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Phospholipase A2 is a Major Component of the Salt-Extractable Pool of Matrix Proteins in Adult Human Articular Cartilage.

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1106
PHOSPHOLIPASE A, IS A MAJOR COMPONENT OF
THE SALT-EXTRACTABLE POOL OF
MATRIX PROTEINS IN
ADULT HUMAN ARTICULAR CARTILAGE
ANNELIESE D. RECKLIES and CHANTAL WHITE
Adult human articular cartilage contains a component with an apparent molecular weight of 16 kd,
which is extractable with high ionic strength buffers.
This protein, which, in addition to lysozyme, is one of
the most prominent components in salt extracts of adult
cartilage, is not detectable in cartilage from newborns.
We performed N-terminal sequence analysis to identify
the protein. The amino acid sequence obtained for the
first 20 residues was identical to that reported for
phospholipase A, (PLA,) from human placenta and
human synovial cells. The extractable PLA, was found
to be active. The lack of PLA, in salt extracts from
newborn cartilage observed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis was confirmed by the very low levels of PLA, activity detectable
in these preparations. PLA, was clearly present in
cartilage extracts from an 18-year-old subject and a
19-year-old subject, suggesting that its accumulation
begins at some stage during the adolescent growth
period. The enzyme does not appear to be released from
cartilage matrix under normal physiologic conditions,
and it is possible that the accumulation of PLA, in
maturing cartilage is a result of the decreased matrix
turnover associated with the termination of skeletal
growth. Whether PLA, is active in the cartilage matrix,
From the Joint Diseases Laboratory, Shriners Hospital for
Crippled Children, and the Department of Surgery, McGill University, Montreal, Quebec, Canada.
Supported by the Shriners of North America and the
Arthritis Society of Canada.
Anneliese D. Recklies, PhD; Chantal White, BSc.
Address reprint requests to Anneliese D. Recklies, PhD,
Joint Diseases Laboratory, Shriners Hospital for Crippled Children,
1529 Cedar Avenue, Montreal, Quebec H3G 1A6, Canada.
Submitted for publication November 6, 1990; accepted in
revised form April 10, 1991.
Arthritis and Rheumatism, Vol. 34, No. 9 (September 1991)
its precise localization, and its effects on the resident
chondrocytes remain to be determined.
The major structural components of cartilage
matrix are the collagens (types 11, VI, IX, and XI) and
the aggregating proteoglycans. In addition, a variety of
other components have been identified and characterized. Some are specific to articular cartilage, while
others have a more ubiquitous distribution (for review,
see ref. 1). These proteins generally bind very strongly
to the cartilage matrix and can be extracted only with
highly chaotropic agents. Cartilage also contains a
number of cationic proteins which are retained due to
their interaction with the highly negatively charged
sulfated glycosaminoglycan side chains of the cartilage
proteoglycans. These components are usually extractable with high ionic strength buffers. The accumulation of lysozyme in cartilage matrix is a good example
of such an interaction (2,3). Lysozyme does not appear to be synthesized by the chondrocytes (4);it is
most likely sequestered from the synovial fluid. A
variety of plasma proteinase inhibitors have also been
shown to be extractable from cartilage with high ionic
strength buffers (5,6), and these, like lysozyme, are
thought to originate from the synovial fluid. While the
salt-extractable pool of cationic proteins contains a
variety of other components, none of these have been
clearly identified, nor has their function in cartilage
been studied.
Salt extracts of adult human articular cartilage
contain 2 major components, with molecular weights
of 14.5 kd and 16 kd, which can account for more than
half of the total extractable protein. Herein we present
evidence that the 16-kd component is phospholipase
PHOSPHOLIPASE A, IN HUMAN ARTICULAR CARTILAGE
A, (PLA,). This enzyme has been shown to be present
in synovial fluid from patients with rheumatoid arthritis (7) and has been implicated in the etiology of this
disease (8,9). While PLA, activity has been demonstrated in crude homogenates from normal and osteoarthritic cartilage (lo), the identity of the enzyme in
cartilage extracts had not been established.
MATERIALS AND METHODS
Materials. Human articular cartilage from femoral
condyles was obtained at autopsy, within 20 hours after
death. Only cartilage from macroscopically normal joints
was harvested. Cartilage from the newborn donor was
harvested from areas excluding the growth plate and the
secondary center of ossification, and represented articular
and resting zone cartilages. The cartilage specimens were
collected under aseptic conditions and transported to the
laboratory in Hanks' balanced salt solution.
