Báo cáo khoa học: Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase docx

Mechanism for the hydrolysis of hyaluronan oligosaccharides by bovine testicular hyaluronidase Ikuko Kakizaki*, Nobuyuki Ibori*, Kaoru Kojima, Masanori Yamaguchi and Masahiko Endo Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, Japan Introduction Bovine testicular hyaluronidase (BTH) (hyaluronoglu- cosaminidase; EC 3.2.1.35) is an endo-b-N-acetyl-d-hex- osaminidase that hydrolyzes hyaluronan (HA) at the b1,4-N-acetylglucosaminide bonds [N-acetylglu- cosamine (GlcNAc)b-(1 fi 4)-glucuronic acid (GlcUA)] [1–4]. In addition, the enzyme also hydrolyzes chondroi- tin sulfates at the b-1,4-N-acetylgalactosaminide bonds, N-acetylgalactosamine (GalNAc)b-(1 fi 4)-GlcUA, but at a lower efficiency, which is dependent on the structure of the chondroitin sulfate [1,5]. The wide substrate speci- ficity of BTH is invaluable for glycotechnological appli- cations, such as the preparation of glycosaminoglycan oligosaccharides of varying chain lengths. Exhaustive digestion with this enzyme yields mainly a mixture of tetrasaccharides and hexasaccharides with GlcUA at the nonreducing end [6]. Hyaluronidases simultaneously display both hydrolytic and transglycosylation activities [7–10]. Indeed, transglycosylation progresses even under Keywords hyaluronan; hydrolysis; oligosaccharide; testicular hyaluronidase Correspondence I. Kakizaki, 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 Tel: +81 172 39 5015 E-mail: kaki@cc.hirosaki-u.ac.jp *These authors contributed equally to this work (Received 18 November 2009, revised 14 January 2010, accepted 29 January 2010) doi:10.1111/j.1742-4658.2010.07600.x Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. The products of the hydrolysis were analyzed by HPLC and ion-spray mass spectroscopy (MS). It was confirmed that the minimum substrate for bovine testicular hyaluronidase is the hyaluronan hexasaccharide, even though it is a poor substrate that is barely cleaved, even on prolonged incubation. When hyal- uronan octasaccharide was the substrate, increasing amounts of tetrasaccha- ride and hexasaccharide were produced with increasing time of incubation. Whereas disaccharide was not detectable in the reaction mixture by HPLC, MS analysis revealed trace amounts. The data suggest that the enzyme gen- erates a disaccharide intermediate from hyaluronan oligosaccharide, the majority of which is transferred to the nonreducing ends of other oligosac- charides, only traces being released as free disaccharide. When hyaluronan octasaccharide, with an unsaturated glucuronic acid at the nonreducing end, was used as a substrate, only a tetrasaccharide was detected by HPLC. How- ever, MS showed that the product was a mixture of equal amounts of two tetrasaccharides, one with and the other without the unsaturated glucuronic acid. This suggests that, in the case of substrates with a double bond at the nonreducing end, a tetrasaccharide is cleaved off instead of a disaccharide. The results of the experiments with pyridylaminated oligosaccharides were entirely consistent with these conclusions, and in addition showed the impor- tance of the reducing end of the substrate for the enzyme to recognize the length of the saccharide. Abbreviations BTH, bovine testicular hyaluronidase; GlcUA, glucuronic acid; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; HA, hyaluronan; PA, 2-pyridylamine. 1776 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS conditions that are optimal for hydrolysis. The optimal conditions for the hydrolysis of HA by commercial BTH are pH 4.0 and the presence of NaCl, whereas for transglycosylation they are pH 7.0 and the absence of NaCl [9,11]. The mechanism by which BTH degrades HA is complex, as the enzyme simultaneously catalyzes deg- radation and elongation of the substrate by hydrolysis and transglycosylation, respectively. Because the oligo- saccharides can act as sequential substrates for both reactions, it is difficult to identify the reaction products and establish how they are generated. The mechanism and kinetics of the hydrolysis by hyaluronidases have been previously investigated by different methods, including colorimetric reactions, capillary zone electro- phoresis, and ion-pair HPLC [6,7,12–15]. In a previous study, we analyzed the products of hydrolysis of HA oligosaccharides of known sizes by BTH using ion- spray MS [6]. In the present study, the mechanism of hydrolysis of pure synthetic oligosaccharide substrates labeled at the reducing and ⁄ or nonreducing ends by BTH was investigated. Results Products of hydrolysis by BTH of high-M r HA Previous studies showed that the optimal conditions for the hydrolysis of HA by BTH are pH 4.0–5.0 in the presence of 150 mm NaCl at 37 °C [9]. The incubation time is dependent on the amounts of substrate and enzyme used. The present study was undertaken to clarify the mechanism of hydrolysis of high-M r HA by BTH and to establish the optimal conditions for the preparation of specific oligosaccharides. In these stud- ies, we used HPLC, on a polyamine II column, to examine the reaction products generated by BTH with varying pH, temperature, and time of incubation, which were far from optimal. To find the characteristic products, at the initial stage of the