hyaluronidase activity is modulated by complex ing

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Matrix Biology 27 (2

Hyaluronidase activity is modulated by complexing with variouspolyelectrolytes including hyaluronan

Brigitte Deschrevel ⁎, Hélène Lenormand, Frédéric Tranchepain, Nicolas Levasseur,Trias Astériou, Jean-Claude Vincent

Laboratoire “Polymères, Biopolymères, Membranes”, UMR 6522 CNRS-Université de Rouen, 76821 Mont-Saint-Aignan Cedex, France

Received 20 June 2007; received in revised form 31 October 2007; accepted 1 November 2007

Abstract

Hyaluronidase (HAase) plays an important role in the control of the size and concentration of hyaluronan (HA) chains, whose biologicalproperties strongly depend on their length. Our previous studies of HA hydrolysis catalyzed by testicular HAase demonstrated that, whilst thesubstrate–dependence curve has a Michaelis–Menten shape with a 0.15 mol L−1 ionic strength, at low ionic strength (5 mmol L−1), a strongdecrease in the initial hydrolysis rate is observed at high substrate concentrations; the HA concentration for which the initial rate is maximumincreases when the HAase concentration is increased. After examination of various hypotheses, we suggested that this could be explained by theability of HA to form non-specific complexes with HAase, which thus becomes unable to catalyze HA hydrolysis. In order to verify thishypothesis, we first showed from turbidimetric measurements that HAase, like albumin, is able to form electrostatic complexes with HA. Albuminthen was used as a non-catalytic protein able to compete with HAase for the formation of non-specific complexes with HA, allowing HAase to befree and catalytically active. The kinetic results showed that the HA–HAase non-specific complex inhibits HAase catalytic activity towards HA.Depending on the albumin concentration with respect to the HAase and HA concentrations, albumin can either remove this inhibition or induceanother type of inhibition. Finally, the extent of such non-specific interactions between polyelectrolytes and proteins in HAase inhibition oractivation, in particular under in vivo conditions, is discussed.© 2007 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Keywords: Hyaluronan; Hyaluronidase; Bovine serum albumin; Hyaluronan–protein complex; Hydrolysis kinetics; Enzymatic inhibition; Enzymatic activation

1. Introduction

Hyaluronan (HA) is a linear high-molar-mass polysaccharidecomposed of repeating disaccharide units of β(1,4)-D-glucuro-nic acid β(1,3)-N-acetyl-D-glucosamine. Since its discovery inthe vitreous humor by Meyer in 1936, it has been well-established that HA is widely distributed in the extra-cellularmatrix (ECM) of vertebrate tissues and that it is involved inmany fundamental biological processes such as cellularadhesion, mobility, proliferation and differentiation (Catterall,

Abbreviations: BSA, bovine serum albumin; CD44, cluster of differentiation44; ECM, extra-cellular matrix; HA, hyaluronan; HAase, hyaluronidase; HAS,hyaluronan synthase; HN, hyaluronectine; MMP, matrix metalloproteinase;TIMP, tissue inhibitor of matrix metalloproteinase.⁎ Corresponding author. Tel.: +33 2 35 14 00 74; fax: +33 2 35 14 67 04.E-mail address: [email protected] (B. Deschrevel).

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1995; Delpech et al., 1997; Laurent, 1987; Rooney et al., 1995;Kennedy et al., 2002). Recently, it has been demonstrated thatthe functions of HA strongly depend on chain size, and that HAfragments and native HA may have opposite roles (Stern et al.,2006). For example, HA fragments containing 4 to 25disaccharides (1.6 103 to 104 g mol−1) have an angiogenicaction on endothelial cells (West and Kumar, 1989a b), contraryto native HA which is anti-angiogenic (Deed et al., 1997). Inaddition, increased HA levels have been observed in response tosevere stress and in tumor progression and invasion (Stern,2004). All this supposes a tight regulation of the HA metabolicpathways.

