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Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates
Biochemical and Biophysical Research Communications 406 (2011) 239–244
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Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates Ikuko Kakizaki a,b,⇑, Isoshi Nukatsuka c, Keiichi Takagaki a, Mitsuo Majima a,d, Mito Iwafune a, Shinichiro Suto e, Masahiko Endo a,b a
Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan Department of Frontier Materials Chemistry, Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan d Department of Health and Nutrition, Faculty of Human Science, Hokkaido Bunkyo University, 196 Kogane-cho, Eniwa 061-1408, Japan e Department of Glycomedicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b c
a r t i c l e
i n f o
Article history: Received 26 January 2011 Available online 12 February 2011 Keywords: Hyaluronidase Transglycosylation Chondroitin sulfate Metal Cation
a b s t r a c t Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Glycosaminoglycans (GAG) are polysaccharide chains composed of repeating disaccharide units of a hexosamine and a non-nitrogenous sugar. They are classified as hyaluronan (HA) chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), heparin (Hep) and keratan sulfate (KS) based on the composition of the disaccharide unit [1,2]. GAG chains, with the exception of HA, occur in conjugation with proteins as proteoglycans. There is considerable structural variation for instance, in sulfation even within one class of glycosaminoglycans. For example, chondroitins exist in a non-sulfated form (Ch), two mono-sulfated forms as chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S) and various polysulfated forms (Ch-D, Ch-E). GAGs even though they are defined based on the composition of their repeating disaccha-
Abbreviations: GAG, glycosaminoglycan; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparin sulfate; Hep, heparin; Ch, chondroitin; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; HA, hyaluronan; BTH, bovine testicular hyaluronidase; GlcNAc, N-acetylglucosamine; GlcUA, glucuronic acid; GalNAc, N-acetylgalactosamine; Der, desulfated dermatan; PA, 2-pyridylamine; GalN, galactosamine; GlcN, glucosamine; HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection. ⇑ Corresponding author at: Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Fax: +81 172 39 5016. E-mail address: [email protected] (I. Kakizaki). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.02.024
ride unit are typically not homogenous molecules. Thus, CS chains may contain disaccharide units of DS in addition to repeating disaccharides of CS. Again HA is an exception since it contains only one type of repeating disaccharide units. The heterogeneity of the GAGs chains is not incidental or without functional significance since specific biological activity has been attributed to the functional motifs in the molecule as in the case of heparin [2–6]. However, there are only very few functional motifs with confirmed correlation between the structure and function. Bovine testicular hyaluronidase (BTH, hyaluronoglucosaminidase, EC 3.2.1.35) is an endoglycosidase that hydrolyzes HA at b1, 4-N-acetylglucosaminide bonds (GlcNAcb–(1 ? 4)–GlcUA) although the details of the reaction mechanism of this enzyme on high molecular weight HA have not been elucidated [7–9]. It is known that this enzyme acts also on internal b1, 4-N-acetylgalactosaminide bonds (GalNAcb–(1 ? 4)–GlcUA) of CSs. The order of action is as follows: HA, Ch, C4S, C6S [7,10]. It has been shown that hyaluronidases, like many other glycosidases, catalyze transglycosylation reaction in addition to the main hydrolysis reaction [11–14]. The optimal conditions for transglycosylation and hydrolysis by BTH are different, pH 7.0 in the absence of NaCl versus pH 4.0 in the presence of NaCl [13,15]. We have used the in vitro transglycosylation activity of BTH to custom synthesize CS oligosaccharides with known carbohydrate sequences for use in structure–function studies of GAGs and proteoglycans [13,16–21]. In these previous investigations we have successfully
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synthesized neo-glycosaminoglycan oligosaccharides consisting of various combinations of C4S, C6S, di-sulfated CSs, chemically desulfated chondroitin sulfate (Ch) and desulfated dermatan sulfate (Der). However, the yields obtained in this approach have been generally low and approaches are necessary to increase the yields but none has been reported. Potential regulators of hyaluronidase activity are mono- or divalent metal cations, multivalent cations such as polyprotamine sulfate and spermine, and inter-a-trypsine inhibitor [22–26]. The effects of cations have been investigated, and most divalent cations other than Cu2+ were reported to have the potential to activate the hydrolysis reaction of BTH [22]. These regulators are probably important in vivo for controlling HA metabolism because HA involves various processes of cell functions and diseases. Studies on the regulators of the transglycosylation reaction by BTH are also invaluable for glycotechnological manipulation of GAGs. Therefore, we have undertaken an investigation of the effect of metal cations on the transglycosylation by BTH using as substrates metal salts of GAG chains and report our findings in this paper. It was found that while barium enhanced the transglycosylation of CSs zinc had an inhibitory effect. In addition, we observed that the degree of the effect by different cations varied depending on the type of GAG used as the donor. 2. Materials and methods
mesh, H-form (Bio-Rad, Richmond, CA), and eluted with distilled water [34]. Then the resulting free acid form of the GAG was incubated for 24 h with a 100 mM solution of MnCl2, BaCl2, ZnCl2 or CuCl2 and the incubation mixture desalted on a PD-10 gel filtration column. The different metal forms of the GAG were recovered by ethanol precipitation and or dialysis against distilled water followed by lyophilization. 2.4. Measurement of the metal binding to GAGs Binding of metal atoms to the GAGs were assessed by atomic absorption using a standard addition method. In the case of barium, atomic absorption was measured by a Hitachi Z-8270 polarized Zeeman atomic absorption spectrophotometer equipped with an SSC-300 auto sampler (Tokyo, Japan) [35]. In the case of all metals except barium, atomic absorption was measured by a Shimadzu AA-6800 atomic absorption spectrophotometer with GFA-EX7 atomizer and ASC-6100 auto sampler (Kyoto, Japan). The molar ratio of bound metal per uronic acid-N-acetylhexosamine disaccharide unit was expressed as the metal ion (lmol/ ml)/hexosamine (lmol/ml). The galactosamine (GalN) or glucosamine (GlcN) content of the metal salts of GAG was determined after hydrolyzes with 4 N HCl at 100 °C for 8 h. The hydrolysate was analyzed by high performance anion exchange chromatography (HPAEC-PAD) on a PA1 column (4 250 mm, Dionex, Sunnyvale, CA) using pulsed amperometric detection.