hydrolysis we trea- ted high-M r HA with different amounts of BTH at pH 4.0 in the presence of 150 mm NaCl for various incubation times (5 min to 48 h) and temperatures (4–50 °C). However, no HA oligosaccharide of a defined size was observed under these conditions. As examples, HPLC elution profiles of the reaction products obtained when the incubation was performed at 4 °C and 37 °C are shown in Fig. 1. It can be seen that when the reaction was performed at 4 °C for only 5 min and with a small amount of enzyme, multiple peaks of similar peak area corresponding to 4-mer to 80-mer HA oligosaccharides were generated (Fig. 1B). When the reaction temperature was increased from 4 °Cto37°C, the peak sizes increased, but the overall size distribution of reaction products remained unchanged (Fig. 1C). The distribution of reaction products and the peak sizes were essentially the same at 37 °C, 42 °C, and 50 °C (data not shown). Increas- ing the amount of enzyme or the incubation time shifted the reaction products to lower-M r species, A B C Fig. 1. Effect of reaction temperature on the hydrolysis of HA by BTH. One milligram of HA (M r = 80 000) was incubated with 0.2 mg of BTH in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at 4 °C (B) or 37 °C (C) for 5 min. One milligram of HA was incubated with 0.2 mg of heat-inactivated BTH at 100 °C for 10 min as the substrate control (A). The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The column was eluted with a linear gradient of 100–246 m M NaH 2 PO 4 at a flow rate of 1.0 mLÆmin )1 over a period of 120 min. Oligosaccharides were detected by UV absorbance at 215 nm. Arrows indicate the elution positions of standard HA oligosaccharides of known chain lengths. I. Kakizaki et al. Hydrolytic mechanism of testicular hyaluronidase FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS 1777 finally yielding a mixture of tetrasaccharides, hexasac- charides, and octasaccharides. We also tested the effect of varying the pH (from 2 to 9 in increments of 0.5) in buffers containing 150 mm NaCl by incubating at 37 °C for 3 h. Again, there was no evidence for the production of a specific product; instead, the reaction mixture consisted of oligosaccharides of various chain lengths (data not shown). Thus, unfortunately, no characteristic peak corresponding to a specific HA oli- gosaccharide was observed under any of the reaction conditions tested. Products of hydrolysis by BTH of 2-pyridylamine (PA)–HA eicosasaccharides We then investigated the mechanism of hydrolysis by BTH, using a substrate of defined chain length and direction. For this purpose, HA eicosasaccharide was fluorescently labeled by attaching PA to the GlcNAc at the reducing end of the saccharide chain. Two micrograms of PA–HA eicosasaccharide was incubated with various amounts of BTH (0.05–5 lg) for different reaction times (5 min to 16 h). The reaction products were analyzed by HPLC to determine their size. Repre- sentative chromatograms are shown in Fig. 2. Peaks corresponding to PA–HA oligosaccharides, ranging from tetrasaccharides to octadecasaccharides, were detected when the substrate was incubated with a small amount of BTH and for a short time (Fig. 2C). How- ever, none of the peaks was prominent, suggesting the absence of a specific oligosaccharide product. The small peak detected at 65 min (Fig. 2B) was apparently not HA octadecasaccharide, because it was also detected in the substrate control consisting of boiled PA–HA eicosasaccharide without enzyme (data not shown). Increasing the amount of BTH or extending the reaction time resulted in a shift indicating the pro- duction of lower-M r species. In particular, hexasaccha- ride and octasaccharide were the predominant products after 16 h of incubation. In the reaction catalyzed by hyaluronidase, transgly- cosylation occurs in equilibrium with the hydrolysis, even under optimal conditions for hydrolysis. In addi- tion, the products become sequential substrates for both reactions. This complicates the interpretation of our results obtained using PA–HA eicosasaccharide as the substrate. Products of hydrolysis by BTH of HA oligosaccharides We also used lower-M r HA oligosaccharides (tetrasac- charide to decasaccharide) as substrates for hydrolysis. A B C D E F G H Fig. 2. HPLC analysis of the products of hydrolysis of PA-HA eico- sasaccharide by BTH. Two micrograms of PA-HA eicosasaccharide was incubated in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 m M NaCl at 37 °C with varying amounts of BTH and for different times as indicated: (B) 0.05 lg of BTH, 5 min incuba- tion; (C) 0.5 lg of BTH, 5 min incubation; (D) 5 lg of BTH, 5 min incubation; (E) 5 lg of BTH, 10 min incubation; (F) 5 lg of BTH, 30 min incubation; (G) 5 lg of BTH, 1 h incubation; (H) 5 lgof BTH, 16 h incubation. Two micrograms of PA-HA eicosasaccharide was incubated with 5 lg of heat-denatured bovine testicular hyaluronidase for 5 min as the substrate control (A). The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The column was eluted with a linear gradient of 50–246 m M NaH 2 PO 4 at a flow rate of 1.0 mLÆmin )1 over a period of 120 min. Oligosaccharides were detected by fluorimetry at exci- tation and emission wavelengths of 320 and 400 nm, respectively. Arrows indicate the elution positions of standard PA-HA oligosaccharides of known chain lengths. Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al. 1778 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS These oligosaccharides, prepared by partial digestion of HA with BTH, have a saturated GlcUA at the non- reducing end and GlcNAc without any label at the reducing end. Fifty micrograms of each of the above oligosaccharides was incubated with 5 lg of BTH under optimal conditions for hydrolysis at 37 °C for 1, 3 or 24 h, and the reaction mixture was analyzed by HPLC using a polyamine II column. The results illus- trated in Fig. 3 show the time sequence changes in the relative peak area of each HA oligosaccharide. The ratios of oligosaccharide after 24 h of incubation with each substrate, as assessed by peak area from the HPLC traces, are shown in Table 1. The reaction mix- tures were also analyzed by ion-spray MS. It is clear that when tetrasaccharide or hexasaccharide was used as substrate, no degradation product was detected by HPLC, even after 24 h of incubation (Fig. 3A,B). The failure to detect degradation products of the hexasac- charide by HPLC was reproducible, and therefore appeared to contradict our previous report [6]. How- ever, the more sensitive MS analysis of the products revealed trace amounts of molecular ions correspond- ing to the disaccharide (at m ⁄ z 395.0) and the tetrasac- charide (at m ⁄ z 775.0) (Fig. 4). This observation is consistent with the slight decrease in the amount of hexasaccharide seen in Fig. 3B. When the octasaccharide was used as the substrate, peaks of tetrasaccharide, hexasaccharide and octasac- Fig. 3. Time course of hydrolysis of HA oligosaccharides by BTH. Fifty micrograms of saturated HA tetrasaccharide (A), hexasaccha- ride (B), octasaccharide (C) or decasaccharide (D) was incubated with 5 lg of BTH in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 m M NaCl at 37 °C for 1, 3 or 24 h. The reaction products at each time point were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The column was eluted with a linear gradient of 50–61.5 m M NaH 2 PO 4 at a flow rate of 1.0 mLÆmin )1 over a period of 30 min. Oligosaccharides were detected by UV absorbance at 215 nm. Relative peak areas corre- sponding to different oligosaccharides are plotted. The peak area for the initial oligosaccharide used as a substrate was defined as 100%. Symbols: filled circles, tetrasaccharide; open circles, hexa- saccharide; filled squares, octasaccharide; open squares, decasac- charide; open triangles, dodecasaccharide. Table 1. Reaction products of HA oligosaccharides after incubation with testicular hyaluronidase for 24 h. Fifty micrograms of saturated HA oligosaccharide was incubated with 5 lg of BTH in 100 lLof 0.1 M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at 37 °C for 24 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The flow rate was set at 1.0 mLÆmin )1 , and elution was performed over 30 min using a lin- ear gradient of 50–61.5 m M NaH 2 PO 4 . Oligosaccharides were detected by UV absorbance at 215 nm. The percentages shown are the relative peak areas of the oligosaccharides when the total peak area of all of the reaction products is defined as 100%. Substrate HA oligosaccharide Reaction products (%) (GlcUA-GlcNAc) 2 (GlcUA-GlcNAc) 2 100 (GlcUA-GlcNAc) 3 (GlcUA-GlcNAc) 3 100 (GlcUA-GlcNAc) 4 (GlcUA-GlcNAc) 2 46.8 (GlcUA-GlcNAc) 3 32.7 (GlcUA-GlcNAc) 4 20.5 (GlcUA-GlcNAc) 5 (GlcUA-GlcNAc) 2 31.5 (GlcUA-GlcNAc) 3 48.3 (GlcUA-GlcNAc) 4 14.7 (GlcUA-GlcNAc) 5 5.50 Fig. 4. Mass spectrum of the HA oligosaccharides produced by the hydrolysis of HA hexasaccharide by BTH. Fifty micrograms of HA hexasaccharide was incubated with 5 lg of BTH in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at 37 °C for 24 h. The reaction products were analyzed by ion-spray MS. HA(2), HA disaccharide; HA(4), HA tetrasaccharide; HA(6), HA hexasaccharide. I. Kakizaki et al. Hydrolytic mechanism of testicular hyaluronidase FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS 1779 charide were detected in the reaction mixture. A longer reaction time decreased the amount of octasaccharide and increased the amounts of tetrasaccharide and hexasaccharide (Fig. 3C). A peak (1.8%) correspond- ing to the decasaccharide, generated by transglycosyla- tion, was detected after 1 h of incubation but disappeared after a longer incubation period. This result was supported by MS analysis (data not shown) and our previous report. When the decasaccharide was used as the substrate, peaks of tetrasaccharide, hexasaccharide, octasaccha- ride and decasaccharide were detected in the reaction mixture, and longer incubation periods decreased the amount of decasaccharide and increased the amounts of tetrasaccharide, hexasaccharide, and octasaccharide (Fig. 3D). After 24 h of incubation, hexasaccharide and tetrasaccharide were detected as the predominant products. A peak (1.4%) corresponding to dodecasac- charide, formed from transglycosylation, was detected after 1 h of incubation, but decreased at 3 h, and was undetectable at 24 h. No disaccharide was detected by HPLC with any of the above oligosaccharides as the substrate. Products of hydrolysis by BTH of HA oligosaccharides with unsaturated GlcUA at the nonreducing end In order to elucidate the rules that