Variations in the chondroitin sulfate-protein linkage region of aggrecans from bovine nasal and human articular cartilages.

Aggrecan-derived chondroitin sulfate (CS) chains, released by β-elimination, were derivatized with p-aminobenzoic acid or p-aminophenol; radioiodinated; and subjected to graded or complete degradations by chondroitin ABC lyase to generate linkage region fragments of the basic structure ΔGlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (where ΔGlyUA represents 4,5-unsaturated glycuronic acid, and R is the adduct), by chondroitin AC lyase to generate the shorter fragment ΔGlyUA-Gal-Gal-Xyl-R, or by chondroitin C lyase to generate the same fragment when it was linked to a 6-O-sulfated or unsulfated GalNAc at the nonreducing end. Fragments were separated by size using gel chromatography, by charge using ion-exchange chromatography, and by size/charge using electrophoresis and then characterized by stepwise degradations from the nonreducing end by using mercuric acetate to remove all terminal ΔGlyUA, by bacterial glycuronidase to remove the same residue when linked to unsulfated or 6-O-sulfated GalNAc/Gal, by mammalian 4-sulfatase to remove sulfate from terminal GalNAc 4-O-sulfate, by chondro-4-sulfatase to remove 4-O-sulfate from other GalNAc/Gal residues, and by β-galactosidase to remove terminal Gal. Results with CS from bovine nasal cartilage aggrecan show that, in nearly all chains, Xyl and probably also the first Gal are unsubstituted, whereas the second Gal is 4-O-sulfated in one CS chain out of five. The first disaccharide repeat is sulfated at C-4 of GalNAc in one chain out of three and unsulfated in the other two. A sulfated first disaccharide is always joined to an unsulfated GlcUA-Gal-Gal sequence. In contrast, CS from human articular cartilage usually has a sulfated first disaccharide repeat. In CS from young human cartilage, sulfate groups are mostly at C-4 of GalNAc in the major part of the chain, but at C-6 in the nonreducing distal portion. In CS from old cartilage, sulfation at C-6 of GalNAc is a major feature from the nonreducing end down to approximately positions 4 and 5 from the linkage region, where GalNAc 4-O-sulfate is common.

Proteoglycans constitute a family of proteins that are characterized by the presence of covalently attached glycosaminoglycan (GAG) 1 side chains and are found at cell surfaces, in pericellular matrices, and especially in the extracellular matrix of connective tissues. Proteoglycans are structurally diverse; core proteins may vary in size from 10 to 400 kDa, and they can contain only a single GAG chain or well over 100. The GAG chains have unique biophysical properties that contribute to the bulk effects of proteoglycans, but they also contain a variety of binding sites for various extracellular cytokines, growth factors, enzymes, and inhibitors (for reviews, see Ref. [1][2][3][4]. The GAG chains are linear polymers of repeating disaccharides containing hexosamine and hexuronic acid (or, in the case of keratan sulfate, galactose). Hexuronic acid-containing GAGs are bound to serine residues in the core protein via the common carbohydrate sequence -4GlcUA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-, forming the so-called GAG-protein linkage region, which was shown by Rodén and co-workers over 30 years ago (for review, see Ref. 5). This structure serves as the primer for chain elongation to form either (-4GlcUA␤1-4GlcNAc␣1-) n , the core polymer in heparan sulfate/heparin, or (-4GlcUA␤1-3GalNAc␤1-) n , the corresponding one in chondroitin sulfate (CS) and dermatan sulfate (DS). Subsequent modifications of galactosaminoglycan chains by different kinds of O-sulfation and by C-5 epimerization of GlcUA to IdoUA, all in an incomplete and sporadic manner, generate a bewildering complexity. Theoretically, such sequences can encode considerable information, but it is not known if mechanisms for deciphering this information exist.
Sequence analysis of GAGs, as of other oligo-and polysaccharides, is hampered by the fact that the monosaccharides, apart from their ability to assume different ring shapes, can be joined via different types of linkages, varying in anomeric con-figuration and position. Hence, a general sequencing strategy (as in polynucleotide and polypeptide sequencing) cannot be applied (6). GAGs can only be sequenced by slow and cumbersome biochemical procedures or by technologies, such as NMR or mass spectrometry, that may not be readily available.