2.1. Chemicals 2.5. Transglycosylation reaction of BTH BTH (type1-S) was purchased from Sigma Chemicals Co. (St. Luis, MO) and further purified as previously reported [27]. HA (from Streptococcus zooepidemicus; average molecular weight, 80,000) was purchased from Kibun Food Chemifa Co., Ltd. (Tokyo, Japan). C4S (from whale cartilage; average molecular weight, 34,000), C6S (from shark cartilage; average molecular weight, 64,000), and DS (from pig skin; average molecular weight, 32,000) were obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Ch and Der were prepared by chemical desulfation of C6S and DS, respectively [28,29] and their unsaturated disaccharide compositions were determined by HPLC after chondroitin ABC lyase digestion [30]. The compositions were as follows; Ddi0S:Ddi4S: Ddi6S = 91.6:0.8:7.6 in Ch, Ddi0S:Ddi4S:Ddi6S = 99.4:0.3:0.3 in Der. Chondroitin ABC lyase and unsaturated disaccharide standards derived from CSs were from Seikagaku Kogyo Co. (Tokyo, Japan). C6S hexasaccharide was prepared by partial digestion of C6S with BTH and purification through a Bio-Gel P-4 column according as reported previously [13,31]. Bio-Gel P-4 (400 mesh, extra fine) and Sephadex G-15 were from Bio-Rad (Richmond, CA) and Pharmacia Biotech Inc. (Uppsala, Sweden), respectively. 2-Pyridylamine (PA) was from Wako Pure Chemical Co. (Osaka, Japan) and was recrystallized from hexane. Other reagents were of analytical grade and obtained from commercial sources. 2.2. Pyridylamination of C6S hexasaccharide Fluorolabeling of the reducing terminus of C6S hesasaccharide with PA was carried out by a modified version of the method of Hase et al. [32] as described in our previous report [33]. The C6S hexasaccharide-PA was used as an acceptor in the transglycosylation reaction of BTH.
Various combinations of C6S hexasaccharide-PA (2 nmol) as an acceptor and 50 lg of GAGs (Na-form or metal-form of HA, Ch, Der, C4S, C6S) as donor substrates were incubated with 1.0 NFU of BTH in 0.1 M Tris–HCl buffer, pH 7.0 at 37 °C for 1 h. The reaction was terminated by boiling for 3 min and the reaction products analyzed by HPLC after centrifugation and filtration. The transglycosylation efficiency, expressed as transglycosylation (%), was estimated based on the amount of the acceptor consumed in the reaction. Thus, transglycosylation% = the amount of acceptor before the reaction minus the amount of acceptor after the reaction 100/ amount of acceptor before the reaction. 2.6. HPLC analysis Normal phase HPLC was carried out on a Hitachi L-6200 equipped with a fluorescence detector (model F-1150, Hitachi, Tokyo, Japan) on a PALPAK Type-S column (4.6 250 mm, Takara Bio Inc., Otsu, Japan) or TSKgel Amide-80 (4.6 250 mm, Tosoh Co., Tokyo, Japan). Two solutions were used: eluent A was a 20:80 (v/ v) mixture of 3% acetic acid and acetonitrile, adjusted to pH 7.3 with triethylamine, while eluent B was a 50:50 (v/v) mixture of 3% acetic acid and acetonitrile. The flow rate was set at 1.0 ml/ min. PA oligosaccharides after transglycosylation were injected onto the column equilibrated with eluent A. Samples were eluted on a linear gradient of 0–100% of eluent B over 60 min. Column temperature was 40 °C. Eluted material was detected at excitation and emission wavelengths of 320 and 400 nm, respectively. 3. Results
2.3. Preparation of the different metal salts of GAG chains
3.1. Effect of the addition of cations to the transglycosylation reaction mixture
The GAGs, which are usually available commercially as their sodium salts, were dissolved in water at 10 mg/ml and passed through a column of AG 50W-X2 cation exchange resin (200–400
It has been shown that divalent cations control the hydrolysis activity of BTH on HA [22]. To study the effect of cations on the transglycosylation reaction of BTH, C6S hexasaccharide-PA
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(acceptor) and C6S (Na-form) (donor) were incubated with BTH in the presence of 1–100 mM of various cations under transglycosylation conditions, i.e. pH 7.0. The products, PA oligosaccharides elongated from C6S hexasaccharide-PA, were monitored by fluorescence of PA. A typical chromatogram of transglycosylation reaction, using C6S hexasaccharide-PA (acceptor) and C6S (Na-form) (donor) without addition of metal, is shown in Fig. 1A. C6S octasaccharide-PA, C6S decasaccharide-PA and longer C6S oligosaccharide-PA, which are transglycosylation products were observed. When metal cations were added in this reaction mixture none of the cations examined here enhanced the transglycosylation activity of BTH, instead some of them inhibited the reaction and the degree of inhibition depended on the concentration of cation
