Glycobiology Advance Access originally published online on June 29, 2007
Glycobiology 2007 17(9):963-971; doi:10.1093/glycob/cwm070
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Kinetics of Hyal-1 and PH-20 hyaluronidases: Comparison of minimal substrates and analysis of the transglycosylation reaction
Lehrstuhl für Pharmazeutische und Medizinische Chemie II, Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany
1 To whom correspondence should be addressed: Tel: +49-941-9434827 Fax: +49-941-9434820 e-mail: armin.buschauer{at}chemie.uni-regensburg.de
Received on April 19, 2007; revised on June 21, 2007; accepted on June 24, 2007
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Abstract |
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The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (
500 µM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action. Key words: capillary electrophoresis / Drosophila Schneider-2 cells / Hyal-1 / PH-20 / transglycosylation
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Introduction |
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Bovine testicular hyaluronidase (BTH) has been studied extensively with respect to its catalytic behavior at various pH values, depending on salt concentrations and the formation of reaction products (Weissmann et al. 1954
; Hoffman et al. 1956; Gorham et al. 1975; Takagaki et al. 1994; Oettl 2000; Hoechstetter 2005). In contrast to BTH, the hyaluronic acid (HA) degradation products of human Hyal-1 and human PH-20 have not been studied in detail yet. Due to the availability of the enzymes from human sources in very low quantities, information about their enzymatic properties was up to now restricted to few basic properties, such as the pH profiles and the nature of the reaction products (Gold 1982; Afify et al. 1993; Gmachl et al. 1993; Frost et al. 1997; Sabeur et al. 1997). Production of pure, recombinant human Hyal-1 (Hofinger et al. 2007) and partially purified, recombinant human PH-20 (described in this paper) facilitated the investigation of more specific properties of Hyal-1 and PH-20 in comparison to BTH.
BTH and, presumably, all mammalian hyaluronidases catalyze the hydrolysis as well as the transglycosylation of HA fragments (Weissmann 1955; Hoffman et al. 1956). In the case of BTH hydrolysis is favored at acidic pH values, while transglycosylation occurs preferentially at neutral pH values and at low NaCl concentrations (Gorham et al. 1975; Saitoh et al. 1995).
The complex reactions catalyzed by hyaluronate hydrolases and the polymeric structure of the substrate severely complicate the analysis of enzymological characteristics by means of Michaelis–Menten kinetics. Although KM and vmax values of hyaluronidase action on high molecular weight HA can be found in literature (Cramer et al. 1994; Vercruysse et al. 1995; Asteriou et al. 2006), the determination of exact Michaelis–Menten kinetics is impeded by the steady increase in the substrate concentration during cleavage of the HA chains. Therefore, small oligosaccharides have been employed enabling an exact monitoring of the reactions catalyzed by BTH (Cramer et al. 1994).
Due to the negatively charged groups present in glycosaminoglycane (GAG) oligosaccharides, capillary zone electrophoresis (CZE) is often applied in the analysis of GAGs in biological or biopharmaceutical preparations (Mao et al. 2002). The analysis of unmodified HA degradation products in biological samples is hampered by the absence of a suitable chromophore in the HA degradation products of hydrolases. In contrast to the mammalian hyaluronidases, which hydrolyze the ß-1,4-glycosidic bond between GlcNAc and glucuronic acid (Weissmann et al. 1954), the bacterial enzymes, the HA lyases, integrate a double bond into the degradation products during an elimination reaction (Stern and Jedrzejas, 2006). This facilitates the detection of the products of lyases by spectrophotometric methods. If detection of hydrolase products is performed by absorbance at low wavelength (200–210 nm) buffer substances as well as protein impurities interfere with the GAG detection. Grimshaw et al. (1994) employed a CZE method with an alkaline phosphate–borate buffer and SDS for the separation of protein impurities from the HA fragments. This method was used for the analysis of HA digestion products formed by the catalysis of pure enzyme samples (BTH, recombinant human PH-20 and recombinant human Hyal-1). The HA hexasaccharide (in the following designated n3, with n representing one HA disaccharide unit) is known to be the minimum substrate for BTH (Highsmith et al. 1975; Cramer et al. 1994). This has been supposed for the human hyaluronidases as well (Stern and Jedrzejas 2006).