The phospholipase A, substrates, l-stearoyl-2-[ 1''Clarachidonyl L-3 phosphatidylcholine and 1,2di['-I4 Clpalmitoyl L-3 phosphatidylcholine were purchased
from Amersham (Oakville, Ontario, Canada) and had a
specific activity of 56 mCi/mmole (2.07 GBq/mmole) and 108
mCi/mmole (4.0 GBq/mmole), respectively. The radiolabeled amino acid precursors 3'S-methionine and 3sS-cysteine
were obtained from New England Nuclear (Lachine, Quebec, Canada). Electrophoresis supplies were from BioRad
(Mississauga, Ontario, Canada), and polyvinylidene difluoride (PVDF) membranes from Millipore (Toronto, Ontario,
Canada). Tissue culture supplies were obtained from Flow
Laboratories (Mississauga, Ontario, Canada).
Preparation of extracts. Cartilage was chopped, and
0.5 gm was extracted with 2 ml of 1M NaCl in 0.1M sodium
acetate buffer (pH 5.5) containing 10 mM EDTA and the
proteinase inhibitors pepstatin A (10 pg/ml), phenylmethylsulfonyl fluoride (1 mM), and iodoacetamide ( 1 mM), with
constant stirring for 48 hours at 4"C, as described by
Killackey et a1 (6). Where indicated, the residue was extracted for 48 hours at 4°C with 4M urea in 0.1M sodium
acetate. A 1-ml aliquot of the extracts was dialyzed against
0.1M ammonium acetate and lyophilized, and the remainder
was dialyzed against 0.1M Tris HCI, pH 9.5, containing
0.1M NaC1, for determination of PLA, activity. Total protein was determined by the method of Bradford (1 l), using a
BioRad protein assay kit. The hydroxyproline content of
cartilage hydrolysates was determined as described by Burleigh et a1 (12).
Gel electrophoresis and autoradiography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed essentially according to the method
of Laemmli (13). Lyophilized extracts were dissolved in
SDS-sample buffer and analyzed under reducing conditions
on 7.5-15% polyacrylamide gradient gels or on 15% polyacrylamide gels. Gels were stained with Coomassie brilliant
blue, and, where indicated, dried and exposed to Kodak
XAR-5 film (Eastman Kodak, Rochester, NY) at room
temperature for 4 days.
1107
Amino acid sequence analysis. N-terminal amino acid
sequence analysis of salt-extractable matrix proteins was
performed according to the method of Matsudaira (14).
Extracts were separated by SDS-PAGE on 7.5-15% gradients and electroblotted onto PVDF membranes. Bands of
interest were identified by brief staining with Coomassie
brilliant blue, excised, and subjected to automated sequence
analysis using an Applied Biosystems 470A gas-phase microsequencer (ABKanada, Mississauga, Ontario, Canada).
Determination of PLA, activity. Extracts were assayed for phospholipase activity as described by Clark et al
L-3 phosphatidyl(151, using I-~tearoy1-2-[l-'~C]arachidonyl
choline as substrate. Prior to use, the substrate was dried
under nitrogen and resuspended, by sonication in an aqueous solution of 5 mM deoxycholate, to a concentration of 50
p M . The assay buffer was 0.1M Tris HCl, pH 9.0, containing
0.1M NaCI, 1 mM CaCl,, and 1 mM deoxycholate. Aliquots
of dialyzed cartilage extracts in a total volume of 10 pl were
added to 40 pl of assay buffer, and the tubes were preincubated for 5 minutes at 37°C. The reaction was started by the
addition of 10 pI of substrate, to give a final concentration of
10 p M . After l-5-minute incubations, lipids were extracted
with chloroform-methanol as described by Clark et al (15)
and analyzed by thin-layer chromatography and autoradiography . For quantitation, bands corresponding to free
arachidonic acid were scraped and counted in a liquid scintillation counter. To demonstrate the formation of lysophosphatidylcholine, 1,2-di['-'4C]palmitoyl L-3 phosphatidylcholine
was used as substrate, and bands comigrating with a lysophophatidylcholine standard were isolated and counted.