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used (data not shown). The results obtained when 10 mM of the different cation was added to the transglycosylation reaction mixture are shown in Fig. 1B. 3.2. Effect of cations bound to GAG on the transglycosylation reaction We then decided to examine the effect of cations on the transglycosylation by using the metal salts of glycosaminoglycans as donors. GAGs (HA, Der, Ch, C4S, C6S) bound with cations (Mn2+, Ba2+, Zn2+, Cu2+) were prepared for use as donors of transglycosylation. The binding efficiency of cations to GAG is shown in Fig. 2. No significant difference in binding efficiency of divalent metal ions to HA was observed. In contrast, there was significant difference in chondroitin sulfates. In the cases of C4S and C6S, which are sulfated GAGs at C4 or C6 of N-acetylgalactosamine, respectively significant binding to divalent cations was observed and the molar ratio of Ba2+ ions bound per uronic acid-N-acetylhexosamine disaccharide unit was the highest of all the metals tested. With C6S, the number of bound cations per disaccharide unit was estimated to be 0.075 for Mn2+, 1.231 for Ba2+, 0.144 for Zn2+, 0.395 for Cu2+. The binding efficiency for C6S was higher than for C4S. These results suggest that divalent cations bind to sulfate groups in addition to other anionic groups and the total binding efficiency is higher at C6 than at C4. When the various metals bound to GAGs were tested as donors it was found that barium bound to CS significantly increased the transglycosylation (Fig. 3). The transglycosylation efficiency of barium salt was increased 1.22-fold for Ch, 1.45-fold for C4S, and 1.46fold for C6S compared to that of sodium salt, the increasing rate is considerable because the transglycosylation efficiency of sodium salt of C6S is much lower than HA. Thus, the barium salt of C6S is a good donor as HA although sodium salt of C6S is a poor donor. A significant increase in transglycosylation was observed when the zinc salt of Ch was used whereas, the zinc salts of C4S and C6S markedly decreased transglycosylation (Fig. 3). When HA, which even though treated with metal cations had almost the same amount of bound metal regardless of the kind of metal, was used as the donor, there was inhibition in the transglycosylation efficiency for Cu and no changes in the others. 3.3. Recovery from inhibition by Zn in transglycosylation In order to examine whether the effect of metals on the degree of the transglycosylation was due to the binding of the cations to
Fig. 1. Effect of adding metal cations to the reaction mixture on the transglycosylation reaction. Two nanomoles of chondroitin 6-sulfate hexasaccharide-PA as an acceptor and 50 lg of chondroitin 6-sulfate (Na-form) as a donor were incubated with 1.0 NFU of bovine testicular hyaluronidase in 0.1 M Tris–HCl buffer, pH 7.0 in the absence (none) or presence of 10 mM of various salt solutions (NaCl, MnCl2, BaCl2, ZnCl2, CuCl2) at 37 °C for 1 h. Reaction products were analysed by HPLC on a TSKgel Amide-80 column by monitoring of fluorescence of PA. (A) HPLC of the transglycosylation products in the absence of metal. (a) Before the reaction; (b) after the reaction. The broken line indicates the gradient curve. Arrows indicate the elution positions of standard PA chondroitin 6-sulfate oligosaccharides (6, hexasaccharide-PA; 8, octasaccharide-PA; 10, decasaccharide-PA; 12, dodecasaccharidePA; 14, tetradecasaccharide-PA). (B) Transglycosylation efficiency in the presence and absence of metals.
Fig. 2. Binding efficiency of divalent cations to chondroitin sulfates. Molar ratios of metallic atoms (Mn, Ba, Zn, Cu) to the hexosamine (GalN or GlcN) content of hyaluronan (A), desulfated dermatan sulfate (B), desulfated chondroitin sulfate (C), chondroitin 4-sulfate (D), or chondroitin 6-sulfate (E) bound were estimated. Metal content was determined by atomic absorption method, and hexosamine content by monosaccharide analysis using the HPAEC-PAD after acidic hydrolysis.
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Fig. 3. Effect of divalent cations bound with donor GAGs on transglycosylation. Transglycosylation reaction was performed using chondroitin 6-sulfate hexasaccharide-PA as an acceptor and hyaluronan (A), desulfated dermatan sulfate (B), desulfated chondroitin sulfate (C), chondroitin 4-sulfate (D), or chondroitin 6-sulfate (E) bound with divalent cations (Mn, Ba, Zn, and Cu) as donors. Transglycosylation efficiency was estimated based on the decrease rate of acceptor.
the donor glycosaminoglycans, ethylenediamine tetra-acetic acid (EDTA) was added to the reaction mixture. Addition of EDTA (5–20 mM) to the reaction mixture containing BTH and C6S hexasaccharide-PA as the acceptor and C6S (Zn-form) as the donor, this was chosen since the change in the case of Zn was the most marked, reversed the strong inhibitory effect of Zn (Fig. 4).
Fig. 4. Effect of EDTA on the inhibition of transglycosylation when chondroitin 6sulfate (Zn-form) was used as a donor. EDTA was added to the reaction mixture of transglycosylation containing chondroitin 6-sulfate hexasaccharide-PA as an acceptor and chondroitin 6-sulfate (Zn-form) as donor. Reaction products were analysed by HPLC on a PALPAK Type-S column by monitoring of fluorescence of PA. The broken line indicates the gradient curve. Arrows indicate elution positions of chondroitin 6-sulfate oligosaccharide-PA (6, hexasaccharide-PA; 8, octasaccharidePA; 10, decasaccharide-PA; 12, dodecasaccharide-PA; 14, tetradecasaccharide-PA). (a) Chondroitin 6-sulfate (Zn-form), EDTA ( ), (b) chondroitin 6-sulfate (Zn-form), 5 mM EDTA, (c) chondroitin 6-sulfate (Zn-form), 20 mM EDTA, and (d) commercially available chondroitin 6-sulfate (Na-form), EDTA ( ).
