Journal of Orthopnedic KeJewrr h 10631437 Rnven Press, Ltd , New York 0 1992 Orthopaedic Research Society
The Dermatan Sulfate Proteoglycans of the Adult Human Meniscus Peter J. Roughley and Robert J. White Genetics Unit, Shriners Hospital for Crippled Children, und Deportment of Surgery, McGill University, Montreal, Quebec, Cunadu
Summary: The dermatan sulfate proteoglycans decorin and biglycan were extracted from pooled adult human menisci with 4 M guanidinium chloride and purified by successive cesium chloride density gradient centrifugation, ion exchange chromatography, and gel filtration. A final yield of about 2 mg of dermatan sulfate proteoglycan per gram of wet tissue was obtained. The proteoglycan is predominantly decorin with some biglycan. and the dermatan sulfate chains contain about 70% of their uronic acid rcsidues as iduronate and possess about three times as much 4-sulfation as 6-sulfation of their N-acetylgalactosamine residues. On gel filtration under associative conditions, about half of the proteoglycan exhibits self-association. This includes most of the biglycan but also a substantial proportion of decorin. The molecules that show self-association appear to have longer dermatan sulfate chains, though there is no apparent difference in their overall composition. The predominance of decorin in the adult meniscus and its ability to interact both with itself and collagen fibrils is compatible with a role in maintaining tissue integrity and meniscus. strength. Key Words: Proteoglycan-Decorin-Biglycan-Human
In man, fibrocartilagenous intra-articular discs or menisci are present in the temporomandibular, sternoclavicular, wrist, and knee joints (16). Each knee has two menisci, whose thick peripheral border is attached to the inside of the joint capsule and that cover about two thirds of the articular surface of the tibia1 plateau (12). The menisci are approximately triangular in cross-section, being flat on their distal surface and concave on their proximal surface where they articulate with the tibia and femur, respectively. As a result of their position and intimate contact with the tibia and femur, the menisci effectively increase the contact area available for load
transmission across the joint (17), and in fact up to 55% of the load is transmitted through the menisci (18). In this way the menisci lessen the compressive forces to which the articular cartilage and the subchondral bone would be otherwise exposed. In addition to this role in weight bearing, the menisci are also important structures in maintaining joint stability. This dual nature of the meniscus is reflected in its fibrocartilagenous nature (2), showing many properties intermediate between a fibrous connective tissue and hyaline cartilage. The cells, which have the appearance of fibroblasts, have been likened to flattened chondrocytes similar to those found in the superficial zone of articular cartilage (1 1). The collagen content of the meniscus is more akin to a fibrous connective tissue with about 90% type I collagen, though 1-2% type 11 collagen characteristic of hyaline cartilage has been reported (14). The gly-
Received July 16, 1991;accepted March 25, 1992. Address correspondence and reprint requests to Dr. P. J. Roughley at Genetics Unit. Shriners Hospital for Crippled Children, 1529 Cedar Avenue. Montreal, Quebec, H3G lA6, Canada.
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cosaminoglycan content of the meniscus is about one tenth that of hyaline cartilage (l), again being more compatible with a fibrous connective tissue, though large proteoglycans similar to those extracted from hyaline cartilage have been detected (26). These large proteoglycans appear to increase in abundance with age in the human meniscus (20), suggesting a possible increased role of the tissue in weight bearing. The large proteoglycans of the adult human meniscus havc been well characterized. They contain chondroitin sulfate, kerdtan sulfate, and oligosaccharides, plus they interact with hyaluronic acid to form aggregates (26). Thus, they appear analogous in structure to the large aggregating proteoglycan of human articular cartilage, aggrecan, and are likely products of the same gene (6). Articular cartilage also contains two dermatan sulfate proteoglycans, termed decorin and biglycan (25,27). Biglycan has two dermatan sulfate chains and decorin has one. Both are the products of distinct but structurally related genes (9,19). These genes are expressed by many connective tissues, and dermatan sulfate proteoglycans may be ubiquitous in their occurrence, though the composition and relative abundance of the two proteoglycans may vary with tissue and age (5,32). It is not, therefore, surprising that the human meniscus has been reported to possess dermatan sulfate proteoglycans (20,26). However, to date there is little structural information available on these meniscal molecules in terms of thcir relationship to decorin and biglycan and the structure of their dermatan sulfate chains. The purpose of this work was to elucidate the identity and composition of the dermatan sulfate proteoglycans that are present in the adult human meniscus. METHODS Materials Cesium chloride was from Terochem Laboratories Ltd. (Edmonton, Alberta, Canada). Guanidinium chloride, agarose, and chondroitin sulfate disaccharides were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Chondroitinase ABC was from ICN Biomedicals Inc. (St. Laurent, Quebec, Canada). Sodium dodecyl sulfate, acrylamide, and methylenebisacrylamide were from Bio-Rad Laboratories (Mississauga, Ontario, Canada). Sepharose CL-4B and Sephacryl S400HR were from Pharmacia Fine Chemicals (Montreal, Quebec, Canada).
