Biochimica et Biophysica Acta, 1033 (1990) 139-147
139
Elsevier BBAGEN 23247
Squid proteoglycans: isolation and characterization of three populations from cranial cartilage Demitrios H. Vynios and Constantine P. Tsiganos Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras (Greece)
(Received 24 May 1989) (Revised manuscript received 5 October 1989)
Key words: Proteoglycan;Cartilage; (Squid)
Squid cranial cartilage is poor in proteoglycans. They were extracted by 2% S D S and purified by isopycnic centrifugation in the presence of detergent. According to their buoyant density and hydrodynamic size they were fractionated into three structurally different populations of M r 1.3 • 10 6, 0.6- 10 6 and 1.0 • 10 6. The proteoglycans of each population differ in the number of oversulphated chondroitin sulphate chains, ranging from two to five, in the nmnber and size of uronic acid and sulphate containing oligosaccharides and in the size of their core protein. The majority, if not all, of the oligosaccharides are linked to the protein via an O-glycosidic bond involving galactosamine and most likely xyiose. The chondroitin sulphate chains are segregated on a small peptide segment of the molecule which also contains a large proportion of the oligosaccharides. The proteoglycans have no tendency to interact with hyaluronate.
Introduction A common feature of proteoglycans from vertebrate cartilage and from chondrocyte cultures is their bottle brush structure and their ability to form large multimolecular complexes with hyaluronate. Thus, a typical proteoglycan from hyaline cartilage consists of a protein core on which are attached up to 100 chondroitin sulphate and 50 keratan sulphate chains of M r 15 000-25 000 and 5000-15 000, respectively, and a large number of oligosaccharides [1]. Recent information from rotary shadowing electron microscopy, together with results of specific enzymic modifications of proteoglycans indicates that the intact proteoglycan monomer contains five distinct structural and perhaps functional regions. Starting from the NH2-terminal side of the molecule there are two globular domains of M r 50 000 and 40 000, respectively [2], a KS-rich region of M r 40000, and adjacent to it a peptide of M r 180 000 bearing the chondroitin sulphate chains in clusters on regions showing great homology
Abbreviations: SDS, sodium dodecyl sulphate; CSE, chondroitin sulphate type E; GdnHCI, guanidine hydrochloride;Zwt, Zwittergent 3-12 Correspondence: C.P. Tsiganos, Laboratory of Biochemistry,Department of Chemistry, Universityof Patras, Patras, Greece.
[3]; the molecule terminates with a globular structure of M r about 30 000 [4]. Depending on the tissue, only a small proportion of the extractable proteoglycans contain the C-terminal globular structure [5]. The first NH2-terminal globular domain, void of glycosaminoglycans and containing all N-linked oligosaccharides [2], is responsible for interacting with hyaluronate and link protein; no special function has yet been found for the other two globules. The structure of the 'hyaluronate-binding region', as both globular domains at the N-terminus have so far been referred to, is remarkably similar in various tissues and it seems that it is highly conserved in interstitial proteoglycans of connective tissue [6]. Little is known about the proteoglycans from invertebrate cartilagenous tissues, which morphologically and histochemically resemble hyaline cartilage. In a previous publication [7] we have shown that squid cranial cartilage behaves quite differently from vertebrate hyaline cartilage toward extracting solvents. Thus, 4 M guanidine hydrochloride extracts from this tissue only 30% of its uronic acid content, whereas 2% SDS removes 85%; the latter solvent extracts only 32% of the chick sternal cartilage uronic acid. A proteoglycan population amounting to 12% of the tissue uronic acid was purified from 4 M guanidine hydrochloride extracts, and it was shown to be of relatively small hydrodynamic size and to contain high amounts of protein
0304-4165/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
140 and hexoses, and 2 - 3 oversulphated chondroitin sulphate chains (CSE). Evidence was also presented that at least 25% of the proteoglycans extracted with 2% SDS do not form aggregates with exogenous hyaluronate. The present study was undertaken to fractionate and elucidate the structure of the majority of proteoglycans extracted from squid cranial cartilage with 2% SDS. Three populations were isolated by a combination of methods. They differed in the size of their protein core, in the number of chains and in the number and type of uronic-acid- and sulphate-containing oligosaccharides; in all samples the glycosaminoglycan chains are segregated in one region of the protein core. Materials and Methods
Chemicals All chemicals were of the best available grade. Guanidine hydrochloride (GdnHC1), CsC1, papain (Thrice crystallized) and trypsin (type III) were from Sigma. Chondroitinase ABC and hyaluronidase from Streptomyces hyalurolyticus were from Miles, Sepharose CL-2B, CL-4B and DEAE-Sephacel from Pharmacia LKB, Bio-Gel P-30 from Bio-Rad, sodium borotritide (19.7 m C i / m m o l ) from Amersham, SDS from Serva and zwittergent 3-12 (Zwt) from Calbiochem.
