature" composition (9), in
keeping with the hypermetabolic activity of the tissue and the
hypertrophic response of the cells. This conclusion was based on
analysis of the cartilage remaining on resected hip joints, which had
an increased content of 4-sulfated disaccharide. However, the study
took no account of the anatomical, morphological, or age patterns
described above and made no attempt to differentiate between anabolic
and catabolic events. The concept of a chondroblastic phenotype in
osteoarthritis may be appealing in relation to tissue repair, but there
is little direct evidence to support it. Clarification of these
experimental findings is urgently required, not just to determine their
place in the pathogenesis of osteoarthritis, but also because
measurements of 4- and 6-sulfated disaccharides in joint fluids are now
being published as "markers" of proteoglycan metabolism without
reference to the normal, macro- or micro-heterogeneity of these
structures (10, 11).
The second of these observations is more recent and concerns the
identification, using monoclonal antibodies, of atypical structures in
the chondroitin sulfate chains of animal and human osteoarthritic
cartilage (12-14). The monoclonal antibodies concerned do not
recognize the regular repeating structure of chondroitin sulfate
chains, but appear to be specific for selected (as yet uncharacterized)
sequences of sulfation both within the chains and at the non-reducing
terminal site of the chains, which are found infrequently. Although
more recent studies have raised doubts about the validity of this
interpretation (15), there is no doubt that in osteoarthritis
abnormalities in the sulfation of chondroitin sulfate chains are
related to attempts at matrix remodeling and repair, and it is an
interesting possibility that these abnormal glycosaminoglycan chains
may contain special properties, such as an increased affinity for
growth factors or other cytokines that may facilitate matrix repair by
the chondrocytes.
The studies described in this report address these questions and
provide an analysis of normal human articular cartilage, which will
enable investigations of diseased tissue to be interpreted more effectively.
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EXPERIMENTAL PROCEDURES |
Intact knee joints were obtained from amputations or massive
replacements at operation for bone tumors not involving the joint space. Fresh specimens (within 1 h of operation) were dissected using sterile techniques, and full-thickness cartilage was removed from
the femoral and tibial articular surfaces as described below.
Total Tissue--
Full-thickness cartilage was removed from all
of the femoral condyle and diced into 1-2 mm2 pieces. The
tissue was thoroughly mixed to ensure that a representative sample
could be taken, and then triplicate aliquots of approximately 50 mg wet
weight were analyzed.
Topographical and Zonal Studies--
A single full-depth plug of
cartilage including the underlying subchondral bone was taken from the
sites indicated in Fig. 2 with a sterile cork borer (3 mm diameter).
After removing the bone, the cartilage plug was sectioned from the
surface on a cryostat at 50 µm, and every four sections were pooled
to give 200-µm zones of tissue.
Radiolabeling of Cartilage Explants--
The glycosaminoglycan
chains of articular cartilage explants were radiolabeled with
[35S]sulfate; the exact conditions of the culture
depended on the subsequent analysis. When the rate of
[35S]sulfate into cartilage explants was determined,
tissue was cultured in the absence of serum, in Dulbecco's modified
Eagle's medium, buffered with HEPES and containing 20 µCi/ml
[35S]sulfate for 4 h (16). Radiolabeling of
cartilage for the purpose of analyzing the newly synthesized
disaccharides of chondroitin sulfate chains or for investigating the
structure of newly synthesized aggrecan, involved culturing tissue in
Ham's F-12 medium containing 100 µCi/ml [35S]sulfate
for 8 h.
Extraction and Purification of Aggrecan--
Aggrecan (A1
fraction) was purified from 4 M guanidine HCl extracts of
cartilage of various ages (newborn, 9, 24, and 47 years) by associative
CsCl equilibrium density gradient centrifugation (17).