HA synthesis occurs on the cytoplasmic face of the plasmamembrane and is catalyzed byHA synthases (HAS) (Weigel et al.,1997). The polysaccharide is transported outside of the cell as it isbeing synthesized (Prehm, 1984). Hyaluronidases (HAase)

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243B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253

catalyze HA cleavage. In the human body, all HAases are of theendo-β-N-acetyl-hexosaminidase type (EC 3.2.1.35) (Csóka et al.,2001; Stern and Jedrzejas, 2006), which means that they catalyzeβ(1,4) bond hydrolysis. In tumor tissues, both HA and HAaseconcentrations are high (Bertrand et al., 1997; Lokeshwar et al.,1996; Victor et al., 1997; Toole, 2002), HA being of rather lowmolarmass (Kumar et al., 1989; Lokeshwar et al., 1997)whichmayfavor angiogenesis and thus cancer progression. In addition, HAoligosaccharides induce proteolytic cleavage of the cancer cellCD44 receptors and thus promote tumor cell migration (Sugaharaet al., 2003, 2006). Although the precise role of HASs and HAasesin the production and/or in the control of the size and concentrationof HA chains has not yet been elucidated, all of this suggests thatHAase may control the balance between long and short HA chains.According to Mio and Stern (2002), regulation of the size andconcentration of HA chains is kinetically and energetically moreefficient through the catabolic rather than the anabolic pathway. Infact, HAase could be present in tissues together with inhibitorswhich may allow HAase to be rapidly activated or inactivated.However, even though some HAase inhibition activities have beendetected in vivo, very little is known about their molecular structureand mechanism of action in vivo (Mio and Stern, 2002).

We examined the kinetics of native HA hydrolysis catalyzedby HAase (Vincent et al., 2003; Astériou et al., 2006) usingbovine testicular HAase as a model, since, like human HAase, itcatalyzes the hydrolysis of the β(1,4) bonds in HA. Using aphysiological-type ionic strength, i.e. 0.15 mol L−1, bovinetesticular HAase obeys the Michaelis–Menten law: the initialreaction rate is a hyperbolic function of the substrate concentra-tion (Vincent et al., 2003). At low ionic strength (close to 5 mmolL−1), the behavior of the reactive system is totally different: forincreasing HA concentrations, the initial hydrolysis rate succes-sively increases, reaches a maximum and then decreases to a verylow level, close to zero, at high substrate concentrations, insteadof reaching a plateau, as it does for a Michaelis–Menten typeenzyme (Astériou et al., 2002; Astériou et al., 2006). In addition,the substrate-dependencies obtained for various HAase concen-trations indicates that the atypical behavior of the HA/HAasesystem observed at high substrate concentrations and at low ionicstrength also depends on the HAase concentration (Astériou et al.,2006). While Bollet et al. (1963) have already observed such alow hydrolysis rate at high HA concentrations with rat kidneyHAase in 0.10 mol L−1 acetate buffer at pH 3.8, they did notprovide any explanation for these results. Similarly, Aronson andDavidson (1967) mention that rat liver HAase in 0.10 mol L−1

acetate buffer at pH 3.9 exhibits inhibition when the HAconcentration is higher than 1.6 g L−1 and that this inhibition isprevented by 0.15 mol L−1 NaCl.

Our detailed study of the HA/HAase reactive system at lowionic strength has led us to examine various hypotheses to explainthe atypical kinetic behavior (Astériou et al., 2006), such as aninhibition by excess of substrate as well defined in basic enzy-mology (Laidler, 1958). Among these hypotheses, the only onewhich could explain all the experimental results is based on theformation of non-specific complexes between HA and HAase(Astériou et al., 2006). According to this hypothesis, in addition tospecific catalytic complexes, HA andHAase are able to form non-

specific complexes by electrostatic interactions. When HAase isnon-specifically complexed with HA, its catalytic activity issuppressed. Thus, with a high ratio for the HA concentration overthe HAase concentration, the formation of non-specific com-plexes leads to a decrease in the concentration of HAase able toform catalytic complexes and so, to a decrease in the initialhydrolysis rate. Conversely, with a low ratio for the HA concen-tration over the HAase concentration, the concentration of HAaseable to catalyze the hydrolysis is high and the HA/HAase systembehaves as a Michealis–Menten system.

The ability of polysaccharides and proteins to form complexeshas been known for a long time and, in the case of HA, it wasshown more than 60 years ago. The Mucin Clot Prevention(Robertson et al., 1940) and the turbidimetric methods (Kass andSeastone, 1944; Dorfman and Ott, 1948) were developed to assayHAase. In the latter method, HAase is assayed by measuring thedisappearance of the turbidity resulting from complexes formedbetween long chains of HA and albumin under acidic conditions.Since then, some investigations (Gold, 1980; Grymonpré et al.,2001) have been devoted to the characterization of the complexesformed between HA and the bovine serum albumin (BSA). Forexample, Xu et al. (2000) studied the influence of pH on thesolubility of HA–BSA electrostatic complexes. Moreover, othersworking on HAase report that serum proteins are able to enhancethe HAase catalytic activity. Gacesa et al. (1981) have shown thatamong the serum proteins, albumin has the greatest effect.According to Maingonnat et al. (1999), other proteins such ashyaluronectin (HN), which is a hyaladherin (i.e. a protein able tospecifically interact with HA), hemoglobin and immunoglobulinsare also able to enhance HAase activity and so, to increase thesensitivity of the HAase detection. However, HAase activityenhancement depends on the protein concentration.