CS and DS chains are present in both large and small proteoglycans. The large cartilage proteoglycan aggrecan contains CS, whereas the small ubiquitous proteoglycans decorin and biglycan can contain either CS or DS: one chain in decorin and two in biglycan. We have recently developed procedures for sequence analysis of CS/DS that include coupling of an iodinatable compound to the reducing end of released GAG chains, followed by radiolabeling of the adduct and graded degradation of the chains by various specific enzymatic or chemical methods. Fragments extending from the radiolabeled reducing end to the point of cleavage are separated by high-resolution gradient polyacrylamide gel electrophoresis and identified after blotting and autoradiography (7). This procedure has been applied to CS from aggrecan (8) and to CS/DS from decorin and biglycan (9). These analyses revealed nonrandom positioning of 4-and 6-O-sulfated GalNAc in CS and a periodic, wave-like, and tissue-specific distribution pattern for both sulfate-and IdoUA-and GlcUA-containing repeats in DS. Similar strategies have been used by Linhardt and co-workers (10) in their sequence analysis of heparin.
The CS-protein linkage region in aggrecan can also contain various types of substitutions. Rat chondrosarcoma aggrecan CS, which has 4-O-sulfated GalNAc moieties, can also carry small amounts of 4-O-sulfate on the second Gal (11, 12) and 2-O-phosphate on the Xyl of the linkage region (11)(12)(13)(14). Shibata et al. (12) also observed that the first disaccharide repeat was mostly unsulfated, and more so if the preceding xylose was phosphorylated. From the CS-substituted trypsin inhibitors of urine and plasma (15,16), a uniformly GalNAc 4-O-sulfated and Gal 4-O-sulfated linkage region was obtained. In shark cartilage aggrecan CS, which has mainly 6-O-sulfated GalNAc, the first Gal of the linkage region and sometimes both the first and second Gal can carry 6-O-sulfate (17,18). The significance (if any) of these variations in the substitution pattern of the linkage region, e.g. for regulation of the assembly of GAG chains, is unknown. Simple methods that permit screening of sequence variation are necessary in order to relate structural microheterogeneity in GAGs to various biological parameters. Such a parameter could be age, as old cartilage contains more 6-O-sulfated CS than young cartilage (19 -21).
In our previous sequencing studies of aggrecan CS (8) and decorin DS (9), we detected charge variants of the linkage region fragments. In this study, such fragments derived from bovine nasal cartilage aggrecan have been characterized by using various specific exo-degrading enzymes and by chemical means. The fragments have then been used as reference compounds in an analysis of aggrecan CS from young and old human articular cartilage.
Preparation of Reducing End, 125 I-Labeled Chondroitin Sulfate-The procedures have been described in detail previously (7)(8)(9). Chondroitin sulfate chains were released by alkaline scission of the Xyl-Ser bond, and the reducing terminal Xyl was coupled by reductive amination to either p-aminobenzoic acid or p-aminophenol. Chains derivatized with p-aminobenzoic acid were radioiodinated under acidic conditions (8), whereas the phenol adduct was iodinated at neutral pH (7).
Separation Methods-Gradient polyacrylamide gel electrophoresis of oligosaccharides, blotting, and autoradiography were carried out as described previously (7). Gel chromatography was carried out on columns (10 ϫ 1000 mm) of Bio-Gel P-2 or P-6 as described (8). Ionexchange chromatography was performed on columns (10 ϫ 100 mm) of Q-Sepharose Fast Flow connected to a Pharmacia chromatography system and eluted with a linear 160-min gradient of 0 -2 M NH 4 HCO 3 at a flow rate of 0.5 ml/min. Column effluents were analyzed for 125 I using a 1271 Ria-Gamma counter.