Agarose/Polyacrylamide Gel Electrophoresis, Autoradiography, and
Western Blotting--
A1 fractions of purified aggrecan (newborn, 9, 24, and 47 years) were dissociated in 8 M urea and
subjected to electrophoresis in large pore, agarose/polyacrylamide
gels, and the outer lanes were stained with toluidine blue to identify
the aggrecan subpopulations (18, 19). Individual subpopulations of
aggrecan were cut from the gel with a scalpel, and the slices were
extracted with 4 M guanidine HCl prior to their analysis by
capillary zone electrophoresis after chondroitin ABC lyase digestion
(see below). Large pore gels were also used to fractionate crude
extracts of different zones of a 9- and a 60-year-old specimen, which
had been cultured in the presence of [35S]sulfate. These
gels were either stained with toluidine blue to localize the endogenous
aggrecan or subjected to autoradiography to identify the newly
synthesized molecules. Autoradiography was carried out by drying the
toluidine-stained gels on Gel Bond and exposing them for various
periods of time (up to 1 week) to x-ray film (Kodak, X-Omat) at room
temperature. The same technique was also used to identify newly
synthesized aggrecan in crude cartilage extracts (2.5, 5, 9, 15, 17, 26, and 65 years) of individual 20-µm sections of radiolabeled
([35S]sulfate) full-depth pieces of tissue. In some cases
(newborn, 5-, 10-, 21-, 31-, 38-, 46-, and 65-year-old cartilage), the
gels were subjected to Western blotting (Hybond N+) and the following polyclonal antisera and monoclonal antibodies were used to localize specific structural components after digesting the blots with chondroitin ABC lyase (25 milliunits) in 50 mM sodium
acetate, pH 7.4 for 3 h at room temperature: HG1, polyclonal
antiserum recognizing the G1 domain of human aggrecan (20); MZ15,
monoclonal antibody recognizing a pentasaccharide sequence present on
keratan sulfate chains (21); 3B3, monoclonal antibody recognizing the terminal unsaturated 6-sulfated disaccharide remaining after digestion of chondroitin sulfate chains with chondroitin ABC lyase (22); 2B6,
monoclonal antibody recognizing the terminal unsaturated 4-sulfated
disaccharide remaining after digestion of chondroitin sulfate chains
with chondroitin ABC lyase (22).
Chondroitin ABC Lyase Digestion--
Chondroitin disaccharides
were separated on a 270A-HT capillary electrophoresis system (Applied
Biosystems, Warrington, Cheshire, United Kingdom (UK)) connected to a
DS4000 chromatography work station (Drew Scientific, London, UK). The
uncoated capillary was initially washed for 2 min with 100 mM NaOH and then conditioned for 5 min with 40 mM phosphate, 40 mM SDS, 10 mM
borate adjusted to pH 9.0. The disaccharides released from chondroitin
sulfate chains with chondroitin ABC lyase were introduced into the
capillary using hydrodynamic loading for 2 s (approximately 8 nl).
A voltage of 15 kV was applied for 15 min, and the disaccharides were
migrated past the detector window by the resultant electro-endo-osmotic flow. Detection was by UV absorbance at 232 nm. Peak identity was
achieved by comparison of migration time with authentic reference standards (
di-0S,1
di-6S,
di-4S) and by overspiking into chondroitin ABC lyase digests. Digest stability was determined by conducting repeat injections at hourly intervals over a 24-h period.
Capillary Electrophoresis--
The disaccharides released from
chondroitin sulfate chains with chondroitin ABC lyase were separated on
a 270A-HT capillary electrophoresis system (Applied Biosystems)
connected to a DS4000 chromatography workstation (Drew Scientific). The
analysis was conducted at pH 9.0 to monitor nonsulfated and
monosulfated disaccharides. The uncoated capillary (72 cm × 50 µm internal diameter, path length to UV window = 50 cm) was
initially washed for 2 min with 100 mM NaOH and the sample
introduced hydrodynamically for 2 s (8 µl), over which time the
capillary eluent was monitored for UV absorbance at 232 nm.
Disaccharides were eluted with 40 mM phosphate, 40 mM SDS, 10 mM borate adjusted to pH 9.0 for 5 min. The concentrations of
di-6S,
di-4S, and
di-0S were summed
and expressed as a proportion of the total disaccharide composition. Recovery of the unsaturated chondroitin disaccharides was investigated by the use of papain. A batch of samples were initially subjected to a
simple chondroitin ABC lyase digestion. After digestion, the tissue was
removed, rinsed in water, dried on paper, and then subjected to papain
digestion. Once the material had been digested, the papain was
denatured by boiling for 5 min at 95 °C. The sample was then treated
with chondroitin ABC lyase overnight. An aliquot of supernatant was
removed and analyzed by capillary electrophoresis. No peaks were
detected for
di-0S,
di-6S, or
di-4S, leading us to conclude
that complete digestion of disaccharides is achieved with chondroitin
ABC lyase treatment alone.