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Results |
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Preparation of partially purified recombinant human PH-20
Human PH-20, also termed sperm adhesion molecule 1 (SPAM1) as its major role is to facilitate the penetration of the sperm through the HA-rich matrix surrounding the oocyte, was expressed as a soluble protein in DS-2 cells with the N-terminal signal peptide exchanged against an insect cell signalling peptide and the C-terminal GPI-anchoring signal peptide missing. Zymography revealed a single protein with hyaluronidase activity at acidic pH (pH 4.0) in the medium of stably transfected and induced DS-2 cells (Figure 1). PH-20 was partially purified by chelating ion metal affinity chromatography (IMAC) utilizing the Cu2+ ions present in the cell medium after induction of the metallothionein promoter. The hyaluronidase activity was determined in the Morgan–Elson assay, a commonly used assay monitoring the glycosidase reaction by quantification of the reducing GlcNAc ends set free during the degradation of HA. Transglycosylase activity, which competes with hydrolase activity, is not detected in this assay. The enzyme exhibited maximum enzymatic activity in the Morgan–Elson assay at pH 4.5 (data not shown) and a molecular mass of 56 kDa (Figure 1). Although the mass differed only slightly from the molecular mass calculated from the amino acid sequence (55 kDa), PH-20 was shown to be glycosylated (Figure 1).
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Though PH-20 was only partially purified to a specific enzymatic activity of 0.5 µmol GlcNAc min–1 mg–1 (compared to 0.23–2.5 µmol GlcNAc min–1 mg–1 for BTH preparations [Oettl et al. 2003
; Hoechstetter 2005] and 10.0 µmol GlcNAc min–1 mg–1 for recombinant PH-20 purified from CHO cells [Bookbinder et al. 2006]), the enzyme was sufficiently active to facilitate the analysis of the HA degradation products by CZE. The large differences in the specific enzymatic activity of the different PH-20 preparations probably originate from varying degrees of purification and, possibly, from a different glycosylation pattern. The glycosylation was shown previously to influence the enzymatic activity of mammalian hyaluronidases (Podyma et al. 1997; Hofinger et al. 2007).
Preparation and quantification of HA oligosaccharides
HA oligosaccharides from an exhaustive digestion mixture of high molecular weight HA were separated in high quantities by gel filtration. The HA fragments were detected at 210 nm due to the lack of a chromophor absorbing at higher wavelength. Detection at this wavelength resulted in a high absorbance of the buffer, leading to a low sensitivity in detection. A volatile incubation buffer system (NH4OAc) was chosen to allow for the lyophilization and concentration of the degradation products after digestion. The components of the lyophilized fractions were identified by ESI-MS (Tawada et al. 2002), and the purity of the oligosaccharides was confirmed by CZE.
The concentration of pure HA oligosaccharides was quantified by the amount of reducing GlcNAc ends by the Morgan–Elson assay and the molecular mass of the respective compound. In CZE the area/time (A/t) ratio of n1, n2, n3 and n4 correlated linearly with the concentration of the respective oligosaccharide in a concentration range between 25 µM and ca. 2.0 mM (data not shown). However, peaks at concentrations >2 mM showed flattened peak tops, thus concentrations above 2.0 mM were not used for quantitative analysis. Triple injection of the same sample confirmed a constant sample volume, injected automatically by pressure (SEM 
5%). Therefore, variations of the injection volume were neglected in the following experiments, and all samples were injected only once.
Degradation of HA hexasaccharide (n3) by BTH at pH 4.0
To investigate, if the pH influences BTH with respect to its degradation kinetics as well as its mechanism of HA degradation, HA hexasaccharide (n3) was used as a substrate at pH 4.0 and at pH 6.0. The conversion of the HA hexasaccharide (n3), the well-known minimum substrate of BTH (Cramer et al. 1994), was monitored over a concentration range (25 µM– 2 mM) to gain information about the catalytic behavior of the hyaluronidase under Michaelis–Menten conditions.
The CZE analysis of the conversion of n3 by BTH at pH 4.0 revealed the formation of n2 as the major reaction product, while all other products, (n1, n4, n5 and n6) were present at very low concentrations. Especially, the formation of the transglycosylation products, n4 and n5, was extremely low. Figure 2A shows an example of an electropherogram after the conversion of 100 µM n3 into n1, n2 and n4 by the action of BTH at pH 4.0.
