In situ interaction of cartilage proteoglycans with matrix proteins

Biochimica etBiophvsicaActa 840 (1985) 170-179

170

Elsevier BBA22051

In situ interaction of cartilage p r o t e o g l y c a n s with matrix proteins Alexis J. A l e t r a s a n d C.P. T s i g a n o s * Laboratory of Biochemistry, Department of Chemist~', University of Patras, Patras (Greece) (Received December 18th, 1984)

Key words: Proteoglycan; Matrix protein; Protein-carbohydrate interaction; (Cartilage)

The interaction of proteoglycans with other matrix proteins via thiol-disulphide interchange was explored. Chick sternal cartilage was extracted with 4 M guanidine hydrochloride in the presence and absence of N-ethylmaleimide and the proteoglycans from the centrifugation A2 fractions were isolated. Those from extracts without N-ethylmaleimide were linked with reducible bonds with 10-15 proteins-glycoproteins including the link proteins, the 148 kDa and 36 kDa proteins. The same was observed with extracts of pig laryngeal and sheep nasal cartilage. The linked proteoglycans from sheep amounted to 2-3% of the extractable uronic acid and belonged to two populations. The major fraction was included by Sepharose 6B (M r 110 000) had twice as long chondroitin sulphate chains, higher 4-sulphated residues and a high content of aspartie acid and leucine-rich protein. The larger proteoglycans had a size and composition similar to those of aggregating proteoglycans.

Introduction Extraction of cartilage with 4 M guanidine hydrochloride followed by centrifugation in a CsC1 linear density gradient has been the most commonly used method for isolating proteoglycans [1]. Under such conditions over 90% of the uronic acid in the gradient is recovered in the bottom highdensity fraction (A1), whereas the rest together with the less buoyant constituents of the tissue are found in the top fraction (A2). In order to avoid proteolytic degradation of the proteoglycans, it has been the practice to include in the extraction mixture the inhibitors, Na2EDTA, 6-aminohexanoic acid and benzamidine hydrochloride [2],

* To whom correspondence should be addressed. Abbreviations: A-di-4S and A-di-6S, 2-acetamido-2-deoxy-3-O(/~-~-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose and 2-acetamido-2-deoxy-3-O-(/~-D-gluco-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose, respectively.

occasionally also iodoacetic and phenylmethanesulphonyl fluoride [3]. Whereas the proteoglycans isolated in the high -density fraction contain no other proteins except the two link proteins, those from the less dense fraction have been shown to be associated with a variety of proteins which may be separated after treatment with 2-mercaptoethanol. Thus Swarm et al. [4] isolated from bovine articular cartilage proteoglycans of relatively low density containing proteins separable only after reduction and alkylation. Similarly, Stanescu and Sweet [5] isolated small proteoglycans from baboon (Papio papio) articular cartilage which contained also reducible proteins. Futhermore, Klein and Singh [6] have shown that proteoglycans from calf rib cartilage extracted with 4 M guanidine hydrochloride and fractionated by gel chromatography on Sepharose 2B under associative or dissociative conditions contained several glycoproteins which were liberated from the aggregates only after treatment with 2-mercaptoethanol. Although the proteo-

0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

171

glycans linked with such proteins may be distinct from the rest with regard to their physical and chemical characteristics [4,5], their association with other proteins may lead to dangerous speculations about their origin and biological function [4-6]. It has been mentioned by Heineg~d and coworkers [7] that there may be some interaction through disulphide exchange between proteoglycans and other proteins since, as they reported in Ref. 7, they occasionally observed protein bands in the bottom fractions of centrifugations at low starting density. This could be avoided by N-ethylmaleimide, and therefore these authors have been including this reagent in the extraction mixture [7,8] as reported by Kimata et al. [9]. In an attempt to isolate proteoglycans directly from sheep nasal cartilage extracts by precipitation with cetylpyridinium chloride we persistently observed several reducible proteins in the proteoglycan preparation presumably due to thiol interaction. To what extent this disulphide exchange occurs and between what components is not known. The object of this study was to explore the extent to which disulphide exchange occurs during extraction of cartilage and to find out between what components this reaction takes place. The evidence we provide indicates that a specific population of proteoglycans reacts mainly with various glycoproteins including the link proteins, and the matrix proteins 148 kDa and 36 kDa [8,10]. A preliminary report has appeared elsewhere [11]. Materials and Methods