govern the hydrolysis mediated by BTH, HA oligosaccharides (tetrasaccha- ride to decasaccharide) with a double bond between C4 and C5 of GlcUA at the nonreducing end were used as substrates. These oligosaccharides were prepared by partial digestion with chondroitin ABC lyase, and purified by HPLC. Fifty micrograms of the unsaturated substrate was incubated with 5 lg of BTH under optimal conditions for hydrolysis for 1, 3 or 24 h, and the reaction mixture was analyzed by HPLC, using a polyamine II column. Because both unsaturated and saturated oligosaccharides are generated from the hydrolysis of unsaturated oligosaccharides, the reaction mixtures were monitored at 215 nm (for general detec- tion) and at 232 nm (specific for unsaturated bonds). The time sequence changes in the reaction mixtures when the unsaturated octasaccharide was used as the substrate are shown in Fig. 5A,B as an example. Plots for the relative peak areas of the HA oligosaccharides in the chromatograms are shown in Fig. 5C,D. The ratio of oligosaccharides in the reaction mixture after 24 h of incubation with each of the substrates, assessed by peak area from the HPLC traces, is shown in Table 2. No degradation products were detected when unsaturated tetrasaccharide or hexasaccharide was used as the substrate, even after 24 h of incubation (Fig. 5Ca,b). When the unsaturated octasaccharide was used as a substrate, an incubation-dependent increase in peak area corresponding to the tetrasaccharide was detected at both 215 nm and 232 nm. A trace amount of mate- rial corresponding to hexasaccharide was detected at 215 nm. The amount of octasaccharide (starting mate- rial) decreased with incubation time, indicating that it was unsaturated, which is consistent with our MS analysis (data not shown). When the unsaturated decasaccharide was used as the substrate, we observed a decrease in the starting material and an increase in tetrasaccharide and hexa- saccharide at both wavelengths. A trace amount of octasaccharide at 215 nm was detected after 1 h and 3 h of incubation, but not after 24 h of incubation (Fig. 5C,D). MS analysis confirmed that unsaturated tetrasaccha- ride and hexasaccharide are not hydrolyzed by BTH, in agreement with the results from HPLC. MS analysis also showed that hydrolysis of unsaturated octasaccha- ride by BTH mainly generated unsaturated and satu- rated tetrasaccharide, with a trace amount of the saturated hexasaccharide. This saturated hexasaccha- ride is presumably a product generated by the transgly- cosylation reaction using saturated oligosaccharides as both acceptor and donor. Although MS analysis is not a quantitative technique, the percentages of unsatu- rated and saturated tetrasaccharide produced from unsaturated octasaccharide were assessed by the mag- nitudes of the fragment ions as 40.8% and 35.8%, respectively (i.e. approximately equal amounts). The unsaturated decasaccharide, however, generated only unsaturated tetrasaccharide and saturated hexasaccha- ride. Products of hydrolysis by BTH of HA oligosaccharides blocked at the reducing ends by PA The reducing ends of saturated and unsaturated oligo- saccharides were pyridylaminated. This resulted in sat- urated oligosaccharides labeled at the reducing end and the unsaturated oligosaccharides having labels at both ends (PA at the reducing end and a double bond at the nonreducing end). Two micrograms of each PA oligosaccharide was incubated with 5 lg of BTH for 1 h under optimal conditions for hydrolysis, and the reaction mixture was analyzed by HPLC. The eluate was monitored by UV absorbance at 215 nm and 232 nm, in addition to fluorescence detection of PA. The ratios of the oligosaccharide products when each Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al. 1780 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS of the PA oligosaccharides was used as the substrate, as assessed by area of the peak in the HPLC trace, are shown in Tables 3 and 4. Saturated and unsaturated PA octasaccharides and smaller oligosaccharides were not degraded. PA oligosaccharides larger than octasac- charides were hydrolyzed, although the degradation pattern was different for saturated and unsaturated substrates. Discussion It has been proposed that the hydrolysis mechanism of BTH involves sequential cleavage of a disaccharide unit, GlcUA-GalNAc, from the nonreducing end of the substrate HA chain [6,9]. Indeed, our HPLC analy- sis of the products of hydrolysis of HA by BTH showed a series of oligosaccharides whose chain lengths differed by a disaccharide unit. However, this observation alone is insufficient proof for this hypothe- sis, because the action of BTH is complicated by several factors. First, free disaccharides are not observed in the HPLC analysis. Second, high-M r HA is quickly degraded to low- M r HA oligosaccharides, which, in turn, can act as substrates for subsequent reactions. Finally, the reaction of hyaluronidase is an equilibrium between hydrolysis and transglycosylation. In the present study, we prepared saturated and unsaturated oligosaccharides and their pyridylaminated derivatives with a high degree of purity, to investigate the hydrolysis reaction mediated by BTH. Our studies resulted in some novel findings, in addition to confirming the previously proposed mechanism for