partial characterization of matrix components interacting with cartilage proteoglycans

a

Archives of Biochemistry and BiophysicsVol. 390, No. 2, June 15, pp. 186–194, 2001doi:10.1006/abbi.2001.2337, available online at http://www.idealibrary.com on

Partial Characterization of Matrix Components Interactingwith Cartilage Proteoglycans

Sandra Hendrickx,1 Pat Thomas,1 Barry N. Preston, Peter G. Stanton,*nd Marie-Paule I. Van Damme2

Department of Biochemistry and Molecular Biology, Monash University, and *Prince Henry’s Institutefor Medical Research, Monash Medical Centre, Clayton 3168, Victoria, Australia

Received September 29, 2000, and in revised form February 23, 2001; published online May 24, 2001

The charge content of aqueous suspensions of milledcartilage samples was determined by a colloid titra-tion technique using a particle charge detector, andthe data were compared with estimates from chemicalanalyses. Results indicated a close correlation be-tween charge content determined by titration andthat estimated by chemical analyses for samples ofnasal septa only (a nonarticular cartilage). Such cor-relation did not hold for articular cartilages (metacar-palphalangeal joint and patella); extraction of thesetissues with 0.1 or 1.2 M NaCl markedly increased theavailability of the negative groups. Protein analysis,by SDS–PAGE, of the 1.2 M extracts indicated the pres-ence of basic proteins, some of collagenous origin,such as chondrocalcin and proline-arginine-rich pro-tein, and some of noncollagenous proteins such aspleiotrophin and histone-H2b. These data thus suggestelectrostatic interactions between these basic pro-teins and the negative groups of proteoglycans. Suchinteractions would have an important effect on theosmotic properties and in the organization of carti-lage. © 2001 Academic Press

Key Words: proteoglycan; charge; basic proteins; car-tilage; interaction.

Cartilage, which is made up of collagen, proteogly-cans (PGs),3 and various matrix proteins/glycoproteins,has an important role in the mechanics of normal joint

1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Fax: 61 3 9905

4699. E-mail: [email protected] Abbreviations used: PG, proteoglycan; MCP, metacarpalphalan-

geal; CMP, cartilage matrix protein; PVDF, polyvinylidene fluoride;
DMEM, Dulbecco’s modifed Eagle’s medium; PCD, particle chargedetector; PARP, proline–arginine-rich protein.
186

movement. The major PG of cartilage, aggrecan, inter-acts noncovalently with hyaluronan to form a complexthat is entrapped within the collagenous framework, afeature that is important in establishing the osmotic/mechanical properties of articular cartilage (1). Theosmotic properties of the tissue are largely determinedby the negative charges associated with aggrecan (2).To date it has been assumed, using a tracer cationmethod (3), that all negative groups of PGs in humanarticular cartilage were freely available and thereforeosmotically active since there was a close agreementbetween charge density measurements and values es-timated by chemical analyses (4). Such agreement wasalso observed when the effective charge content of na-sal septa (a nonarticular cartilage) was determinedusing a colloid titration technique. Correlation be-tween charge density and chemical analyses was ob-served when titration was performed on either suspen-sions of milled bovine nasal septa (5), or on matricesnewly synthesised by chondrocytes isolated from nasalsepta and grown in culture for several weeks (6). How-ever, such correlation did not apply when titratingmatrices synthesized by chondrocytes isolated from bo-vine articular cartilage (metacarpalphalangeal joints,MCP) and grown in culture for several weeks (7). In-deed, less than 10% of the total negative groupspresent could be titrated, even after digestion of thematrix with papain, indicating the possible involve-ment of these negative groups in electrostatic interac-tions with basic matrix components.

There is evidence to suggest that basic proteins in-teract with the negative groups of cartilage proteogly-cans. Van Lent et al. (8–10) and Fassbender et al. (11)have shown that cationic molecules up to 150 kDa areable to diffuse into the tissue and bind to matrix com-ponents. Furthermore, the low concentration of salt in
the microenvironment of the PGs (2, 3) would favor
0003-9861/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

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187CARTILAGE MATRIX PROTEINS INTERACTING WITH PROTEOGLYCANS

interactions between the negative groups of PGs andbasic matrix components.