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Considering n5 as the largest reaction product, and omitting hydrolysis reactions of HA fragments larger than n3, the following reactions can be assumed to occur at the initial stage of the degradation of n3 at pH 4.0:
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Previous data (Highsmith et al. 1975) indicated that n1 and n2 are too small to bind to the active site. The low amounts of n1 compared to n2 indicate that both, hydrolysis [Eq. (1)] and transglycosylation [Eqs. (2) and (3)], seem to contribute to the decrease in n3. As the octasaccharide (n4) is known to be a better substrate than n3 (Highsmith et al. 1975) the high concentrations of n2 may be caused by hydrolysis of n4 into 2 n2 at an advanced stage of the reaction. Intriguingly, the degradation of n3 slowed down at concentrations >300 µM and came to a complete halt at concentrations >500 µM, indicating substrate inhibition of the enzyme at higher concentrations. It should be noted that under physiological conditions n3 is the degradation product of high molecular weight HA rather than the substrate. Thus, inhibition can be looked upon as substrate and/or product inhibition.
Octa- (n4) and decasaccharides (n5) produced by transglycosylation at the initial stage of the reaction were observed to be preferentially degraded in the course of the incubation period even in the presence of a high excess of hexasaccharide (n3) (e.g. 100 µM in Figure 3). The initial linear part of the degradation curve of n3 at various concentrations facilitated the calculation of initial degradation velocities as a factor of the substrate concentration (Figures 3 and 4).
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Degradation of HA hexasaccharide (n3) by BTH at pH 6.0
Previously, experiments with high molecular weight HA had indicated a difference in the degradation mode of BTH at acidic and neutral pH (Hoechstetter et al. 2001
). Therefore, the degradation of n3 was analyzed at pH 6.0 by analogy with the CZE experiments performed at pH 4.0.
The incubation of BTH with n3 at pH 6.0 preferentially resulted in oligosaccharides of higher molecular weight (n4 and n5) than the substrate. Intriguingly, no n1 was produced during the degradation of n3 at pH 6.0 (as an example shown for 100 µM n3 in Figure 2B). Considering the reactions involved in the degradation process of n3 shown in Eqs. (1)–(3)
, the hydrolysis of n3 into n1 and n2 [Eq. (1)] can therefore be excluded from the processes occurring at pH 6.0. Thus, the decrease in n3 at the initial stage of the reaction was exclusively caused by the transglycosylase activity of BTH, which is known to occur preferentially at increased pH values (Gorham et al. 1975).
At pH 6.0 an increase in the initial degradation velocity of n3 was observed up to concentrations of ca. 800 µM, then the velocity of degradation decreased fast until at concentrations >1 mM no conversion of the substrate was observed any more (Figure 4). These results are consistent with the substrate/product inhibition observed at pH 4.0. However, at pH 4.0 the inhibition occurred at lower substrate concentrations (>200 µM). A correct calculation of KM and vmax values was therefore not possible.
Degradation of n3 and n4 by human PH-20 at pH 4.5
Partially purified human PH-20, expressed in DS-2 cells, was analyzed for its degradation behavior with respect to low molecular weight HA oligosaccharides at pH 4.5, its pH optimum, measured in the Morgan–Elson assay. Surprisingly, the PH-20 incubation mixtures analyzed after different periods of incubation by CZE revealed no conversion of the HA hexasaccharide. Although BTH and PH-20 belong to the same hyaluronidase subtype, recombinant human PH-20 did not recognize the HA hexasaccharide as a substrate molecule. Incubation with increasing concentrations of HA hexasaccharide (25 µM–2 mM) incubated for various time periods with PH-20 resulted in no conversion of the potential minimum substrate molecule.
Therefore, the HA octasaccharide (n4) was offered as a substrate and was found to be degraded very efficiently. Intriguingly, the conversion of HA octasaccharide (n4) proceeded very quickly, with hexa- (n3) and tetrasaccharides (n2) forming the major final reaction products (Figure 5, t = 60 min). In the course of the degradation significant amounts of HA fragments larger than the octasaccharide (n4) were formed, indicating the occurrence of transglycosylation reactions. Yet, these deca- (n5) and dodecasaccharides (n6) were obviously preferred as substrate molecules, since they were again hydrolyzed during the incubation period (Figure 5, t = 10 min and 60 min).
