Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/d, 41100 Modena, Italy
Received on January 27, 2003; revised on March 26, 2003; accepted on March 27, 2003
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Abstract |
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Key words: chondroitin / glycosaminoglycans / K4 polysaccharide / oligosaccharides
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Introduction |
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K4d is useful to gain detailed insight into the mode of action of several enzymes, such as the C-5 epimerase involved in dermatan sulfate biosynthesis (Hannesson et al., 1996) and both the GlcUA- and the GalNAc-transferases in chondroitin sulfate formation (Lidholt and Fjelstad, 1997
). Therefore for this purpose native K4 is defructosylated to produce the polysaccharide possessing the chondroitin backbone.
Nonsulfated oligosaccharides and low-molecular-mass derivatives show a great variety of biological and potentially pharmacological actions. Hyaluronic acid (HA), a nonsulfated unbranched polysaccharide composed of repeating disaccharides possessing the structure of GlcUA acid (ß13) and N-acetyl-D-glucosamine (ß1
4), differs from K4d by the nature of N-acetyl-D-exosamine. HA oligosaccharides stimulate angiogenesis (West et al., 1985
), induce many kinds of inflammatory factors (Horton et al., 1998
), and inhibit tumor growth in vivo (Zeng et al., 1998
), probably due to the presence of specific receptors on the surface of cells (Kincade et al., 1997
; Entwistle et al., 1996
). These results suggest that nonsulfated oligosaccharides and low-molecular-mass derivatives besides HA may acquire new activities and functions after depolymerization.
Various methods for the production of nonsulfated oligomers, in particular from HA, have been described. Most of these involve the digestion of polysaccharide with endohydrolase or testicular hyaluronidase followed by purification by size-exclusion chromatography (Lesley et al., 2000; Tammi et al., 1998
) and/or ion-exchange chromatography (Tammi et al., 1998
; Chai et al., 2001
). These methods result in even-numbered oligosaccharides for which the minimal size of HA is 2 disaccharide units in length (4-mers). Gram/mg-scale HA oligosaccharides were obtained in pure, uniform-size form available for investigating new biological funtions of this polyanion (Mahoney et al., 2001
; Tawada et al., 2002
).
In this article, we describe mg-scale isolation and purification of K4 and K4d oligosaccharides and their subsequent analyses by size-exclusion high-performance liquid chromatography (HPSEC), ion-exchange HPLC, and fluorophore-assisted carbohydrate electrophoresis (FACE). These molecules may be used for a variety of biological studies.
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Results |
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Figures 1A and 1B respectively show SAX-HPLC analyses of K4 and K4d oligosaccharides produced after 8 h of hyaluronidase treatment, compared with HA oligosaccharides (Figure 1C). Unfortunately, under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, that is, pH 5.2 at 37°C, fructose is slowly liberated forming the defructosylated K4, as already observed by Rodriguez et al. (1988). As a consequence, a mixture of K4 and K4d oligosaccharide species of decreasing length, from
20- to 4-mers, are generated and separated by strong anion-exchange (SAX)-HPLC. In fact, the chromatogram of Figure 1A shows for each oligosaccharide species of definite length two peaks, corresponding to the K4 and its defructosylated product. In a previous paper (Volpi, 2003
) I reported that unsaturated K4 and K4d disaccharides produced by the action of chondroitin ABC lyase can be separated by SAX-HPLC with the first species being eluted before the fructosylated one. The same analytical approach was used to verify the composition of each size-uniform species using as standard of the defructosylated species purified K4d oligosaccharides. As expected, for each couple of peaks of oligosaccharides of various lengths separated by SAX-HPLC (Figure 1A), the first (lower retention times) was constituted by the fructosylated species and the second (greater retention times) by the defructosylated one (not shown). Furthermore, the two unsaturated disaccharides derived from K4 and K4d uniform-size mixture,
HexUAFru-GalNAc for K4 and
HexUA-GalNAc for defructosylated K4, produced by the action of chondroitinase ABC were separated by using high-performance capillary electrophoresis (HPCE; electrokinetic chromatography with sodium dodecyl sulfate) (Volpi, 2003
) confirming that each K4 size-separated oligosaccharide was composed of K4 and its defructosylated species (not shown).
