A~xwves Vol.
OF BIOCHEMISTI~Y 189, No. 1, July,
Anatomically
AND p. 28-36,
Determined
M. B. E. SWEET, Orthopaedic
Research
Bro~~~r~src~s 1978
Polydispersity of Proteoglycans Articular Cartilage
E. J-M. A. THONAR,
Laboratories,
AND
Department of Orthopaedic Surgery, Johannesburg, South Africa
Received
November
9, 1977; revised
February
of immature
A. R. IMMELMAN University
of the Witwatersrand,
27, 1978
Proteoglycans of the articulating and growing zones of minimumand maximum-contact areas of calf articular cartilage were studied. Material was extracted sequentially (0.15 M sodium acetate, 2 M CaCL, and 4 M guanidinium chloride) in the presence of protease inhibitors. The very small proportion of material extracted by 0.15 M sodium acetate was poor in carbohydrates, but rich in serine, glycine, and glutamic acid, and had a K;,,. of 0.42 on Sepharose 2B. Proteoglycan extracted from the articulating zone was of smaller average hydrodynamic size (K,, of monomer, 0.42) than that from the growing zone (K.,, of monomer, 0.321, but the attached chondroitin sulfate chains were of similar size. Proteoglycan prepared from the articulating area of minimum contact was chondroitin sulfate enriched (molar ratio of GalN:GlcN, 27) in comparison to that prepared from other regions of the articular cartilage (GalN:GlcN, 9-12). It is suggested that age-related maturation may be modified by physiological load or stress. EXPERIMENTAL
Proteoglycans of hyaline cartilage are composite molecules consisting of chondroitin sulfate and keratan sulfate covalently linked to protein. One end of the protein core interacts specifically with hyaluronate (1, 2) to form aggregates. Adjacent to the hyaluronate-binding region is a polypeptide sequence substituted with keratan sulfate (3). The remainder of the core protein is of variable length and contains the majority of the chondroitin sulfate, attached singly or in clusters of up to 10 chains (4). Proteoglycans are polydisperse mainly because of variations in the length of the chondroitin sulfate attachment region (5) and, as suggested by their extractability profiles, may exist at different levels of organization in the tissues (6). The latter may be assessed by the quantitative analysis of proteoglycans extracted by a standardized procedure using solutions of increasing ionic strength and/or denaturing ability (6, 7). The present paper describes the application of these methods to a study of the articulating and growing zones of calf articular cartilage in which variations in glycosaminoglycan content are known to occur 03).
’ Abbreviations used: DEAE, diethylaminoethyl; Al, aggregated; Dl, subunit; A,,,,, and G,,,,,,, articulating and growing zones of the minimum-contact area; A.,;,, and G,,,;,,, articulating and growing zones of the maximum-contact area. 28
0003.9861/78/1891-0028$02.00/0 Copyright All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
PROCEDURES
Materials. Apart from the following chemicals of reagent grade, all reagents were of analytical grade: glucosamine hydrochloride, galactosamine hydrochloride, glucuronolactone, carbazole, guanidinium chloride, and urea. Twice-crystallized papain was from British Drug Houses, Poole, England. DEAE’-cellulose was from Merck, Darmstadt, Germany, and Sephadex G200 and Sepharose CL-2B (Batch 1836) were from Pharmacia Fine Chemicals, Uppsala, Sweden. Analytical methods. Uranic acid was measured by an automated modification (9) of the carbazole-borosulfuric acid method (IO) with glucuronolactone as standard. Glucosamine and galactosamine were quantitatively determined on a 20-cm column of Beckman M81 resin on a Beckman Model 116 amino acid analyzer. Specimens were hydrolyzed in 6 M HCI in sealed tubes flushed with nitrogen, at 100°C for 4 h. Protein was measured by an automated modification (11) of the method of Lowry et al. (IZ), with bovine serum albumin as the standard, or by the summation of amino acids. Neutral sugars were measured by the anthrone procedure (13). Tissue. The hind limbs of calves, I month of age, were obtained fresh from the abattoir. The articular