Isolation of proteoglycans Squid (Illex illecebrosus coidentii) were obtained from local fishermen immediately after being caught in the Corinth bay and transferred to the laboratory packed in ice. The cranial cartilage was immediately removed, cut into slices of about 1 mm thick, suspended in 10-fold of its weight 2% SDS/0.04 M phosphate buffer (pH 7.4) containing the proteinase inhibitors, 0.1 M E-amino-nhexanoic acid, 0.01 M disodium EDTA, 0.005 M benzamidine-HC1 [19] and 0.01 M N-ethylmaleimide [20] and shaken gently at room temperature for three 24 h periods. The extracts were pooled and the macromolecules were precipitated with 75% (v/v) ethanol/2% (v/v) propionic acid. The precipitate was dissolved in 4 M GdnHC1/4% Zwt/0.05 M acetate buffer (pH 5.8) ( = 10 m g / m l , 250 /~g uronic acid/ml) containing the above proteinase inhibitors. The density was adjusted to 1.5 m g / m l by the addition of solid CsC1 and the solutions were centrifuged at 95000 x gav for 72 h at 10 ° C in a Beckman Ty65 rotor. The tubes were cut into a high-density fraction, D1 (8 ml), and a low density fraction, D2 (4 ml). Because fraction D1 contained some protein impurities as detected by SDS-electrophoresis, it was subjected to a second ultracentrifugation as above. The tubes this time were cut into three equal volume fractions, D1D1, D1D2 and DID3, from bottom to top. Fraction D1D3 was found to contain the protein impurities and was not studied any further. The other two fractions were extensively dialysed against water and freeze-dried.
Analytical methods Uronic acid and protein were quantitated by an automated version [8] of the borate-carbazole [9] and folin [10] reactions, respectively. Total hexoses were measured by the anthrone reaction [11], using galactose as standard and analysing solutions of glucuronolactone of comparable concentrations to account for interference of uronic acid. Hydroxyproline was quantitated by the method of Woessner [12], after hydrolysis of samples in 6 M HC1 at 110 ° C for 20 h under nitrogen. Collagen was calculated by multiplying by 11 the amount of hydroxyproline [13]. Amino-acid analyses were performed on a Beckman 120C amino-acid analyser after hydrolysis of the samples in 6 M HC1 (100/zg/ml) for 20 h at 1 1 0 ° C under nitrogen. The analysis for hexosamines and hexosaminitols was performed on the amino acid analyser (AA-15 resin) with elution buffer of 0.15 M boric acid/0.35 M sodium chloride/0.067 M citrate (pH 5.28) [14] after hydrolysis of the samples in 8 M HC1 (100/~g/ml) for 3 h at 95 ° C under nitrogen. Sialic acids were measured by the periodate-resorcinol reaction [15]. Individual neutral sugars were quantitated by HPLC as DNP-derivatives [16] and sulphate and phosphate ester groups were determined by the benzidine reaction [17] and by the method of Fiske and SubbaRow [18], respectively.
Enzymic degradations (1) Samples of wet tissue in 0.1 M Tris-HC1 (pH 7.2) (100 m g / m l ) containing 0.01 M disodium EDTA and 0.005 M cysteine-HC1 were incubated at 60 ° C for 18 h in the presence of 20 ~g of papain/ml. At the end, aliquots of the tissue digest were analysed for uronic acid, galactosamine, glucosamine, total hexoses and hydroxyproline. (2) Solutions of proteoglycans (5 m g / m l ) in 0.1 M Tris-acetate (pH 7.3) were incubated overnight at 37 ° C in the presence of 0.5 units of chondroitinase ABC per ml and the digests were chromatographed on a Sepharose CL-2B eluted with 4 M GdnHC1/0.1% Zwt/0.05 M acetate buffer (pH 5.8). (3) Solutions of proteoglycans (5 m g / m l ) in 0.1 M Tris-acetate (pH 7.3) were digested with 10 /~g/ml of trypsin at 3 7 ° C for varying lengths of time up to 20 h with fresh addition of trypsin every 5 h. The digests were applied directly to a Sepharose CL-2B column eluted with 0.2 M ammonium bicarbonate. (4) Solutions of proteoglycans (3 m g / m l ) in 0.1 M sodium acetate/0.15 M sodium chloride (pH 5.2) were incubated at 37 ° C for 8 h in the presence of I T R U / m l of Streptomyces hyaluronidase. The digests were applied
141 directly to a Sepharose CL-2B column eluted with 0.5 M sodium acetate (pH 7.0).
Isolation of the carbohydrate moieties Samples of each proteoglycan preparation in 0.05 M N a O H / 1 M sodium borohydride/0.5 mM sodium borotritide (10 m g / m l ) were incubated for 48 h at 45 ° C under vacuum [21]. The reaction was stopped by the dropwise addition of 4 M acetic acid, and ethanol up to 66% (v/v) was added to precipitate the CSE chains. The precipitates were further chromatographed on a DEAE-Sephacel column equilibrated with 0.02 M LiC1 and eluted with 3 bed volumes of 0.02 M, 1 M and 6 M LiC1. Only the last fraction contained uronic acid, hexoses and radioactivity, from which the CSE was recovered by ethanol precipitation. The 66% ethanol supernatants were evaporated to dryness with repeated addition of methanol and will be referred to as the oligosaccharide fraction.
