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Evidence for the presence of a low molecular weight proteoglycan aggregation factor in rabbit ear cartilage
Biochimica et Btophysica Acta, 320 (1973) 442-452 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 27215
EVIDENCE FOR THE PRESENCE OF A LOW MOLECULAR WEIGHT PROTEOGLYCAN AGGREGATION FACTOR IN RABBIT EAR CARTILAGE
BOHUMILA ROKOSOVA, ALBERT N. HANSON and J. PETER BENTLEY
The Department of Blochemi~try, University of Oreoon Medical School, Portland, Oreo. 97201 (U.S.A.) (Received March 26th, 1973)
SUMMARY
Proteoglyeans were extracted from rabbit ear cartilage with 0.15 M NaCI, and with 4 M guanidine. HCI. They were subjected to gel chromatography on Sepharose 2B and 6B either before or after centrifugation in CsCI density gradients. This was to remove a previously described proteoglycan aggregation factor named glycoprotein link (Sajdera, S. W. and Haseall, V. C. (1969) J. Biol. Chem. 244, 77-87 and Hascall, V. C. and Sajdera, W. S. (1969) J. Biol. Chem. 244, 2384-2396). Evidence for at least three aggregation states is presented. The presence of glycoprotein link and an additional aggregation factor (X) is required for the largest aggregates to be formed. In the absence of Factor X, which precipitates at low ionic strength, large aggregates cannot be formed even in the presence of glycoprotein link. In the absence of glycoprotein link, smaller species are formed which can be further dissociated into even smaller units if Factor X is removed. The smallest unit is the only species which can be found in the 0.15 M NaC! extract while in the o/ 4 M guanidine • HCI extract it represents only a minor portion (about 10/o) of the total proteoglycan. Studies on radioactively labeled cartilage suggest that this smallest unit is more recently synthesized than the larger aggregates.
INTRODUCTION
It is generally agreed that different preparations of cartilage proteoglycans may exhibit widely varying molecular weights. The molecular weight reported by various investigators covers the range from several hundred thousands to several millions. Meyer I suggested that proteoglycan complexes of molecular weight above 1 • 106 could be made up of smaller complexes bridged by a basic protein and a number of fractionation techniques have been employed in an effort to define the proteoglycan fraction representing the fundamental unit, however, despite many efforts, such fractions have not been satisfactorily defined. As has been pointed out by Muir and coworkers 2,3, different methods of preparation of connective-tissue proteoglycan may select varying proportions of different molecular species present in the same tissue.
PROTEOGLYCAN AGGREGATION FACTOR IN CARTILAGE
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Sajdera and Hascall 4'5 suggested that the lower molecular weight proteoglycan fractions, such as PPL3 (ref. 6), may arise as a result of high shear forces involved in the preparation procedure and, to avoid scission of macromolecules during extraction, they introduced the so-called dissociative procedure. This employs solutions of 4 M guanidine • HCI or of 3 M MgC! 2 to dlsaggregate the proteoglycan complexes existing in situ and to extract the so called proteoglycan subunits from the tissue. Sajdera and Hascall "'s also showed that the isolated subunits could reassociate to proteoglycan complexes upon dilution of the extract (under so-called associative conditions) and the reformed complex could be isolated by equilibrium centrifugation in CsCI density gradients. Subsequently, the isolated proteoglycan complex can be again disaggregated into proteoglycan subunit and a linking glycoprotein when the guanidine concentration is increased again to dissociative conditions. Purified proteoglycan subunit is then isolated by the second density gradient centrifugation4' s. Although the purified subunit, the fraction with the highest buoyant density, represents the bulk fraction, it does not represent the entire population of proteoglycan originally present in the dissociative extract. The dissociative extraction is a highly efficient method which mobilizes more than 80 % of the uronic acid containing material present in the tissue and brings all this material into the same solution. It is obvious that in addition to the subunits released from proteoglycan complex and capable of re-aggregation under associative conditions, the original extract may also contain proteoglycans which were free in the tissues and which may possess molecular characteristics different from those of the subunits. Indeed, the presence of high molecular weight proteoglycan with high protein:uronic acid ratios in the fractions of lower buoyant densities have been reported by Tsiganos et al. 7 and Mashburn and Hoffmana. In addition, low molecular weight proteoglycans which are not degradation products, can be extracted from cartilagea'9'1 o. We have found that these minor components isolated from rabbit ear cartilage are highly active metabolically1L~2 and here we present evidence for their involvement in the formation of higher aggregates and for the existence of an additional linking factor which is necessary for this aggregation to proceed. MATERIALSAND METHODS Tissue source
New Zealand white rabbits approximately 6 weeks of age were used. Ears were removed and cleaned of skin. The cartilage tissue was crushed in a stainless steel mortar after freezing in liquid nitrogen. In some experiments the tissue was sliced finely using a Mickle chopper (Brinkman Instruments, Great Neck, N.Y., U.S.A.) and incubated with [U-14C]glucose in Krebs-Ringer bicarbonate buffer for 4 h at 37 °C as previously described 12. Following incubation the tissue was rapidly washed and crushed after freezing in liquid nitrogen as above. The crushed tissue was subjected to one of the extraction procedures outlined below. ( A ) Direct extraction
Direct extraction with 4 M guanidine • HCI in 0.05 M sodium acetate buffer, pH 5.8, was carried out as described by Sajdera and Hascall 4. The extraction procedure was repeated once and the extracts pooled. The combined extracts were clarified by centrifugation at 10 000×g.
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(B) Pre-extraction with salt solution For some of the experiments the tissue was extracted three times overnight with 0.15 M NaCI, pH 7.4, at 4 °C in the presence of thymol prior to the 4 M guanidine • HCI extraction. The respective extracts were combined and clarified as above. The clarified 0.15 M NaCI extracts were dialyzed salt free and the entire contents o f the dialysis sac were lyophilised. The lyophilised material was redissolved in 0.025 M Na2SO4 and precipitated with cetyl pyridinium chloride. The precipitate, after redissolving in 2 M NaCI was precipitatext with 70 % ethanol and dried in vacuo. We have previously shown 12 that this salt extraction procedure is not accompanied by proteoglycan degradation brought about by release of autolytic enzymes from the tissue.
