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Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilageI. Physical properties related to age

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1239
HETEROGENEITY OF PROTEOGLYCANS EXTRACTED
BEFORE AND AFTER COLLAGENASE TREATMENT
OF HUMAN ARTICULAR CARTILAGE
I. Physical Properties Related to Age
ROB J.
VAN DE
STADT, ROEL KUIJER, G. P. JOS VAN KAMPEN, MARGRET H. M. T.
ELS VAN DE VOORDE-VISSERS, and JAN K. VAN DER KORST
Proteoglycans were isolated from young and mature human articular cartilage 4 different ways: by
direct extraction with 4M guanidine hydrochloride
(GuHC1); after digestion of the residue from this first
extraction with collagenase, by extraction with 4M
GuHCI; associatively with 0.5M GuHCl after digestion
of the cartilage with collagenase; and dissociatively with
4M GuHCl after digestion of the cartilage with collagenase. The structural properties of these proteoglycans were compared. Proteoglycan aggregates and
monomers isolated from second extractions and from
young cartilage were of larger hydrodynamic size than
proteoglycans isolated from first extractions and mature
Cartilage, respectively. The same applied to the
chondroitin sulfate chain lengths of these proteoglycans.
The proteoglycan fraction from second extractions of
Cartilage contained a larger proportion of monomers
than the fraction from first extractions. Associative
extraction of mature collagenase-digested cartilage
yielded mainly proteoglycan monomers, whereas an
appreciable amount of proteoglycan aggregate was also
liberated from young collagenase-digested cartilage.
Our results indicate that, because of their larger size,
From the Jan van Breemen Institute, Center for Rheumatology and Rehabilitation, Amsterdam, The Netherlands.
Supported by grant ZWO nr. 95-120 from the Netherlands
Organization of Pure Scientific Research and by a grant from The
Netherlands League Against Rheumatism.
Rob J. van de Stadt, PhD: Biochemist; Roe1 Kuijer, PhD:
Biochemist; G . P. Jos van Kampen, PhD: Biologist; Margret
H. M. T. de Koning: Technician; Els van de Voorde-Vissers:
Technician; Jan K. van der Korst, PhD, MD: Professor of
Rheumatology .
Address reprint requests to Rob J. van de Stadt, PhD, Jan
van Breemen Institute. Adm. Helfrichstraat 1, 1056 AB Amsterdam,
The Netherlands.
Submitted for publication July 18, 1985; accepted in revised
form May 13, 1986.
Arthritis and Rheumatism, Vol. 29, No. 10 (October 1986)
DE
KONING,
proteoglycans from second extractions of cartilage are
more entrapped in the collagen network. These large
proteoglycans can only be liberated from the matrix
after extraction of the smaller proteoglycans, followed
by digestion of the residue with collagenase. This indicates that proteoglycans overlap and entangle with the
collagen and protect it from degradation by collagenase.
The major functions of articular cartilage are to
act as a shock absorber during weight-bearing and to
act as a bearing surface during motion (1). These
properties are related to the structure of the extracellular matrix of cartilage (2). The 2 major macromolecular components of this matrix, collagen and
proteoglycans, account for some 60% and 35% of the
dry weight, respectively (3). Cartilage proteoglycans
are composed of the glycosaminoglycans, chondroitin
sulfate and keratan sulfate, which are covalently
bound to the central core protein (4).The core protein
of proteoglycan monomers may bind to hyaluronic
acid to form multimolecular aggregates. This interaction is stabilized by the binding of a link protein (5).
Because their large size and high charge density give
rise to strong water binding and electrostatic interactions, proteoglycan aggregates are mainly responsible
for the viscoelastic properties of cartilage.
Several types of proteoglycans have been detected in different cartilage tissues (6-8). Besides,
cartilage proteoglycans are highly polydispersed and
their physical properties differ when they are isolated
from different sources within the same species, or
even from different cartilage layers (9-1 1).