the hydrolysis of HA by BTH. AB C a b cd a b cd D Fig. 5. HPLC analysis of the products of hydrolysis of unsaturated HA oligosaccharides by BTH. Fifty micrograms of unsaturated tetra- saccharide (Ca and Da), hexasaccharide (Cb and Db), octasaccha- ride (A, B, Cc and Dc), or decasaccharide (Cd and Dd) was incubated with 5 lg of BTH in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 m M NaCl at 37 °C for 1, 3 or 24 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The column was eluted with a linear gradient of 50–61.5 m M NaH 2 PO 4 at a flow rate of 1.0 mLÆmin )1 over a period of 30 min. Oligosaccharides were detected by UV absorbance at both 215 nm (A, C) and 232 nm (B, D). The chromatogram shows the results obtained when unsaturated octasaccharide was used as a substrate (A, B). Arrows indicate the elution positions of standard unsaturated HA oligosaccharides numbered by sugar length. Relative peak areas corresponding to each oligosaccharide are plotted (C, D). In each case, the peak area for the initial oligosaccharide used as a substrate was defined as 100%. Symbols: filled circles, tetrasaccharide; open circles, hexa- saccharide; filled squares, octasaccharide; open squares, decasac- charide; open triangles, dodecasaccharide. I. Kakizaki et al. Hydrolytic mechanism of testicular hyaluronidase FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS 1781 The HA hexasaccharide was found to be the mini- mum substrate for BTH [6,7,12,16]. In contrast, a kinetic study using recombinant hyaluronidases had suggested that HA octasaccharide was the minimum substrate [14]. The analytical methods used to analyze the reaction mixtures in the above studies are different from each other and from those used in the present study. In this investigation, using a combination of HPLC and MS analyses, we confirmed our previous finding that hexasaccharide is the minimum substrate for BTH [6]. Oligosaccharides of low M r are more readily ionized than those of high M r . Consequently, MS data do not quantitatively reflect the ratio of oligosaccharides in the reaction mixture. Nevertheless, an approximate ratio can be deduced from the relative intensities of the detected ions. The ratio of oligo- saccharides in the reaction mixture after 24 h of incu- bation of saturated hexasaccharide was assessed by MS to be 91.3% hexasaccharide, 4.5% tetrasaccharide, and 4.2% disaccharide. These results suggest that hexasac- charide is quite resistant to degradation by BTH but is converted to disaccharide and tetrasaccharide on pro- longed incubation. Indeed, in our experiments, hexasac- charide was often more abundant than tetrasaccharide in the final reaction mixture of BTH-mediated hydroly- sis of high-M r HA. This finding is entirely consistent with these conclusions. In the case of the unsaturated HA oligosaccharides, octasaccharide is the minimum substrate for BTH, rather than the hexasaccharide. When saturated HA octasaccharide was used as the substrate for BTH, about 30% hexasaccharide was detected in the reaction mixture. By contrast, when unsaturated octasaccharide was used as the substrate, the product detected by Table 3. Reaction products of pyridylaminated HA oligosaccharides after incubation with testicular hyaluronidase for 1 h. Two micro- grams of saturated oligosaccharide was incubated with 5 lgof testicular hyaluronidase in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 m M NaCl at 37 °C for 1 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The flow rate was set at 1.0 mLÆmin )1 , and elution was performed over 30 min, using a linear gradient of 50–61.5 m M NaH 2 PO 4 . Oligosaccharides were detected by UV absorbance at 215 nm. The percentages shown are the relative peak areas of the oligosaccharides when the total peak area of all reaction products is defined as 100%. Substrate hyaluronen oligosaccharide Reaction products (%) (GlcUA-GlcNAc) 2 -PA (GlcUA-GlcNAc) 2 -PA 100 (GlcUA-GlcNAc) 3 -PA (GlcUA-GlcNAc) 3 -PA 100 (GlcUA-GlcNAc) 4 -PA (GlcUA-GlcNAc) 4 -PA 100 (GlcUA-GlcNAc) 5 -PA (GlcUA-GlcNAc) 3 -PA 42.9 (GlcUA-GlcNAc) 4 -PA 13.7 (GlcUA-GlcNAc) 5 -PA 18.3 (GlcUA-GlcNAc) 2 20.1 (GlcUA-GlcNAc) 3 5.00 (GlcUA-GlcNAc) 6 -PA (GlcUA-GlcNAc) 3 -PA 30.9 (GlcUA-GlcNAc) 4 -PA 27.1 (GlcUA-GlcNAc) 5 -PA 3.30 (GlcUA-GlcNAc) 6 -PA 1.50 (GlcUA-GlcNAc) 2 17.9 (GlcUA-GlcNAc) 3 16.3 (GlcUA-GlcNAc) 4 3.00 Table 2. Reaction products of unsaturated HA oligosaccharides after incubation with testicular hyaluronidase for 24 h. Fifty micrograms of unsaturated oligosaccharide was incubated with 5 lg of BTH in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 mM NaCl at 37 °C for 24 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The flow rate was set at 1.0 mLÆ- min )1 , and the elution was performed over 30 min using a linear gradient of 50–61.5 mM NaH 2 PO 4 . Oligosaccharides were detected by UV absorbance at both 215 nm and 232 nm. The percentages shown are the relative peak areas of the oligosaccharides when the total peak area of all reaction products is defined as 100%. D 4 GlcUA-GlcNAc-GlcUA-GlcNAc, D 4 -unsaturated HA tetrasaccharide; D 4 GlcUA-GlcNAc- (GlcUA-GlcNAc) 2 , D 4 -unsaturated HA hexasaccharide; D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 3 , D 4 -unsaturated HA octasaccharide; D 4 GlcUA-Glc- NAc-(GlcUA-GlcNAc) 