(Received for publication, August 18, 1994; and in revised form, November 3, 1994)
From the
A rat chondrosarcoma cell line and primary bovine chondrocytes
have been used to study cell-mediated aggrecan catabolism. Addition of
1 µM retinoic acid to chondrosarcoma cultures resulted in
aggrecan proteolysis with the release of greater than 90% of the cell
layer aggrecan into the medium within 4 days. NH-terminal
sequencing of chondroitin sulfate-substituted catabolic products gave a
single major NH
-terminal sequence of ARGNVILTXK,
initiating at Ala
. This showed that the proteinase,
commonly referred to as ``aggrecanase,'' which cleaves the
Glu
-Ala
bond of the interglobular domain of
aggrecan (Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C.
R.(1990) J. Biol. Chem. 266, 8683-8685), is active in
this cell system.
Aggrecan G1 domain, generated by cleavage of the
interglobular domain, was also liberated during catabolism and this was
characterized with three antipeptide antisera. Anti-CDAGWL was used as
a general probe for G1 domain. Anti-FVDIPEN was used to specifically
detect G1 domain with COOH terminus of Asn, the form
which is readily generated by cleavage of aggrecan by a wide range of
matrix metalloproteinases. Anti-NITEGE antiserum was used to
specifically detect G1 domain with COOH terminus of Glu
,
the form which is the expected product of
``aggrecanase''-mediated cleavage of aggrecan. Western blot
analysis indicated that a single form of G1 domain of about 60 kDa was
formed. G1 domain of this size reacted with both anti-CDAGWL and
anti-NITEGE but not with anti-FVDIPEN. Similar experiments with primary
bovine chondrocyte cultures, treated with either retinoic acid or
interleukin 1, showed that two forms of catabolic G1 domain, of about
62 and 66 kDa, were formed. Both of these forms reacted on Western
blots with anti-CDAGWL and also with anti-NITEGE.
It is suggested
that cell-mediated catabolism of the aggrecan interglobular domain in
these culture systems, whether promoted by retinoic acid or interleukin
1, primarily involves cleavage of the Glu-Ala
bond by aggrecanase. The accumulation of G1 domain with a
COOH-terminal of Glu
shows that such aggrecanase-mediated
cleavage can occur independent of the cleavage of the
Asn
-Phe
bond by matrix metalloproteinases.
Aggrecan is the major osmotically active component of cartilage
matrix, and it confers on the tissue a capacity to resist compression
under load. The aggrecan core protein has two globular domains near the
amino terminus which are referred to as G1 and G2 and these are
connected by a proteolytically sensitive segment known as the
interglobular domain (IGD). ()The ternary complex formed by
the interaction of the G1 domain with hyaluronan and link protein
anchors the aggrecan within the tissue. Accelerated catabolism of
matrix components in articular cartilage appears to be a hallmark of
cartilage loss in joint diseases such as osteoarthritis; however, the
specific proteinase(s) responsible for aggrecan catabolism have yet to
be conclusively identified(1) .
Analysis of aggrecan
fragments from a range of experimental systems has recently indicated
that a novel proteinase (``aggrecanase''), which cleaves the
Glu-Ala
(see Footnote 2) bond of the IGD of
aggrecan, plays a central role in the catabolic process. CS-substituted
catabolic products which initiate at Ala
are released
from articular cartilage explants treated with RA or
IL-1(2, 3, 4) , growth plate explants treated
with RA (5) , and are present in human synovial fluids from
patients with both osteoarthritis (6) and inflammatory joint
diseases(7) . However, attempts to generate such cleavage
products with a range of purified proteinases (8, 9, 10, 11) and with subcellular
fractions from chondrocytes (8) have been unsuccessful, so
that the identity of this activity remains obscure. The finding that
aggrecan catabolism in cartilage explants can be slowed by addition of
specific inhibitors of both cysteine and metalloproteinases has however
lead to the suggestion that the activity forms part of a complex
proteolytic cascade responsible for aggrecan catabolism in
vivo(12) .