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At the initial stage of the reaction, i.e., if merely octasaccharide (n4) is present as a substrate, the following reactions can occur:
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The hydrolysis of n4 [Eqs. (4) and (5)] seems to be of minor importance as concluded from the formation of low concentrations of n1 and n2 during the initial phase of degrada- tion (Figure 5A, t = 10 min and Figure 5B). Moreover, the transglycosylation reactions [Eqs.(6) and (7)] seem to account for the major decrease in the concentration of n4, as the reaction products observed after 10 min of incubation were n3 and n5, and only to a minor extent n6 and n2 (Figure 5, t = 10 min). At increased substrate concentrations (>500 µM) higher molecular weight oligosaccharides (up to n8) were detected (data not shown), confirming the preference of transglycosylation reactions catalyzed by PH-20. During the processing of the octasaccharide (n4) the HA disaccharide (n1) was detected only at very low concentrations, as shown in Figure 5, or not at all.
Degradation of the HA hexa- (n3) and octasaccharide (n4) by Hyal-1
Recombinant human Hyal-1 produced by DS-2 cells was incubated with n3 as a substrate at pH 3.5, the optimum pH of Hyal-1 activity (Hofinger et al. 2007). Similar to PH-20, the HA hexasaccharide (n3) was not converted by Hyal-1 at any of the concentrations used (20 µM–6 mM).
Therefore, various concentrations of HA octasaccharide (n4) were used as substrate of Hyal-1 at pH 3.5. In contrast to the hexasaccharide (n3), the octasaccharide (n4) was converted quickly at substrate concentration between 25 µM and 1 mM (Figures 6A and 7A). At concentrations above 1 mM weak substrate inhibition was observed (Figures 4 and 7B). Furthermore, the reaction products varied significantly depending on the substrate concentration. At low n4 concentrations (<500 µM) no transglycosylation products were observed (Figures 6A and 7A), while at higher n4 concentrations (>500 µM) fragments larger than the substrate appeared, and the disaccharide (n1) was present at concentrations close to the CZE detection limit (Figures 6B and 7B). At low substrate concentrations the reactions catalyzed by Hyal-1 seem to be restricted to hydrolysis of n4 [Eqs. (4) and (5)]. When the substrate concentration increased above ca. 500 µM transglycosylation reactions occurred at a higher frequency than at low substrate concentrations, enabling the production of HA fragments larger than the substrate [Eqs. (6) and (7)].
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Kinetics of substrate degradation
The time-consumption curves of the hexasaccharide (n3) and the octasaccharide (n4) by the action of the hyaluronidases exhibited linear parts at the initial stage of the reactions, referring to the steady-state conditions of the Michaelis–Menten kinetics. Quantification of the substrate peaks was performed using CZE calibration curves. The slope of the linear part of the time-consumption curves was used as the initial degradation velocity of the substrate.
Figure 4 shows the initial reaction velocity at increasing substrate concentrations for BTH at pH 4.0 and 6.0, for Hyal-1 at pH 3.5 and for PH-20 at pH 4.5. Both, BTH and Hyal-1, were inhibited by the substrate at increasing concentrations: BTH was inhibited by n3 concentrations above 200 µM at pH 4.0 and at pH 6.0 by n3 concentrations above 800 µM, Hyal-1 was inhibited by n4 concentrations above 1 mM. In contrast to the other hyaluronidases, PH-20 was not inhibited by the HA octasaccharide (n4) at concentrations up to 2 mM. Due to the occurrence of substrate inhibition KM and vmax values could only be obtained for the degradation of n4 by PH-20 (KM = 130 ± 18 µM, vmax = 1.6 ± 0.7 nmol/min) (Figure 4).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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The availability of the human hyaluronidases Hyal-1 and PH-20 from recombinant expression systems facilitated the observation of details of the catalytic behavior of these poorly characterized enzymes. Similar to the results described previously for human Hyal-1 (Hofinger et al. 2007
), the DS-2 cells proved to be an efficient expression system for the production of an enzymatically active, glycosylated human hyaluronidase PH-20. However, one has to keep in mind that the recombinant enzymes might not be identical to the endogenous human enzymes in terms of post-translational modifications (glycosylations, intramolecular proteolytic cleavage).