I’HOTEOGLYCANS
OF
CALF
FK:. I. Calf femur. (Left) Diagrammatic representation of the distal femoral epiphysis with the regions studied indicated. MIN. minimum-contact area; MAX, maximum-contact area (shaded), (Bight) Minimumand maximum-contact areas in lateral projection. surfaces were wiped clean of synovial fluid. Full-thickness cores of articular cartilage, 3 mm’, were removed from the area of maximum contact on the condylar summits and from the area of minimum contact lateral and medial to the patellar groove (Fig. 1). The superficial articulating zone (l-mm thickness) and the deep growing zone (l-mm thickness), taken 1 mm above the zone of provisional calcification, were removed and treated separatelg in each case. Cartilage was washed very briefly in ice-cold 0.15 M NaCI. The four samples were labeled A ,,11,,and G,,,,, (articulating and growing zones of the minimum-contact area) and A,,,, and G.,,, (articulating and growing zones of the maximum-contact area). Extraction of proteo&ycans. Slices of cartilage were transferred to and extracted in 10 times their weight of 0.15 M sodium acetate, pH 6.8, on a rotating vertical stage (10 rpm) at 2°C for 3 h. The extract was removed and the remaining cartilage washed with 3 ml of the same solution. Extract and washings were combined and clarified by centrifugation (10,009~ for 30 min). The cartilage was then extracted with 10 times its weight of 2 M CaClz in 0.05 M sodium acetate, pH 6.8, at 2°C for 24 h (14). Extract and washings were treated as before and the residual cartilage was extracted in 10 times its weight of 4 M guanidinium chloride in 0.05 IM sodium acetate, pH 5.8, at 2°C for 48 h (1, 7). Extract and washings were treated as before. The remaining cartilage was washed three times in 0.15 M sodium acetate (4°C) and digested with papain as described below. All extracting solutions contained the following ptoteolytic enzyme inhibitors: 0.01 M EDTA (disodium salt) (not present in 2 M CaCL), 0.1 M 6-aminohexanoic acid, and 5 mM benzamidinium chloride (15). centrifugution. Equilibrium density-gradient From each extract, aggregated (Al) proteoglycans were prepared using associative density gradients (0.5 M guanidinium chloride-CsCl in 0.05 M sodium acetate, pH 6.8; starting density, 1.69 g/ml; 40,000 rpm,
AHTICULAK
CAHTILAGE
29
20°C 48 h; Beckman 75-Ti rotor) (16). Aliquots of the Al preparations were subjected to dissociative CsCl density-gradient centrifugation in the presence of 4 M guanidinium chloride (starting density, 1.5 g/ml; 40 000 rpm, 2O”C, 48 h; Beckman 75-Ti rotor) for the preparation of proteoglycan subunit (AIDl) (16). All solutions included the protease inhibitors listed above. I’roteoglycans were dialyzed, freeze-dried, and stored at -75°C until required. I’roteoglycan subunit was also prepared by ion-exchange chromatography on DEAE-cellulose in the presence of 7 M urea from the Al preparation (17). Columns were eluted with 3 bed volumes of 7 M urea, 0.3 M NaCl in 7 M urea, and 3 M NaCl in 7 M urea, all in 0.05 M Tris-HCI, pH 6.8; proteoglycan subunit was recovered from the 3 M NaCl fraction. Amino acid analysis. Samples of AID1 proteoglycans were hydrolyzed in 6 M HCl at 106°C in sealed tubes flushed with nitrogen for 24 h. Excess acid was removed in LJ~CUO over NaOH and PiOr,. Amino acids were determined on a Beckman Model 1 I6 amino acid analyzer. Cysteine was estimated as cysteic acid following oxidation with performic acid prior to hydtolysis (18). No corrections were made for losses occutring during hydrolysis. Enzymatic digestion. Cartilage of the inextractable residue was digested with crystalline papain using 1 mg/g of wet tissue in 0.05 M sodium acetate, pH 5.8. containing 0.02 M I.