Approximately one-half of the proteoglycans in
adult normal cartilage can be extracted with high
molar chaotropic salt solutions that dissociate the
proteoglycan aggregates (2). The remaining proteo-
VAN DE STADT ET AL
1240
glycans are more resistant to extraction and cannot be
extracted without prior degradation of collagen
(12,13). In contrast with adult cartilage, a larger part of
the proteoglycans of young cartilage can be extracted
directly. The biochemical and physical properties of
the more readily extractable proteoglycans have been
studied extensively in many species, including humans, in relation to age and diseases such as osteoarthritis (4,1416). The proteoglycans that are more
effectively entrapped in the collagen network, and
hence are not extracted directly by high molar salt
solutions, have received less attention so far. Because
these proteoglycans are numerous, it is important to
investigate whether they are comparable with those
that can be extracted directly.
In this paper, the structure of the less readily
extractable proteoglycans is described, compared with
the structure of the more readily extracted proteoglycans, and related to age. The chemical properties of
these proteoglycans are discussed in an accompanying
paper (1 1).
MATERIALS AND METHODS
Materials. All chemicals used were of analytical
grade unless otherwise specified. Guanidine hydrochloride
(GuHCI), special Aristar grade, and CsCl were obtained
from BDH Ltd. (Poole, UK). Highly purified collagenase
from Clostridium histolyticum (326 unitslmg, lot 5275) was
purchased from Worthington Biochemicals (Freehold, NJ).
Hyaluronic acid from human umbilical cord and proteinaseK were obtained from Sigma (St. Louis, MO). Benzamidine
hydrochloride and 6-aminocaproic acid were purchased from
Aldrich Europe (Beerse, Belgium) and trypsin inhibitor, as
well as Fractogel HW-75S, were from Merck (Darmstadt,
FRG). The polyol Si-300 high performance liquid chromatography (HPLC) column was obtained from Serva
(H&idelberg, FRG).
Source of tissues. Human articular samples were
obOained at autopsy, within 12 hours of death. Most of the
selected individuals died from heart failure or accidents. The
cause of death in many of the infants was unknown at the
time of autopsy. None of the autopsy findings showed
evidence of connective tissue disease, malignancy, or joint
damage due to trauma or other joint diseases. Macroscopically, all selected cartilage samples looked normal; fibrillated
samples were discarded. The complete distal femur and
proximal tibia were dissected and, if necessary, stored at
-85°C before removal of the cartilage. Ten joint specimens
from individuals ages 3-12 months (denoted 0.5-year-old)
were pooled. Cartilage in this pool was obtained from the
epiphyses of the tissues with careful exclusion of growth
plates and ossified nuclei. Knee cartilage samples from
subjects 60 and 63 years of age were treated individually.
Cartilage was dissected with careful exclusion of adhering
tissues. After a brief washing with physiologic salt solution,
the cartilage samples were frozen in liquid nitrogen and
mechanically powdered as described previously (17). The
resulting cartilage powder was used immediately or was
stored at -85°C for future use. The extraction efficiency of
proteoglycans appears to vary with tissue geometry (10).
The results obtained in this study may not be similar to
results obtained when other methods are used for the homogenization of cartilage.
Isolation of proteoglycans. Proteoglycans were extracted 4 different ways. They were extracted with 4M
GuHCI, 0.15M potassium acetate, pH 5.8, at 4°C for 48
hours in the presence of one-tenth volume of a proteinaseinhibitor cocktail containing 0.lM EDTA, O.05M iodoacetic
acid, 1M 6-aminocaproic acid, O.05M benzamidine hydrochloride, and 50 mg/liter trypsin inhibitor, as described
elsewhere (18). The suspension was centrifuged at 56,OOOg at
4°C for 1 hour, then the supernatant was retrieved and
dialyzed against 19 volumes of O.15M potassium acetate
buffer, pH 5.8, containing one-tenth volume of the
proteinase-inhibitor cocktail without trypsin inhibitor (fraction 1). The cartilage residue was washed 4 times with
distilled water, suspended in O.05M Tris-C1, pH 7.5, 0.001M
CaCI2, and digested with 50 units of highly purified collagenase/gm of wet residue at 37°C for 30 hours. Then 4M
GuHCl and one-tenth volume of the proteinase-inhibitor
cocktail were added and proteoglycans were extracted at 4°C
for 48 hours (fraction 2).