Density gradient centrifugation Equilibrium centrifugation in a CsC1 density gradient was performed in a Spinco Model L ultracentrifuge with a Spinco SW 39 rotor for 74 h at 33 500 rev./min at 10 °C. The gradient was partitioned from the top into 0.4-ml fractions with an Auto Densi-Flow instrument (Buchler Instruments, Inc., Fort Lee, N.J., U.S.A.). The density of each fraction was determined by the use of a 200-pl capillary pipette as a pycnometer. The original density for 4 M guanidine • HCI/CsCI was 1.50 g/ml, and for 0.4 M guanidine • HCi/CsC! it was 1.69 g/ml (ref. 4). Solid CsCI was added directly to the guanidine solutions. The 4 M guanidine • HCI extract (dissociative conditions) was transferred to 0.4 M guanidine • HCI (associative conditions) by dialysis against 9 vol. of 0.05 M sodium acetate 4. This was always accompanied by the formation of a very fine precipitate which required centrifugation at 27 000 × g for sedimentation. If, however, KCI to 0.3 M was added or if the 4 M guanidine • HCI solution was dialyzed exhaustively against 0.15 M NaCI, no precipitate was formed.
Gel chromatography Samples of the various proteoglycan extracts were subjected to gel filtration on Sepharose 2B or 6B. Attempts to determine the void volume of Sepharose 2B columns were made using blue dextran (Pharmacia Fine Chemicals). More than 90 % of the blue dextran was retarded by these columns, making the definition of void volume quite arbitrary. For this reason all results are expressed in terms of volume of eluent. Identical columns (1.5 cm × 75 cm) fitted with flow adaptors were used and gel beds of the same height were prepared under the same packing pressure. Under these conditions the void volume was approx. 32 ml. The columns were equilibrated and eluted with either 4 M guanidine • HCI in 0.05 M sodium acetate, pH 5.8, 0.4 M guanidine • HCI in the same buffer or 0.15 M NaC1. Fractions eluted with 4 M guanidine • HCI were dialyzed before further analysis. The analytical procedures used have been described in the previous paper t2. Data on the characterization of the glycosaminoglycan moiety of proteoglycans isolated from rabbit ear cartilage by the procedures used in this study have been reported previously. These were typical of chondroitin sulfate (mixed 4- and 6-sulfate isomers) and no keratan sulfate could be detected t3.
PROTEOGLYCAN AGGREGATION FACTOR IN CARTILAGE
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RESULTS
Analysis of total proteoglycans A portion of the 4 M guanidine extract of cartilage was dialyzed against water to remove the guanidine • HCI. A fine precipitate occurred in the dialysis bag. For the sake of brevity we will refer to this precipitate as X. This subsequently redissolved upon the addition of solid KCI to a final concentration of 0.3 M. Chondroitin sulfate proteoglycan was precipitated from the clear KCI solution with cetyl pyridinium chloride. The proteoglycan was recovered from the precipitate by dissolving in 2.0 M KCi and reprecipitated from this solution with ethanol at 70 % final concentration. A sample of this "total" proteoglycan preparation was dissolved in 0.15 M NaCI and applied to a Sepharose 2B column which was eluted with 0.15 M NaCI. Uronic acid and protein (A2a o .m) were measured in the effluent and the results are presented in Fig. I. First a sharp peak of essentially excluded material with a high protein: uronic acid ratio was seen. This was immediately followed by a very broad peak with a relatively low protein content. This retarded material is distributed over a wide region covering the total capacity of the column. In other words, it is markedly heterogeneous in size, even though most of it comes out o f the column immediately after the exclusion volume.
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Gel filtration in dissociative conditions A second portion o f the initial" 4 M guanidine • HCI extract was fractionated by equilibrium centrifugation in a CsCI density gradient under the dissociative conditions of Sajdera and Hascalr. Fig. 2 shows that after the centrifugation was complete relatively little uronic acid containing material was distributed through the gradient, most of this material was recovered from the bottom fraction with the highest density. However, the uronic acid content distributed throughout the gradient in the lower density fractions represented 14-15 % of the total recovered uronate. Protein, as evidenced by absorbance at 280 nm, was found both at the top and bottom of the tube. A third protein peak was found in fractions with a density distributed around 1.45 g/ml. The bottom fraction from this dissociative density gradient profile was corn-
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I=ig. 2. Distribution of uronic acid and protein in 4 M guanidine • HCI extracted proteoglycans after equilibrium density gradient centrifugation carried out under dissociative conditions. O-O, uronic acid; A---A, protein (A28o,m); A-A, density g/ml. pletely excluded from Sepharose 6B and nothing was retarded (Fig. 3B). This was not the case when the total 4 M guanidine extract was applied to Sepharose 6B prior to the density gradient centrifugation step. Fig. 3A shows that in this uncentrifuged material a certain proportion of the proteoglycan penetrated the gel and in addition, a component with a relatively high protein content appeared towards the end of the elution profile. The proteoglycan which penetrated the gel (Fig. 3A) represents only a minor portion of the total uronate, but possesses a higher specific radioactivity (3900 dpm/ #mole of uronate) than does the excluded fraction (1100 dpm/#mole of uronate). Virtually the same distribution on Sepharose 6B as that obtained for the 4 M guanidine • HCI extract purified by density gradient centrifugation, was obtained for the nonpurified 4 M guanidine extract which had been preceded by a 0.15 NaCl extraction (Fig. 3C). The material extracted with 0.15 M NaCI and recovered by cetyl pyridinium chloride precipitation was retarded on Sepharose 6B, as had been found previously 12. When the material recovered from the bottom fraction after density gradient centrifugation in dissociative conditions (PGS of Sajdera and Hascall 4) was fractionated on Sepharose 2B in 4 M guanidine • HCI buffered to pH 5.8, (Fig. 4a), no excluded material could be demonstrated but the uronic acid profile covered largely the region immediately after the exclusion volume. Gel filtration after density gradient centrifugation in associative conditions A third portion of the original guanidine extract was transferred by dialysis into associative conditions to allow aggregation. Again, a small amount of fine precipitate was observed in the dialysis bag. At this time, the precipitate (X) was removed by centrifugation at 27 000 × g and was found to contain approx. 2-3 % of the total uronic acid present prior to the dialysis step. The proteoglycans remaining in the
