in situ cross-linking of cartilage proteoglycans

Journal of Orthopaedic Research 8:18%198 Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society

In Situ Cross-Linking of Cartilage Proteoglycans

Frank M. Phillips, Lawrence A. Pottenger, and "Rick V. Hay

Departments of Orthopaedic Surgery and Rehabilitation and *Pathology, The University of Chicago, Chicago, Illinois, U . S . A .

~~~~~ ~

Summary: Although the in vitro interactions between purified cartilage matrix components have been studied extensively, little is known about these interactions in situ. In this study, cartilage was treated with a cross-linking reagent with a span of 1.2 nm between its reactive terminal groups in order to preserve the native relationships between closely associated matrix components throughout extraction, purification, and preparation for electron microscopy. After in situ cross-linking, electron microscopy and gel chromatography revealed that about one-half of the guanidine hydrochloride extractable proteoglycans were polymeric, usually with two to five proteoglycan subunits in each polymer. Cross-linking consistently involved the thin segments of the proteoglycan subunits. Some of the proteoglycan polymers were capable of binding hyaluronic acid and were parts of aggregates under associative conditions. SDS-polyacrylamide gel electrophoresis revealed that link proteins were present within the polymers, and studies in which purified proteoglycans were cross-linked in vitro confirmed that the link proteins increased the proportion of polymeric proteoglycans. These findings suggest that individual proteoglycans within cartilage have intimate associations with other proteoglycans that are mediated by link proteins. Key Words: Proteoglycan-Link protein- Aggregate-Cross-linking-Poly mer.

The biomechanical properties of cartilage result in part from the structure formed when proteoglycans are enmeshed within and constrained by a dense mesh of collagen fibers (27). The proteoglycans exist in an underhydrated, highly concentrated state and as such exert considerable osmotic pres- sure, assuring resiliency of the tissue to external compressive forces (14,19). Proteoglycans are im- mobilized within the cartilage extracellular matrix by attachments to matrix components as well as by being physically trapped by the collagen mesh

Received October 26, 1988; accepted June 27, 1989. Address correspondence and reprint requests to Dr. L. A.

Pottenger at Section of Orthopaedics, The University of Chicago Medical Center, Box 102, 5841 South Maryland Ave., Chicago, IL 60637, U.S.A.

(21,24). In osteoarthritis (18) and rheumatoid arthri- tis (1 l), cartilage proteoglycan concentrations fall, perhaps due to altered interactions between proteoglycans and other matrix proteins.

Interactions between the various cartilage matrix components have stimulated considerable study. Extracted cartilage proteoglycans are able to bind to hyaluronic acid and to link and other matrix proteins, and are thought to attach to collagen (12,33). Link proteins are able to attach to collagen (7), to self-associate into polymers, and to bind simulta- neously to proteoglycan monomers and hyaluronic acid (33). Interaction between proteoglycans, link proteins, and hyaluronic acid allows formation of aggregates in which many proteoglycans attach to individual hyaluronic acid chains (33).

Two previous studies using light scattering techniques have shown that purified cartilage proteogly-

189

190 F . M. PHILLIPS ET AL.

cans are able to form dimers at 150 mM ionic strength, but these tend to dissociate at higher ionic strengths (26,30). Self-associated proteoglycans appear to attach through their protein cores rather than their carbohydrate side chains. It has not been determined whether self-associated proteoglycans are able to bind link proteins or hyaluronic acid. Proteoglycan aggregate morphology has also been studied extensively with electron microscopic monolayer techniques (3,4,19,35). However, using these techniques, large numbers of proteoglycan dimers have not been observed. Most electron microscopic monolayer techniques involve placing proteoglycans in high ionic strength solutions such as 1M ammonium acetate prior to grid preparation (4,19,35). This treatment may cause artifactual disruption of physiologic, ionic strength dependent proteoglycan interactions, such as proteoglycan self-association.

Efficient extraction and purification of the matrix components from cartilage requires dissociation of in situ interactions with chaotropic agents such as 4M guanidine hydrochloride. While disruption of some intermolecular interactions may be reversible, others may be permanently destroyed by the dena- turation process. One method of studying interactions between molecules within tissue is to co- valently cross-link the molecules prior to extraction from the tissue. This approach tends to preserve the native interactions in spite of subsequent dissociation and purification procedures.

