ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 198, No. 2, December, pp. 439-448, 1979
Age-Related
Changes in Proteoglycan
M. B. E. SWEET,* *Orthopaedic
Research Johannesburg,
E. J-M. A. THONAR,”
Structure
AND J. MARSH?
Laboratories, Department of Orthopaedic Surgery, University of the Witwatersrand, and 7Department of Medical Biochemistry, University of Cape Town, Cape Town, South Africa
Received April 21, 1979 Proteoglycans of calf and steer articular cartilage were studied with a view of assessing structure and changes occurring as a result of the aging process. The average reduction in hydrodynamic size noted in steer was associated with a diminution in size ofthe chondroitin sulfate-rich region of the core protein as well as the chondroitin sulfate chains themselves. By contrast the keratan sulfate-rich region was hydrodynamically larger in steer although the keratan sulfate chains were only slightly longer than in calf. The proteoglycans showed a maturation-related decrease in chondroitin sulfate content (shorter chains, fewer chains, smaller chondroitin sulfate-rich region) and an enrichment in keratan sulfate chains in both the chondroitin sulfate-rich and keratan sulfate-rich regions. Proteoglycans from both age groups contained an oligosaccharide which was recovered mainly from outside of the keratan sulfate-rich region. There were no significant differences in size between keratan sulfate chains recovered from the keratan sulfate-rich region and the chondroitin sulfate-rich region.
Proteoglycans are major components of hyaline cartilage. They consist of a central core protein to which chondroitin sulfate, keratan sulfate, and a small proportion of oligosaccharides are covalently linked (l-3). One end of the core protein is not substituted with glycosaminoglycans and has some tertiary structure permitting the formation of a noncovalent complex with hyaluronate (4, 5). Adjacent to the hyaluronate-binding region is a peptide sequence largely substituted with keratan sulfate (6). To the remainder of the core protein is attached chondroitin sulfate and the rest of the keratan sulfate (6). The normal maturation process in articular cartilage is characterized by a fall in water content (‘7) and a decreased extractability of proteoglycans which become richer in keratan sulfate and smaller in hydrodynamic size (8). These changes may be related, inter alia, to a greater degree of crosslinking of the collagen network (9) and to an apparent shortening of the chondroitin sulfate-rich region of the core protein (10). The aim of this study was to examine the question of aging as it affects proteoglycans of articular cartilage by assessing the in439
dividual fragments derived from the parent macromolecules. In particular, keratan sulfate chains have been characterized and the distribution of the oligosaccharide along the core protein has been studied. EXPERIMENTAL
PROCEDURES
Materials All chemicals were of the best available grade: benzamidine HCl and 6-aminohexanoic acid were from E. Merck, Darmstadt; diphenylcarbamyl chloride-treated trypsin and a-chymotrypsin were from Sigma; chondroitinase ABC (P. vulgaris) was from Miles; Bio-Gel P-2, P-10, and P-30 were from Bio-Rad; Sephadex G-75 and G-200, Sepharose 2B, CL2B, 4B, and 6B, and blue dextran were from Pharmacia.
Analytical
Methods
Uranic acid was determined by an automated modification (11) of the carbazole-borosulfuric acid method (12) with glucuronolactone as standard. Protein was determined by the Folin method (13) or by summation of amino acids. Sialic acid, as a monitor for keratan sulfate and the oligosaccharide (3,14), was determined by the method of Jourdian et al. (15). Amino acids and hexosamines were determined on a Beckman 116 amino acid analyzer after hydrolysis under nitrogen of samples in 6 M HCl for 24 h at 100°C or in 4 M HCl for 4 h 0003-9861/79/140439-10$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
440
SWEET, THONAR,
at lOO”C, respectively. Hexosaminitols were determined by the method of Downs and Pigman (16). Neutral sugars were determined by the anthrone procedure (17). Specific enzymatic determination of galactose was performed as described elsewhere (3).
Tissue Articular cartilage of calves (4 to 8 weeks of age) and steers (3 to 5 years of age) was dissected out immediately after slaughter. Because of its vascularity the deepest 2 mm adjacent to the zone of provisional calcification of calf cartilage was discarded. The remaining cartilage and that of the steers was diced and frozen in liquid nitrogen for transportation to the laboratory.
Extraction
of Proteoglycans
Finely diced cartilage (1 to 2 mm thick) was transferred from liquid nitrogen into lo-fold its weight of 4 M guamdine. HCl in 0.05 M sodium acetate, pH 5.8, and stirred at 2°C for 48 h. The extracting solution contained the following proteolytic enzyme inhibitors: 0.01 M disodium EDTA, 0.005 M benzamidine.HCl, and 0.1 M 6aminohexanoic acid (18).
Preparation
of Al and Dl Proteoglycanl
The extracts were filtered through glass wool and 2 vol ethanol was added, with constant stirring. The resulting precipitates, which contained all three components of proteoglycan aggregate (unpublished work), were collected by centrifugation (25,OOOg, 2”C, 20 min), washed in 70% ethanol, recollected and dissolved in 0.5 M guanidine.HCl or 4 M guanidine.HCl, both in 0.05 M sodium acetate, pH 5.8, containing the protease inhibitors listed above. CsCl was added to the two solutions to give a starting density of 1.69 g/ml (0.5 M guanidine.HCl for Al preparation) or 1.50 g/ml (4 M guanidine. HCl for Dl preparation) (20). CsCl density gradient centrifugation was carried out at 40,060 rpm for 48 h at 10°C in a Beckman 75Ti rotor. The tubes were sliced in a Beckman tube slicer. Al samples were recovered from the bottom quarter of tubes containing the associative solution (0.5 M guanidine. HCl) and Dl samples from the bottom two-fifths of those containing the dissociative solution (4 M guanidine. HCl) by dialysis and lyophilization.
AND MARSH rich region, the keratan sulfate-rich region, chondroitin sulfate chains, keratan sulfate chains, and the oligosaccharide. Treatment with hydroxylamine. Al proteoglycan was fractionated on a preparative column of Sepharose 2B. Material eluting in the void volume was collected, dialyzed, and lyophilized. Samples (10 mg/ml) were incubated in 1 M hydroxylamine, 0.05 M 2-(N-morpho1ino)ethanesulfonic acid, pH 6.5, for 120 h at 23°C (6). Following dialysis and lyophilization, samples were assessed on an analytical column of Sepharose 2B. Papain digestion. Dl proteoglycans were digested with 2x crystalline papain (British Drug Houses) (1 pg/mg proteoglycan) in 0.05 M sodium acetate, pH 5.8, containing 0.02 M L-cysteine and 0.005 M disodium EDTA at 60°C for 24 h (21). Aliquots of the digests were assessed on an analytical column of Sephadex G-200 or Bio-Gel P-30. Chondroitinase ABC. Dl proteoglycan was digested with chondroitinase ABC (0.05 unit/mg proteoglycan) in 0.1 M Tris-acetate buffer, pH 7.3, at 37°C for 5 h. This was followed by digestion with trypsin and chymotrypsin in the same buffer (10 pg/mg proteoglycan) at 37°C for 12 h (22). Digests were fractionated on both analytical and preparative columns of Sepharose 6B (see Fig. 6); materials from peaks I and II were collected for further analysis, digestion with papain, or treatment with alkaline borohydride. Samples of Dl proteoglycan were digested with chondroitinase ABC only or with trypsin and chymotrypsin and chromatographed on Sepharose 4B and 6B, respectively. Alkaline bwohydride. Samples of Dl proteoglycan or aliquots of material digested and fractionated as in chondroitinase ABC section above were desalted on Bio-Gel P-2 and treated (-10 mg/ml) with 0.05 M NaOH in 1 M sodium borohydride at 45°C for 48 h under nitrogen (3, 23, 24). The borohydride was destroyed by the dropwise addition of concentrated acetic acid, following which samples were freeze dried and desalted on Bio-Gel P-2 as described below. Reduction of Al proteoglycans. In order to dissociate proteoglycan aggregates and to inhibit their reformation with hyaluronate, Al proteoglycan was dissolved in 1% sodium dodecyl sulfate (SDS)V5% mercaptoethanol and kept at 65°C for 45 min. An aliquot of the sample was assessed on the Sepharose CLZB SDS analytical column (see below).
Gel Chromatography Enzymatic and Chemical of Proteoglycan
Degradation
Al and Dl proteoglycans were degraded by a variety of means in order to assess the relative sizes or proportions of the core protein, the chondroitin sulfate* The Al, Dl notation was described by Heinegard w.
Analytical columns of Sepharose 2B, 4B, and 6B, Sephadex G-75, Bio-Gel P-2, P-10, and P-30 (0.6 x 145 cm) and Sephadex G-200 (0.8 x 120 cm) were eluted with 0.5 M sodium acetate, pH 6.8 (or pH 5.8 for Sepharose 2B), at 1.0-2.0 ml/h at 18°C. Identical sized fractions (0.7 ml) were collected with the aid of fraction 2 Abbreviation
used: SDS, sodium dodecyl sulfate.