Determination of PLA, protein levels in cartirage
extracts. To estimate the amount of PLA, protein in cartilage
extracts, aliquots were separated by SDS-PAGE on 7.5-15%
gradient gels and transferred to PVDF membranes. Under
these conditions PLA, is separated from lysozyme, the other
major component in this molecular weight range. The band
corresponding to PLA, was excised, hydrolyzed by gasphase hydrolysis, and the amino acid composition was
determined using a Dionec Bio-LC amino acid analyzer
(Dionec, Canada, Mississauga, Ontario, Canada).
Explant culture and biosynthetic labeling. Explant
cultures of adult human articular cartilage were established
in T25 tissue culture flasks, at 0.5 gm of cartilage, in 3 ml
Dulbecco's modified Eagle's medium (DMEM), per flask. To
determine release of PLA, from cartilage explants, the
culture medium was collected after 24 hours or 48 hours of
culture and the cartilage extracted with 1M NaCl as described above. Aliquots of the culture medium and salt
extracts were assayed for PLA, activity, while the remainder was dialyzed against 0.1M ammonium acetate, lyophilized, and analyzed by SDS-PAGE. For biosynthetic labeling, cartilage explants were equilibrated in cysteine-free or
methionine-free medium for two l-hour periods, and 35Scysteine or "S-methionine was added, at 30 pCi/ml, in
DMEM containing a total concentration of 20 pM cysteine
or methionine, respectively. 'The culture medium was harvested after 24 hours and the explants extracted as above.
Both extracts and media were exhaustively dialyzed against
0.1M ammonium acetate to remove unincorporated radioac-
RECKLIES AND WHITE
1108
tivity and processed for analysis by SDS-PAGE on 7.5-15%
gradients, followed by autoradiography.
RESULTS
Extraction of cartilage with 1M NaCl removes
components that are bound to the cartilage matrix
mainly by electrostatic interactions. When such extracts from adult articular cartilage were analyzed by
SDS-PAGE, they were found to contain 2 prominent
low molecular weight bands, while only the smaller of
the 2 was evident in extracts from neonatal cartilage
(Figure 1). The molecular weight of the smaller component suggested that it was lysozyme; it has been
known for some time that cartilage from many species
contains large quantities of this enzyme, as determined
by activity measurements (2) and immunologic assays
(3). The identity of the lower molecular weight component as lysozyme was confirmed by N-terminal
sequence analysis.
The identity of the larger component (molecular
weight -16 kd) was unknown. We therefore determined the amino acid sequence of this component, by
electrophoretic transfer of proteins separated on SDSpolyacrylamide gradient gels onto PVDF membranes
Figure 1. Protein composition of 1M NaCl extracts of human
cartilage from subjects of various ages. Salt extracts from cartilage
were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gels were stained with Coomassie brilliant blue.
The age of the cartilage donor (in years) is shown below each lane
(NB = newborn; 2 different specimens). A prominent band comigrating with the hen eggwhite lysozyme marker (14.4 kd) can be seen
with all specimens. A second band, migrating just above lysozyme,
is present in salt extracts from adult cartilage only. Molecular weight
standards are shown on the left and right.
IbkDacomponent:
N L V N F H R MI K L T T G K E A A L S
Synovial fluid PLA,: N L V N F H R M I K L T T G K E A A L S Y E F
.
Figure 2. N-terminal amino acid sequence of the 16-kd component
of adult human articular cartilage. Salt extracts of the specimen from
the 19-year-old subject shown in Figure 1 were used for the analysis.
The molar yields (in pmoles) for the first 5 cycles were as follows:
cycle 1 (N) 11.6, cycle 2 (L) 33.0, cycle 3 (V)27.0, cycle 4 (N) 13.2,
cycle 5 (F) 26.0. No minor sequences were detected in this analysis.
The sequence for the human synovial cell-associated phospholipase
A, (PLA,) is that predicted from the reported complementary DNA
sequence (19).
and microsequencing of the isolated band corresponding to the 16-kd species. The N-terminal amino acid
sequence (Figure 2) was found to be identical to the
sequence reported for human platelet (16), placental
(17), and synovial fluid PLA, (16,18). Salt extracts of
the specimen from the 19-year-old donor shown in
Figure 1 were used for the analysis. The molar yields
(in pmoles) for the first 5 cycles were as follows: cycle
1 (N) 11.6, cycle 2 (L) 33.0, cycle 3 (V) 27.0, cycle 4
(N) 13.2, cycle 5 (F) 26.0. No minor sequences were
detected by this analysis. The initial cycle yields
represented -20% of the total loaded protein, as
determined by amino acid analysis of material blotted
onto PVDF membranes; this is similar to sequencing
yields for PVDF blots of pure proteins as reported by
the manufacturer.