Column chromatography Gel chromatography on Sepharose CL-2B, CL-4B and Bio-Gel P-30 was performed at 4 ° C , using the eluants mentioned in the legends to the figures. The flow rates were 7 m l / c m 2 per h and void and total volume of the columns were determined with sheep nasal cartilage proteoglycan aggregates and tritiated water, respectively. For the determination of molecular weight, analytical columns of Sepharose CL-2B were calibrated with sheep nasal cartilage proteoglycans of M r 1 . 5 • 1 0 6 and 0.9.10 6 and of Sepharose CL-4B with sheep nasal cartilage chondroitin sulphate and squid skin chondroitin and oversulphated chondroitin sulphate chains of M r 15 000, 80000 and 40000, respectively. Results
The chemical composition of squid cranial cartilage and of the pooled 2% SDS extracts are shown in Table I. The tissue, like that of other sources of cartilage, is
TABLE I
Chemical analysis of squid cranial cartilage and of the pooled 2 % SDS extracts, expressed as mg per g wet weight of tissue Compound
Tissue
Extract
Water U r o n i c acid Protein a Galactosamine Glucosamine T o t a l hexose Collagen b
820.00 3.35 13.73 0.32 2.52 71.64
2.98 84.57 6.61 0.32 0.69 10.04
Sialic acids
-
a Measured by the folin reaction. b C a l c u l a t e d f r o m h y d r o x y p r o l i n e x 11 [13].
-
characterized by a high content of water, but, in contrast to hyaline cartilage, it contains appreciably lower amounts of uronic acid and collagen [22,23]. Its galactosamine content is higher than that of uronic acid, suggesting the presence of significant amounts of structures other than CSE. Interestingly, no sialic acid was detected in this tissue in contrast to hyaline cartilage from vertebrates. From the dry weight, the collagen, uronic acid and hexosamine content of the tissue residue (not shown) it was estimated that nearly all non-collagenous non-proteoglycan protein was extracted by 2% SDS. In the linear density gradient centrifugation about 70% of the extracted uronic acid (60% of the tissue content) was centrifuged to the lower 8 ml of the tube, fraction D1, while the remainder, together with various other proteins, was recovered in fraction D2. Because fraction D1 was found by SDS electrophoresis in 5% polyacrylamide gels to contain proteins, it was subjected to a second centrifugation under the same conditions. All of the uronic acid was distributed in the bottom and middle 4 ml fractions, DID1 (42%, p = 1.6 g / m l ) and D I D 2 (58%, O---1.48 g/ml), respectively, leaving the protein impurities in the top fraction. Attempts to isolate the proteoglycans in fraction D2 were unsuccessful. In particular, when this fraction was centrifuged in a lower starting density, 1.35 g/ml, and 2 ml fractions were collected, less than 10% of the uronic acid was obtained free from other proteins in the bottom 2 ml of the tube. The remainder, together with proteins and collagen, was distributed throughout the rest of the tube. At least 50% of the uronic acid in fraction D2 was found to belong to oligosaccharides which were liberated by digestion with papain and might not all of them be part of the proteoglycan structure (see below). Furthermore, when D2 was subjected to chromatography on DEAE-Sephacel in the presence of 6 M urea and Triton X-100 about 90% of the uronic acid could not be eluted from the column. Therefore, all of our studies were performed on the proteoglycans of fractions D1D1 and D1D2, which would represent more than 60% of the tissue proteoglycans if it is assumed that part of the extracted uronic acid is on glycoproteins.
Gel chromatography of proteoglycans Gel chromatography of D1D1 on Sepharose CL-2B eluted with 0.5 M sodium acetate (pH 7.0) revealed that this fraction contained two populations of macromolecules (Fig. 1A), neither of which has been shown to interact with hyaluronate or tritiated hyaluronate decasaccharides [7]. However, when 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8) was used as eluant, the excluded molecules became retarded whereas the retarded remain unaffected (Fig. 1B). Similar elution profile was obtained when the column was eluted with 0.1% SDS (not shown). In contrast, fraction D1D2 was quite
142 C 03
4/~o_v_o.c_o.~o_
D
02
..% 01
\"
8c
"%
°'°'0"°"
/I ot
I
°'°'o"c')~-o'o-o'Q-o
8m
0.2
7
q~ [];k
01
,'I %o t
,o' .=,ion
vo
";
t
t
v~
vo
.oo,ioo
o.o o.o.o. ,o-Ooo t vt
Fig. 1. Gel chromatography of D 1 D I A (A,B) and D1 D2 (C,D) on Sepharose CL-2B (140 × 0.8 cm). The elution was performed with 0.5 M sodium acetate (pH 7.0) (A,C) and 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8) (B,D). Fractions of 1.2 ml were collected and analysed for uronic acid (11) and protein (o). Horizontal bars indicate the fractions which were pooled for the isolation of D 1 D 1 A and D1D1B populations. Arrows indicate the elution position of standard cartilage proteoglycans of M r 1.5.106 and 0.9.106. V0 and Vt, void and total volume of the column, respectively.
homogeneous when chromatographed on Sepharose CL-2B in 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8), suggesting the presence of a single polydisperse population (Fig. 1D). Upon chromatography of this fraction in 0.5 M sodium acetate (pH 7.0) the proteoglycans became also excluded by the gel (Fig. 1C) and remained unaffected after either digestion with hyaluronidase from Streptomyces or after mixing with
T A B L E II
Chemical composition of proteoglycans (% dry weight) The amino acids are expressed as residues per 1000 residues.