441
AGE-RELATEDCHANGESINPROTEOGLYCANSTRUCTURE collectors equipped with drop counters. An analytical column of Sepharose CL2B (0.6 x 145 cm) was eluted with 0.05 M sodium phosphate buffer in 0.1% SDS, pH 7.3, at 0.6 ml/h at 23°C (25). Fractions of 0.8 ml were collected as before. All columns were calibrated with Al proteoglycan or high molecular weight hyal~ nate and glucuronolactone. Preparative columns of Sepharose 2B (1.5 x 120 cm) and 6B (1.0 x 145 cm) were eluted with 0.5 Msodium acetate, pH 5.8 or 6.8, respectively, at about 3 ml/cmVh. Respective fractions of 5 and 3 ml were collected. Column effluents were analyzed for uranic acid, sialic acid, and protein. Sialic acid determination was found to be preferable to the anthrone procedure as a monitor for keratan sulfate and the oligosaccharlde (3, 14).
Desalting
TABLE
I
COMPOWPIONOF CALFANDSTEER ARTICULAR CARTILAGES Calf Water content GalN:GlcN Hexosamine (I*rmol/lOO mg tissue) Chondroitin-SO1
75.1 7.9 12.0 10.5 (87.2%) 0.9 (7.5%) 0.3 (2.1%)
Keratan-SO, Hyaluronate
on Bio-Gel P-2
The high concentration of salts in alkaline bomhydridetreated samples does not allow the application of material in small volumes to analytical columns. A preliminary investigation had established the presence of a pentasaccharide in proteoglycans of bovine articular cartilage (3). Therefore it was decided to desalt the samples on a gel of lowest exclusion limit rather than to employ dialysis. Columns of Bio-Gel P-2 (1.5 x 25 cm) were recalibrated with blue dextran after every five runs. Samples (0.8 ml) were applied and eluted with 0.5 M pyridine acetate, pH 7.0. Material was collected to the end of the blue dextran profile. The desalted material was subsequently lyophilized.
Electrophoresis
of Glycosaminoglycans
Glycosaminoglycans were recovered from papain digests of hydroxylamine-treated Dl proteoglycan following chromatography on Sepharose 2B. Samples were subjected to electrophoresis on cellulose acetate strips before and after speciiic enzymatic digestion as described elsewhere (7). International reference standards of glycosaminoglycans were a gift of Dr. Martin B. Mathews, Department of Paediatrics, University of Chicago, Chicago, Illinois. RESULTS
AND DISCUSSION
The general characteristics of the articular cartilage used in this study are outlined in Table I. Adult articular cartilage was less hydrated and contained less glycosaminoglycan but more keratan sulfate than immature cartilage. Intact monomeric proteoglycans (Dl) were assessed on Sepharose 2B: those of steer were more retarded by the gel than those of calf (K,, 0.42 against 0.34) (Figs. la and b). This point was further examined
Steer 67.9 2.2 7.6 5.2 (68.1%) 2.0 (26.6%) 0.3 (3.6%)
n Water content and glycosaminoglycan concentrations (as pmol hexosamine/lOO mg dry tissue) were estimated as described elsewhere (7, 14).
by chromatographing Al proteoglycans treated with 1% SDS/5% mercaptoethanol, on Sepharose CLBB: The results confirmed that adult proteoglycans were smaller than those of calf (Figs. lc and d). It is noteworthy that the sialic acid peaks of both Al SDS preparations, particularly in the adult, eluted behind the uronate peaks. This is in agreement with the explanation for proteoglycan polydispersity suggested by Heinegard (10): The smaller the proteoglycan the higher the ratio of keratan sulfate to STEER
;::~‘A,,id&\> J
I
,325
.? .9
1 .3 .5 .7 .9 Kd
FIG. 1. Gel filtration of proteoglycans on Sepharose 2B or CLZB (0.6 x 145 cm). Dl proteoglycans were prepared from steer (a) and calf(b) articular cartilage. Al proteoglycans were reduced in 1% SDS/58 mercaptoethanol and chromatographed on Sepharose CL2B in 0.1% SDS (c, steer; d, calf). Column effluents were analyzed for uranic acid (-) and/or sialic acid (- - -).
442
SWEET, .3
1
THONAR,
Al-OHNH2-2B 1
.2 .4 .6
.a 1.0
Kd FIG. 2. Gel titration of hydroxylamine-treated Al proteoglycan on Sepharose 2B. Al proteoglycan (a, calf; b, steer) excluded from Sepharose 2B (1.5 x 120 cm) was treated with 1 M hydroxylamine as described in the text and chromatographed on an analytical column of Sepharose 2B (0.6 x 145 cm). Fractions were analyzed for uranic acid: calf (-); steer (- - -).
chondroitin sulfate. The small retarded peak (Figs. lc and d) may represent the link proteins (25), which were not studied further. No hyaluronate was detectable in the void volume of the CLBB SDS column effluents, suggesting it was small enough to be retarded by the gel. Because of compositional changes it has been suggested that proteoglycans of hyaline cartilage become smaller with age as a result of a progressive shortening of the chondroitin sulfate-rich region (8). Electron microscopy of proteoglycans has confirmed the existence of a range of sizes within an individual preparation, but no similar evidence has been obtained for an age-related decrease in size. Kimura et al. (26) found average monomer lengths of 343 and 310 nm in aggregates from bovine nasal septum and chick embryo epiphyses, respectively. In order to test the hypothesis the experiments described below were performed. In contrast to material from bovine nasal septum, Al preparations from cartilage structures subject to high stress contain a large proportion of proteoglycans retarded by Sepharose 2B: e.g., calf articular cartilage 55% (27), calf annulus fibrosus 50%, calf nucleus pulposus 50% (G. Lyons, unpublished work). This suggests either the existence of large numbers of nonaggregating proteoglycans or that much of the hyaluronate in these tissues is of relatively low molecular weight permitting aggregation with only one proteoglycan monomer. In
AND MARSH
view of the above, we selected those Al proteoglycans excluded from Sepharose 2B for treatment with hydroxylamine assuming however, that this sample would be representative of the total population. Treatment with hydroxylamine cleaves off the chondroitin sulfate-rich region, leaving the remainder of the molecule still attached to hyaluronate (10). Assessment of material treated in this way on Sepharose 2B, should give an indication of the relative size of the chondroitin sulfate-rich region. The uronate profiles in Fig. 2 indicate that the chondroitin sulfate-rich region was of larger hydrodynamic size in the calf (K,, 0.50 calf; 0.67 steer). Qualitative analysis of the excluded and retarded fractions confirmed the predominance of keratan sulfate in the void volume and chondroitin sulfate in the retarded volume. Traces of hyaluronate (originally excluded from the preparative Sepharose 2B column as part of the starting Al preparations) were identified in both fractions, suggesting the possible existence of short chain hyaluronate bound to one or a few hyaluronate-binding regions. Chondroitin sulfate chains were prepared by papain digestion and/or by alkaline borohydride degradation of Dl monomers, and assessed on Sephadex G-200 (Fig. 3). The K,, of 0.58 (steer) and 0.51 (calf) would indicate molecular weights of 8900 and 10,400, respectively, assuming the batch of Sephadex G-200 had the same elution properties as that used by Wasteson (28). This difference would certainly contribute to the difference in hydrodynamic size of the Dl monomers. (Dl-OHBH4-GSOO(
.1 .3 .5 7 .Q Kd FIG. 3. Gel chromatography of chondroitin sulfate chains on Sephadex G-200 (0.8 x 120 cm). Chondroitin sulfate chains were liberated from calf (-) and steer (- - -) Dl proteoglycans by alkaline borohydride as described in the text. Column effluents were analyzed for uranic acid.
443
AGE-RELATEDCHANGESINPROTEOGLYCANSTRUCTURE
Chondroitin sulfate is attached in clusters of one to eight polysaccharide chains to the core protein (22). Clusters were prepared by digestion of Dl monomers by trypsin and chymotrypsin (22) and assessed on Sepharose 6B (Fig. 4). The results (K,, 0.39) for calf and (K,, 0.49) for steer are consistent with molecular weights of 52,000 and 36,000, respectively, as calculated from the data of Heinegard and Hascall (22). The core proteins, free of chondroitin sulfate chains, were assessed on Sepharose 4B (Fig. 5). The results (K,, 0.30 for calf and 0.36 for steer) support the idea of a decrease in core protein size with age (8). However the elution profile for steer was much broader than for calf, possibly because of a greater proportion of keratan sulfate. The existence of an age-related decrease in core protein size is supported by the amino acid composition of calf and steer Dl monomers (Table II). Those of the former were richer in serine and glycine, amino acids associated with the chondroitin sulfate linkage region (29). The molar ratios of galactosamine/X amino acids indicate the presence of 2.6-fold as much galactosamine containing glycosaminoglycan per gram of protein in calf than in steer. Corrections for the galactosamine originating from the linkage region of keratan sulfate and for average chondroitin sulfate chain size yield figures which indicate that one gram of protein core in calf is substituted with 2.3-fold as many chondroitin sulfate chains as is the case in steer. The data presented (Table II, Dl-CB-4B) suggest a modest decrease in
Dl -T,C-6B I
0 .2 .4 .6 .6
1.0
Kd FIG. 4. Gel filtration of chondroitin sulfate clusters on Sepharose 6B (0.6 x 145 cm). Dl proteoglycans were digested with trypsin and chymotrypsin as described in the text. Column effluents were analyzed for uranic acid: calf (-); steer (- - -).