Several basic matrix proteins have been isolatedfrom cartilage of various sources (12–17). Most studiesto date investigating cartilage matrix proteins haveused as extractants chaotropic agents, such as guani-dinium chloride (GuHCl), which completely disrupt thematrix. Some of the proteins extracted in this manner,i.e., chondronectin (18) and cartilage matrix protein(CMP), a slightly basic protein found in bovine nasalsepta but not in articular cartilage (19, 20) have beeninvestigated for possible interaction with PGs. Otherstudies have used low salt solutions (,1 M NaCl) toxtract matrix proteins (21, 22) or NaCl concentrations1 M to extract more strongly interacting matrix com-onents (23, 24).In this study, the possible binding of basic matrix

roteins to the negative groups of articular cartilageamples was investigated by extracting the tissuesith increasing salt concentration and establishing,sing a titration technique, if such a procedure in-reased the availability of the negative groups of PGs.ome of the proteins, which have been identified in theaCl extracts, were characterized and analyzed for

heir biosynthetic activity.

MATERIALS AND METHODS

Papain (from papaya latex type II, EC 3.4.22.2, P3125, SigmaChemical Co.). Pure CsCl was obtained from Novachem Pty. Ltd.(Australia). Poly-diallyl-dimethyl-ammonium chloride (Poly-Dad-mac) was from Mutek GmbH (Germany). Proteinase inhibitors wereas follows: 0.1 M 6-amino-n-hexanoic acid, 0.01 M ethylenedia-mineacetic acid (EDTA), 0.001 M benzamidine hydrochloride, 0.0001M phenylmethylsulfonyl fluoride, 0.02 M iodoacetic acid, 0.02% so-dium azide, and 8 mg/L soybean trypsin inhibitor.

Preparation of Milled Cartilage Samples

Bovine adult cartilages (nasal septa, MCP joints, and patellae)were obtained within 1 h of slaughter from the abattoir (G. and K.

’Connor, Pakenham, Victoria, Australia). Dried cartilage was fro-en in solid carbon dioxide and pulverized in liquid nitrogen for noonger than 1 min in a Spex freezer/mill (Spex Industries Inc.,

etuchen, MA), following the method of Bayliss and Ali (25, 26).illed bovine cartilages were stored at 220°C in sealed containers

ntil required.

Determination of the Charge Content of CartilagePreparations using a Particle Charge Detector

Charge content was estimated by measuring streaming potentialsusing a Mutek particle charge detector (PCD 02) and a PCD-Titrator(Mutek). Titration of the solution/suspension with a polycation (PolyDADMAC, 0.001N) was performed until the streaming potentialreached a zero value, whereby the suspension is then at its isoelectricpoint. Aliquots of each sample were titrated in triplicate.

Titration of Cartilage Samples in Water Suspensions
Cartilage samples (10 mg dry weight) were suspended in water for
24 h and the charge content estimated as described above.

Titration of Cartilage Samples after Extraction withNaCl (0–4 M)

Cartilage samples (10 mg dry weight) were extracted for 24 h withvarious “fixed” concentrations of NaCl (0.05, 0.1, 0.5, 1.2, and 4 M) inthe presence of proteinase inhibitors. Samples were centrifuged andthe pellets were washed with water (two times) to eliminate contam-ination with NaCl which interferes with the titration technique. Thecharge content of the pellet was determined as described above. Thesupernatant of each wash was added to the NaCl extractant andused to determine the amount of PGs and protein released into theNaCl/water extracts, after dialysis of the NaCl extracts againstdistilled water until the retentate had a salt concentration of lessthan 5 mM.

Extraction of Matrix Proteins at Various NaCl Concentrationsand Purification by SDS–PAGE

Extraction at Fixed Salt Concentrations

Milled cartilages (1 g) were extracted for 24 h at 4°C under con-stant mixing in 10 vol/g wet weight of distilled water, or NaCl ateight fixed concentrations (0.02, 0.05, 0.08, 0.1, 0.2, 0.5, 1.2, and 4M). Total protein content and PG content (as uronic acid) weremeasured using tissue extracted with 4 M GuHCl for 48 h at 4°C(27). All extractions were carried out in the presence of 0.02 Mphosphate buffer, pH 7.0, and proteinase inhibitors.

Sequential Extractions

Tissues were sequentially extracted six times with 0.015 M NaClfor 24 h at 4°C to remove weakly interacting matrix proteins andthen with 1.2 M NaCl.