Chemicals Guanidine hydrochloride (Grade I), benzamidine hydrochloride, 6-aminohexanoic acid, N-ethylmaleimide, dithiothreitol, DEAE-cellulose (fibrous form, capacity 0.9 meq/g), chondroitinase ABC and AC and Schiff's reagent were purchased from Sigma, St. Louis, MO, U.S.A. Solutions of guanidine hydrochloride were passed through a column of activated charcoal (BDH) before buffering. Iodoacetamide specially purified for biochemical work and CsC1 AnalaR were from BDH Chemicals, Poole, Dorset, U.K. Sephadex G-200, Sepharose CL-6B and CL-2B from Pharmacia, Uppsala, Sweden; urea of analytical grade (Serva) was purified by passing 8 M solutions through a

column of mixed bed resin (Zerolit DM-F, BDH) at 4°C and used within 4 days. All other chemicals used throughout this study were of highest available purity.

Analytical methods Uronic acid and protein was determined by the carbazole [12] and the Folin method [13], respectively; glucuronolactone and bovine serum albumin being used as standards. Total hexose was determined by the anthrone method [14] using galactose as standard and sialic acids by the method of Jourdian et al. [15], N-acetyl neuraminic acid being the standard. Amino acid, glucosamine and galactosamine analyses were performed on a Beckman model 120C amino acid analyzer after hydrolysing the samples in 6 M HCI at ll0°C for 20 h [16] or in 8 M HCI at 95°C for 3 h [17] under nitrogen, respectively. In order to study the sulphation pattern on the chondroitin sulphate chains, samples of proteoglycans were digested with chondroitinase ABC and AC [18] and the A-disaccharides were separated by HPLC [19].

Extraction of proteoglycans Sheep nasal, pig laryngeal and chick sternal cartilage was used for this study. Nasal and laryngeal cartilage from animals 10-12 months old was obtained from the local abattoirs immediately after slaughter, cleaned from surrounding tissue and perichondrium and cut into slices of about 1 mm thick. Sterna from 18-20-days-old white Leghorn chicks were removed immediately after death, cleaned and cut as above. The cartilage was extracted twice for 24 h at 4°C with 10 vol. each time of 4 M guanidine hydrochloride/0.05 M sodium acetate buffer pH 5.8 containing the proteinase inhibitors 5 mM benzamidine hydrocloride, 100 mM 6-aminohexanoic acid and 10 mM Na2EDTA [2]. In another set of experiments with chick cartilage the extraction mixture contained also 10 or 50 mM N-ethylmaleimide and the extraction was performed in the dark.

Density gradient centrifugations The extracts were dialysed against 7 vol. of 0.05 M acetate buffer (pH 5.8) containing the inhibitors

172 a n d / o r 10 mM N-ethylmaleimide overnight at 4°C. The final concentration of uronic acid was adjusted to 1.2 m g / m l by ultrafiltration and the density of the solutions to 1.6 g / m l by the addition of solid CsC1. The solutions were then centrifuged in a Beckman L2-65B ultracentrifuge in a 8 x 12 ml T-65 fixed angle rotor at 95 000 x g~v for 50 h at 10°C. At the end of the run the tubes were frozen in an ethanol/solid CO 2 bath and cut to a high-density fraction A1 (3.5 ml) and to a low-density fraction A2 (8.5 ml). The excluded fractions from dissociative chromatography on Sepharose CL-6B of A2 from sheep extracts were concentrated by ultrafiltration to uronic acid concentration of 1.2 mg/ml, their density adjusted to 1.5 g / m l and then centrifuged as above. The tubes were cut into three fractions: bottom, E-1 (3 ml), middle, E-2 (6 ml) and top E-3 (3 ml).

CL-6B and on Sephadex G-200 was performed at 4°C with 4 M guanidine hydrochloride/0.05 M sodium acetate (pH 5.8), or 0.5 M sodium acetate (pH 7.0) or 0.2 M NaC1 being the eluants. When fractions A2 from extracts in the presence or absence of N-ethylmaleimide were chromatographed the elution buffer contained also 100 mM 6aminohexanoic acid and 10 mM NazEDTA. The fractions were analysed for uronic acid and protein (absorbance at 280 nm). Various fractions were pooled as shown in the respective figures and studied further.