Dl-CB-4B I
.I .3 .5 .7
.Q
Kd FIG. 5. Gel filtration of core proteins on Sepharose 4B. Dl proteoglycans (~30 mg) were digested with chondroitinase ABC, lyophilized, and applied to an analytical Sepharose 4B column (0.6 x 145 cm). Column effluents were analyzed for uranic acid: calf (-); steer (- - -).
core protein size in the mature animal. Consequently, the difference in degree of substitution with chondroitin sulfate may be even greater when expressed on a molar basis. TABLE
II
AMINOACID COMPOSITION ANDMOLARRATIOSOF GALACTOSAMINEANDGLUCOSAMINETOXAMINO ACIDSOFDl MONOMERSFROM~ALFAND STEERARTICULARCARTILAGE" Amino acid
Calf
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ‘h Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Hexosamine/Z amino acids GalN/P amino acids GlcN/Z amino acids
55 127 139 73 121 65 13 47 5 35 79 29 44 51 14 28 3.86 3.59 0.31
75
Steer 86 51 99 142 75
109 86 12 58 8 31 87 33 49 28 13 33 1.94 1.39 0.51
a Amino acids are expressed as residues per thousand residues.
444
SWEET,
THONAR,
Calf
.2 .4 .6 .6 1.0 Kd
FIG. 6. Gel filtration of enzyme-generated fragments of proteoglycans on Sepharose 6B. Calf and steer Dl monomers were sequentially degraded with chondroitinase ABC, trypsin, and chymotrypsin (see the text), lyophilized, and applied to an analytical column of Sepharose 6B (0.6 x 145 cm). Column effluents were analyzed for sialic acid (-) and by the anthrone procedure (- - -). Material equivalent to peaks I and II was recovered after gel f&ration on a larger Sepharose 6B column (1.0 x 145 cm) for further analysis.
Dl proteoglycans from both age groups were sequentially digested with chondroitinase ABC, trypsin, and chymotrypsin and the resulting fragments chromatographed on Sepharose 6B (Fig. 6). This procedure has been designed for the recovery of the keratan sulfate-rich region (peak I, Fig. 6) separate from the remainder of the core protein (peak II, Fig. 6). The results of this experiment indicate that the keratan sulfate-rich region becomes larger with age (K,, 0.29 for steer and 0.35 for calf). Despite the quantitative increase in keratan sulfate with age (Table I) the relative proportions of sialic acid-positive material in the keratan sulfate-rich region (-40%) and in the remainder of the core protein (-60%) remained constant. The amino acid composition of the keratan sulfate-rich peptides resembled data given by Heinegard (Table III) (10). The protein core in the keratan sulfate-rich region was richer in glutamic acid, proline, and phenylalanine and poorer in aspartic acid, serine, and glycine than in the remainder of the proteoglycan.
AND MARSH
Smaller keratan sulfate-bearing peptides originating from the chondroitin sulfate-rich region of the core protein (peak II, Fig. 6) (6) were also of larger average size in adult. Furthermore the adult material emerged as a much broader peak than that of calf (Fig. 6). The ratio of glucosamine to protein from both the keratan sulfate-rich region and the chondroitin sulfate-rich region was higher in adult (Table III). These results suggest an increase in the substitution of the core protein with keratan sulfate and/or an increase in the size of the chains. As will be shown below, the enrichment of the proteoglycan with glucosamine-containing glycosaminoglycan results from increased substitution with keratan sulfate chains rather than from increased glucosaminoglycan chain size. To reduce keratan sulfate-peptides to a still smaller size, Dl proteoglycan was TABLE
III
AMINO ACID AND AMINO SUGAR COMPOSITION FRAGMENTS RELEASED BY CHONDROITINASE ABC, TRYPSIN, AND CHYMOTRYPSIN DIGESTION (SEE FIG. 6) OF CALF AND STEER Dl PROTEOGLYCANS”
Calf Ammo acid Aspartic acid Threonine Serine Glutamic acid Prolme Glycine Alanine ?4 Cystine Vahne Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine GaIN/GlcN GlcN/protein
Peak I 44 40 75
151 1’76 81 88 1 46
9 29 79 26
82 43 12
18 0.19 1.02
OF
Steer
Peak II
Peak I
Peak II 110
74 62
53 48
139 125
96 175
85 74 20 68 4 40
175 72 66 2 34 6 24
a4 19
64
91
22
29 20 11 14
46 8 18
32 42 39 15 36 0.23 0.67
132
91
1.45
0.09
0.27
2.41
60
78 124 70 99 82 23 56 9 33
a Amino acids are expressed as residues per thousand residues.
445
AGE-RELATEDCHANGESINPROTEOGLYCANSTRUCTURE IDl-C&P-P30
Pkl -P I Pkll-P
1
___ ____-__
.l .3 .5 .7 .9 .l .3 .5 .? .9 Kd FIG. 7. Gel filtration of keratan sulfate-peptides on Bio-Gel P-30. Dl proteoglycans were digested with chondroitinase ABC followed by papain before being assessed on Bio-Gel P-30 (0.6 x 145 cm). Column effluents were analyzed for sialic acid: calf (-); steer (- - -).
treated with chondroitinase ABC followed by papain. The digest was assessed on BioGel P-30 (Fig. 7). Each preparation eluted as a large peak in or near the void volume with a distinct shoulder ( Kd 0.50) indicating a more retarded component. This latter may have represented single keratan sulfate chains linked to a short peptide (compare below with alkaline borohydride-treated Dl) while the larger material (K,, 0.22 in calf and 0.14 in steer), a greater proportion of which was present in steer, probably consisted of peptides with two or more keratan sulfate chains. Greater amounts of these larger peptides seem to be recovered from regions of the core protein heavily substituted with keratan sulfate. For example, when a papain digest of peak I (Fig. 6) (calf material) was compared with a similar digest of peak II on Sephadex G-75 (Fig. S), material from peak I was mostly excluded and clearly larger than that from peak II. It seems likely therefore that keratan sulfate chains are linked to the core protein particularly close together on the keratan sulfate-rich region. Gel filtration of the papain digests (Dl-P) on Bio-Gel P30 did not reveal any material resembling the oligosaccharide (3). Significant quantities of oligosaccharide could however be released from papain-generated fragments by treatment with alkaline borohydride after acetylation (unpublished observations). The results presented have indicated a relative increase in glucosaminoglycan
Kd
Gel filtration of calf keratan sulfate-peptides on Sephadex G-75 (0.6 x 145 cm). Fragments generated by the chondroitinase ABC, trypsm, chymotrypsin sequence were recovered after chromatography on Sepharose 6B (see Fig. 6). Material from peaks I and II was separately digested with papain and assessed on Sephadex G-75. Column effluents were analyzed by the anthrone procedure. FIG. 8.
content of adult proteoglycan. Dl proteoglycans were treated with alkaline borohydride to release intact glycosaminoglycans (3, 14) prior to determining the relative sizes of calf and steer keratan sulfate. In addition, fragments generated by the chondroitinase ABC, trypsin, chymotrypsin sequence were treated in the same way so as to obtain information regarding the relative sizes of keratan sulfate in different regions of the molecule. The results presented below (Fig. 9) indicate a moderate increase in average chain size of keratan sulfate in
PlO B
c
D
L!?h
-7 -
4
B c
i\;\ .1
3.5.7.!
Kd
FIG. 9. Gel filtration of sialic acid-containing polysaccharides and oligomers. Dl proteoglycans were degraded with alkaline borohydride as described in the text. Material was assessed on analytical columns of Bio-Gel P-2 (P2), on Bio-Gel P-30 after desalting on Bio-Gel P-2 (P2P30), on Bio-Gel P-30 without prior desalting (P30), and on B&Gel P-10 without prior desalting (PlO). Column effluents were analyzed for sialic acid. The peaks marked A, B, C, and D are described in the text.
446
SWEET, THONAR,
steer, and an associated decrease in proportion of oligosaccharide recovered. When alkaline borohydride-treated Dl was desalted on Bio-Gel P-2 prior to gel filtration on Bio-Gel P-30, three sialic acidpositive components were obtained (Fig. 9): (A) a minor component eluting near the void volume (& = 0.2), particularly in adult material, which was not examined further. It may have represented very large keratan sulfate or material not completely p-eliminated. (B) a major component slightly larger in steer (K,, 0.48) than in calf (0.52). (C) a smaller component eluting in both cases at K,, O&L 0.70 was present in greater proportion in the young animal (Fig. 9). When material was not desalted on BioGel P2 a fourth very small component (D) appeared in calf material (K,, 0.80 on BioGel P-30). Data in Fig. 9 indicate a small amount of sialic acid-positive material is retarded by Bio-Gel P-2 (K,, 0.2 on analytical column of Bio-Gel P-2). Strict adherence to desalting conditions as described under Experimental Procedures normally leads to the loss of this minor component, which was not studied further. The use of an analytical column of Bio-Gel P-10 further resolved the smaller components described above, clearly indicating the presence of oligosaccharide (component Dl-OHMI‘,-
EtOH
PPT
SNF
.l .3 .5 .7
.l .3 .5 I .s Kd
FIG. 10. Gel filtration of glycosaminoglycans on B&Gel P-30. Calf Dl proteoglycan was degraded with alkaline borohydride, desalted on Bio-Gel P2, lyophilized and dissolved in 0.9% (w/v) NaCl. Material precipitated by the addition of 2 vol ethanol (PPT) and that remaining in solution (SNF) was recovered and assessed on Bio-Gel P-30 (0.6 x 145 cm). Column effluents were analyzed for uranic acid (- - -) and sialic acid (-).
AND MARSH IPk I-OHBH4-P30
/ b
0.2 s: 4” 0.4-
Pk WOHBH4c
P30 D
c
e
d
0.2.