-cysteine and 0.005 M EDTA (disodium salt) at 60°C for 4 h (19). After clarification by centrifugation (10 OOOg, 30 min), glycosaminoglycans were recovered from the supernatant by precipitation with 3 vol of 0.5 M potassium acetate in ethanol and then analyzed for uranic acid, galactosamine, and glucosamine. Glycosaminoglycans were also prepared by treating ptoteoglycans with 0.5 M NaOH in 0.02 M sodium borohydride (20) at 22°C under nitrogen for 24 h. Electrophoresis. Samples of Al and AlDl proteoglycan were subjected to electrophoresis on large-pore polyacrylamide/agarose gels before and after fractionation on Sepharose CL 2B. A modification (21) of the method of McDevitt and Muir (22) was used: Flat-bed gels of 1.2% (w/v) acrylamide and 0.6% agarose were prepared in 0.4 M Tris-acetate containing 1 mM sodium sulfate, pH 6.8. The gels were equilibrated in the same buffer overnight. For electrophoresis, the buffer was diluted 1:4 and contained I mM EDTA. Chondroitin sulfate was run as a standard in each gel. Gel chromatography. Samples of Al (before and after fractionation on DEAE-cellulose) and AlDl proteoglycan (about 0.2 mg of uronate in 0.25 ml of 0.5 M sodium acetate, pH 6.8) were applied to an analytical column (100 x 0.6 cm) of Sepharose CL-2B (Batch 1836) which was eluted downward with the same buffer at 18“C and a rate of 1 ml/h using a peristaltic pump. Fractions of 0.5 ml were analyzed for utonic acid. Free chondroitin sulfate chains (0.2 mg of uronate)
30
SWEET,
THONAB,
AND
from the articulating than from the growing zones (Table I). Although the proportion of total uranic acid in the inextractable residue was very similar in each tissue, there were differences in the dissociative extraction profiles between the articulating and growing zones. Thus 2 M CaCL extracted more proteoglycan from the articulating than from the growing zone. Only about 50% of the total uranic acid was extracted with this solvent from tissue G,,,,. These results suggest that the proteoglycans of the growing zone may be more resistant to extraction than those of the articulating zone. Sodium acetate extract. The composition of the material extracted by 0.15 M
were applied in 0.25 ml of 0.5 M sodium acetate, pH 6.8, to a column (100 x 0.5 cm) of Sephadex G20O and eluted downward with the same buffer at a rate of 1 ml/h using a peristaltic pump. Fractions of 0.5 ml were analyzed for uranic acid. Both columns were calibrated with blue dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) and glucuronolactone. BESlJLTS
Extraction of proteoglycans. Proteoglycans were sequentially extracted from anatomically distinct areas of immature articular cartilage with 0.15 M sodium acetate, 2 M CaCl2, and 4 M guanidinium chloride. A very small proportion of total proteoglycan (as uranic acid) was extractable with isoosmotic sodium acetate, slightly more TABLE SRQ~JF:NTIAI.
EXTI~AWION
IMMEI,MAN
I
OF PIWTEO(;I.Y(~AN
FIWM
IMMATIIIE
~~___ ‘%GlcA _~~~~ 0.15 M Sodium tate 2 M Ca& 4 M Guanidinium Residue Starting material
A llllli
A ,,I111 GalN/ GlCN
%GlcA
GalN/ GICN
~~~ %GlcA
G,,,.,, GalN/ GlcN
4.2
1.32
1.9
2.36
6.2
0.95
7.82
HCl
70.13 10.29 16.91
12.6 5.8 12.5 11.7
72.47 13.02 13.19
6.5 4.3 7.2 6.4
54.55 23.59 19.50
8.7 7.8 13.7 9.5
55.57 23.32 20.16
8.42 6.64 9.03 8.25
DENSITY-GIIADIRNT
II
CENTI~IFIKATION
OF 0.15 M SOIHIIM
ACETATE
Articulating
EXTI~AC’I-s
Growing
~Uronate (%)”
(1 Expressed from maximum-
G #1/d/1 ~__ :;a GlcA GalN/ GlcN
2.67
TABLE
1.655 1.617 1.584 I.558 1.534 1.513 1.496 1.477 1.459 1.444 1.427 1.414 1.399 Starting material