Analysis of the degradation products of the HA hexasaccharide (n3) by BTH confirmed the ability of the enzyme to catalyze hydrolysis as well as transglycosylation reactions. The transfer of disaccharide (n1) units of HA or chondroitin sulfate through the transglycosylative activity of BTH is well known from earlier studies (Weissmann 1955; Hoffman et al. 1956; Takagaki et al. 1994). However, this study revealed pH-dependent differences in the catalytic behavior of the enzyme with respect to its minimum substrate, the HA hexasaccharide (n3).
Although an X-ray structure of recombinant human hyaluro- nidase Hyal-1 has been published very recently (Chao et al. 2007) no experimental data on the binding of the substrate are available in the literature. Therefore, a model of the specific binding sites of BTH was considered, which was proposed by Highsmith et al. on an experimental basis (Highsmith et al. 1975; Takagaki et al. 1994). The current concept of the catalytic site of BTH comprises the active site with a Glu residue acting as an acid/base catalyst and several binding sites (S) on both sides of the cleavage site, each occupied by one disaccharide unit of the HA chain. Highsmith et al. (1975) proposed a site S2–S1–S1'–S2'–S3' according to the terminology of Schechter and Berger (1966) with the reducing terminus of the sugar chain located on the right hand side and the cleavage occurring between S1 and S1'.
Based on the observation at pH 5.0, HA tetrasaccharides (n2) are insufficient substrate molecules for BTH, hexasaccharides (n3) are the minimum substrate moieties and octasaccharides (n4) are better substrates than hexasaccharides, differential affinities of the disaccharide binding sites were proposed: weak binding affinity of the sites S1 and S1', high affinity of S2' in contrast to a very weak binding affinity of S2 and an important, but not essential binding affinity of S3' (Highsmith et al. 1975) (Figure 8).
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Binding of n3 encompasses the sites S1–S1'–S2' (Highsmith et al. 1975
) with n1 leaving the site S1 and n2 the sites S1'–S2' after hydrolysis at pH 4.0. At pH 6.0, however, n1 does not, or only to a minor extent, leave the site S1, but remains bound until transglycosylation to another acceptor molecule like n3 has occurred. Therefore, the increased transglycosylation activity at nearly neutral pH might be explained by an increase in the affinity of the site S1 at pH 6.0 compared to pH 4.0 (Figure 8).
In contrast to the degradation at pH 4.0, we detected no disaccharide (n1) units as reaction products at pH 6.0. Although the degradation of HA oligosaccharides by BTH or ovine testicular hyaluronidase has often been observed before, the reaction products as well as the kinetic parameters determined differ significantly due to the use of various detection methods and buffer conditions. The formation of the HA disaccharide (n1) as a reaction product was reported by several authors (Weissmann 1955; Takagaki et al. 1994; Saitoh et al. 1995), while others could not detect any n1 (Highsmith et al. 1975; Cramer et al. 1994; Kinoshita et al. 2001). As all the results were obtained in buffer systems at pH 5.0–5.3 other factors such as the nature of the buffering ions as well as the overall salt concentrations seem to influence the degradation behavior of BTH (Saitoh et al. 1995; Asteriou et al. 2006).
Asteriou et al. (2006) describe substrate inhibition for the degradation of high molecular weight HA by BTH at low ionic strengths, but no substrate inhibition has been described up to now for the degradation of low molecular weight HA oligosaccharides (Gorham et al. 1975; Highsmith et al. 1975; Cramer et al. 1994). The detection of CZE kinetics at higher salt concentrations (>0.1 M) was impossible due to interference of the CZE method with elevated salt concentrations.
The recombinant human enzymes PH-20 and Hyal-1 exhibited a completely different catalytic behavior than BTH with respect to HA oligosaccharides. Both enzymes were not able to catalyze any conversion or degradation of the supposed minimum substrate, the HA hexasaccharide (n3). Although experimental evidence for the identity of the minimum substrate of human hyaluronidases was missing, the HA hexasaccharide (n3) is generally believed to be the smallest substrate for all mammalian-type hyaluronidases (Stern and Jedrzejas 2006). However, the HA octasaccharide (n4) could be proven in this study to be the minimum substrate for both, recombinant human PH-20 and Hyal-1.