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Source of Meniscus Lateral and medial menisci were removed from both knees of four individuals (a 71-year-old woman, a 77-year-old man, a 79-year-old man, and an 81-year-old man) at the time of autopsy, within 20 h of death. In no case was there a clinical history of joint disease, though focal areas of fibrillation were apparent on the articular surfaces. The menisci were dissected free of their horns and of the peripheral region through which capsular attachment occurs. The remaining tissue was pooled for proteoglycan extraction. Extraction and Purification of Proteoglycans The meniscal tissue (21 g) was diced into small pieces (about 1 mm in each dimension) and extracted with 4 M guanidinium chloride and purified as described previously for articular cartilage (25,27). Briefly, the filtered extract was subjected to direct disyociative CsCl density gradient centrifugation at a starting density of 1.4 giml. The gradients were divided into three pools of decreasing density, termed D1, D2, and D3, where molecules in the D2 preparation had buoyant densities between 1.481.32 g/ml. The D1 preparation contains all the meniscal aggregating proteoglycan, the D2 preparation contains the dermatan sulfate proteoglycans, and the D3 preparation is rich in meniscal proteins. The yield of macromolecules in the three preparations was 74, 155, and 256 mg for D1, D2, and D3, respectively. The D2 preparation was then subjected to diethylaminoethyl (DEAE)-ion exchange chromatography to remove contaminating meniscal proteins. The dermatan sulfate proteoglycans underwent elution with 0.3 M NaCl and were obtained with a yield of 59 mg. Finally, contaminating aggregating proteoglycans and hyaluronic acid were removed by dissociative Sepharose CL-413 chromatography. The dermatan sulfate proteoglycans eluted with a K,, of 0.57 and were recovered with a yield of 41 mg. AgarosePolyacrylamide Gel Electrophoresis The sedimentation position of the dermatan sulfate proteoglycans in CsCl density gradients and their elution position on chromatography was monitored by agarose/polyacrylamide gel electrophoresis, using a slab gel system (13). Proteoglycans were identified by staining with Toluidine blue. In this system the dermatan sulfate proteoglycans migrate as a single band with a mobility greater than the aggregating proteoglycans (27).
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Sepharose CL-4B Chromatography The ability of the dermatan sulfate proteoglycans to self-associate was assessed by chromatography through Sepharose CL-4B under associative conditions, using 0.2 M sodium acetate, pH 5.5, as the eluent (25,27). These chromatography conditions were used in a preparative manner to separate the dermatan sulfate proteoglycans into two pools, the larger of which contained molecules that had undergone self-association (pool l), and the smaller of which contained molecules that remained in a monomeric form (pool 2). The core protein and dermatan sulfate composition of the molecules in these pools was studied. SDS/Polyacrylamide Gel Electrophoresis The size of the intact proteoglycans and their core proteins were estimated by SDSipolyacrylamide gel electrophoresis using 4 2 0 % gradient slab gels (25). Gels were stained successively with Coomassie brilliant blue R-250 for protein and Alcian blue for proteoglycan (8). Core proteins were released by treatment of the proteoglycans with chondroitinase ABC (27). Prior to electrophoretic analysis, samples were dialyzed into 0.125 M Tris/ HCI, pH 6.8, containing 0.1% SDS. Protein Sequence Analysis Confirmation of the presence of decorin and biglycan was performed by N-terminal amino acid sequence analysis. Samples were dissolved in water and applied to a glass fiber filter coated with Biobrene for analysis on an Applied Biosystems 470A gas phase sequencer. Resulting phenylthiohydantoin amino acid derivatives were resolved using an on-line Applied Biosystems 120A analyzer. The relative amounts of decorin and biglycan in samples was estimated from the repetitive yield of alanine at residue 3 in decorin and residue 4 in biglycan. Residues 1 and 2 in the two proteoglycans are identical and are therefore not of use for differential quantitation. This calculation assumes that any blockage of the N-terminal amino acids is similar for decorin and biglycan. Alkali Digestion The size of the dermatan sulfate chains on the proteoglycans was determined by Sephacryl
S400HR chromatography under dissociative conditions, following their release from the proteoglycans by treatment with 50 mM NaOH in the presence of 1 M NaBH, (27). The elution position of the dermatan sulfate was monitored by uronic acid analysis (3). Chondroitinase Digestion The composition of the dermatan sulfate chains was determined by analysis of the disaccharides released following digestion with chondroitinase AC or ABC (27). The disaccharides released by chondroitinase ABC were used to quantitate the proportion of 4- and 4-sulfated galactosamine residues following their resolution by high performance liquid chromatography. The proportion of iduronic acid was estimated from the difference in the absorbance at 232 nm generated by treatment with chondroitinase AC and ABC. The latter enzyme generates a chromophore in every uronic acid residue, whereas the former enzyme generates a chromophore only in glucuronic acid residues. The difference between the two absorbances therefore can be used as an estimate of iduronic acid content.
RESULTS Dermatan sulfate proteoglycans were obtained with a yield of 2 mgig of wet meniscus by a combination of density gradient centrifugation, ion exchange chromatography, and gel filtration, by an identical protocol to that previously used in the preparation of dermatan sulfate proteoglycans from human articular cartilage (27). The final purification step of dissociative Sepharose CL-4B chromatography yielded a single symmetrical peak of K,, 0.57 (Table 1). On chromatography through Sepharose CL-4B under associative conditions this preparation resolved into a number of components (Fig. l ) , including minor peaks at void volume (V,) and total volume (V,) and a major asymmetric included peak. Analysis of the column eluent by SDS/polyacrylTABLE 1. Chromatography of dermatan suljate proteoglycans through Sepharose CL-4B Conditions
Pool"
Dissociative Associative Associative
1 2
Pools are delineated in Fig. 1.
K,
Yield (mg)
0.57 0.20
41 16
0.50
20
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P . J . ROUGHLEY A N D R. J . WHITE
0.15-
,
pool1
pool2
H F - + l
W Q)
0
g 0.1 e 2rn 0.050
2o
43'
40
50
60
70
Fraction number
"0
80
4
90
"1
FIG. 1. Chromatography of meniscal dermatan sulfate proteoglycans through Sepharose CL-4B under associative conditions. The void volume (V,) and total volume (V,) of the column are indicated. Fractions 33-47 (pool 1) and 48-62 (pool 2) were collected for further analysis. Protein content of the fractions was monitored by absorbance at 280 nm (EZ8J.