Reduction and alkylation Samples, about 0.5 mg protein/ml in 4 M guanidine hydrochloride/0.05 M Tris-HC1 (pH 9.0) were reduced with 10 mM dithiothreitol at 37°C for 3 h under nitrogen and alkylated with 40 mM iodoacetamide for 16 h at 20°C in the dark [211.

Isolation of proteoglycans linked with proteins Pooled fractions E-2 and E-3 from sheep nasal extracts were reduced, alkylated and then chromatographed on Sepharose CL-2B eluted with 4 M guanidine hydrochloride; the uronic acid profile being similar to that in Fig. 5B. The fractions of each mode were pooled separately, dialysed against 8 M urea/0.05 M Tris-HC1 (pH 6.8) and then chromatographed on a DEAE-cellulose column eluted as described below. The proteoglycan fractions (3 M NaC1) were retained and used for further analysis.

Ion-exchange chromatography Ion-exchange chromatography of proteoglycans before and after reduction and alkylation on DEAE-cellulose (C1-) equilibrated with 8 M urea/0.05 M Tris-HC1 (pH 6.8) was performed according to Antonopoulos et al. [20]. Samples of 80-100 ~tg uronic acid/ml cellulose were applied and the columns were eluted with three bed volumes of equilibration buffer, 0.3 M and 3 M NaCI both containing 8 M urea/0.05 M Tris-HC1 (pH 6.8). The fractions were dialysed and analysed for uronic acid, protein and subjected to electrophoresis.

Gel chromatography Gel chromatography on Sepharose CL-2B or

Alkaline borohydride treatment Free chondroitin sulphate chains were prepared from proteoglycans by treating the samples (5 m g / m l ) with 50 mM N a O H / 1 M NaBH 4 at 45°C for 48 h [22]. After neutralization with acetic acid the mixture was chromatographed on Sepharose CL-6B and Sephadex G-200 eluted with 0.5 M sodium acetate (pH 7.0) and 0.2 M NaC1 [23], respectively.

Gel electrophoresis Polyacrylamide gel electrophoresis in 0.1% SDS was performed according to Laemmli [24]. The running and the stacking gels were 8 and 4%, respectively. Samples containing up to 100 ~tg protein in 100 /xl of 2% SDS with or without 5% ( v / v ) 2-mercaptoethanol were incubated at 37°C for 3 h and then electrophoresed at 2 m A / g e l for 6 h. The gels were fixed in 12% (w/v) trichloroacetic acid overnight at 4°C, washed three times with 7% (v/v) acetic acid, stained with Coomassie brilliant blue R-250 (BDH) (0.25% w / v ) in 43% methanol/7% acetic acid (v/v) and then destained in 7% (v/v) acetic acid with gentle shaking. Reference proteins (Sigma) human IgG, bovine serum albumin, egg albumin and egg lysozyme were electrophoresed in an identical manner. When the zones were stained for glycoproteins

173

0.4

the gels were fixed and washed as before and then stained with Schiff's reagent for 2 h at 4°C in the dark as described by Glossman and Neville [25].

Evidence that proteoglycans interact with proteins during extraction The in situ formation of complexes of proteoglycans with other components of cartilage through disulphide exchange was investigated by comparing less buoyant centrifugation fractions from extracts in the presence and absence of N-ethylmaleimide. Chick sternal cartilage was used for this set of experiments. When fraction A2 from the extract in the absence of N-ethylmaleimide was chromatographed on Sepharose CL-6B in 4 M guanidine hydrochloride containing the proteinase inhibitors, about 60% of the uronic acid was excluded by the gel (Fig. 1A). This amount was observed to increase when the sample had stood in the cold for a few days before chromatography, indicating some kind of interaction. Furthermore, a broad included protein peak containing little or no uronic acid was obtained suggesting the presence of appreciable amounts of non-proteoglycan proteins of relatively low molecular weight. The material eluted in the total volume of the column is residual benzamidine in the sample. In contrast, the elution profile under the same conditions of the same fraction from extract in the presence of N-ethylmaleimide was quite different particularly with respect to the protein distribution (Fig. 1B). The excluded fractions lacked considerable amounts of protien which appeared in the retarded fractions.