4 , D 4 -unsaturated HA decasaccharide. Substrate hyaluronen oligosaccharide Reaction products (%) At 215 nm At 232 nm D 4 GlcUA-GlcNAc-GlcUA-GlcNAc Tetrasaccharide 100 Tetrasaccharide 100 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 2 Hexasaccharide 100 Hexasaccharide 100 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 3 Tetrasaccharide 74.3 Tetrasaccharide 76.6 Hexasaccharide 3.00 Hexasaccharide 0.70 Octasaccharide 22.7 Octasaccharide 22.7 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 4 Tetrasaccharide 65.3 Tetrasaccharide 86.6 Hexasaccharide 27.3 Hexasaccharide 6.00 Octasaccharide 0 a Octasaccharide 0 Decasaccharide 7.40 Decasaccharide 7.40 a The peak of HA octasaccharide was observed after 1 h and 3 h of incubation, but disappeared after 24 h of incubation (see Fig. 3). Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al. 1782 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS HPLC was almost exclusively tetrasaccharide. In MS analysis, unsaturated and saturated tetrasaccharides can be distinguished by the mass of their corresponding deprotonated molecular ions (m ⁄ z 757.0 and m ⁄ z 775.0, respectively). MS analysis of the products of hydrolysis of the unsaturated octasaccharide by BTH showed an approximate 50 : 50 ratio of unsaturated and saturated tetrasaccharide. This suggests that if the substrate has an unsaturated bond at the nonreducing end, BTH rec- ognizes the second rather than the first N-acetylglucos- aminide bond from the nonreducing end. Therefore, only unsaturated tetrasaccharides are generated, and not unsaturated disaccharides. Furthermore, our data suggest that the unsaturated tetrasaccharide thus gener- ated can be transferred to the nonreducing ends of other saturated oligosaccharides. However, the result- ing unsaturated oligosaccharide does not become the acceptor for further transglycosylation, because it now has an unsaturated GlcUA at the nonreducing end. Our experiments with unsaturated HA oligosaccha- rides as substrates suggest a general rule for BTH-med- iated hydrolysis; namely, BTH recognizes the first N-acetylglucosaminide bond from the nonreducing end of saturated hexasaccharides or larger oligosaccharides, and cleaves a saturated disaccharide, GlcUA-GalNAc. However, in the case of an unsaturated octasaccharide or larger oligosaccharide, BTH recognizes only the second N-acetylglucosaminide bond. Therefore, an unsaturated tetrasaccharide (D 4 GlcUA-GlcNAc- GlcUA-GlcNAc), instead of a saturated disaccharide (GlcUA-GalNAc), is liberated. A schematic diagram of the enzymatic reaction when octasaccharide with or without unsaturated GlcUA (D 4 GlcUA) was used as a substrate is shown in Fig. 6A,B. The saturated disac- charide cleaved from the nonreducing end of a satu- rated oligosaccharide is immediately transferred to the nonreducing end of another saturated oligosaccharide (the acceptor) in the active site of BTH. The saturated disaccharide is thought to be converted to an interme- diate in the active site of BTH before it is transferred to the hydroxyl group at C4 of the GlcUA at the non- reducing end of the acceptor [17]. If the acceptor mole- cule is water instead of an oligosaccharide, then disaccharide is released into the reaction mixture. Indeed, saturated disaccharide is detected in trace amounts by MS analysis. It appears that any free satu- rated disaccharide produced is immediately consumed as an acceptor for the disaccharide intermediate held in the active site of BTH, thereby generating the satu- rated tetrasaccharide. This could explain why only trace amounts of saturated disaccharides are detectable in the reaction mixture. The above findings suggest that the initial products of hydrolysis of HA octasaccharide are a disaccharide and a hexasaccharide. The disaccharide, retained via its intermediate form in the active site of BTH, is immediately transferred to a different disaccharide to generate a tetrasaccharide by the transglycosylation activity of BTH. Alternatively, it is immediately trans- ferred to other oligosaccharides in the reaction mix- ture, thereby elongating the acceptor oligosaccharides by a disaccharide unit. Thus, tetrasaccharide and deca- saccharide are generated (mostly as transglycosylation products) in addition to hexasaccharide (mostly an initial hydrolysis product). Any octasaccharides still found in the reaction mixture are unhydrolyzed Table 4. Reaction products of pyridylaminated unsaturated HA oligosaccharides after incubation with testicular hyaluronidase for 1 h. Two micrograms of saturated oligosaccharide was incubated with 5 lg of testicular hyaluronidase in 100 lL of 0.1 M sodium acetate buffer (pH 4.0) containing 150 m M NaCl at 37 °C for 1 h. The reaction products were analyzed by HPLC on a polyamine II column (4.6 · 250 mm). The flow rate was set at 1.0 mLÆmin )1 , and elution was performed over 30 min, using a linear gradient of 50–61.5 mM NaH 2 PO 4 . Oligosac- charides were detected by UV absorbance at 215 nm. The percentages shown are the relative peak areas of the oligosaccharides when the total peak area of all the reaction products is defined as 100%. Substrate hyaluronen oligosaccharide Reaction products (%) D 4 GlcUA-GlcNAc-GlcUA-GlcNAc-PA D 4 GlcUA-GlcNAc-GlcUA-GlcNAc-PA 100 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 