The atypical behavior of the HA/HAase system at low ionicstrength together with both the ability of albumin to formcomplexes withHA and the effect of added proteins on theHAaseactivity have led us to suggest that the addition of a non-catalyticprotein, such as BSA, induces a competition between HAase andthe non-catalytic protein for the formation of non-specificcomplexes with HA. Consequently, this addition of the non-catalytic protein could allow the concentration of free, and thuscatalytically active, HAase, to increase, leading to an increase inthe HA hydrolysis rate. In order to test this hypothesis, we choseto use BSA as the non-catalytic protein. The results of this studyshow how non-specific complexes between HA and HAaseinhibited the HAase catalytic activity towards HA and how,depending on the BSA concentration with respect to the HAaseand HA concentrations, BSA either removed this inhibition orinduced inhibition of the hydrolysis of HA catalyzed by HAase.

2. Results

2.1. Existence of HA–protein electrostatic complexes

The formation of non-specific complexes between HA and thenon-catalytic protein has been studied by using spectrophoto-metric measurements. Spectra of a 0.73 g L−1 HA solution, a 1 gL−1 BSA solution and a 0.73 g L−1 HAplus 1 g L−1 BSAmixture

244 B. Deschrevel et al. / Matrix Biology 27 (2008) 242–253

at low ionic strength and at physiological-type ionic strength, allat pH 4, were carried out. For the sake of clarity, throughout thepaper, low ionic strength signifies that no salt was added in themedium and that the ionic strength, mainly due to the buffer, wasclose to 5 mmol L−1 and, physiological-type ionic strengthsignifies that 0.15mol L−1 salt were added to themedium and thatits ionic strength was thus close to 0.155 mol L−1, a valuecorresponding approximately to what is usually called physiolo-gical ionic strength. As expected (Xu et al., 2000), Fig. 1 showsthat at low ionic strength, the HA and BSA spectra were clearlynot additive; the spectrum of the mixture, with values of absor-bancemuch higher than those corresponding to the sum of theHAand BSA spectra, was characteristic of turbidity. Conversely, atphysiological-type ionic strength, the spectrum of the mixtureclosely resembled to the sum of the HA and BSA spectra.

Absorbance at 400 nm of mixtures containing 0.73 g L−1 HAand 2 g L−1 BSA were recorded over time. At pH 4 (Fig. 2), atphysiological-type ionic strength, the absorbance did not vary as afunction of time and was equal to the sum of the individualabsorbances of HA and BSA, while at low ionic strength, theabsorbance strongly increased in less than 1 min and then quicklydecreased and stabilized at a value close to 0.8. At physiological-type ionic strength, we observed the same behavior at pH 5 (Fig. 2)as at pH 4. At low ionic strength, the absorbance increase wasextremely low at pH 5 (Fig. 2) compared to that measured at pH 4.The same experiments were performed with HAase instead ofBSA. At physiological-type ionic strength, at pH 4 as well as at pH5, the absorbance increased a bit immediately after the addition ofHAase. and then it stayed nearly constant (Fig. 3). This increasecorresponded to the absorbance of theHAase solution.At low ionicstrength, the same behaviorwas observed at pH4 and 5 (Fig. 3): theabsorbance increased, then decreased and finally tended to stabilizeat a value higher than that measured at physiological-type ionicstrength. However, the rates of absorbance increase and decreasewere lower at pH 5 than at pH 4.

Fig. 1. Spectra between 250 and 650 nm of a 0.73 g L−1 HA solution, a 1 g L−1

BSA solution and a 0.73 g L−1 HA plus 1 g L−1 BSA mixture at low ionicstrength and at pH 4.