Kd
FIG. 11. Gel filtration of keratan sulfate and sialic acid-containing oligosaccharide on Bio-Gel P-30. Material corresponding to peaks I and II (see Fig. 6) was recovered and treated with alkaline borohydride before being assessed on Bio-Gel P-30 (0.6 x 145 cm) without desalting on Bio-Gel P-2. Peak I, calf; a; peak I, steer; b; peak II, calf; c; peak II, steer; d. Material labeled B, C, and D is described in the text.
C) in steer. Components B and C were purified and studied in greater detail. The data in Fig. 10 indicate the sialic acidcontaining material could be satisfactorily separated from chondroitin sulfate chains by precipitation of the latter (in 0.9% NaCl, w/v) with 2 vol ethanol. The glucosaminoglycans, which remained in solution, were then purified by fractionation on Bio-Gel P-30, recovery of the major keratan sulfate peak and the oligosaccharide, and rechromatography on Bio-Gel P-30. Analysis of these components indicates that adult keratan sulfate was, on average, one disaccharide unit longer than calf keratan sulfate. Molar ratios of galactosaminitol:glucosamine:galactose:sialic acid were 1:7:7:2 (adult keratan sulfate, component B), 1:6:6:2 (calf keratan sulfate, component B), and 1:1:1:2 (oligosaccharide, component C). Fragments resulting from the chondroitinase ABC, trypsin, chymotrypsin sequence were treated with alkaline borohydride to assess the relative proportions of the larger (component B) and smaller (component C) sialic acid-bearing glucosaminoglycans in peak I and peak II (from Fig. 6). The data in Fig. 11 indicate the presence of both components in peaks I (keratan sulfate-rich region) and II (chondroitin sulfate-rich region) but that the smaller oligosaccharide (component C) was present in greater pro-
AGE-RELATED
CHANGES
IN PROTEOGLYCAN
portions in peak II in both calf and steer. The slight difference between the elution position of the large keratan sulfate (K,, 0.55, peak I, and 0.52, peak II calf; 0.48, peak I, and 0.46, peak II steer) might be accounted for by the failure of the alkaline borohydride to produce p-elimination of some keratan sulfate chains attached to terminal amino acids of peptides produced by the enzyme action (30). GENERAL
DISCUSSION
A comparison of bovine articular cartilage from immature (l-2 months) and adult (3- 5 years) animals indicates the following maturation-related changes occur in the matrix proteoglycans: (1) Proteoglycans become smaller. (2) The chondroitin sulfate-rich region becomes smaller because both the chondroitin sulfate chains and core protein are shorter. Several other changes occur: (3) Dl monomers become enriched with keratan sulfate, as evidenced by a rise in the glucosamine/protein ratio. There is only a modest increase in keratan sulfate chain size. The results showed that increased substitution occurs in both the keratan sulfaterich and the chondroitin sulfate-rich regions of the core protein (see Table III). (4) The keratan sulfate-rich region becomes larger. (5) Proteoglycans have less chondroitin sulfate (shorter chains, fewer chains). (6) The oligosaccharide, a prominent feature of calf proteoglycans, does not undergo the same age-related increase as the other sialic acid-bearing component, i.e., keratan sulfate. Some of the changes noted are a result of a probable shortening of the core protein, as suggested by others (8, 31). This is more likely due to post-translational modification than to synthesis of a shorter range of proteoglycans in the older animal. The increased proportion of smaller proteoglycans in adult suggests the proteoglycans, modified possibly by extracellular proteases, may remain in situ for longer than in immature cartilage. Other changes however result from qualitative and presumably quantitative differences in the attached
STRUCTURE
447
carbohydrate chains. Taking into account published values for molecular weights of the core protein and it’s three regions (10,32) and the data presented in this paper, we calculate the respective numbers of carbohydrate side chains per molecule of proteoglycan on average as: chondroitin sulfate; 180 in calf and 80 in steer; keratan sulfate; 67 in calf and 83 in steer; oligosaccharide; 10 in calf and 5 in steer. Physiological implications of a change in proteoglycan size and composition have not received much attention. Cartilage stiffness and resistance to deformation are directly related to fixed charge density and glycosaminoglycan content (33). Equivalent amounts of keratan sulfate influence stiffness more than chondroitin sulfate (34), suggesting a possible advantage of keratan sulfaterich proteoglycans in adult cartilage subject to high load. It may be that the immature form of matrix synthesized in some osteoarthrotic cartilage (35) fails because it is inappropriate for adult requirements. ACKNOWLEDGMENTS We thank Dr. Vincent C. Hascall for helpful advice. This work was supported by the South African Medical Research Council, the Arthritis Foundation, and the Medical Faculty Research Endowment Fund. We thank Ferelyth Douglas for preparing the manuscript. REFERENCES 1. HASCALL, 2. 3. 4. 5. 6. 7.
8. 9. 10.
V. C., AND SAJDERA, S. W. (1969) J. Bid. Chem. 244,2384-2396. HASCALL, V. C., AND RIOLO, R. L. (19’72)J. Bid. Chm. 247, 4529-4538. THONAR, E. J-M. A., AND SWEET, M. B. E. (1979) Biochim. Biophys. Acta 584, 353-357. HARDINGHAM, T. E., AND MUIR, H. (1974) Biothem. J. 139, 565-581. HASCALL, V. C., AND HEINEGARD, D. (1974) J. Bid. Chem. 249, 4242-4249. HEINEGARD, D., AND AXELSSON, I. (1977) J. Bi01. Ch.em. 252, 1971-1979. THONAR, E. J-M. A., SWEET, M. B. E., IMMELMAN, A. R., AND LYONS, G. (1978) Calcif. Tissue Res. 26, 19-21. INEROT, S., HEINEGARD, D., AUDELL, L., AND OLSSEN, S-E. (1978) B&hem. J. 169,143156. MAROUDAS, A. (1976) Nature (London) 260, 80% 809. HEINEGARD, D. (1977)J. Bid. Chem. 252, 19801989.
448
SWEET,
THONAR,
11. IMMELMAN, A. R., SWEET, M. B. E., AND THONAR, E. J-M. A. (19’78)s. Afr. J. Med. Lab. Techlzol. 24, 35-36. 12. BITTER, T., AND MUIR, H. (1962)Anul. Biochem. 4, 330-334. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 14. THONAR, E. J-M. A., SWEET, M. B. E., IMMELMAN, A. R., AND LYONS, G. (1979) Arch. Biochem. Biophys. 194, 179-189. 15. JOURDIAN, G. W., DEAN, L., AND ROSEMAN, S. (1971) J. Biol. Chem. 246, 430-435. 16. DOWNS, F., AND PIGMAN, W. (1976) in Methods in Carbohydrate Chemistry, Vol VII: General Methods, Glycosaminoglycans, and Glycoproteins (Whistler, R. L., and BeMiller, J. N., eds.), pp. 244-248, Academic Press, New York. 17. TREVELYAN, W. E., AND HARRISON, J. S. (1952) B&hem. J. 50, 298-303. 18. OEGEMA, T. R., HASCALL, V. C., AND DZIEWIATKOWSKI, D. D. (1975) J. Bill. Chem. 250,61516159. 19. HEINEGARD, D. (1972) Biochim. Biophys. Acta 285, 181- 192. 20. SAJDERA, S. W., AND HASCALL, V. C. (1969) J. Biol. Chem. 244, 77-87. 21. SCOTT, J. E. (1960) Methods B&hem. Anal. 8, 145-197. 22. HEINEGARD, D., AND HASCALL, V. C. (1974) Arch. B&hem. Biaphys. 165, 427-441. 23. YANAGASHITA, M., RODBARD, D., AND HASCALL, V. C. (1979) J. Bill. Chem. 254, 911-920.
AND
MARSH
24. CARLSON, D. M. (1968) J. Biol. Chm. 243, 616626. 25. BAKER, J., AND CATERSON, B. (1977) Biochem. Btiphys. Res. Commun. 77, l-10. 26. KIMURA, J. H., OSDOBY, P., CAPLAN, A. I., AND HASCALL, V. C. (1978) J. Biol. Chem. 253, 4721-4729. 27. SWEET, M. B. E., THONAR, E. J-M. A., AND IMMELMAN, A. R. (1978) Arch. Biochem. Biophys. 189, 28-36. 28. WASTESON, A. (1975) B&hem. J. 122, 477-485. 29. MATHEWS, M. B. (1975) Molecular Biology Biochemistry and Biophysics, Vol. 19: Connective Tissue: Macromolecular Structure and Evolution, pp. 106- 107, Springer-Verlag, New York. 30. NEUBERGER, A., G~TTSCHALK, A., AND MARSHALL, R. D. (1966) in Glycoproteins. Their Composition, Structure and Function (Gottschslh, A., ed.), B. B. A. Library series, Vol. 5, pp. 273-295, Elsevier, Amsterdam. 31. BAYLISS, M. T., AND ALI, S. Y. (1978) B&hem. J. 176, 683-693. 32. HEINEGARD, D., AND HASCALL, V. C. (1974) J. Bial. Chem. 249, 4250-4259. 33. MAROUDAS, A. (1973) in Adult Articular Cartilage (Freeman, M. A. R., ed.), pp. 131-170, Pitman Medical, London. 34. KEMPSON, G. E., MUIR, H., SWANSON, S. A. V., AND FREEMAN, M. A. R. (1970) Biochim. Biophys. Acta 215, 70-77. 35. SWEET, M. B. E., THONAR, E. J-M. A., IMMELMAN, A. R., AND SOLOMON, L. (1977) Ann. Rheum. Dis. 36, 387-398.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 198, No. 2, December, pp. 439-448, 1979
Age-Related
Changes in Proteoglycan
M. B. E. SWEET,* *Orthopaedic
Research Johannesburg,
E. J-M. A. THONAR,”
Structure
AND J. MARSH?