4. Discussion It has been reported that cations play an important role in hydrolysis of HA by hyaluronidase [22,36]. Sodium chloride is commonly used to facilitate the hydrolytic activity of testicular hyaluronidase. Further, most divalent cations except Cu facilitate hydrolysis reaction of this enzyme [22]. In this study, we have demonstrated that divalent cations could also control transglycosylation activity of testicular hyaluronidase. We have shown that cations could either facilitate or inhibit transglycosylation depending on the kind of metal ion and donor GAG used. It is suggested that the effect of cations on the hydrolytic activity of BTH on HA is due to the influence of the cations on the substrate rather than the enzyme [26]. Our results show that inclusion of cations in the reaction mixture had no influence of the transglycosylation activity of BTH. However, the activity was affected when cation bound to C6S was used as the donor. This suggests that cations bound to the GAG apparently alter its 3dimensional structure and thereby facilitate the activity of hyaluronidase on the polysaccharide. In fact, it has been reported that binding of certain cation change the 3-dimensional structure of GAG [37]. It is also known that carboxylate groups on GlcUA of HA show a chelation effect at pH 6 [38] and as a result the carboxylate groups may affect hydrolysis activity of BTH by binding with cations. Further, in CS species functional groups contributing to the binding of cations are carboxylate groups in neutral and acidic aqueous [39]. In the present study, effect of cations on transglycosylation by BTH was investigated using five different GAGs as donors and the effects differed depending on the kind of GAG. The binding efficiency of HA was similar among metals but differed significantly in CS. Der differs from CS in that it has iduronic acid in addition to GlcUA in the repeating disaccharide units of the polysaccharide chains. It is known that GAGs having iduronic acids are structurally more flexible than those having only GlcUA and are therefore capable of associating strongly with cations [40]. Accordingly, an explanation can be provided that the differences in the effect of cations on the transglycosylation behavior of Der and CSs are due to the differences in the 3-dimensional conformation of the carboxylate groups on uronic acids. From the relationships between the metal binding and transglycosylation efficiency, it can be deduced that
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sulfate groups are also important for metal binding. Especially, the association between this bulky group and Ba, whose ion radius is relatively big, can bring changes in 3-dimensional conformation of GAGs. The radii of the metal ions tested in this study are as follows: Na+, 0.97, Mn2+, 0.80, Ba2+, 1.34, Zn2+, 0.74, Cu2+, 0.72. In the reaction of hyaluronidase, hydrolysis and transglycosylation occurs as an equilibrium reaction. First we predicted that the effects of metal on transglycosylation correlate with that on hydrolysis. However, it was shown that there is less correlation between the effects on the two reactions: both barium salts and zinc salts of CSs up-regulated hydrolysis compared to their sodium salts when they were used as substrates (data not shown), in contrast, barium salts enhanced while zinc salt inhibited transglycosylation. CSs, which are the sugar moieties of CS–proteoglycans, and HA coexist in extracellular matrices or surfaces of cells. In vitro transglycosylation using BTH synthesises hybrid sugar chains of these different glycosaminoglycans [41]. It has not yet been investigated whether hybrid sugar chains consisting of HA and CS is actually generated in living body by the transglycosylation. It is also not known whether artificial HA/CS hybrid oligosaccharides have significant biological activity. However, cations should be paid attention to as control factors of transglycosylation both in vitro and in vivo. Our glycotechnological approach using the systematic transglycosylation of BTH has made it possible to custom-synthesize hybrid GAGs among HA, Ch (desulfated CS), C4S, C6S, Der (desulfated DS) [13,16–21]. However, the yields were low when sulfated substrates such as C4S, C6S, were used in transglycosylation reaction. The present study helps to explain the reason. Our findings also provide an approach to control transglycosylation activity of hyaluronidase by reversible modification of the GAG donor with metals and thereby increase yields. Acknowledgments This work was supported by Grants-in Aid (Nos. 15370041, 17770106, and 19570119) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by Grants for Priority Research Designated by the President of Hirosaki University, and by the Fund for Cooperation for Innovative Technology and Advanced Research in Evolution Area (CITY AREA). We would like to thank Prof. V.P. Bhavanandan for his interest and editorial help with the manuscript. References [1] L. Rodén, Structure and metabolism of connective tissue proteoglycans, in: W.J. Lannarz (Ed.), The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press, New York, 1980, pp. 267–371. [2] L. Kjellen, U. Lindahl, Proteoglycans: structures and interactions, Annu. Rev. Biochem. 60 (1991) 443–475. [3] C. Malavaki, S. Mizumoto, N. Karamanos, K. Sugahara, Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease, Connect. Tissue Res. 49 (2008) 133–139. [4] U. Lindahl, G. Backstrom, L. Thunberg, I.G. Leder, Evidence for a 3-O-sulfated Dglucosamine residue in the antithrombin-binding sequence of heparin, Proc. Natl. Acad. Sci. USA 77 (1980) 6551–6555. [5] R.N. Achur, I. Kakizaki, S. Goel, K. Kojima, S.V. Madhunapantula, A. Goyal, M. Ohta, S. Kumar, K. Takagaki, D.C. Gowda, Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria, Biochemistry 47 (2008) 12635–12643. [6] H. Habuchi, S. Suzuki, T. Saito, T. Tamura, T. Harada, K. Yoshida, K. Kimata, Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor, Biochem. J. 285 (Pt 3) (1992) 805–813. [7] K. Meyer, Hyaluronidases, in: P.D. Boyer, E.G. Krebs (Eds.), The Enzymes, Academic Press, New York, 1971, pp. 307–320. [8] K. Takagaki, I. Kakizaki, Degradation of glycosaminoglycans, in: J.P. Kamerling, G.J. Boons, Y.C. Lee, A. Suzuki, N. Taniguchi, A.G.J. Voragen (Eds.), Comprehensive Glycoscience – From Chemistry to Systems Biology, Elsevier Science B.V., Amsterdam, The Netherlands, 2007, pp. 171–192.