HPLC Separation of Radiolabeled Disaccharides--
Radiolabeled
cartilage was digested with papain in 0.1 M sodium acetate,
2.4 mM EDTA, and 10 mM cysteine, pH 5.8, at
65 °C for 24 h. Samples were then dialyzed into 0.1 M Tris acetate, pH 8.0, and concentrated in a Centricon
ultrafiltration device containing a membrane with
Mr 10,000 exclusion. Concentration was achieved
by centrifugation at 4600 × g for 40-45 min.
Following digestion of the samples with 25 milliunits of chondroitin
ABC lyase, they were mixed with four volumes of cold ethanol and stored overnight at 4 °C. After removal of a small pellet containing the
enzyme and any undigested material, the supernatants were eluted from a
Partisil 5-PAC column equilibrated in Tris borate buffer as described
previously (23). The
di-6S and
di-4S disaccharides released from
endogenous chondroitin sulfate chains were monitored by their UV
absorbance at 232 nm, and the radiolabeled ([35S]sulfate)
di-6S and
di-4S disaccharides were analyzed using an in-line
monitor (Berthold HPLC radioactivity monitor; LB 506 C-1) and their
proportions were calculated relative to each other.
Immunohistochemistry--
Full-depth blocks (1 mm × 2 mm)
pieces of articular cartilage including the subchondral bone were taken
from the femoral condyle with a scalpel and snap-frozen in
n-hexane that was prechilled in a CO2/ethanol
slurry. Cryosections (7 µm) of the cartilage, mounted on glass
microscope slides, were digested at room temperature for 30 min with 25 milliunits of chondroitin ABC lyase, and the terminal disaccharides
remaining on the chondroitin sulfate chains were localized with the
monoclonal antibodies 3B3 (6-sulfated) and 2B6 (4-sulfated) and
reaction products were detected using a gold-enhancement-of-silver kit
marketed by Amersham International Ltd. The specificity of the
immunostaining was determined by digesting the tissue sections with
chondroitin ACII lyase, which removes the final disaccharide remaining
associated with the core protein of the proteoglycan after it is
treated with chondroitin ABC lyase. This procedure completely abolished
all of the 3B3(+) and 2B6(+) immunoreactivity.
 |
RESULTS |
Effect of Age on the Sulfation of Chondroitin Sulfate--
The
unsaturated disaccharides of chondroitin sulfate released from
full-thickness slices of normal human cartilage by chondroitin ABC
lyase were mainly the monosulfated and nonsulfated disaccharides
di-6S,
di-4S, and
di-0S. Disulfated and higher sulfated
products were also identified, but they only accounted for a small
proportion of the total disaccharide pool (<1%).
The
di-6S content of cartilage increased with age up to 20 years of
age, whereas the
di-4S decreased immediately after birth, up to 20 years of age (Fig. 1). During subsequent
aging (20-85 years), there was little change in the overall 6- or
4-sulfation. In contrast, the content of
di-0S was relatively
constant throughout life (2-5%), as was the proportion of higher
sulfated products (results not shown).

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Fig. 1.
The effect of age on the
di-6S ( ) and di-4S
( ) content of full-thickness human articular cartilage.
Full-thickness articular cartilage was removed from the entire surface
of femoral condyles of different ages. A representative sample of
tissue was digested with chondroitin ABC lyase, and the disaccharides
released were quantified by capillary electrophoresis.
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Topographical and Zonal Variations in Sulfation--
The schematic
shown in Fig. 2 illustrates the different
areas of the knee joint selected for analysis. Although all areas of
cartilage expressed age-related changes, there was considerable variation in the
di-6S:
di-4S molar ratio of full-thickness
cartilage and the value obtained depended on the region of the joint
surface sampled (results not shown). These topographical differences in composition were more obvious in tissue obtained from immature joints,
but they were also evident in mature specimens. The extent to which
these variations reflected changes in the thickness of cartilage and,
therefore, zonal changes in composition, was investigated by sectioning
cartilage from the articular surface through the tissue depth and
analyzing consecutive 200-µm layers. As an example of the
site-dependent variability in composition, the results obtained for the posterior tip of the lateral femoral condyle from knee
joints of four different ages, are shown in Fig.
3. It is apparent that
di-6S and
di-4S concentrations are not evenly distributed in different zones.
In immature (9-year-old) cartilage, chondroitin sulfate chains in the
upper half of the tissue were
di-6S-rich, whereas the lower half of
the tissue contained chains that were predominantly
di-4-sulfated.