In these experiments, turbidity indicated that non-specificcomplexes had formed following mixing of HA and protein.The fact that turbidity appeared only under low ionic strengthconditions indicated the electrostatic nature of the interactionsestablished between HA and protein. At physiological-typeionic strength, the concentration of small ions was sufficientlyhigh to screen at least part of the electric charges borne by HAand protein and thus prevented the formation of a suspension ofHA–protein complexes. With a pKa value close to 2.9 (Berriaudet al., 1998; Cleland et al., 1982), at pH 4 and above, HA is apolyanionic molecule. According to the isoelectric pH (pI)value for BSA, estimated to be close to 5.0 (Peters, 1975; Wanget al., 1996; Xu et al., 2000), the net charge of BSA is positive atpH 4 and close to zero at pH 5, while the net charge of HAase,whose pI is around 6.8 (Astériou, 2002), is positive at pH 4 aswell as at pH 5. Knowing the charge state of HA, HAase andBSA according to the pH allows us to explain why, at low ionicstrength, HAase was able to form with HA complexes insuspension at pH 5 as well as at pH 4, whereas BSAwas able toform such complexes only at pH 4. Therefore, in order to studythe effect of complex formation between BSA and HA on thekinetics of HA hydrolysis catalyzed by HAase, kinetic expe-riments were performed at pH 4 instead of pH 5, as was done inour previous study (Astériou et al., 2006).

The major difference between the HA/BSA and the HA/HAase systems is that HAase, contrary to BSA, is able to formwith HA specific complexes associated with its catalytic acti-vity. In other words, the development of the composition of thereaction medium as a function of time for the HA/HAase systemincludes the formation of specific and non-specific complexes,while for the HA/BSA system, this development is due only tothe formation of non-specific complexes. In the case of the HA/HAase mixtures (Fig. 3), the absorbance increase was due to theformation of non-specific complexes between HA and HAase.The subsequent absorbance decrease was due to both the stabi-lization of the heterogeneous system, as observed for the HA/BSA system, and a progressive decrease in HA chain sizeresulting from the catalytic activity of HAase. Indeed, it hasbeen reported (Meyer and Rapport, 1952) that short HA chains(lower than 6 103–8 103 g mol−1) are not able to form withproteins complexes in suspension. Thus, for the HA/HAasemixtures at low ionic strength, the fact that the rate of absor-bance decrease was higher at pH 4 than at pH 5 (Fig. 3) is incomplete agreement with the kinetic results according to whichthe HAase-catalyzed HA hydrolysis rate is higher at pH 4 thanat pH 5 (results not shown).

2.2. Effect of BSA addition on the kinetics of the HA hydrolysiscatalyzed by HAase

In order to test the effect of BSA on the kinetics of HAhydrolysis by HAase, a preliminary experiment was performedat low ionic strength and at pH 4.85. The HA concentration was0.73 g L−1 and HAase was 0.5 g L−1. The ratio of the HA andHAase concentrations was high enough for non-specific com-plex formation to occur, and low enough for enzymatic hy-drolysis to be measurable. Fig. 4 shows that, as expected,

Fig. 2. Absorbance at 400 nm, as a function of time, of mixtures containing 0.73 g L−1 HA and 2 g L−1 BSA at low ionic strength (●) and at physiological-type ionicstrength (■), at pH 4 and pH 5.


enzymatic hydrolysis occurred but the reaction rate was verylow. After 50 min, a small volume of concentrated BSA solutionwas added, so that the BSAwas 2 g L−1. We observed (Fig. 4)that the addition of BSA gave rise to a strong and immediateenhancement of the hydrolysis rate, elevated from 0.7 to17 μmol L−1 min−1, or 25-fold. This result was in agreementwith our hypothesis based on the formation of non-specificcomplexes between HA and proteins and on the existence of acompetition between BSA and HAase to form those complexes.A more detailed study of the effect of BSA on the kinetics of theHA hydrolysis catalyzed by HAase was thus carried out.

2.3. Influence of the BSA concentration on the kinetics of theHA hydrolysis catalyzed by HAase

A series of HA enzymatic hydrolysis experiments wereperformed with various BSA concentrations, at low ionic

Fig. 3. Absorbance at 400 nm, as a function of time, of mixtures containing 0.73 g L−1

strength (■), at pH 4 and pH 5.

strength and at pH 4. The concentration of HA in the reactionmedium was 0.73 g L−1, that of HAase was 0.5 g L−1 and theBSA concentration ranged from 0 to 4 g L−1. For eachcondition, the experimental points, obtained from our improvedversion of the Reissig assay (Astériou et al., 2001; Reissig et al.,1955), were fitted by the bi-exponential model developed byVincent et al. (2003). The initial hydrolysis rate was thencalculated.