Purification by SDS–PAGE

Samples were prepared by desalting extracts on a Sephadex G50column (1 3 15 cm) equilibrated in 0.05 M NH4 HCO3. Proteins were

urified under reducing conditions by SDS–PAGE (28) in 13% acryl-mide gels (1.5 3 150 3 150 mm) in a discontinuous buffer systemsing the Protean II apparatus (Bio-Rad Laboratories, Richmond,A). Densitometric scanning analysis of silver stain intensities waserformed using a Hewlett Packard flatbed Scanjet II scanner. Anal-sis of concentration and molecular weight of individual bands waserformed using MCID-M44 software (Imaging Research, Brockniversity, Canada).Gels were electroeluted onto Immobilon–PVDF membranes in a

rans-Blot cell (Bio-Rad Laboratories) at 100 V (0.3 milliamps) for8 h at 4°C (29). Protein bands, identified by silver stain, werexcised from the membranes (29) and N-terminal amino acid se-uences were determined by Edman degradation on an Appliediosystems Model 470A/120A gas phase protein sequencer. Aminocid sequences were compared to published sequences held at theustralian National Genomic Information Service (Sydney, Austra-

ia).

Chemical Analyses

The uronic acid content of PGs was determined using the modifiedcarbazole method of Bitter and Muir (30). Chemical amounts wereconverted to units of charge equivalence, assuming 1 mEq of chargefor every 194 mg of uronic acid measured.

Amino acid composition of samples was determined on hydroly-sates (24 h at 100°C in constant boiling HCl) using the reversed-
phase high performance liquid chromatography (HPLC) Picotagmethod (Waters, Division of Millipore, Milford, MA). Protein content

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188 HENDRICKX ET AL.

was determined using the Bio-Rad protein assay (Bio-Rad Labora-tories), using bovine serum albumin as standard.

Biosynthetic Studies

Radioactive Labeling of Cartilage

Cartilage ('1 g wet weight) was finely diced and placed in organculture at 37°C in 10 mL Dulbecco’s modified Eagle’s medium(DMEM, CSL, Melbourne, Australia) supplemented with Eagle’snonessential amino acids (31). After incubation for 24 h, the tissuewas incubated for 1 h in lysine-depleted medium and then furtherncubated for 6 h in lysine-depleted medium containing [L-14C]lysine

(Dupont, NEN Research Products, Boston, MA) at 200 mCi/g wetweight tissue. The medium was removed and collected for analysis ofradiactively labeled proteins.

The tissue was then suspended for 24 h at 37°C in DMEM con-taining proteinase inhibitors. After centrifugation at 1700g for 15min, the supernatant was removed for further analysis. The pelletwas then further extracted for 24 h at 4°C with 1.2 M NaCl contain-ing 0.02 M phosphate buffer and proteinase inhibitors. The super-natants obtained after extraction with medium and 1.2 M NaCl arereferred to as medium extracts and 1.2 M NaCl extracts, respec-tively. To determine the amount of radioactivity remaining in thetissue, pellets were extracted for 48 h at 4°C with 4 M GuHClcontaining 0.02 M phosphate buffer and proteinase inhibitors.

Analysis of Radiolabelled Proteinsa. Determination of percentage incorporation. Aliquots of me-

dium, medium extracts, and 1.2 M NaCl extracts were purified by gelchromatography on Sephadex G-50 in 4 M GuHCl/0.05 M sodiumacetate (pH 6.1) buffer. The excluded volume fractions were collectedand assayed for radioactivity.

b. SDS–PAGE and fluorography. Aliquots of purified medium,medium extracts, and 1.2 M NaCl extracts were treated overnightwith 5 vol of ethanol at 4°C (32) and the precipitates dissolved in 40ml of SDS–PAGE loading buffer. After electrophoresis, gels werebriefly rinsed in water and immersed in Amplify (Amersham, En-gland) or En3hance (Dupont, Boston, MA) for 20 min prior to being

ried under vacuum and then exposed to Kodak X-Omat AR film forp to 21 days at 270°C. Densitometric analysis was performed asescribed above.

RESULTS

Determination of the Charge Content of Articular andNonarticular Cartilage Samples in WaterSuspensions

The charge content of aqueous suspensions of milledcartilage samples was estimated by a titration tech-nique using a particle charge detector (PCD), whichdetects only “free” charged groups, and the data com-pared with those determined by chemical analyses.Results indicate that, for nasal septa, close correlationwas observed between charge content determined byPCD and chemical analyses, as previously reported (6,7). However, with milled suspensions of articular car-tilage, the PCD detected only 34% (MCP) and 64%(patella) of the expected charge (Table I). Two consec-utive extractions with water resulted in a residual 11%
(MCP) and 4% (patella), bringing the final charge con-
tent to 45% (MCP) and 68% (patella) of the total chargeexpected.