Furthermore, the concentration of
di-6S increased from the mid-zones
of the tissue toward the articular surface and the highest
concentration of this disaccharide was located in the upper quartile of
the tissue (800-1200 µm). The decreased content of 6-sulfation
observed at the surface of cartilage was a consistent finding at all
ages. By 14 years of age, the thickness of cartilage at this site had
decreased by 50% to approximately 3 mm and there was a concomitant
loss of the deeper,
di4-S-rich region observed in the younger
specimen, except for the zones directly adjacent to the subchondral
bone. The remainder of the tissue retained a
di-6S:
di-4S ratio
that was similar to the distribution measured in the upper half of the
9-year-old specimen. By 43 years of age, the cartilage at this site had
reduced in thickness even further and the
di-6S content of the
middle and deep zones of the tissue had increased by 10-30%; the
composition of the surface layers remained unchanged. These trends
continued with advancing age until, at 60 years of age, the cartilage
was only 1.25 mm thick and all zones had a
di-6S content of
approximately 85%, with the exception of the 200-µm surface layer of
the tissue.

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Fig. 2.
Schematic showing the sites sampled for the
topographical and zonal analysis of human articular
cartilage.
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Fig. 3.
The zonal distribution of
di-6S ( ), di-4S ( ),
and di-0S ( ) through the depth of articular
cartilage at the posterior tip of the lateral femoral condyle (site 5),
in specimens of different ages. A single full-depth sample of
cartilage was removed from the posterior tip of the lateral femoral
condyle of 9-year-old (a), 14-year-old (b),
43-year-old (c), and 60-year-old (d) specimens.
Each plug of cartilage was sectioned at 50 µm from the articular
surface to the subchondral bone, and every four sections were pooled.
The disaccharides of chondroitin sulfate released by digestion of the
tissues with chondroitin ABC lyase were analyzed by capillary
electrophoresis.
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When this detailed analysis of disaccharide composition was extended to
the other eleven areas of the joint, age-related variations were
observed at all of these sites (Fig. 4).
In addition, at both ages, there was a topographical variation in the
zonal distribution of chondroitin sulfation. This was more pronounced
in immature cartilage, where cartilage thickness in different areas of
the joint was also most variable, but the same phenomenon was also evident at different sites on the adult knee joint.

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Fig. 4.
The zonal distribution of
di-6S (closed symbols)
and di-4S (open
symbols) through the depth of articular cartilage at
12 sites on the knee joint, in specimens of 9 years ( , ) and 60 years ( , ) of age. Full-depth samples of articular cartilage
were taken from defined anatomical sites on the femoral condyles (as
illustrated in Fig. 2), and each plug of tissue was sectioned at 50 µm. The disaccharides of chondroitin sulfate released by digestion of
the tissues with chondroitin ABC lyase were analyzed by capillary
electrophoresis.
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The content of the 0-sulfated disaccharides generated by chondroitinase
ABC digestion of chondroitin sulfate was also measured for the
specimens described above. Although this disaccharide comprised only a
small proportion of the total disaccharide pool, it also exhibited
zonally related changes. These were mainly confined to the articular
surface where they were present at a higher concentration compared with
the remainder of the cartilage (results not shown).
Synthesis of Chondroitin Sulfate Chains--
The rates of
[35S]sulfate incorporation into full-depth cartilage
pieces (i.e. glycosaminoglycan synthesis) confirmed that the biosynthetic activity of the chondrocyte was higher during the years
just prior to and during puberty (Fig.
5). This anabolic response decreased
between 20 and 30 years of age and, although there was a transient
increase in activity, at an age usually associated with the time of
menopause in women (40-60 years), the rates of synthesis remained
fairly constant during adult life. In order to determine the extent to
which the aging and zonal changes described in the previous section
were a consequence of anabolic or catabolic events, cartilage explants
were radiolabeled with [35S]sulfate and the disaccharide
composition of the endogenous and newly synthesized chondroitin sulfate
chains were compared. The results indicated that, at all ages,
chondroitin sulfate chains from both pools have a similar
disaccharide composition. Furthermore, when the same analysis was
applied to different zones of cartilage, the
di-6S and
di-4S
content of endogenous and newly synthesized chains had a similar
composition (Fig. 6, a-c) at
all ages.

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Fig. 5.