Laboratories, Department of Orthopaedic Surgery, University of the Witwatersrand, and 7Department of Medical Biochemistry, University of Cape Town, Cape Town, South Africa
Received April 21, 1979 Proteoglycans of calf and steer articular cartilage were studied with a view of assessing structure and changes occurring as a result of the aging process. The average reduction in hydrodynamic size noted in steer was associated with a diminution in size ofthe chondroitin sulfate-rich region of the core protein as well as the chondroitin sulfate chains themselves. By contrast the keratan sulfate-rich region was hydrodynamically larger in steer although the keratan sulfate chains were only slightly longer than in calf. The proteoglycans showed a maturation-related decrease in chondroitin sulfate content (shorter chains, fewer chains, smaller chondroitin sulfate-rich region) and an enrichment in keratan sulfate chains in both the chondroitin sulfate-rich and keratan sulfate-rich regions. Proteoglycans from both age groups contained an oligosaccharide which was recovered mainly from outside of the keratan sulfate-rich region. There were no significant differences in size between keratan sulfate chains recovered from the keratan sulfate-rich region and the chondroitin sulfate-rich region.
Proteoglycans are major components of hyaline cartilage. They consist of a central core protein to which chondroitin sulfate, keratan sulfate, and a small proportion of oligosaccharides are covalently linked (l-3). One end of the core protein is not substituted with glycosaminoglycans and has some tertiary structure permitting the formation of a noncovalent complex with hyaluronate (4, 5). Adjacent to the hyaluronate-binding region is a peptide sequence largely substituted with keratan sulfate (6). To the remainder of the core protein is attached chondroitin sulfate and the rest of the keratan sulfate (6). The normal maturation process in articular cartilage is characterized by a fall in water content (‘7) and a decreased extractability of proteoglycans which become richer in keratan sulfate and smaller in hydrodynamic size (8). These changes may be related, inter alia, to a greater degree of crosslinking of the collagen network (9) and to an apparent shortening of the chondroitin sulfate-rich region of the core protein (10). The aim of this study was to examine the question of aging as it affects proteoglycans of articular cartilage by assessing the in439
dividual fragments derived from the parent macromolecules. In particular, keratan sulfate chains have been characterized and the distribution of the oligosaccharide along the core protein has been studied. EXPERIMENTAL
PROCEDURES
Materials All chemicals were of the best available grade: benzamidine HCl and 6-aminohexanoic acid were from E. Merck, Darmstadt; diphenylcarbamyl chloride-treated trypsin and a-chymotrypsin were from Sigma; chondroitinase ABC (P. vulgaris) was from Miles; Bio-Gel P-2, P-10, and P-30 were from Bio-Rad; Sephadex G-75 and G-200, Sepharose 2B, CL2B, 4B, and 6B, and blue dextran were from Pharmacia.
Analytical
Methods
Uranic acid was determined by an automated modification (11) of the carbazole-borosulfuric acid method (12) with glucuronolactone as standard. Protein was determined by the Folin method (13) or by summation of amino acids. Sialic acid, as a monitor for keratan sulfate and the oligosaccharide (3,14), was determined by the method of Jourdian et al. (15). Amino acids and hexosamines were determined on a Beckman 116 amino acid analyzer after hydrolysis under nitrogen of samples in 6 M HCl for 24 h at 100°C or in 4 M HCl for 4 h 0003-9861/79/140439-10$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
440
SWEET, THONAR,
at lOO”C, respectively. Hexosaminitols were determined by the method of Downs and Pigman (16). Neutral sugars were determined by the anthrone procedure (17). Specific enzymatic determination of galactose was performed as described elsewhere (3).
Tissue Articular cartilage of calves (4 to 8 weeks of age) and steers (3 to 5 years of age) was dissected out immediately after slaughter. Because of its vascularity the deepest 2 mm adjacent to the zone of provisional calcification of calf cartilage was discarded. The remaining cartilage and that of the steers was diced and frozen in liquid nitrogen for transportation to the laboratory.
Extraction
of Proteoglycans
Finely diced cartilage (1 to 2 mm thick) was transferred from liquid nitrogen into lo-fold its weight of 4 M guamdine. HCl in 0.05 M sodium acetate, pH 5.8, and stirred at 2°C for 48 h. The extracting solution contained the following proteolytic enzyme inhibitors: 0.01 M disodium EDTA, 0.005 M benzamidine.HCl, and 0.1 M 6aminohexanoic acid (18).
Preparation
of Al and Dl Proteoglycanl
The extracts were filtered through glass wool and 2 vol ethanol was added, with constant stirring. The resulting precipitates, which contained all three components of proteoglycan aggregate (unpublished work), were collected by centrifugation (25,OOOg, 2”C, 20 min), washed in 70% ethanol, recollected and dissolved in 0.5 M guanidine.HCl or 4 M guanidine.HCl, both in 0.05 M sodium acetate, pH 5.8, containing the protease inhibitors listed above. CsCl was added to the two solutions to give a starting density of 1.69 g/ml (0.5 M guanidine.HCl for Al preparation) or 1.50 g/ml (4 M guanidine. HCl for Dl preparation) (20). CsCl density gradient centrifugation was carried out at 40,060 rpm for 48 h at 10°C in a Beckman 75Ti rotor. The tubes were sliced in a Beckman tube slicer. Al samples were recovered from the bottom quarter of tubes containing the associative solution (0.5 M guanidine. HCl) and Dl samples from the bottom two-fifths of those containing the dissociative solution (4 M guanidine. HCl) by dialysis and lyophilization.
AND MARSH rich region, the keratan sulfate-rich region, chondroitin sulfate chains, keratan sulfate chains, and the oligosaccharide. Treatment with hydroxylamine. Al proteoglycan was fractionated on a preparative column of Sepharose 2B. Material eluting in the void volume was collected, dialyzed, and lyophilized. Samples (10 mg/ml) were incubated in 1 M hydroxylamine, 0.05 M 2-(N-morpho1ino)ethanesulfonic acid, pH 6.5, for 120 h at 23°C (6). Following dialysis and lyophilization, samples were assessed on an analytical column of Sepharose 2B. Papain digestion. Dl proteoglycans were digested with 2x crystalline papain (British Drug Houses) (1 pg/mg proteoglycan) in 0.05 M sodium acetate, pH 5.8, containing 0.02 M L-cysteine and 0.005 M disodium EDTA at 60°C for 24 h (21). Aliquots of the digests were assessed on an analytical column of Sephadex G-200 or Bio-Gel P-30. Chondroitinase ABC. Dl proteoglycan was digested with chondroitinase ABC (0.05 unit/mg proteoglycan) in 0.1 M Tris-acetate buffer, pH 7.3, at 37°C for 5 h. This was followed by digestion with trypsin and chymotrypsin in the same buffer (10 pg/mg proteoglycan) at 37°C for 12 h (22). Digests were fractionated on both analytical and preparative columns of Sepharose 6B (see Fig. 6); materials from peaks I and II were collected for further analysis, digestion with papain, or treatment with alkaline borohydride. Samples of Dl proteoglycan were digested with chondroitinase ABC only or with trypsin and chymotrypsin and chromatographed on Sepharose 4B and 6B, respectively. Alkaline bwohydride. Samples of Dl proteoglycan or aliquots of material digested and fractionated as in chondroitinase ABC section above were desalted on Bio-Gel P-2 and treated (-10 mg/ml) with 0.05 M NaOH in 1 M sodium borohydride at 45°C for 48 h under nitrogen (3, 23, 24). The borohydride was destroyed by the dropwise addition of concentrated acetic acid, following which samples were freeze dried and desalted on Bio-Gel P-2 as described below. Reduction of Al proteoglycans. In order to dissociate proteoglycan aggregates and to inhibit their reformation with hyaluronate, Al proteoglycan was dissolved in 1% sodium dodecyl sulfate (SDS)V5% mercaptoethanol and kept at 65°C for 45 min. An aliquot of the sample was assessed on the Sepharose CLZB SDS analytical column (see below).
Gel Chromatography Enzymatic and Chemical of Proteoglycan
Degradation
Al and Dl proteoglycans were degraded by a variety of means in order to assess the relative sizes or proportions of the core protein, the chondroitin sulfate* The Al, Dl notation was described by Heinegard w.
Analytical columns of Sepharose 2B, 4B, and 6B, Sephadex G-75, Bio-Gel P-2, P-10, and P-30 (0.6 x 145 cm) and Sephadex G-200 (0.8 x 120 cm) were eluted with 0.5 M sodium acetate, pH 6.8 (or pH 5.8 for Sepharose 2B), at 1.0-2.0 ml/h at 18°C. Identical sized fractions (0.7 ml) were collected with the aid of fraction 2 Abbreviation
used: SDS, sodium dodecyl sulfate.