Uronicacid Pr°teina
Galactosamine
hyaluronate decasaccharides (not shown). This chromatographic behaviour of the proteoglycans, which appears not to be due to hyaluronate-mediated aggregation, is under investigation. The excluded and retarded populations of D1D1
Glucosamine
were isolated by pooling the respective fractions from preparative chromatography on Sepharose CL-2B in 0.5 M sodium acetate (pH 7.0) and the preparations will be referred to as D1D1A and D1D1B, respectively. Both preparations were chromatographed on a Sepharose CL-2B analytical column in 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8) as single polydisperse populations (not shown) with K D values of 0.27 and
Mannose Galactose Glucose Asp Thr
0.5, respectively; fraction D1D2 had a KD value of 0.35. These KD values of D1D1A, D1D1B and D1D2 correspond to M r 1.3-10 6, 0 . 6 - 1 0 6 and 1.0-10 6, r e spectively, when nasal cartilage proteoglycans are used as reference compounds. They are expected to be approximate, because of lack of reference compounds of exactly the same structure.
Chemical composition of proteoglycans The chemical composition of the proteoglycan fractions is shown in Table II. The proteoglycans contain
Sulphate Phosphate Total hexose Xylose Fucose
Ser
Glu Pro Gly Ala
1/2Cys
D1D1A 14.40 29.50
D1D1B 18.30 22.40
D1D2 11.90 34.30
0.55 15.10 0.29
0.69 18.60 0.42
13.40 0.45
15.80
19.80
14.00 0.49
6.70
6.50
5.10
0.90
0.76
0.18
0.15 1.20 1.90 106 106 76 113
0.07 0.98 2.20 104 102 65 110
0.26 1.62 1.72 107 104 61 114
100 74 83
107 67
1.56
88 64 82
traces
1.04
traces
1.2l
82 traces
Val
89
76
76
Met lie Leu Tyr Phe
4 51 66 20 46
5 50 68 21 36
6 51 67 19 35
Lys His Arg
41 15 33
44 30 32
43 10 41
a Determined by the folin reaction.
143 TABLE III
Chemical compositions Chemical composition of chondroitin sulphate and the oligosaccharide fraction from each proteoglycan population expressed as percentage of the proteoglycan dry weight. Numbers in parentheses refer to residues per condroitin sulphate chain. DIDIA
DID1B
CSE Uronic acid Galactosamine Galactosaminitol Glucosamine Glucosaminitol Sulphate Phosphate Sialic acids Total hexose Xylose Fucose Mannose Galactose Glucose
13.09 13.31 12.30 0.15 3.52 0.11 0.12 1.46
(223) (223)
(384) (5)
(2) (2) (24)
DID2
oligos.
SE
oligos.
CSE
1.31 1.88 0.58 0.49 0.09 2.80 0.16 3.20 0.08 1.44 0.14 1.08 0.44
17.20 (220) 17.50 (220) 16.16 (379) 0.18 (4)
1.10 1.37 1.13 0.57 0.08 2.44 0.21
10.89 11.08 10.23 0.28
4.29 0.14
2.25 traces 0.90 0.09 0.79 0.27
(2)
0.16 (2) 1.95 (24)
high amounts of protein, which explains their relatively low buoyant density. In addition to glucosamine, they contain more galactosamine, xylose and other neutral sugars than can be accounted for by the presence of only CSE chains [24-26]. In view of the absence of keratan sulphate from squid cranial cartilage [26], this suggests the presence of other structures discussed below. Interestingly, the proteoglycans contain appreciable amounts of phosphate groups which, in the absence of ribose or deoxyribose (Table II), make them in this respect similar to the proteoglycans from other sources [27]. About 50% of the phosphatase were found on the CSE chains (Table III). Despite the rather profound differences in sugar composition, the proteoglycans show small differences in that of their amino acids; the most prominent being between Ser, Pro and His content. They contain, however, significantly less Ser and Gly and more Asp and Pro than the proteoglycans from bovine or sheep hyaline cartilage [28,29].