RESULTS AND DISCUSSION
Chondroitin Sulfate-Protein Linkage Region of Bovine Nasal Cartilage Aggrecan-CS chains released by ␤-elimination, derivatized with p-aminobenzoic acid, and radioiodinated were degraded by treatment with chondroitin ABC or AC (type I or II) lyase, followed by gel chromatography on Bio-Gel P-6 ( Fig.  1). A nearly complete ABC lyase digest (Fig. 1a) contained saccharides of the general carbohydrate backbone structure ⌬GlyUA-(GalNAc-GlcUA) n -Gal-Gal-Xyl-R (where n ϭ 1, 2, 3, etc., and R ϭ radioiodinated reducing terminal adduct). Exhaustive digestions with AC lyase generated a shorter fragment, corresponding to n ϭ 0 in the above structure, in accordance with the known specificity of the enzyme. To separate charge variants of the linkage region saccharides, they were subjected to ion-exchange chromatography on Q-Sepharose (Fig. 2). The saccharide pool ⌬GlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (n ϭ 1) (Fig. 2a) separated into one low-charged (pool A) and one high-charged (pool B) fraction in approximately equal proportions. The saccharide pool ⌬GlyUA-Gal-Gal-Xyl-R (n ϭ 0) (Fig. 2b) also yielded two fractions (pools A and B), but in a ratio of ϳ4:1.
The low-charged linkage region tetrasaccharide ⌬GlyUA-Gal-Gal-Xyl-R (n ϭ 0) (pool A in Fig. 2b) was treated with glycuronidase to remove the charged, nonreducing terminal residue and then rechromatographed on Q-Sepharose (Fig. 2c). It is shown that the saccharide was completely degraded to a component of even lower charge density, which should correspond to Gal-Gal-Xyl-R with one remaining negative charge in R. The structure of the tetrasaccharide was confirmed by stepwise removal of monosaccharides from the nonreducing end, followed by gel chromatography on Bio-Gel P-2. As shown in Fig. 3, the size was gradually diminished by successive treatments with glycuronidase (panel b) and then with ␤-galactosidase (panel c). The final product, the expected Xyl-R, emerged in the same position as phenyl-O-␤-D-galactopyranoside. The various fragments were also subjected to electrophoresis, which separates both according to size and charge (Fig. 4). The intact, low-charged linkage region saccharide ⌬GlyUA-Gal-Gal-Xyl-R (n ϭ 0) (pool A) is shown in lane 1. After removal of the charged, nonreducing terminal residue ⌬GlyUA using glycuronidase, the trisaccharide product Gal-Gal-Xyl-R was obtained (lane 2). Although it is smaller, it migrated more slowly because it had a much lower charge density (Ϫ1/3) than the initial tetrasaccharide (Ϫ2/4). After further degradation using ␤-galactosidase (lane 3), the Xyl-R product migrated faster than the previous ones because it both was smaller and had an increased charge density (Ϫ1/1).
The less abundant, high-charged forms of the linkage region tetrasaccharide ⌬GlyUA-Gal-Gal-Xyl-R (n ϭ 0) (pool B in Fig.  2b) had a high mobility on electrophoresis (Fig. 4, lane 4). Two minor, even faster moving components were also observed. All CS was released from aggrecan by alkaline elimination, derivatized with p-aminobenzoic acid, and radioiodinated as described under "Experimental Procedures." Material corresponding to 5 mg and containing 0.5-1.5 ϫ 10 8 cpm of 125 I was digested with chondroitin lyase and subjected to gel chromatography on Bio-Gel P-6. a, chondroitin ABC lyase digest (ChЈABCase; 50 milliunits of enzyme in 100 l of buffer for 4 h at 37°C); b, chondroitin AC-I lyase digest (ChЈAC-I-ase; 65 milliunits of enzyme in 200 l of buffer at 37°C overnight). The same result as in b was also obtained with chondroitin AC-II lyase. The saccharides have the general carbohydrate structure ⌬GlyUA-(GalNAc-GlcUA) n -Gal-Gal-Xyl-R (where n ϭ 0, 1, 2, 3, etc., and R ϭ radioiodinated reducing terminal adduct). Saccharide with n ϭ 0 was pooled as indicated and freeze-dried.

FIG. 2. Isolation of charge variants of chondroitin sulfate-protein linkage region saccharides from bovine nasal cartilage aggrecan.