441
AGE-RELATEDCHANGESINPROTEOGLYCANSTRUCTURE collectors equipped with drop counters. An analytical column of Sepharose CL2B (0.6 x 145 cm) was eluted with 0.05 M sodium phosphate buffer in 0.1% SDS, pH 7.3, at 0.6 ml/h at 23°C (25). Fractions of 0.8 ml were collected as before. All columns were calibrated with Al proteoglycan or high molecular weight hyal~ nate and glucuronolactone. Preparative columns of Sepharose 2B (1.5 x 120 cm) and 6B (1.0 x 145 cm) were eluted with 0.5 Msodium acetate, pH 5.8 or 6.8, respectively, at about 3 ml/cmVh. Respective fractions of 5 and 3 ml were collected. Column effluents were analyzed for uranic acid, sialic acid, and protein. Sialic acid determination was found to be preferable to the anthrone procedure as a monitor for keratan sulfate and the oligosaccharlde (3, 14).
Desalting
TABLE
I
COMPOWPIONOF CALFANDSTEER ARTICULAR CARTILAGES Calf Water content GalN:GlcN Hexosamine (I*rmol/lOO mg tissue) Chondroitin-SO1
75.1 7.9 12.0 10.5 (87.2%) 0.9 (7.5%) 0.3 (2.1%)
Keratan-SO, Hyaluronate
on Bio-Gel P-2
The high concentration of salts in alkaline bomhydridetreated samples does not allow the application of material in small volumes to analytical columns. A preliminary investigation had established the presence of a pentasaccharide in proteoglycans of bovine articular cartilage (3). Therefore it was decided to desalt the samples on a gel of lowest exclusion limit rather than to employ dialysis. Columns of Bio-Gel P-2 (1.5 x 25 cm) were recalibrated with blue dextran after every five runs. Samples (0.8 ml) were applied and eluted with 0.5 M pyridine acetate, pH 7.0. Material was collected to the end of the blue dextran profile. The desalted material was subsequently lyophilized.
Electrophoresis
of Glycosaminoglycans
Glycosaminoglycans were recovered from papain digests of hydroxylamine-treated Dl proteoglycan following chromatography on Sepharose 2B. Samples were subjected to electrophoresis on cellulose acetate strips before and after speciiic enzymatic digestion as described elsewhere (7). International reference standards of glycosaminoglycans were a gift of Dr. Martin B. Mathews, Department of Paediatrics, University of Chicago, Chicago, Illinois. RESULTS
AND DISCUSSION
The general characteristics of the articular cartilage used in this study are outlined in Table I. Adult articular cartilage was less hydrated and contained less glycosaminoglycan but more keratan sulfate than immature cartilage. Intact monomeric proteoglycans (Dl) were assessed on Sepharose 2B: those of steer were more retarded by the gel than those of calf (K,, 0.42 against 0.34) (Figs. la and b). This point was further examined
Steer 67.9 2.2 7.6 5.2 (68.1%) 2.0 (26.6%) 0.3 (3.6%)
n Water content and glycosaminoglycan concentrations (as pmol hexosamine/lOO mg dry tissue) were estimated as described elsewhere (7, 14).
by chromatographing Al proteoglycans treated with 1% SDS/5% mercaptoethanol, on Sepharose CLBB: The results confirmed that adult proteoglycans were smaller than those of calf (Figs. lc and d). It is noteworthy that the sialic acid peaks of both Al SDS preparations, particularly in the adult, eluted behind the uronate peaks. This is in agreement with the explanation for proteoglycan polydispersity suggested by Heinegard (10): The smaller the proteoglycan the higher the ratio of keratan sulfate to STEER
;::~‘A,,id&\> J
I
,325
.? .9
1 .3 .5 .7 .9 Kd
FIG. 1. Gel filtration of proteoglycans on Sepharose 2B or CLZB (0.6 x 145 cm). Dl proteoglycans were prepared from steer (a) and calf(b) articular cartilage. Al proteoglycans were reduced in 1% SDS/58 mercaptoethanol and chromatographed on Sepharose CL2B in 0.1% SDS (c, steer; d, calf). Column effluents were analyzed for uranic acid (-) and/or sialic acid (- - -).
442
SWEET, .3
1
THONAR,
Al-OHNH2-2B 1
.2 .4 .6
.a 1.0
Kd FIG. 2. Gel titration of hydroxylamine-treated Al proteoglycan on Sepharose 2B. Al proteoglycan (a, calf; b, steer) excluded from Sepharose 2B (1.5 x 120 cm) was treated with 1 M hydroxylamine as described in the text and chromatographed on an analytical column of Sepharose 2B (0.6 x 145 cm). Fractions were analyzed for uranic acid: calf (-); steer (- - -).
chondroitin sulfate. The small retarded peak (Figs. lc and d) may represent the link proteins (25), which were not studied further. No hyaluronate was detectable in the void volume of the CLBB SDS column effluents, suggesting it was small enough to be retarded by the gel. Because of compositional changes it has been suggested that proteoglycans of hyaline cartilage become smaller with age as a result of a progressive shortening of the chondroitin sulfate-rich region (8). Electron microscopy of proteoglycans has confirmed the existence of a range of sizes within an individual preparation, but no similar evidence has been obtained for an age-related decrease in size. Kimura et al. (26) found average monomer lengths of 343 and 310 nm in aggregates from bovine nasal septum and chick embryo epiphyses, respectively. In order to test the hypothesis the experiments described below were performed. In contrast to material from bovine nasal septum, Al preparations from cartilage structures subject to high stress contain a large proportion of proteoglycans retarded by Sepharose 2B: e.g., calf articular cartilage 55% (27), calf annulus fibrosus 50%, calf nucleus pulposus 50% (G. Lyons, unpublished work). This suggests either the existence of large numbers of nonaggregating proteoglycans or that much of the hyaluronate in these tissues is of relatively low molecular weight permitting aggregation with only one proteoglycan monomer. In
AND MARSH
view of the above, we selected those Al proteoglycans excluded from Sepharose 2B for treatment with hydroxylamine assuming however, that this sample would be representative of the total population. Treatment with hydroxylamine cleaves off the chondroitin sulfate-rich region, leaving the remainder of the molecule still attached to hyaluronate (10). Assessment of material treated in this way on Sepharose 2B, should give an indication of the relative size of the chondroitin sulfate-rich region. The uronate profiles in Fig. 2 indicate that the chondroitin sulfate-rich region was of larger hydrodynamic size in the calf (K,, 0.50 calf; 0.67 steer). Qualitative analysis of the excluded and retarded fractions confirmed the predominance of keratan sulfate in the void volume and chondroitin sulfate in the retarded volume. Traces of hyaluronate (originally excluded from the preparative Sepharose 2B column as part of the starting Al preparations) were identified in both fractions, suggesting the possible existence of short chain hyaluronate bound to one or a few hyaluronate-binding regions. Chondroitin sulfate chains were prepared by papain digestion and/or by alkaline borohydride degradation of Dl monomers, and assessed on Sephadex G-200 (Fig. 3). The K,, of 0.58 (steer) and 0.51 (calf) would indicate molecular weights of 8900 and 10,400, respectively, assuming the batch of Sephadex G-200 had the same elution properties as that used by Wasteson (28). This difference would certainly contribute to the difference in hydrodynamic size of the Dl monomers. (Dl-OHBH4-GSOO(
.1 .3 .5 7 .Q Kd FIG. 3. Gel chromatography of chondroitin sulfate chains on Sephadex G-200 (0.8 x 120 cm). Chondroitin sulfate chains were liberated from calf (-) and steer (- - -) Dl proteoglycans by alkaline borohydride as described in the text. Column effluents were analyzed for uranic acid.
443
AGE-RELATEDCHANGESINPROTEOGLYCANSTRUCTURE
Chondroitin sulfate is attached in clusters of one to eight polysaccharide chains to the core protein (22). Clusters were prepared by digestion of Dl monomers by trypsin and chymotrypsin (22) and assessed on Sepharose 6B (Fig. 4). The results (K,, 0.39) for calf and (K,, 0.49) for steer are consistent with molecular weights of 52,000 and 36,000, respectively, as calculated from the data of Heinegard and Hascall (22). The core proteins, free of chondroitin sulfate chains, were assessed on Sepharose 4B (Fig. 5). The results (K,, 0.30 for calf and 0.36 for steer) support the idea of a decrease in core protein size with age (8). However the elution profile for steer was much broader than for calf, possibly because of a greater proportion of keratan sulfate. The existence of an age-related decrease in core protein size is supported by the amino acid composition of calf and steer Dl monomers (Table II). Those of the former were richer in serine and glycine, amino acids associated with the chondroitin sulfate linkage region (29). The molar ratios of galactosamine/X amino acids indicate the presence of 2.6-fold as much galactosamine containing glycosaminoglycan per gram of protein in calf than in steer. Corrections for the galactosamine originating from the linkage region of keratan sulfate and for average chondroitin sulfate chain size yield figures which indicate that one gram of protein core in calf is substituted with 2.3-fold as many chondroitin sulfate chains as is the case in steer. The data presented (Table II, Dl-CB-4B) suggest a modest decrease in
Dl -T,C-6B I
0 .2 .4 .6 .6
1.0
Kd FIG. 4. Gel filtration of chondroitin sulfate clusters on Sepharose 6B (0.6 x 145 cm). Dl proteoglycans were digested with trypsin and chymotrypsin as described in the text. Column effluents were analyzed for uranic acid: calf (-); steer (- - -).