Determination of Charge Content and PG and ProteinContent of Articular and Nonarticular CartilageSamples after Extraction of the Tissues with NaCl

To determine if the results presented above could beattributed to the masking of the negative groups bycationic matrix components, cartilage samples wereextracted with various “fixed” concentrations of NaCland the charge content was estimated on the extractedtissues, after several washes with water to eliminateany contamination with NaCl. The amount of PGs andproteins released into the extractanct was determinedby chemical analyses.

i. PG and protein content. PGs and proteins werereleased to differing extents in the various NaCl solu-tions, for the three different tissues as indicated inTable II. Results are expressed as micrograms ofuronic acid or protein per milligram dry weight and aspercentage of the total PG or protein present in a 4 MGuHCl extract.

In nasal cartilage, PG content was highest in waterextracts (85%); the value decreased markedly usinglow salt concentrations (0.05–0.5 M) and increasedagain with 1.2 and 4 M NaCl (37 and 48%, respec-tively). For articular cartilage samples, the amount ofPGs extracted using 0.5 or 1.2 M NaCl was slightlyhigher than that released in lower salt concentrationsor in 4 M NaCl. The total amount of PGs extracted with4 M GuHCl (mg/mg tissue) was highest in nasal septa.

The percentage of proteins released in water extractsof nasal septa was higher than that observed in the saltextracts and much higher than the values observed forarticular cartilages. The latter did not markedly vary

TABLE I

Total Charge Content of Milled Articular and NonarticularCartilage Samples Determined by a Titration

Technique and by Chemical Analyses

Nasal septa(mEq/mg)a

MCP(mEq/mg)a

Patella(mEq/mg)a

Titrationb 0.98 6 0.10 0.17 6 0.02 0.40 6 0.01Chemical analysisc 1.00 6 0.07 0.50 6 0.04 0.59 6 0.05

a The charge content is expressed per mg dry weight.b Titration experiments were performed using a particle charge

detector equipped with an automatic titrator. Values are expressedas means 6 standard deviation (n 5 3).

c Chemical analyses were performed on papain digests by uronicacid analysis and chemical amounts were converted to units ofcharge equivalence assuming 1 mEq of charge for every 194 mg of

ronic acid. Values are expressed as means 6 standard deviationn 5 3) (S. Blackwell, pers. commun.).

with the concentration of the extractant. However, the

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total amount of proteins present in MCP and patella,as determined after extraction of the tissue with 4 MGuHCl, was nearly twice that determined for nasalsepta.

The ratio of protein to proteoglycan content in the 4M GuHCl of articular cartilage samples was approxi-mately three times higher than that of nasal septa, ascalculated from Tables IIA and IIB (protein/PG ratiosof 0.45, 1.68, and 1.40 were obtained for nasal septa,MCP and patella samples, respectively).

ii. Charge content. The charge content on the ex-tracted tissue determined by PCD titration was ex-pressed as a percentage of the “original charge content”(i.e., before extraction) estimated by chemical analysisof PG (as shown in Table I).

For both MCP and patella, there was a marked in-crease in charge content after extraction with 0.1 MNaCl with 84 and 82%, respectively, of the total chargebeing detected and 1.2 M NaCl with 72 and 93%, re-spectively, being detected (Fig. 1a). On the other hand,the charge content of nasal septa decreased with in-creasing concentration of the extractant (52% with 4 MNaCl). The low value observed when titrating in waterwas due to the release of PGs into the extractant (85%;

TAB

Proteoglycan and Protein Content in ExtractsUsing “Fixed” NaCl C

NaCl (M)

Nasal

mg/mg %

A: Proteogl

0 72.3 6 5.2 85.05 13.9 6 1.0 16.1 12.4 6 0.9 14.5 5.9 6 0.4 7.2 31.9 6 2.1 37.0 40.9 6 3.2 48M GuHClb 85.1 6 6.5 100

B: Prote

0 37.2 6 2.2 96.05 20.5 6 1.2 53.1 22.1 6 1.3 57.5 26.8 6 1.4 69.2 27.5 6 1.7 71

26.5 6 1.8 68M GuHClb 38.7 6 2.8 100

a Cartilage samples were extracted with fixed concentrations of Naroteins determined in the extracts from uronic acid analysis (30) an

uronic acid/mg dry weight for PGs and mg protein/mg dry weightextracted with 4 M GuHCl (see b).

b Tissue was solubilized for 24 h in 4 M GuHCl containing proteinasas 100%.