Cross-linking reagents are short molecules with reactive functional groups at each terminal. Since the reactive groups are between 0.5 and 1.2 nm apart depending on the reagent used, they are able to cross-link proteins that are intimately associated. The use of bifunctional cross-linking reagents has been applied to a wide variety of macromolecules for the purpose of determining the “nearest- neighbor” relationships on cell membranes (16), the number of protomers in an assembly (31), and the self-association properties of molecules (32). To our knowledge, this technique has not been applied to the problem of cartilage structure.

In this study, proteoglycan interactions in cartilage were stabilized with bifunctional cross-linking reagents prior to disruption with guanidine hydrochloride. Extracted cross-linked complexes containing proteoglycans were analyzed and compared to proteoglycans that were subjected to cross- linking after purification. The cross-linking reagent, dithiobis(succinimidy1 propionate), was employed.

It reacts with lysine residues, has a cross-linking range of approximately 1.2 nm, and contains a re- ducible disulfide bond that can be chemically cleaved so that proteins cross-linked to each other can be reconverted to their monomeric forms (16,17).

MATERIALS AND METHODS

Fresh bovine nasal cartilage (BNC) was obtained from a local slaughterhouse, immediately placed on ice, and frozen within 2 h. Some of the tissue was used for in situ cross-linking studies. Proteoglycans and link proteins were purified from the remainder of the tissue for studies of cross-linking of purified cartilage components.

The guanidine hydrochloride (GdnHC1) was the “ultrapure” grade from Schwarz/Mann Biotech. (Cleveland, OH, U.S.A.). The cesium chloride (CsC1) (Grade l), 2-(N-morpholino)-ethanesulfonic acid (MES), dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride, benzamine hydrochloride, and cytochrome c were purchased from Sigma (St. Louis, MO, U.S.A.); the dithiobis (succinimidyl propionate) (DTSP) was purchased from Fluka (Ronkonkoma, NY, U.S.A.) and the 6-aminohexanoic acid from Eastman (Rochester, NY, U.S.A.). Dialysis tubing (12,00CL14,000 MW cutoff) was purchased from Spectrapor (Los Angeles, CA, U.S.A.). All other materials were of reagent grade.

DTSP Cross-Linking in situ

Forty micron cartilage sections were prepared with a cryostat. In order to reduce proteoglycan extraction during the cross-linking reaction, which would lead to proteoglycan cross-linking outside of the cartilage, the sections were pre-extracted for 24 h at 4°C with 15 volumes of standard buffer containing inhibitors of proteolysis (0.05 ionic strength phosphate buffer, pH 7.4, with 100 mM NaCl, 10 mM EDTA, 5 mM benzamidine-HC1, 100 mM 6- aminohexanoic acid, 1 mM phenylmethylsulfonyl fluoride, and 10 mM N-ethylmaleimide). Pre- extracted cartilage samples were thoroughly rinsed in 150 mM NaC1, then mixed with 10 volumes of MES buffer [lo0 mM NaCl and 50 mM MES (pH 7.5)]. Cross-linking was performed at room temperature for 6 h using 300 pg of DTSP (dissolved in DMSO to 4 mg/ml) per ml of cartilage-buffer mixture. Unreacted DTSP was inactivated by addition of 50 pl of 1M ammonium acetate/ml of reaction

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IN SITU PROTEOGL YCAN CROSS-LINKING 191

mixture. The control samples were treated similarly, but without DTSP.

After quenching the cross-linking reaction, the fluid was decanted and the cartilage sections were extracted with 4M GdnHCl in standard buffer for 24 h. Proteoglycan aggregates (Al) and monomers (AlDID1) were purified as described below. Pro- teoglycan concentrations were estimated by uronic acid analysis (2) at all stages of the cross-linking reaction and proteoglycan purification, for both the control and cross-linked samples.