While, similar to lysozyme, most of the PLA,
could be extracted with 1M salt, a residual quantity
was extracted with 4M urea (Figure 3). In neonatal
cartilage, however, even the urea extracts did not
contain any PLA,, when analyzed by SDS-PAGE.
Prominent bands corresponding to PLA, were seen in
cartilage extracts from 18-year-old and 19-year-old
subjects, approximately equal in intensity to the lysozyme band (Figure 1). In cartilage specimens from
older donors, the intensity was quite variable, but in
all cases the PLA, band appeared to be more intense
than the lysozyme band.
To determine whether the extractable PLA,
was enzymatically active, both salt and urea extracts
of the cartilage specimens shown in Figure 3 (from
3-day-old, 38-year-old, and 72-year-old donors) were
assayed for activity using sn-2 arachidonyl phosphatidylcholine as substrate and determining the rate of
release of free arachidonic acid. Figure 4 shows the
time course of PLA, activity obtained with the salt
extracts from the 3 cartilages. PLA, activity was
detectable in all preparations; however, the activity in
the salt extract from the 3-day-old subject was -100-
PHOSPHOLIPASE A, IN HUMAN ARTICULAR CARTILAGE
1109
amino acid sequence of synovial fluid PLA, (19),
suggesting that PLA, is the major component of these
bands. The PLA, content of the cartilage from the
38-year-old donor was estimated to be 220-240 pg/gm
wet weight, while the value for the cartilage from the
72-year-old donor was found to be 46 pg/gm wet
weight (Table 1). Based on the estimates of the PLA,
protein content of the salt and urea extracts, a specific
activity of 1.1 nmole/minute/pg PLA, was calculated
for the various preparations (Table 1). There was no
difference in the specific activities calculated for the
salt extract and the urea extract, suggesting that no
loss of activity occurred during urea extraction, relative to salt extraction.
To confirm that the hydrolysis of the arachidonyl phosphatidylcholine was due to PLA,, concomitant formation of lysophosphatidylcholine and free
fatty acids was analyzed using 1,2-di[l-'4C]palmitoyl
L-3 phosphatidylcholine as the assay substrate. Approximately equal molar quantities of both hydrolysis
products, free palmitoic acid and lysophosphatidylcholine, could be recovered, suggesting that the major
hydrolysis reaction occurred at the C-2 position of the
subst rate.
Since the newborn cartilage used for extraction
consisted of both articular surface and epiphyseal
-
Figure 3. Protein composition of 1M NaC1, and subsequent 4M
urea, extracts of human cartilage. Extracts from a newborn infant
(lanes 1 and 4), a 38-year-old subject (lanes 2 and S ) , and a
72-year-old subject (lanes 3 and 6) were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and gels were stained
with Coomassie brilliant blue. Approximately equal amounts of
protein were loaded in each lane. Molecular weight standards are
shown on the left.
fold lower than in the adult cartilages. Extracts from 3
additional neonatal cartilage specimens showed similar low activity levels and lacked a 16-kd band on SDS
gels, indicating that the lack of PLA, in neonatal
cartilage is a general phenomenon.
In this analysis as well, some residual PLA,
was detectable in subsequent urea extracts after the
initial salt extraction of adult cartilage, but not of
newborn cartilage. Extraction with lower ionic
strength buffers (O.1M or 0.2M NaCl) resulted in
negligible recovery of activity (data not shown). The
PLA, activity determined in the extracts from the 3
cartilage specimens is summarized in Table 1. Adult
cartilage contained much higher levels of PLA, activity, based on wet weight of the cartilage, than did
cartilage from the newborn. Similar results were obtained when activity levels were based on total hydroxyproline content (data not shown).