2 -PA D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 2 -PA 100 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 3 -PA D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 3 -PA 100 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 4 -PA D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 4 -PA 15.6 (GlcUA-GlcNAc) 3 -PA 43.6 D 4 GlcUA-GlcNAc-GlcUA-GlcNAc 40.8 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 5 -PA D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 5 -PA 5.5 (GlcUA-GlcNAc) 4 -PA 44.7 (GlcUA-GlcNAc) 5 -PA 23.3 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 2 9.8 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 3 12.2 D 4 GlcUA-GlcNAc-(GlcUA-GlcNAc) 4 4.5 I. Kakizaki et al. Hydrolytic mechanism of testicular hyaluronidase FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS 1783 substrates. Similarly, series of oligosaccharides elon- gated by a disaccharide unit are generated from satu- rated oligosaccharides larger than octasaccharides. The results of our experiments with PA oligosaccha- rides suggest that the reducing end of the substrate is also important for recognition of the chain length by the enzyme, although the cleavage occurs only from the nonreducing end. Our results also suggest that BTH does not recognize the length of the saccharide chain if the monosaccharide at the reducing end is replaced by another nonsugar molecule such as PA. The complex reaction catalyzed by BTH appears to be repeated in an extremely rapid sequence between hydrolysis and transglycosylation. Thus, the reaction mechanism for HA, a high-M r substrate, is likely to be chaotic. Nevertheless, using highly purified oligosac- charides appropriately modified at the reducing or nonreducing ends, we have been able to elucidate the sites in the oligosaccharide substrates that are recog- nized by the enzyme. Experimental procedures Materials HA (from Streptococcus zooepidemicus, average M r of 80 000) was purchased from Kibun Food Chemifa Co., Ltd (Tokyo, Japan). BTH (type 1-S) was from Sigma-Aldrich (St Louis, MO, USA), and chondroitin ABC lyase was from Seikagaku Kogyo Co. (Tokyo, Japan). Other reagents were of analytical grade and obtained from commercial sources. Preparation of HA oligosaccharides Saturated HA oligosaccharides were prepared by partial digestion with BTH as follows. One gram of HA was incu- bated with 200 mg of BTH in 100 mL of 0.1 m sodium acetate buffer (pH 4.0) containing 150 mm NaCl at 37 °C for 3 h, and the reaction was stopped by boiling for 10–15 min. The mixture was then clarified by centrifugation for 10 min at 10 000 g at 4 °C, concentrated to about 20 mL, and desalted on a Sephadex G-25 column equili- brated with distilled water. Fractions determined to be posi- tive for uronic acid using the carbazole sulfate method were pooled and concentrated to 20 mL. The resulting mix- ture of saturated HA oligosaccharides was fractionated by perfusion chromatography performed on a Perceptive Bio- systems Bio Cad Perfusion Chromatography Workstation (GMI, Inc., Minneapolis, MN, USA). The sample was applied to a Q Sepharose high-performance column (dimen- sions, 50 · 100 mm; Pharmacia, Uppsala, Sweden), which was pre-equilibrated with 20 mm acetic acid (pH 6.5). The oligosaccharides were eluted, over a 100 min period, using a linear gradient of 0 m to either 0.1 m, 0.2 m or 0.5 m NaCl at a flow rate of 2 mLÆmin )1 . The eluate was monitored by UV absorbance at 215 nm. The uronic acid A B Fig. 6. Schematic diagram of the action of BTH on HA octasaccharide with or without an unsaturated GlcUA at the nonreducing end. (A) BTH recognizes the first N-acetylglucosaminide bond and cleaves it. The disaccharide, GlcUA-GlcNAc, is retained in the active site, and the hexasaccharide is released into the reaction mixture. The disaccharide in the active site of the enzyme is immediately transferred to the non- reducing end of other oligosaccharides (acceptors) in the reaction mixture. (B) BTH recognizes the second N-acetylglucosaminide bond and cleaves it to produce two tetrasaccharides, one with and one without an unsaturated bond. Unsaturated disaccharide is not generated. Some of the unsaturated HA tetrasaccharides become donors and can be transferred to the nonreducing ends of other oligosaccharides (acceptors) in the reaction mixture. Hydrolytic mechanism of testicular hyaluronidase I. Kakizaki et al. 1784 FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS content of specific fractions was determined with the carbazole sulfate method. The fractions were pooled according to the elution profiles, desalted, concentrated, and subjected to ion-spray MS to identify the peaks as sat- urated oligosaccharides (tetrasaccharide, hexasaccharide, octasaccharide, decasaccharide, or dodecasaccharide) with GlcUA at the nonreducing ends. Instead of preparing chemically labeled oligosaccharides at the nonreducing ends, we prepared HA oligosaccharides with an unsaturated GlcUA at the nonreducing end for sub- strates of BTH. HA oligosaccharides, with an unsaturated double bond between C4 and C5 of the GlcUA nonreducing end, were prepared by partial digestion with chondroitin ABC lyase [18,19]. Two hundred milligrams of HA was incubated with 1.5 U (0.5 U per 24 h) of chondroitin ABC