Isolation and Purification of Proteoglycans

Bovine nasal cartilage was extracted with 4M GdnHCl for 48 h. The extracts were dialyzed against nine volumes of buffer to promote aggregation and then subjected to CsCl density gradient centrifugation under associative conditions. Puri- fied aggregates (A1 fraction) were collected as previously described (13). A1 fractions were subjected to two dissociative CsCl density gradient centrifu- gations in 4M GdnHCl to purify proteoglycan monomers (AlDlDl) (13). Link proteins, which were separated from the proteoglycans by the second centrifugation, were further purified by another dissociative CsCl density gradient centrifugation and two gel permeation chromatographies on a Sephacryl S-200 column (33). Protein concentrations were determined by the Lowry procedure (1). When proteoglycans and link protein were com- bined, mixtures contained molar equivalents of proteoglycan monomers (AlDlDl) and link proteins, assuming a proteoglycan monomer molecular weight of 2.5 x lo6 Da.

DTSP Cross-Linking of Purified Proteoglycans

Cross-linking reactions using similar techniques were performed on (i) A1 fraction, (ii) purified proteoglycans (AlDlDI), and (iii) purified proteoglycan (AlDlDl)-link protein mixtures. The A1 fraction was obtained from cartilage that had been pre- extracted in buffer, so that the results would be comparable with the in situ cross-linking experi- ments. Purified proteoglycans for cross-linking were derived from non-pre-extracted cartilage, so the proteoglycans would be similar to those used in previous published studies.

Proteoglycan solutions (approximately 20 mg/ml) that had been dialyzed against 150 mM NaCl were diluted with equal volumes of MES buffer. Cross-

linking was performed at room temperature for 4 min to 6 h using 150 pg/ml of DTSP (dissolved to 4 mg/ml in DMSO) (16). The reaction was stopped by adding 50 ~1 of 1M ammonium acetate/ml of reaction mixture. Incubation of samples for 6 h gave results identical with those obtained on incubation for 4 min. Control samples were treated in the same fashion except that DTSP was omitted.

Electron Microscopy

All fractions were dialyzed against 150 mM NaCl prior to preparation for electron microscopy (EM). A modification of the Kleinschmidt protein monolayer electron microscopic technique was used, in which proteoglycan solutions were diluted with 1M ammonium acetate in cytochrome c to produce a solution containing 4 Fg/ml of proteoglycan and 0.1% cytochrome c (4,28). The proteoglycan- cytochrome c solution was released on the surface of a 0.3M ammonium acetate solution, and copper grids coated with 3% nitrocellulose in amyl acetate were used to pick up the proteoglycan-cytochrome c film. Electron micrographs were taken with a Siemens 101 electron microscope at 4 to 20,000 times magnification. Grid windows were selected randomly and photographed.

All proteoglycans in the photographs, including monomers, polymers, and proteoglycans within aggregates, were counted blindly. Proteoglycan subunits that appeared to attach to each other without any hyaluronic acid discernable were counted as polymeric proteoglycans. Any polymers containing more than 20 proteoglycan subunits, or having an obvious central hyaluronic acid core, or having the typical morphology of an aggregate, were consid- ered to be aggregates. These accounted for less than 1% of the proteoglycans seen in AlDlDl fractions. Numerous photographs of each sample were studied. Each photograph of a particular sample was treated as a separate collection of data, and a Mi- crosoft Excel computer program was used to calcu- late the percentages, means, and standard errors of the electron microscopic proteoglycan counts.

Column Chromatography

AlDlDl fractions of in situ cross-linked proteoglycans and controls were placed on a Sepharose CL-2B column (120 x 0.8 cm), and eluted with 490 mM ammonium acetate, 10 mM MES solution (pH 7.0) at a rate of 1.2 ml/h. The column was calibrated

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192 F. M . PHILLIPS ET AL.

with glucuronolactone. The column effluent was monitored for uronic acid (2 ) .

SDSPolyacrylamide Gel Electrophoresis

The AlDlDl fractions purified from BNC cross- linked in situ as well as from non-cross-linked BNC, the DTSP-treated A1 and nontreated control A1 fractions, and link proteins purified from non- cross-linked BNC were subjected to sodium dodec- yl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on slab gels cast with 10% acryla- mide:bisacrylamide (at a ratio of 37.5: 1 WIW) stabilized with linear polyacrylamide (9). Cross-linked species were either left intact as nonreduced polymers, or reduced to component monomers by cleavage of the DTSP disulfide bond with 10% 2- mercaptoethanol (16) prior to electrophoresis. Gels were fixed and stained with Coomassie brilliant blue R-250.