The total amount of PLA, protein in salt and
urea extracts of adult cartilage was estimated from the
total amino acid content of the bands corresponding to
PLA,, after separation by SDS-PAGE. As shown in
Table 2, the amino acid composition of the bands was
generally consistent with that determined from the
I--]
5000
0.0
1.0
2.0 3.0 4.0
TIME (Minutes)
5.0
Figure 4. Time course of phospholipase A, activity in 1M NaCl
extracts of human cartilage. The preparations used were the same as
those shown in Figure 3. The newborn cartilage extract (0)was
assayed undiluted, while the extracts from the 38-year-old (W) and
72-year-old ( 0 )subjects were assayed at a 10-fold dilution in assay
buffer. Activity is shown as cpm of arachidonic acid (free fatty acid)
released.
RECKLIES AND WHITE
1110
Table 1. Phospholipase A, (PLA,) levels in cartilage extracts*
Recovery of activity
(% of total activity)$
Age of donor
Activity
(per gm wet weight)?
PLA, protein
(pdgm wet weight)
1.1
277.9
65.3
ND
220, 2388
46
Newborn
38 years
72 years
Newborn superficial7
Newborn deep7
0.2
2.70
1M salt
extract
>95
95 (1.08)
>95 (1.34)
4M urea
extract
5 (1.01)
-
ND
ND
* PLA, activity levels were determined in salt, and subsequent urea, extracts from cartilage specimens
from donors of the indicated ages. Extracts from the 38-year-old and the 72-year-old subjects were
diluted 10-fold for assay; extracts from the newborn cartilage were assayed undiluted. Incubation time
was 5 minutes. PLA, protein in cartilage extracts was estimated by amino acid analysis as described
in Materials and Methods. Amino acid analysis was not performed on extracts from the newborn
subject, or on the urea extract from the 72-year-old subject because the amounts of protein in the PLA,
bands were too low for detection. PLA, activity is expressed as nmoles arachidonic acid released/
minute. ND = not determined; - = not detectable.
t The total activity was calculated from the activities determined for the salt and urea extracts.
$ Numbers in parentheses represent the specific PLA, activities calculated from activity measurements and estimates of the PLA, protein content. Values are nmoleslminute/pg PLA, protein.
8 Values are from 2 separate determinations.
IT See Results.
cartilage whereas the adult material used for analysis
was strictly articular cartilage, the possibility that
PLA, might be more concentrated at the articular
surface was investigated. The articular cartilage is a
relatively small proportion of the total joint cartilage in
Table 2. Amino acid composition of the 16-kd component blotted
onto .polvvinvlidene
difluoride (PVDF)*
.
~
Residues/100
Residue
Calculated
from cDNA
AspIAsn
Thr
Ser
GluIGln
Pro
GIY
Ala
Val
Ile
Leu
TYr
Phe
His
LYS
Arg
10.1
9.2
9.2
5.5
1.8
10.1
7.3
2.8
1.8
5.5
7.3
4.6
3.7
11.9
9.2
Determined
from PVDF
10.1
8.3
9.1
8.6
2.2
12.0
7.5
3.5
2.1
6.3
5.6
4.0
2.9
8.2
9.6
* The 16-kd component was isolated by separating salt or urea
extracts on sodium dodecyl sulfate-polyacrylamide gradient gels
and electrophoretically transferring onto PVDF membranes. The
data shown are from the 1M salt extract from a 38-year-old cartilage
donor. The calculated amino acid composition was determined for
the mature protein based on the complementary DNA (cDNA)
sequence reported by Seilhammer et al (18).
the newborn, and this might have accounted for the
overall very low levels of PLA, found in this age
group. Cartilage was harvested from the articular
surface to a depth of -0.4 mm (superficial) and from
the deep zone (deep). Salt extracts and subsequent
urea extracts from these preparations were assayed for
PLA, activity. The enzyme levels, shown as total
activity/gm wet weight, are presented in Table 1.
Extracts from the articular surface contained essentially no PLA, activity; most of the total PLA, found in
newborn cartilage was located in extracts from the
deep layers. Thus, the low levels of PLA, in newborn
cartilage could not be attributed to a differential distribution of the enzyme which was masked when total
joint cartilage was extracted.
The extractability of PLA, with 1M salt suggests that the interaction with the matrix is of an ionic
nature and hence some exchange should occur, as has
been observed with lysozyme. The release of PLA,
from cartilage explants was therefore investigated.