CAIWII.A(:I
OF BIOCHEMISTI~Y 189, No. 1, July,
Anatomically
AND p. 28-36,
Determined
M. B. E. SWEET, Orthopaedic
Research
Bro~~~r~src~s 1978
Polydispersity of Proteoglycans Articular Cartilage
E. J-M. A. THONAR,
Laboratories,
AND
Department of Orthopaedic Surgery, Johannesburg, South Africa
Received
November
9, 1977; revised
February
of immature
A. R. IMMELMAN University
of the Witwatersrand,
27, 1978
Proteoglycans of the articulating and growing zones of minimumand maximum-contact areas of calf articular cartilage were studied. Material was extracted sequentially (0.15 M sodium acetate, 2 M CaCL, and 4 M guanidinium chloride) in the presence of protease inhibitors. The very small proportion of material extracted by 0.15 M sodium acetate was poor in carbohydrates, but rich in serine, glycine, and glutamic acid, and had a K;,,. of 0.42 on Sepharose 2B. Proteoglycan extracted from the articulating zone was of smaller average hydrodynamic size (K,, of monomer, 0.42) than that from the growing zone (K.,, of monomer, 0.321, but the attached chondroitin sulfate chains were of similar size. Proteoglycan prepared from the articulating area of minimum contact was chondroitin sulfate enriched (molar ratio of GalN:GlcN, 27) in comparison to that prepared from other regions of the articular cartilage (GalN:GlcN, 9-12). It is suggested that age-related maturation may be modified by physiological load or stress. EXPERIMENTAL
Proteoglycans of hyaline cartilage are composite molecules consisting of chondroitin sulfate and keratan sulfate covalently linked to protein. One end of the protein core interacts specifically with hyaluronate (1, 2) to form aggregates. Adjacent to the hyaluronate-binding region is a polypeptide sequence substituted with keratan sulfate (3). The remainder of the core protein is of variable length and contains the majority of the chondroitin sulfate, attached singly or in clusters of up to 10 chains (4). Proteoglycans are polydisperse mainly because of variations in the length of the chondroitin sulfate attachment region (5) and, as suggested by their extractability profiles, may exist at different levels of organization in the tissues (6). The latter may be assessed by the quantitative analysis of proteoglycans extracted by a standardized procedure using solutions of increasing ionic strength and/or denaturing ability (6, 7). The present paper describes the application of these methods to a study of the articulating and growing zones of calf articular cartilage in which variations in glycosaminoglycan content are known to occur 03).
’ Abbreviations used: DEAE, diethylaminoethyl; Al, aggregated; Dl, subunit; A,,,,, and G,,,,,,, articulating and growing zones of the minimum-contact area; A.,;,, and G,,,;,,, articulating and growing zones of the maximum-contact area. 28
0003.9861/78/1891-0028$02.00/0 Copyright All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
PROCEDURES
Materials. Apart from the following chemicals of reagent grade, all reagents were of analytical grade: glucosamine hydrochloride, galactosamine hydrochloride, glucuronolactone, carbazole, guanidinium chloride, and urea. Twice-crystallized papain was from British Drug Houses, Poole, England. DEAE’-cellulose was from Merck, Darmstadt, Germany, and Sephadex G200 and Sepharose CL-2B (Batch 1836) were from Pharmacia Fine Chemicals, Uppsala, Sweden. Analytical methods. Uranic acid was measured by an automated modification (9) of the carbazole-borosulfuric acid method (IO) with glucuronolactone as standard. Glucosamine and galactosamine were quantitatively determined on a 20-cm column of Beckman M81 resin on a Beckman Model 116 amino acid analyzer. Specimens were hydrolyzed in 6 M HCI in sealed tubes flushed with nitrogen, at 100°C for 4 h. Protein was measured by an automated modification (11) of the method of Lowry et al. (IZ), with bovine serum albumin as the standard, or by the summation of amino acids. Neutral sugars were measured by the anthrone procedure (13). Tissue. The hind limbs of calves, I month of age, were obtained fresh from the abattoir. The articular