amide gel electrophoresis (Fig. 2) indicated that only the major included peak contained the dermatan sulfate proteoglycans. The migration pattern on electrophoresis suggested that both decorin and biglycan were present but that decorin was the major species. The biglycan, which migrates more slowly on electrophoresis, predominated in the early part of the elution profile (pool 1). This was not too surprising as biglycan has been shown to undergo selfassociation under these conditions (25). However, this region of the column profile (pool 1) aho contained substantial amounts of a proteoglycan having a mobility similar to that of decorin in all the fractions, which has been reported not to undergo selfassociation (25). The presence of decorin throughout the column profile would suggest true interaction, rather than fortuitous overlap from a predominant monomeric population. It is possible that biglycan may exist in both double and single chain forms, and that the latter might have an electrophoretic mobility similar to decorin. Thus, the properties of the core protein of the dermatan sulfate proteoglycans were studied in more detail. For this purpose the proteoglycans were divided into predominantly self-associated (pool 1) and monomeric (pool 2) preparations (Fig. 1). The self-associating pool had a K,, of 0.20 and represented 45% of the total proteoglycan by weight, whereas the monomeric pool had a K,, of 0.50 (Table 1). On SDSipolyacrylamide gel electrophoresis after treatment with chondroitinase ABC, both proteoglycan pools showed the doublet pattern at about M, 45,000, characteristic of decorin (Fig. 3).
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Biglycan, isolated from human articular cartilage, yields a single core protein on chondroitinase treatment (27). The predominance of decorin in pool 1 was confirmed by N-terminal protein sequence analysis, as was the presence of biglycan as a relatively minor component. The amino acid yield on sequencing indicated that biglycan accounted for about 30% of the proteoglycan molecules in this pool (Table 2). Sequence analysis of pool 2 indicated the presence of mainly decorin, which accounts for about 85% of the proteoglycan molecules in the pool. To determine whether the self-associating proteoglycan pool had a different dermatan sulfate composition to the monomeric pool, the structure of the glycosaminoglycan chains was studied. On gel filtration, following release of the chains by alkaline borohydride, the dermatan sulfate from the monomeric pool 2 showed a broad size range with a K,,
94,000--m
68,000+ 45,000-
25,700 +
13,70037 40 45 50 55 Fraction number FIG. 2. SDSipolyacrylamide gel electrophoresis of fractions from the associative Sepharose CL-4B chromatography of the meniscal proteoglycans (Fig. 1). The migration position of standard proteins of known molecular weight are indicated.
635
PROTEOGLYCANS OF THE HUMAN MENISCUS 0.2
,
1 """
+DS-PG
I
Pool 1
0
2 0.15 W
e D S - P G II
M, 45,000-+
40
50
60
70
80
90
Fraction number FIG. 4. Chromatography of meniscal dermatan sulfate proteoglycans through Sephacryl S400HR after incubation in the presence of alkaline borohydride. The two profiles represent pool 1 and pool 2 isolated by associative Sepharose CL-4B chromatography of the intact proteoglycans (Fig. 1). Uronic acid content of the fractions was monitored by absorbance at 530 nm (E530).
Pool Chase A5C
1 2 1 2
+ + - -
FIG. 3. SDSipolyacrylamide gel electrophoresis of pools from the associative Sepharose CL-4B chromatography of the meniscal proteoglycans (Fig. 1) after incubation in the presence or absence of chondroitinase ABC. The migration position of ovalbumin (M,45,000) is indicated. DS-PG, dermatan sulfate proteoglycan.
in the degree of epimerization of uronic acid residues in the two preparations. In both cases, iduronic acid, measured by its resistance to modification by chondroitinase AC, represented about 70% of the total uronic acid residues (Table 2). It should be pointed out, however, that similarity in average composition does not necessarily preclude differences in monosaccharide distribution along the chains, which would not be detectable by these techniques. DISCUSSION
of 0.50 (Fig. 4 and Table 2). The dermatan sulfate from the pool 1 proteoglycans showed a prevalence of the larger-size ranges observed in pool 2 (Fig. 4), with a K,, of 0.45 (Table 2). Analysis of the component disaccharides following chondroitinase ABC treatment indicated that there was no difference in the average sulfation pattern in the two preparations. In both caseb 4-sulfation predominated over 6-sulfation in about a 3: 1 ratio (Table 2). Furthermore, there was also no apparent difference TABLE 2. Analysis of proteoglycan composition Pool
K,""
1:llb
% IdA
6:4SO,
1 2
0.45
1:2
0.50
1:s
71 70
0.35 0.35
ldA, iduronic acid. K,, is evaluated on Sephacryl S400HR. Molar ratio of higlycan (1)idecorin (11) is calculated from protein sequencing data. a
'
The adult human meniscus yielded about 2 mg/g of wet weight of dermatan sulfate proteoglycan, of which about 75% is decorin. This is similar to the yield of dermatan sulfate proteoglycan obtained from newborn human articular cartilage (27) and is also similar to the yield of aggregating proteoglycan previously purified from the adult meniscus (26). These data indicate that aggrecan, decorin, and biglycan are extracted in a weight ratio of about 4:3: 1, respectively. The presence of both aggregating proteoglycans and dermatan sulfate proteoglycans has also been reported in the articular disc of the adult bovine temporomandibular joint (22,3 1) and the chicken meniscus (24) and may be a common feature of fibrocartilages, reflecting their physiological role of having to resist both compressive and tensile forces. Indeed, fibrous connective tissues such as adult bovine tendons, which contain predominantly dermatan sulfate proteoglycans in regions subjected