lyase in 12 mL of 33.3 mm Tris ⁄ HCl buffer (pH 8.0) con- taining 33.3 mm sodium acetate at 37 °C for 72 h, and the reaction was stopped by boiling for 5 min. The reaction mix- ture was clarified by centrifugation for 10 min at 10 000 g at 4 °C and fractionated by HPLC, using a YMC-Pack poly- amine II column (10 · 250 mm; YMC Co., Tokyo, Japan). Fractions were pooled, desalted, concentrated, and subjected to ion-spray MS to identify the unsaturated oligosaccharides (disaccharide, tetrasaccharide, hexasaccharide, octasaccha- ride, decasaccharide, or dodecasaccharide). Pyridylamination of HA oligosaccharides Fluorolabeling of the reducing ends of saturated ⁄ unsatu- rated HA oligosaccharides with PA was performed by a modification of the method of Hase et al. [20], as described in our previous report [21]. In order to eliminate contami- nation of the substrates used with non-PA oligosaccharides, PA oligosaccharides were purified by HPLC, using a poly- amine II column. The individual PA oligosaccharides were collected, and their purity was verified by ion-spray MS; they were then used as substrates in the hydrolysis reaction of BTH. Hydrolysis reaction of BTH Each HA oligosaccharide (100–200 lg) or PA–HA oligosac- charide (2–10 lg) was incubated with a suitable amount of BTH in 0.1 m sodium acetate buffer (pH 4.0), containing 150 mm NaCl, at 37 °C for the indicated time. For pH experiments, the buffers were glycine ⁄ HCl for pH 2.0–3.0, citrate ⁄ NaOH for pH 3.5–4.0, acetate ⁄ NaOH for pH 4.5–5.5, Mes ⁄ NaOH for pH 6.0–6.5, phosphate ⁄ NaOH for pH 7.0–7.5, and Tris ⁄ HCl for pH 8.0–9.0. The amounts of substrate and enzyme varied, depending on the specific experiment. The reaction was stopped by boiling for 5 min, and the reaction mixture was clarified by centrifugation for 5 min at 8000 g at 4 °C and filtered. The filtrate was then analyzed by HPLC, using a polyamine II column, and ⁄ or by ion-spray MS. HPLC analysis HPLC was performed on an Hitachi L-6200 system equipped with a UV detector (model L-7400; Hitachi, Tokyo, Japan) and ⁄ or a fluorescence detector (model F-1150; Hitachi). A polyamine II column (10 · 250 mm; YMC Co.) was used for the fractionation of unsaturated HA oligosaccharides produced by chondroitin ABC lyase and for the purification of PA oligosaccharides. A smaller polyamine II column (4.6 · 250 mm; YMC Co.) was used for analysis of the hydrolysis products of BTH. Conditions for the fractionation of unsaturated HA oligosaccharides were as follows. Two solutions were prepared for this col- umn: solution A was 16 mm NaH 2 PO 4 , and solution B was 500 mm NaH 2 PO 4 . Chondroitin ABC lyase digests were injected onto the column equilibrated with solution A at a flow rate of 1.0 mLÆmin )1 , and then eluted over 60 min with a linear gradient of solution B from 0% to 94%. The eluate was monitored for UV absorbance at 232 nm. Con- ditions for the analysis of BTH-digested HA oligosaccha- rides were as follows. Two solutions with different concentrations of NaH 2 PO 4 were prepared for this column (solutions A and B). The linear gradient varied, depending on the size range of oligosaccharides to be analyzed (see fig- ure legends for details). BTH digests containing only satu- rated HA oligosaccharides were monitored by UV absorbance at 215 nm. BTH digests containing both satu- rated and unsaturated HA oligosaccharides were monitored by UV absorbance using two detectors set at 215 nm and 232 nm; the latter wavelength detected the unsaturated bonds. PA-HA oligosaccharides were monitored by fluores- cence detection (excitation at 320 nm; emission at 400 nm) and by UV absorbance at either 232 nm or 215 nm. Ion-spray MS Mass spectra of HA oligosaccharides were obtained on a PE-Sciex API-100 single-quadrupole mass spectrometer (Thornhill, Ontario, Canada), equipped with an atmo- spheric pressure ionization source, as described previously [6,22]. The mass spectrometer was operated in the negative ion mode. Samples dissolved in 50% of isopropanol were ionized by ESI and continuously infused into the ESI chamber at a flow rate of 5 lL min )1 . Acknowledgements This work was supported by Grants-in Aid (Nos. 17770106 and 19570119) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by the Fund for Cooperation for Innovative Technology and Advanced Research in Evolution Area (CITY AREA). We would like to thank V. P. Bhavanandan for his interest and editorial help with the manuscript. I. Kakizaki et al. Hydrolytic mechanism of testicular hyaluronidase FEBS Journal 277 (2010) 1776–1786 ª 2010 The Authors Journal compilation ª 2010 FEBS 1785 [...]... 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January 2010) doi:10.1111/j.1742-4658.2010.07600.x Synthetic hyaluronan oligosaccharides with defined structures and their pyridylaminated derivatives were used to investigate the mechanism of hydrolysis of hyaluronan by bovine testicular hyaluronidase. . to confirming the previously proposed mechanism for the hydrolysis of HA by BTH. AB C a b cd a b cd D Fig. 5. HPLC analysis of the products of hydrolysis of unsaturated HA oligosaccharides by BTH any of the reaction conditions tested. Products of hydrolysis by BTH of 2-pyridylamine (PA)–HA eicosasaccharides We then investigated the mechanism of hydrolysis by BTH, using a substrate of defined