Dl-CB-4B I
.I .3 .5 .7
.Q
Kd FIG. 5. Gel filtration of core proteins on Sepharose 4B. Dl proteoglycans (~30 mg) were digested with chondroitinase ABC, lyophilized, and applied to an analytical Sepharose 4B column (0.6 x 145 cm). Column effluents were analyzed for uranic acid: calf (-); steer (- - -).
core protein size in the mature animal. Consequently, the difference in degree of substitution with chondroitin sulfate may be even greater when expressed on a molar basis. TABLE
II
AMINOACID COMPOSITION ANDMOLARRATIOSOF GALACTOSAMINEANDGLUCOSAMINETOXAMINO ACIDSOFDl MONOMERSFROM~ALFAND STEERARTICULARCARTILAGE" Amino acid
Calf
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine ‘h Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Hexosamine/Z amino acids GalN/P amino acids GlcN/Z amino acids
55 127 139 73 121 65 13 47 5 35 79 29 44 51 14 28 3.86 3.59 0.31
75
Steer 86 51 99 142 75
109 86 12 58 8 31 87 33 49 28 13 33 1.94 1.39 0.51
a Amino acids are expressed as residues per thousand residues.
444
SWEET,
THONAR,
Calf
.2 .4 .6 .6 1.0 Kd
FIG. 6. Gel filtration of enzyme-generated fragments of proteoglycans on Sepharose 6B. Calf and steer Dl monomers were sequentially degraded with chondroitinase ABC, trypsin, and chymotrypsin (see the text), lyophilized, and applied to an analytical column of Sepharose 6B (0.6 x 145 cm). Column effluents were analyzed for sialic acid (-) and by the anthrone procedure (- - -). Material equivalent to peaks I and II was recovered after gel f&ration on a larger Sepharose 6B column (1.0 x 145 cm) for further analysis.
Dl proteoglycans from both age groups were sequentially digested with chondroitinase ABC, trypsin, and chymotrypsin and the resulting fragments chromatographed on Sepharose 6B (Fig. 6). This procedure has been designed for the recovery of the keratan sulfate-rich region (peak I, Fig. 6) separate from the remainder of the core protein (peak II, Fig. 6). The results of this experiment indicate that the keratan sulfate-rich region becomes larger with age (K,, 0.29 for steer and 0.35 for calf). Despite the quantitative increase in keratan sulfate with age (Table I) the relative proportions of sialic acid-positive material in the keratan sulfate-rich region (-40%) and in the remainder of the core protein (-60%) remained constant. The amino acid composition of the keratan sulfate-rich peptides resembled data given by Heinegard (Table III) (10). The protein core in the keratan sulfate-rich region was richer in glutamic acid, proline, and phenylalanine and poorer in aspartic acid, serine, and glycine than in the remainder of the proteoglycan.
AND MARSH
Smaller keratan sulfate-bearing peptides originating from the chondroitin sulfate-rich region of the core protein (peak II, Fig. 6) (6) were also of larger average size in adult. Furthermore the adult material emerged as a much broader peak than that of calf (Fig. 6). The ratio of glucosamine to protein from both the keratan sulfate-rich region and the chondroitin sulfate-rich region was higher in adult (Table III). These results suggest an increase in the substitution of the core protein with keratan sulfate and/or an increase in the size of the chains. As will be shown below, the enrichment of the proteoglycan with glucosamine-containing glycosaminoglycan results from increased substitution with keratan sulfate chains rather than from increased glucosaminoglycan chain size. To reduce keratan sulfate-peptides to a still smaller size, Dl proteoglycan was TABLE
III
AMINO ACID AND AMINO SUGAR COMPOSITION FRAGMENTS RELEASED BY CHONDROITINASE ABC, TRYPSIN, AND CHYMOTRYPSIN DIGESTION (SEE FIG. 6) OF CALF AND STEER Dl PROTEOGLYCANS”
Calf Ammo acid Aspartic acid Threonine Serine Glutamic acid Prolme Glycine Alanine ?4 Cystine Vahne Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine GaIN/GlcN GlcN/protein
Peak I 44 40 75
151 1’76 81 88 1 46
9 29 79 26
82 43 12
18 0.19 1.02
OF
Steer
Peak II
Peak I
Peak II 110
74 62
53 48
139 125
96 175
85 74 20 68 4 40
175 72 66 2 34 6 24
a4 19
64
91
22
29 20 11 14
46 8 18
32 42 39 15 36 0.23 0.67
132
91
1.45
0.09
0.27
2.41
60
78 124 70 99 82 23 56 9 33
a Amino acids are expressed as residues per thousand residues.
445
AGE-RELATEDCHANGESINPROTEOGLYCANSTRUCTURE IDl-C&P-P30
Pkl -P I Pkll-P
1
___ ____-__
.l .3 .5 .7 .9 .l .3 .5 .? .9 Kd FIG. 7. Gel filtration of keratan sulfate-peptides on Bio-Gel P-30. Dl proteoglycans were digested with chondroitinase ABC followed by papain before being assessed on Bio-Gel P-30 (0.6 x 145 cm). Column effluents were analyzed for sialic acid: calf (-); steer (- - -).
treated with chondroitinase ABC followed by papain. The digest was assessed on BioGel P-30 (Fig. 7). Each preparation eluted as a large peak in or near the void volume with a distinct shoulder ( Kd 0.50) indicating a more retarded component. This latter may have represented single keratan sulfate chains linked to a short peptide (compare below with alkaline borohydride-treated Dl) while the larger material (K,, 0.22 in calf and 0.14 in steer), a greater proportion of which was present in steer, probably consisted of peptides with two or more keratan sulfate chains. Greater amounts of these larger peptides seem to be recovered from regions of the core protein heavily substituted with keratan sulfate. For example, when a papain digest of peak I (Fig. 6) (calf material) was compared with a similar digest of peak II on Sephadex G-75 (Fig. S), material from peak I was mostly excluded and clearly larger than that from peak II. It seems likely therefore that keratan sulfate chains are linked to the core protein particularly close together on the keratan sulfate-rich region. Gel filtration of the papain digests (Dl-P) on Bio-Gel P30 did not reveal any material resembling the oligosaccharide (3). Significant quantities of oligosaccharide could however be released from papain-generated fragments by treatment with alkaline borohydride after acetylation (unpublished observations). The results presented have indicated a relative increase in glucosaminoglycan
Kd
Gel filtration of calf keratan sulfate-peptides on Sephadex G-75 (0.6 x 145 cm). Fragments generated by the chondroitinase ABC, trypsm, chymotrypsin sequence were recovered after chromatography on Sepharose 6B (see Fig. 6). Material from peaks I and II was separately digested with papain and assessed on Sephadex G-75. Column effluents were analyzed by the anthrone procedure. FIG. 8.
content of adult proteoglycan. Dl proteoglycans were treated with alkaline borohydride to release intact glycosaminoglycans (3, 14) prior to determining the relative sizes of calf and steer keratan sulfate. In addition, fragments generated by the chondroitinase ABC, trypsin, chymotrypsin sequence were treated in the same way so as to obtain information regarding the relative sizes of keratan sulfate in different regions of the molecule. The results presented below (Fig. 9) indicate a moderate increase in average chain size of keratan sulfate in
PlO B
c
D
L!?h
-7 -
4
B c
i\;\ .1
3.5.7.!
Kd
FIG. 9. Gel filtration of sialic acid-containing polysaccharides and oligomers. Dl proteoglycans were degraded with alkaline borohydride as described in the text. Material was assessed on analytical columns of Bio-Gel P-2 (P2), on Bio-Gel P-30 after desalting on Bio-Gel P-2 (P2P30), on Bio-Gel P-30 without prior desalting (P30), and on B&Gel P-10 without prior desalting (PlO). Column effluents were analyzed for sialic acid. The peaks marked A, B, C, and D are described in the text.
446
SWEET, THONAR,
steer, and an associated decrease in proportion of oligosaccharide recovered. When alkaline borohydride-treated Dl was desalted on Bio-Gel P-2 prior to gel filtration on Bio-Gel P-30, three sialic acidpositive components were obtained (Fig. 9): (A) a minor component eluting near the void volume (& = 0.2), particularly in adult material, which was not examined further. It may have represented very large keratan sulfate or material not completely p-eliminated. (B) a major component slightly larger in steer (K,, 0.48) than in calf (0.52). (C) a smaller component eluting in both cases at K,, O&L 0.70 was present in greater proportion in the young animal (Fig. 9). When material was not desalted on BioGel P2 a fourth very small component (D) appeared in calf material (K,, 0.80 on BioGel P-30). Data in Fig. 9 indicate a small amount of sialic acid-positive material is retarded by Bio-Gel P-2 (K,, 0.2 on analytical column of Bio-Gel P-2). Strict adherence to desalting conditions as described under Experimental Procedures normally leads to the loss of this minor component, which was not studied further. The use of an analytical column of Bio-Gel P-10 further resolved the smaller components described above, clearly indicating the presence of oligosaccharide (component Dl-OHMI‘,-
EtOH
PPT
SNF
.l .3 .5 .7
.l .3 .5 I .s Kd
FIG. 10. Gel filtration of glycosaminoglycans on B&Gel P-30. Calf Dl proteoglycan was degraded with alkaline borohydride, desalted on Bio-Gel P2, lyophilized and dissolved in 0.9% (w/v) NaCl. Material precipitated by the addition of 2 vol ethanol (PPT) and that remaining in solution (SNF) was recovered and assessed on Bio-Gel P-30 (0.6 x 145 cm). Column effluents were analyzed for uranic acid (- - -) and sialic acid (-).
AND MARSH IPk I-OHBH4-P30
/ b
0.2 s: 4” 0.4-
Pk WOHBH4c
P30 D
c
e
d
0.2.