)

2

I~

3

it

4

t

A 0.3

Results and Discussion Extraction with 4 M guanidine hydrochloride of sheep nasal and pig laryngeal or chick sternal cartilage with or without N-ethylmaleimide yielded about 85% of the uronic acid in the tissue. Upon centrifugation of the sheep or pig cartilage extracts under associative conditions and in a starting density 1.6 g/ml, fractions A1 and A2 contained 94 and 6% of the total uronic acid, respectively, and 45.5 and 54.5% of the protein. Those from the chick extracts (both with and without N-ethylmaleimide) contained 96 and 4% of uronic acid, respectively. The analyses were performed on samples after exhaustive dialysis.

t

~ /

~=0.1°'2 J:

o

0.3

-

< 0.2 ~-

0"t1 30

20

t Vo

Fraction

40 Number

50

t Vt

Fig. 1. Gel chromatographyon Sepharose CL-6B. Samples of A2 density gradient fractions of chick sternal cartilageextracts in the absence (A) and in the presence(B) of 10 mM N-ethylmaleimide were chromatographed on columns (0.8× 150 cm) eluted with 4 M guanidine hydrochloride/0.05 M sodium acetate (pH 5.8) containing proteinase inhibitors and fractions of 1.4 ml were collected. Fractions were pooled as indicated, C) O, A53o (uronic acid); • •, A2so. V0 and Vt, void and total volume, respectively.

The fractions along the elution profiles were pooled as indicated (Fig. 1A and B) dialysed and equal amounts in protein were subjected to SDSpolyacrylamide gel electrophoresis before and after reduction with 2-mercaptoethanol. A variety of protein bands (10-15) of apparent molecular weight 14000-150000 appeared to enter the gel only after reduction of the first largest (excluded) fraction of preparation A2 (Fig. 2A, gel 1). None of these bands belonged to proteoglycans which remained on top of the gels as ascertained by Toluidine blue staining (not shown). The intense band in the reduced samples 1, 2 and 3, between the 45 and 66 kDa marker appears to correspond to the subunit of the 148 kDa matrix protein [8];

174 1 -

2 +

-

3 +

-

1

4 +

_

+

-

2 +

-

3 +

-

4 +

-

+

A

148 kDa

S u b ~ L i n kI:Z:= 36

k D a ~

Fig. 2. SDS-8% polyacrylamide gel electrophoresis of pooled fractions of Fig. 1; numbering corresponds to that of Fig. 1. (A) and (B) pools of Fig, 1A and Fig. 1B, respectively: ( - ) before and ( + ) after treatment with 2-mercaptoethanol.

the 36 kDa protein [10] is also recognized. With column fractions of smaller hydrodynamic size and poorer in uronic acid some bands entered the gels before reduction some of which disappeared upon this treatment (Fig. 2A, gels 2 and 3). The main band above the region of molecular weight 150000 (gels 2 ( - ) and 3 ( - )) seems to correspond to the 148 kDa matrix protein from bovine tracheal extracts [8], since upon reduction it disappeared giving rise most likely to a subunit of molecular weight about 60000. Free link proteins appeared in pool 3(gel 3 ( - ) ) . They became slower upon reduction and then had the same mobilities as two bands derived after reduction of the pools preceeding pool 3. An entirely different distribution of the various proteins was revealed when the corresponding pooled fractions of A2 from the extract in the presence of N-ethylmaleimide were electrophoresed (Fig. 2B). The excluded fractions contained only traces of about five bands in the region of molecular weight 45 000-66 000, visible only when large amounts were used. The included fractions were also different from those of A2 from the extract in the absence of N-ethylmaleimide in that they contained several free protein bands all but one being unaffected after treatment with 2-mercaptoethanol. This particular band near the top of gels 2 and 3, dissappearing upon reduction, seems to correspond to the 148 kDa trimer. It was nearly absent from the corresponding pools of