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Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates Ikuko Kakizaki a,b,⇑, Isoshi Nukatsuka c, Keiichi Takagaki a, Mitsuo Majima a,d, Mito Iwafune a, Shinichiro Suto e, Masahiko Endo a,b a
Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan Department of Frontier Materials Chemistry, Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan d Department of Health and Nutrition, Faculty of Human Science, Hokkaido Bunkyo University, 196 Kogane-cho, Eniwa 061-1408, Japan e Department of Glycomedicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan b c
a r t i c l e
i n f o
Article history: Received 26 January 2011 Available online 12 February 2011 Keywords: Hyaluronidase Transglycosylation Chondroitin sulfate Metal Cation
a b s t r a c t Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Glycosaminoglycans (GAG) are polysaccharide chains composed of repeating disaccharide units of a hexosamine and a non-nitrogenous sugar. They are classified as hyaluronan (HA) chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), heparin (Hep) and keratan sulfate (KS) based on the composition of the disaccharide unit [1,2]. GAG chains, with the exception of HA, occur in conjugation with proteins as proteoglycans. There is considerable structural variation for instance, in sulfation even within one class of glycosaminoglycans. For example, chondroitins exist in a non-sulfated form (Ch), two mono-sulfated forms as chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate (C6S) and various polysulfated forms (Ch-D, Ch-E). GAGs even though they are defined based on the composition of their repeating disaccha-
Abbreviations: GAG, glycosaminoglycan; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparin sulfate; Hep, heparin; Ch, chondroitin; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; HA, hyaluronan; BTH, bovine testicular hyaluronidase; GlcNAc, N-acetylglucosamine; GlcUA, glucuronic acid; GalNAc, N-acetylgalactosamine; Der, desulfated dermatan; PA, 2-pyridylamine; GalN, galactosamine; GlcN, glucosamine; HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection. ⇑ Corresponding author at: Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Fax: +81 172 39 5016. E-mail address: [email protected] (I. Kakizaki). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.02.024
ride unit are typically not homogenous molecules. Thus, CS chains may contain disaccharide units of DS in addition to repeating disaccharides of CS. Again HA is an exception since it contains only one type of repeating disaccharide units. The heterogeneity of the GAGs chains is not incidental or without functional significance since specific biological activity has been attributed to the functional motifs in the molecule as in the case of heparin [2–6]. However, there are only very few functional motifs with confirmed correlation between the structure and function. Bovine testicular hyaluronidase (BTH, hyaluronoglucosaminidase, EC 3.2.1.35) is an endoglycosidase that hydrolyzes HA at b1, 4-N-acetylglucosaminide bonds (GlcNAcb–(1 ? 4)–GlcUA) although the details of the reaction mechanism of this enzyme on high molecular weight HA have not been elucidated [7–9]. It is known that this enzyme acts also on internal b1, 4-N-acetylgalactosaminide bonds (GalNAcb–(1 ? 4)–GlcUA) of CSs. The order of action is as follows: HA, Ch, C4S, C6S [7,10]. It has been shown that hyaluronidases, like many other glycosidases, catalyze transglycosylation reaction in addition to the main hydrolysis reaction [11–14]. The optimal conditions for transglycosylation and hydrolysis by BTH are different, pH 7.0 in the absence of NaCl versus pH 4.0 in the presence of NaCl [13,15]. We have used the in vitro transglycosylation activity of BTH to custom synthesize CS oligosaccharides with known carbohydrate sequences for use in structure–function studies of GAGs and proteoglycans [13,16–21]. In these previous investigations we have successfully
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synthesized neo-glycosaminoglycan oligosaccharides consisting of various combinations of C4S, C6S, di-sulfated CSs, chemically desulfated chondroitin sulfate (Ch) and desulfated dermatan sulfate (Der). However, the yields obtained in this approach have been generally low and approaches are necessary to increase the yields but none has been reported. Potential regulators of hyaluronidase activity are mono- or divalent metal cations, multivalent cations such as polyprotamine sulfate and spermine, and inter-a-trypsine inhibitor [22–26]. The effects of cations have been investigated, and most divalent cations other than Cu2+ were reported to have the potential to activate the hydrolysis reaction of BTH [22]. These regulators are probably important in vivo for controlling HA metabolism because HA involves various processes of cell functions and diseases. Studies on the regulators of the transglycosylation reaction by BTH are also invaluable for glycotechnological manipulation of GAGs. Therefore, we have undertaken an investigation of the effect of metal cations on the transglycosylation by BTH using as substrates metal salts of GAG chains and report our findings in this paper. It was found that while barium enhanced the transglycosylation of CSs zinc had an inhibitory effect. In addition, we observed that the degree of the effect by different cations varied depending on the type of GAG used as the donor. 2. Materials and methods
mesh, H-form (Bio-Rad, Richmond, CA), and eluted with distilled water [34]. Then the resulting free acid form of the GAG was incubated for 24 h with a 100 mM solution of MnCl2, BaCl2, ZnCl2 or CuCl2 and the incubation mixture desalted on a PD-10 gel filtration column. The different metal forms of the GAG were recovered by ethanol precipitation and or dialysis against distilled water followed by lyophilization. 2.4. Measurement of the metal binding to GAGs Binding of metal atoms to the GAGs were assessed by atomic absorption using a standard addition method. In the case of barium, atomic absorption was measured by a Hitachi Z-8270 polarized Zeeman atomic absorption spectrophotometer equipped with an SSC-300 auto sampler (Tokyo, Japan) [35]. In the case of all metals except barium, atomic absorption was measured by a Shimadzu AA-6800 atomic absorption spectrophotometer with GFA-EX7 atomizer and ASC-6100 auto sampler (Kyoto, Japan). The molar ratio of bound metal per uronic acid-N-acetylhexosamine disaccharide unit was expressed as the metal ion (lmol/ ml)/hexosamine (lmol/ml). The galactosamine (GalN) or glucosamine (GlcN) content of the metal salts of GAG was determined after hydrolyzes with 4 N HCl at 100 °C for 8 h. The hydrolysate was analyzed by high performance anion exchange chromatography (HPAEC-PAD) on a PA1 column (4 250 mm, Dionex, Sunnyvale, CA) using pulsed amperometric detection.