RESULTS

In situ Proteoglycan Cross-Linking

The proteoglycan extraction profiles of the cross- linked and control samples are shown in Table 1. After cross-linking within cartilage, 30% fewer proteoglycans were extracted from the cartilage with 4M GdnHCl than in the non-cross-linked control. The structure of these unextractable proteoglycans is the subject of further research, but in the present study only those proteoglycans that could be extracted with 4M GdnHCl after cross-linking were studied further.

Typical elution profiles for AlDlDl fractions derived from the DTSP-treated and control cartilage

TABLE 1. Proteoglycan extraction from cartilage cross-linked with DTSP

Untreated DTSP Reaction control” cross-linked”

Buffer extracta 30.7% 32.2% Cross-linking reaction 7.5%‘ 2.5% 4M GdnHCl extract 49.8% 25.3% Residual uronic acid

in cartilage 10.0% 40.0%

Buffer extract represents those proteoglycans pre-extracted

% of total cartilage uronic acid extracted at the various

Exposed to reagents of cross-linking reaction without DTSP.

from cartilage prior to the cross-linking reactions.

stages.

samples subjected to Sepharose CL-2B gel chromatography are given in Fig. 1. The cross-linked sample contained an early peak that suggests the presence of larger molecular weight cross-linked proteoglycan complexes. This impression was confirmed by EM. The proteoglycans that eluted in the control samples and in the later peak of the cross-linked samples were found to be predominantly monomeric.

When AlDlDl fractions in situ cross-linked proteoglycans were first reduced with 2-mercaptoethanol in order to split the cross-linkages, and then subjected to SDS-PAGE, stainable bands corresponding to the link proteins were observed (Fig. 2, lane 4) . Two bands (M, of 34 and 26 kDa) were also noted but were too faint to be photographed. These bands may correspond to link protein frag- ments previously reported (23). No protein bands of M , greater than the link proteins were observed even on gels capable of resolving proteins up to approximately 600 kDa (data not shown). No link proteins were dissociated by reduction of non- cross-linked AlDlDl (Fig. 2 , lane 3) , and no link proteins were recovered when AlDlDl of in situ cross-linked proteoglycans were not reduced prior to electrophoresis (Fig. 2, lane 9).

When the non-cross-linked A1 fraction was elec- trophoresed, stainable bands corresponding to the link proteins were observed in both the reduced (Fig. 2 , lane 1) and nonreduced (Fig. 2, lane 6) samples. When the cross-linked A1 fraction was analyzed before reduction, faint bands corresponding to small amounts of link proteins were observed

Sepharose CL-PB _ _ _ DTSP cross-linked proteoglycans

c5

non cross-linked proteoglycans Q,

A c

50

0 vo

Tube Number FIG. 1. Gel chromatography of purified proteoglycans. Seph- arose CL-2B gel chromatography of A l D l D l fractions extracted from DTSP- treated (broken line) or -untreated (solid line) cartilage. Fractions were assayed for uronic acid.

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97,

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1 2 3 4 5 6 7 8 9 1 0 FIG. 2. SDS-PAGE analysis of cross-linked cartilage components. Purified A1 that was cross-linked in vitro as described in the text as well as a non-cross-linked control A1 sample were subject to SDS-PAGE. Proteoglycans (A1 D l D1) were purified from BNC that was cross-linked in situ with DTSP (BNCDTSP). As controls, proteoglycans and link proteins were separately purified from non-cross-linked BNC. For lanes 1-5, samples were dissolved in electrophoresis sample buffer at 95°C for 3 min and then reduced with 10% 2- mercaptoethanol prior to electrophoresis. For lanes 6-10, samples were dissolved in buffer but not reduced with 2- mercaptoethanol. A composite photograph of the gels is shown. Lanes correspond to samples as follows: non- cross-linked Al, lanes 1 and 6 (0.5 mg proteoglycans); cross- linked A l , lanes 2 and 7 (0.5 mg); proteoglycan from non- cross-linked BNC, lanes 3 and 8 (0.5 mg); proteoglycan from BNC-DTSP, lanes 4 and 9 (0.5 mg); and purified link proteins, lanes 5 and 10 (5 pg). M, values and reference mobilities for standard proteins (phosphorylase b, bovine serum albumin, ovalbumin, and carbonic anhydrase) are given left.