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only to tension, also contain about half their proteogl ycan as large aggregating proteoglycans in regions that are exposed to compression as they pass over bone (33). The adult meniscal dermatan sulfate proteoglycans are composed mainly of decorin, with biglycan representing about 20% of the molecules. The adult bovine temporomandibular disc also possesses a high proportion of decorin (31), as does adult bovine tendon (32) and skin (23) and adult human articular cartilage (28). In contrast, young connective tissues appear to contain both decorin and biglycan as major components (5,7,27,32). At least in human articular cartilage, the deficiency in matrix biglycan in the adult can be related to a deficiency in the synthesis of this molecule by the adult chondrocytes (21). The reason for this deficiency is not clear but may relate to the functional ability of decorin to interact with collagen fibrils, a property not shared by biglycan (4). The functional role of biglycan is not known, but its need may be more important in growing rather than mature tissues, whereas the need for decorin may be essential throughout life. The dermatan sulfate chains in thc adult human meniscus contain about 70% of their uronic acid as iduronate and about 75% of their sulfate on the 4-position of the N-acetylgalactosamine. The proportion of iduronic acid in decorin or biglycan varies considerably with tissues. In bovine bone the uronic acid is all glucuronate, irrespective of age (29), whereas in bovine skin even the fetal tissue has greater than 80% iduronate (5). In bovine tendon there is about 60% iduronate and it changes little with age (15). In human articular cartilage, the proportion of iduronate would appear to increase with age from 20 to 50% (27,28), whereas in pig laryngeal cartilage there is no iduronate (28). While there is great variation in iduronate content between t he dermatan sulfate proteoglycans of different tissues, they all show considerably more 4-sulfation relative to 6-sulfation (15,21,28,29). This is even true in a tissue such as adult human articular cartilage, where the aggregating proteoglycans , presumably produced by the same cells, are predominantly 6-sulfated (21). Some connective tissues do, however, show a slight increase in the proportion of 6-sulfate with age in their dermatan sulfate proteoglycans (15). The reason for this vast array of dermatan sulfate structures is at present unclear but may relate to the ability of dermatan sulfate chains to self-associate (10). This property is not common to all dermatan
J Orthop R e s , Vol. 10. No. 5 , 1992
sulfate chains, but appears to require sequences where both iduronate and glucuronate are present. In immature bovine articular cartilage, selfassociation appears to be a property unique to biglycan but not decorin (3,and this may relate to the reported differences in their chain composition. In neonatal human articular cartilage, biglycan also appears to self-associate to a much greater degree than decorin (27). In the adult meniscus, a substantial proportion of decorin appears to self-associate. This is not too surprising if self-association is via the dermatan sulfate chains, as it is likely that some such chains on decorin may have the appropriate sequence for interaction. However, one cannot exclude the possibility that self-association is mediated by protein-protein interactions. In this respect it is of interest to note that decorin from bovine skin, which does not normally self-associate, can be induced to do so by removal of its N-linked oligosaccharides, which presumably induces an appropriate conformational change (30). Irrespective of its origin, it is possible that the self-association of decorin could relate to adult connective tissues where biglycan is deficient, as it could potentially provide a mechanism for the interaction of adjacent collagen fibrils in a tight lateral array, presumably giving the tissues increased mechanical strength. Acknowledgment: We thank the departments of pathology at the Royal Victoria Hospital and the Montreal General Hospital for the provision of autopsy facilities, Ms. N . Nikolajew for typing the manuscript, and Ms. J. Wishart for preparing the figures. This work was supported bv the Medical Research Council of Canada and the Shriners of North America.
REFERENCES 1. Arnoczky S , Adams M, DeHaven K , Eyre U, Mow V: Meniscus. In: Injury and Repair of the Musculoskeletal Soft Tissues. ed by SLY Woo. JA Buckwalter, Park Ridge, I l k nois, American Academy of Orthopaedic Surgeons, 1988, pp 487-537 2. Benjamin M, Evans EJ: Fibrocartilage. J Anat 171:l-15, 1990 3. Bitter T, Muir H: A modified uronic acid carbazole reaction. A n d Biochern 4:330-334. 1962 4. Brown DC, Vogel KG: Characteristics of the in vitro interaction of a small proteoglycan (PGTI) of bovine tendon with type I collagen. Matrix 9:468478. 1989 5. Choi HC, Johnson TL, Pal S. Tang LH, Rosenberg L , Neame PJ: Characterization of the dermatan sulfate proteoglycans, DS-PG I and DS-PG IT, from bovine articular cartilage and skin isolated by octyl-Sepharose chromatography. J niol Chem 264:287&2884, 1989 6. Doege KJ, Saski M, Kimura T, Yamada Y: Complete coding sequence and deduced primary structure of the human large aggregating proteoglycan, aggrecan. Human-specific re-
PROlEOGLYCANS OF THE HUMAN MENISCUS
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peals, and additional alternatively spliced forms. J Biol Chem 266:89&902, 1991 Fisher LW, Hawkins GR, Tuross N. Termine JD: Purification and partial characterization of small proteoglycans 1 and 11, bone sialoproteins I and 11, and osteonectin from the mineral compartment of developing human bone. J Biol Chenz 262:9702-9708, 1987 Fisher LW, Termine JD, Dejter SW, et al.: Proteoglycans of developing bone. J Biol Clzem 258:65884594. 1983 Fisher LW, Termine JD. Young MF: Deduced protein sequence of bone small proteoglycan 1 (biglycan) shows homology with proteoglycan I1 (decorin) and several nonconnective tissue proteins in a variety of species. J Biol CAem 264:4571-4576, 1989 Fransson LA, Coster L: Interaction between dermatan sulphate chains. 11. Structural studies on aggregating glycan chains and oligosaccharides with affinity for dermatansulphate substituted agarose. Biochirn Biophys Actn 582: 132-144, 1979 Ghadially FN: Fine Structure of Syncrviul Joints. London, Butterworth, 1983 Gray H: Gray’s Anatomy, 29th ed. ed by CM Goss. Philadelphia, Lea and Febiger, 1973, pp 346-355 Heinegard D, Sommarin Y, Hedbom E. Wieslandcr J . Larsson B: Assay of proteoglycan preparations using agarosepolyacrylamide gel electrophoresis. Anal Biochem 151:4148, 198.5 Herbage D: Les collagenes de I’articulation. Ann Biol Clin 44:152-155, 1986 Honda T, Katagiri K, Kuroda A, Matsunaga E, Shinkai H: Age-related changes of the dermatan sulfate containing small proteoglycans of bovine tendon. Collagen Re/ Rrs 7:171184, 1987 Jaffe HL: Metabolic, Degenerative, and 1nfTuwmatot-y Diseases of Bones and Joints. Philadelphia, Lea and Fehiger, 1975, pp 97-101 Kettlekamp DB. Jacobs AW: Tibiofernoral contact areadetermination and implications. J Bone Join1 Surg 54A:349356, 1972 Krause WR. Pope MH, Johnson RJ. Wilder DG: Mechanical changes in the knee after menisectomy. J Bone Joint Surg 58A:599404. 1976 Krusius T. Kuoslahti E: Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci USA 83:7683-7687, 1986 McNicol D, Roughley PJ: Extraction and characterization of