In all of these studies, evidence for this
activity has been obtained by isolation of high molecular weight
CS-bearing core protein products and detection of the new NH terminus of Ala
by automated sequence analysis.
This approach has, however, not clarified the precise role of this
proteinase in chondrocyte-mediated aggrecan catabolism. For example, it
is not known whether cleavage of the Glu
-Ala
bond alone is sufficient for catabolism of the aggrecan IGD by
chondrocytes in situ. This is an important point, since
inhibitor studies (12) have suggested that a proteolytic
cascade may be involved. In addition, many matrix metalloproteinases
(MMP-1, -2, -3, -7, -8, and -9) have been shown to cleave the IGD at
the Asn
-Phe
bond (8, 9, 10, 11) , which is 32
residues NH
-terminal of the aggrecanase site, and the G1
domain generated by this cleavage has been isolated from human
cartilage(10) . Moreover, evidence has recently been presented
to show that although neutrophil collagenase (MMP-8) preferentially
cleaves the Asn
-Phe
bond (9) ,
cleavage of the Glu
-Ala
bond also occurs
following prolonged incubation of an aggrecan G1-G2 fragment with high
concentrations of MMP-8(13) .
In order to understand the
precise cellular mechanisms of aggrecan catabolism, it is therefore
critical to determine whether one or both of these cleavages, or indeed
any other cleavages, occur in a cell-dependent catabolic system.
Clarification of this issue can be obtained by examining the structure
of the G1 domain liberated in this process. Thus, if the activity
referred to as aggrecanase can act alone, and cleaves only the
Glu-Ala
bond, then the G1 domain generated
should bear a COOH terminus of Glu
and nonaggregating
fragments should bear an NH
-terminal of Ala
.
On the other hand, if proteolysis at site(s) other than the aggrecanase
site also occur, such as the Asn
-Phe
bond,
then other separable forms of G1 domain should be generated. Therefore,
in the present study we have used a chondrosarcoma culture
system(14) , and primary bovine chondrocyte cultures, in which
aggrecan catabolism is accompanied by the liberation of G1 domain. The
nonaggregating fragments have been analyzed by NH
-terminal
analysis and the structure of the G1 domain has been examined by
Western analysis with antisera which are specific for a COOH terminus
of either Asn
or Glu
.
All-trans-retinoic acid and insulin (from bovine
pancreas) were from Sigma. Dulbecco's modified Eagle's
medium culture medium (Cellgro) was from Mediatech, Washington, DC
Fetal bovine serum (FBS) was from HyClone, Logan, UT. Human recombinant
IL-1 was from Genzyme, Boston, MA. Goat-anti-rabbit IgG
(peroxidase-conjugated) was from Calbiochem Corp. The ECL detection
system, biotinylated molecular weight markers, and
streptavidin-horseradish peroxidase conjugate were from Amersham Corp.
and were used according to the manufacturers instructions. Materials
and procedures used for the isolation, deglycosylation, and
NH
-terminal sequencing of aggrecan fragments were as
described previously(2, 6) . Aggrecan was assayed as
CS equivalents by dimethylmethylene blue (15) with shark
chondroitin sulfate as standard. Protein was assayed by the
bicinchoninic acid kit from Pierce, with bovine serum albumin as
standard. Purified samples of bovine G1 domain
(Val
-Arg
) and link protein were a gift
from Dr. L. Rosenberg, Montefiore Hospital, New York. Antibody 8-A-4
was obtained from the Developmental Studies Hybridoma Bank, Baltimore,
MD.
