Abstract

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology, 4, 333–342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to ∼0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sβ(1–3)GalNAc6S structure.

Received on July 23, 1999; revised on October 11, 1999; accepted on October 14, 1999.

Introduction

The sulfated anionic polysaccharide chondroitin sulfate (CS) is, along with other glycosaminoglycans (GAGs) an abundant component of extracellular matrices, especially cartilage. However, it is also found on cell surfaces (Fransson, 1987), in neural tissues (Margolis and Margolis, 1997) and in invertebrates (Nader et al., 1984; Oliveira et al., 1994; Kim et al., 1996; Mourao et al., 1996). Several workers have identified specific CS functions which suggest a direct role in macro­molecular interactions; the presence of 4-sulfates is important in the cytoadherence of malaria-infected red blood cells (Cooke et al., 1996) and the binding of the malaria parasite to placenta (Fried and Duffy, 1998; Fried et al., 1998). It has also been shown (Herndon and Lander, 1990; Mark et al., 1990) that the expression of some CS epitopes in the rodent is developmentally regulated and a Δdi-DiSD containing epitope of the proteoglycan (PG) DSD-1 is reported to influence neurite outgrowth (Nadanaka et al., 1998).

Chondroitin sulfate chains comprise a linkage region, a chain cap, and a repeat region. The repeat region is a repeating disaccharide [-4)GlcAβ(1–3)GalNAcβ(1-]n which may be sulfated on the C4 and/or C6 of GalNAc and C2 of GlcA; the sulfation pattern is known to vary depending on the source of the CS. Dermatan sulfates (DS) contain IdoA rather than GlcA and have a very low incidence of 6-sulfation. Recently CSs with 3-sulfated GlcA residues (Sugahara et al., 1996b) and fucose branches (Mourao et al., 1996) have been identified. A sulfated GalNAc residue has been shown to be the major chain cap of human articular cartilage aggrecan CS chains (Plaas et al., 1997) with GalNAc4,6S, which is very rare in the repeat region, increasing from being almost absent in fetal material, to representing ∼60% of chain termini in the normal adult (Plaas et al., 1997) but only ∼30% in osteoarthritic cartilage (Plaas et al., 1998).

The linkage region, through which CS is O-linked to a serine of a protein core, has the general structure –4)GlcAβ(1–3)Galβ(1–3)Galβ(1–4)Xylβ(1-O)-Ser (Roden, 1980). Sulfation on C4 or 6 of either or both Gal residues has been observed (de Waard et al., 1992; Shibata et al., 1992; Sugahara et al., 1992; Cheng et al., 1996; de Beer et al., 1996; Lauder et al., unpublished observations). Sugahara et al. (1995) have identified, in aorta, a DS-specific linkage region containing iduronic acid, i.e., –4)IdoAα(1–3)Galβ(1–3)Galβ(1–4)Xylβ(1-O)-Ser. Linkage regions have, in contrast to the chain cap, been shown to be undersulfated with respect to the repeat region (Lauder et al., 1999). Thus, it is clear that the structure of CS chains varies between tissue sources and within a chain. Further, it is known that the ratio of 4- to 6-sulfation in CS chains changes with development (Kimata et al., 1973), tumor progression (Edwards and Wagner, 1988), and atherosclerosis (Adany et al., 1990) along with age (Brown et al., 1998). Apart from systems such as the binding of antithrombin III to heparin and heparin co-factor II to DS, few structure/function relationships of GAGs have been elucidated.

Hyaluronan (HA) is composed of the repeating disaccharide [-4)GlcAβ(1–3)GlcNAcβ(1-]n and there are no reports of any substitutions or variance in the linear sequence of this GAG which is not synthesized attached to a core protein.

Chondroitin lyase enzymes are eliminases which cleave the –3)GalNAcβ(1–4)GlcA/IdoAα(1- bond in CS/DS in the case of chondroitin ABC lyase, while chondroitin AC lyases act on CS alone. These enzymes convert the nonreducing terminal uronic acid residue of the oligosaccharides generated to a Δ4,5-unsaturated uronic acid (ΔUA). A recent study of the specificity of chondroitin ABC lyase preparations (Hamai et al., 1997) revealed that conventional preparations contained two distinct chondroitin ABC lyase enzymes, an endo- and an exo-lyase, differing in their affinity for substrates based upon size. The chondroitin ABC endolyase was not able to cleave a tetrasaccharide while the exolyase could. The authors found that “Protease Free” chondroitin ABC lyase, as produced by Seikagaku Corp. has a single activity, containing exclusively the endolyase enzyme. Whereas previously the isolation of tetrasaccharides from the repeat region was believed to rely upon non-limit digests, it is now clear that the use of chondroitin ABC endolyase allows the isolation of such oligosaccharides from a limit digest. Hexasaccharide linkage regions, retaining the first repeat region disaccharide, have been isolated following digestion with chondroitin ABC lyase which is unable to cleave the bond between the disaccharide and the linkage region, whereas chondroitin AC lyase enzymes generate tetrasaccharide linkage regions (de Waard et al., 1992).

There are a variety of hyaluronidases derived from animal and microbial sources, the latter being eliminases generating oligosaccharides with a nonreducing terminal ΔUA. In contrast, those from animal sources generate a saturated product. Chondroitin ABC endolyase will also act, at a low rate, upon HA, generating an unsaturated terminal ΔUA.

HPAEC methodologies are widely used within the carbo­hydrate field, and other GAGs have also been examined using such methodologies including keratan sulfate (Whitham et al., 1999).