Age-related changes in the rate of sulfate
incorporation by full-thickness samples of normal human articular
cartilage. Triplicate samples of full-depth cartilage were
obtained from "normal" femoral condyles within 1 h of
amputation of the limb. These were incubated for 4 h in the
presence of 20 µCi of [35S]sulfate and subsequently
stored at 20 °C prior to analysis. The incorporation of radiolabel
into glycosaminoglycan chains was calculated and expressed as nanomoles
of sulfate incorporated/h/g wet weight.
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Fig. 6.
The zonal distribution of
di-6S ( , ) and di-4S
( , ) through the depth of articular cartilage at the posterior
tip of the femoral condyle (site 5), in specimens aged 14 years
(a), 30 years (b), and 60 years
(c). Full-thickness plugs of fresh cartilage were
incubated for 8 h in the presence of 100 µCi of
[35S]sulfate and then sectioned at 50 µm from the
articular surface pooling every four sections to give zones equivalent
to 200 µm. The disaccharides released from the endogenous
glycosaminoglycan chains, by the action of chondroitin ABC lyase, were
determined by capillary electrophoresis using an UV monitor.
Radiolabeled disaccharides were fractionated by ion exchange
chromatography, and the fractions were analyzed by scintillation
counting, The closed symbols indicate the
distribution of disaccharides released from endogenous chondroitin
sulfate chains and open symbols those released
from newly synthesized ([35S]sulfate-labeled)
molecules.
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Analysis of Aggrecan Fractionated by Agarose/PAGE--
Purified
aggrecan preparations were fractionated by electrophoresis in
large-pore, agarose/polyacrylamide gels (Fig.
7). All of the toluidine blue-stained
bands were immunoreactive with a polyclonal antiserum raised against
the G1 domain of human aggrecan (Fig. 8).
Whereas extracts of newborn cartilage (4 h old) contained only one
major aggrecan species (band 1a), which migrated at a slower rate than
band 1b in the preparations from the 9-, 24-, and 47-year-old
specimens, with advancing age, bands 2 and 3 accounted for a higher
proportion of the total aggrecan pool. The "free G1" domain, a
normal turnover product of aggrecan that accumulates in cartilage
during aging, was also identified as a diffuse, slower migrating,
immunoreactive band in the gels. The presence of keratan sulfate in
each aggrecan species was also confirmed by immunoreactivity with the
monoclonal antibody MZ15.

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Fig. 7.
Agarose/polyacrylamide gel fractionation of
aggrecan purified from normal human articular cartilage of different
ages. Cartilage extracts were prepared by extracting the tissue
with 4 M guanidine HCl and purifying aggrecan on
equilibrium CsCl density gradients. After electrophoresis, the gels
were stained with toluidine blue. Lane 1,
newborn; lane 2, 9 years; lane
3, 24 years; lane 4, 47 years.
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Fig. 8.
Agarose/polyacrylamide gel fractionation of
cartilage extracts of different ages, Western blotted, and
immunolocalized with a range of polyclonal and monoclonal
antisera. In each case the antisera recognized was: G1 domain of
human aggrecan (lane 1), keratan sulfate (MZ15,
lane 2), or unsaturated reducing termini of
chondroitin sulfate chains remaining after chondroitin ABC lyase
digestion of the blot di-6S (3B3, lane 3) and
di-4S (2B6, lane 4).
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The individual bands 1a, 1b, 2, and 3 from each specimen shown in Fig.
7 were extracted from the gels and re-electrophoresed (results not
shown) to ensure that they were homogeneous, before releasing the
sulfated disaccharides from the constituent chondroitin sulfate chains
with chondroitinase ABC. As expected, an age-related increase in the
concentration of
di-6S and in the ratio of
di-6S:
di-4S was
measured, in keeping with the total tissue analysis, but this was
generally higher than that obtained after digesting intact tissue
samples (Table I). This finding may
indicate that the proteoglycans remaining in the tissue residue after
extraction (10-15% of the total glucuronate), presumably those that
are intimately associated with the collagenous network, are
di-4S-rich. There was very little difference in the disaccharide
composition of bands 1b, 2, and 3 at any one age. Furthermore, at each
age there was also very little difference in the composition of each
band indicating that, if they arise primarily by extracellular cleavage of the core protein, then the 6- and 4-sulfated disaccharides are very
evenly distributed on the protein core of aggrecan at all ages.