Fig. 5 gives the initial hydrolysis rate plotted against BSAconcentrations and demonstrates that three domains should beconsidered with respect to the BSA concentration. In the firstdomain, corresponding to BSA concentrations between 0 and0.1 g L−1, the initial hydrolysis rate remained very low. TheBSA concentration was thus too low for competition betweenBSA and HAase to occur for non-specifically complexed HA;the HA concentration was high enough to complex both HAaseand BSA. Under these conditions, the concentration of free and

HA and 2 g L−1 HAase at low ionic strength (●) and at physiological-type ionic

Fig. 4. HA reducing end concentration plotted against the reaction time for the HA(0.73 g L−1) hydrolysis catalyzed by HAase (0.5 g L−1) in 5 mmol L−1 phosphatebuffer at pH 4.85 and 37 °C. After 50 min of reaction, BSA was added to thereaction medium; the BSA concentration in the reaction medium was 2 g L−1.

Fig. 5. Initial hydrolysis rate plotted against the BSA concentration in thereaction medium for the HA (0.73 g L−1) hydrolysis catalyzed by HAase (0.5 gL−1) in 5 mmol L−1 phosphate buffer, at pH 4 and 37 °C.


catalytically active HAase, and thus the initial reaction rate, didnot increase. In the second domain, corresponding to BSAconcentrations between 0.1 and 0.5 g L−1, the initial hydrolysisrate greatly increased up to a maximum value. The initialreaction rate was 25 times higher in the presence of 0.5 g L−1

BSA than without BSA. In that BSA concentration range, thecompetition between BSA and HAase to form non-specificcomplexes with HA occurred, led to an increase in the fractionof free and catalytically active HAase molecules and thus, to anincrease in the initial hydrolysis rate: the higher the BSA con-centration, the higher the concentration of free HAase and thehigher the initial hydrolysis rate. For a BSA concentration of0.5 g L−1, the concentration of free HAase reached its maxi-mum value, which probably corresponded to a value close to thetotal HAase concentration. In the third domain, correspondingto BSA concentrations higher than 0.5 g L−1, the initialhydrolysis rate slowly decreased and, for a BSA concentrationequal to 4 g L−1, reached a value slightly higher than thatobtained without added BSA. As the BSA concentration wasincreased, HA complexed more and more with BSA. Thus,although the concentration of free and catalytically activeHAase was maximum, the ability of HAase to form a specificenzyme–substrate complex became less and less because ofsteric hindrance, due to the increasing levels of complex forma-tion between HA and BSA.

2.4. Influence of BSA on the substrate–dependence of the HAhydrolysis catalyzed by HAase

HA hydrolysis experiments were performed with different HAconcentrations, ranging from 0 to 1.46 g L−1, at low ionicstrength, at pH 4 and with a 0.5 g L−1 HAase concentration. Forone series of experiments, the BSA concentration in the reactionmediumwas 0.25 g L−1, and for the other, noBSAwas added. Foreach hydrolysis experiment, the initial reaction rate was deter-

mined from the kinetic curve obtained by fitting the experimentalpoints. The substrate–dependence curves (i.e. initial hydrolysisrate plotted against HA concentration) obtained with 0.25 g L−1

BSA andwithout added BSA are given in Fig. 6. In the absence ofBSA, the substrate–dependence curve obtained at pH 4 had thesame shape as that previously obtained at pH 5 (Astériou et al.,2006): for increasing HA concentrations, the initial hydrolysisrate increased, reached a maximum at an HA concentration of0.37 g L−1, then decreased and finally reached a value close tozero for HA concentrations above 0.73 g L−1. As mentionedabove, according to our hypothesis, the atypical behaviorobserved at high substrate concentrations was due to non-specificcomplex formation between HA and HAase, which suppressesHAase enzymatic activity: the higher the HA concentration, thelower the concentration of free and catalytically active HAase.

In the presence of 0.25 g L−1 BSA, the initial hydrolysis rateincreased as the HA concentration increased, and reached amaximum at an HA concentration close to 0.73 g L−1. Themaximum initial reaction rate was thus obtained for a higher HAconcentration in the presence of 0.25 g L−1 than in the absenceof BSA. For HA concentrations ranging from 0 to 0.73 g L−1,the higher the HA concentration, the higher the enhancement ofthe initial hydrolysis rate resulting from addition of BSA. ForHA concentrations up to 0.37 g L−1, even though there was freeand catalytically active HAase in the absence of BSA, the factthat initial reaction rates were enhanced by addition of BSAshowed that, even in these cases, a portion of HAase wascomplexed to HA and thus inactive. By forming complexeswith HA, the added BSA allowed HAase to be released and thisin turn increased the initial reaction rate. In fact, in the absenceof BSA, the increase in the initial rate was due only to theincrease in the substrate concentration; this increase in HAconcentration also led to a greater non-specific complex forma-tion with HAase and thus to a decrease in the concentration ofactive HAase. In the absence of BSA, for HA concentrations

Fig. 6. Influence of the HA concentration on the initial hydrolysis rate of the HAhydrolysis catalyzed by HAase (0.5 g L−1) in 5 mmol L−1 phosphate buffer, atpH 4 and 37 °C. HA enzymatic hydrolysis performed in the absence (●) and inthe presence of 0.25 g L−1 (■) BSA.