Saccharides with the carbohydrate backbone ⌬GlyUA-Gal-NAc-GlcUA-Gal-Gal-Xyl-R (n ϭ 1) and ⌬GlyUA-Gal-Gal-Xyl-R (n ϭ 0) were obtained by exhaustive digestions with chondroitin ABC lyase and chondroitin AC lyase (either type I or II), respectively, followed by gel chromatography on Bio-Gel P-6 (as described in the legend to Fig. 1). Material derived from 3 mg of CS and containing 6 ϫ 10 7 cpm of 125 I was used. Separation into charge variants was achieved by ion-exchange chromatography on Q-Sepharose Fast Flow, yielding two major forms (pools A and B) in each case (pooled as indicated by bars and freezedried). a, saccharide with n ϭ 1; b, saccharide with n ϭ 0; c, saccharide with n ϭ 0 (pool A) further digested with glycuronidase (1 unit of enzyme in 100 l of buffer for 72 h at 37°C).
high-charged forms of saccharide (n ϭ 0), except the one with the highest charge, were resistant to digestion with glycuronidase (lane 5), suggesting a preponderance of 4-O-sulfate on the second Gal. This was confirmed by treatment with chondro-4sulfatase (lane 6). All three components lost one charge, and the major one migrated like unsulfated ⌬GlyUA-Gal-Gal-Xyl-R (see also Ref. 31). The charged, nonreducing terminal residue ⌬GlyUA of the high-charged saccharides was removed by treatment with Hg(OAc) 2 , and products with slightly retarded mobilities were obtained (lane 7). Repeated digestions of the mercury-treated material with excessive amounts of mammalian exo-4-sulfatase (lane 8) generated only trace amounts of Gal-Gal-Xyl-R, suggesting that mammalian exo-4-sulfatase is specific for GalNAc-4S.
The linkage region hexasaccharide ⌬GlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R (n ϭ 1) obtained after digestion with chondroitin ABC lyase comprised approximately equal proportions of low-and high-charged variants (pools A and B in Fig. 2a). Electrophoretic analysis (Fig. 5) revealed that each pool contained one major, slow-moving (lane 1) or fast-moving (lane 4) component. The slow-moving, low-charged hexasaccharide (lane 1) was sensitive to glycuronidase, and the pentasaccharide product obtained migrated to a more retarded position (lane 2). Apparently, reduction in charge density (from Ϫ3/6 to Ϫ2/5, i.e. a 20% change) had a greater effect on electrophoretic mobility than reduction in size, which was 16%. The terminal ⌬GlyUA-Gal-NAc disaccharide repeat was removed by treatment with chondroitin AC-II lyase (lane 3), and the tetrasaccharide product obtained migrated to the same position as ⌬GlyUA-Gal-Gal-Xyl-R (Fig. 4, lane 1). Removal of one charged and one uncharged residue reduces size (by 33%), but does not alter charge density. The conclusion from these experiments is that ϳ50% of the linkage region in bovine nasal cartilage aggrecan CS consists of an unsulfated hexasaccharide sequence, -GlcUA-Gal-NAc-GlcUA-Gal-Gal-Xyl-(Structure I in Table I), and was obtained as the low-charged hexasaccharide pool A in Fig. 2a. The other 50% of the linkage region was obtained in the highcharged hexasaccharide pool B in Fig. 2a (Fig. 5, lane 4). The charged, nonreducing terminal residue ⌬GlyUA of the highcharged variants was removed by treatment with Hg(OAc) 2 , and the resulting pentasaccharide products migrated to more retarded positions (lane 5). The major variant appeared to be monosulfated, either at GalNAc or at the second Gal (see structures on the right in Fig. 5). The pentasaccharides were then treated exhaustively with exo-4-sulfatase, but only a partial effect was obtained (lane 6). We therefore conclude that the original high-charged hexasaccharide pool comprised two major isomers, one with sulfate at C-4 of GalNAc and another with sulfate at C-4 of the second Gal; the former was sensitive to exo-4-sulfatase and generated an unsulfated pentasaccharide, GalNAc-GlcUA-Gal-Gal-Xyl-R (see also structures on the right in Fig. 5). Both the high-charged hexasaccharide isomer with GalNAc-4S and the entire pool of unsulfated hexasaccharide (the latter amounted to 50% of total linkage region material) would generate unsulfated tetrasaccharide linkage region fragments after treatment with chondroitin AC lyase (see above). As the unsulfated tetrasaccharide products amounted to 80% of the linkage region material, the two monosulfated hexasaccharide isomers should represent 30% (the GalNAc 4-O-sulfated isomer) and 20% (the Gal 4-O-sulfated isomer) of the total, respectively (Structures II and III in Table I). Di-, tri-, or tetrasulfated hexasaccharides (Structures IV-VI) are rare. As 50% of the linkage region in bovine nasal cartilage aggrecan CS was unsulfated (Structure I), half of the chains should be sensitive to chondroitin C lyase, which cleaves at both unsulfated and 6-O-sulfated GalNAc (32). However, as shown previously (8), very little linkage region tetrasaccharide (n ϭ 0) is released by C lyase. This suggests that C lyase requires a 6-O-sulfate on the second GalNAc in the sequence -GlcUA-GalNAc-GlcUA-GalNAc-GlcUA-Gal-Gal-Xyl-in order to cleave between an unsulfated first GalNAc and GlcUA linked to Gal.