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Table I
The disaccharide composition of the toluidine blue staining bands
fractionated by agarose/polyacrylamide gel electrophoresis shown in
Fig. 7
Aggrecan populations were extracted from the gels with 4 M
guanidine HCl prior to digestion of the extracts with chondroitin ABC
lyase and fractionation of the disaccharides released by capillary
electrophoresis.
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Age-related changes in disaccharide composition were also observed when
Western blots of the large pore gels were probed with the monoclonal
antibodies 3B3 and 2B6 after chondroitinase ABC digestion of the
membranes. These antibodies recognize the terminal unsaturated 6- or
4-sulfated disaccharide that remains associated with the protein core
after digestion of aggrecan with chondroitinase ABC. Thus, there was an
increase in the intensity of
di-6S staining of all bands with
advancing age, indicating that the disaccharide in this position of the
chondroitin sulfate chain is predominantly 6-sulfated (Fig. 8).
Immunohistochemical Localization of the Reducing Terminal
Disaccharide of Chondroitin Sulfate Chains--
The monoclonal
antibodies 3B3 and 2B6 were also used to immunostain chondroitinase
ABC-digested histological sections of cartilage in order to determine
if there was any variation in the spatial distribution (pericellular
versus intercellular) of chondroitin sulfate chains
substituted with terminal unsaturated
di-6S or
di-4S
disaccharides. The findings obtained for adult cartilage (23 years)
demonstrated that the 4-sulfated disaccharide recognized by 2B6 was
only localized intercellularly in the top and bottom 20 µm of the
tissue, where the staining was very strong. The matrix in the remainder
of the cartilage did not stain, and reaction product was confined to a
pericellular location through most of the tissue depth. The 6-sulfated
disaccharide recognized by 3B3 was also unevenly distributed. It showed
both intercellular and pericellular staining at the surface and it was
also concentrated in a region in the middle zones of the cartilage.
The Composition of Newly Synthesized and Endogenous Chondroitin
Sulfate Chains Associated with Aggrecan--
The similarity in the
disaccharide composition of the endogenous and newly synthesized
chondroitin sulfate chains, suggested that the same results should be
expected for purified aggrecan. Thus, the metabolic relationship
between newly synthesized aggrecan and the pool of proteoglycan
pre-existing in the tissue, was investigated by pulse-labeling
full-thickness samples of cartilage from donors of different ages with
[35S]sulfate. The labeling period was restricted to
4 h in order to minimize turnover of aggrecan and avoid the
production of degradation products. At all ages, autoradiography of the
agarose/polyacrylamide gels that were used to fractionate aggrecan,
showed that over 90% of the radioactivity was restricted to the slower
migrating band 1 in each case (Fig. 9).
However, extracts of the mature specimens did contain some labeled
proteoglycan migrating at the position of bands 2 and 3. The latter
finding was more pronounced when extracts were analyzed from different
zones of cartilage. Whereas zones 2-8 (400 µm each) of the
9-year-old cartilage only contained one radiolabeled aggrecan band,
zones 2-7 (200 µm each) from the 60-year-old specimen contained two
bands, and in zone 8 of the 60-year-old specimen, three labeled bands
were identified (Fig. 10). In marked
contrast, three radiolabeled proteoglycan bands were observed in the
surface zone of both the 9- and the 60-year-old specimens of cartilage.
The specific banding pattern of the surface zone of the tissue was
observed for all specimens regardless of their age (results not shown).
In addition, the [35S]sulfate radiolabel in the surface
slices was more evenly distributed between the three bands, compared
with the bands from the other zones of the tissue.

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Fig. 9.
Agarose/polyacrylamide gel fractionation of
cartilage extracts of different ages. Proteoglycans were
radiolabeled in culture with [35S]sulfate before
extraction, electrophoresis, and localization of newly synthesized
molecules by autoradiography of the dried gels.
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Fig. 10.