The inability of Hyal-1 and PH-20 to degrade n3 indicates either a very low affinity of HA fragments of three disaccharide units or a low catalytic activity towards bound n3. However, two facts hint to a low affinity of n3 towards the binding site: firstly, BTH is known to exhibit a lower affinity towards n3 than n4, suggesting the existence of a site S3' being relevant for binding (Highsmith et al. 1975). A second hint can be drawn from the observation, that n3 inhibited the degradation of high molecular weight HA by Hyal-1 very weakly (30% inhibition at 3 mM, unpublished result). Thus, the affinity of the subsite S2', which provides the essential affinity for binding of the hexasaccharide to BTH (Highsmith et al. 1975), is significantly decreased in PH-20 and Hyal-1.
The conversion products of the octasaccharide by PH-20 can be explained by assuming a shift of the essential binding site from position S2' to position S3' (Figure 8). The site S1 seems to exhibit considerable affinity since transglycosylation reactions were preferentially catalyzed by PH-20. However, the model cannot provide a satisfactory explanation for the formation of n2 and n3 as the major reaction products, but n1 in extremely low concentrations. Presumably, the enzyme catalyzed mainly the transglycosylation reaction shown in Eq. (6) as can be concluded from the occurrence of the decasaccharide (n5) as the main transglycosylation product. The fast degradation of the decasaccharide (n5) in the course of time indicates a preferential hydrolysis of n5, which could provide the major reaction products n2 and n3.
The following binding site model is proposed to explain the reactions catalyzed by Hyal-1: the octasaccharide (n4) was degraded to di-, tetra- and hexasaccharides, thus suggesting the binding of the octasaccharide in two different positions, one covering the sites S2–S1–S1'–S2' [Eq. (5)] and the other one covering the sites S1–S1'–S2'–S3' [Eq. (4)]. Assuming that the affinity of the hexasaccharide is insufficient for binding, the binding affinity of the octasaccharide might be dependent on the occupation of at least two binding sites with medium affinity, i.e., two of the sites S2, S2' and S3' (Figure 8).
Hyal-1 was able to catalyze transglycosylation of the HA octasaccharide (n4), albeit only in the presence of high substrate concentrations (n4 > 500 µM). In contrast to Hyal-1, the transglycosylase action of BTH was pH-dependent, but without any significant dependency on the substrate concentration.
An explanation for the concentration-dependent changes in the catalytic process might be provided by the affinity of the disaccharide unit occupying the subsite S1 (and S2) in the binding model. For transglycosylation at least the site S1 has to stay occupied, while the product on the other side of the cleavage site leaves the site S1'–S2'–S3' and a new acceptor molecule is bound. Thus, if the rate constant for the release of the disaccharide unit in position S1 (koff) is on the same time scale as the diffusion-controlled encounter and binding of enzyme and acceptor molecule, the occurrence of concentration-dependent transglycosylation events might be explained as follows: at low substrate concentrations (<500 µM) the position S1 releases the disaccharide unit before another acceptor molecule is bound; at high substrate concentrations (>500 µM), however, the probability for the encounter and binding of another acceptor molecule within the time-range of koff is increased, resulting in the occurrence of transglycosylative events.
The question remains if the concentration-dependent shift from mere hydrolysis to more transglycosylative events has any biological importance in the catabolism of HA. In contrast to the PH-20 isoenzymes the main function of Hyal-1 seems to be the hydrolysis of high molecular weight HA to low molecular weight oligosaccharides at acidic pH values.
The analysis of the degradation of HA oligosaccharides provided new insights into the catalytic mechanism of different hyaluronidase isoenzymes within the family of mammalian HA hydrolases. The differences between the hyaluronidase subtypes and isoenzymes were much more distinctive than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of HA degradation.
The suggested binding site model in combination with three-dimensional structural data (Jedrzejas and Stern 2005; Chao et al. 2007) should facilitate the target-based design of selective inhibitors of mammalian hyaluronidases.
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Materials and methods |
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Chemicals
HA from Streptococcus zooepidemicus was purchased from Aqua Biochem GmbH (Dessau, Germany). Bovine testicular hyaluronidase (BTH, Neopermease) was a gift from Sanabo (Vienna, Austria), Hyal-1 was produced by DS-2 cells and purified as described previously (Hofinger et al. 2007
). Molecular cloning materials, bacterial strains, vectors and cells used for expression of PH-20 were identical to those described by Hofinger et al. (2007). BSA was purchased from Serva (Heidelberg, Germany), 4-dimethylaminobenzaldehyde (DMAB) from Sigma-Aldrich (Munich, Germany) and all other chemicals were from Merck (Darmstadt, Germany). Water was purified by a Milli-Q system (Millipore, Eschborn, Germany).