Fig. 7. Influence of the HAase concentration on the initial hydrolysis rate of theHA (0.73 g L−1) hydrolysis catalyzed by HAase in 5 mmol L−1 phosphatebuffer, at pH 4 and 37 °C. HA enzymatic hydrolysis performed in the absence(▲) and in the presence of 1 g L−1 (▼) BSA.


ranging from 0.37 to approximately 0.73 g L−1, the increase inthe substrate concentration was not sufficient to compensate forthe decrease in the active HAase concentration and thus, theinitial reaction rate decreased. When 0.25 g L−1 of BSA wasadded, sufficient HAase was released to increase the initialreaction rate. For HA concentrations ranging from approxi-mately 0.73 to 1.46 g L−1, the addition of BSA still had apositive effect on the initial hydrolysis rate, but this effectdiminished as the HA concentration was increased and becamenearly non-existent for an HA concentration of at least 1.46 gL−1. In that range of HA concentrations, the fact that, in theabsence of BSA, the initial rate was close to zero, indicated thatnearly all HAase was non-specifically complexed to HA. Fig. 5indicates that, with an HA concentration as high as 0.73 g L−1,0.5 g L−1 HAase and 0.25 g L−1 BSA, the portion of HAasereleased was not maximum but was high. Thus, in the presenceof 0.25 g L−1 of BSA, the maximum of the initial rate for thesubstrate–dependence curve (Fig. 6) probably corresponded toan HA concentration somewhat higher than 0.73 g L−1. More-over, with a total concentration of proteins (0.5 g L−1 HAaseand 0.25 g L−1 BSA) maintained at a constant level, increasingthe HA concentration above that corresponding to the maxi-mum of the initial rate led to a progressive decrease in thecompetition between BSA and HAase in forming non-specificcomplexes with HA. Consequently, although all the BSA wascomplexed to HA, the portion of released HAase decreased andthe concentration of free and active HAase tended towards zeroat an HA concentration at least equal to 1.46 g L−1. In otherwords, with an HA concentration at least equal to 1.46 g L−1

and above, and an HAase concentration equal to 0.5 g L−1, a0.25 g L−1 BSA concentration was not sufficiently high toinduce any release of HAase.

In order to verify the last point, two additional experimentswere performed with 1 and 4 g L−1 of BSA respectively, the HAconcentration being 1.46 g L−1 and that of HAase 0.5 g L−1. In

the presence of 1 g L−1 of BSA, the initial reaction rate was33 μmol L−1 min−1 and with 4 g L−1, it was 17 μmol L−1

min−1. As expected, 1 g L−1 of BSA clearly enhanced the initialhydrolysis rate, compared to that obtained with 0.25 g L−1 ofBSA or without BSA. This confirmed that the BSA concentra-tion necessary to give rise to HAase release increased withincreasing HA concentrations. However, for a given HA con-centration, to avoid HA becoming too greatly complexed withBSA and thus unable to form catalytic complexes with HAase,the BSA concentration must not be too high. This was partiallythe case when using 4 g L−1 of BSA: although the maximumHAase was free, the high level of BSA complexed with HAsuppressed HAase catalytic activity.

2.5. Influence of BSA on the enzyme–dependence of the HAhydrolysis catalyzed by HAase

In order to study the influence of BSA on the enzyme–dependence curve of the HA hydrolysis catalyzed by HAase atpH 4 and at low ionic strength, two series of experiments wereperformed with reaction media containing 0.73 g L−1 HA. Forthe first series, HAase concentrations ranged from 0 to 3 g L−1

and no BSA was added, and for the second series, HAaseconcentrations ranged from 0 to 2 g L−1 and 1 g L−1 of BSAwasadded. At low ionic strength and without added BSA, as for thesubstrate–dependence curve, an atypical shape was observed forthe enzyme–dependence curve of HA hydrolysis catalyzed byHAase (Fig. 7). For HAase concentrations equal to 0.5 g L−1 andbelow, the initial hydrolysis rate remained very low and close tozero, instead of increasing with increasing HAase concentrationas it should normally do. In good agreement with previousresults, under these conditions, the ratio of the HA and HAaseconcentrations was too high for the concentration of free andcatalytically active HAase to be significant; HAase formed non-specific complexes with HA. Above an HAase concentration of


0.5 g L−1, the shape of the enzyme–dependence curve becamenormal: the initial reaction rate linearly increased when theHAase concentration was increased. With 0.73 g L−1 HA, thenon-specific complex formation between HA and HAase hadreached its maximum level and thus, the HAase concentrationwas sufficiently high for a portion of HAase to be free and active.This portion of free HAase increased linearly with increasing theHAase concentration, and so did the initial hydrolysis rate.