When concentrated culture medium was analyzed by
SDS-PAGE and compared with salt extracts of the
cartilage explants at the end of the culture period, no
evidence for release of PLA, was obtained, whereas a
band migrating in a position
to lysozyme was
readily seen (Figure 5). Culture media from cartilage
explants were also analyzed for PLA, activity a&
found to be negative
not shown).
activity was readily detected when an aliquot of a salt
PHOSPHOLIPASE A, IN HUMAN ARTICULAR CARTILAGE
1111
readily identifiable by the relative incorporation of
cysteine as compared with methionine.)
Autoradiographs of salt extracts from cartilage
explants labeled with 35S-cysteine showed a diffuse
band (Figure 61, which comigrated with the intense
PLA, band seen after staining of the gel with
Coomassie brilliant blue (Figure 5). (The autoradiography results shown in Figures 6a and c are from the
same gels used in Figures 5a and b, respectively.) No
radioactivity corresponding to PLA, was observed in
culture medium from these explants. Newly synthesized proteins extracted from cartilage explants with
1M NaCl and those found in the corresponding culture
medium are shown in Figure 6a, while Figures 6b and
c show only the salt-extractable material, since no
Figure 5. Protein composition of media and salt extracts from
cultured cartilage explants. Explants were established from cartilage
from an 18-year-old subject (a) and a 48-year-old subject (b). Culture
media and salt extracts were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and the gels were stained with
Coomassie brilliant blue. For each specimen, results from 2 separate
explants are shown. The lane to the right in a shows the migration of
molecular weight standards (lysozyme 14.4 kd, soybean trypsin
inhibitor 21.5 kd, carbonic anhydrase 31 kd, ovalbumin 42.7 kd,
bovine serum albumin 66.2 kd, phosphorylase B 97.4 kd). The
migration position of phospholipase A, is indicated by arrows.
extract from adult cartilage was added to the culture
medium, ruling out the possibility that an inhibitor of
PLA, is released into the culture media. Thus, the
PLA, contained in cartilage is not readily released,
and therefore might not contribute significantly to the
pool of PLA, found in synovial fluid.
Since PLA, has been shown to be synthesized
and released from synovial fibroblasts, it is possible
that it is sequestered from the synovial fluid, similar to
lysozyrne, and accumulates in the cartilage. Alternatively, it could be synthesized in situ by the articular
chondrocytes. To investigate whether biosynthesis of
PLA, occurs in human articular cartilage , explants
were labeled with 35S-cysteine or 35S-methionine.
Newly synthesized proteins in the culture medium, as
well as in salt extracts, were analyzed by SDS-PAGE,
followed by autoradiography . (PLA, contains 14 cysteine residues and 1 methionine, and thus should be
Figure 6. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) analysis of newly synthesized proteins of cultured
cartilage explants. Cartilage explants were cultured in the presence
of either 35S-cysteine( C ) or "S-methionine (M) for 24 hours, and
culture media and salt extracts were analyzed by SDS-PAGE.
Extracts were established from cartilage from an 18-year-old subject
(a), a 54-year-old subject (b), and a 48-year-old subject (c). Autoradiographs in a and c are from the Coomassie brilliant blue-stained
gels shown in Figures 5a and b, respectively. The lanes to the left in
a and c show high molecular weight standards (as listed in Figure 5);
the lane to the left in b shows low molecular weight standards
(carbonic anhydrase 30 kd, soybean trypsin inhibitor 21.5 kd,
cytochrome c 12.5 kd, aprotinin 6.5 kd, insulin 3.5 kd). The
migration position of phospholipase A, is indicated by arrows.
RECKLIES AND WHITE
1112
radioactive band corresponding to PLA, was detected
in culture medium from these explants. Thus, similar
to the resident material, any newly synthesized PLA,
is not released from cartilage. These findings suggest
that at least part of the salt-extractable PLA, could be
synthesized by the articular chondrocytes.
DISCUSSION
Our findings in the present study demonstrate
that phospholipase A, is a major component of the
salt-extractable protein pool of adult human articular
cartilage, whereas it is essentially absent in neonatal
cartilage. The data presented were derived from 7
different adult and 4 different newborn cartilage specimens. The band identified as PLA, was present at
varying intensity in salt extracts of all the adult specimens, but in none of the newborn specimens. It can
thus be concluded that PLA, is a component of
macroscopically normal adult human cartilage.