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0.4 M guanidine • HCI supernatant were subjected to density gradient centrifugation in CsC1 and the fraction with the highest buoyant density recovered from the bottom of the tube was applied to a Sepharose 2B column after removal of CsCI by dialysis against 0.15 M NaCI. The column was eluted with 0.15 M NaCI buffered to pH 5.8. This fraction should correspond to the proteoglycan complex (PGC of Sajdera and HascalP) since the link protein was not removed under the associative conditions 4'5. However, the results of gel chromatography shown in Fig. 5b indicate that under these conditions the material recovered as proteoglycan complex from the associative density gradient penetrated the gel to a much greater extent than did the proteoglycan subunit preparation made under dissociative conditions. It eluted in a region much closer to the total volume and is therefore of considerably smaller size than the material prepared under dissociative conditions. In another portion of the 4 M guanidine .HCI extract, the precipitate (X)
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PROTEOGLYCAN AGGREGATION FACTOR IN CARTILAGE
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which occurred upon dilution was not removed and was redissolved when CsCI was added prior to density gradient centrifugation in associative conditions. As a result, the elution profile on Sepharose 2B eluted with 0.15 M NaCI (Fig. 5a) of the highest buoyant density fraction is strikingly different than that seen in Fig. 5b. Fig. 5 demonstrates that the same proteoglycan preparation can, under associative conditions give rise to large complexes which are excluded from Sepharose 2B (Fig. 5a) when X is present but exist as considerably smaller species in the absence of X (Fig. 5b). Dissociation of PGS by removal of X Proteoglycan subunit prepared from the initial 4 M guanidine HCI extract by dissociative density gradient centrifugation eluted from Sepharose 2B in the region immediately following the exclusion volume. This was true when columns were eluted with either 4 M guanidine. HC! or 0.15 M NaCI (Fig. 4a). Fractions from this separation were pooled, dialyzed in the cold and lyophilized. The dry material was redissolved in a small volume of 4 M guanidine • HCI and diluted by dialysis to 0.4 M guanidine • HCI. A small amount of precipitate (X) formed and was removed by centrifugation. Material rem~..aing in solution was applied to a column of Sepharose 2B an deluted with 0.15 M NaCI. Fig. 4b shows that the uronic acid elution profile is markedly shifted to the region much closer to the total volume than the non-dialyzed proteogiycan subunit preparation. Thus, Fig. 4 indicates that dissociatively prepared proteoglycan subunits can be further dissociated by the removal of X. •
The salt extractable fractions As shown in Fig. 6, this preparation was retarded by Scpharose 2B and eluted at a position close to the total volume. Because of the methods used in the preparation we do not know whether X was originally present in the extract and lost upon purification or whether the proteoglycans existed in the low molecular weight state in situ. The specific radioactivity of the salt extractable proteoglycan is, however, about three times that of the unextractable material 11,12 which suggests the two are from distinct tissue pools.
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DISCUSSION
The work of Sajdera and Hascall 4'5 described in the introduction led them to suggest that "proteoglycan subunit" or PGS was probably the basic unit produced by the chondrocyte and that its aggregation into larger complexes gave rise to the very high molecular weight species which could be isolated from cartilage. This was somewhat modified by Rosenberg et a l . ' 4.t s who were able to demonstrate the presence of a smaller unit than proteoglycan subunit in dissociative extracts of cartilage. This smaller unit was identical to material referred to as PPL 3 isolated from cartilage by Pal et al. 6 using high speed homogenisation or disruptive procedures. Rosenberg et a l ) 4 , i s considered it to be a monomer which could form dimers (or PGS). By electron microscopic technique they were able to show that the dimers but not monomers were capable of further aggregation provided a linking glycoprotein was present. The results presented here are in agreement with the work of Rosenberg e t al. ~4, ~5 in that we show that proteoglycan can be isolated from tissue in at least three aggregation states (Figs 4a, 5a and 6) and in our demonstration of the presence of a proteoglycan species of a lower molecular weight than proteoglycan subunit (perhaps a monomer). Whether the "monomer" exists as such in the tissue cannot be determined if dissociative extraction is used. Its presence in the extract may merely represent dissociation (by the extractant) of higher aggregates existing in vivo. However, its presence can be demonstrated also in tissue extracts made with 0.15 M NaCI and the highest rate of precursor uptake into this low molecular weight proteoglycan 12 defines this as a product of synthesis rather than of degradation. In addition, on the basis of results obtained by a combination of density gradient centrifugation and gel chromatography, our results suggest that another linking factor, distinct from the linking glycoprotein of Hascall and Sajdera s mediates aggregation of smaller proteoglycans into PGS-like macromolecules. For simplicity, we have referred to this factor as X. Proteoglycans from the same dissociative extract can form large complexes excluded from Sepharose 2B when transferred into associative conditions, provided that the glycoprotein link 4's and X are present (Figs 1 and 5a). Removal of the glycoprotein link by density gradient centrifugation in dissociative conditions gives rise predominantly to species of the proteoglycan subunit type 4'5 (Fig. 4a). When X is removed from the isolated proteoglycan subunit then the elution profile on Sepharose 2B is shifted toward the region of a smaller molecular size as shown in Fig. 4b. This indicates that the presence of X was required for the subunit PGS to retain its molecular weight characteristics when analyzed by gel filtration. In the absence of this factor proteoglycan complex cannot be formed and even under the favorable associative conditions smaller proteoglycans predominate (Fig. 5b). Whether a similar X factor is required for proteoglycan aggregation in other tissues than rabbit ear cartilage is not known at this time, but preliminary data indicate that a similar phenomenon occurs in bovine tracheal cartilage. Other than the determination of uronic acid, no further characterization of X was attempted because of the extremely small amount of a fine precipitate which can be recovered. This may also be a reason why it remained undetected by others. Larger scale preparations are being carried out to enable further analysis.