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Discussion |
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E. coli K4 produces a chondroitin-like molecule with fructose side branches on the 3-position of the glucuronic residues (Rodriguez et al., 1988) with a repetitive disaccharidic sequence composed of
4)-GlcUAFru-ß(1
3)GalNAc-ß(1
. This polysaccharide can be defructosylated under acid hydrolysis producing a polymer with the backbone composed of disaccharides
4)-GlcUA-ß(1
3)GalNAc-ß(1
having the same structure as chondroitin (Rodriguez et al., 1988
). K4 polysaccharide has a molecular mass of about 200300 kDa evaluated by HPSEC and submarine agarose-gel electrophoresis (Lee and Cowman, 1994
), and no appreciable modification of its mass was observed after its complete defructosylation (not shown; Volpi, 2003
). Under the experimental conditions used, K4 and K4d were degraded by testicular hyaluronidase to produce oligosaccharide species ranging approximately from 4- to 20-mers after 8 h treatment. Under the same conditions, HA from rooster comb of
1.000 kDa was degraded to produce oligosaccharides of 3035-mers. These differences in the progression of the enzymatic reaction were probably due to the greater molecular mass of HA than K4 and K4d, also considering that the three polyanions are equally sensitive to the action of the hyaluronidase (Rodriguez et al., 1988
).
Rodriguez et al. (1988) reported for the first time that K4d polysaccharide is sensitive to the action of testicular hyaluronidase, which causes it to produce oligomers. Furthermore, Rodriguez et al. (1988)
also noted that under the experimental conditions used to perform the testicular hyaluronidase treatment of K4, specifically, low pH at 37°C for long periods of time, the fructose was slowly liberated and the defructosylated product formed was a substrate for the enzyme. I was able to purify oligosaccharides from 4-mers to 16-mers composed of mixtures of fructosylated and defructosylated species after treatment with hyaluronidase and to perform their separation by preparative anion-exchange chromatography. However, I was unable to obtain in pure form oligosaccharides possessing the backbone structure of K4.
The purification methods employed here allowed the production on mg-scale of a wide range of K4d oligosaccharide sizes (oligosaccharides having the structure of a chondroitin, 4)-GlcUA-ß(1
3)GalNAc-ß(1
), in particular from 4-mers to 16-mers, but it is expected to obtain species possessing larger homogeneous molecular masses by varying the conditions of the enzymatic treatment, as also reported for HA (Mahoney et al., 2001
; Tawada et al., 2002
).
The defructosylated product of K4 has been used to gain detailed insight into the mode of action of several enzymes, such as the C-5 epimerase involved in dermatan sulfate biosynthesis (Hannesson et al., 1996) and both the GlcUA- and the GalNAc-transferases in chondroitin sulfate formation (Lidholt and Fjelstad, 1997
). The pure, uniform-size K4d oligosaccharides, having the structure of chondroitin in a wide variety of sizes, are now available for investigating new biological functions, as reported for HA (Termeer et al., 2000
. West et al., 1985
). In fact, at the moment, no procedure has been available to produce pure oligosaccharides of definite length having the structure of
4)-GlcUA-ß(1
3)GalNAc-ß(1
. Inoue and Nagasawa (1981)
reported the possibility of producing uniform-size chondroitin species by chemical desulfatation and depolymerization of chondroitin-6-sulfate and further separation of partially desulfated oligosaccharides by anion-exchange chromatography. However, this approach can produce partial desulfatation of the polysaccharide with the preservation of part of sulfate groups and can induce chemical modification in the backbone structure of the chondroitin. On the contrary, K4d can be obtained from K4 by an acid treatment that does not introduce chemical modifications in the structure of the polysaccharide (Rodriguez et al., 1988
). Furthermore, pure uniform-size oligosaccharides possessing the structure of the chondroitin could be chemically sulfated to obtain fully O-sulfated chondro-oligosaccharides useful to evaluate their biological properties as compared with O-sulfated hyaluro-oligosaccharides having an inhibitory activity on hyaluronidase (Suzuki et al., 2002).
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Materials and methods |
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AG 1-X2 anion-exchange resin, dry mesh size 200400 and wet bead size 75180 µm, and ion-exchange resin AG 501-X8(D), dry mesh size 2050 and wet bead size 3001.180 µm, were from Bio-Rad (Hercules, CA). Cellulose membrane dialysis, MWCO of 100 Da, were from Spectrum (Tokyo, Japan). All the other reagents were of analytical grade.
Preparation of the defructosylated polysaccharide
The nonsulfated fructosylated K4 was defructosylated by acid treatment at pH 3.0, according to Rodriguez et al. (1988) and as previously reported (Volpi, 2003
). Twenty-five milliliters of the K4 polysaccharide (50 mg) in distilled water were stirred at room temperature with about 50 mg ion-exchange resin AG 501-X8(D) for 24 h. After removal of the resin by filtration, the mixture was neutralized with sodium hydroxide and dyalized against water for 2 days. The dialysate was lyophilized to yield K4d. The progression of the defructosylation process was monitored by HPCE, as previously reported (Volpi, 2003
).