Preparation of recombinant human PH-20
Human PH-20 cDNA was amplified by PCR from the I.M.A.G.E. clone IMAGp998N2310771 using 5'-ACGCC- TAGATCTCTGAATTTCAG-3' as forward-primer generat- ing a BglII cleavage site and 5'-TAAATTGGGCCCAGA- TAGTGTGGA-3' as reverse-primer with an additional ApaI cleavage site. The PCR product was ligated into the expression vector pMT/Hygro using cloning procedures as described (Hofinger et al. 2007). PH-20 was expressed into the medium of DS-2 cells as performed previously with Hyal-1 (Hofinger et al. 2007).
Purification of PH-20 was achieved by a HiTrap® Chelating Column (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden) equilibrated in 50 mM sodium phosphate, 0.5 M NaCl, 0.1% Triton X-100, pH 6.5. Before loading to the column, the clarified cell medium was supplemented with 0.1% Triton X-100. The column was washed with two column volumes of equilibration buffer and PH-20 was eluted by a decrease of the pH to 4.5.
Fractions were monitored for their hyaluronidase activity by the Morgan–Elson assay using the following incubation mixture: 150 µL of water, 100 µL of BSA (0.2 mg mL–1), 100 µL of McIlvaine's buffer, 50 µL of hyaluronic acid (5 mg mL–1) and 50 µL of enzyme solution. McIlvaine's buffer was prepared by mixing solution A (0.2 M Na2HPO4, 0.1 M NaCl) and solution B (0.1 M citric acid, 0.1 M NaCl) in the appropriate proportions to reach the desired pH.
Additionally, hyaluronidase activity was shown by zymography (Cherr et al. 1996; Oettl et al. 2003) and the protein content was determined in the bicinchoninic acid (BCA) assay (Smith et al. 1985; Hofinger et al. 2007). SDS-PAGE and periodic-acid Schiff's (PAS) staining were performed as described previously (Hofinger et al. 2007).
Preparation of HA oligosaccharides
High molecular weight HA was exhaustively digested by BTH for 48 h at 37°C. 1 mL of BTH (50,000 IE mL–1, according to the supplier, solubilized in 0.2 mg mL–1 BSA) was added to an incubation mixture containing 50 mL of incubation buffer (0.1 M HOAc/NaOAc, 0.1 M NaCl, pH 4.0), 50 mL of HA solution (5 mg mL–1), 25 mL of BSA solution (0.2 mg mL) and 74 mL of water. At the end of the incubation time the solution was boiled for 10 min and centrifuged for 20 min at 5000 x g. The supernatant was lyophilized and the remaining oligosaccharides dissolved in Milli-Q water to gain a concentration of 40 mg mL–1. The oligosaccharides were filtered (GH Polypro membrane disc filter, 0.45 µm, Pall Life Sciences, Dreieich, Germany) and separated on a HiLoad 16/60 SuperdexTM 30 prep grade (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden) at a flow rate of 1 mL min–1 with a maximum sample volume of 1.5 mL. The column was operated by a Millipore® Waters solvent delivery module, Model 590, connected to a Merck Hitachi L-4000 UV detector. Detection was carried out at 210 nm and fractions were collected manually. Di- and tetrasaccharides were sufficiently pure after one separation step, but hexa- and octasaccharides had to be purified twice over the Superdex 30 column. Fractions were pooled, lyophilized and dissolved in Milli-Q water. Purity and identity of the fractions was determined by mass spectrometry and capillary zone electrophoresis, respectively. Oligosaccharide solutions were stored at –20°C.
ESI-MS
HA oligosaccharides were analyzed by ESI-MS on a ThermoQuest Finnigan TSQ 7000 spectrometer (Thermo Electron GmbH, Dreieich, Germany). Lyophilized oligosaccharide samples were dissolved in a 1:10 mixture of water and methanol containing 10 mM NH4OAc and were manually injected into the ion source. Analysis was performed in the negative ion mode with a spray voltage of 3 kV and a capillary temperature of 200°C.
Morgan–Elson assay (colorimetric hyaluronidase activity assay)
For quantification of HA oligosaccharides 0.05–0.1 µmol of GlcNAc were dissolved in 450 µL water to obtain a calibration curve. Unknown oligosaccharide concentrations were determined in triplicates.