Previously a high pH anion exchange chromatography (HPAEC) fingerprinting method was developed for the analysis of CS/DS and HA disaccharides (Midura et al., 1994), which, along with other methodologies including capillary zone electrophoresis (Karamanos, et al., 1995a), ion exchange chromatography (Karamanos, et al., 1994), and ion pair chromatography (Karamanos et al., 1995b), has allowed the determination of the disaccharide profile of these GAGs. However, none of these methods allows the characterization of oligo­saccharides other than disaccharides. Methods allowing the analysis of larger oligosaccharides are essential for the analysis of GAG structure and for the development of sequencing strategies. These considerations highlight the need for extended chromatographic analysis methods.

In this work we report an HPAEC fingerprinting methodology for the analysis of CS/DS oligosaccharides of di-, tetra-, and hexa-saccharide in size generated by the action of chondroitin ABC endolyase, and HA oligosaccharides up to hexadecasaccharide generated by bacterial hyaluronate lyase.

Results and discussion

We have isolated a series of CS and DS derived di-, tetra-, and hexa-saccharides by depolymerization of the parent chain using chondroitin ABC endolyase and chondroitin ACII lyase, and HA oligosaccharides up to hexadecasaccharide by depolymerization with bacterial hyaluronate lyase. The purified oligosaccharides have been used to calibrate an HPAEC fingerprinting method which now allows a detailed quantitative examination of the abundance and structure of CS, DS, and HA chains.

The analysis of these oligosaccharides by strong anion exchange chromatography using a Spherisorb 5S column, although suitable for preparative purposes, suffered from poor reproducibility and resolution, therefore an analytical HPAEC method was developed. A complete list of the elution times of each oligosaccharide examined is shown in Table I and Table II.

Size exclusion chromatography of CS chains depolymerized in a limit digest by chondroitin ABC endolyase reveals abundant di- and tetra-saccharides (data not shown), confirming the observations of Hamai et al. (1997) and Lauder et al. (1999) that chondroitin ABC endolyase is unable to cleave tetrasaccharides to disaccharides.

It is clear from an inspection of the HPAEC profiles of CS/DS chains (Figure 1, Table I) that the fingerprinting method resolves each di- and tetra-saccharide without the need to parse them into pools of different size (Figure 1), however linkage region octa- and hexa-saccharides co-elute with the di- and tetra-saccharides reported here (Lauder et al., 1999). There are elution zones within which oligo­saccharides with the same number of sulfate groups elute; unsulfated CS/DS oligosaccharides elute between 20 and 21 min, monosulfated between 29 and 37 min, disulfated between 47 and 66 min, trisulfated between 61.5 and 75 min, and tetrasulfated at 78 min. Thus, given only an HPAEC elution position is it possible to derive information about the sulfation status of CS/DS oligosaccharides. There is some overlap between short highly sulfated oligosaccharides and longer more lowly sulfated oligosaccharides, i.e., ΔHexa-6S/6S/6S elutes significantly in advance of other shorter trisulfated oligosaccharides such as Δtetra-DiSD/4S and even elutes before disulfated disaccharides, demonstrating the importance of charge density in the resolution of these oligosaccharides. A prior SEC step will allow the size of the oligosaccharide to be determined and, allied with HPAEC data the charge density of an unknown oligosaccharide may be determined. The significantly increased body of data available following a chondroitin ABC endolyase digestion compared to a conventional disaccharide analysis may be seen by comparison of the chondroitin ACII lyase digest profile shown in the inset of Figure 1a.

We have isolated a series of HA oligosaccharides of up to hexadecasaccharide and have determined their elution position using the same fingerprinting method (Table II, Figure 2). Hyaluronan oligosaccharides of increasing length, although unsulfated, cross into zones of elution associated with smaller mono- and disulfated CS/DS oligosaccharides. However, hyaluronate lyase from Streptomyces sp. will not act upon CS and therefore oligosaccharide identity may be confirmed by enzyme specificity. Price et al. (1997) previously examined HA oligosaccharides up to hexadecasaccharide by HPAEC, however a prior SEC step was required as differently sized HA oligosaccharides coelute.

Using this method, it is possible to resolve CS oligosaccharides which differ only in the position of a sulfate; compare Δdi-4S and Δdi-6S. Further, the position of a 4- or 6-sulfate within an oligosaccharide has a bearing on the elution position. A 6-sulfated reducing terminal GalNAc-ol residue retards the elution of the oligosaccharide compared to a 4-sulfate at this position; compare Δdi-4S (29.7 min) with Δdi-6S (36.3 min), Δdi-DiSB (57.1 min) with Δdi-DiSD (65.6 min), Δtetra-6S/4S (47.8 min) with Δtetra-6S/6S (53.4 min), and Δtetra-DiSD/4S (70.1 min) with Δtetra-DiSD/6S (72 min). Conversely, an internal GalNAc 6-sulfate reduces the elution time compared to a 4-sulfate; compare Δtri-4S/GlcA (37.9 min) with Δtri-6S/GlcA (35.7 min) and Δtetra-4S/4S (49.6 min) with Δtetra-6S/4S (47.8 min). This may be due to differences in the conformation of the sulfate on C6 and C4, however, the reason for the reversal of the effect when considering an internal GalNAc is not clear.