oligos. (223) (223)
(384) (11)
1.01 1.97 0.88 0.46 3.17 0.20
2.75 0.09 (2) 0.10 (2) 1.21 (24)
2.30 0.03 1.09 0.26 1.52 0.48
chains of CSE and not 'doublets' with oligosaccharides as well. Fucose branches have been identified on chondroitin sulphate from sea cucumber [30]. The CSE was found to contain phosphates ranging from 4 to 11 residues per chain. These amounts exceed their content in xylose where the phosphates have been located in glycosaminoglycans from other sources [27], suggesting their presence on other sugars, too. Gel chromatography on Sepharose CL-4B (Fig. 2) indicated that the CSE chains in the three proteoglycan populations are of the same hydrodynamic size ( K D = 0.52), of M r about 150000. An average chain length of
0.2
c t_
,
139
Elsevier BBAGEN 23247
Squid proteoglycans: isolation and characterization of three populations from cranial cartilage Demitrios H. Vynios and Constantine P. Tsiganos Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras (Greece)
(Received 24 May 1989) (Revised manuscript received 5 October 1989)
Key words: Proteoglycan;Cartilage; (Squid)
Squid cranial cartilage is poor in proteoglycans. They were extracted by 2% S D S and purified by isopycnic centrifugation in the presence of detergent. According to their buoyant density and hydrodynamic size they were fractionated into three structurally different populations of M r 1.3 • 10 6, 0.6- 10 6 and 1.0 • 10 6. The proteoglycans of each population differ in the number of oversulphated chondroitin sulphate chains, ranging from two to five, in the nmnber and size of uronic acid and sulphate containing oligosaccharides and in the size of their core protein. The majority, if not all, of the oligosaccharides are linked to the protein via an O-glycosidic bond involving galactosamine and most likely xyiose. The chondroitin sulphate chains are segregated on a small peptide segment of the molecule which also contains a large proportion of the oligosaccharides. The proteoglycans have no tendency to interact with hyaluronate.
Introduction A common feature of proteoglycans from vertebrate cartilage and from chondrocyte cultures is their bottle brush structure and their ability to form large multimolecular complexes with hyaluronate. Thus, a typical proteoglycan from hyaline cartilage consists of a protein core on which are attached up to 100 chondroitin sulphate and 50 keratan sulphate chains of M r 15 000-25 000 and 5000-15 000, respectively, and a large number of oligosaccharides [1]. Recent information from rotary shadowing electron microscopy, together with results of specific enzymic modifications of proteoglycans indicates that the intact proteoglycan monomer contains five distinct structural and perhaps functional regions. Starting from the NH2-terminal side of the molecule there are two globular domains of M r 50 000 and 40 000, respectively [2], a KS-rich region of M r 40000, and adjacent to it a peptide of M r 180 000 bearing the chondroitin sulphate chains in clusters on regions showing great homology
Abbreviations: SDS, sodium dodecyl sulphate; CSE, chondroitin sulphate type E; GdnHCI, guanidine hydrochloride;Zwt, Zwittergent 3-12 Correspondence: C.P. Tsiganos, Laboratory of Biochemistry,Department of Chemistry, Universityof Patras, Patras, Greece.
[3]; the molecule terminates with a globular structure of M r about 30 000 [4]. Depending on the tissue, only a small proportion of the extractable proteoglycans contain the C-terminal globular structure [5]. The first NH2-terminal globular domain, void of glycosaminoglycans and containing all N-linked oligosaccharides [2], is responsible for interacting with hyaluronate and link protein; no special function has yet been found for the other two globules. The structure of the 'hyaluronate-binding region', as both globular domains at the N-terminus have so far been referred to, is remarkably similar in various tissues and it seems that it is highly conserved in interstitial proteoglycans of connective tissue [6]. Little is known about the proteoglycans from invertebrate cartilagenous tissues, which morphologically and histochemically resemble hyaline cartilage. In a previous publication [7] we have shown that squid cranial cartilage behaves quite differently from vertebrate hyaline cartilage toward extracting solvents. Thus, 4 M guanidine hydrochloride extracts from this tissue only 30% of its uronic acid content, whereas 2% SDS removes 85%; the latter solvent extracts only 32% of the chick sternal cartilage uronic acid. A proteoglycan population amounting to 12% of the tissue uronic acid was purified from 4 M guanidine hydrochloride extracts, and it was shown to be of relatively small hydrodynamic size and to contain high amounts of protein
0304-4165/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
140 and hexoses, and 2 - 3 oversulphated chondroitin sulphate chains (CSE). Evidence was also presented that at least 25% of the proteoglycans extracted with 2% SDS do not form aggregates with exogenous hyaluronate. The present study was undertaken to fractionate and elucidate the structure of the majority of proteoglycans extracted from squid cranial cartilage with 2% SDS. Three populations were isolated by a combination of methods. They differed in the size of their protein core, in the number of chains and in the number and type of uronic-acid- and sulphate-containing oligosaccharides; in all samples the glycosaminoglycan chains are segregated in one region of the protein core. Materials and Methods
Chemicals All chemicals were of the best available grade. Guanidine hydrochloride (GdnHC1), CsC1, papain (Thrice crystallized) and trypsin (type III) were from Sigma. Chondroitinase ABC and hyaluronidase from Streptomyces hyalurolyticus were from Miles, Sepharose CL-2B, CL-4B and DEAE-Sephacel from Pharmacia LKB, Bio-Gel P-30 from Bio-Rad, sodium borotritide (19.7 m C i / m m o l ) from Amersham, SDS from Serva and zwittergent 3-12 (Zwt) from Calbiochem.