To determine if the first Gal and/or Xyl was substituted with charged groups, a different approach was taken. CS chains were coupled to an uncharged iodinatable compound, i.e. paminophenol, to avoid repulsion from possible phosphate esters on Xyl. The end-labeled CS was treated with alkaline phosphatase (33) after digestion with chondroitin AC-II lyase. Analysis by electrophoresis (data not shown) indicated that the linkage region saccharides were the same in phosphatase-treated and in untreated samples. The low-charged tetrasaccharide ⌬GlyUA-Gal-Gal-Xyl-RЈ, where RЈ is now uncharged, was isolated as in Fig. 2b, treated with glycuronidase, and subjected to electrophoresis, followed by direct autoradiography, omitting Stepwise exo-degradation of the low-charged linkage region saccharide ⌬GlyUA-Gal-Gal-Xyl-R from bovine nasal cartilage aggrecan. The low-charged variant of saccharide with n ϭ 0 was eroded stepwise by using first glycuronidase and then glycuronidase ϩ ␤-galactosidase, each followed by gel chromatography on Bio-Gel P-2. Material corresponding to 0.4 -0.6 mg of starting material and containing 1 ϫ 10 6 cpm. of 125 I was used. a, intact saccharide (pool A in Fig. 2b); b, saccharide treated with glycuronidase (pool A in Fig. 2c) the transfer-blotting step. The uncharged trisaccharide product migrated only a short distance, and had the same mobility as 125 I-labeled p-hydroxyphenyl-O-␤-D-xylopyranoside (data not shown). Hence, Xyl and the first Gal in bovine nasal aggrecan CS were mostly unsubstituted (see also Table I). It is possible that phosphorylation of Xyl is required for chain biosynthesis and/or intracellular progression and that it is followed by dephosphorylation just before or shortly after secretion.
Chondroitin Sulfate-Protein Linkage Region of Human Articular Cartilage Aggrecan-CS chains derived from aggrecan of young or old cartilage were released by ␤-elimination, derivatized with p-aminobenzoic acid, radioiodinated, and subjected to degradation by chondroitin AC-I or C lyase, followed by gel chromatography on Bio-Gel P-6 ( Fig. 6). AC lyase cleaves hexosaminidic bonds to GlcUA when GalNAc is either 4-or 6-O-sulfated, whereas C lyase cleaves only when GalNAc is unsulfated or 6-O-sulfated (32). Exhaustive digestion with AC-I lyase generated the linkage region tetrasaccharide ⌬GlyUA-Gal-Gal-Xyl-R (n ϭ 0) in both cases (Fig. 6, a and c). In contrast, chondroitin C lyase degraded CS from young cartilage less extensively (Fig. 6b) than did AC lyase (Fig. 6a), in keeping with the presence of substantial amounts of GalNAc-4S in CS from young cartilage. CS from old cartilage (Fig. 6d), which contains a greater proportion of 6-O-sulfated GalNAc, was degraded more extensively. The latter results indicated that Gal-NAc-4S was concentrated to the first three disaccharide repeats of CS from old cartilage (represented by fragments n ϭ 1, 2, and 3 in Fig. 6d), whereas 6-O-sulfated (or unsulfated) Gal-NAc was dominant from the fourth position onward.