Following the depolymerization of both CS and DS a single species of nonreducing Δ-uronic acid is generated from both IdoA (DS) and GlcA (CS) resulting in identical disaccharides generated from CS and DS. However, longer oligosaccharides have an internal uronic acid retaining this conformational distinction between IdoA and GlcA, which has a slight effect upon the elution of an oligosaccharide; compare the CS Δtetra-4S/4S (49.6 min) and the DS derived ΔtetraDS-4S/4S (48.9 min). Merry et al. (1999) also report the elution of HS oligosaccharides containing IdoA earlier than related oligo­saccharides containing GlcA.

Information on the nature of an unknown oligosaccharide may be derived from enzymatic desulfation by chondro 4- or 6-sulfatase and comparison of the modified elution position. Both enzymes specifically remove the 4- or 6-sulfate respectively from the reducing terminal GalNAc residue (Sugahara et al., 1996a), while chondro 4-sulfatase alone acts upon the adjacent internal GalNAc residue. The 4-sulfatase will not act upon a disulfated GalNAc4S,6S residue, while chondro 6-sulfatase will if it is at the reducing terminus (Sugahara et al., 1996a). These enzymes should be allowed to act upon the oligosaccharides prior to reduction as the presence of excess reductant and the products of reduction inhibits enzyme activity (data not shown).

The utility of this fingerprinting method in the analysis of the sequence of longer oligosaccharides was confirmed. (Figure 3). Following incubation of a CS hexasaccharide with chondroitin ACII lyase Δdi-6S and Δdi-4S were observed with a ratio of 2:1, while following incubation with chondroitin ABC endolyase Δdi-6S and Δtetra-6S/4S were observed in equal amounts. It is known that chondroitin ABC endolyase will remove the nonreducing terminal disaccharide from an isolated hexasaccharide (Sugahara et al., 1996a); the oligosaccharide clearly has the structure ΔHexa-6S/6S/4S.

The monosulfated CS trisaccharides, Δtri-4S/GlcA and Δtri-6S/GlcA, reported in this work are not the products of chondroitin ABC endolyase, which will only cleave a -GalNAcβ1–4GlcAβ1- bond leaving a reducing terminal GalNAc residue. These oligosaccharides, which have a reducing terminal GlcA residue, may have been generated by breakdown of the CS chain in the tissue or during commercial processing; they are only found in abundance in commercial preparations of CS. Both oligosaccharides have been previously observed by Sugahara et al. (1994).

The A-lyase activity, which acts on 4-sulfated GalNAc residues, is an order of magnitude greater in chondroitin ABC endolyase than the C-lyase which acts upon 6-sulfated GalNAc residues. Thus, during cleavage, bonds involving a 4-sulfated GalNAc residue will be cleaved more often, forcing them to appear at the reducing terminus of the resulting oligosaccharides. This significantly reduces the number of oligosaccharides actually observed.

The sulfation pattern within CS chains is non-random. Glucuronic acid sulfation occurs only between a 4-sulfated GalNAc residue on the nonreducing side and a 6-sulfated GalNAc residue on the reducing side (Nadanaka and Sugahara, 1997; Chai et al., 1998). Our data are in agreement with these observations as 2-sulfated GlcA residues were only found at the nonreducing end of an oligosaccharide adjacent to an 6-sulfated GalNAc and cleaved from the 4-sulfated GalNAc residue which was present on the nonreducing side in the parent polymer. It is not possible to estimate the ratio of GlcAβ(1–3)GalNAc4Sβ(1–4)GlcAβ(1–3)GalNAc6S to that which is 2-sulfated upon the internal GlcA. The unsulfated form has been detected in the linkage regions of bovine articular cartilage aggrecan (Lauder et al., unpublished observations), which are undersulfated.

Oligosaccharides containing a Δdi-DiSD unit are of interest as this has been found to be a part of the MO-225 epitope (Yamagata et al., 1987) expressed transiently during odontoblast development (Mark et al., 1990) and the mAb 473HD epitope (Faissner et al., 1994). The neurite outgrowth activity of DSD-1-PG is inhibited by mAb 473HD and this inhibition is attenuated by shark cartilage (Nadanaka et al., 1998). In this work we show that fresh shark cartilage contains abundant Δdi-DiSD containing oligosaccharides derived from GlcAβ(1–3)GalNAc4Sβ(1–4)GlcA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S and GlcAβ(1–3)GalNAc4Sβ(1–4)GlcA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S (Table I). Shark cartilage CS has also been shown to inhibit the CS mediated interaction of 6B4 CSPG/phosphacan and pleiotrophin.

The method reported here employs elution conditions similar to that reported by Midura et al. (1994); only in the final section of the gradient, beyond 1 M NaCl, do the elution conditions differ. Examination of the elution times for disaccharides from both sets of data shows very close agreement, confirming the reproducibility of the method and its portability between laboratories. It is noteworthy that the same chromatography method is also used in the analysis of CS linkage regions (Lauder et al., 1999).

Monitoring the elution of CS/DS and HA oligosaccharides depolymerized by an eliminase, using absorbance at 232 nm, requires no further modification of the oligosaccharide, employs widely available equipment, is very sensitive (ca. ε 5100–5700 M-1cm-1) (Yamagata et al., 1968) and provides a molar response. However, the oligosaccharides must be reduced to prevent “peeling” due to the high pH during chromatography. The method may be calibrated with either commercial CS and HA disaccharides, or, a standard mixture generated from CS by chondroitin ABC endolyase digestion.

To determine the molar response of CS oligosaccharides a series of Δdi-6S standards were prepared and aliquots examined by HPAEC as described. The peak areas were determined and comparison with molar amounts of oligosaccharide provided a linear response from 10 pmol to 1 µmol with a molar peak area of 3.32604E+14 units.