(Fig. 2, lane 7). After reduction of the DTSP cross- linkage, large amounts of link proteins were seen (Fig. 2, lane 2), with the amount seen constituting an approximately equivalent proportion by mass to that seen in the non-cross-linked A1 fraction (Fig. 2,

lane 1). Cross-linked link protein dimers and tet- ramers were never observed. Nonreduced link proteins exhibited faster mobilities than reduced link proteins as has been noted previously (33).

When the AlDlDl fractions were studied by EM, proteoglycans that were attached to other proteoglycans to form polymers were seen in all of the samples studied. Proteoglycan subunits of these polymers demonstrated a morphology similar to the proteoglycan components of aggregates. This con- sisted of “thick” and “thin” segments that were similar to those previously reported (4). Dimers contained proteoglycan subunits consistently linked to one another at their thin segments with wide spacing between their thick segments (Fig. 3). Tri- mers and more complex polymers had stellate ap- pearances, the binding again involving the thin segments, such that the thick segments were peripher- ally oriented. This type of binding was observed in proteoglycan polymers, in both the presence and absence of DTSP. Only proteoglycans with clearly visible thick and thin segments were counted as polymeric subunits. Occasionally, a thin strand without a contiguous thick segment was seen to emerge from a polymer (Fig. 3). These thin strands, apparently participating in polymerization, may represent partially digested proteoglycans that maintain their ability to associate with other proteoglycans .

Occasional polymers in which the terminal part of the thin segment of one proteoglycan was linked to the chondroitin sulfate-rich carboxy-terminal end of

FIG. 3. Electron micrograph of the A1 D1 D1 fraction extracted from DTSP-treated cartilage. Pro- teoglycans appear to be linked together at their thin segments (arrow) to form polymers. Dimers (D) and trimers (T) were the most frequently seen polymers. Occasional thin strands without contiguous thick segments were seen to emerge from polymers (arrowheads). Bar = 500 nm.

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194 F. M. PHILLIPS ET AL.

a second proteoglycan, or in which two subunits appeared to be linked at their carboxy-terminal ends, were seen. These less common types of linkages seemed to link proteoglycans of different polymers together (Fig. 4). Linkages did not appear to involve the thick segments of proteoglycans other than at their carboxy-terminal ends.

Forty-seven percent of all of the proteoglycans counted in the AlDlDl fraction derived from the DTSP-treated cartilage were in polymeric form (Ta- ble 2). Eighty-seven percent of these polymers contained five or fewer proteoglycan subunits, with dimers predominating. The larger polymers, partic- ularly those containing more than eight proteoglycan subunits, appeared as though they were formed by two or more smaller polymers linked together (Fig. 4). In the non-cross-linked but similarly extracted and purified cartilage control, only 12.6% of proteoglycans were polymeric. The difference between polymer counts in the in situ cross-linked and control samples cannot be explained by differential extractibility of proteoglycans with GdnHCl. Even if it were assumed that all of the proteoglycans that were not extracted from the cartilage because of in situ cross-linking were monomers, the proportion of polymeric proteoglycans from the in situ cross- linked cartilage would still be approximately twice that of the control.

In order to determine whether these cross-linked proteoglycans existed as discrete nonaggregated polymers within cartilage or were in some way capable of binding hyaluronic acid and therefore part of aggregates, the A1 fraction derived from the in situ cross-linked sample was studied by EM (Table

3). A total of 72.8% of the proteoglycans in the A1 fraction appeared to be involved in aggregation with hyaluronic acid, 20.4% appeared to exist as nonaggregated monomers, and the remaining 6.9% were in nonaggregated polymeric forms composed of up to seven proteoglycans. Since 47.1% of the purified proteoglycans (AlDlDl) was cross-linked, a large proportion of these cross-linked proteoglycans must have been aggregating with hyaluronic acid while in the A1 fraction.