Aggrecan fragments present in medium samples were prepared for
analysis by three methods. For NH-terminal sequencing, D1
samples were prepared and processed as described
previously(2) . For SDS-PAGE analysis of unfractionated medium,
25-µl portions containing about 5 µg of CS were dried,
dissolved in 25 µl of 50 mM Tris, 50 mM sodium
acetate, 10 mM EDTA, pH 7.6, and digested with chondroitinase
ABC (0.1 unit/mg CS) for 1 h at 37 °C. Alternatively, medium
samples containing about 1 mg of CS were taken for CsCl gradient
centrifugation under associative conditions (starting density 1.45
g/ml), and the A1 through A4 fractions were dialyzed sequentially
against water, 1 M NaCl/50 mM Tris acetate, pH 7.6,
and water. The A1 fraction contained greater than 95% of the recovered
aggrecan in each case. For SDS-PAGE, portions were deglycosylated with
chondroitinase ABC and keratanase as described (2) and in some
cases with stromelysin (MMP-3) as described(10) .
Anti-FVDIPEN serum was prepared
similarly but using the peptide conjugate
thyroglobulin-Cys-NLeu-FVDIPEN(19) . The peptide
FVDIPEN corresponds to the 7-residue carboxyl-terminal
sequence of the G1 species generated by cleavage of the
Asn
-Phe
bond of both the rat and human
aggrecan core proteins(16, 17) . Anti-FVDIPEN serum
batch 1776 was used throughout this study.
Anti-CDAGWL antiserum (previously termed anti-HAL) was raised against a keyhole limpet hemocyanin conjugate of the aggrecan G1 peptide (CDAGWLADQTVRYPI) which corresponds to residues 180 through 194 of both the human and rat sequences (16, 17) and was affinity-purified for use as described previously (20) .
Figure 1: Specificity of anti-CDAGWL, anti-FVDIPEN, and anti-NITEGE. Aggrecan preparations from the Swarm rat chondrosarcoma tumor were chondroitinase digested, electrophoresed on a 4-12% gradient gel, and blotted to nitrocellulose. The sample analyzed and the antiserum used for each lane were as follows: lane 1, A1 fraction, anti-CDAGWL; lane 2, A1 fraction, anti-NITEGE; lane 3, A1 fraction, anti-FVDIPEN; lane 4, A1D1 fraction, anti-CDAGWL; lane 5, A1D1 fraction, MMP-3-digested, anti-CDAGWL; lane 6, A1D4 fraction, anti-CDAGWL; lane 7, A1D4 fraction, MMP-3-digested, anti-CDAGWL.
Figure 2:
Schematic of G1 domain species under
investigation. The structure of the 55-kDa form of G1
(Glu-Asn
) detected specifically by the
anti-FVDIPEN serum, and the 60-kDa form of G1
(Glu
-Glu
) detected specifically by the
anti-NITEGE serum are shown in relation to the position of the
disulfide-bonded loops of the aggrecan G1
domain.
The reactivity of both forms of G1 with
anti-CDAGWL was supported by studies with MMP-3 which cleaves the
Asn-Phe
bond and so converts all core
species, including the 60-kDa G1 into the 55-kDa G1. Thus when rat A1D1 (lane 4) was treated with MMP-3, all high molecular weight
species disappeared and the only detectable product was the 55 kDa G1 (lane 5). Likewise, when rat A1D4 (lane 6) was
treated with MMP-3, high molecular weight species including the 60-kDa
G1 disappeared, and the amount of 55-kDa G1 present was markedly
increased (lane 7). Reactivity of anti-CDAGWL with these
multiple size forms of aggrecan core (lane 1) is predicted
from the presence of four regions of aggrecan (residues 180 through 194
and 278 through 292 in the G1 domain; residues 514 through 528 and 617
through 631 in the G2 domain) which show high sequence similarity to
the immunizing peptide (residues 180 through 194 in the G1 domain).
Reactivity of anti-CDAGWL with link protein (lanes 1, 6, and 7) is predicted from the presence of two regions of link
protein (residues 259 through 273 and 358 through 372) which show high
sequence similarity (21) to the immunizing peptide.