P R O T E O G L Y C A N A G G R E G A T I O N FACTOR IN C A R T I L A G E
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The presence of a small amount of a lower molecular weight proteoglycan can be demonstrated in 4 M guanidine extracts. These can be detected when the extract is subjected to gel filtration on Sepharose 6B (Fig. 3A) but they are absent if the tissue has been pre-extracted with 0.15 M NaCI (Fig. 3C) and they were not found in the proteoglycan subunit recovered from the two bottom fractions after density gradient centrifugation (Fig. 3B). On the other hand, extraction of cartilage with isotonic solutions of NaC1 brings into solution proteoglycans which are exclusively of the small molecular weight type and which represent less than 10 % of the total proteoglycan12. Thus salt extractable low molecular weight material may be present in the tissue in addition to that which is aggregated into the larger complexes of the "proteoglycan subunit" and "proteoglycan complex" type. We have previously shown that this extractable material and its chondroitin sulfate side chains had much higher specific activities than the residual or unextractable proteoglycan when cartilage preincubated with labeled glucose was used tt'12. This implies that the lower molecular weight, salt extractable fraction represents more recently synthesized material. Since the aggregated forms (Figs 4a and 5a) can (in the absence of X) be dissociated into similar low molecular weight material (Figs 4b, 5b and 6) which can associate in vitro into larger complexes in the presence of X (Figs 5a and 5b) it is logical to suppose that newly synthesized units can aggregate in vioo into larger forms. This aggregation is probably a fast process, since the highly labeled low molecular weight proteoglycan represents only a minor portion of the total. Other observations of greater incorporation of radioactive precursors into low molecular weight proteoglycans have been made t6'~7. Attempts to demonstrate a precursor-product relationship between these fractions and the more highly aggregated proteoglycans were unsuccessful 16, t s perhaps because the extraction procedures were dissociative or disruptive which may have mixed newly synthesized low molecular weight proteoglycan with older material which existed in aggregate form prior to extraction. That such mixing can occur was observed by Campo et al. i s who added radioactive PPL3 to nonradioactive cartilage and homogenized the mixture. Upon subsequent fractionation the label was found to be randomly distributed between all of the proteoglycan subfractions. The demonstration that isotonic salt extracts contain only the low molecular weight type of proteoglycan will permit us to approach the question of any precursor product relationships without the complications mentioned above. Fig. 6 indicates a good deal of heterogeneity in the low molecular weight 0.15 M NaCI extractable proteoglycan and it is possible that even this fraction does not represent the smallest elementary particle. The recent work of Woodward et al. 19 suggests that this may in fact be the case. On the basis of ultracentrifuge and viscosity studies carried out in high salt concentrations they found that the monomeric unit of dissociatively extracted proteoglycan may have a molecular weight as low as 40 000 to 60 000 even when stringent precautions were taken against degradation. Even though the results reported here have much in common with those of workers using other cartilage tissues, they also differ in several respects and we should always bear in mind that phenomena observed in rabbit ear cartilage may not be the same as those occurring in bovine nasal or pig laryngeal cartilage. The converse is also true.
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ACKNOWLEDGEMENTS
The technical assistance of Mrs Kathryn Johnson and Mrs Barbara Jelen are gratefully acknowledged. Supported in part by N.I.H. grant HL-14126-12 and by a grant from the American Heart Association. REFERENCES 1 2 3 4 5 6 7 8 9 10 !1 12 13 14 15 16 17 18 19
Meyer, K. (1966) Fed. Proc. 34, 1046 Muir, H. and Jacobs, S. (1967) Biochem. J. 103, 367-374 Brandt, K. D. and Muir, H. (1969)Blochem. J. 114, 871-876 Sajdera, S. W. and Hascall, V. C. (1969) J. Biol. Chem. 244, 77-87 Hascall, V. C. and Sajdera, S. W. (1969) J. Biol. Chem, 244, 2384-2396 Pal, S., Doganges, P. T. and Schubert, M. (1966) J. Biol. Chem. 241, 4261-4266 Tsiganos, C. P., Hardingham, T. E. and Muir, H. (1971) Btochim. Biophys. Acta 229, 529-534 Mashburn, T. A. and Hoffman, P. (1971) J. Biol. Chem. 246, 6497-6506 Tsiganos, C. P. and Muir, H. (1969) Biochem. J. !13, 885-894 Seraflni-Francassini, A. and Stimson, W. (1971) FEBS Lett. 15, 245-248 Rokosova, B. and Bentley, J. P. (1972) Blochlm. Btophys. Acta 264, 98-102 Rokosova, B. and Bentley, J. P. (1973) Biochim. Biophys. Acta 297, 473--485 Bentley, J. P. and Rokosova, B. (1970) Biochem. J. 116, 329-336 Rosenberg, L., Pal, S., Beale, R. and Schubert, M. (1970) J. Biol. Chem. 245, 4112-4122 Rosenberg, L., Hellman, W. and Kleinschmidt, A. K. (1970) J. Biol. Chem. 245, 4123-4130 Hardingham, T. E. and Muir, H. (1972) Biochem. J. 126, 791-803 Serafini-Francassini, A. and Stimson, W. H. (1972) FEBS Lett. 26, 336-340 Campo, R. D., Bielen, R. J. and Hetherington, J. (1972) Biochim. Biophys. Acta 261, 136-142 Woodward, C. B., Hranisavljevic, J. and Davidson, E. A. (1972) Biochemistry 11, 1168-1176
EVIDENCE FOR THE PRESENCE OF A LOW MOLECULAR WEIGHT PROTEOGLYCAN AGGREGATION FACTOR IN RABBIT EAR CARTILAGE
BOHUMILA ROKOSOVA, ALBERT N. HANSON and J. PETER BENTLEY
The Department of Blochemi~try, University of Oreoon Medical School, Portland, Oreo. 97201 (U.S.A.) (Received March 26th, 1973)
SUMMARY
Proteoglyeans were extracted from rabbit ear cartilage with 0.15 M NaCI, and with 4 M guanidine. HCI. They were subjected to gel chromatography on Sepharose 2B and 6B either before or after centrifugation in CsCI density gradients. This was to remove a previously described proteoglycan aggregation factor named glycoprotein link (Sajdera, S. W. and Haseall, V. C. (1969) J. Biol. Chem. 244, 77-87 and Hascall, V. C. and Sajdera, W. S. (1969) J. Biol. Chem. 244, 2384-2396). Evidence for at least three aggregation states is presented. The presence of glycoprotein link and an additional aggregation factor (X) is required for the largest aggregates to be formed. In the absence of Factor X, which precipitates at low ionic strength, large aggregates cannot be formed even in the presence of glycoprotein link. In the absence of glycoprotein link, smaller species are formed which can be further dissociated into even smaller units if Factor X is removed. The smallest unit is the only species which can be found in the 0.15 M NaC! extract while in the o/ 4 M guanidine • HCI extract it represents only a minor portion (about 10/o) of the total proteoglycan. Studies on radioactively labeled cartilage suggest that this smallest unit is more recently synthesized than the larger aggregates.