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proteoglycan from human meniscus. Biochem J 185:705713, 1980 Melching LI, Roughley PJ: The synthesis of dermatan sulphate proteoglycans by fetal and adult human articular cartilage. Biochem J 261:501-508, 1989 Nakano T, Scott PG: Proteoglycans of the articular disc of the bovine temporomandibular joint. I. High molecular weight chondroitin sulphate proteoglycan. Matrix 9:277283, 1989 Pearson CH, Winterbottom N, Fackre DS, Scott PG, Carpenter MR: The NH2-terminal amino acid sequence of bovine skin proteodermatan sulfate. J Biol Chem 258:1510115104, 1983 Pedrini-Mille A, Pedrini VA, Maynard JA, Vailas AC: Response of immature chicken meniscus to strenuous exercise: biochemical studies of proteoglycan and collagen. J Orthop Re.? 6:196-204, 1988 Rosenberg LC, Choi HU, Tang LH, et al.: Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilage. J Biol Chern 260:6304-6313, 1985 Roughley PJ. McNicol D, Santer V, Buckwalter J: The presence of a cartilage-like proteoglycan in the adult human meniscus. Bicrchrm J 197:77-83, 1981 Roughley PJ, White RJ: Derniatan sulfate proteoglycans of human articular carlilage. The properties of dermatan sulfate proteoglycans 1 and 11. Aiochem J 262323427, 1989 Sampaio LO, Bayliss MT, Hardingham TE, Muir H: Dermatan sulphate proteoglycans from human articular cartilage. Variation in its content with age and its structural comparison with a small chondroitin sulphate proteoglycan from pig laryngeal cartilage. Bioclwm J 254:757-764, 1988 Sato S. Kahemtulla F, Prince CW, Tomano M, Butler WT: Proteoglycans of adult bovine compact bone. Connect Tissue Res 14165-76, 1985 Scott PC, Dodd CM: Self-aggrcgation of bovine skin proteodermatan sulphate promoted by removal of the three N-linked oligosaccharides. Connect Tissue Res 24:225-236, 1990 Scott PG, Nakano T. Dodd CM. Prigle GA, Kuc IM: Protcoglycans of the articular disc of the bovine temporomandibular joint. 11. Low molecular weight dermatan sulphate proteoglycan. Mutrix 9:28&29?. 1989 Vogel KC. Evanko SP: Proteoglycans of fetal bovine tendon. J Biol Chem 262:13607-13613, 1987 Vogel K, Heinegard D: Characterization of proteoglycans from the adult bovine tendon. J B i d Chem 260:9298-9306, 1985
J Orthop Res, Vol. 10, NO. 5, I992
The Dermatan Sulfate Proteoglycans of the Adult Human Meniscus Peter J. Roughley and Robert J. White Genetics Unit, Shriners Hospital for Crippled Children, und Deportment of Surgery, McGill University, Montreal, Quebec, Cunadu
Summary: The dermatan sulfate proteoglycans decorin and biglycan were extracted from pooled adult human menisci with 4 M guanidinium chloride and purified by successive cesium chloride density gradient centrifugation, ion exchange chromatography, and gel filtration. A final yield of about 2 mg of dermatan sulfate proteoglycan per gram of wet tissue was obtained. The proteoglycan is predominantly decorin with some biglycan. and the dermatan sulfate chains contain about 70% of their uronic acid rcsidues as iduronate and possess about three times as much 4-sulfation as 6-sulfation of their N-acetylgalactosamine residues. On gel filtration under associative conditions, about half of the proteoglycan exhibits self-association. This includes most of the biglycan but also a substantial proportion of decorin. The molecules that show self-association appear to have longer dermatan sulfate chains, though there is no apparent difference in their overall composition. The predominance of decorin in the adult meniscus and its ability to interact both with itself and collagen fibrils is compatible with a role in maintaining tissue integrity and meniscus. strength. Key Words: Proteoglycan-Decorin-Biglycan-Human
In man, fibrocartilagenous intra-articular discs or menisci are present in the temporomandibular, sternoclavicular, wrist, and knee joints (16). Each knee has two menisci, whose thick peripheral border is attached to the inside of the joint capsule and that cover about two thirds of the articular surface of the tibia1 plateau (12). The menisci are approximately triangular in cross-section, being flat on their distal surface and concave on their proximal surface where they articulate with the tibia and femur, respectively. As a result of their position and intimate contact with the tibia and femur, the menisci effectively increase the contact area available for load
transmission across the joint (17), and in fact up to 55% of the load is transmitted through the menisci (18). In this way the menisci lessen the compressive forces to which the articular cartilage and the subchondral bone would be otherwise exposed. In addition to this role in weight bearing, the menisci are also important structures in maintaining joint stability. This dual nature of the meniscus is reflected in its fibrocartilagenous nature (2), showing many properties intermediate between a fibrous connective tissue and hyaline cartilage. The cells, which have the appearance of fibroblasts, have been likened to flattened chondrocytes similar to those found in the superficial zone of articular cartilage (1 1). The collagen content of the meniscus is more akin to a fibrous connective tissue with about 90% type I collagen, though 1-2% type 11 collagen characteristic of hyaline cartilage has been reported (14). The gly-
Received July 16, 1991;accepted March 25, 1992. Address correspondence and reprint requests to Dr. P. J. Roughley at Genetics Unit. Shriners Hospital for Crippled Children, 1529 Cedar Avenue. Montreal, Quebec, H3G lA6, Canada.