A2 from the extract in the absence of N-ethylmaleimide. It appears that in the absence of Nethylmaleimide, the matrix protein is involved in larger aggregates and breaks into its subunit upon reduction (Fig. 2A, gel 1). The liberation by reduction of various proteins from the fractions with most of the uronic acid and their absence in the fractions from extract in the presence of N-ethylmaleimide suggested that these proteins were either linked to proteoglycans or to each other or both via reducible bonds and that this interaction occurs during extraction or standing. In order to clarify these possibilities samples of the Sepharose CL-6B excluded fractions of A2 from extract in the absence of N-ethylmaleimide were chromatographed on DEAE-cellulose and the various fractions were compared. About 30% of the protein but no uronic acid was eluted with 8 M urea and 0.3 M NaC1/8 M urea, whereas the proteoglycans were recovered in the 3 M NaC1/8 M urea fraction. The protein bands were detected in all frac-

1

2

3

PAS456

7

150

!i mm

--"

i-.--14.3

Fig. 3. SDS-8% polyacrylamide gel electrophoresis of fractions from DEAE-cellulose. (1-3) Fractions from chromatography of chick A2-Sepharose CL-6B excluted fraction, eluted with 8 M urea, 0.3 M and 3 M NaC1 in 8 M urea, respectively, after treatment with 2-mercaptoethanol; (4-6) fractions from rechromatography of fraction 3 after reduction and alkylation; PAS, gel 3 stained for glycoproteins; (7) 3 M NaC1/8 M urea fraction from chromatography of Sepharose CL-6B excluded fraction of A2 from extract in the presence of N-ethylmaleimide after treatment with 2-mercaptoethanol.

175 TABLE I CARBOHYDRATE COMPOSITION LULOSE FRACTIONS The Fig. and The

OF

DEAE-CEL-

proteoglycans in the Sepharose CL-6B excluded fraction of 1A were isolated by chromatography on DEAE-cellulose, then reduced, alkylated and chromatographed as before. results are expresssed as mg/g Lowry protein.

Component

8 M urea

0.3 M NaCI/8 M urea

Sialic acids Glucosamine Galactosamine Total hexose

5.8 13 8.4 21.2

13.1 32.4 12.1 85.3

tions after reduction, thus confirming the presence of reducible oligomers of the proteins and also their association with proteoglycans (Fig. 3, gels 1, 2, 3). Kleine and Singh [6] have also reported the presence of reducible aggregates of individual glycoproteins in extracts from calf rib cartilage. When the proteoglycan fraction was reduced and alkylated and chromatographed as before, 47 and 19% of the protein was eluted now with the first two solvents respectively and the proteoglycans again with 3 M NaC1/8 M urea. Only the first two fractions contained the protein bands (Fig. 3, gels 4, 5, 6), providing further evidence of their covalent linkage with the proteoglycans and in amounts (Lowry protein) about three times that of the uronic acid. The gross chemical composition of the first two DEAE-cellulose fractions (Table I) together with the periodate Schiff's staining results (Fig. 3, PAS) indicate that at least seven of these proteins are glycoproteins. In contrast, when the Sepharose CL-6B excluded fractions of A2 from extract with N-ethylmaleimide were chromatographed on DEAE-cellulose the first two fractions contained no proteins and that of the proteoglycans (3 M NaC1/8 M urea) traces of five bands (Fig. 3, gel 7) as before chromatography (Fig. 2B, gel 1 (+)). They were eluted, however, with the first two solvents when the proteoglycan fraction was reduced and alkylated before chromatography, confirming their association with the proteoglycans. Although the amounts of these proteins are small (about onefifth or less that of uronic acid) they were also identified on proteoglycans even when higher con-

centrations of N-ethylmaleimide (50 mM) were used. Whether this reflects high reactivity of the proteoglycans or some other phenomenon is not clear. Similar observations have been reported by Noro et al. [26], who have isolated a proteoglycan from chick embryo epiphyseal cartilage in the presence of N-ethylmaleimide-containing disulphide-bonded collagenous protein. The studies on the chick extracts in the absence of N-ethylmaleimide were extended to similar preparations from sheep nasal and pig laryngeal cartilage. An almost identical profile of uronic acid and protein to that of chick A2 (Fig. 1A) was obtained upon chromatography on Sepharose CL6B of sheep and pig A2 fractions (not shown). Similarly, SDS-polyacrylamide gel electrophoresis of the Sepharose CL-6B excluded fractions in the presence of 2-mercaptoethanol revealed the presence of a variety of proteins (Fig. 4) with the link proteins, the 148 kDa subunit and the 36 kDa protein being quite prominent.