To
examine the peptide structure required for optimal reactivity with
anti-NITEGE, a radioimmunoassay was established using radioiodinated
YPLPRNITEGE. The assay detected the unlabeled form of this peptide with
a log-linear range of detection of about 0.3-8 nM. Next,
a series of COOH-terminal truncation peptides including YPLPRNITEG,
YPLPRNITE, YPLPRNIT, and YPLPRNI were prepared and assayed (Fig. 3A). Consistent with the peptide orientation of
the immunogen, the anti-NITEGE antiserum clearly possessed a very high
degree of specificity for the COOH-terminal Glu residue. Thus, when the
COOH-terminal Glu was truncated to generate YPLPRNITEG, there was a
greater than 10,000-fold loss in recognition by the antiserum. Indeed,
none of the truncated peptides at concentrations up to about 500 nM were recognized by the antiserum, confirming the very strong
requirement for the Glu residue at the carboxyl terminus of the NITEGE
sequence. Thus YPLPRNITE was not recognized by the antiserum at any
concentration tested, even though this closely related peptide contains
a COOH-terminal Glu residue. The apparent immunoreactivity of YPLPRNIT
at concentrations above 1000 nM was not further investigated.
The effect of extension of the peptide with aggrecan core sequence
beyond the carboxyl-terminal Glu residue was also examined with the
peptides YPLPRNITEGEA, YPLPRNITEGEARG, and YPLPRNITEGEARGS (Fig. 3B). In each case there was at least a 1000-fold
reduction in reactivity with the antiserum. When taken together, these
data indicate that the COOH-terminal Glu residue bearing
an unsubstituted
-carboxyl group is required for optimal
recognition by the antiserum and that proteases cleaving
NH
- or COOH-terminal to the Glu
-Ala
bond of the aggrecan IGD will generate fragments recognized
100-10,000-fold less well than those terminating in
Glu
. To further examine the importance of the
carboxyl-terminal Glu residue, the effect of COOH-terminal
substitutions on antiserum reactivity was also determined (data not
shown). When the COOH-terminal L-Glu was replaced with the
stereoisomer D-Glu, the amide of glutamic acid, glutamine,
aspartic acid, or asparagine, the antiserum also recognized the
peptides with markedly reduced sensitivity. The reduction in
sensitivity was very marked (about 10,000-fold) on substitution with
asparagine or aspartic acid, but somewhat less marked
(100-1000-fold) on substitution with the glutamate analogues.
Figure 3:
Peptide specificity of anti-NITEGE: the
effect of carboxyl-terminal truncations and extensions. Peptides with
COOH-terminal truncations and extensions were compared for their
ability to compete with I-YPLPRNITEGE for antibody.
Competition curves are shown in A (top) for the
control peptide YPLPRNITEGE (
) and the peptides YPLPRNITEG
(
), YPLPRNITE (
), YPLPRNIT (
), and YPLPRNI
(
). Competition curves are shown in B (bottom)
for the control peptide YPLPRNITEGE (
) and the peptides
YPLPRNITEGEA (
),YPLPRNITEGEARG (
), and YPLPRNITEGEARGS
(
). Assay details and calculations are given under
``Experimental Procedures.''
The specificity of anti-FVDIPEN was examined in a similar set of
experiments with COOH-terminal truncations extensions and modifications
of the peptide YTGEDFVDIPEN(19) . These results indicated that
the COOH-terminal Asn residue bearing an unsubstituted
carboxyl group is required for optimal recognition by the antiserum and
that proteases cleaving NH
- or COOH-terminal of the
Asn
-Phe
bond of the aggrecan IGD will
generate fragments recognized 40-10,000-fold less well than those
terminating in Asn
. Consistent with this high specificity
for the respective COOH-terminal residues with an unsubstituted
carboxyl group, neither anti-NITEGE nor anti-FVDIPEN antisera showed
reactivity on Western blots with those forms of rat aggrecan core
protein which are known to contain these sequences internally (see Fig. 1and Fig. 6-8).