Isolation of proteoglycans Squid (Illex illecebrosus coidentii) were obtained from local fishermen immediately after being caught in the Corinth bay and transferred to the laboratory packed in ice. The cranial cartilage was immediately removed, cut into slices of about 1 mm thick, suspended in 10-fold of its weight 2% SDS/0.04 M phosphate buffer (pH 7.4) containing the proteinase inhibitors, 0.1 M E-amino-nhexanoic acid, 0.01 M disodium EDTA, 0.005 M benzamidine-HC1 [19] and 0.01 M N-ethylmaleimide [20] and shaken gently at room temperature for three 24 h periods. The extracts were pooled and the macromolecules were precipitated with 75% (v/v) ethanol/2% (v/v) propionic acid. The precipitate was dissolved in 4 M GdnHC1/4% Zwt/0.05 M acetate buffer (pH 5.8) ( = 10 m g / m l , 250 /~g uronic acid/ml) containing the above proteinase inhibitors. The density was adjusted to 1.5 m g / m l by the addition of solid CsC1 and the solutions were centrifuged at 95000 x gav for 72 h at 10 ° C in a Beckman Ty65 rotor. The tubes were cut into a high-density fraction, D1 (8 ml), and a low density fraction, D2 (4 ml). Because fraction D1 contained some protein impurities as detected by SDS-electrophoresis, it was subjected to a second ultracentrifugation as above. The tubes this time were cut into three equal volume fractions, D1D1, D1D2 and DID3, from bottom to top. Fraction D1D3 was found to contain the protein impurities and was not studied any further. The other two fractions were extensively dialysed against water and freeze-dried.
Analytical methods Uronic acid and protein were quantitated by an automated version [8] of the borate-carbazole [9] and folin [10] reactions, respectively. Total hexoses were measured by the anthrone reaction [11], using galactose as standard and analysing solutions of glucuronolactone of comparable concentrations to account for interference of uronic acid. Hydroxyproline was quantitated by the method of Woessner [12], after hydrolysis of samples in 6 M HC1 at 110 ° C for 20 h under nitrogen. Collagen was calculated by multiplying by 11 the amount of hydroxyproline [13]. Amino-acid analyses were performed on a Beckman 120C amino-acid analyser after hydrolysis of the samples in 6 M HC1 (100/zg/ml) for 20 h at 1 1 0 ° C under nitrogen. The analysis for hexosamines and hexosaminitols was performed on the amino acid analyser (AA-15 resin) with elution buffer of 0.15 M boric acid/0.35 M sodium chloride/0.067 M citrate (pH 5.28) [14] after hydrolysis of the samples in 8 M HC1 (100/~g/ml) for 3 h at 95 ° C under nitrogen. Sialic acids were measured by the periodate-resorcinol reaction [15]. Individual neutral sugars were quantitated by HPLC as DNP-derivatives [16] and sulphate and phosphate ester groups were determined by the benzidine reaction [17] and by the method of Fiske and SubbaRow [18], respectively.
Enzymic degradations (1) Samples of wet tissue in 0.1 M Tris-HC1 (pH 7.2) (100 m g / m l ) containing 0.01 M disodium EDTA and 0.005 M cysteine-HC1 were incubated at 60 ° C for 18 h in the presence of 20 ~g of papain/ml. At the end, aliquots of the tissue digest were analysed for uronic acid, galactosamine, glucosamine, total hexoses and hydroxyproline. (2) Solutions of proteoglycans (5 m g / m l ) in 0.1 M Tris-acetate (pH 7.3) were incubated overnight at 37 ° C in the presence of 0.5 units of chondroitinase ABC per ml and the digests were chromatographed on a Sepharose CL-2B eluted with 4 M GdnHC1/0.1% Zwt/0.05 M acetate buffer (pH 5.8). (3) Solutions of proteoglycans (5 m g / m l ) in 0.1 M Tris-acetate (pH 7.3) were digested with 10 /~g/ml of trypsin at 3 7 ° C for varying lengths of time up to 20 h with fresh addition of trypsin every 5 h. The digests were applied directly to a Sepharose CL-2B column eluted with 0.2 M ammonium bicarbonate. (4) Solutions of proteoglycans (3 m g / m l ) in 0.1 M sodium acetate/0.15 M sodium chloride (pH 5.2) were incubated at 37 ° C for 8 h in the presence of I T R U / m l of Streptomyces hyaluronidase. The digests were applied
141 directly to a Sepharose CL-2B column eluted with 0.5 M sodium acetate (pH 7.0).
Isolation of the carbohydrate moieties Samples of each proteoglycan preparation in 0.05 M N a O H / 1 M sodium borohydride/0.5 mM sodium borotritide (10 m g / m l ) were incubated for 48 h at 45 ° C under vacuum [21]. The reaction was stopped by the dropwise addition of 4 M acetic acid, and ethanol up to 66% (v/v) was added to precipitate the CSE chains. The precipitates were further chromatographed on a DEAE-Sephacel column equilibrated with 0.02 M LiC1 and eluted with 3 bed volumes of 0.02 M, 1 M and 6 M LiC1. Only the last fraction contained uronic acid, hexoses and radioactivity, from which the CSE was recovered by ethanol precipitation. The 66% ethanol supernatants were evaporated to dryness with repeated addition of methanol and will be referred to as the oligosaccharide fraction.