In some experiments, prior to proteoglycan extraction, the starting cartilage powder was first digested with
highly purified collagenase as described above. Then
proteoglycans were extracted with 0.5M GuHCl in 0.15M
potassium acetate, pH 5.8, yielding fraction A (associative
extraction), or with 4M GuHCl in 0.15M potassium acetate,
pH 5.8, at 4°C for 48 hours, yielding fraction B (dissociative
extraction). Fraction B was dialyzed to associative conditions. Finally, all fractions were subjected to CsCl density
gradient centrifugation with a starting density of 1.47 gmlml
(19). The bottom one-fifth fraction (Al) of the gradient with
a density of about 1.60 was collected, dialyzed against
potassium acetate buffer and water, and lyophilized.
Isolation of 35S-proteoglycans.35S04-labeledproteoglycans were isolated from rabbit articular chondrocyte
monolayer cultures by a method described elsewhere (18).
Briefly, chondrocytes from rabbit articular cartilage were
grown in monolayer cultures in Dulbecco’s modified Eagle’s
medium containing 10% fetal calf serum (20). Starting just
before confluency (7 days), the cells were labeled with 25
pCi Na235S04/10ml medium (New England Nuclear, Boston, MA). After 24 hours, the medium was replaced by fresh
medium containing 25 pCi Na235S04.This procedure was
repeated 6 times. One milliliter of the proteinase-inhibitor
cocktail was added per 10 ml of the collected medium, and
proteoglycans were precipitated with one-fourth volume of
5% cetylpyridinium chloride for 20 minutes. The precipitate
was washed twice with 3 ml 0.1M K2S04 containing onetenth volume of the proteinase-inhibitor cocktail, and then
suspended in 3 ml 1.25M MgCI2 and incubated at 4°C
overnight (21). Proteoglycans were precipitated with 4 volumes of ethanol (-20°C) overnight and dissolved in the 4M
GuHCl extract of the cell layers, which was dialyzed to
associative conditions at 4°C overnight. CsCl was added
1241
PHYSICAL PROPERTIES OF CARTILAGE PROTEOGLYCANS
until a density of 1.47 gm/ml was achieved, and density
centrifugation, dialysis of the A1 fraction, and lyophilization
occurred as described above.
Radioactively labeled proteoglycans were used primarily as internal standards for chromatographic procedures.
Column chromatography. Proteoglycan samples
were subjected to Fractogel HW-75s chromatography to
determine the relative hydrodynamic sizes of proteoglycan
aggregates and monomers. Gel permeation chromatography
of proteoglycans using Fractogel HW-75s resulted in findings similar to those obtained with chromatography with the
classic soft gels, such as Sepharose CL-’LB, but it has some
important advantages (22). On Fractogel HW-75S, proteoglycan aggregates, as well as proteoglycan monomer
elutes, were included. The size distributions and the relative
amounts of both these molecular complexes can be studied,
and the method is about 3 times faster than the chromatographic procedures normally used.
A Fractogel HW-75s column (0.7 x 120 cm) was
packed according to instructions of the manufacturer. This
column was equilibrated and eluted with 0.5M K2S04,O.05M
Tris-S04, pH 7.0, for associative runs and with 2M GuHCI,
O.OSM Tris-S04, pH 7.0, for dissociative runs. The flow rate
was 0.2 ml/minute. Proteoglycan samples were dissolved at
3 mg/ml in 0.15M K2S04,O.OSM Tris-S04, pH 7.0 (associative runs) or in 4M GuHC1, O.OSM Tris-S04, pH 7.0 (dissociative runs). As an internal standard, small aliquots of
35S04-labeled proteoglycans were added, containing about
20,000 disintegrations per minute. Prior to chromatography
under associative conditions, the proteoglycan samples were
incubated with 1% hyaluronic acid (by dry weight of
proteoglycan) at 4°C overnight. Fractions were collected and
monitored for uronic acid (23) and radioactivity, using
Picofluor-15 and a Tricarb 300CD scintillation counter
(United Technologies, Packard Instruments Co., Downers
Grove, IL). The relative amounts of aggregated proteoglycans and nonaggregated proteoglycans were estimated by
cutting and weighing traces of the uronic acid profiles and, in
some cases, of the 35S-proteoglycan dpm profiles (24).
Determination of chondroitin sulfate chain lengths.