2.1. Chemicals 2.5. Transglycosylation reaction of BTH BTH (type1-S) was purchased from Sigma Chemicals Co. (St. Luis, MO) and further purified as previously reported [27]. HA (from Streptococcus zooepidemicus; average molecular weight, 80,000) was purchased from Kibun Food Chemifa Co., Ltd. (Tokyo, Japan). C4S (from whale cartilage; average molecular weight, 34,000), C6S (from shark cartilage; average molecular weight, 64,000), and DS (from pig skin; average molecular weight, 32,000) were obtained from Seikagaku Kogyo Co. (Tokyo, Japan). Ch and Der were prepared by chemical desulfation of C6S and DS, respectively [28,29] and their unsaturated disaccharide compositions were determined by HPLC after chondroitin ABC lyase digestion [30]. The compositions were as follows; Ddi0S:Ddi4S: Ddi6S = 91.6:0.8:7.6 in Ch, Ddi0S:Ddi4S:Ddi6S = 99.4:0.3:0.3 in Der. Chondroitin ABC lyase and unsaturated disaccharide standards derived from CSs were from Seikagaku Kogyo Co. (Tokyo, Japan). C6S hexasaccharide was prepared by partial digestion of C6S with BTH and purification through a Bio-Gel P-4 column according as reported previously [13,31]. Bio-Gel P-4 (400 mesh, extra fine) and Sephadex G-15 were from Bio-Rad (Richmond, CA) and Pharmacia Biotech Inc. (Uppsala, Sweden), respectively. 2-Pyridylamine (PA) was from Wako Pure Chemical Co. (Osaka, Japan) and was recrystallized from hexane. Other reagents were of analytical grade and obtained from commercial sources. 2.2. Pyridylamination of C6S hexasaccharide Fluorolabeling of the reducing terminus of C6S hesasaccharide with PA was carried out by a modified version of the method of Hase et al. [32] as described in our previous report [33]. The C6S hexasaccharide-PA was used as an acceptor in the transglycosylation reaction of BTH.
Various combinations of C6S hexasaccharide-PA (2 nmol) as an acceptor and 50 lg of GAGs (Na-form or metal-form of HA, Ch, Der, C4S, C6S) as donor substrates were incubated with 1.0 NFU of BTH in 0.1 M Tris–HCl buffer, pH 7.0 at 37 °C for 1 h. The reaction was terminated by boiling for 3 min and the reaction products analyzed by HPLC after centrifugation and filtration. The transglycosylation efficiency, expressed as transglycosylation (%), was estimated based on the amount of the acceptor consumed in the reaction. Thus, transglycosylation% = the amount of acceptor before the reaction minus the amount of acceptor after the reaction 100/ amount of acceptor before the reaction. 2.6. HPLC analysis Normal phase HPLC was carried out on a Hitachi L-6200 equipped with a fluorescence detector (model F-1150, Hitachi, Tokyo, Japan) on a PALPAK Type-S column (4.6 250 mm, Takara Bio Inc., Otsu, Japan) or TSKgel Amide-80 (4.6 250 mm, Tosoh Co., Tokyo, Japan). Two solutions were used: eluent A was a 20:80 (v/ v) mixture of 3% acetic acid and acetonitrile, adjusted to pH 7.3 with triethylamine, while eluent B was a 50:50 (v/v) mixture of 3% acetic acid and acetonitrile. The flow rate was set at 1.0 ml/ min. PA oligosaccharides after transglycosylation were injected onto the column equilibrated with eluent A. Samples were eluted on a linear gradient of 0–100% of eluent B over 60 min. Column temperature was 40 °C. Eluted material was detected at excitation and emission wavelengths of 320 and 400 nm, respectively. 3. Results
2.3. Preparation of the different metal salts of GAG chains
3.1. Effect of the addition of cations to the transglycosylation reaction mixture
The GAGs, which are usually available commercially as their sodium salts, were dissolved in water at 10 mg/ml and passed through a column of AG 50W-X2 cation exchange resin (200–400
It has been shown that divalent cations control the hydrolysis activity of BTH on HA [22]. To study the effect of cations on the transglycosylation reaction of BTH, C6S hexasaccharide-PA
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(acceptor) and C6S (Na-form) (donor) were incubated with BTH in the presence of 1–100 mM of various cations under transglycosylation conditions, i.e. pH 7.0. The products, PA oligosaccharides elongated from C6S hexasaccharide-PA, were monitored by fluorescence of PA. A typical chromatogram of transglycosylation reaction, using C6S hexasaccharide-PA (acceptor) and C6S (Na-form) (donor) without addition of metal, is shown in Fig. 1A. C6S octasaccharide-PA, C6S decasaccharide-PA and longer C6S oligosaccharide-PA, which are transglycosylation products were observed. When metal cations were added in this reaction mixture none of the cations examined here enhanced the transglycosylation activity of BTH, instead some of them inhibited the reaction and the degree of inhibition depended on the concentration of cation