In this work we have established the elution position, following HPAEC, of a series of CS and DS oligosaccharides of di-, tetra-, and to a limited extent hexa- saccharide in size, and HA oligosaccharides up to hexadecamer. Allied with recent work by Plaas et al. (1997), 1998) and Lauder et al. (unpublished observations) who report methods for HPAEC analysis of the nonreducing termini and octa-, hexa-, and tetra-saccharide linkage regions of CS, respectively, the work reported here facilitates detailed structural analysis of CS/DS and HA chains, allowing analysis of rare biological samples and CS, DS, and HA structures found to have important biological properties such as acting as binding partners or epitopes.

In allied areas other workers have developed related, HPLC based, methodologies for the analysis and sequencing of heparin and HS. Vives et al. (1999) report an SAX based HPLC technique for the sequencing which utilizes a 3H label in the reducing sugar. Merry et al. (1999) report a method enabling the analysis of small aliquots of metabolically labeled HS oligosaccharides which, following initial preparation by enzymatic depolymerization, have a ΔUA nonreducing terminal residue. Both techniques examine the products of the action of chemical and enzymatic methods to modify the structure of the parent oligosaccharide confirming that it is possible to discriminate between oligosaccharides, derived from HS, which differ by a single residue or the position of sulfation.

Analysis of CSs

To confirm the utility of this methodology several CS samples were examined following chondroitin ABC endolyase digestion, the results are shown in Figure 1 and Table III.

Both freshly prepared bovine tracheal cartilage (BTC) and commercial CS(A), derived from BTC, have a similar abundance of 4-sulfated GalNAc residues (40% and 44%, respectively). Neither showed evidence for GlcA 2-sulfation.

Human articular cartilage comprises mainly 6-sulfated GalNAc residues (Table III) and the majority of the ∼6% 4-sulfated GalNAc residues are found as Δdi-4S, only a small percentage being found in tetrasaccharides. There is no evidence of significant levels of GlcA 2-sulfation, although there is clear evidence for the presence of a small amount of Δdi-DiSE.

The commercial sample of mixed whale and shark cartilage CS is mainly 6-sulfated (70%), and has a high level of GlcA 2-sulfation (14%). Significantly Δtetra-DiSD/4S is almost as abundant as Δtetra-DiSD/6S and will be part of GlcAβ(1–3)GalNAc4Sβ(1–4)GlcA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S observed by Nadanaka and Sugahara (1997) and Chai et al. (1998). Whale cartilage alone lacks the abundant GlcA 2-sulfation seen in the mixed sample and is largely 4-sulfated (65%) showing that the Δdi-DiSD containing oligosaccharides derive from the shark cartilage.

Commercial shark cartilage, CS(C), comprises mainly 6-sulfated GalNAc residues with only ∼10% 4-sulfation, all of the 4-sulfated GalNAc residues appearing within a disaccharide or Δtetra-6S/4S, suggesting that there are no long segments of 4-sulfated GalNAc residues. There is only ∼3% GlcA 2-sulfation and there is no evidence of the oligosaccharides Δtetra-DiSD/4S or Δtetra-4S/4S, found in the mixed whale and shark cartilage and also absent from the whale sample. Interestingly, fresh shark scapular and fin cartilage do contain significant amounts of these biologically important Δdi-DiSD containing oligosaccharides. This difference may reflect differences in tissue source or in tissue processing; however, it does highlight the importance of fingerprinting methods in the characterization of GAGs, especially during studies relating the structure of CS to function.

Conclusions

We have developed a simple CS/DS and HA fingerprinting method which, within 80 min (Figure 1), allows a full determination of the sulfation profile and the identification of significant structures with a sensitivity of ∼0.1 nmol. The method will be useful for the elucidation of the structure of long CS oligosaccharides which are the product of non-limit digests or binding sequences.

Materials and methods

A Spherisorb S5 SAX column was purchased from Phase Separations Ltd. (Deeside, Clwyd, UK), the Toyoperl HW-40s resin was purchased from Anachem Ltd. (Luton, UK) and the BioGel-P2 resin was purchased from Bio-Rad (Watford, Herts, UK). Diphenyl carbamyl chloride (DPCC) treated trypsin (bovine pancreas, EC 3.4.21.4), guanidine hydrochloride (practical grade), and mixed whale and shark cartilage CS, DS, CS(A), and CS(C) were purchased from Sigma Chemical Co. (Poole, Dorset, UK). Chondroitin ABC endolyase (Protease free) (Proteus vulgaris, EC 4.2.2.4), chondroitin ACII lyase (Arthrobacter aurescens, EC 4.2.2.5), and streptococcal hyaluronate lyase (EC 4.2.2.1) were obtained from Seikagaku Corp. (Japan) via ICN Biomedicals Ltd. (High Wycombe, Bucks, UK). Cesium chloride was from Fluka Chemicals (Gillingham, Dorset, UK), lithium perchlorate (ACS grade), sulfur tri-oxide pyridine complex, and piperazine were from Aldrich Chemical Co. (Gillingham, Dorset, UK), and sodium hydroxide (A.R. 46/48%) was from Fisons Scientific Equipment (Loughborough, Leicester, UK). All other chemicals were of analytical grade.