Proteoglycans were dissolved at 4 mglml in 0.OSM Tris-C1,
0.001M CaC12, pH 7.5, and digested with proteinase-K (1
unW2.5 mg proteoglycans) overnight at 60°C. The relative
chain lengths of chondroitin sulfate were determined using a
Polyol Si-300 HPLC column (0.7 x 30 cm) equilibrated in
and eluted with 0.5M K2S04, O.OSM Tris-S04, pH 7.0, at a
flow rate of 0.2 mYminute. Fractions were collected and
monitored for uronic acid and radioactivity. No standards
for chondroitin sulfate were available to calibrate the column. Only relative chain lengths could be determined.
RESULTS
Nonspecific enzyme activity of collagenase.
Proteoglycans isolated from articular cartilage that
was predigested with collagenase were studied. Because proteoglycans are rather sensitive to proteinase
activity, it is essential to determine if such activity is
present in the collagenase preparation used. This was
.-V
C
0
L
Y
time
(min)
Figure 1. Effect of collagenase digestion on isolated proteoglycans
(A1 fraction). A, Fractogel HW-75s chromatogram of proteoglycans
isolated from the knee cartilage of a 63-year-old subject, after
incubation with 1% hyaluronic acid (by dry weight of proteoglycans). B, Fractogel HW-75s chromatogram of the same proteoglycan fraction, after digestion with collagenase at 37°C for 40 hours,
followed by incubation with 1% hyaluronic acid.
tested by digestion of proteoglycans (A1 fraction) that
were isolated from 63-year-old knee cartilage with
collagenase, and by comparison of the Fractogel
HW-75s chromatograms of the treated and untreated
material (Figure 1). No destruction of proteoglycans
was observed, because no uronic acid-containing material appeared in or near the bed volume of the
column and the proteoglycan profile did not change.
Although the collagenase itself did not contain
detectable proteoglycanase activity, it is possible that
during collagenase digestion of the cartilage residues
obtained after a first extraction with 4M GuHC1, some
residual autolytic proteinase activity remained, which
could have become active under the conditions applied
for collagenase digestion (37°C for 30 hours). Therefore, a second test was performed by adding radioactively labeled proteoglycans to the residue retrieved
from the first extraction. This suspension was digested
with collagenase as described in Materials and Methods, after which proteoglycans were isolated and
subjected to Fractogel HW-75s chromatography under associative conditions (Figure 2).
No radioactive material was detected in or near
1242
VAN DE STADT ET AL
100
0
200
time (minl
Figure 2. Effect of collagenase digestion on 35S04-labeled
proteoglycans in the presence of the cartilage residue from the first
extraction. A, Fractogel HW-75s chromatograms of a mixture of
first extraction proteoglycans isolated from the knee cartilage of a
63-year-old subject and 35S04-labeledproteoglycans isolated from
rabbit articular chondrocyte monolayer cultures, after incubation
with 1% hyaluronic acid (by dry weight of proteoglycans). B,
Fractogel HW-75s chromatograms of a mixture of proteoglycans
obtained by adding 35S04-labeledproteoglycans to the residue of the
first extraction, after which this mixture was digested with
collagenase and the proteoglycans were isolated as described in
Maaerials and Methods. The resulting proteoglycan sample was
incubated with 1% hyaluronic acid prior to chromatography. Fractions of 0.6 ml were collected and monitored for uronic acid and
radioactivity
.
the bed volume of the column. Only a small (5-10%)
shift of radioactively labeled proteoglycans, from aggregates to monomers, was observed. Since proteoglycans were incubated with hyaluronic acid prior to
chromatography, this small shift is not likely to be
caused by degradation of hyaluronate during the
collagenase treatment and the second extraction. Apparently, a small proportion of the proteoglycans lost
thdr ability to interact with hyaluronic acid, possibly
by degradation of the hyaluronic acid binding region of
thdr core proteins by residual endogenous proteinase
activity. It was concluded that incubations of residues
of first extractions with collagenase under the isolation
conditions may affect the hyaluronic acid binding
region of the core protein of proteoglycans to a smaIl
extent, and may result in a shift of 5-10% of proteoglycan aggregates to proteoglycan monomers. This
disadvantage is considered in the results presented
henceforth.