Figure 6: Western blot of chondrosarcoma medium products with anti-NITEGE. Medium A1 samples from a CsCl gradient (see ``Experimental Procedures'') were prepared from cultures before RA addition (control) and after different periods of treatment (days 1-4). Portions were chondroitinase digested, electrophoresed on 4-12% gradient gels, and blotted to nitrocellulose for detection with anti-NITEGE. See text for description.
To examine the structure of the
fragments, aggrecan was prepared from a day 3 medium sample which
contained the bulk of the released product. NH-terminal
analysis showed a major sequence of ARGNVILTXK with a minor
sequence of EEVPXXXNS. The major sequence, which initiates at
Ala
, showed that aggrecan catabolism in this system
clearly involves cleavage of aggrecan at the
Glu
-Ala
bond of the aggrecan interglobular
domain. Furthermore, the yield of the Ala
NH
terminus (about 0.3 mol of NH
terminus/mol of core)
was similar to that obtained from both bovine (2) and human (7) fragment preparations in which this product would appear to
account for a high percentage of the degraded core protein present.
Detection of the minor sequence, which initiates at Glu
,
was consistent with the presence of about 20% aggregatable species
which would be expected to contain intact G1 domain and so initiate at
the native NH
-terminal of Glu
. Additionally, in
two experiments with chondrocytes prepared from freshly excised Swarm
rat chondrosarcoma tumor (details not shown), it was found that
aggrecan catabolism could be induced by RA treatment and also that the
major CS-bearing catabolic products released into the medium initiated
at Ala
.
Figure 4: Western blot of chondrosarcoma cell layer products with anti-CDAGWL. Cell layer extracts (see ``Experimental Procedures'') prepared from cultures before RA addition (control) and after different periods of treatment (days 1-5) were electrophoresed on 4-12% gradient gels and blotted to nitrocellulose for detection with anti-CDAGWL. See text for description.
When the corresponding medium samples were fractionated on a CsCl gradient and the A1 fractions were analyzed (Fig. 5) a very similar pattern of products to that seen in the cell layer was observed. In addition to the 350-kDa species, prominent immunoreactive species were also seen at about 250 and 125 kDa, and these probably represent aggrecan core species which have been generated from the 350-kDa species by cleavage within the CS attachment region. The precise structure of these products and the identity of the proteinase(s) involved in their generation was not investigated. In these medium samples, however, the major new immunoreactive species generated during the catabolic process was clearly the 60-kDa species (see days 2-4, Fig. 5). The size and immunoreactivity of this 60-kDa species, found in both the medium A1 samples (Fig. 5) and cell layer (Fig. 4), suggested that it was the discrete form of G1 domain (Fig. 2) liberated during RA-induced catabolism by these cells.
Figure 5: Western blot of chondrosarcoma medium products with anti-CDAGWL. Medium A1 samples from a CsCl gradient (see ``Experimental Procedures'') were prepared from cultures before RA addition (control) and after different periods of treatment (days 1-4). Portions were chondroitinase-digested, electrophoresed on 4-12% gradient gels, and blotted to nitrocellulose for detection with anti-CDAGWL. See text for description.
To
characterize this material further the same medium A1 samples were
electrophoresed and probed with anti-NITEGE (Fig. 6); a highly
specific and strong reaction was also obtained with a 60-kDa species
present in the day 2, 3, and 4 samples only. This suggested that the
60-kDa species detected by anti-CDAGWL ( Fig. 4and Fig. 5) was, in fact, G1 domain with a COOH terminus of
Glu which therefore had been generated by
aggrecanase-mediated cleavage of the Glu
-Ala
bond.