Isolation of chondroitin sulfate from cartilages

Chondroitin sulfate was isolated from fresh cartilages using a protocol similar to that previously described for keratan sulfate (KS) (Dickenson et al., 1990). Briefly, the diced cartilage was digested by papain (1 U/100 mg tissue) in 0.1 M sodium acetate pH 6.8 with 2.4 mM EDTA and 10 mM cysteine HCl, added just prior to digestion, for 24 h at 65°C. The GAGs were precipitated from the soluble fraction by the addition of four volumes of ethanol and the solution was cooled to 6°C and allowed to stand overnight. The precipitate was resuspended in a minimum volume of 50 mM sodium acetate and the CS precipitated by the drop-wise addition of 2 volumes of ethanol while the solution was stirred. The solution was cooled to 6°C and allowed to stand overnight before recovery of the CS-rich precipitate, which was dialyzed overnight against distilled water and lyophilized.

The CS chains were released from the attached amino acids by β-elimination with 0.05 M NaOH containing 1 M sodium borohydride at 45°C for 12 h (Carlsson, 1968). The reaction was terminated by the careful addition of 1 M acetic acid.

Modification of sulfation status

Chondroitin sulfate disaccharides with zero (Δdi-0S) or three (Δdi-TriS) sulfates were prepared chemically. An aliquot of CS was desulfated by the method of Nagasawa et al. (1977). Briefly, to 20 mg of the pyridine salt of CS was added 0.5 ml of 5% methanol in DMSO, and the mixture heated to 80°C for 6 h. The desulfated CS was recovered by dialysis or desalted by chromatography on a Bio-Gel P-2 column then lyophilized.

Additional sulfate esters were added to CS chains by the method of Bossennec et al. (1990). Briefly, to 200 mg of the tri-butylamine salt of CS was added 100 mg of sulfur tri-oxide pyridine complex and the solution cooled to 4°C. After 1 h the pH of the solution was raised to pH 9 by the addition of NaOH and the oversulfated CS was recovered by the addition of 4 volumes of ethanol and dialyzed or desalted by chromatography on a Bio-Gel P-2 column then lyophilized.

Depolymerization of glycosaminoglycans

Samples of CS or DS were depolymerized by 1 U/100 mg of chondroitin ABC endolyase or chondroitin ACII lyase in 2 ml of 0.1M Tris/HCl pH 8 at 37°C for 15 h. Hyaluronan was depolymerized by 5000 U/10 mg of bacterial hyaluronate lyase in 1 ml of 0.1 M Tris/HCl, 0.15 M NaCl pH 5, at 37°C for 15 h. In each case the enzyme was inactivated by heating at 100°C for 1 min and the oligosaccharides generated were reduced by the addition of NaBH4 to 25 mM.

An aliquot (1 nmol as determined by absorbance at 232 nm; Yamagata et al., 1968) of a known hexasaccharide ΔUAβ(1–3)[GalNAc6Sβ(1–4)GlcAβ(1–3)]2GalNAc4S-ol (ΔHexa-6S/6S/4S) was resuspended in 10 µl of 0.1 M ammonium acetate pH 8 and digested by 1 mU of chondroitin ABC endolyase in a total volume of 15 µl. The hexasaccharide was digested at 37°C for 15min before the enzyme was inactivated by heating at 100°C for 1 min and the oligosaccharides generated were reduced by the addition of 5 µl of 100 mM sodium borohydride. The oligosaccharides present were identified by HPAEC as described below.

Isolation of standard oligosaccharides

To calibrate the HPAEC fingerprinting method an example of each CS, DS, and HA oligosaccharide reported was isolated and characterized as follows. Reduced CS/DS oligosaccharides were subjected to size exclusion chromatography (SEC) on a Toyoperl HW40s column (50 cm × 1 cm) eluted in 0.5 M ammonium acetate at 0.4 ml/min, the eluent being monitored by absorbance at 232nm. Di-, tetra-, and hexa-saccharides separately pooled. Larger HA oligosaccharides were pooled from the Vo of the column. The pools were subject to repeated lyophilization and then stored lyophilized at –20°C.

The individual oligosaccharides were purified, from the CS/DS di-, tetra-, and hexa-saccharide pools recovered following HW-40 SEC and the total HA digest, by strong anion exchange chromatography. In each case a 10 mg aliquot was resuspended in 500 µl of 2 mM LiClO4, pH 5.0, and chromatographed on a Spherisorb S5 column (25 cm × 1 cm). Bound material was eluted by a linear gradient of 0.002–0.25 M LiClO4, pH 5.0, and the column eluate was monitored on-line at 232 nm. Individual factions were pooled, desalted, and lyophilized. The structure and purity of each oligosaccharide was confirmed by 1D and 2D 600 MHz 1H and 13C NMR experiments (unpublished observations).

Linkage region oligosaccharide

The linkage region tetrasaccharides ΔUAβ(1–3)Gal[±(6S)]β(1–3)Galβ(1–4)Xyl-ol were isolated from bovine articular cartilage aggrecan CS by exhaustive digestion with chondroitin sulfate ACII lyase followed by size exclusion chromatography to separate the resistant tetrasaccharides from the repeat region derived disaccharides (Lauder et al., 1999).

High pH anion exchange chromatography

The individual CS/DS and HA oligosaccharides were used to calibrate the HPAEC fingerprinting method using a modification of the method of Midura et al. (1994) (Figure 1).

Analysis of biological samples

Samples of CS for analysis were subject to digestion by chondroitin ABC endolyase and the reduced oligosaccharides examined by HPAEC without further treatment.

Quantification

The relative abundance of each oligosaccharide identified was determined by estimation of the peak area and the application of a response factor equivalent to the inverse of the molar extinction coefficient (Yamagata et al., 1968).