The enzymatic activity of the hyaluronidase samples used for CZE analysis was determined in the incubation mixtures used for the CZE containing 555 µg mL–1 high molecular weight HA instead of low molecular weight oligosaccharides. The detection of reducing GlcNAc ends was performed as described by Muckenschnabel et al. (1998). Enzymatic activity is given as nmol reducing GlcNAc ends liberated per minute under defined assay conditions.
Capillary zone electrophoresis (CZE)
Incubation mixtures
Various concentrations of pure HA hexasaccharides (n3) and octasaccharides (n4) were incubated with BTH, Hyal-1 and PH-20 solubilized in the following incubation mixtures:
BTH, pH 4.0: 44 µg mL BSA, 22 mM HOAc/NaOAc, pH 4.0, 22 mM NaCl, 6.1 nmol GlcNAc min–1 mL–1 BTH determined in the Morgan–Elson reaction at pH 4.0.
BTH, pH 6.0: analogous to BTH, pH 4.0, but using 22 mM HOAc/NaOAc, pH 6.0, 6.1 nmol GlcNAc min–1 mL–1 BTH as determined in the Morgan–Elson assay at pH 6.0.
PH-20: analogous to BTH, pH 4.0, but using 6.0 nmol GlcNAc min–1 mL–1 PH-20 as determined in the Morgan–Elson reaction at pH 4.0.
Hyal-1: analogous to BTH, pH 4.0, but using 22 mM formic acid/Na-formate, pH 3.5, and 10.0 nmol GlcNAc min–1 mL–1 Hyal-1 as determined in the Morgan–Elson reaction at pH 3.5.
For the calculation of the NaCl concentration in the incubation mixtures, it was considered that Hyal-1 and PH-20 were both dissolved in 50 mM Na-phosphate, 0.5 M NaCl, 0.1% Triton X-100, pH 4.5. Hyaluronidase activity was determined directly before the kinetic measurement in the Morgan–Elson assay using incubation mixtures analogous to the CZE mixtures.
For each oligosaccharide concentration an incubation mixture was preincubated for 5 min at 37°C, then the enzyme was added and samples of 25–100 µL were taken at defined time points (0–60 min). The enzymatic reaction was stopped by mixing the sample with twice the volume of ice-cold acetonitrile. All samples were then dried in the vacuum and re-dissolved in 100 µL of 10-fold diluted CZE operating buffer. After centrifugation (10 min, 12,000 x g) the supernatant was analyzed by CZE.
CZE calibration curves
After determination of the concentration in the Morgan–Elson assay, n1, n2, n3 and, if necessary, n4 were mixed in the same buffer systems as used for the respective kinetics. The calibration mixtures were treated in an identical way as the samples, i.e., they were mixed with twice the volume of acetonitrile, dried in the vacuum and solubilized in 10-fold diluted CZE operating buffer (see below).
CZE conditions
All CZE experiments were carried out in the normal mode on a Biofocus 3000 capillary electrophoresis system (Biorad, München, Germany) equipped with an uncoated fused silica capillary (75 cm x 50 µm, Chrompack). Buffer conditions were adapted from Grimshaw et al. (1994). The CZE operating buffer contained 50 mM Na2HPO4, 20 mM Na2B4O7, pH 9. The sample was injected by pressure (20 psi s) and electrophoresis was performed in the normal polarity mode at a capillary temperature of 15°C with a run voltage of 15 kV applied to the capillary. HA fragments were detected by absorption at 200 nm. The capillary was washed after each run with 0.1 M NaOH (200 s), Milli-Q water (200 s) and the operating buffer (300 s).
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Graduate Training Program (Graduiertenkolleg) GRK 760 "Medicinal Chemistry: Molecular Recognition–Ligand-Receptor Interactions" of the Deutsche Forschungsgemeinschaft (DFG).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We thank S. Bollwein for excellent technical assistance.
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Abbreviations |
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BSA, bovine serum albumin; BTH, bovine testicular hyaluronidase; CZE, capillary zone electrophoresis; DMAB, 4-dimethylaminobenzaldehyde; DS-2 cells, Drosophila Schneider-2 cells; GAG, glycosaminoglycane; GlcNAc, N-acetylglucosamine; HA, hyaluronic acid; IMAC, ion metal affinity chromatography; n, hyaluronic acid disaccharide unit
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