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used (data not shown). The results obtained when 10 mM of the different cation was added to the transglycosylation reaction mixture are shown in Fig. 1B. 3.2. Effect of cations bound to GAG on the transglycosylation reaction We then decided to examine the effect of cations on the transglycosylation by using the metal salts of glycosaminoglycans as donors. GAGs (HA, Der, Ch, C4S, C6S) bound with cations (Mn2+, Ba2+, Zn2+, Cu2+) were prepared for use as donors of transglycosylation. The binding efficiency of cations to GAG is shown in Fig. 2. No significant difference in binding efficiency of divalent metal ions to HA was observed. In contrast, there was significant difference in chondroitin sulfates. In the cases of C4S and C6S, which are sulfated GAGs at C4 or C6 of N-acetylgalactosamine, respectively significant binding to divalent cations was observed and the molar ratio of Ba2+ ions bound per uronic acid-N-acetylhexosamine disaccharide unit was the highest of all the metals tested. With C6S, the number of bound cations per disaccharide unit was estimated to be 0.075 for Mn2+, 1.231 for Ba2+, 0.144 for Zn2+, 0.395 for Cu2+. The binding efficiency for C6S was higher than for C4S. These results suggest that divalent cations bind to sulfate groups in addition to other anionic groups and the total binding efficiency is higher at C6 than at C4. When the various metals bound to GAGs were tested as donors it was found that barium bound to CS significantly increased the transglycosylation (Fig. 3). The transglycosylation efficiency of barium salt was increased 1.22-fold for Ch, 1.45-fold for C4S, and 1.46fold for C6S compared to that of sodium salt, the increasing rate is considerable because the transglycosylation efficiency of sodium salt of C6S is much lower than HA. Thus, the barium salt of C6S is a good donor as HA although sodium salt of C6S is a poor donor. A significant increase in transglycosylation was observed when the zinc salt of Ch was used whereas, the zinc salts of C4S and C6S markedly decreased transglycosylation (Fig. 3). When HA, which even though treated with metal cations had almost the same amount of bound metal regardless of the kind of metal, was used as the donor, there was inhibition in the transglycosylation efficiency for Cu and no changes in the others. 3.3. Recovery from inhibition by Zn in transglycosylation In order to examine whether the effect of metals on the degree of the transglycosylation was due to the binding of the cations to
Fig. 1. Effect of adding metal cations to the reaction mixture on the transglycosylation reaction. Two nanomoles of chondroitin 6-sulfate hexasaccharide-PA as an acceptor and 50 lg of chondroitin 6-sulfate (Na-form) as a donor were incubated with 1.0 NFU of bovine testicular hyaluronidase in 0.1 M Tris–HCl buffer, pH 7.0 in the absence (none) or presence of 10 mM of various salt solutions (NaCl, MnCl2, BaCl2, ZnCl2, CuCl2) at 37 °C for 1 h. Reaction products were analysed by HPLC on a TSKgel Amide-80 column by monitoring of fluorescence of PA. (A) HPLC of the transglycosylation products in the absence of metal. (a) Before the reaction; (b) after the reaction. The broken line indicates the gradient curve. Arrows indicate the elution positions of standard PA chondroitin 6-sulfate oligosaccharides (6, hexasaccharide-PA; 8, octasaccharide-PA; 10, decasaccharide-PA; 12, dodecasaccharidePA; 14, tetradecasaccharide-PA). (B) Transglycosylation efficiency in the presence and absence of metals.
Fig. 2. Binding efficiency of divalent cations to chondroitin sulfates. Molar ratios of metallic atoms (Mn, Ba, Zn, Cu) to the hexosamine (GalN or GlcN) content of hyaluronan (A), desulfated dermatan sulfate (B), desulfated chondroitin sulfate (C), chondroitin 4-sulfate (D), or chondroitin 6-sulfate (E) bound were estimated. Metal content was determined by atomic absorption method, and hexosamine content by monosaccharide analysis using the HPAEC-PAD after acidic hydrolysis.
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Fig. 3. Effect of divalent cations bound with donor GAGs on transglycosylation. Transglycosylation reaction was performed using chondroitin 6-sulfate hexasaccharide-PA as an acceptor and hyaluronan (A), desulfated dermatan sulfate (B), desulfated chondroitin sulfate (C), chondroitin 4-sulfate (D), or chondroitin 6-sulfate (E) bound with divalent cations (Mn, Ba, Zn, and Cu) as donors. Transglycosylation efficiency was estimated based on the decrease rate of acceptor.
the donor glycosaminoglycans, ethylenediamine tetra-acetic acid (EDTA) was added to the reaction mixture. Addition of EDTA (5–20 mM) to the reaction mixture containing BTH and C6S hexasaccharide-PA as the acceptor and C6S (Zn-form) as the donor, this was chosen since the change in the case of Zn was the most marked, reversed the strong inhibitory effect of Zn (Fig. 4).
Fig. 4. Effect of EDTA on the inhibition of transglycosylation when chondroitin 6sulfate (Zn-form) was used as a donor. EDTA was added to the reaction mixture of transglycosylation containing chondroitin 6-sulfate hexasaccharide-PA as an acceptor and chondroitin 6-sulfate (Zn-form) as donor. Reaction products were analysed by HPLC on a PALPAK Type-S column by monitoring of fluorescence of PA. The broken line indicates the gradient curve. Arrows indicate elution positions of chondroitin 6-sulfate oligosaccharide-PA (6, hexasaccharide-PA; 8, octasaccharidePA; 10, decasaccharide-PA; 12, dodecasaccharide-PA; 14, tetradecasaccharide-PA). (a) Chondroitin 6-sulfate (Zn-form), EDTA ( ), (b) chondroitin 6-sulfate (Zn-form), 5 mM EDTA, (c) chondroitin 6-sulfate (Zn-form), 20 mM EDTA, and (d) commercially available chondroitin 6-sulfate (Na-form), EDTA ( ).