Table II).

However, if the charge content determined by titra-tion was expressed as a percentage of the chemicalamount of PG remaining in the pellet after salt extrac-tion (Fig. 1b), over 90% of the remaining negativegroups in nasal septa were freely detectable by titra-tion at most salt concentrations. For articular samples,on the other hand, unmasking of the negative groupscould be achieved only by extraction with 0.1 and 1.2 Msalt, where over 90% of the negative groups becamedetectable by titration, indicating that at other saltconcentrations some masking of the negative groupsstill occurred.

Analysis by SDS–PAGE and Partial Characterizationof Proteins in NaCl Extracts

To determine the strength of interaction with carti-lage PGs, matrix proteins were extracted from articu-lar cartilage using nine fixed NaCl concentrations (0–4M). SDS–PAGE (13%) revealed the presence of severalbands in all extracts of MCP joints, in the molecularweight range of 13–120 kDa (Fig. 2); a similar patternwas observed in the patella extracts (results not pre-sented). In the 1.2 M extracts, several intense staining

II

Articular and Nonarticular Cartilage Samplescentrations (0–4 M)

MCP Patella

mg/mg % mg/mg %

n contenta

.4 6 0.2 9 3.0 6 0.2 6

.0 6 0.2 8 2.2 6 0.2 4

.0 6 0.3 10 7.3 6 0.5 14

.3 6 0.6 19 1.8 6 0.2 3

.4 6 0.6 19 6.2 6 0.5 12

.7 6 0.4 12 3.9 6 0.3 8

.2 6 3.1 100 50.0 6 0.4 100

ontenta

.7 6 0.9 22 17.1 6 1.3 24

.4 6 0.9 20 12.2 6 1.0 17

.7 6 1.1 27 10.6 6 0.7 15

.7 6 1.0 27 16.4 6 1.1 23

.9 6 7.3 23 6.0 6 0.5 9

.9 6 0.9 20 9.7 6 0.7 14

.0 6 4.1 100 70.0 6 5.2 100

0–4 M) containing proteinase inhibitors and the content of PGs andhe Bio-Rad protein assay, respectively. Results are expressed as mgD, n 5 3) and as percentage of the total amount of PGs/proteins

hibitors. The total amount extracted for PGs and proteins were used

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visible in extracts from lower NaCl concentrations cor-responding to approximate molecular weights of 37, 35,33, 19, and 18 kDa (Fig. 2). These bands representaround 50% of the total density of silver staining pro-tein bands within the lane.

On the basis of these results, cartilage samples weresuccessively extracted six times with 0.15 M NaCl toremove “weakly interacting” proteins and then ex-tracted with 1.2 M NaCl. Samples from articular aswell as nasal septa were used in this case for compar-ison. These sequential extracts were analyzed by SDS–PAGE (Fig. 3) and the bands present specifically and/ormost abundantly in the 1.2 M extracts (i.e., 37–25, 19,and 18 kDa) were analyzed by N-terminal amino acidsequencing. In articular cartilage these bands wereidentified (Table III) as chondrocalcin (minor peptideat 37 kDa and major peptide at 35 kDa) (22, 33, 34),proline–arginine-rich protein (PARP, 29 kDa) (15, 35),pleiotrophin (19 kDa) (16), and histone H2B (18 kDa)(16) (Table III). A faint band at 25 kDa was identifiedas ubiquitin. Protein corresponding to 33 kDa could notbe identified by N-terminal amino acid sequencing.The major bands (37, 35, and 33 kDa, 19 and 18 kDa)were also present in nonarticular cartilage; PARP (29

FIG. 1. Determination of the “effective anionic” charge content ofroteoglycans after extraction of cartilage with “fixed” NaCl concen-rations. Cartilage samples (10 mg dry weight) were suspended for4 h in 3 ml NaCl at various concentrations (0–4 M). Samples wereentrifuged, the pellets washed (two times) with water, and theharge content of the pellet estimated by a titration technique usingparticle charge detector. Values, expressed as means 6 standard

deviation (n 5 3) were calculated as a percentage of the “original”charge concentration (i.e., before extraction; see Table I), determinedby chemical analysis of PG (a) and as a percentage of the chemicalamount of PG “remaining in the pellet” after salt extraction (b).

kDa), however, appeared to be present only in trace

amount. Although the same basic proteins were de-tected for both articular and nonarticular samples, theratio of protein to PG is three times higher for articularcartilages than for nasal septa, as stated earlier, indi-cating that masking of the charge in articular samplescould be a consequence of the amount of basic proteinpresent.