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cosaminoglycan content of the meniscus is about one tenth that of hyaline cartilage (l), again being more compatible with a fibrous connective tissue, though large proteoglycans similar to those extracted from hyaline cartilage have been detected (26). These large proteoglycans appear to increase in abundance with age in the human meniscus (20), suggesting a possible increased role of the tissue in weight bearing. The large proteoglycans of the adult human meniscus havc been well characterized. They contain chondroitin sulfate, kerdtan sulfate, and oligosaccharides, plus they interact with hyaluronic acid to form aggregates (26). Thus, they appear analogous in structure to the large aggregating proteoglycan of human articular cartilage, aggrecan, and are likely products of the same gene (6). Articular cartilage also contains two dermatan sulfate proteoglycans, termed decorin and biglycan (25,27). Biglycan has two dermatan sulfate chains and decorin has one. Both are the products of distinct but structurally related genes (9,19). These genes are expressed by many connective tissues, and dermatan sulfate proteoglycans may be ubiquitous in their occurrence, though the composition and relative abundance of the two proteoglycans may vary with tissue and age (5,32). It is not, therefore, surprising that the human meniscus has been reported to possess dermatan sulfate proteoglycans (20,26). However, to date there is little structural information available on these meniscal molecules in terms of thcir relationship to decorin and biglycan and the structure of their dermatan sulfate chains. The purpose of this work was to elucidate the identity and composition of the dermatan sulfate proteoglycans that are present in the adult human meniscus. METHODS Materials Cesium chloride was from Terochem Laboratories Ltd. (Edmonton, Alberta, Canada). Guanidinium chloride, agarose, and chondroitin sulfate disaccharides were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Chondroitinase ABC was from ICN Biomedicals Inc. (St. Laurent, Quebec, Canada). Sodium dodecyl sulfate, acrylamide, and methylenebisacrylamide were from Bio-Rad Laboratories (Mississauga, Ontario, Canada). Sepharose CL-4B and Sephacryl S400HR were from Pharmacia Fine Chemicals (Montreal, Quebec, Canada).
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Source of Meniscus Lateral and medial menisci were removed from both knees of four individuals (a 71-year-old woman, a 77-year-old man, a 79-year-old man, and an 81-year-old man) at the time of autopsy, within 20 h of death. In no case was there a clinical history of joint disease, though focal areas of fibrillation were apparent on the articular surfaces. The menisci were dissected free of their horns and of the peripheral region through which capsular attachment occurs. The remaining tissue was pooled for proteoglycan extraction. Extraction and Purification of Proteoglycans The meniscal tissue (21 g) was diced into small pieces (about 1 mm in each dimension) and extracted with 4 M guanidinium chloride and purified as described previously for articular cartilage (25,27). Briefly, the filtered extract was subjected to direct disyociative CsCl density gradient centrifugation at a starting density of 1.4 giml. The gradients were divided into three pools of decreasing density, termed D1, D2, and D3, where molecules in the D2 preparation had buoyant densities between 1.481.32 g/ml. The D1 preparation contains all the meniscal aggregating proteoglycan, the D2 preparation contains the dermatan sulfate proteoglycans, and the D3 preparation is rich in meniscal proteins. The yield of macromolecules in the three preparations was 74, 155, and 256 mg for D1, D2, and D3, respectively. The D2 preparation was then subjected to diethylaminoethyl (DEAE)-ion exchange chromatography to remove contaminating meniscal proteins. The dermatan sulfate proteoglycans underwent elution with 0.3 M NaCl and were obtained with a yield of 59 mg. Finally, contaminating aggregating proteoglycans and hyaluronic acid were removed by dissociative Sepharose CL-413 chromatography. The dermatan sulfate proteoglycans eluted with a K,, of 0.57 and were recovered with a yield of 41 mg. AgarosePolyacrylamide Gel Electrophoresis The sedimentation position of the dermatan sulfate proteoglycans in CsCl density gradients and their elution position on chromatography was monitored by agarose/polyacrylamide gel electrophoresis, using a slab gel system (13). Proteoglycans were identified by staining with Toluidine blue. In this system the dermatan sulfate proteoglycans migrate as a single band with a mobility greater than the aggregating proteoglycans (27).
633
PROTEOGLYCANS OF THE HUMAN MENISCUS
Sepharose CL-4B Chromatography The ability of the dermatan sulfate proteoglycans to self-associate was assessed by chromatography through Sepharose CL-4B under associative conditions, using 0.2 M sodium acetate, pH 5.5, as the eluent (25,27). These chromatography conditions were used in a preparative manner to separate the dermatan sulfate proteoglycans into two pools, the larger of which contained molecules that had undergone self-association (pool l), and the smaller of which contained molecules that remained in a monomeric form (pool 2). The core protein and dermatan sulfate composition of the molecules in these pools was studied. SDS/Polyacrylamide Gel Electrophoresis The size of the intact proteoglycans and their core proteins were estimated by SDSipolyacrylamide gel electrophoresis using 4 2 0 % gradient slab gels (25). Gels were stained successively with Coomassie brilliant blue R-250 for protein and Alcian blue for proteoglycan (8). Core proteins were released by treatment of the proteoglycans with chondroitinase ABC (27). Prior to electrophoretic analysis, samples were dialyzed into 0.125 M Tris/ HCI, pH 6.8, containing 0.1% SDS. Protein Sequence Analysis Confirmation of the presence of decorin and biglycan was performed by N-terminal amino acid sequence analysis. Samples were dissolved in water and applied to a glass fiber filter coated with Biobrene for analysis on an Applied Biosystems 470A gas phase sequencer. Resulting phenylthiohydantoin amino acid derivatives were resolved using an on-line Applied Biosystems 120A analyzer. The relative amounts of decorin and biglycan in samples was estimated from the repetitive yield of alanine at residue 3 in decorin and residue 4 in biglycan. Residues 1 and 2 in the two proteoglycans are identical and are therefore not of use for differential quantitation. This calculation assumes that any blockage of the N-terminal amino acids is similar for decorin and biglycan. Alkali Digestion The size of the dermatan sulfate chains on the proteoglycans was determined by Sephacryl
S400HR chromatography under dissociative conditions, following their release from the proteoglycans by treatment with 50 mM NaOH in the presence of 1 M NaBH, (27). The elution position of the dermatan sulfate was monitored by uronic acid analysis (3). Chondroitinase Digestion The composition of the dermatan sulfate chains was determined by analysis of the disaccharides released following digestion with chondroitinase AC or ABC (27). The disaccharides released by chondroitinase ABC were used to quantitate the proportion of 4- and 4-sulfated galactosamine residues following their resolution by high performance liquid chromatography. The proportion of iduronic acid was estimated from the difference in the absorbance at 232 nm generated by treatment with chondroitinase AC and ABC. The latter enzyme generates a chromophore in every uronic acid residue, whereas the former enzyme generates a chromophore only in glucuronic acid residues. The difference between the two absorbances therefore can be used as an estimate of iduronic acid content.