INTRODUCTION
It is generally agreed that different preparations of cartilage proteoglycans may exhibit widely varying molecular weights. The molecular weight reported by various investigators covers the range from several hundred thousands to several millions. Meyer I suggested that proteoglycan complexes of molecular weight above 1 • 106 could be made up of smaller complexes bridged by a basic protein and a number of fractionation techniques have been employed in an effort to define the proteoglycan fraction representing the fundamental unit, however, despite many efforts, such fractions have not been satisfactorily defined. As has been pointed out by Muir and coworkers 2,3, different methods of preparation of connective-tissue proteoglycan may select varying proportions of different molecular species present in the same tissue.
PROTEOGLYCAN AGGREGATION FACTOR IN CARTILAGE
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Sajdera and Hascall 4'5 suggested that the lower molecular weight proteoglycan fractions, such as PPL3 (ref. 6), may arise as a result of high shear forces involved in the preparation procedure and, to avoid scission of macromolecules during extraction, they introduced the so-called dissociative procedure. This employs solutions of 4 M guanidine • HCI or of 3 M MgC! 2 to dlsaggregate the proteoglycan complexes existing in situ and to extract the so called proteoglycan subunits from the tissue. Sajdera and Hascall "'s also showed that the isolated subunits could reassociate to proteoglycan complexes upon dilution of the extract (under so-called associative conditions) and the reformed complex could be isolated by equilibrium centrifugation in CsCI density gradients. Subsequently, the isolated proteoglycan complex can be again disaggregated into proteoglycan subunit and a linking glycoprotein when the guanidine concentration is increased again to dissociative conditions. Purified proteoglycan subunit is then isolated by the second density gradient centrifugation4' s. Although the purified subunit, the fraction with the highest buoyant density, represents the bulk fraction, it does not represent the entire population of proteoglycan originally present in the dissociative extract. The dissociative extraction is a highly efficient method which mobilizes more than 80 % of the uronic acid containing material present in the tissue and brings all this material into the same solution. It is obvious that in addition to the subunits released from proteoglycan complex and capable of re-aggregation under associative conditions, the original extract may also contain proteoglycans which were free in the tissues and which may possess molecular characteristics different from those of the subunits. Indeed, the presence of high molecular weight proteoglycan with high protein:uronic acid ratios in the fractions of lower buoyant densities have been reported by Tsiganos et al. 7 and Mashburn and Hoffmana. In addition, low molecular weight proteoglycans which are not degradation products, can be extracted from cartilagea'9'1 o. We have found that these minor components isolated from rabbit ear cartilage are highly active metabolically1L~2 and here we present evidence for their involvement in the formation of higher aggregates and for the existence of an additional linking factor which is necessary for this aggregation to proceed. MATERIALSAND METHODS Tissue source
New Zealand white rabbits approximately 6 weeks of age were used. Ears were removed and cleaned of skin. The cartilage tissue was crushed in a stainless steel mortar after freezing in liquid nitrogen. In some experiments the tissue was sliced finely using a Mickle chopper (Brinkman Instruments, Great Neck, N.Y., U.S.A.) and incubated with [U-14C]glucose in Krebs-Ringer bicarbonate buffer for 4 h at 37 °C as previously described 12. Following incubation the tissue was rapidly washed and crushed after freezing in liquid nitrogen as above. The crushed tissue was subjected to one of the extraction procedures outlined below. ( A ) Direct extraction
Direct extraction with 4 M guanidine • HCI in 0.05 M sodium acetate buffer, pH 5.8, was carried out as described by Sajdera and Hascall 4. The extraction procedure was repeated once and the extracts pooled. The combined extracts were clarified by centrifugation at 10 000×g.
444
B. ROKOSOVA et aL
(B) Pre-extraction with salt solution For some of the experiments the tissue was extracted three times overnight with 0.15 M NaCI, pH 7.4, at 4 °C in the presence of thymol prior to the 4 M guanidine • HCI extraction. The respective extracts were combined and clarified as above. The clarified 0.15 M NaCI extracts were dialyzed salt free and the entire contents o f the dialysis sac were lyophilised. The lyophilised material was redissolved in 0.025 M Na2SO4 and precipitated with cetyl pyridinium chloride. The precipitate, after redissolving in 2 M NaCI was precipitatext with 70 % ethanol and dried in vacuo. We have previously shown 12 that this salt extraction procedure is not accompanied by proteoglycan degradation brought about by release of autolytic enzymes from the tissue.