Structural differences of proteoglycans from first
and second extractions. Proteoglycans from 0.5- and
60-year-old cartilage, isolated from second extractions
after collagenase digestion of the first cartilage residue, contained 24% and 21% more proteoglycan
monomers than proteoglycans isolated from first extractions, respectively (Table 1). The extent of this
shift was much greater than could be expected from
possible proteolytic degradation. The results implicate
the presence of more nonaggregating proteoglycans in
the proteoglycan monomer fraction from the second
extraction compared with the fraction from the first
extraction.
Proteoglycan aggregates from 0.5-year-old cartilage had a larger hydrodynamic size than proteoglycan aggregates from 60-year-old cartilage as can be
seen by the relative position of the aggregate peaks
with regard to the aggregate peak of the internal
standard (35S04-labeled proteoglycans) (Figures 3
and 4).
Chromatography of dissociated proteoglycans
from first and second extractions of 0.5- and 60-yearold cartilage in the presence of 2M GuHCl on
Fractogel HW-75s revealed that dissociated proteoglycans from second extractions and those from young
cartilage were larger in hydrodynamic size than the
ones from first extractions and mature cartilage (Figures 3 and 4). The difference in hydrodynamic size
between proteoglycans from first and second extrac-
Table 1. Extraction yields and proportions of proteoglycan (PG)
aggregates and monomers under various extraction conditions*
Cartilage specimens
0.5-y ear-old
First extraction
Second extraction
60-year-old
First extraction
Second extraction
0.5-year-old (collagenasepretreated)
0.5M GuHCl
4M GuHCl
60-year-old (collagenasepretreated)
0.5M GuHCl
4M GuHCl
% uronic
acid
extracted
% PG
%Fa
aggregates
monomers
78
12
73
49
27
51
53
26
77
56
23
42
85
63
62
37
38
18
69
13
57
87
43
44
* To estimate the proportions of proteoglycan aggregates and monomers in the A1 fractions, a tracing of the uronic acid elution profile
was cut and weighed (24). GuHCl = guanidine hydrochloride.
PHYSICAL PROPERTIES OF CARTILAGE PROTEOGLYCANS
0. E
-
1243
ioo
n
1
"t
0. E
500
2
0
m
-a
ul
c
E
a
-
'0
'0
u
0
.-vC
0.:
500
0
v)
c
3
ul
0
500
0.5
c
100
200
time
300
(min)
Figure 3. Fractogel HW-75s chromatograms of proteoglycans isolated from 0.5-year-old articular knee cartilage. 1 = fraction 1:
proteoglycans isolated from the first extract with 4M guanidine
hydrochloride (GuHCI). 2 = fraction 2: proteoglycans isolated from
the residue of the first extraction after digestion with collagenase.
The upper panels show proteoglycans eluted under associative
conditions; the lower panels show proteoglycans eluted under
dissociative conditions. Prior to chromatography under associative
conditions, proteoglycans were incubated with 1% hyaluronic acid
(by dry weight of proteoglycans). V,= total column volume. See
Materials and Methods for details.
tions was larger in mature cartilage compared with that
in young cartilage.
The proteoglycan fractions from first and from
second extractions both contain link proteins, as was
revealed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (data not shown).
100
I
0
200
time
300
Imin)
Figure 4. Fractogel HW-75s chromatograms of proteoglycans isolated from 60-year-old articular knee cartilage. 1 = fraction 1:
proteoglycans isolated from the first extract with 4M guanidine
hydrochloride (GuHC1).2 = fraction 2: proteoglycans isolated from
the residue of the first extraction after digestion with collagenase.
The upper panels show proteoglycans eluted under associative
conditions; the lower panels show proteoglycans eluted under
dissociative conditions. Prior to chromatography under associative
conditions, proteoglycans were incubated with 1% hyaluronic acid
(by dry weight of proteoglycans). V, = total column volume. See
Materials and Methods for details.