Biosynthesis of Cartilage Matrix Proteins

In order to determine which matrix proteins found incartilage are synthesized by chondrocytes, bovine MCPjoints were incubated with [14C]lysine. After 6 h label-ng, the tissue was extracted several times with me-ium followed by extraction with 1.2 M NaCl and iden-ification of the proteins by SDS–PAGE. The remain-ng proteins were then extracted with 4 M GuHCl.

As indicated in Table IV, 41.7% of labeled macromol-cules were released into the medium. Labeled macro-olecules derived from the extraction of the tissueith medium and 1.2 M NaCl accounted for 10.7 and.8%, respectively, with the remaining 41.8% beingxtracted with 4 M GuHCl (Table IV).Analysis of these extracts on 13% SDS–PAGE indi-

ates the presence of several radioactively labeled pro-eins in the culture medium and after extraction of theissue with medium and with 1.2 M NaCl (Fig. 4). Inarticular, labeled proteins were observed in the 1.2 MaCl extracts with molecular sizes of 35 and 29 kDa,

orresponding to chondrocalcin and PARP, respec-

FIG. 2. SDS–PAGE of proteins extracted from articular cartilage(metacarpalphalangeal joint, MCP) using various “fixed” NaCl con-centrations (0, 0.02, 0.05, 0.08, 0.1, 0.2, 0.5, 1.2, and 4 M) in 0.02 Mphosphate buffer (pH 7) containing proteinase inhibitors. Electro-phoresis was performed on 13% acrylamide gels under reducingconditions. Samples corresponding to material isolated from 10 mgwet weight tissue were loaded per well. Proteins bands were visual-ized by silver staining and compared with standard molecular weight
proteins. Arrows indicate approximated molecular weight of proteinbands visualized in 1.2 M NaCl extracts.

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tively; 56 and 46 kDa, which could possibly correspondto link protein LP2 (36) and proline–arginine-richleucine-rich repeat protein (PRELP) (37), respectively.Bands corresponding to histone H2b and pleiotrophinhad a lower specific radioactivity in the 1.2 M NaClextracts (Fig. 4). The band corresponding to 44 kDacould not be identified.

Interestingly, the 35-kDa protein, corresponding to themajor peptide of chondrocalcin (35 kDa) was predomi-nantly extracted with 1.2 M NaCl, while the 37-kDaprotein, possibly the minor peptide of chondrocalcin, wasreleased in the medium or extracted with medium.

DISCUSSION

In articular cartilage, the ability of the tissue towithstand high shear and compressive loads depends

FIG. 3. SDS–PAGE of proteins extracted from articular (metacarwith 0.015 M NaCl (bands A–F) followed by extraction with 1.2 Mindicate approximated molecular weight of protein bands visualizeremoved with 0.15 M NaCl.

TAB

Characterization of the Major Proteins from

MW (kDa) Identity Homology to seq

37 Chondrocalcin minor peptide 82% (over 13 amino35 Chondrocalcin major peptide 100% (over 14 amin33 Blocked29 Proline–Arginine rich protein 86% (over 14 amino25 Ubiquitin 93% (over 15 amino19 Pleiotrophin 93% (over 15 amino18 Histone H2B 92% (over 13 amino

a Proteins were prepared from 1.2 M NaCl extracts of MCP jointxtraction with 0.15 M NaCl. Proteins were separated on 13% SDS–ere excised from the membrane and subject to N-terminal sequencb Staining density determined from relative optical density versus

lane, using MCID-M4 software (Imaging Research, Brock University, Cc No amino acid identified.

on the high water content of the cartilage matrix,which is thought to be regulated by the effective con-centration of the negatively charged groups of theGAGs (38, 39).