To assess the proportion of proteoglycans within reconstituted aggregates that were able to be cross- linked, the A1 fraction from a 4M GdnHCl extract of cartilage that had been pre-extracted with standard buffer was cross-linked. The A1 fraction under associative conditions was reacted with DTSP and then subjected to two dissociative CsCl centrifuga- tions. A total of 22.3% of the proteoglycans in the resulting AID ID1 fraction were polymeric (Ta- ble 2).

Cross-Linking of Purified Proteoglycans

EM revealed that 7.5% of proteoglycans in the AlDlD 1 fractions derived from untreated cartilage that had not been pre-extracted were polymeric (Table 4). This percentage was unchanged by cross- linking of the purified proteoglycans with DTSP. When combinations of purified proteoglycans and link proteins were studied, 10.8% of proteoglycans were polymeric in control samples, and 15.3% of proteoglycans were seen to exist as polymers after DTSP cross-linking. In all of the samples studied, dimers were the most frequent polymeric form,

FIG. 4. Electron micrograph of the A1 D1 D1 fraction extracted from DTSP-treated cartilage. A more complex polymer (P) seems to be formed by the association between two simpler polymers (arrows). Other polymeric forms are also present. Bar = 500 nm.



TABLE 2. Percentage polymers in AlDlDl fractions counted on monolayer electron microscopy

Polymer distribution according to number of subunits

Number of Polymers 2 3 4 5 6-10 Sample DTSP n PGs counted (% i- SEM) (%I (%I (%) (%I (%I > 10

Cartilage" + 5 1,097 47.1 f 4.0 19.2 11.2 6.4 4.3 6.0 0.0 Cartilage - 4 903 12.6 f 0.8 7.3 3.7 0.4 0.6 0.6 0.0 Alb + 5 1,513 22.3 f 1.9 13.4 5.4 2.5 1 .o 0.0 0.0

+ or - denotes the presence or absence of DTSP; PGs, proteoglycans. " Cartilage was treated with DTSP and then purified to the AlDlDl fraction.

A1 fraction derived from pre-extracted cartilage was treated with DTSP and then purified to the AlDlDl fraction.

with lesser proportions of more complex polymers (Tables 2 and 4).

DISCUSSION

In situ cross-linking of proteoglycans enables one to overcome some of the limitations imposed by conventional dissociative extraction of proteoglycans from cartilage. The proteoglycans were cross- linked in cartilage so that when they were extracted, their native relationships to one another were maintained. The decreased extraction after cross-linking suggests that a proportion of proteoglycans in cartilage may be so closely associated with insoluble matrix proteins, such as collagen, that once cross-linked to these proteins, the proteoglycans are no longer extractable with 4M GdnHC1. It is also possible that cross-linking may have caused proteoglycans to become entangled with the unextractable components, without actually being directly cross-linked to each other.

Electron microscopy and gel chromatography confirmed that about one-half of the proteoglycans that were extracted after cross-linking were polymeric, with dimers being the predominant form. Polyacrylamide electrophoresis of the purified extracts after reduction of the cross-linkages revealed that link proteins had been cross-linked to the polymeric proteoglycans in situ (Fig. 2). A large proportion of these polymeric proteoglycans were components of aggregates under associative conditions, indicating that at least some of the polymeric proteoglycans could bind hyaluronic acid.

A major objection to the usefulness of cross- linking techniques has been the possibility that random collision-dependent cross-linking could occur at significant frequencies (20). However, studies employing concentrated hemoglobin solutions as a model system demonstrate that such collisional cross-linking occurs under extreme conditions such as very high protein concentrations and long time periods of exposure to the cross-linking reagent (15). In the present study, nonspecific cross-linking because of random collisions of unassociated proteoglycans appears unlikely. Proteoglycans that are strongly negatively charged would tend to repel one another rather than have a high frequency of random collisions. Addition of DTSP to purified proteoglycans (AlDlDl) did not result in cross-linking of proteoglycan monomers (Table 4), which would be predicted in the event of random cross-linkages. In addition, only link proteins were found to be cross-linked to proteoglycans in significant amounts in situ, whereas in the event of random cross- linkages, one would have expected other soluble proteins in cartilage to have been cross-linked to the proteoglycans as well. Also, transient random collisions of monomers causing nonspecific cross- linking tends to result in larger polymeric species (32) , whereas proteoglycan dimers were the predominant polymeric form in the present study. The extremely consistent site of cross-linkage on the proteoglycans also makes a random process seem less likely.