Column chromatography Gel chromatography on Sepharose CL-2B, CL-4B and Bio-Gel P-30 was performed at 4 ° C , using the eluants mentioned in the legends to the figures. The flow rates were 7 m l / c m 2 per h and void and total volume of the columns were determined with sheep nasal cartilage proteoglycan aggregates and tritiated water, respectively. For the determination of molecular weight, analytical columns of Sepharose CL-2B were calibrated with sheep nasal cartilage proteoglycans of M r 1 . 5 • 1 0 6 and 0.9.10 6 and of Sepharose CL-4B with sheep nasal cartilage chondroitin sulphate and squid skin chondroitin and oversulphated chondroitin sulphate chains of M r 15 000, 80000 and 40000, respectively. Results
The chemical composition of squid cranial cartilage and of the pooled 2% SDS extracts are shown in Table I. The tissue, like that of other sources of cartilage, is
TABLE I
Chemical analysis of squid cranial cartilage and of the pooled 2 % SDS extracts, expressed as mg per g wet weight of tissue Compound
Tissue
Extract
Water U r o n i c acid Protein a Galactosamine Glucosamine T o t a l hexose Collagen b
820.00 3.35 13.73 0.32 2.52 71.64
2.98 84.57 6.61 0.32 0.69 10.04
Sialic acids
-
a Measured by the folin reaction. b C a l c u l a t e d f r o m h y d r o x y p r o l i n e x 11 [13].
-
characterized by a high content of water, but, in contrast to hyaline cartilage, it contains appreciably lower amounts of uronic acid and collagen [22,23]. Its galactosamine content is higher than that of uronic acid, suggesting the presence of significant amounts of structures other than CSE. Interestingly, no sialic acid was detected in this tissue in contrast to hyaline cartilage from vertebrates. From the dry weight, the collagen, uronic acid and hexosamine content of the tissue residue (not shown) it was estimated that nearly all non-collagenous non-proteoglycan protein was extracted by 2% SDS. In the linear density gradient centrifugation about 70% of the extracted uronic acid (60% of the tissue content) was centrifuged to the lower 8 ml of the tube, fraction D1, while the remainder, together with various other proteins, was recovered in fraction D2. Because fraction D1 was found by SDS electrophoresis in 5% polyacrylamide gels to contain proteins, it was subjected to a second centrifugation under the same conditions. All of the uronic acid was distributed in the bottom and middle 4 ml fractions, DID1 (42%, p = 1.6 g / m l ) and D I D 2 (58%, O---1.48 g/ml), respectively, leaving the protein impurities in the top fraction. Attempts to isolate the proteoglycans in fraction D2 were unsuccessful. In particular, when this fraction was centrifuged in a lower starting density, 1.35 g/ml, and 2 ml fractions were collected, less than 10% of the uronic acid was obtained free from other proteins in the bottom 2 ml of the tube. The remainder, together with proteins and collagen, was distributed throughout the rest of the tube. At least 50% of the uronic acid in fraction D2 was found to belong to oligosaccharides which were liberated by digestion with papain and might not all of them be part of the proteoglycan structure (see below). Furthermore, when D2 was subjected to chromatography on DEAE-Sephacel in the presence of 6 M urea and Triton X-100 about 90% of the uronic acid could not be eluted from the column. Therefore, all of our studies were performed on the proteoglycans of fractions D1D1 and D1D2, which would represent more than 60% of the tissue proteoglycans if it is assumed that part of the extracted uronic acid is on glycoproteins.
Gel chromatography of proteoglycans Gel chromatography of D1D1 on Sepharose CL-2B eluted with 0.5 M sodium acetate (pH 7.0) revealed that this fraction contained two populations of macromolecules (Fig. 1A), neither of which has been shown to interact with hyaluronate or tritiated hyaluronate decasaccharides [7]. However, when 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8) was used as eluant, the excluded molecules became retarded whereas the retarded remain unaffected (Fig. 1B). Similar elution profile was obtained when the column was eluted with 0.1% SDS (not shown). In contrast, fraction D1D2 was quite
142 C 03
4/~o_v_o.c_o.~o_
D
02
..% 01
\"
8c
"%
°'°'0"°"
/I ot
I
°'°'o"c')~-o'o-o'Q-o
8m
0.2
7
q~ [];k
01
,'I %o t
,o' .=,ion
vo
";
t
t
v~
vo
.oo,ioo
o.o o.o.o. ,o-Ooo t vt
Fig. 1. Gel chromatography of D 1 D I A (A,B) and D1 D2 (C,D) on Sepharose CL-2B (140 × 0.8 cm). The elution was performed with 0.5 M sodium acetate (pH 7.0) (A,C) and 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8) (B,D). Fractions of 1.2 ml were collected and analysed for uronic acid (11) and protein (o). Horizontal bars indicate the fractions which were pooled for the isolation of D 1 D 1 A and D1D1B populations. Arrows indicate the elution position of standard cartilage proteoglycans of M r 1.5.106 and 0.9.106. V0 and Vt, void and total volume of the column, respectively.
homogeneous when chromatographed on Sepharose CL-2B in 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8), suggesting the presence of a single polydisperse population (Fig. 1D). Upon chromatography of this fraction in 0.5 M sodium acetate (pH 7.0) the proteoglycans became also excluded by the gel (Fig. 1C) and remained unaffected after either digestion with hyaluronidase from Streptomyces or after mixing with
T A B L E II
Chemical composition of proteoglycans (% dry weight) The amino acids are expressed as residues per 1000 residues.