I’HOTEOGLYCANS
OF
CALF
FK:. I. Calf femur. (Left) Diagrammatic representation of the distal femoral epiphysis with the regions studied indicated. MIN. minimum-contact area; MAX, maximum-contact area (shaded), (Bight) Minimumand maximum-contact areas in lateral projection. surfaces were wiped clean of synovial fluid. Full-thickness cores of articular cartilage, 3 mm’, were removed from the area of maximum contact on the condylar summits and from the area of minimum contact lateral and medial to the patellar groove (Fig. 1). The superficial articulating zone (l-mm thickness) and the deep growing zone (l-mm thickness), taken 1 mm above the zone of provisional calcification, were removed and treated separatelg in each case. Cartilage was washed very briefly in ice-cold 0.15 M NaCI. The four samples were labeled A ,,11,,and G,,,,, (articulating and growing zones of the minimum-contact area) and A,,,, and G.,,, (articulating and growing zones of the maximum-contact area). Extraction of proteo&ycans. Slices of cartilage were transferred to and extracted in 10 times their weight of 0.15 M sodium acetate, pH 6.8, on a rotating vertical stage (10 rpm) at 2°C for 3 h. The extract was removed and the remaining cartilage washed with 3 ml of the same solution. Extract and washings were combined and clarified by centrifugation (10,009~ for 30 min). The cartilage was then extracted with 10 times its weight of 2 M CaClz in 0.05 M sodium acetate, pH 6.8, at 2°C for 24 h (14). Extract and washings were treated as before and the residual cartilage was extracted in 10 times its weight of 4 M guanidinium chloride in 0.05 IM sodium acetate, pH 5.8, at 2°C for 48 h (1, 7). Extract and washings were treated as before. The remaining cartilage was washed three times in 0.15 M sodium acetate (4°C) and digested with papain as described below. All extracting solutions contained the following ptoteolytic enzyme inhibitors: 0.01 M EDTA (disodium salt) (not present in 2 M CaCL), 0.1 M 6-aminohexanoic acid, and 5 mM benzamidinium chloride (15). centrifugution. Equilibrium density-gradient From each extract, aggregated (Al) proteoglycans were prepared using associative density gradients (0.5 M guanidinium chloride-CsCl in 0.05 M sodium acetate, pH 6.8; starting density, 1.69 g/ml; 40,000 rpm,
AHTICULAK
CAHTILAGE
29
20°C 48 h; Beckman 75-Ti rotor) (16). Aliquots of the Al preparations were subjected to dissociative CsCl density-gradient centrifugation in the presence of 4 M guanidinium chloride (starting density, 1.5 g/ml; 40 000 rpm, 2O”C, 48 h; Beckman 75-Ti rotor) for the preparation of proteoglycan subunit (AIDl) (16). All solutions included the protease inhibitors listed above. I’roteoglycans were dialyzed, freeze-dried, and stored at -75°C until required. I’roteoglycan subunit was also prepared by ion-exchange chromatography on DEAE-cellulose in the presence of 7 M urea from the Al preparation (17). Columns were eluted with 3 bed volumes of 7 M urea, 0.3 M NaCl in 7 M urea, and 3 M NaCl in 7 M urea, all in 0.05 M Tris-HCI, pH 6.8; proteoglycan subunit was recovered from the 3 M NaCl fraction. Amino acid analysis. Samples of AID1 proteoglycans were hydrolyzed in 6 M HCl at 106°C in sealed tubes flushed with nitrogen for 24 h. Excess acid was removed in LJ~CUO over NaOH and PiOr,. Amino acids were determined on a Beckman Model 1 I6 amino acid analyzer. Cysteine was estimated as cysteic acid following oxidation with performic acid prior to hydtolysis (18). No corrections were made for losses occutring during hydrolysis. Enzymatic digestion. Cartilage of the inextractable residue was digested with crystalline papain using 1 mg/g of wet tissue in 0.05 M sodium acetate, pH 5.8. containing 0.02 M I.