The results presented in this study indicate that inarticular cartilage (MCP and patella) a fraction ofthese negative groups appear to be bound to basicmatrix components as their negative charge content,determined by PCD on aqueous suspensions of milledcartilage, was lower than those estimated by chemicalanalyses. Only 34% (MCP) and 68% (patella) of thetotal charge content could be detected. In contrast, innonarticular cartilage (nasal septa) there was an ex-cellent correlation between charge determined by titra-tion and that estimated by chemical analyses, asshown previously (6).

phalangeal joints) and nasal cartilages using sequential extractionCl (band G). Electrophoresis was performed as for Fig. 2. Arrows1.2 M NaCl extracts after weakly interacting proteins have been

III

.2 M NaCl Extracts of Articular Cartilagea

ce Sequence % of Total densityb

ids) D-E-A-A-G-N-L-R-Q-E-D-A-R- 6.0cids) D-A-E-V-D-A-T-L-K-S-L-N-N-Q- 9.8

2.8ids) G-T-A-P-V-D-V-L-R-A-Xc-F-P-A- 6.6ids) M-Q-I-F-V-K-T-L-T-G-K-T-D-T-L- 1.8ids) G-K-K-E-K-P-E-K-K-V-V-K-S-D-D- 18.1ids) G?-E-P-A-K-S-A-P-A-P-T-K-G- 5.1

fter removal of weakly interacting protein species with successiveGE and then electroblotted to PVDF membranes. Individual bandsnalysis for protein identification.els (arbitrary units) and expressed as percentage of total staining in

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To investigate if the negative groups in articularcartilage are involved in electrostatic interactions withbasic matrix components, samples were extracted withincreasing NaCl concentrations. NaCl was chosen asan extractant, as it enables the release of individualcomponents/proteins from the matrix, which may in-teract electrostatically with the negative charge of thePGs while retaining the integrity of the matrix. Inmost studies to date, however, chaotrophic agents suchas GuHCl, which disrupt the cartilage matrix, havebeen used to analyze matrix macromolecules (27, 40).

The results presented in this study clearly indicatethat the charge content of MCP and patella, deter-mined by PCD, increased after extracting the tissuewith NaCl (Fig. 1a). The charge was highest at 0.1 M(84 and 82% of values estimated by chemical analyses,

TAB

Percentage of Radioactively-Labeled Macromolecules Relea

Treatment

Macromolecules released in mediumb

acromolecules successively extracted from tissue with:Medium1.2 M NaCl4 M GuHCl

otal

a The proportion of radioactively labeled macromolecules was deteras assayed for radioactivity.b Cartilage samples were incubated for 6 h with [14C]lysine in Du

FIG. 4. Fluorograph of SDS–PAGE of proteins extracted from ar-ticular cartilage. Tissue was labelled with [14C]lysine for 24 h beforeextraction with medium or 1.2 M NaCl. Extracts were prepared forSDS–PAGE by precipitation of material in 5 vol of ethanol. Lane A,proteins released into the culture medium; lane B, proteins extracted
from tissue with medium containing proteinase inhibitors; lane C,proteins extracted from tissue with 1.2 M NaCl.
for MCP and patella, respectively) and 1.2 M NaCl (72and 93%, respectively). Further increasing the salt con-centration to 4 M decreased the values of the effectivecharge (61 and 75%, respectively). This decrease wasnot due to a loss of PGs into the 4 M NaCl extract as forarticular cartilage (Fig. 1b), there was only a slightdecrease in the chemical amount of PGs released whenincreasing the NaCl concentration from 0.5/1.2 to 4 M(Table IIA).

When taking into account the values of the amountof PGs released into the NaCl extracts, it can be con-cluded from Fig. 2 and Table IIA that, after extractionof the tissues with 1.2 M NaCl, all negative groups ofarticular cartilage samples are “free” (Fig. 1b). Theseresults thus indicate that these groups are involved inelectrostatic interactions with basic matrix compo-nents in the tissue.

With nasal septa, there was a constant decrease inthe charge content with increasing concentration of theextractant, with a minimum value (52%) at 4 M NaCl.Similarly, the chemical content of PGs extracted with 4M NaCl (48.1%) was lower than that obtained whenthe tissue was extracted with water (85%). This differ-ence could be attributed to conformational changes inthe PG as proposed by Eyring and Yang (41), whoobserved that the protein–polysaccharide complex pu-rified from bovine nasal septa contracted in the pres-ence of salts. In this study, only 7–16% PGs are ex-tracted from nasal septa with lower salt concentrations(0.05–0.5 M). These data correlate well with previousstudies by Robinson and Hopwood (42), who demon-strated that only 15% of the total hexuronic acid ma-terial could be released from bovine nasal septa usingNaCl (0.15–5 M). Other electrolytes, such as CaCl2 (2M) and LiBr (4 M) extracted 80–90% of the total hexu-ronate after 30 h extraction.