--

A

S u b "--~ Link~

--

4.

A

'

4-

B

t

36 k D a.--~-

Fig. 4. SDS-8% polyacrylamide gel electrophoresis of Sepharose CL-6B excluded fractions of sheep nasal (A) and pig laryngeal (B) A2 fractions; ( - ) before and ( + ) after treatment with 2-mercaptoethanol.

176

When A1 fraction from extracts in the absence of N-ethylmaleimide was electrophoresed only the link proteins and a diffused band with an apparent molecular weight 70000-80000, which appears to represent the hyaluronate binding region [8,27], were detected with and without reduction (not shown). However, when high amounts of the top protein-rich fraction (AI-D3) derived from centrifugation of A1 in 4 M guanidine hydrochloride were electrophoresed a faint band of M r about 60000 was additionaly observed only after reduction. This band which appears to correspond to the 148 kDa monomer could have been bonded either with the link proteins or with the proteoglycans which are found in the top fraction without affecting their binding with hyaluronate, and hence they separate when centrifugation is performed under dissociative conditions. Combining the results outlined so far, it appears that during extraction of cartilage and perhaps during dialysis of the extracts a series of reactions takes place between individual proteinsglycoproteins alone and with proteoglycans giving rise to aggregates of varying sizes. This phenomenon may be common to most types of cartilage, and because it can be prevented by N-ethylmaleimide which blocks free thiol groups, it appears to proceed through disulphide exchange.

Characterization of the proteoglycans linked with proteins In order to find out what proportion of the proteoglycans had reacted with the proteins-glycoproteins and to isolate sufficient amounts of these proteoglycans for further characterization, large amounts of A2-Sepharose CL-6B excluded fraction from sheep nasal cartilage extracted without N-ethylmaleimide were prepared and subjected to dissociative centrifugation in a starting density 1.5 g/ml, Three fractions, bottom 3 ml, E-l, middle 6 ml, E-2, and top 3 ml, E-3, were obtained containing 26, 11 and 63% of the applied uronic acid, respectively. Only the top two fractions and particularly E-3 contained the protein bands (not shown). The top two fractions were pooled and upon chromatography on Sepharose CL-2B in 4 M guanidine hydrochloride appeared heterogeneous with about 55% of the uronic acid in the void volume of the column (Fig. 5A). The protein bands

0.6 r 0.5

1

2

3

4

5

A

0.4~"

0.3~ ~0.2 ,~0.1 0

"~

~

--

o

~,0.2 ~ ,~0.1

0.1, 0~]

20

--

30

Fraction

Vo

40

50

Number

Vt

Fig. 5. Gel chromatography on Sepharose CL-2B of sheep A2-Sepharose CL-6B excluded fraction and fractions thereof. The columns (0.8×150 cm) were eluted with 4 M guanidine hydrochloride/0.05 M sodium acetate buffer (pH 5.8) and fractions of 1.4 ml were collected. Fractions were pooled as indicated. O, A530 (uronic acid); e, /1250. V0 and Vt void and total volume, respectively. (A) A2-Sepharose CL-6B excluded fraction; (B) and (C), the excluded and retarded fractions from (A) (vertical line), respectively, after reduction and alkylation.

were detected after reduction in all fractions (Fig. 6). Upon reduction and alkylation and rechromatography of the excluded and retarded fractions (vertical line Fig. 5A) the proteoglycans were of smaller hydrodynamic size (Fig. 5B and C) belonging to two distinct populations, Furthermore, all protein bands were found now only in the main protein peak (fractions 37-50). The effect of reduction on the hydrodynamic size of proteoglycans suggested that the whole population was involved in complexes with the proteins and perhaps with each other as it has been reported for proteoheparan sulphate from fibroblasts [28]. The possibility of the presence of disulphide linked proteoglycan aggregates was investigated by

t77

centrifuging a sample of pooled E-2 and E-3 under the conditions they were obtained and after reduction and alkylation. After reduction, about 95% of the uronic acid was found in the bottom 9 ml of the gradient, whereas with the unreduced sample 85% was found on the top 3 ml, indicating that it was the liberated proteins which had made the proteoglycans large and of low buoyant density. However, the possibility that a very small proportion of the proteoglycans may be bonded with each other via disulphide bonds still cannot be excluded since a small proportion had the same buoyant density after reduction, and hence possible changes could not be seen. The two populations of proteoglycans linked to proteins were isolated from reduced and alkylated pooled fractions E-2 and E-3 by chromatography