Acknowledgements

We thank the Arthritis Research Campaign for support (Grant N0511).

Abbreviations

CS, chondroitin sulfate; DS, dermatan sulfate; 6S, 4S, and 2S, O-ester sulfate group on C6, C4 and C2, respectively; HPAEC, high pH anion exchange chromatography; ΔUA, delta 4,5-unsaturated uronic acid; GlcA, glucuronic acid; GalNAc(-ol), 2-deoxy-2-N-acetylamino-d-galactose (-galactitol); HA, hyaluronan; Δdi-0S, ΔUAβ(1–3)GalNAc-ol; Δdi-4S, ΔUAβ(1–3)GalNAc4S-ol; Δdi-UA2S, ΔUA2Sβ(1–3)GalNAc-ol; Δdi-6S, ΔUAβ(1–3)GalNAc6S-ol; Δdi-DiSE, ΔUAβ(1–3)GalNAc4,6S-ol; Δdi-DiSB, ΔUA2Sβ(1–3)GalNAc4S-ol; Δdi-DiSD, ΔUA2Sβ(1–3)GalNAc6S-ol; Δdi-triS, ΔUA2Sβ(1–3)GalNAc4,6S-ol; Δtri-4S/GlcA, ΔUAβ(1–3)GalNAc4Sβ(1–4)GlcA-ol; Δtri-6S/GlcA, ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcA-ol; Δtetra-6S/6S, ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S-ol; Δtetra-6S/4S, ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol; Δtetra-4S/4S, ΔUAβ(1–3)GalNAc4Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol; Δtetra-DiSD/4S, ΔUA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol; Δtetra-DiSD/6S, ΔUA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S-ol; Δtetra-DiSE/DiSD, ΔUAβ(1–3)Gal­NAc4,6Sβ(1–4)GlcA2Sβ(1–3)GalNAc6S-ol; Δhexa-6S/6S/6S, ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6Sβ(1–4)Glc­Aβ(1–3)GalNAc6S-ol; Δhexa-6S/6S/4S, ΔUAβ(1–3)Gal­NAc6Sβ(1–4)GlcAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)Gal­NAc4S-ol.

1

To whom correspondence should be addressed

Fig.1. HPAEC of the oligosaccharides derived from chondroitin ABC endolyase digestion of (a) mixed whale and shark CS and (b) human articular cartilage CS. The oligosaccharides were chromatographed on a CarboPac PA1 column (250 mm × 4 mm) maintained at 30°C. Elution was at 1 ml/min; a 12 min isocratic period of 98% eluent A (0.1 M NaOH)/2% eluent B (1.3 M NaCl in 0.1 M NaOH) was followed by a linear gradient of 2–46% eluent B over 50 min; 46–87% eluent B over 8 min, 87–100% eluent B over 6 min followed by a 4 min isocratic phase of 100% eluent B. The eluent was monitored by absorbance at 232 nm. In (a) the elution position of each disaccharide is shown and the insert shows the data derived from disaccharide analysis (chondroitin ACII lyase digestion alone). Some artifactual peaks may be observed, especially with eluents which have not been recently prepared, including a broad hump around 15–20 min and a sharper peak at around 27 min. Each of the di-, tetra-, and hexa-saccharides reported in this work are well resolved by the elution protocol.

Fig.1. HPAEC of the oligosaccharides derived from chondroitin ABC endolyase digestion of (a) mixed whale and shark CS and (b) human articular cartilage CS. The oligosaccharides were chromatographed on a CarboPac PA1 column (250 mm × 4 mm) maintained at 30°C. Elution was at 1 ml/min; a 12 min isocratic period of 98% eluent A (0.1 M NaOH)/2% eluent B (1.3 M NaCl in 0.1 M NaOH) was followed by a linear gradient of 2–46% eluent B over 50 min; 46–87% eluent B over 8 min, 87–100% eluent B over 6 min followed by a 4 min isocratic phase of 100% eluent B. The eluent was monitored by absorbance at 232 nm. In (a) the elution position of each disaccharide is shown and the insert shows the data derived from disaccharide analysis (chondroitin ACII lyase digestion alone). Some artifactual peaks may be observed, especially with eluents which have not been recently prepared, including a broad hump around 15–20 min and a sharper peak at around 27 min. Each of the di-, tetra-, and hexa-saccharides reported in this work are well resolved by the elution protocol.

Fig.2. HPAEC of the oligosaccharides derived from hyaluronate lyase digestion of hyaluronan. For elution protocol see Figure 1. *, Artifactual peak observed at ∼32 min.

Fig.2. HPAEC of the oligosaccharides derived from hyaluronate lyase digestion of hyaluronan. For elution protocol see Figure 1. *, Artifactual peak observed at ∼32 min.

Fig.3. Sequence determination of a hexasaccharide derived from CS. Samples of an unknown hexasaccharide were incubated with chondroitin lyase enzymes as described in methods and the products examined: solid line, parent oligosaccharide; dashed line, following digestion by chondroitin ACII lyase; and dottedline, following digestion by chondroitin ABC endolyase. For elution protocol see Figure 1.

Fig.3. Sequence determination of a hexasaccharide derived from CS. Samples of an unknown hexasaccharide were incubated with chondroitin lyase enzymes as described in methods and the products examined: solid line, parent oligosaccharide; dashed line, following digestion by chondroitin ACII lyase; and dottedline, following digestion by chondroitin ABC endolyase. For elution protocol see Figure 1.

Table I.