Agarose/polyacrylamide gel fractionation of
radiolabeled ([35S]sulfate) cartilage extracts from
different zones of cartilage. Gels were either stained with
toluidine blue to identify the endogenous aggrecan pool, or gels were
subjected to autoradiography to identify the newly synthesized
molecules. a, 9 years; b, 60 years. Note that the
immature cartilage was sliced into 400-µm zones, whereas the adult
tissue was analyzed as 200-µm slices.
|
|
 |
DISCUSSION |
Proteoglycans have a major role in maintaining the
physicochemical, mechanical, and metabolic homeostasis of articular
cartilage. In this report, we have concentrated on the age-related
changes occurring in the major chondroitin sulfate containing
proteoglycan, aggrecan. The constituent chondroitin sulfate chains of
aggrecan impart the negative fixed charge density that is the basis of many of the molecule's properties (1). By inference, it is generally
accepted that normal age-related changes in the composition of
chondroitin sulfate chains (e.g. sulfation pattern) reflect important, but as yet poorly understood, changes in the properties of
the extracellular matrix. These changes are initiated by the resident
chondrocytes, presumably to enable the tissue to respond more
effectively to the altered biological environment that it encounters
with advancing age. There are some published studies of the chondroitin
sulfate-disaccharide composition of normal human articular cartilage
(4, 8, 24). However, these either investigated a very limited age
range, or they were primarily aimed at describing the changes in the
osteoarthritic femoral head cartilage and suffered from the design
flaws highlighted in the Introduction. Sophisticated methods have also
been developed to enable the chondroitin sulfate chain size and the
number of chains/protein core to be determined and major changes in
these parameters in bovine cartilage were identified (25). In the study
described here, no attempt was made to determine the size or number of
chondroitin sulfate chains at different ages, but it is clear that
changes in these parameters do occur in human articular cartilage (4).
It is therefore likely that the physicochemical properties of aggrecan
are influenced by these structural events.
We have shown that the major changes in sulfation pattern occur during
maturation of the cartilage (up to approximately 20 years of age) and
that this corresponds to the period when the chondrocyte is most
actively synthesizing glycosaminoglycan chains. However, the more
detailed analysis of regional and zonal changes emphasized how these
parameters can influence the data. It was clearly shown that
age-related changes in composition at different sites and in different
compartments of the tissue continued to take place beyond the age
indicated by the measurements of randomized, full-depth tissue samples.
The data shown in Fig. 4 indicate that the thickness of the tissue may
correlate with the mechanical load to which the different topographical
regions of the joint are subjected and in this way the composition of
the tissue may be affected. However, although there was very little
difference in tissue thickness at sites 10 and 11 for the 9- and
60-year-old specimens, there was a dramatic change in the
di-6S:
di-4S ratio at these sites. Thus, metabolic differences
cannot be attributed only to mechanical loading, and they often likely
reflect metabolic changes that are a consequence of normal aging. It is
of interest that Plaas et al. (26) also observed changes in
the sulfation of the non-reducing termini of chains, later in life than
the alterations in 4- and 6-monosulfation of the internal repeating disaccharides. Whether this biological feature of aging represents two different phases of tissue metabolism, which are influenced to
different extents by anabolic and catabolic events, remains to be established.
Another anomalous result was obtained when the biosynthesis of
chondroitin sulfate chains was studied. The similar disaccharide composition of newly synthesized and endogenous chains, analyzed in
full-depth cartilage, suggested that most age-related and zonal changes
were biosynthetic in origin, but the agarose/polyacrylamide gels (Figs.
7 and 9) indicated otherwise. The purified, newly synthesized aggrecan
was largely confined to a single molecular species, whereas the
pre-existing aggrecan was represented by up to three molecular species
with different electrophoretic mobilities. Thus, it is coincidental
that the composition of these different molecular pools were similar,
and we conclude that the most likely mechanism giving rise to the
heterogeneity observed in aggrecan molecular size is catabolic in
origin. This would be in keeping with many other studies, which have
provided structural and enzymatic evidence that is consistent with this
hypothesis. Even so, it can be confidently stated that the data are
consistent with the replacement of a considerable proportion of the
chondroitin sulfate pool during the period from before to after
puberty. This analysis also supports the hypothesis put forward by
Lohmander et al. (27), which incorporated the concept of
multiple turnover pools of proteoglycan in articular cartilage.
Furthermore, the appearance of multiple
[35S]sulfate-labeled bands in the composite gels (Figs. 9
and 10) suggests that the structural changes can also arise via
anabolic processes. It is also possible that the role of each of these
metabolic processes will depend not only on the age of the
individual, but also on the tissue compartment from which the molecule
is derived (pericellular versus intercellular) and the zone
of tissue analyzed. This hypothesis is certainly supported by the
immunohistochemical studies and the Western blots of composite gels. It
is worth noting that in both cases the distribution was very different
to that obtained by chemical analysis of the tissue or analysis of
purified aggrecan, suggesting that the terminal unsaturated
disaccharide remaining after digestion of the chondroitin sulfate
chains does not necessarily reflect the composition of the remainder of
the chain (28). These studies also support the conclusions of Plaas
et al. (15), who showed that the use of monoclonal
antibodies alone can be very misleading and that, regardless of the
important structural information they can impart, monoclonal antibody
technology should only be used as an adjunct of detailed chemical analyses.