Many factors reduce the efficiency of the cross- linking reagents. Hydrolysis of one end of the bi-

TABLE 3. Proteoglycan distribution in A1 fractions

Number of Aggregate subunits Nonaggregated monomers Nonaggregated polymers Sample DTSP n PGscounted (% f SEM) (% f SEM) (% 2 SEM)

Cartilage + 4 2,216 72.8 2 1.25 20.4 f 1.03 6.9 * 0.73 Cartilage - 3 1,000 11.8 f 4.31 16.7 2 3.38 5.5 f 0.92

+ or - denotes the presence or absence of DTSP; PGs, proteoglycans.

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TABLE 4. Percentage of polymers in AlDIDl fraction of purified proteoglycans

Polymer distribution according to number of subunits

Number of Polymers 2 3 4 5 6-10 >10 Sample DTSP n PGscounted (% i SEM) (%) (%) (%) (%) (%) (%I

AlDlDl + 6 89 1 7.5 i 1.2 7.2 0.3 0.0 0.0 0.0 0.0 AlDlDl - 14 2,085 7.5 i 1.6 4.7 0.6 0.0 0.5 0.7 1 .0 AlDlDl + link + 12 2,181 15.9 2 2.0 8.2 0.8 0.6 0.5 4.8 1 .0 AlDlDl + link - 14 2,361 10.8 i 0.9 9.4 1.4 0.0 0.0 0.0 0.0

+ or - denotes the presence or absence of DTSP; PGs, proteoglycans; link, link proteins.

functional reagent can occur, as well as intramolec- ular cross-link formation at the site of potential intermolecular cross-linking (32). Therefore, the percentages of cross-linked proteoglycans found are probably less than the actual amount of proteoglycans close enough together to be linked. The percentages may have been further reduced by our ultrastructural analysis. Unless a contact site between proteoglycans was seen on the electron micrographs, the proteoglycans were counted as monomers regardless of their proximity to one another. Some of the thin segments of the proteoglycans within a photograph are slightly out of focus and thus difficult to discern. Those proteoglycans may actually have been cross-linked at their thin segments, which were not visualized, but were counted as monomers, thereby reducing the estimated percentage of polymers. Therefore, the actual amount of potentially cross-linkable proteoglycans within cartilage may be greater than 50%.

Structures similar to the polymers identified in the present study have been observed in previous electron microscopic studies of cartilage proteoglycans. In the first report of the electron microscopic appearance of purified cartilage proteoglycans, Rosenberg et al. described monomers, dimers, and “first order aggregates” consisting of proteoglycans radiating outward in starlike fashion from a central locus (28). In a recent study on interverte- bra1 discs, Buckwalter et al. reported the presence of “clusters” of proteoglycan monomeric subunits attaching to a central point with no identifiable central hyaluronic acid filament (6). A similar arrange- ment has also been reported in dogfish cartilage (34). These “first order aggregates” and proteoglycan “clusters” closely resemble the proteoglycan polymers observed in this study and may in fact represent the same structure. Alternatively, they have been thought to represent small aggregates ( 3 , in spite of the fact that they are present under dis- aggregating conditions.

When bovine nasal cartilage is studied by conventional electron microscopic monolayer techniques without cross-linking agents, polymers are seen infrequently and represent only a small proportion of the total proteoglycans. The low proportion of polymers may be due to artifactual disruption caused by dissociative extraction of the proteoglycans from cartilage, or the high ionic strength ammonium acetate used in the electron microscopic monolayer technique. In the present study, the cross-linking reagent maintained in situ relationships during proteoglycan extraction and preparation for EM, which could account for the higher frequency of polymeric proteoglycans observed after cross-linking.