Preparation of K4 and K4d oligosaccharides
K4 and K4d (and HA used as standard) were partially degraded by bovine testicular hyaluronidase. To a solution containing 50 mg polysaccharides in 5 ml 100 mM Na-acetate buffer adjusted to pH 5.2 with acetic acid containing 150 mM NaCl, 5000 U hyaluronidase was added, and enzymatic digestion was performed at 37°C for 18 h. The degradation of HA used as standard was performed on 20 mg. The incubation time of the hyaluronidase varied according to the sizes of K4 and K4d oligosaccharides to be obtained, assessed at various time points by running 20 µl of the reaction mixture on SAX-HPLC. The reactions were stopped by boiling for 20 min. The samples were centrifuged at 10,000 rpm for 30 min at 5°C, and the supernatants were lyophilized. The lyophilized samples were dissolved in distilled water, and each K4 and K4d oligosaccharide was isolated from the parent digest by anion-exchange chromatography on a column (2 x 90 cm) of AG l-X2 anion-exchange resin (Inoue and Nagasawa, 1981) using low-pressure liquid chromatography (Biologic LP chromatography system from BioRad) at a flow of 1 ml/min. Oligosaccharides were eluted at room temperature with a linear gradient of 0.050.5 M NaCl buffered at pH 4.0 with 0.1 M HCl. The eluant was monitored at 214 nm, and a fraction of 2 ml was collected and further analyzed for uronic acid content (Bitter and Muir, 1962
). Each uniform-sized K4 and K4d oligosaccharide fraction was desalted by using cellulose membrane dialysis and freeze-dried to give the final products.
Quantitative analysis of each size-uniform oligosaccharide obtained was performed by weight.
SAX-HPLC
HPLC equipment was from Jasco (pump mod. PU-1580, UV detector mod. UV-1570, Rheodyne injector equipped with a 100-µl loop, software Jasco-Borwin rel. 1.5; Tokyo, Japan). The K4 and K4d oligosaccharides were analyzed by SAX-HPLC separation using a 150 x 4.6-mm stainless-steel column spherisorb 5-SAX (5 µm, trimethylammoniopropyl groups Si-CH2-CH2-CH2-N+(CH3)3 in Cl form, from Phase Separations Limited (Deeside Industrial Park, Deeside Clwyd, UK) and detection at 214 nm. Isocratic separation was performed using 50 mM NaCl pH 4.00 for 5 min followed by a 560-min linear gradient from 100% 50 mM NaCl, pH 4.00, to 100% 1.2 M NaCl, pH 4.00, at a flow rate of 1.5 ml/min.
FACE analyses
For the FACE analyses, the method of Tawada et al. (2002) was utilized. Derivatization of K4 and K4d oligosaccharides with ANTS used 2 nmol of each oligosaccharide lyophilized in a 0.5-ml tube. All samples were then derivatized by adding of 5 µl ANTS reagent solution (Glyko L2, Part 50058, Novato, CA) followed by incubation for 15 min at room temperature. Then 5 µl sodium cyanoborohydride solution (Glyko L1, Part 50056) was added, followed by incubation for 16 h at 37°C. After incubation, all samples were centrifuged briefly and adjusted to a final volume of 20 µl with distilled water. For electrophoresis, OLIGO Gel Running Buffer was dissolved in water and cooled on ice. Gels were thoroughly cleaned with distilled water and the wells of each gel were rinsed extensively with running buffer immediately prior to use. The assembled electrophoresis apparatus containing the electrophoresis buffer, and one gel was placed in the Glyko Gel Box and cooled on ice to equilibrate the buffer to 4°C.
A 2-µl aliquot of each fluorotagged K4 or K4d oligosaccharide was mixed with 3 µl distilled water and 5 µl Glyko loading buffer. Four microliters of each sample were loaded and electrophoresed for 150 min at a constant 1000 V. After electrophoresis, the gel was removed from the apparatus, and the covering glass plates were thoroughly cleaned with pure water. The gel was illuminated with UV light (365 nm) and photographed.
HPSEC
HPLC mod. LC-1500 was from Jasco (pump mod. PU-1580, UV detector mod. UV-1570, software Jasco-Borwin rel. 1.5). The mobile phase was composed of a 125 mM Na2SO4 and 2 mM NaH2PO4 adjusted to pH 6.0 with 0.1 N NaOH. Flow rate was 0.9 ml/min with a back pressure of 40 kg/cm2. Samples were solubilized in the mobile phase at a concentration of 5 mg/ml. Ten microliters (50 µg) were injected in HPLC. Columns were Protein Pak 125 (Waters, cod. 84601, 7.8 mm x 30 cm, native globular 280 kDa, and random coil 130 kDa; Milford, CT) and Protein Pak 300 (Waters, cod. T72711, 7.5 mm x 30 cm, native globular 10400 kDa, and random coil 2150 kDa) alone or assembled in series. The eluent was monitored at 214 nm.