Further evidence to support the identification of G1
domain with a COOH terminus of Glu was obtained as
follows. First, reactivity with anti-NITEGE (Fig. 7, lane
1) was completely eliminated (Fig. 7, lane 2) when
the medium A1 sample was digested with MMP-3 before SDS-PAGE to remove
the COOH-terminal extension of Phe
-Glu
(see Fig. 2for schematic). Second, reactivity with anti-NITEGE was
completely blocked (Fig. 7, lane 3) by preincubation of
the serum with 10 µg/ml of the immunizing peptide (YLPLPRNITEGE),
but it was unaffected (Fig. 7, lane 4) by preincubation
with the same concentration of the peptide (YLPLPRNITEGEARGS) which
spans the cleavage site.
Figure 7: Western blot identification of chondrosarcoma medium products with specific antisera. Chondroitinase-digested products were electrophoresed on 4-12% gradient gels and blotted to nitrocellulose for detection with one of the three anti-peptide sera. The sample analyzed and the antiserum used for each lane were as follows: lane 1, day 3 medium, A1 fraction, anti-NITEGE; lane 2, day 3 medium, A1 fraction, MMP-3-digested, anti-NITEGE; lane 3, day 2 medium, A1 fraction, anti-NITEGE preadsorbed with YLPLPRNITEGE; lane 4, day 2 medium, A1 fraction, anti-NITEGE preadsorbed with YLPLPRNITEGEARGS; lane 5, day 3 medium, A1 fraction, anti-FVDIPEN; lane 6, day 3 medium, A1 fraction, MMP-3-digested, anti-FVDIPEN; lane 7, unfractionated day 3 medium, anti-CDAGWL.
Next, to examine these samples for the
possible presence of G1 domain with a COOH terminus of Asn (see Fig. 2), Western blots of the same A1 preparations
were probed with anti-FVDIPEN (Fig. 7, lane 5). In none
of the medium A1 samples was immunoreactive protein detected. This
absence of reactivity was not however due to poor sensitivity of
detection with this antiserum, since when the same amount of A1 sample
was digested with MMP-3 before SDS-PAGE, a single strongly
immunoreactive product of about 55 kDa was detected in all samples with
anti-FVDIPEN (Fig. 7, lane 6). Furthermore, this
product was also detected by this method in A1 and A1D4 samples of rat
chondrosarcoma tumor extracts (Fig. 1), and it therefore clearly
represents the 55-kDa G1 with a COOH terminus of Asn
shown schematically in Fig. 2. Finally, when
unfractionated catabolic medium was electrophoresed and probed with
anti-CDAGWL (Fig. 7, lane 7), the only G1 domain
detected corresponded to the 60-kDa species, and there was no evidence
for the presence of the 55-kDa species. This clearly indicated that the
60-kDa G1 species which was isolated in the medium A1 fractions ( Fig. 5and Fig. 6), and which was also apparently reactive
with anti-NITEGE (Fig. 7), appeared to be the only G1 species
released into the medium during RA-induced catabolism in this system.
The amount of 60-kDa G1 present in these samples was estimated by standardization of the anti-CDAGWL Western blot with variable loadings (0.1-1.0 µg) of purified bovine G1 domain (not shown). This visual comparison indicated that the amount of G1 present in the medium A1 samples run (Fig. 5) was about 1 µg. Since these A1 samples contained an estimated total of 3 µg of nonaggregating core protein, it appears that a major proportion of the G1 domain product expected to be generated by this catabolic process was indeed present in the culture medium.
To examine the structure of the G1 domain generated in these cultures, A1 preparations were made from the collected medium of control, RA-treated, and IL-1-treated cells and portions were taken for Western blot analysis with both anti-CDAGWL and anti-NITEGE (Fig. 6). In control medium (lane 1), the major components detected with anti-CDAGWL were two high molecular core species, at about 250 and 350 kDa, and a link protein doublet at about 46 kDa. Predictably, none of these species reacted with anti-NITEGE (lane 2). In the medium from RA-treated cells (lane 3), two new prominent species, at about 62 and 66 kDa were detected with anti-CDAGWL. Both of these species also appeared to react specifically and to the same relative intensity with anti-NITEGE (lane 4). The immunoreactivity patterns obtained with the medium from IL-1-treated cells (lanes 5 and 6) could not be distinguished from those obtained with the RA-treated cells.