Elution position of CS/DS oligosaccharides following analysis by HPAEC

Oligosaccharide Code Generated by the action of:   Elution (min) 
  ACII ABC  
      
ΔUAβ(1–3)GalNAc-ol Δdi-0S ✓ ✓  21.3 
ΔUA2Sβ(1–3)GalNAc-ol Δdi-UA2S ✓ ✓  33.2 
ΔUAβ(1–3)GalNAc4S-ol Δdi-4S ✓ ✓ 29.7 
ΔUAβ(1–3)GalNAc6S-ol Δdi-6S ✓ ✓ ✓ 36.3 
      
ΔUA2Sβ(1–3)GalNAc4S-ol Δdi-DiSB ✓ ✓ 57.1 
ΔUA2Sβ(1–3)GalNAc6S-ol Δdi-DiSD ✓ ✓ ✓ 65.6 
      
ΔUAβ(1–3)GalNAc4,6S-ol Δdi-DiSE ✓ ✓  56.4 
ΔUA2Sβ(1–3)GalNAc4,6S-ol Δdi-TriS ✓ ✓  75.4 
      
ΔUAβ(1–3)GalNAc4Sβ(1–4)GlcA-ol Δtri-4S/GlcA See Text   37.9 
ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcA-ol Δtri-6S/GlcA See Text   35.7 
      
ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol Δtetra-6S/4S ✓ 47.8 
ΔUAβ(1–3)GalNAc4Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol Δtetra-4S/4S ✓ 49.6 
ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S-ol Δtetra-6S/6S ✓ ✓ 53.4 
      
ΔUAβ(1–3)GalNAc4Sβ(1–4)IdoAα(1–3)GalNAc4S-ol ΔtetraDS-4S/4S ✓ 48.9 
      
ΔUA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol Δtetra-DiSD/4S ✓ 70.1 
ΔUA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S-ol Δtetra-DiSD/6S ✓ ✓ 72.0 
      
ΔUAβ(1–3)GalNAc4,6Sβ(1–4)GlcA2Sβ(1–3)GalNAc6S-ol Δtetra-DiSE/DiSD ✓ ✓ 77.8 
      
ΔUAβ(1–3)[GalNAc6Sβ(1–4)GlcAβ(1–3)]2GalNAc4S-ol ΔHexa-6S/6S/4S ✓ 61.5 
ΔUAβ(1–3)[GalNAc6Sβ(1–4)GlcAβ(1–3)]2GalNAc6S-ol ΔHexa-6S/6S/6S ✓ ✓ 62.7 
      
ΔUAβ(1–3)[GalNAc4Sβ(1–4)IdoAβ(1–3)]2GalNAc4S-ol ΔHexa-DS 4S/4S/4S ✓ 62.8 
      
ΔUAβ(1–3)Galβ(1–3)Galβ(1–4)Xyl-ol Link ✓ ✓ 20.4 
ΔUAβ(1–3)Gal6Sβ(1–3)Galβ(1–4)Xyl-ol Link(6S) ✓ ✓ 31.6 
Oligosaccharide Code Generated by the action of:   Elution (min) 
  ACII ABC  
      
ΔUAβ(1–3)GalNAc-ol Δdi-0S ✓ ✓  21.3 
ΔUA2Sβ(1–3)GalNAc-ol Δdi-UA2S ✓ ✓  33.2 
ΔUAβ(1–3)GalNAc4S-ol Δdi-4S ✓ ✓ 29.7 
ΔUAβ(1–3)GalNAc6S-ol Δdi-6S ✓ ✓ ✓ 36.3 
      
ΔUA2Sβ(1–3)GalNAc4S-ol Δdi-DiSB ✓ ✓ 57.1 
ΔUA2Sβ(1–3)GalNAc6S-ol Δdi-DiSD ✓ ✓ ✓ 65.6 
      
ΔUAβ(1–3)GalNAc4,6S-ol Δdi-DiSE ✓ ✓  56.4 
ΔUA2Sβ(1–3)GalNAc4,6S-ol Δdi-TriS ✓ ✓  75.4 
      
ΔUAβ(1–3)GalNAc4Sβ(1–4)GlcA-ol Δtri-4S/GlcA See Text   37.9 
ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcA-ol Δtri-6S/GlcA See Text   35.7 
      
ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol Δtetra-6S/4S ✓ 47.8 
ΔUAβ(1–3)GalNAc4Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol Δtetra-4S/4S ✓ 49.6 
ΔUAβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S-ol Δtetra-6S/6S ✓ ✓ 53.4 
      
ΔUAβ(1–3)GalNAc4Sβ(1–4)IdoAα(1–3)GalNAc4S-ol ΔtetraDS-4S/4S ✓ 48.9 
      
ΔUA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc4S-ol Δtetra-DiSD/4S ✓ 70.1 
ΔUA2Sβ(1–3)GalNAc6Sβ(1–4)GlcAβ(1–3)GalNAc6S-ol Δtetra-DiSD/6S ✓ ✓ 72.0 
      
ΔUAβ(1–3)GalNAc4,6Sβ(1–4)GlcA2Sβ(1–3)GalNAc6S-ol Δtetra-DiSE/DiSD ✓ ✓ 77.8 
      
ΔUAβ(1–3)[GalNAc6Sβ(1–4)GlcAβ(1–3)]2GalNAc4S-ol ΔHexa-6S/6S/4S ✓ 61.5 
ΔUAβ(1–3)[GalNAc6Sβ(1–4)GlcAβ(1–3)]2GalNAc6S-ol ΔHexa-6S/6S/6S ✓ ✓ 62.7 
      
ΔUAβ(1–3)[GalNAc4Sβ(1–4)IdoAβ(1–3)]2GalNAc4S-ol ΔHexa-DS 4S/4S/4S ✓ 62.8 
      
ΔUAβ(1–3)Galβ(1–3)Galβ(1–4)Xyl-ol Link ✓ ✓ 20.4 
ΔUAβ(1–3)Gal6Sβ(1–3)Galβ(1–4)Xyl-ol Link(6S) ✓ ✓ 31.6 
Table II.