Charge variants of these linkage region tetrasaccharides were detected by electrophoresis. The tetrasaccharides (n ϭ 0) released by AC-I lyase (Fig. 7a) comprised one dominating component (marked with 0) with the mobility of unsubstituted ⌬GlyUA-Gal-Gal-Xyl-R (cf. Fig. 4, lane 1) and two to three minor, high-mobility variants, which may contain sulfated Gal as well as other charged substituents. Their relative mobilities suggested that the double band represented isomeric, monosulfated variants and that the fast-moving one was disulfated. In CS from old cartilage, the high-mobility variants were somewhat more prominent. Corresponding tetrasaccharides (n ϭ 0) released by C lyase were almost exclusively of the low-mobility type (Fig. 7b), indicating that sulfated -GlcUA-Gal-Gal-Xyl-Ser sequences must be preferentially extended with 4-O-sulfated disaccharide repeats in position 1.
Despite exhaustive treatment with AC-I lyase (Fig. 6, a and  c), small amounts of the linkage region hexasaccharide ⌬GlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-R remained undegraded, more so in material from young cartilage. When these were examined by electrophoresis (Fig. 7c), several components were detected. There were two minor, slow-moving components (the top two bands marked with 1) with the mobilities of unsulfated and monosulfated hexasaccharide, respectively (cf. Fig. 5, lanes  1 and 4). These components were also generated by cleavage with C lyase (Fig. 7d), suggesting that they carried 6-O-sulfated GalNAc also in the succeeding disaccharide (position 2). Both components could be the result of incomplete digestion. However, the most prominent components in the linkage region hexasaccharide pool (Fig. 7c) were very fast moving (see, for example, the bottom band marked with 1), suggesting sulfation also on the first GlcUA and a high degree of sulfation in the first disaccharide repeat, both of which may decrease the rate of cleavage by AC-I lyase (32). Very little high-mobility hexasaccharide was released by C lyase (see the bottom band marked with 1 in Fig. 7d), indicating that an oversulfated linkage region hexasaccharide sequence can be succeeded by largely 4-O-sulfated disaccharides in position 2. When C lyase cleaves between GalNAc-6S and GlcUA, there is no need for 6-Osulfate in the disaccharide linked at the nonreducing side (32). Linkage region extended with two consecutive GalNAc 4-Osulfated disaccharide repeats was analyzed in Fig. 7e, and four to six major components were observed. At least half of them (migrating as the component marked with 2 or faster) must contain oversulfated disaccharides. Analogous results were obtained with saccharides containing three consecutive GlcUA-GalNAc-4S repeats (Fig. 7f). The small amounts of material obtained precluded further detailed analysis.
To further examine the linkage region and the first disaccharide repeat in aggrecan CS from old cartilage, we used a chain preparation that was derivatized with the uncharged adduct p-aminophenol, radioiodinated, and then exhaustively degraded with AC-II lyase (to obtain the tetrasaccharide ⌬GlyUA-Gal-Gal-Xyl-RЈ) or with ABC lyase (to obtain the hexasaccharide ⌬GlyUA-GalNAc-GlcUA-Gal-Gal-Xyl-RЈ). The tetrasaccharide was subjected to ion-exchange chromatography on Q-Sepharose (as in Fig. 2; data not shown), and the major charge variant (90% of total material) migrated as a tetrasaccharide with one negative charge, i.e. the carboxylate on ⌬GlyUA (data not shown). This indicates that both the Xyl and Gal residues in aggrecan CS from old cartilage were mostly unsubstituted. The hexasaccharide linkage region fragments that included the first disaccharide repeat comprised one major and a few minor components (data not shown). The major component migrated as a monosulfated hexasaccharide. The minor components included unsulfated, low-mobility hexasaccharide and a couple of presumably oversulfated, high-mobility ones. The nonreducing terminal ⌬GlyUA-GalNAc repeat in the major hexasaccharide component was degraded by either Hg(OAc) 2 treatment or glycuronidase digestion. Mercury treatment yielded pentasaccharide that migrated to a more retarded position. Digestion with glycuronidase generated two components; one was resistant to the enzyme, and the other was a pentasaccharide (data not shown). The pentasaccharide generated by glycuronidase should be 6-O-sulfated at GalNAc, whereas the resistant hexasaccharide should contain 4-O-sulfated GalNAc. Hence, the first disaccharide repeat in human articular cartilage aggrecan CS is mostly sulfated, either at C-4 or C-6 of GalNAc.