Density gradient centrifugation Equilibrium centrifugation in a CsC1 density gradient was performed in a Spinco Model L ultracentrifuge with a Spinco SW 39 rotor for 74 h at 33 500 rev./min at 10 °C. The gradient was partitioned from the top into 0.4-ml fractions with an Auto Densi-Flow instrument (Buchler Instruments, Inc., Fort Lee, N.J., U.S.A.). The density of each fraction was determined by the use of a 200-pl capillary pipette as a pycnometer. The original density for 4 M guanidine • HCI/CsCI was 1.50 g/ml, and for 0.4 M guanidine • HCi/CsC! it was 1.69 g/ml (ref. 4). Solid CsCI was added directly to the guanidine solutions. The 4 M guanidine • HCI extract (dissociative conditions) was transferred to 0.4 M guanidine • HCI (associative conditions) by dialysis against 9 vol. of 0.05 M sodium acetate 4. This was always accompanied by the formation of a very fine precipitate which required centrifugation at 27 000 × g for sedimentation. If, however, KCI to 0.3 M was added or if the 4 M guanidine • HCI solution was dialyzed exhaustively against 0.15 M NaCI, no precipitate was formed.
Gel chromatography Samples of the various proteoglycan extracts were subjected to gel filtration on Sepharose 2B or 6B. Attempts to determine the void volume of Sepharose 2B columns were made using blue dextran (Pharmacia Fine Chemicals). More than 90 % of the blue dextran was retarded by these columns, making the definition of void volume quite arbitrary. For this reason all results are expressed in terms of volume of eluent. Identical columns (1.5 cm × 75 cm) fitted with flow adaptors were used and gel beds of the same height were prepared under the same packing pressure. Under these conditions the void volume was approx. 32 ml. The columns were equilibrated and eluted with either 4 M guanidine • HCI in 0.05 M sodium acetate, pH 5.8, 0.4 M guanidine • HCI in the same buffer or 0.15 M NaC1. Fractions eluted with 4 M guanidine • HCI were dialyzed before further analysis. The analytical procedures used have been described in the previous paper t2. Data on the characterization of the glycosaminoglycan moiety of proteoglycans isolated from rabbit ear cartilage by the procedures used in this study have been reported previously. These were typical of chondroitin sulfate (mixed 4- and 6-sulfate isomers) and no keratan sulfate could be detected t3.
PROTEOGLYCAN AGGREGATION FACTOR IN CARTILAGE
445
RESULTS
Analysis of total proteoglycans A portion of the 4 M guanidine extract of cartilage was dialyzed against water to remove the guanidine • HCI. A fine precipitate occurred in the dialysis bag. For the sake of brevity we will refer to this precipitate as X. This subsequently redissolved upon the addition of solid KCI to a final concentration of 0.3 M. Chondroitin sulfate proteoglycan was precipitated from the clear KCI solution with cetyl pyridinium chloride. The proteoglycan was recovered from the precipitate by dissolving in 2.0 M KCi and reprecipitated from this solution with ethanol at 70 % final concentration. A sample of this "total" proteoglycan preparation was dissolved in 0.15 M NaCI and applied to a Sepharose 2B column which was eluted with 0.15 M NaCI. Uronic acid and protein (A2a o .m) were measured in the effluent and the results are presented in Fig. I. First a sharp peak of essentially excluded material with a high protein: uronic acid ratio was seen. This was immediately followed by a very broad peak with a relatively low protein content. This retarded material is distributed over a wide region covering the total capacity of the column. In other words, it is markedly heterogeneous in size, even though most of it comes out o f the column immediately after the exclusion volume.
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Gel filtration in dissociative conditions A second portion o f the initial" 4 M guanidine • HCI extract was fractionated by equilibrium centrifugation in a CsCI density gradient under the dissociative conditions of Sajdera and Hascalr. Fig. 2 shows that after the centrifugation was complete relatively little uronic acid containing material was distributed through the gradient, most of this material was recovered from the bottom fraction with the highest density. However, the uronic acid content distributed throughout the gradient in the lower density fractions represented 14-15 % of the total recovered uronate. Protein, as evidenced by absorbance at 280 nm, was found both at the top and bottom of the tube. A third protein peak was found in fractions with a density distributed around 1.45 g/ml. The bottom fraction from this dissociative density gradient profile was corn-
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I=ig. 2. Distribution of uronic acid and protein in 4 M guanidine • HCI extracted proteoglycans after equilibrium density gradient centrifugation carried out under dissociative conditions. O-O, uronic acid; A---A, protein (A28o,m); A-A, density g/ml. pletely excluded from Sepharose 6B and nothing was retarded (Fig. 3B). This was not the case when the total 4 M guanidine extract was applied to Sepharose 6B prior to the density gradient centrifugation step. Fig. 3A shows that in this uncentrifuged material a certain proportion of the proteoglycan penetrated the gel and in addition, a component with a relatively high protein content appeared towards the end of the elution profile. The proteoglycan which penetrated the gel (Fig. 3A) represents only a minor portion of the total uronate, but possesses a higher specific radioactivity (3900 dpm/ #mole of uronate) than does the excluded fraction (1100 dpm/#mole of uronate). Virtually the same distribution on Sepharose 6B as that obtained for the 4 M guanidine • HCI extract purified by density gradient centrifugation, was obtained for the nonpurified 4 M guanidine extract which had been preceded by a 0.15 NaCl extraction (Fig. 3C). The material extracted with 0.15 M NaCI and recovered by cetyl pyridinium chloride precipitation was retarded on Sepharose 6B, as had been found previously 12. When the material recovered from the bottom fraction after density gradient centrifugation in dissociative conditions (PGS of Sajdera and Hascall 4) was fractionated on Sepharose 2B in 4 M guanidine • HCI buffered to pH 5.8, (Fig. 4a), no excluded material could be demonstrated but the uronic acid profile covered largely the region immediately after the exclusion volume. Gel filtration after density gradient centrifugation in associative conditions A third portion of the original guanidine extract was transferred by dialysis into associative conditions to allow aggregation. Again, a small amount of fine precipitate was observed in the dialysis bag. At this time, the precipitate (X) was removed by centrifugation at 27 000 × g and was found to contain approx. 2-3 % of the total uronic acid present prior to the dialysis step. The proteoglycans remaining in the