Uronicacid Pr°teina
Galactosamine
hyaluronate decasaccharides (not shown). This chromatographic behaviour of the proteoglycans, which appears not to be due to hyaluronate-mediated aggregation, is under investigation. The excluded and retarded populations of D1D1
Glucosamine
were isolated by pooling the respective fractions from preparative chromatography on Sepharose CL-2B in 0.5 M sodium acetate (pH 7.0) and the preparations will be referred to as D1D1A and D1D1B, respectively. Both preparations were chromatographed on a Sepharose CL-2B analytical column in 4 M GdnHC1/0.1% Zwt/0.05 M acetate (pH 5.8) as single polydisperse populations (not shown) with K D values of 0.27 and
Mannose Galactose Glucose Asp Thr
0.5, respectively; fraction D1D2 had a KD value of 0.35. These KD values of D1D1A, D1D1B and D1D2 correspond to M r 1.3-10 6, 0 . 6 - 1 0 6 and 1.0-10 6, r e spectively, when nasal cartilage proteoglycans are used as reference compounds. They are expected to be approximate, because of lack of reference compounds of exactly the same structure.
Chemical composition of proteoglycans The chemical composition of the proteoglycan fractions is shown in Table II. The proteoglycans contain
Sulphate Phosphate Total hexose Xylose Fucose
Ser
Glu Pro Gly Ala
1/2Cys
D1D1A 14.40 29.50
D1D1B 18.30 22.40
D1D2 11.90 34.30
0.55 15.10 0.29
0.69 18.60 0.42
13.40 0.45
15.80
19.80
14.00 0.49
6.70
6.50
5.10
0.90
0.76
0.18
0.15 1.20 1.90 106 106 76 113
0.07 0.98 2.20 104 102 65 110
0.26 1.62 1.72 107 104 61 114
100 74 83
107 67
1.56
88 64 82
traces
1.04
traces
1.2l
82 traces
Val
89
76
76
Met lie Leu Tyr Phe
4 51 66 20 46
5 50 68 21 36
6 51 67 19 35
Lys His Arg
41 15 33
44 30 32
43 10 41
a Determined by the folin reaction.
143 TABLE III
Chemical compositions Chemical composition of chondroitin sulphate and the oligosaccharide fraction from each proteoglycan population expressed as percentage of the proteoglycan dry weight. Numbers in parentheses refer to residues per condroitin sulphate chain. DIDIA
DID1B
CSE Uronic acid Galactosamine Galactosaminitol Glucosamine Glucosaminitol Sulphate Phosphate Sialic acids Total hexose Xylose Fucose Mannose Galactose Glucose
13.09 13.31 12.30 0.15 3.52 0.11 0.12 1.46
(223) (223)
(384) (5)
(2) (2) (24)
DID2
oligos.
SE
oligos.
CSE
1.31 1.88 0.58 0.49 0.09 2.80 0.16 3.20 0.08 1.44 0.14 1.08 0.44
17.20 (220) 17.50 (220) 16.16 (379) 0.18 (4)
1.10 1.37 1.13 0.57 0.08 2.44 0.21
10.89 11.08 10.23 0.28
4.29 0.14
2.25 traces 0.90 0.09 0.79 0.27
(2)
0.16 (2) 1.95 (24)
high amounts of protein, which explains their relatively low buoyant density. In addition to glucosamine, they contain more galactosamine, xylose and other neutral sugars than can be accounted for by the presence of only CSE chains [24-26]. In view of the absence of keratan sulphate from squid cranial cartilage [26], this suggests the presence of other structures discussed below. Interestingly, the proteoglycans contain appreciable amounts of phosphate groups which, in the absence of ribose or deoxyribose (Table II), make them in this respect similar to the proteoglycans from other sources [27]. About 50% of the phosphatase were found on the CSE chains (Table III). Despite the rather profound differences in sugar composition, the proteoglycans show small differences in that of their amino acids; the most prominent being between Ser, Pro and His content. They contain, however, significantly less Ser and Gly and more Asp and Pro than the proteoglycans from bovine or sheep hyaline cartilage [28,29].
oligos. (223) (223)
(384) (11)
1.01 1.97 0.88 0.46 3.17 0.20
2.75 0.09 (2) 0.10 (2) 1.21 (24)
2.30 0.03 1.09 0.26 1.52 0.48
chains of CSE and not 'doublets' with oligosaccharides as well. Fucose branches have been identified on chondroitin sulphate from sea cucumber [30]. The CSE was found to contain phosphates ranging from 4 to 11 residues per chain. These amounts exceed their content in xylose where the phosphates have been located in glycosaminoglycans from other sources [27], suggesting their presence on other sugars, too. Gel chromatography on Sepharose CL-4B (Fig. 2) indicated that the CSE chains in the three proteoglycan populations are of the same hydrodynamic size ( K D = 0.52), of M r about 150000. An average chain length of
0.2
c t_
,