An additional interesting finding that has emerged from the current
investigation is a zonal variation in disaccharide composition and the
fact that this is not constant with age or topographical location on
the joint surface. These analyses confirm that all three parameters can
influence the sulfotransferase(s) activity of chondrocytes. In
particular, the relatively high
di-4S content of the deep layers of
articular cartilage before puberty suggests that this composition is a
consequence of the hypertrophic properties of the chondrocyte and thus,
the mineralization of the tissue, which is still active at this age.
Analyses of the
di-6S:
di-4S ratio of articular cartilage and the
growth (epiphyseal) cartilage from an 8-year-old specimen (1.90 and 0.77, respectively) also support this hypothesis, as do the
measurements that Deutsch et al. (25) made of bovine tibial
growth plates. However, the latter investigation again
highlighted the problems that are encountered if the
metabolic heterogeneity of chondrocytes is not taken into account. Deutsch et al. (25) found that, although the
total tissue content of growth cartilage is rich in
di-4S, this
composition is confined to the resting and upper proliferative zones of
the tissue and the hypertrophic zones are in fact
di-6S-rich. It should be appreciated that these cartilages reside on opposite sides of
the secondary center of ossification and that they are likely to exist
under different mechanical and biological conditions that could
influence the metabolic state of the resident chondrocytes. There are
also changes in the chemistry of the growth plate depending on its
stage of development. It is known that closure of the growth plate is
not an even process, and the variation in
di-6S:
di-4S ratio
measured in zones of cartilage derived from different topographical locations is consistent with this explanation. Furthermore, it is
likely that there are also species and joint specificity associated with the chemistry. For example, Lemperg et al. (29) showed that articular cartilage from calves and heifers was almost exclusively chondroitin 4-sulfate and Platt et al. (30) demonstrated
that the
di-6S:
di-4S ratio of normal articular cartilage from
equine metacarpophalangeal joints decreased with advancing age, unlike that of the middle carpal joints, where there was an age-related increase in the ratio (31). Thus, our hypothesis concerning a simple
association between mineralization of cartilage and the presence of
di-4S-rich chondroitin sulfate chains has not been adequately tested
in the present study and will require further investigation.
Disaccharide measurement of tissue slices and the structural studies of
aggrecan fractionated on composite gels also indicated that the
synthesis products of chondrocytes in the surface layers of cartilage
were different. First, it has been shown that decorin is enriched in
these zones of cartilage and the chondroitin sulfate/dermatan sulfate
chains associated with this core protein are known to be
di-4S-rich
(32). Second, a recent publication from Schumacher et al.
(33) described the purification of a high molecular weight proteoglycan
from extracts of the surface layers of human articular cartilage, and
it is likely that its presence in tissue digests contributed to the
lower
di-6S:
di-4S at this site.
The main objectives of the present investigation were (i) to
characterize the age-related, topographical, and zonal changes in the
chondroitin sulfate disaccharide composition of human normal articular
cartilage and to determine the extent to which this reflects the
composition of the chains associated with aggrecan and (ii) to
ascertain the extent to which these changes were anabolic or catabolic
in origin. These objectives have been achieved, and the findings
reported here provide a useful reference for characterizing the changes
in composition and metabolism observed in osteoarthritic cartilage.
 |
ACKNOWLEDGEMENT |
We thank Dr. J. Dudhia for discussion of the data.
 |
FOOTNOTES |
*
This work was supported by the Arthritis Research Campaign,
United Kingdom.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Corresponding author: Professor M T Bayliss The Royal Veterinary
College Royal College Street London NW1 OTU Tel: 0044 171 468 5268 FAX:
0011 171 388 1027 email: mbayliss{at}rvc.ac.uk
 |
ABBREVIATIONS |
The abbreviations used are:
di-0S,
di-6S,
and
di-4S, unsaturated 0-sulfated, 6-sulfated, and 4-sulfated
disaccharides, respectively, released from chondroitin sulphate by the
action of chondroitin ABC lyase;
HPLC, high performance liquid
chromatography.
 |
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