Determination of the relative chain lengths of
chondroitin sulfate of proteoglycans from first and
second extractions of 0.5- and 60-year-old cartilage
revealed that chondroitin sulfate chains of proteoglycans from second extractions and from young cartilage were larger than those of proteoglycans from
VAN DE STADT ET AL
1244
digested cartilage with 4M GuHCl yielded 85% and
69% of the tissue uronic acid, respectively. These
latter extraction yields are higher compared with the
extraction yields obtained when cartilage was incu-
M60-1
60-2
M05-1
U05-2
E
~
P
000
500
A
c
A
V
m
In
m
000 7
500
B
0
I
0
50
0
100
150
time
-
2 00
E
a
Iminl
-0
Figwe 5. Relative chain lengths of chondroitin sulfates. Isolated
proteoglycans (A1 fractions) were obtained from 60-year-old and
0.5-year-old knee cartilage by a first extraction with 4M guanidine
hydkochloride (GuHCl) (6&1 and (0.5-1). The resulting cartilage
residues were digested with collagenase and additional amounts of
proteoglycans were isolated from second extractions with 4M
GuHCl (60-2 and 0.5-2). To the isolated proteoglycan fractions a
small amount of 35S04-labeledproteoglycans isolated from rabbit
articular chondrocyte monolayer cultures was added. The samples
were digested with proteinase-K and chromatographed as described
in Materials and Methods. V, = total volume column; V, = void
volume.
first extractions and from older cartilage, respectively,
and chondroitin sulfate chains of proteoglycans from
older cartilage were more polydispersed than those of
proteoglycans from young cartilage (Figure 5). Thus,
proteoglycans from second extractions have larger
hydrodynamic sizes than those from first extractions,
which can be explained by their larger chondroitin
sulfate chains. Whether variations in the length of the
proteoglycan core protein also contribute to the difference in hydrodynamic size is as yet unknown.
Structural differences between proteoglycans
from associatively and dissociatively extracted cartilage
prdreated with collagenase. After collagenase digestion of initial cartilage samples, an associative incubation with 0.5M GuHCl extracted 42% of the tissue
uronic acid from 0.5-year-old cartilage and 18% from
60-year-old cartilage (Table 1). This can be explained
by differences in the structure of the collagen network
that occur with maturation and result in a far better
entrapment of proteoglycan aggregates.
Extraction of young and mature collagenase-
900
m
v)
m
900
I
0
100
2 00
time
300
Imin)
Figure 6. Fractogel HW-75s chromatograms of proteoglycans isolated from collagenase-digested 60-year-old articular knee cartilage.
Cartilage was first digested with collagenase in 0.05M Tris-C1,
0.001M CaCI2,pH 7.5, at 37°C for 30 hours, after which proteoglycans were extracted with 0.5M guanidine hydrochloride (GuHCI) at
4°C for 30 hours and isolated as described in Materials and Methods
(A), or proteoglycans were extracted with 4M GuHCl at 4°C for 30
hours and isolated as described in Materials and Methods (B). The
upper panels show proteoglycans eluted under associative conditions; the lower panels show proteoglycans eluted under dissociative conditions. Prior to chromatography under associative conditions, proteoglycans were incubated with 1% hyaluronic acid (by
dry weight of proteoglycans). V,=total column volume. See Materials and Methods for details.
PHYSICAL PROPERTIES OF CARTILAGE PROTEOGLYCANS
100
200
time
300
(min)
Figure 7. Fractogel HW-75s chromatograms of proteoglycans isolated from collagenase-digested0.5-year-oldarticular knee cartilage.
Cartilage was first digested with collagenase in 0.OSM Tris-C1,
0.001M CaC12, pH 7.5, at 37°C for 30 hours, after which proteoglyCMS were extracted with 0.5Mguanidine hydrochloride (GuHC1) at
4'C for 30 hours and isolated as described in Materials and Methods
(A), or proteoglycans were extracted with 4M GuHCl at 4°C for 30
hours and isolated as described in Materials and Methods (B). The
upper panels show proteoglycans eluted under associative conditions; the lower panels show proteoglycans eluted under dissociative conditions. Prior to chromatography under associative conditions, proteoglycans were incubated with 1% hyaluronic acid (by
dry weight of proteoglycans). V, = total column volume. See
Materials and Methods for details.
bated with 4M GuHCl without prior collagenase digestion (78% and 53%, respectively), which indicates that
collagenase digestion of cartilage renders more proteoglycans accessible for extraction.