The N-terminal amino acid sequence of the
cartilage PLA, indicates that the enzyme belongs to
the group of extracellular phospholipases, or group I1
enzymes, which have been associated with inflammatory conditions in various species. These enzymes are
characterized by the lack of a cysteine residue at
position 11, which is present in all known sequences of
pancreatic PLA,, as well as in the forms found in
cobra and sea snake venom (group I PLA,). To date, it
appears that only 1 form of extracellular group I1 PLA,
is present in humans, since the reported amino acid
sequences for PLA, from platelets (16), synovial fluid
(1618), and placenta (17) are identical. While phospholipase activity has been shown to be present in
crude homogenates of human articular cartilage
(10,19), neither its identity nor its predominantly extractable nature has been clearly demonstrated previously. This enzyme has also been shown to be present
in a cell-associated form in cultured rabbit chondrocytes (20,21), as well as in a free form in synovial fluid
(7,221.
Estimates of the protein content of the PLA,
band after SDS-PAGE and amino acid analysis by
electroblotting suggest that the PLA, content in cartilage is 40-200 pg/gm wet weight. Results of the amino
acid analysis are generally fairly consistent, within the
limitations of the technique, with the suggestion that
PLA, is the major component in the 16-kd band.
Specific activity of PLA, was calculated to be -1.1
nmole/minute/pg for the cartilage preparations. There
was no significant difference between the specific
activity calculated for the salt and urea extracts,
suggesting that no loss of activity occurred during
exposure to urea. In studies using 2-linoleoyl-phosphatidylcholine, Franson et a1 (23) found a specific activity
of 0.53 nmoles/minute/pg protein for highly purified
PLA, preparations from platelets, macrophages, or
polymorphonuclear celis. These values are well within
the range of activity observed for the cartilage preparations.
The source of the enzyme in the salt-extractable
protein pool of articular cartilage is unclear at present.
The identity of the N-terminal sequence of the cartilage PLA, with that of the human synovial fluid
enzyme (1618) would suggest that the PLA, might be
absorbed from synovial fluid, which has been shown to
contain PLA,, particularly following inflammatory episodes (7,22). Adsorption to cartilage matrix components could occur, leading to a build-up of enzyme
levels. Although this requires further investigation in
explant culture studies using purified secretory PLA,,
considering the small size of the enzyme and its basic
nature, it is very likely that this process does occur.
Local synthesis by chondrocytes stimulated by
interleukin-1 (IL-1) could also contribute to the PLA,
pool: There is clear evidence that isolated articular
chondrocytes stimulated with IL-1 in culture can synthesize PLA, (20,21). In addition, our biosynthesis
data suggest that synthesis of this enzyme could occur
in cultured cartilage explants. Thus, one would expect
a progressive increase in the PLA, content, perhaps
parallel to the course of trauma and/or inflammation in
the joint. This might explain the variable levels of
extractable PLA, observed in adult cartilage. While
the rate of PLA, clearance from cartilage matrix is not
known at present, its tight binding to the matrix would
suggest a slow turnover.
Increased PLA, activity has been reported in
osteoarthritic cartilage, as compared with agematched normal cartilage, in studies using crude cartilage hornogenates in low ionic strength buffer as
source of the enzyme (10). Our observations indicate
that the PLA, is fairly tightly bound to the cartilage
matrix and is released only by extraction with 1M salt
solutions, and not with low ionic strength buffers or
culture medium. In addition, we have found that while
activity can be quantitated fairly accurately in dialyzed
salt extracts, this is not the case with dialyzed guanidine extracts, unless proteoglycans are removed by
centrifugation in CsCl density gradients. PLA, can be
recovered in the low buoyant density fractions (Recklies AD: unpublished observations). Whether the de-
PHOSPHOLIPASE A, I N HUMAN ARTICULAR CARTILAGE
crease in activity is due to the association of the
enzyme with the cartilage proteoglycans remains to be
determined.
The cause of the accumulation of PLA, in
cartilage is probably its electrostatic interaction with
the highly negatively charged sulfated glycosaminoglycans since, similar to lysozyme, PLA, contains a large
number of basic amino acids and has a PI of 10 (24).
However, the association of the PLA, with the plasma
membrane of articular chondrocytes cannot at present
be ruled out as the source of the extractable material,
since membrane-associated PLA, can be extracted
with 1M salt (25). A PLA, band is not readily detectable by SDS-PAGE in salt extracts from isolated
chondrocytes, nor is there any clear incorporation of
radiolabeled cysteine into a component comigrating
with PLA, in such preparations (Recklies AD: unpublished observations). Unstimulated rabbit chondrocytes have been shown to contain barely detectable
levels of PLA, activity (20). These observations make
the chondrocyte plasma membrane an unlikely source
of the large quantities of PLA, that can be extracted
from adult articular cartilage.