Elution position of hyaluronan oligosaccharides following analysis by HPAEC

Oligosaccharide Code Elution time (min) 
ΔUAβ(1–3)GlcNAc-ol Δdi-HA 22.9 
ΔUAβ(1–3)GlcNAcβ(1––4)GlcAβ(1–3)GlcNAc-ol Δtetra-HA 28.4 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]2GlcNAc-ol Δhexa-HA 31.7 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]3GlcNAc-ol Δocta-HA 34.6 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]4GlcNAc-ol Δdeca-HA 36.5 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]5GlcNAc-ol Δduodeca-HA 38.3 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]6GlcNAc-ol Δtetradeca-HA 39.7 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]7GlcNAc-ol Δhexadeca-HA 40.8 
Oligosaccharide Code Elution time (min) 
ΔUAβ(1–3)GlcNAc-ol Δdi-HA 22.9 
ΔUAβ(1–3)GlcNAcβ(1––4)GlcAβ(1–3)GlcNAc-ol Δtetra-HA 28.4 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]2GlcNAc-ol Δhexa-HA 31.7 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]3GlcNAc-ol Δocta-HA 34.6 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]4GlcNAc-ol Δdeca-HA 36.5 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]5GlcNAc-ol Δduodeca-HA 38.3 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]6GlcNAc-ol Δtetradeca-HA 39.7 
ΔUAβ(1–3)[GlcNAcβ(1––4)GlcAβ(1–3)]7GlcNAc-ol Δhexadeca-HA 40.8 
Table III.

Results of HPAEC analysis of CSs

 Commercially available CS  Prepared in the laboratory   
Oligosaccharide Whale/ shark Whale  Shark CS(C)  BTCa CS(A)  BTCa CS(A)  HACb CS Shark fin Shark cartilage 
 Percentage abundance      
Δdi-0S 0.1 1.8 0.7 0.3 0.7 1.1 
Δdi-4S 18 54 6.7 35.3 38.4 5.0 21.7 21.8 
Δdi-6S 39.9 29.5 73.7 48.9 45.9 85.0 15.2 17.0 
Δdi-DiSD 10.9 0.3 21.0 20.5 
Δdi-DiSE 0.6 0.3 0.9 
         
Δtri-4S/GlcA 0.5 
Δtri-6S/GlcA 1.5 
         
Δtetra-6S/4S 8.1 2.8 0.3 4.8 5.5 0.4 8.7 8.6 
Δtetra-4S/4S 4.9 9.2 3.1 2.8 3.5 0.7 9.0 8.0 
Δtetra-6S/6S 8.9 4.2 13.0 6.5 6.0 8.5 3.2 3.7 
Δtetra-DiSD/4S 3.3 0.5 13.7 12.8 
Δtetra-DiSD/6S 3.8 2.5 5.8 6.5 
0-Sulfation 0.1 1.6 0.6 0.3 0.5 0.8 
4-Sulfation 30.5 64.7 10.3 40.1 44.2 6.4 44.6 42.4 
6-Sulfation 69.5 35.3 89.6 58.4 55.2 93.3 54.9 56.8 
2-Sulfation 13.9 0.3 3.0 29.0 28.5 
 Commercially available CS  Prepared in the laboratory   
Oligosaccharide Whale/ shark Whale  Shark CS(C)  BTCa CS(A)  BTCa CS(A)  HACb CS Shark fin Shark cartilage 
 Percentage abundance      
Δdi-0S 0.1 1.8 0.7 0.3 0.7 1.1 
Δdi-4S 18 54 6.7 35.3 38.4 5.0 21.7 21.8 
Δdi-6S 39.9 29.5 73.7 48.9 45.9 85.0 15.2 17.0 
Δdi-DiSD 10.9 0.3 21.0 20.5 
Δdi-DiSE 0.6 0.3 0.9 
         
Δtri-4S/GlcA 0.5 
Δtri-6S/GlcA 1.5 
         
Δtetra-6S/4S 8.1 2.8 0.3 4.8 5.5 0.4 8.7 8.6 
Δtetra-4S/4S 4.9 9.2 3.1 2.8 3.5 0.7 9.0 8.0 
Δtetra-6S/6S 8.9 4.2 13.0 6.5 6.0 8.5 3.2 3.7 
Δtetra-DiSD/4S 3.3 0.5 13.7 12.8 
Δtetra-DiSD/6S 3.8 2.5 5.8 6.5 
0-Sulfation 0.1 1.6 0.6 0.3 0.5 0.8 
4-Sulfation 30.5 64.7 10.3 40.1 44.2 6.4 44.6 42.4 
6-Sulfation 69.5 35.3 89.6 58.4 55.2 93.3 54.9 56.8 
2-Sulfation 13.9 0.3 3.0 29.0 28.5 

CS was isolated from a variety of sources, digested with chondroitin ABC endolyase and following reduction was examined by HPAEC as described.

aBTC, bovine tracheal cartilage.

bHAC, human articular cartilage.

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