The results presented in this study thus indicatethat changes in the physical properties of cartilagesamples occur with different salt concentrations andthat these changes differ between articular and nonar-

IV

d into the Medium or Extracted from Articular Cartilagea

m/g wet weight (3103) % of [14C]lysine-labeled proteins

5793 41.7

1489 10.7803 5.8

5807 41.813891 100

ed by separation on Sephadex G50 and the excluded volume fraction

co’s modified Eagle’s medium.

LE

se

dp

min

ticular cartilages. It is becoming evident that the mac-

3csbasioct

rutlo


romolecular composition of articular and nonarticularcartilages differs. The PG content in MCP and patellais approximately half that of nasal septa while theprotein content is nearly double. Similarly, the colla-gen content (mg/mg dry weight) of articular cartilage istwice that of nasal septa (S. Blackwell, pers. commun.).Furthermore, the collagen meshwork in the latter hasa much more open arrangement of its collagen fibrils,as indicated by ultrastructural analyses using polariz-ing light microscopy (S. Blackwell, pers. commun.).

It could therefore be argued that the tighter mesh-work organization of the collagen fibrils in articularcartilage could be responsible for the lower charge con-tent observed by PCD as it could preclude the accessi-bility of the polycation to the negative groups of thePGs. However, extensive studies by van Lent et al.(8–10) have shown that cationic molecules up to 150kDa are able to diffuse into articular cartilage and thatproteins with the highest isoelectric point were re-tained to the greatest level. Consequently, the titrationresults obtained for milled samples of MCP and patellaare not likely to be explained by any steric exclusion ofthe probe by the large-molecular-weight PGs or thecollagenous network.

SDS–PAGE analysis has indicated that there was arelease of proteins in 1.2 M salt extracts that were not(or slightly) visible in the lower NaCl extracts. The fivemain bands apparent only in the 1.2 M NaCl extractswere of approximate molecular weight between 37 and18 kDa. They were identified as cationic proteins, someof collagenous origin, such as chondrocalcin, the C-terminal propeptide of type II collagen (22, 34), andPARP, the NH2-terminal domain of collagen 1(XI) (15,5). Most proteins were identified in both articularartilage and nasal septa; in the latter PARP waseemingly present at lower concentration. However, asoth the protein content and the collagen content ofrticular cartilage are approximately twice that of na-al septa, while the PG content is approximately half,t could be postulated that the extent to which bindingf these proteins to PGs affects the negative “free”harge content of PGs (and therefore the masking ofhese charges) will be different for both cartilages.

Several studies have previously shown a similarange of extractable matrix proteins varying in molec-lar weight between 120 and 18 kDa (43). These pro-eins were extracted successively from human articu-ar cartilage using phosphate-buffered saline and cha-trophic agents (0.5 and 4 M GuHCl). PARP,

pleiotrophin, and histone H2b have been previouslyidentified as components of extracts of fetal epiphysealcartilage and nasal cartilage using 0.5 or 4 M GuHCl(16). Zhidkova et al. (35) have also isolated PARP fromhuman chondrocytes.

Some of the proteins found in the 1.2 M NaCl ex-
tracts, such as chondrocalcin, were also present in the
0.15 M NaCl extracts. These proteins may representthe existence of two distinct pools with different inter-active properties. It has been reported that PGs canexist in different stages of interaction and require,under culture conditions, a maturation process of 12 hbefore maximal binding to hyaluronan is obtained, sug-gesting a process of structural change, possibly disul-fide or noncovalent bond formation (12). Chondrocalcinhas been shown to be a component of 0.15 M NaClextracts of articular cartilage (22), but not as a compo-nent of two pools of proteins with differing salt extract-abilities.

Biosynthetic studies have confirmed that severalproteins, synthesized by chondrocytes, were releasedin the 1.2 M NaCl extracts, particularly chondrocalcinand PARP. The interactive properties of these basicproteins with cartilage proteoglycans need to be fur-ther analyzed.

CONCLUDING REMARKS

This study has demonstrated that basic proteins,some of collagenous origin, are involved in electrostaticinteractions with the negative groups of PGs as theyrequire high salt concentration (1.2 M NaCl) to bereleased from the tissue. Such interactions would havean important effect on the osmotic properties and inthe organization of cartilage, in particular when ma-trix degeneration occurs, such as in arthritic cartilage.

ACKNOWLEDGMENTS

This research was supported by an Australian Research Grant.The authors thank Prof. C. Handley and Dr. Robinson for usefuldiscussions and Mrs. D. Tweedale for Amino Acid Sequence analy-ses. Two of the authors were recipients of a Monash Graduate Schol-arship.

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