RESULTS Dermatan sulfate proteoglycans were obtained with a yield of 2 mgig of wet meniscus by a combination of density gradient centrifugation, ion exchange chromatography, and gel filtration, by an identical protocol to that previously used in the preparation of dermatan sulfate proteoglycans from human articular cartilage (27). The final purification step of dissociative Sepharose CL-4B chromatography yielded a single symmetrical peak of K,, 0.57 (Table 1). On chromatography through Sepharose CL-4B under associative conditions this preparation resolved into a number of components (Fig. l ) , including minor peaks at void volume (V,) and total volume (V,) and a major asymmetric included peak. Analysis of the column eluent by SDS/polyacrylTABLE 1. Chromatography of dermatan suljate proteoglycans through Sepharose CL-4B Conditions
Pool"
Dissociative Associative Associative
1 2
Pools are delineated in Fig. 1.
K,
Yield (mg)
0.57 0.20
41 16
0.50
20
634
P . J . ROUGHLEY A N D R. J . WHITE
0.15-
,
pool1
pool2
H F - + l
W Q)
0
g 0.1 e 2rn 0.050
2o
43'
40
50
60
70
Fraction number
"0
80
4
90
"1
FIG. 1. Chromatography of meniscal dermatan sulfate proteoglycans through Sepharose CL-4B under associative conditions. The void volume (V,) and total volume (V,) of the column are indicated. Fractions 33-47 (pool 1) and 48-62 (pool 2) were collected for further analysis. Protein content of the fractions was monitored by absorbance at 280 nm (EZ8J.
amide gel electrophoresis (Fig. 2) indicated that only the major included peak contained the dermatan sulfate proteoglycans. The migration pattern on electrophoresis suggested that both decorin and biglycan were present but that decorin was the major species. The biglycan, which migrates more slowly on electrophoresis, predominated in the early part of the elution profile (pool 1). This was not too surprising as biglycan has been shown to undergo selfassociation under these conditions (25). However, this region of the column profile (pool 1) aho contained substantial amounts of a proteoglycan having a mobility similar to that of decorin in all the fractions, which has been reported not to undergo selfassociation (25). The presence of decorin throughout the column profile would suggest true interaction, rather than fortuitous overlap from a predominant monomeric population. It is possible that biglycan may exist in both double and single chain forms, and that the latter might have an electrophoretic mobility similar to decorin. Thus, the properties of the core protein of the dermatan sulfate proteoglycans were studied in more detail. For this purpose the proteoglycans were divided into predominantly self-associated (pool 1) and monomeric (pool 2) preparations (Fig. 1). The self-associating pool had a K,, of 0.20 and represented 45% of the total proteoglycan by weight, whereas the monomeric pool had a K,, of 0.50 (Table 1). On SDSipolyacrylamide gel electrophoresis after treatment with chondroitinase ABC, both proteoglycan pools showed the doublet pattern at about M, 45,000, characteristic of decorin (Fig. 3).
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Biglycan, isolated from human articular cartilage, yields a single core protein on chondroitinase treatment (27). The predominance of decorin in pool 1 was confirmed by N-terminal protein sequence analysis, as was the presence of biglycan as a relatively minor component. The amino acid yield on sequencing indicated that biglycan accounted for about 30% of the proteoglycan molecules in this pool (Table 2). Sequence analysis of pool 2 indicated the presence of mainly decorin, which accounts for about 85% of the proteoglycan molecules in the pool. To determine whether the self-associating proteoglycan pool had a different dermatan sulfate composition to the monomeric pool, the structure of the glycosaminoglycan chains was studied. On gel filtration, following release of the chains by alkaline borohydride, the dermatan sulfate from the monomeric pool 2 showed a broad size range with a K,,
94,000--m
68,000+ 45,000-
25,700 +
13,70037 40 45 50 55 Fraction number FIG. 2. SDSipolyacrylamide gel electrophoresis of fractions from the associative Sepharose CL-4B chromatography of the meniscal proteoglycans (Fig. 1). The migration position of standard proteins of known molecular weight are indicated.
635
PROTEOGLYCANS OF THE HUMAN MENISCUS 0.2
,
1 """
+DS-PG
I
Pool 1
0
2 0.15 W
e D S - P G II
M, 45,000-+
40
50
60
70
80
90
Fraction number FIG. 4. Chromatography of meniscal dermatan sulfate proteoglycans through Sephacryl S400HR after incubation in the presence of alkaline borohydride. The two profiles represent pool 1 and pool 2 isolated by associative Sepharose CL-4B chromatography of the intact proteoglycans (Fig. 1). Uronic acid content of the fractions was monitored by absorbance at 530 nm (E530).