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0.4 M guanidine • HCI supernatant were subjected to density gradient centrifugation in CsC1 and the fraction with the highest buoyant density recovered from the bottom of the tube was applied to a Sepharose 2B column after removal of CsCI by dialysis against 0.15 M NaCI. The column was eluted with 0.15 M NaCI buffered to pH 5.8. This fraction should correspond to the proteoglycan complex (PGC of Sajdera and HascalP) since the link protein was not removed under the associative conditions 4'5. However, the results of gel chromatography shown in Fig. 5b indicate that under these conditions the material recovered as proteoglycan complex from the associative density gradient penetrated the gel to a much greater extent than did the proteoglycan subunit preparation made under dissociative conditions. It eluted in a region much closer to the total volume and is therefore of considerably smaller size than the material prepared under dissociative conditions. In another portion of the 4 M guanidine .HCI extract, the precipitate (X)
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PROTEOGLYCAN AGGREGATION FACTOR IN CARTILAGE
449
which occurred upon dilution was not removed and was redissolved when CsCI was added prior to density gradient centrifugation in associative conditions. As a result, the elution profile on Sepharose 2B eluted with 0.15 M NaCI (Fig. 5a) of the highest buoyant density fraction is strikingly different than that seen in Fig. 5b. Fig. 5 demonstrates that the same proteoglycan preparation can, under associative conditions give rise to large complexes which are excluded from Sepharose 2B (Fig. 5a) when X is present but exist as considerably smaller species in the absence of X (Fig. 5b). Dissociation of PGS by removal of X Proteoglycan subunit prepared from the initial 4 M guanidine HCI extract by dissociative density gradient centrifugation eluted from Sepharose 2B in the region immediately following the exclusion volume. This was true when columns were eluted with either 4 M guanidine. HC! or 0.15 M NaCI (Fig. 4a). Fractions from this separation were pooled, dialyzed in the cold and lyophilized. The dry material was redissolved in a small volume of 4 M guanidine • HCI and diluted by dialysis to 0.4 M guanidine • HCI. A small amount of precipitate (X) formed and was removed by centrifugation. Material rem~..aing in solution was applied to a column of Sepharose 2B an deluted with 0.15 M NaCI. Fig. 4b shows that the uronic acid elution profile is markedly shifted to the region much closer to the total volume than the non-dialyzed proteogiycan subunit preparation. Thus, Fig. 4 indicates that dissociatively prepared proteoglycan subunits can be further dissociated by the removal of X. •
The salt extractable fractions As shown in Fig. 6, this preparation was retarded by Scpharose 2B and eluted at a position close to the total volume. Because of the methods used in the preparation we do not know whether X was originally present in the extract and lost upon purification or whether the proteoglycans existed in the low molecular weight state in situ. The specific radioactivity of the salt extractable proteoglycan is, however, about three times that of the unextractable material 11,12 which suggests the two are from distinct tissue pools.
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450
B. ROKOSOVA et al.
DISCUSSION
The work of Sajdera and Hascall 4'5 described in the introduction led them to suggest that "proteoglycan subunit" or PGS was probably the basic unit produced by the chondrocyte and that its aggregation into larger complexes gave rise to the very high molecular weight species which could be isolated from cartilage. This was somewhat modified by Rosenberg et a l . ' 4.t s who were able to demonstrate the presence of a smaller unit than proteoglycan subunit in dissociative extracts of cartilage. This smaller unit was identical to material referred to as PPL 3 isolated from cartilage by Pal et al. 6 using high speed homogenisation or disruptive procedures. Rosenberg et a l ) 4 , i s considered it to be a monomer which could form dimers (or PGS). By electron microscopic technique they were able to show that the dimers but not monomers were capable of further aggregation provided a linking glycoprotein was present. The results presented here are in agreement with the work of Rosenberg e t al. ~4, ~5 in that we show that proteoglycan can be isolated from tissue in at least three aggregation states (Figs 4a, 5a and 6) and in our demonstration of the presence of a proteoglycan species of a lower molecular weight than proteoglycan subunit (perhaps a monomer). Whether the "monomer" exists as such in the tissue cannot be determined if dissociative extraction is used. Its presence in the extract may merely represent dissociation (by the extractant) of higher aggregates existing in vivo. However, its presence can be demonstrated also in tissue extracts made with 0.15 M NaCI and the highest rate of precursor uptake into this low molecular weight proteoglycan 12 defines this as a product of synthesis rather than of degradation. In addition, on the basis of results obtained by a combination of density gradient centrifugation and gel chromatography, our results suggest that another linking factor, distinct from the linking glycoprotein of Hascall and Sajdera s mediates aggregation of smaller proteoglycans into PGS-like macromolecules. For simplicity, we have referred to this factor as X. Proteoglycans from the same dissociative extract can form large complexes excluded from Sepharose 2B when transferred into associative conditions, provided that the glycoprotein link 4's and X are present (Figs 1 and 5a). Removal of the glycoprotein link by density gradient centrifugation in dissociative conditions gives rise predominantly to species of the proteoglycan subunit type 4'5 (Fig. 4a). When X is removed from the isolated proteoglycan subunit then the elution profile on Sepharose 2B is shifted toward the region of a smaller molecular size as shown in Fig. 4b. This indicates that the presence of X was required for the subunit PGS to retain its molecular weight characteristics when analyzed by gel filtration. In the absence of this factor proteoglycan complex cannot be formed and even under the favorable associative conditions smaller proteoglycans predominate (Fig. 5b). Whether a similar X factor is required for proteoglycan aggregation in other tissues than rabbit ear cartilage is not known at this time, but preliminary data indicate that a similar phenomenon occurs in bovine tracheal cartilage. Other than the determination of uronic acid, no further characterization of X was attempted because of the extremely small amount of a fine precipitate which can be recovered. This may also be a reason why it remained undetected by others. Larger scale preparations are being carried out to enable further analysis.