1

2

3

4

5

on Sepharose CL-2B. A bimodal distribution of uronic acid, similar to that in Fig. 5B was obtained and the proteoglycans of each population, designated 2B-I and 2B-II (Fig. 5B) were freed from other proteins by chromatography on DEAE-cellulose. The small proteoglycans (2B-II), amounting to about 75% of the uronic acid, appeared quite homogenous on Sepharose CL-6B (Fig. 7) with a distribution constant (Kd) 0.240. Using spherical proteins as standards an average molecular weight of 200 000 was calculated. However, a lower figure, 110000, was obtained when two fractions of chondroitin sulphate chains were used as standards (Fig. 7), which may be more correct. The larger proteoglycan population, appeared more polydispersed on Sepharose CL-2B, similar to peak I in Fig. 5B, like the bulk of the tissue proteoglycan subunit (not shown), with a K d (0.250) slightly smaller than that (0.280) of the subunit. The chondroitin sulphate chains of the proteoglycans were liberated by alkaline borohydride, and upon chromatography on Sephadex G-200 (not shown) or Sepharose CL-6B (Fig. 7) were found to be different. From their chromatography on Sephadex G-200 and using the data of Wasteson [23], an average molecular weight of 28 000 and 14000 was calculated for those from 2B-II

0.4 E = o 0.3

0.2 J= o

0.1

0

t/ 20 t Vo

Fig. 6. SDS-8% polyacrylamide gel electrophoresis of Sepharose CL-2B pooled fractions of Fig. 5A, after reduction; numbering corresponds to that of Fig. 5A.

30 Fraction

40 Number

50

60

l Vt

Fig. 7. Gel chromatography on Sepharose CL-6B of small proteoglycans linked with proteins (2B-II) and chondroitiu sulphate chains. The column (0.8 × 150 cm) was eluted w i t h 0.5 M sodium acetate (pH 7.0), and fractions of 1.25 ml were collected, e, 2B-II; A, chains of 2B-If; II, chains of large proteoglycans (2B-I). V0 and V~ void and total volume respectively.

178

and 2B-I, respectively; the latter figure being the same with that for chains form aggregable proteoglycans. These data and the uronic acid to protein ratio (Table II) suggest that each molecule of small proteoglycans would have 2 or 3 chondroitin sulphate chains. Apart from the difference in the size of the chondroitin sulphate chains the two populations differed also in the relative amounts of 4- and 6-sulphated disaccharides, the small proteoglycans containing higher amounts of 4sulphated residues (Table II). The proteoglycans differed also in the composition of their protein core thus making them distinctly different. The small proteoglycans contained high amounts of aspartic acid and leucine and low serine and

T A B L E 11 C O M P O S I T I O N OF P R O T E O G L Y C A N S L I N K E D W I T H PROTEINS The two populations of proteoglycans 2B-I1 and 2B-I, linked with matrix proteins were isolated by gel and ion exchange chromatography; A-PG is aggregable proteoglycans prepared by gel chromatography (unpublished). The values of amino acids are expressed as residues/1000 residues and are the averages of two independent determinations. 2B-II Asp Thr Ser Glu Pro Gly Ala Val Cys a Met Ile Leu Tyr Phe His Lys Arg Uronic a c i d / protein ( w / w ) A-di-4S (%) A-di-6S (%) Molar ratio G a l N / G l c N Mol.weight (chains)

112 44 84 112 62 98 52 53 14 2 44 105 27 31 35 70 50 0.43 92 5.6 7.5 28000

a Measured as carboxymethyl cysteine.