1 To whom correspondence should be addressed; e-mail: volpi{at}unimo.it
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Abbreviations |
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References |
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Calabro, A., Benavides, M., Tammi, M., Hascall, V.C., and Midura, R.J. (2000) Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulphate by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology, 10, 273281.
Chai, W., Beeson, J.G., Kogelberg, H., Brown, G.V., and Lawson, A.M. (2001) Inhibition of adhesion of Plasmodium falciparum-infected erythrocytes by structurally defined hyaluronic acid dodecasaccharides. Infect. Immun., 69, 420425.
Entwistle, J., Hall, C.L., and Turley, E.A. (1996) HA receptors: regulators of signalling to the cytoskeleton. J. Cell Biochem., 61, 569577.[CrossRef][ISI][Medline]
Hannesson, H.H., Hagner-McWhirter, A., Tiedemann, K., Lindahl, U., and Malmstrom, A. (1996) Biosynthesis of dermatan sulphate. Defructosylated Escherichia coli K4 capsular polysaccharide as a substrate for the D-glucuronyl C-5 epimerase, and an indication of a two-base reaction mechanism. Biochem. J., 313, 589596.[ISI][Medline]
Horton, M.R., McKee, C.M., Bao, C., Liao, F., Farber, J.M., Hodge-DuFour, J., Pure, E., Oliver, B.L., Wright, T.M., and Noble, P.W. (1998) Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10 in mouse macrophages. J. Biol. Chem., 273, 3508835094.
Inoue, Y. and Nagasawa, K. (1981) Depolymerization of glycosaminoglycuronans into di- and higher molecular-weight oligo-saccharides: improved preparation of N-acetyldermosine and oligomeric N-acetylchondrosines. Carbohydr. Res., 97, 263278.[CrossRef][ISI][Medline]
Kincade, P.W., Zheng, Z., Katoh, S., and Hanson, L. (1997) The importance of cellular environment to function of the CD44 matrix receptor. Curr. Opin. Cell Biol., 9, 635642.[CrossRef][ISI][Medline]
Lee, H.G. and Cowman, M.K. (1994) An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal. Biochem., 219, 278287.[CrossRef][ISI][Medline]
Lesley, J., Hascall, V.C., Tammi, M., and Hyman, R. (2000) Hyaluronan binding by cell surface CD44. J. Biol. Chem., 275, 2696726975.
Lidholt, K. and Fjelstad, M. (1997) Biosynthesis of the Escherichia coli K4 capsule polysaccharide. A parallel system for studies of glycosyltransferases in chondroitin formation. J. Biol. Chem., 272, 26822687.
Mahoney, D.J., Aplin, R.T., Calabro, A., Hascall, V.C., and Day, A.J. (2001) Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length. Glycobiology, 11, 10251033.
Rodriguez, M.L., Jann, B., and Jann, K. (1988) Structure and serological characteristics of the capsular K4 antigen of Escherichia coli O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone. Eur. J. Biochem., 177, 117124.[Abstract]
Suzuki, A., Toyoda, H., Toida, T., and Imanari, T. (2001) Preparation and inhibitory activity on hyaluronidase of fully O-sulphated hyaluro-oligosaccharides. Glycobiology, 11, 5764.
Tammi, R., MacCallum, D., Hascall, V.C., Pienimaki, J.P., Hyttinen, M., and Tammi, M. (1998) Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides. J. Biol. Chem., 273, 2887828888.
Tawada, A., Masa, T., Oonuki, Y., Watanabe, A., Matsuzaki, Y., and Asari, A. (2002) Large-scale preparation, purification, and characterization of hyaluronan oligosaccharides from 4-mers to 52-mers. Glycobiology, 12, 421426.
Termeer, C.C., Hennies, J., Voith, U., Ahrens, T., Weiss, J.M., Prehm, P., and Simon, J.C. (2000) Oligosaccharides of hyaluronan are potent activators of dendritic cells. J. Immunol., 165, 18631870.
Volpi, N. (2003) Separation of capsular polysaccharide K4 and defructosylated K4 derived disaccharides by high-performance capillary electrophoresis and high-performance liquid chromatography. Electrophoresis, 24, 10631068.[CrossRef][ISI][Medline]
West, D.C., Hampson, I.N., Arnold, F., and Kumar, S. (1985) Angiogenesis induced by degradation products of hyaluronic acid. Science, 228, 13241326.[ISI][Medline]
Zeng, C., Toole, B.P., Kinney, S.D., Kuo, J.W., and Stamenkovic, I. (1998) Inhibition of tumor growth in vivo by hyaluronan oligomers. Int. J. Cancer, 77, 396401.[CrossRef][ISI][Medline]