It therefore appears that with primary bovine chondrocytes, under
either RA or IL-1 treatment, the major G1 domain generated has a
COOH-terminal of Glu, and it is therefore also a specific
product of aggrecanase-mediated cleavage of the IGD. Interestingly,
with bovine chondrocytes this catabolic G1 domain is itself generated
in two discrete forms. Although the source of this heterogeneity has
not been investigated here, it seems likely that it is a result of
variable carbohydrate substitution, which is an established feature of
bovine G1 domain structure. (
)
The results presented clearly indicate that cleavage of the
Glu-Ala
bond is the primary proteolytic
event in catabolism of the interglobular domain of aggrecan by both
chondrosarcoma cells and primary bovine chondrocytes. This follows from
the observation that G1 domain(s) with a COOH terminus of Glu
and CS-bearing fragments with NH
-terminal Ala
are the major catabolic products which can be detected in these
systems. Furthermore, since products from both sides of the
Glu
-Ala
bond have now been identified in
two separate culture systems, it can be concluded that the activity
responsible (aggrecanase) is an endopeptidase.
Identification of the
60-kDa product of the chondrosarcoma cell line as aggrecan G1 was based
on the following observations. First, the protein appeared on day 2 of
RA treatment in both the cell layer (Fig. 4) and medium (Fig. 5) and was therefore generated along with the bulk of the
high molecular weight nonaggregating core species identified by
Sepharose CL-2B chromatography. Second, the protein showed very similar
electrophoretic properties to keratanase-treated bovine hyaluronan
binding region (Val-Arg
) which is detected on
Western blots with anti-CDAGWL as a doublet in the 55-60 kDa
range (not shown). Third, the protein could be isolated in the A1
fraction from a CsCl gradient fractionation of culture medium,
consistent with the hyaluronan-binding properties of G1 domain.
The 60-kDa rat G1 detected by anti-CDAGWL (Fig. 5) appeared to be the same species detected by anti-NITEGE (Fig. 6), since the electrophoretic properties and relative abundance between samples of the material detected by the two antisera appeared to be identical. By the same reasoning, the two G1 species generated by bovine chondrocytes and detected by the anti-CDAGWL serum (Fig. 8, lanes 3 and 5) appeared to be the same species detected by the anti-NITEGE serum (Fig. 8, lanes 4 and 6). If this interpretation is correct, it would follow that all of the detectable G1 domain generated in these systems is a product of aggrecanase action.
Figure 8: Western blot identification of bovine chondrocyte medium products with specific antisera. Deglycosylated A1 medium aggrecan samples (in each case, day 3 plus day 4 combined) were electrophoresed on 4-12% gradient gels and blotted to nitrocellulose for detection with different antisera. The sample analyzed and the antiserum used for each lane were as follows: lane 1, control medium, anti-CDAGWL; lane 2, control medium, anti-NITEGE; lane 3, RA medium, anti-CDAGWL; lane 4, RA medium, anti-NITEGE; lane 5, IL-1 medium, anti-CDAGWL; lane 6, IL-1 medium, anti-NITEGE.
On the other hand, since the present data are
qualitative only, it cannot be entirely excluded that a proportion of
the G1 species detected by the anti-CDAGWL serum have a COOH-terminal
other than Glu 373. Such a ``hidden'' product would
necessarily have a COOH-terminal in close proximity to
Glu, since this electrophoretic system ( Fig. 1and Fig. 7) can readily separate the 60- and 55-kDa forms of G1
domain which differ in size by only 32 amino acid residues (see Fig. 2). A definitive conclusion on this point will require the
development of standardized immunoassays for G1 with the different
antibodies used here.