-cysteine and 0.005 M EDTA (disodium salt) at 60°C for 4 h (19). After clarification by centrifugation (10 OOOg, 30 min), glycosaminoglycans were recovered from the supernatant by precipitation with 3 vol of 0.5 M potassium acetate in ethanol and then analyzed for uranic acid, galactosamine, and glucosamine. Glycosaminoglycans were also prepared by treating ptoteoglycans with 0.5 M NaOH in 0.02 M sodium borohydride (20) at 22°C under nitrogen for 24 h. Electrophoresis. Samples of Al and AlDl proteoglycan were subjected to electrophoresis on large-pore polyacrylamide/agarose gels before and after fractionation on Sepharose CL 2B. A modification (21) of the method of McDevitt and Muir (22) was used: Flat-bed gels of 1.2% (w/v) acrylamide and 0.6% agarose were prepared in 0.4 M Tris-acetate containing 1 mM sodium sulfate, pH 6.8. The gels were equilibrated in the same buffer overnight. For electrophoresis, the buffer was diluted 1:4 and contained I mM EDTA. Chondroitin sulfate was run as a standard in each gel. Gel chromatography. Samples of Al (before and after fractionation on DEAE-cellulose) and AlDl proteoglycan (about 0.2 mg of uronate in 0.25 ml of 0.5 M sodium acetate, pH 6.8) were applied to an analytical column (100 x 0.6 cm) of Sepharose CL-2B (Batch 1836) which was eluted downward with the same buffer at 18“C and a rate of 1 ml/h using a peristaltic pump. Fractions of 0.5 ml were analyzed for utonic acid. Free chondroitin sulfate chains (0.2 mg of uronate)
30
SWEET,
THONAB,
AND
from the articulating than from the growing zones (Table I). Although the proportion of total uranic acid in the inextractable residue was very similar in each tissue, there were differences in the dissociative extraction profiles between the articulating and growing zones. Thus 2 M CaCL extracted more proteoglycan from the articulating than from the growing zone. Only about 50% of the total uranic acid was extracted with this solvent from tissue G,,,,. These results suggest that the proteoglycans of the growing zone may be more resistant to extraction than those of the articulating zone. Sodium acetate extract. The composition of the material extracted by 0.15 M
were applied in 0.25 ml of 0.5 M sodium acetate, pH 6.8, to a column (100 x 0.5 cm) of Sephadex G20O and eluted downward with the same buffer at a rate of 1 ml/h using a peristaltic pump. Fractions of 0.5 ml were analyzed for uranic acid. Both columns were calibrated with blue dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) and glucuronolactone. BESlJLTS
Extraction of proteoglycans. Proteoglycans were sequentially extracted from anatomically distinct areas of immature articular cartilage with 0.15 M sodium acetate, 2 M CaCl2, and 4 M guanidinium chloride. A very small proportion of total proteoglycan (as uranic acid) was extractable with isoosmotic sodium acetate, slightly more TABLE SRQ~JF:NTIAI.
EXTI~AWION
IMMEI,MAN
I
OF PIWTEO(;I.Y(~AN
FIWM
IMMATIIIE
~~___ ‘%GlcA _~~~~ 0.15 M Sodium tate 2 M Ca& 4 M Guanidinium Residue Starting material
A llllli
A ,,I111 GalN/ GlCN
%GlcA
GalN/ GICN
~~~ %GlcA
G,,,.,, GalN/ GlcN
4.2
1.32
1.9
2.36
6.2
0.95
7.82
HCl
70.13 10.29 16.91
12.6 5.8 12.5 11.7
72.47 13.02 13.19
6.5 4.3 7.2 6.4
54.55 23.59 19.50
8.7 7.8 13.7 9.5
55.57 23.32 20.16
8.42 6.64 9.03 8.25
DENSITY-GIIADIRNT
II
CENTI~IFIKATION
OF 0.15 M SOIHIIM
ACETATE
Articulating
EXTI~AC’I-s
Growing
~Uronate (%)”
(1 Expressed from maximum-
G #1/d/1 ~__ :;a GlcA GalN/ GlcN
2.67
TABLE
1.655 1.617 1.584 I.558 1.534 1.513 1.496 1.477 1.459 1.444 1.427 1.414 1.399 Starting material
CAIWII.A(:I