4. Discussion It has been reported that cations play an important role in hydrolysis of HA by hyaluronidase [22,36]. Sodium chloride is commonly used to facilitate the hydrolytic activity of testicular hyaluronidase. Further, most divalent cations except Cu facilitate hydrolysis reaction of this enzyme [22]. In this study, we have demonstrated that divalent cations could also control transglycosylation activity of testicular hyaluronidase. We have shown that cations could either facilitate or inhibit transglycosylation depending on the kind of metal ion and donor GAG used. It is suggested that the effect of cations on the hydrolytic activity of BTH on HA is due to the influence of the cations on the substrate rather than the enzyme [26]. Our results show that inclusion of cations in the reaction mixture had no influence of the transglycosylation activity of BTH. However, the activity was affected when cation bound to C6S was used as the donor. This suggests that cations bound to the GAG apparently alter its 3dimensional structure and thereby facilitate the activity of hyaluronidase on the polysaccharide. In fact, it has been reported that binding of certain cation change the 3-dimensional structure of GAG [37]. It is also known that carboxylate groups on GlcUA of HA show a chelation effect at pH 6 [38] and as a result the carboxylate groups may affect hydrolysis activity of BTH by binding with cations. Further, in CS species functional groups contributing to the binding of cations are carboxylate groups in neutral and acidic aqueous [39]. In the present study, effect of cations on transglycosylation by BTH was investigated using five different GAGs as donors and the effects differed depending on the kind of GAG. The binding efficiency of HA was similar among metals but differed significantly in CS. Der differs from CS in that it has iduronic acid in addition to GlcUA in the repeating disaccharide units of the polysaccharide chains. It is known that GAGs having iduronic acids are structurally more flexible than those having only GlcUA and are therefore capable of associating strongly with cations [40]. Accordingly, an explanation can be provided that the differences in the effect of cations on the transglycosylation behavior of Der and CSs are due to the differences in the 3-dimensional conformation of the carboxylate groups on uronic acids. From the relationships between the metal binding and transglycosylation efficiency, it can be deduced that
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sulfate groups are also important for metal binding. Especially, the association between this bulky group and Ba, whose ion radius is relatively big, can bring changes in 3-dimensional conformation of GAGs. The radii of the metal ions tested in this study are as follows: Na+, 0.97, Mn2+, 0.80, Ba2+, 1.34, Zn2+, 0.74, Cu2+, 0.72. In the reaction of hyaluronidase, hydrolysis and transglycosylation occurs as an equilibrium reaction. First we predicted that the effects of metal on transglycosylation correlate with that on hydrolysis. However, it was shown that there is less correlation between the effects on the two reactions: both barium salts and zinc salts of CSs up-regulated hydrolysis compared to their sodium salts when they were used as substrates (data not shown), in contrast, barium salts enhanced while zinc salt inhibited transglycosylation. CSs, which are the sugar moieties of CS–proteoglycans, and HA coexist in extracellular matrices or surfaces of cells. In vitro transglycosylation using BTH synthesises hybrid sugar chains of these different glycosaminoglycans [41]. It has not yet been investigated whether hybrid sugar chains consisting of HA and CS is actually generated in living body by the transglycosylation. It is also not known whether artificial HA/CS hybrid oligosaccharides have significant biological activity. However, cations should be paid attention to as control factors of transglycosylation both in vitro and in vivo. Our glycotechnological approach using the systematic transglycosylation of BTH has made it possible to custom-synthesize hybrid GAGs among HA, Ch (desulfated CS), C4S, C6S, Der (desulfated DS) [13,16–21]. However, the yields were low when sulfated substrates such as C4S, C6S, were used in transglycosylation reaction. The present study helps to explain the reason. Our findings also provide an approach to control transglycosylation activity of hyaluronidase by reversible modification of the GAG donor with metals and thereby increase yields. Acknowledgments This work was supported by Grants-in Aid (Nos. 15370041, 17770106, and 19570119) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, by Grants for Priority Research Designated by the President of Hirosaki University, and by the Fund for Cooperation for Innovative Technology and Advanced Research in Evolution Area (CITY AREA). We would like to thank Prof. V.P. Bhavanandan for his interest and editorial help with the manuscript. References [1] L. Rodén, Structure and metabolism of connective tissue proteoglycans, in: W.J. Lannarz (Ed.), The Biochemistry of Glycoproteins and Proteoglycans, Plenum Press, New York, 1980, pp. 267–371. [2] L. Kjellen, U. Lindahl, Proteoglycans: structures and interactions, Annu. Rev. Biochem. 60 (1991) 443–475. [3] C. Malavaki, S. Mizumoto, N. Karamanos, K. Sugahara, Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease, Connect. Tissue Res. 49 (2008) 133–139. [4] U. Lindahl, G. Backstrom, L. Thunberg, I.G. Leder, Evidence for a 3-O-sulfated Dglucosamine residue in the antithrombin-binding sequence of heparin, Proc. Natl. Acad. Sci. USA 77 (1980) 6551–6555. [5] R.N. Achur, I. Kakizaki, S. Goel, K. Kojima, S.V. Madhunapantula, A. Goyal, M. Ohta, S. Kumar, K. Takagaki, D.C. Gowda, Structural interactions in chondroitin 4-sulfate mediated adherence of Plasmodium falciparum infected erythrocytes in human placenta during pregnancy-associated malaria, Biochemistry 47 (2008) 12635–12643. [6] H. Habuchi, S. Suzuki, T. Saito, T. Tamura, T. Harada, K. Yoshida, K. Kimata, Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor, Biochem. J. 285 (Pt 3) (1992) 805–813. [7] K. Meyer, Hyaluronidases, in: P.D. Boyer, E.G. Krebs (Eds.), The Enzymes, Academic Press, New York, 1971, pp. 307–320. [8] K. Takagaki, I. Kakizaki, Degradation of glycosaminoglycans, in: J.P. Kamerling, G.J. Boons, Y.C. Lee, A. Suzuki, N. Taniguchi, A.G.J. Voragen (Eds.), Comprehensive Glycoscience – From Chemistry to Systems Biology, Elsevier Science B.V., Amsterdam, The Netherlands, 2007, pp. 171–192.
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