P R O T E O G L Y C A N A G G R E G A T I O N FACTOR IN C A R T I L A G E
451
The presence of a small amount of a lower molecular weight proteoglycan can be demonstrated in 4 M guanidine extracts. These can be detected when the extract is subjected to gel filtration on Sepharose 6B (Fig. 3A) but they are absent if the tissue has been pre-extracted with 0.15 M NaCI (Fig. 3C) and they were not found in the proteoglycan subunit recovered from the two bottom fractions after density gradient centrifugation (Fig. 3B). On the other hand, extraction of cartilage with isotonic solutions of NaC1 brings into solution proteoglycans which are exclusively of the small molecular weight type and which represent less than 10 % of the total proteoglycan12. Thus salt extractable low molecular weight material may be present in the tissue in addition to that which is aggregated into the larger complexes of the "proteoglycan subunit" and "proteoglycan complex" type. We have previously shown that this extractable material and its chondroitin sulfate side chains had much higher specific activities than the residual or unextractable proteoglycan when cartilage preincubated with labeled glucose was used tt'12. This implies that the lower molecular weight, salt extractable fraction represents more recently synthesized material. Since the aggregated forms (Figs 4a and 5a) can (in the absence of X) be dissociated into similar low molecular weight material (Figs 4b, 5b and 6) which can associate in vitro into larger complexes in the presence of X (Figs 5a and 5b) it is logical to suppose that newly synthesized units can aggregate in vioo into larger forms. This aggregation is probably a fast process, since the highly labeled low molecular weight proteoglycan represents only a minor portion of the total. Other observations of greater incorporation of radioactive precursors into low molecular weight proteoglycans have been made t6'~7. Attempts to demonstrate a precursor-product relationship between these fractions and the more highly aggregated proteoglycans were unsuccessful 16, t s perhaps because the extraction procedures were dissociative or disruptive which may have mixed newly synthesized low molecular weight proteoglycan with older material which existed in aggregate form prior to extraction. That such mixing can occur was observed by Campo et al. i s who added radioactive PPL3 to nonradioactive cartilage and homogenized the mixture. Upon subsequent fractionation the label was found to be randomly distributed between all of the proteoglycan subfractions. The demonstration that isotonic salt extracts contain only the low molecular weight type of proteoglycan will permit us to approach the question of any precursor product relationships without the complications mentioned above. Fig. 6 indicates a good deal of heterogeneity in the low molecular weight 0.15 M NaCI extractable proteoglycan and it is possible that even this fraction does not represent the smallest elementary particle. The recent work of Woodward et al. 19 suggests that this may in fact be the case. On the basis of ultracentrifuge and viscosity studies carried out in high salt concentrations they found that the monomeric unit of dissociatively extracted proteoglycan may have a molecular weight as low as 40 000 to 60 000 even when stringent precautions were taken against degradation. Even though the results reported here have much in common with those of workers using other cartilage tissues, they also differ in several respects and we should always bear in mind that phenomena observed in rabbit ear cartilage may not be the same as those occurring in bovine nasal or pig laryngeal cartilage. The converse is also true.
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B. ROKOSOVA et al,
ACKNOWLEDGEMENTS
The technical assistance of Mrs Kathryn Johnson and Mrs Barbara Jelen are gratefully acknowledged. Supported in part by N.I.H. grant HL-14126-12 and by a grant from the American Heart Association. REFERENCES 1 2 3 4 5 6 7 8 9 10 !1 12 13 14 15 16 17 18 19
Meyer, K. (1966) Fed. Proc. 34, 1046 Muir, H. and Jacobs, S. (1967) Biochem. J. 103, 367-374 Brandt, K. D. and Muir, H. (1969)Blochem. J. 114, 871-876 Sajdera, S. W. and Hascall, V. C. (1969) J. Biol. Chem. 244, 77-87 Hascall, V. C. and Sajdera, S. W. (1969) J. Biol. Chem, 244, 2384-2396 Pal, S., Doganges, P. T. and Schubert, M. (1966) J. Biol. Chem. 241, 4261-4266 Tsiganos, C. P., Hardingham, T. E. and Muir, H. (1971) Btochim. Biophys. Acta 229, 529-534 Mashburn, T. A. and Hoffman, P. (1971) J. Biol. Chem. 246, 6497-6506 Tsiganos, C. P. and Muir, H. (1969) Biochem. J. !13, 885-894 Seraflni-Francassini, A. and Stimson, W. (1971) FEBS Lett. 15, 245-248 Rokosova, B. and Bentley, J. P. (1972) Blochlm. Btophys. Acta 264, 98-102 Rokosova, B. and Bentley, J. P. (1973) Biochim. Biophys. Acta 297, 473--485 Bentley, J. P. and Rokosova, B. (1970) Biochem. J. 116, 329-336 Rosenberg, L., Pal, S., Beale, R. and Schubert, M. (1970) J. Biol. Chem. 245, 4112-4122 Rosenberg, L., Hellman, W. and Kleinschmidt, A. K. (1970) J. Biol. Chem. 245, 4123-4130 Hardingham, T. E. and Muir, H. (1972) Biochem. J. 126, 791-803 Serafini-Francassini, A. and Stimson, W. H. (1972) FEBS Lett. 26, 336-340 Campo, R. D., Bielen, R. J. and Hetherington, J. (1972) Biochim. Biophys. Acta 261, 136-142 Woodward, C. B., Hranisavljevic, J. and Davidson, E. A. (1972) Biochemistry 11, 1168-1176