The presence of aggrecanase-generated G1 in A1 fractions (density > 1.56 g/ml) of culture medium ( Fig. 6and Fig. 8) suggests that this protein is released from cell layers bound to hyaluronan in aggregate structures which contain one or more high buoyant density aggrecan molecules. This implies, much as previously indicated with IL-1-treated cartilage explants(22) , that aggrecanase-mediated cleavage of the IGD does not markedly interfere with the hyaluronan-binding properties of the G1 domain. Whereas aggregate turnover in steady-state cartilage explants appears to involve a co-ordinated process involving the removal of hyaluronan and release of aggrecan chondroitin sulfate(23) , the fate of the G1 domain generated under steady-state conditions is unknown. Since chondrocytes can exhibit receptor-mediated uptake of hyaluronan(24) , it seems possible that hyaluronan, along with catabolic G1 domain, might be endocytosed under steady state. The present study however suggests that in cell culture systems where rapid aggrecan catabolism has been induced with RA or IL-1, a sizable proportion of the catabolic G1 domain is released, along with the CS-bearing products, into the culture medium. The role of hyaluronan and its metabolic fate in the present systems remain to be investigated.
Although fragments with an NH terminus of
Ala
appear to be the major CS-bearing products released
into synovial fluid from articular cartilage in
vivo(6, 7) , the enzyme responsible (aggrecanase)
is not the only proteinase which cleaves aggrecan in vivo.
Thus, our previous studies (10) on human articular cartilage
showed that a proportion of the G1 domain found in this tissue is the
product of metalloproteinase-mediated cleavage of the
Asn
-Phe
bond (see Fig. 2).
Furthermore, this 55-kDa form of G1 domain can be detected with
anti-FVDIPEN on Western blots of human articular cartilage extracts. (
)It is therefore possible that a proportion of the aggrecan
catabolized in vivo undergoes an initiating cleavage of the
Asn
-Phe
bond by a matrix metalloproteinase.
On the other hand, the data in the present paper clearly shows that the
activity (aggrecanase) which is responsible for aggrecan catabolism in
these culture systems can cleave the Glu
-Ala
bond of the IGD without a prior cleavage of the
Asn
-Phe
bond. Thus if cleavage of any IGD
bond on the NH
-terminal side of the aggrecanase site was
absolutely required to allow subsequent cleavage of the
Glu
-Ala
bond then G1 domain with a
COOH-terminal of Glu
would not be generated in any
catabolic system. In this regard it therefore seems likely that much of
the aggrecan catabolized in vivo undergoes an initiating
cleavage of the Glu
-Ala
bond and that the
55-kDa form of G1 domain present in human cartilage (10) results from secondary proteolysis of this 60-kDa form
(see Fig. 2). Indeed this idea is supported by the finding that
G1 domain with COOH terminus of Glu
, as detected with
anti-NITEGE on Western blots, is also present in rat chondrosarcoma
extracts (Fig. 1), human articular cartilage extracts, and human
synovial fluids.
Since the cell-mediated catabolism of
the aggrecan interglobular domain described here apparently involves
cleavage of the Glu-Ala
bond alone, the
activity responsible clearly exhibits a very high level of specificity
for this cleavage site under these conditions. This specificity is
unusual when compared with the multiple cleave sites within the
interglobular domain generated on incubation of aggrecan in solution
with a range of MMPs (8, 9, 10, 11) . In this regard, it
is interesting that although MMP-1, -2, -3, -7, -8, and -9 have been
shown to readily cleave the Asn
-Phe
bond,
cleavage of the Glu
-Ala
bond also appears
to occur following prolonged incubation of aggrecan with high
concentrations of MMP-8(13) . It is therefore tempting to
speculate that aggrecanase is a new member of the MMP family with an
unusually narrow cleavage specificity for aggrecan. Alternatively, the
site(s) of cleavage of aggrecan by such proteinases may be markedly
influenced by the precise conditions of substrate structure or
presentation which operate in cell culture and in vivo. Such
conditions may be difficult to reproduce in solution incubations with
purified components.