1245
Associative extractions of mature cartilage liberated primarily proteoglycan monomers (Figure 6),
whereas associative extractions from young cartilage
yielded large amounts of proteoglycan aggregates as
well (Figure 7). Dissociative extractions of mature
collagenase-treated cartilage yielded both aggregating
proteoglycan monomers and nonaggregating proteoglycans, as did dissociative extraction of young
collagenase-digested cartilage (Figures 6 and 7). These
results suggest that most of the aggregating proteoglycans did not lose their aggregating capacity. Therefore, the proteoglycan monomers retrieved after associative extraction of collagenase-digested mature
cartilage are probably not derived from aggregation of
proteoglycans by degradation.
Collagenase digestion of both young and mature
cartilage prior to dissociative extraction of proteoglycans yielded 11% and 20% more proteoglycan
monomers, respectively, compared with the direct
dissociative extraction without prior collagenase digestion (Table 1). This is only partly due to proteolytic
activity, as discussed above. Incubation of coliagenase-digested cartilage with 4M GuHCl resulted in the
extraction of more nonaggregating proteoglycans in
both young and mature cartilage.
Collagenase digestion of cartilage prior to extraction of proteoglycans did not alter the difference in
hydrodynamic size of dissociated proteoglycan monomers from young and mature cartilage, nor did it result
in more polydispersed proteoglycans (Figures 3, 4, 6,
and 7). However, dissociated proteoglycans isolated
from the collagenase-digested residue of the first
extraction of 60-year-old cartilage were of larger hydrodynamic size than associatively or dissociatively
extracted proteoglycans isolated from collagenasedigested 60-year-old cartilage. Therefore, it appears
that proteoglycans isolated in second extractions of
the cartilage residue, after digestion of this residue
with collagenase, can only be extracted when the more
readily extractable proteoglycans are first removed
from the tissue. Apparently, proteoglycans entangle
and overlap with collagen and with each other (lo),
and thus protect collagen from degradation by
collagenase.
DISCUSSION
The used collagenase preparation proved to be
free of detectable proteoglycanase activity, and only a
few of the added 35S-proteoglycans(5-10%) are affected by endogenous proteolytic activity when
1246
proteoglycans are derived from collagenase-digested
cartilage residues (Figure 2). Thus, degradation of
3sS-proteoglycans by endogenous proteolytic activity
during collagenase treatment cannot be totally excluded. However, it seems reasonable to think that
this will affect only a small number of the proteoglycans. Although tests were performed with added
heterologous proteoglycans, it is also likely that the
endogenous proteoglycans are not affected to an appreciable extent by proteolytic activity during the
second extractions. The results indicated that relatively more nonaggregating proteoglycans are extracted from collagenase-digested cartilage and from
collagenase-digested residues of cartilage after first
extractions of proteoglycans. The chemical composition of these proteoglycans also supports the view that
this fraction is enriched in nonaggregating proteoglycans (1 1).
Decrease in size of the proteoglycan monomers
and chondroitin sulfate chains with age has been
reported before (25-28). The very large exclusion limit
of Fractogel HW-75S, however, makes it possible to
show that the size of proteoglycan aggregates also
decreases with age. According to Roughley et a1 (29),
the decrease in size of proteoglycan aggregates in adult
cartilage may be caused by displacement of proteoglycan monomers from hyaluronic acid by a hyaluronic acid-binding protein. However, we observed an
age-dependent decrease in the size of both dissociated
proteoglycans and of their chondroitin sulfate chains,
which led us to believe that the general decrease in
sizie of proteoglycan aggregates that occurs with age is
not singularly caused by displacement of proteoglycan
monomers.
Because proteoglycans isolated from collagenase-digested cartilage residues are of larger hydrodynamic size, it is probable that they are physically more
entrapped in the collagen network. However, the
possibility of the strong binding of these proteoglycans
to collagen, which prevents them from being extracted
in the first instance, cannot be excluded. These results
corroborate data from Bayliss et a1 (lo), who found
uniform chondroitin sulfate chain size throughout cartilage in the directly extracted proteoglycans, but
found a larger chain size in the proteoglycans that
were not extracted.
Together with the results described in the accompanying paper ( l l ) , it can be stated that differences in chemical composition of proteoglycans from
first and from second extractions are due to differences
VAN DE STADT ET AL
in proteoglycan size and the relative proportion of
nonaggregating proteoglycans.
ACKNOWLEDGMENT
All tissues of human origin were kindly supplied by
the Institute for Pathology of the Vrije Universiteit in
Amsterdam.
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