With an HA concentration of 0.73 g L−1 and an HAaseconcentration of 0.5 g L−1, according to the results on Fig. 5, aBSA concentration of 1 g L−1 corresponded to a level of BSAcomplexed with HA slightly higher than that necessary to obtainthe maximum concentration of free and active HAase. In otherwords, with 0.73 g L−1 HA, a concentration of BSA of 1 g L−1

must be high enough for a maximum of HAase to be free andcatalytically active whatever the enzyme concentration used.Fig. 7 shows that, as expected, in the presence of 1 g L−1 BSA,the enzyme–dependence curve had a normal shape: the initialhydrolysis rate linearly increased as the enzyme concentrationwas increased.

3. Discussion

The ability of proteins to form complexeswith polyelectrolytessuch as polysaccharides has been recognized for a long time andhas various applications. The spectrophotometric results of thepresent study indicated that, at low ionic strength and at pH 4,BSA and HA formed non-specific complexes, as revealed by theappearance of turbidity uponmixing these two species. In fact, theability of proteins to produce complexes with HA has been usedsince 1940, when Robertson et al. (1940) described the MucinClot Prevention method for HAase assay, which was based on aprecipitate formation when adding acidified serum to HA. In1944, Kass and Seastone (1944) published an HAase assaymethod in which addition of acidified horse serum to HAsolutions gave a turbid suspension. Later, Dorfman andOtt (1948)suggested substituting horse serum albumin for serum. Thisturbidimetric method was further developed (Tolksdorf et al.,1949), introducing the definition of the turbidity reducing unitbased on the formation of a turbid solution when BSA is mixedwith HA at pH 3.8. It constitutes the current United StatesPharmacopeia XXII assay for HAase (US Pharmacopeia, 1990).Moreover, our spectrophotometric results are in agreement withthose of Xu et al. (2000) who have shown that, under 10−3 molL− 1 ionic strength, according to the pH and the BSAconcentration, HA and BSA may form soluble or insolublecomplexes. On the basis of these observations, themost importantresult from our spectrophotometric measurements is that HAasetoo was able to form non-specific complexes with HA. The factthat both pH and ionic strength influence the formation of the non-specific complexes between HA and BSA or HAase clearlyindicates the electrostatic nature of the interactions involved inthese complexes. In addition, the turbidimetric behavior of eachHA/protein system studied with respect to pH agrees with the pKa

value ofHA and the pI values of the two proteins, the difference inthe behavior between the two proteins being related to thedifference between their pI values.

As mentioned above, at low ionic strength, at pH 4 as well asat pH 5, HA and HAase form non-specific complexes. For agivenHAase concentration, the higher the HA concentration, thelower the portion of free HAase, up to an HA concentrationabove which the portion of free HAase became very close tozero. Thus, when studying the substrate–dependence of theHAase-catalyzed hydrolysis of HA under these conditions,increasing the HA concentration consisted in both an increase inthe substrate concentration and a decrease in the portion of freeand catalytically active HAase. In fact, the substrate–depen-dence curves obtained clearly showed that, when involved in acomplex with HA, HAase is unable to catalyze the enzymatichydrolysis. Indeed, if HAase in the non-specific complexes HA–HAase was catalytically active, the initial hydrolysis rate shouldnot decrease and should not reach a value near zero at high HAconcentrations. The atypical shape of the substrate–dependencecurves can thus be explained as the result of two opposite effectsassociated to the increase in the HA concentration: i) an increasein the substrate concentration, which allows the initial reactionrate to increase, and ii) a decrease in the concentration of thecatalytically active enzyme, which leads to a decrease in theinitial reaction rate. The fact that HAase in the non-specificcomplex HA–HAase is catalytically inactive means that HAmay behave as an inhibitor of HAase in addition to being itsusual substrate. This original result brings up a question aboutthe value of the stability constant for the non-specific complexHA–HAase as compared to that of the specific catalytic complexHA–HAase. Indeed, the kinetic results suggest that the non-specific complex is more stable than the specific one.