The salt-extractable PLA, is closely associated
with the cartilage matrix, since we could not detect
any release of PLA, from cultured cartilage explants,
unlike the release of lysozyme, which was readily
observed within a 24-hour culture period. In addition,
we did not detect any release of PLA, from cartilage
explants cultured in the presence of IL-I for up to 3
days, and there appeared to be no obvious loss of
PLA, from the matrix as judged by the amount of
PLA, extractable from the cartilage after short-term
IL-1 treatment (Recklies AD: unpublished observations). Thus, although cartilage contains large quantities of PLA,, this probably does not contribute greatly
to the pool of PLA, in synovial fluid from patients with
rheumatoid arthritis. Whether the prolonged action of
degradative enzymes such as the metalloproteinases,
which have been implicated as the major degradative
forces in cartilage matrix destruction, can cause release of PLA, remains to be established.
Our data show that neonatal cartilage contains
very low levels of PLA,, compared with adult cartilage. To eliminate the possibility that levels of PLA,
similar to those found in adult cartilage were present
close to the articular surface in neonatal tissue, a depth
study was performed, and no large differences in PLA,
activity where detected. Though extracts from the
deeper zone showed higher activity levels than those
from the articular surface zone, these levels were still
-
1113
very low compared with adult cartilage. The lack of
PLA, in neonatal cartilage thus appears to be due to
lower levels of supply, either from the synovial fluid or
from synthesis by the neonatal chondrocytes, or both.
While PLA, has been shown to be associated with
matrix vesicles and growth plate chondrocytes (26,27),
the enzyme appears to remain localized in the growth
plate. Alternatively, the lack of accumulation could be
due to increased turnover of matrix components in the
neonatal cartilage as compared with adult cartilage.
The levels of synthesis of PLA, in neonatal and adult
cartilage, as well as adsorption of exogenous PLA, by
cartilage matrix, require further investigation. The
exact time point between birth and cessation of
growth, when PLA, starts to accumulate in the articular cartilage remains to be established. We have
shown that only very low levels of the enzyme are
present at birth, but that it is readily detectable at the
end of the secondary growth period. It is thus tempting
to speculate that this accumulation might reflect a
decrease in the turnover rate of matrix components,
once growth has ceased.
The PLA, extractable from cartilage matrix is
enzymatically active, though we cannot say to what
extent. Whether the enzyme is active in situ in the
cartilage matrix is not known at present. However,
considering the large quantities of PLA, protein
present in the matrix, one would assume that the
enzyme activity has to be very low to allow maintenance of the integrity of the plasma membranes and
survival of the chondrocytes. Further work is needed
to elucidate the role of PLA, in cartilage matrix.
There has recently been considerable interest in
the role of PLA, in relation to inflammation, particularly in rheumatoid arthritis (8,9). Vadas et a1 (28) have
shown that intraarticular injection of PLA, into rabbit
knee joints caused a proliferative synovitis, suggesting
a role for extracellular PLA, in the development of
inflammatory joint lesions. PLA, has been implicated
as a control element in the 'synthesis of inflammatory
mediators (29), such as eicosanoids, prostaglandins,
and platelet-activating factor, providing a pool of free
arachidonic acid for the cyclooxygenase pathway.
Lysophosphatidylcholine, a product of the PLA,catalyzed hydrolysis of phospholipids, has been
shown to be cytolytic and fusogenic (30). More recently, it has also been reported to be chemotactic for
monocytes (31). Thus, uncontrolled activity of the
resident PLA, in the cartilage matrix could have quite
detrimental effects on the resident cells. Liberation of
PLA, and generation of an environment that allows its
RECKLIES AND WHITE
1114
increased activity during inflammatory episodes might
contribute to the destruction of cartilage in rheumatoid
arthritis.
ACKNOWLEDGMENTS
We thank Dr. P. J. Roughley for providing the cartilage specimens used in this study and Elisa DiMiguel for
her skillful assistance with the protein sequencing. Mary
Speagle provided excellent technical assistance. Mark Lepic
and Jane Wishart prepared the figures.
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