The structures proposed for various linkage region fragments obtained from aggrecan CS of young and old human articular cartilage are summarized in Table II. Structures VII-XI represent segments that, upon degradation by C lyase, would generate linkage region fragments extended with no (VII), one (VIII), two (IX), or three (X) 4-O-sulfated disaccharide repeats. From the results obtained in Figs. 6 and 7, the amounts of saccharides derived from these structures were calculated. In CS from young cartilage, Structures VII-IX accounted for 18, 16, and 14% of the linkage region variants, respectively, whereas the majority of the linkage region was recovered in long 4-O-sulfated segments like Structure X (24%) or higher saccharides (28%). In aggrecan CS from old cartilage, the 6-O-sulfated GalNAc moieties were more frequent and dominated from the nonreducing end down to the vicinity of the linkage region. The majority of the linkage region fragments were recovered as Structures VII-X (81%). Small amounts of Structure XI with, most likely, additional sulfate in one of the Gal moieties and possibly also on GlcUA should also be included.
By using the different charge variants of linkage region saccharides as reference compounds, we examined the electrophoretic banding patterns obtained from whole graded enzymatic digests of articular cartilage aggrecan CS (Fig. 8). CS from young cartilage was treated with AC-II lyase (Fig. 8a), and as expected, bands representing the shortest fragments, i.e. tetrasaccharides (n ϭ 0), increased in intensity with time. Most of these were unsubstituted (see right side of panels a and b in Fig. 8), but minor bands representing tetrasaccharides with, most likely, one, two, or three sulfate groups were also observed. The saccharide with two sulfates appeared as a double band, probably because there were two isomers. A saccharide with three sulfates must be sulfated on both Gal residues and on GlcUA. Saccharides with n ϭ 1 persisted for up to 4 h of digestion and then disappeared; saccharides with n ϭ 2, 3, etc. disappeared gradually at earlier time points. The electrophoretogram thus revealed the presence of three overlapping series of saccharides. One started with monosulfated ⌬GlyUA-Gal-Gal-Xyl-R (see right side of panel b in Fig. 8) and included a series of saccharides extended with n ϭ 1, 2, 3, etc. sulfated disaccharide repeats. A second one appeared to be based on monosulfated (or possibly more highly sulfated) ⌬GlyUA-Gal-Gal-Xyl-R extended with sulfated disaccharides (numbers in-  Fig. 6) were subjected to gradient polyacrylamide gel electrophoresis. The saccharides have the general carbohydrate structure ⌬GlyUA-(GalNAc-Gl-cUA) n -Gal-Gal-Xyl-R (where n ϭ 0, 1, 2, 3, etc., and R ϭ radioiodinated reducing terminal adduct). a, saccharide with n ϭ 0 obtained after cleavage by AC lyase; b, corresponding saccharide generated by C lyase; c, saccharide with n ϭ 1 obtained after cleavage by AC lyase; d, corresponding saccharide generated by C lyase; e and f, saccharides with n ϭ 2 and 3, respectively, obtained after cleavage by C lyase. The migration positions of the major charge variants in each population are indicated. Quantification was carried out by scanning densitometry. The direction of migration is from top to bottom. dicated in brackets). A third one could be an extension of unsulfated ⌬GlyUA-Gal-Gal-Xyl-R with likewise unsulfated disaccharide repeats (numbers indicated in parentheses). However, the vast majority of the chains must consist of unsulfated ⌬GlyUA-Gal-Gal-Xyl-R extended with sulfated disaccharide repeats. Similar time course analyses were made with C lyase digests of CS from young (data not shown) or old (Fig. 8b) cartilage. CS from young cartilage was rapidly degraded from the nonreducing end, indicating that 6-O-sulfated GalNAc moi-eties were concentrated to the peripheral end of the chains. In CS from old cartilage (Fig. 8b), the patterns confirmed the preponderance of 6-O-sulfated disaccharides from positions 4 and 5 onward as well as a high degree of oversulfation in and near the linkage region.
The present strategy for sequence analysis of glycosaminoglycan chains should permit screening of glycans obtained from different sources and produced under various biological circumstances. Microheterogeneity due to variable substitution  of Xyl with phosphate at C-2, of the Gal residues with sulfate at C-4 or C-6, of the first GlcUA with sulfate at C-2 or C-3, as well as un-, mono-, or disulfation of the first repeating GlcUA-GalNAc disaccharide, can be detected. It should be of interest to examine how (and if) microheterogeneity, especially in segments near the core protein, is related to nonrandom variations in the distal part of a chain.