Kd
FIG. 11. Gel filtration of keratan sulfate and sialic acid-containing oligosaccharide on Bio-Gel P-30. Material corresponding to peaks I and II (see Fig. 6) was recovered and treated with alkaline borohydride before being assessed on Bio-Gel P-30 (0.6 x 145 cm) without desalting on Bio-Gel P-2. Peak I, calf; a; peak I, steer; b; peak II, calf; c; peak II, steer; d. Material labeled B, C, and D is described in the text.
C) in steer. Components B and C were purified and studied in greater detail. The data in Fig. 10 indicate the sialic acidcontaining material could be satisfactorily separated from chondroitin sulfate chains by precipitation of the latter (in 0.9% NaCl, w/v) with 2 vol ethanol. The glucosaminoglycans, which remained in solution, were then purified by fractionation on Bio-Gel P-30, recovery of the major keratan sulfate peak and the oligosaccharide, and rechromatography on Bio-Gel P-30. Analysis of these components indicates that adult keratan sulfate was, on average, one disaccharide unit longer than calf keratan sulfate. Molar ratios of galactosaminitol:glucosamine:galactose:sialic acid were 1:7:7:2 (adult keratan sulfate, component B), 1:6:6:2 (calf keratan sulfate, component B), and 1:1:1:2 (oligosaccharide, component C). Fragments resulting from the chondroitinase ABC, trypsin, chymotrypsin sequence were treated with alkaline borohydride to assess the relative proportions of the larger (component B) and smaller (component C) sialic acid-bearing glucosaminoglycans in peak I and peak II (from Fig. 6). The data in Fig. 11 indicate the presence of both components in peaks I (keratan sulfate-rich region) and II (chondroitin sulfate-rich region) but that the smaller oligosaccharide (component C) was present in greater pro-
AGE-RELATED
CHANGES
IN PROTEOGLYCAN
portions in peak II in both calf and steer. The slight difference between the elution position of the large keratan sulfate (K,, 0.55, peak I, and 0.52, peak II calf; 0.48, peak I, and 0.46, peak II steer) might be accounted for by the failure of the alkaline borohydride to produce p-elimination of some keratan sulfate chains attached to terminal amino acids of peptides produced by the enzyme action (30). GENERAL
DISCUSSION
A comparison of bovine articular cartilage from immature (l-2 months) and adult (3- 5 years) animals indicates the following maturation-related changes occur in the matrix proteoglycans: (1) Proteoglycans become smaller. (2) The chondroitin sulfate-rich region becomes smaller because both the chondroitin sulfate chains and core protein are shorter. Several other changes occur: (3) Dl monomers become enriched with keratan sulfate, as evidenced by a rise in the glucosamine/protein ratio. There is only a modest increase in keratan sulfate chain size. The results showed that increased substitution occurs in both the keratan sulfaterich and the chondroitin sulfate-rich regions of the core protein (see Table III). (4) The keratan sulfate-rich region becomes larger. (5) Proteoglycans have less chondroitin sulfate (shorter chains, fewer chains). (6) The oligosaccharide, a prominent feature of calf proteoglycans, does not undergo the same age-related increase as the other sialic acid-bearing component, i.e., keratan sulfate. Some of the changes noted are a result of a probable shortening of the core protein, as suggested by others (8, 31). This is more likely due to post-translational modification than to synthesis of a shorter range of proteoglycans in the older animal. The increased proportion of smaller proteoglycans in adult suggests the proteoglycans, modified possibly by extracellular proteases, may remain in situ for longer than in immature cartilage. Other changes however result from qualitative and presumably quantitative differences in the attached
STRUCTURE
447
carbohydrate chains. Taking into account published values for molecular weights of the core protein and it’s three regions (10,32) and the data presented in this paper, we calculate the respective numbers of carbohydrate side chains per molecule of proteoglycan on average as: chondroitin sulfate; 180 in calf and 80 in steer; keratan sulfate; 67 in calf and 83 in steer; oligosaccharide; 10 in calf and 5 in steer. Physiological implications of a change in proteoglycan size and composition have not received much attention. Cartilage stiffness and resistance to deformation are directly related to fixed charge density and glycosaminoglycan content (33). Equivalent amounts of keratan sulfate influence stiffness more than chondroitin sulfate (34), suggesting a possible advantage of keratan sulfaterich proteoglycans in adult cartilage subject to high load. It may be that the immature form of matrix synthesized in some osteoarthrotic cartilage (35) fails because it is inappropriate for adult requirements. ACKNOWLEDGMENTS We thank Dr. Vincent C. Hascall for helpful advice. This work was supported by the South African Medical Research Council, the Arthritis Foundation, and the Medical Faculty Research Endowment Fund. We thank Ferelyth Douglas for preparing the manuscript. REFERENCES 1. HASCALL, 2. 3. 4. 5. 6. 7.
8. 9. 10.
V. C., AND SAJDERA, S. W. (1969) J. Bid. Chem. 244,2384-2396. HASCALL, V. C., AND RIOLO, R. L. (19’72)J. Bid. Chm. 247, 4529-4538. THONAR, E. J-M. A., AND SWEET, M. B. E. (1979) Biochim. Biophys. Acta 584, 353-357. HARDINGHAM, T. E., AND MUIR, H. (1974) Biothem. J. 139, 565-581. HASCALL, V. C., AND HEINEGARD, D. (1974) J. Bid. Chem. 249, 4242-4249. HEINEGARD, D., AND AXELSSON, I. (1977) J. Bi01. Ch.em. 252, 1971-1979. THONAR, E. J-M. A., SWEET, M. B. E., IMMELMAN, A. R., AND LYONS, G. (1978) Calcif. Tissue Res. 26, 19-21. INEROT, S., HEINEGARD, D., AUDELL, L., AND OLSSEN, S-E. (1978) B&hem. J. 169,143156. MAROUDAS, A. (1976) Nature (London) 260, 80% 809. HEINEGARD, D. (1977)J. Bid. Chem. 252, 19801989.
448
SWEET,
THONAR,
11. IMMELMAN, A. R., SWEET, M. B. E., AND THONAR, E. J-M. A. (19’78)s. Afr. J. Med. Lab. Techlzol. 24, 35-36. 12. BITTER, T., AND MUIR, H. (1962)Anul. Biochem. 4, 330-334. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 14. THONAR, E. J-M. A., SWEET, M. B. E., IMMELMAN, A. R., AND LYONS, G. (1979) Arch. Biochem. Biophys. 194, 179-189. 15. JOURDIAN, G. W., DEAN, L., AND ROSEMAN, S. (1971) J. Biol. Chem. 246, 430-435. 16. DOWNS, F., AND PIGMAN, W. (1976) in Methods in Carbohydrate Chemistry, Vol VII: General Methods, Glycosaminoglycans, and Glycoproteins (Whistler, R. L., and BeMiller, J. N., eds.), pp. 244-248, Academic Press, New York. 17. TREVELYAN, W. E., AND HARRISON, J. S. (1952) B&hem. J. 50, 298-303. 18. OEGEMA, T. R., HASCALL, V. C., AND DZIEWIATKOWSKI, D. D. (1975) J. Bill. Chem. 250,61516159. 19. HEINEGARD, D. (1972) Biochim. Biophys. Acta 285, 181- 192. 20. SAJDERA, S. W., AND HASCALL, V. C. (1969) J. Biol. Chem. 244, 77-87. 21. SCOTT, J. E. (1960) Methods B&hem. Anal. 8, 145-197. 22. HEINEGARD, D., AND HASCALL, V. C. (1974) Arch. B&hem. Biaphys. 165, 427-441. 23. YANAGASHITA, M., RODBARD, D., AND HASCALL, V. C. (1979) J. Bill. Chem. 254, 911-920.
AND
MARSH
24. CARLSON, D. M. (1968) J. Biol. Chm. 243, 616626. 25. BAKER, J., AND CATERSON, B. (1977) Biochem. Btiphys. Res. Commun. 77, l-10. 26. KIMURA, J. H., OSDOBY, P., CAPLAN, A. I., AND HASCALL, V. C. (1978) J. Biol. Chem. 253, 4721-4729. 27. SWEET, M. B. E., THONAR, E. J-M. A., AND IMMELMAN, A. R. (1978) Arch. Biochem. Biophys. 189, 28-36. 28. WASTESON, A. (1975) B&hem. J. 122, 477-485. 29. MATHEWS, M. B. (1975) Molecular Biology Biochemistry and Biophysics, Vol. 19: Connective Tissue: Macromolecular Structure and Evolution, pp. 106- 107, Springer-Verlag, New York. 30. NEUBERGER, A., G~TTSCHALK, A., AND MARSHALL, R. D. (1966) in Glycoproteins. Their Composition, Structure and Function (Gottschslh, A., ed.), B. B. A. Library series, Vol. 5, pp. 273-295, Elsevier, Amsterdam. 31. BAYLISS, M. T., AND ALI, S. Y. (1978) B&hem. J. 176, 683-693. 32. HEINEGARD, D., AND HASCALL, V. C. (1974) J. Bial. Chem. 249, 4250-4259. 33. MAROUDAS, A. (1973) in Adult Articular Cartilage (Freeman, M. A. R., ed.), pp. 131-170, Pitman Medical, London. 34. KEMPSON, G. E., MUIR, H., SWANSON, S. A. V., AND FREEMAN, M. A. R. (1970) Biochim. Biophys. Acta 215, 70-77. 35. SWEET, M. B. E., THONAR, E. J-M. A., IMMELMAN, A. R., AND SOLOMON, L. (1977) Ann. Rheum. Dis. 36, 387-398.