Pool Chase A5C
1 2 1 2
+ + - -
FIG. 3. SDSipolyacrylamide gel electrophoresis of pools from the associative Sepharose CL-4B chromatography of the meniscal proteoglycans (Fig. 1) after incubation in the presence or absence of chondroitinase ABC. The migration position of ovalbumin (M,45,000) is indicated. DS-PG, dermatan sulfate proteoglycan.
in the degree of epimerization of uronic acid residues in the two preparations. In both cases, iduronic acid, measured by its resistance to modification by chondroitinase AC, represented about 70% of the total uronic acid residues (Table 2). It should be pointed out, however, that similarity in average composition does not necessarily preclude differences in monosaccharide distribution along the chains, which would not be detectable by these techniques. DISCUSSION
of 0.50 (Fig. 4 and Table 2). The dermatan sulfate from the pool 1 proteoglycans showed a prevalence of the larger-size ranges observed in pool 2 (Fig. 4), with a K,, of 0.45 (Table 2). Analysis of the component disaccharides following chondroitinase ABC treatment indicated that there was no difference in the average sulfation pattern in the two preparations. In both caseb 4-sulfation predominated over 6-sulfation in about a 3: 1 ratio (Table 2). Furthermore, there was also no apparent difference TABLE 2. Analysis of proteoglycan composition Pool
K,""
1:llb
% IdA
6:4SO,
1 2
0.45
1:2
0.50
1:s
71 70
0.35 0.35
ldA, iduronic acid. K,, is evaluated on Sephacryl S400HR. Molar ratio of higlycan (1)idecorin (11) is calculated from protein sequencing data. a
'
The adult human meniscus yielded about 2 mg/g of wet weight of dermatan sulfate proteoglycan, of which about 75% is decorin. This is similar to the yield of dermatan sulfate proteoglycan obtained from newborn human articular cartilage (27) and is also similar to the yield of aggregating proteoglycan previously purified from the adult meniscus (26). These data indicate that aggrecan, decorin, and biglycan are extracted in a weight ratio of about 4:3: 1, respectively. The presence of both aggregating proteoglycans and dermatan sulfate proteoglycans has also been reported in the articular disc of the adult bovine temporomandibular joint (22,3 1) and the chicken meniscus (24) and may be a common feature of fibrocartilages, reflecting their physiological role of having to resist both compressive and tensile forces. Indeed, fibrous connective tissues such as adult bovine tendons, which contain predominantly dermatan sulfate proteoglycans in regions subjected
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P . J. ROUGHLEY AND R . J. WHITE
only to tension, also contain about half their proteogl ycan as large aggregating proteoglycans in regions that are exposed to compression as they pass over bone (33). The adult meniscal dermatan sulfate proteoglycans are composed mainly of decorin, with biglycan representing about 20% of the molecules. The adult bovine temporomandibular disc also possesses a high proportion of decorin (31), as does adult bovine tendon (32) and skin (23) and adult human articular cartilage (28). In contrast, young connective tissues appear to contain both decorin and biglycan as major components (5,7,27,32). At least in human articular cartilage, the deficiency in matrix biglycan in the adult can be related to a deficiency in the synthesis of this molecule by the adult chondrocytes (21). The reason for this deficiency is not clear but may relate to the functional ability of decorin to interact with collagen fibrils, a property not shared by biglycan (4). The functional role of biglycan is not known, but its need may be more important in growing rather than mature tissues, whereas the need for decorin may be essential throughout life. The dermatan sulfate chains in thc adult human meniscus contain about 70% of their uronic acid as iduronate and about 75% of their sulfate on the 4-position of the N-acetylgalactosamine. The proportion of iduronic acid in decorin or biglycan varies considerably with tissues. In bovine bone the uronic acid is all glucuronate, irrespective of age (29), whereas in bovine skin even the fetal tissue has greater than 80% iduronate (5). In bovine tendon there is about 60% iduronate and it changes little with age (15). In human articular cartilage, the proportion of iduronate would appear to increase with age from 20 to 50% (27,28), whereas in pig laryngeal cartilage there is no iduronate (28). While there is great variation in iduronate content between t he dermatan sulfate proteoglycans of different tissues, they all show considerably more 4-sulfation relative to 6-sulfation (15,21,28,29). This is even true in a tissue such as adult human articular cartilage, where the aggregating proteoglycans , presumably produced by the same cells, are predominantly 6-sulfated (21). Some connective tissues do, however, show a slight increase in the proportion of 6-sulfate with age in their dermatan sulfate proteoglycans (15). The reason for this vast array of dermatan sulfate structures is at present unclear but may relate to the ability of dermatan sulfate chains to self-associate (10). This property is not common to all dermatan
J Orthop R e s , Vol. 10. No. 5 , 1992
sulfate chains, but appears to require sequences where both iduronate and glucuronate are present. In immature bovine articular cartilage, selfassociation appears to be a property unique to biglycan but not decorin (3,and this may relate to the reported differences in their chain composition. In neonatal human articular cartilage, biglycan also appears to self-associate to a much greater degree than decorin (27). In the adult meniscus, a substantial proportion of decorin appears to self-associate. This is not too surprising if self-association is via the dermatan sulfate chains, as it is likely that some such chains on decorin may have the appropriate sequence for interaction. However, one cannot exclude the possibility that self-association is mediated by protein-protein interactions. In this respect it is of interest to note that decorin from bovine skin, which does not normally self-associate, can be induced to do so by removal of its N-linked oligosaccharides, which presumably induces an appropriate conformational change (30). Irrespective of its origin, it is possible that the self-association of decorin could relate to adult connective tissues where biglycan is deficient, as it could potentially provide a mechanism for the interaction of adjacent collagen fibrils in a tight lateral array, presumably giving the tissues increased mechanical strength. Acknowledgment: We thank the departments of pathology at the Royal Victoria Hospital and the Montreal General Hospital for the provision of autopsy facilities, Ms. N . Nikolajew for typing the manuscript, and Ms. J. Wishart for preparing the figures. This work was supported bv the Medical Research Council of Canada and the Shriners of North America.
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PROlEOGLYCANS OF THE HUMAN MENISCUS
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