2B-I 75 55 168 137 58 165 69 52 5 traces 24 50 14 24 18 66 25 1.82 78 16 15.6 14000

A-PG 62 56 137 152 89 129 66 61 8 2 26 66 16 34 16 41 16 2.75 74 22 11.2 14000

glycine, whereas the larger fraction had an amino acid composition comparable to that of aggregable proteoglycans (Table II). Both' populations had a relatively high content of protein (Table II) which would make them less buoyant. During centrifugation in a starting density 1.6 g / m l these proteoglycans would be distributed in the top A2 fraction even if no complexes with proteins (presence of N-ethylmaleimide) had formed. The small proteoglycans have many chemical and macromolecular characteristics similar to those of a low-molecular weight proteoglycan isolated from articular [4], baboon [5] and bovine nasal cartilage [7]. The larger proteoglycans, on the other hand, appear to be rather a subpopulation of the aggregable proteoglycans; this is also supported by immunological cross reaction with antiserum to the hyaluronate binding region of aggregable proteoglycans (unpublished data). On the basis that fraction A2 contained 6% of the extracted uronic acid, and since about 60% of this fraction (Sepharose CL-6B excluded) was subjected to centrifugation, it appears that 2-3% of the extracted proteoglycans (based on uronic acid) are linked with other matrix proteins via reducible bonds. It is noteworthy that material absorbing at 280 nm was eluted in the void volume of Sepharose CL-2B column when the excluded fraction of proteoglycans was chromatographed after reduction and alkylation (Fig. 5B). This material contains no hydroxyproline, has a high absorbance at 260 nm and from other data (unpublished) it appears to be a nucleoprotein, Furthermore, this nucleoprotein does not participate in aggregates with proteoglycans since when A2-Sepharose CL-6B excluded fraction was chromatographed on DEAE-cellulose it was recovered in the 8 M urea fraction. General discussion

The object of this study was to explore the extent of disulphide exchange which takes place during extraction of cartilage. The evidence is quite clear that a plethora of proteins-glycoproteins interact with each other and with proteoglycans and this may lead to missinterpretation of the results [4-6]. Not only a good proportion of proteoglycans may be taken as existing in the tissue as covalent complexes with other constituents but

!79 also the isolation of the various p r o t e i n s - g l y c o p r o teins in the form that they exist in the tissue m a y be i m p e d e d . F u r t h e r m o r e , even small a m o u n t s of n o n p r o t e o g l y c a n p r o t e i n s in the p r o t e o g l y c a n prep a r a t i o n s m a y lead to ironeous results when these p r e p a r a t i o n s are s t u d i e d with sensitive techniques such as i m m u n o l o g i c . It a p p e a r s therefore, necessary that reagents which b l o c k free thiol g r o u p s should be i n c l u d e d in the e x t r a c t i o n solvent as it has been p r a c t i c e d b y few investigators [7,9,26], p a r t i c u l a r l y when c o m p o n e n t s , i n c l u d i n g p r o t e o glycans, of low b u o y a n t d e n s i t y are to be isolated a n d studied, T h e r e a p p e a r s to be some degree of specificity in the p r o t e o g l y c a n s which react with the glycoproteins. A t least 75% of them are of small h y d r o d y n a m i c size, have high a m o u n t s of aspartic acid a n d leucine rich p r o t e i n a n d c h o n d r o i t i n s u l p h a t e chains twice as long c o n t a i n i n g a high ratio of 4to 6-sulphation. F r o m the size of the p r o t e o g l y c a n - g l y c o p r o t e i n aggregates a n d that of the i n d i v i d u a l c o m p o n e n t s (Fig. 1 a n d Fig. 5) it a p p e a r s that each p r o t e o g!ycan molecule should have on it either several of the g l y c o p r o t e i n m o n o m e r s or several m o n o m e r s of the s a m e type, or both. In either case the g l y c o p r o t e i n s need not be linked singly o n t o the p r o t e o g l y c a n molecule since they can form aggregates of their own a n d therefore they m a y be t r a n s f e r r e d en block. The g l y c o p r o t e i n s include k n o w n c o m p o n e n t s of cartilage such as the link proteins, the 148 k D a a n d 36 k D a proteins; w h e t h e r the r e m a i n d e r are also synthesized by the c h o n d r o c y t e s or are derived from s e r u m r e m a i n s to be d e t e r m i n e d .

Acknowledgement W e t h a n k Mrs. M a r i a A n g e l o p o u l o u for t y p i n g this m a n u s c r i p t .

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