In the present electron microscopic study, 7.5% of purified proteoglycans were found to be polymeric in the absence of cross-linking (Table 4). These polymeric proteoglycans may represent a subset of proteoglycans that either self-associate or are naturally polymeric, with the polymers stable in 1M ammonium acetate. A higher proportion of these polymers were found in extracts of pre- extracted cartilage, compared to extracts from non- pre-extracted cartilage (Tables 2 and 4). Buffer pre- extraction of cartilage preferentially extracts non- aggregating proteoglycan monomers, which are smaller and may not possess the regions of the proteoglycan molecule where polymerization takes place (25). Therefore, proteoglycans remaining after pre-extraction would contain a greater proportion of proteoglycans that could form polymers. Our studies do not confirm the widespread self- association of purified proteoglycans seen with light scattering techniques (26,30).

SDS-PAGE revealed that link proteins can be cross-linked to the polymeric proteoglycans within cartilage and that large proportions of the link proteins present in purified A1 fractions are able to be cross-linked to the polymeric proteoglycans. This does not necessarily imply that the link proteins are



cross-linked to the proteoglycans at their sites of polymerization. However, in vitro addition of link proteins to AlDlDl significantly increased the percentage of polymeric proteoglycans seen with cross-linking (Table 4). A previous study has also shown that purified link proteins are able to be cross-linked to proteoglycans at the hyaluronic acid binding region by dimethylsuberimidate (DMS), a cross-linking reagent with a span similar to DTSP (10). Link proteins are able to self-associate into polymers that can bind to hyaluronic acid (33). The proteoglycan polymers observed in the present study may occur when link proteins self-associate, with the proteoglycans binding only to the self- associated link protein polymers, i.e., with no direct association between the proteoglycans themselves. It is also possible that link proteins act by attaching to the hyaluronic acid binding region so as to align the proteoglycan monomers in such a fashion that the proteoglycans themselves are associated and able to be directly cross-linked.

Recent studies have identified two domains within the thin segment of the proteoglycan subunit (8,23,29). The first domain at the amino terminus of the thin segment is thought to represent the hyaluronic acid binding region of the proteoglycan (8,23). The second domain is found within 25 nm of the first domain on the thin segment of the proteoglycan (23) and has amino acid sequences that match the two repeated disulfide loops of link proteins (8,22). It has been suggested that the second domain is able to bind link protein but probably does not have hyaluronic acid binding activity (8). This domain may represent the site of proteoglycan polymerization or another site for link protein at- tachment to proteoglycans.

In situ studies reveal a higher proportion of cross- linked proteoglycans than that seen after in vitro cross-linking of the purified A1 fraction (Table 2). Proteoglycans in cartilage are highly concentrated and this increase in polymers may be due in part to concentration-dependent interactions between proteoglycans. This type of pattern is also described for apolipoprotein self-association, where polymerization increases with concentration (3 1). Previous in vitro ultrastructural studies have reported that there is an average separation between proteoglycan subunits on an aggregate of 20 to 47 nm (4,28). In situ, proteoglycans within aggregates may, however, be more tightly packed along the hyaluronic acid chain than on in vitro reconstituted aggregates. Thus, some of the polymeric proteoglycans from

the in situ studies may represent cross-linking of immediately adjacent proteoglycan-link protein complexes that are each attached to the same hyaluronic acid chain. This would require that adjacent proteoglycan-link protein complexes be within 1.2 nm of each other on the hyaluronic acid chain, which would suggest that the complexes are in some way associated with each other.

Alternatively, only some of the proteoglycan subunits of the polymer may be directly attached to the hyaluronic acid chain of an aggregate. Previous EM of aggregates has shown proteoglycans that were apparently linked to other proteoglycans at their thin segments with only one of the thin segments actually attaching to a hyaluronic acid chain (4). Aggregation of proteoglycan polymers with hyaluronic acid would increase the charge density of the aggregates and provide an additional mechanism for the immobilization of proteoglycans within cartilage.

Cartilage matrix components exist in such high concentrations within cartilage that it is difficult to recreate similar conditions with purified components in vitro. In situ cross-linking appears to offer an excellent method for stabilizing native interactions so that they can be preserved during extraction from cartilage. Although this evidence for close proximity of molecules is grounds for suspecting a natural interaction, additional evidence is needed for

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proof of a natural bonding.

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in situ cross-linking of cartilage proteoglycans

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