All the kinetic results of the present study show that BSAwasable to compete with HAase to form non-specific complexeswith HA, with, as a consequence, the release of free HAasewhich thus regained its catalytic activity. This means that thestability of the non-specific complex HA–BSAwas higher thanthat of the non-specific complex HA–HAase. In fact, as long asthe added BSA allowed the release of HAase by forming non-specific complexes with HA, it induced an increase in the initialrate of the HA hydrolysis catalyzed by HAase. Under theseconditions, BSA acted as an activator of HAase or, in otherwords, it prevented the inhibition of HAase by HA. Conversely,when the maximum HAase was released after the addition ofenough BSA, further addition of BSA led to an increase in thelevel of BSA complexed with HA: the more BSA complexedwith HA, the lower the initial reaction rate, because thecomplexed BSA hinders the formation of specific complexesbetween HA and HAase. In these cases, BSA behaved as aninhibitor of HAase. Thus, according to its concentration withrespect to the HA and HAase concentrations, BSA may acteither as an activator or as an inhibitor of HAase.

The effects of proteins on HAase activity have already beenreported. The first studies, reviewed by Mathews and Dorfman(1955), date back to the 1940s and concern the influence of serumonHAase activity. According to these studies, serum,whatever itsorigin, inhibited HAase. As early as 1941, Hobby et al. (1941)ascribed this inhibition to salt formation between serum albuminand HA. Gacesa et al. (1981) noted that the extent of inhibitionwas largely dependent upon the ratio of enzyme over serum


quantities. These results may be explained by the fact that the totalprotein concentration in serum was high compared to the HAaseconcentration used, so that serum proteins, by allowing for a highlevel of proteins complexed with HA, behave as HAase inhibitor.Another explanation given for HAase inhibition by serum is thepresence in serum of an HAase inhibitor whose level is increasedin the sera of patients with cancer, liver disease and dermatolo-gical disorders (Mio et al., 2000). Attempts to characterize thisserum inhibitor conclude that it is a high-molar mass thermolabileglycoprotein requiring magnesium for its inhibiting activity (Mioet al., 2000;Mathews andDorfman, 1955; Kolarova, 1975)whichis observed at pH values above 5 (Kolarova, 1975; Gacesa et al.,1981; Mio et al., 2000). The fact that this serum inhibitor has noeffect on Streptomyces HAase whilst it inhibits bovine testicular,snake and bee venom HAases (Mio et al., 2000) indicates that itsinhibition activity is not by complexing to HA. This seruminhibitor, identified byMio et al. (2000) as belonging to the inter-α-inhibitor family, may thus be a more specific HAase inhibitorthan other serum proteins and be an inhibitor whose activity maydirectly concern HAase. The results reported by Fiszer-Szafarz(1968) show that the sera of cancer patients have a greaterinhibitory activity towards HAase than normal sera. However, theinhibition activity of cancerous sera is magnesium-independent.In fact, the high serum protein contents used in this study suggestthat this inhibition could, at least partly, be due to a high level ofproteins complexed with HA.

At pH values below 5, Gacesa et al. (1981) observe a markedenhancement of bovine testicular HAase activity upon additionof serum to incubation mixtures. They further show that thegreatest activation effect is obtained by using BSA as well ashuman serum albumin. Gold (1982) shows that both bovinetesticular and human liver HAases exhibit increased activity inthe presence of BSA at pH 4. More recently, Maingonnat et al.(1999) report a dependence of HAase activity with respect toprotein concentration at pH 3.8 that is very similar to that shownin the present study. Our experimental results allowed us toconfirm these authors regarding the origin of the inhibitoryeffect of high protein content. However, this is not the caseconcerning the activation effect of proteins. Indeed, accordingto Maingonnat et al. (1999), the protein activation effect may bedue to the fact that HA can bind small amounts of proteins thatfacilitate the opening of the HA random coil, thus facilitatingHAase accessibility to HA. But, if such is the case, the inhibi-tion we observed at low ionic strength and at high substrateconcentration should not depend on enzyme concentration(Asteriou et al., 2006). An interesting result of the study ofMaingonnat et al. (1999) is that the protein effect on the HAaseactivity is observed not only with BSA, but also with humanserum albumin, immunoglobulins, hemoglobin, transferrin and,interestingly, with HNwhich is a hyaladherin. It should be notedhere that the dissociation constant for the specific complex HA–HN, equal to 10−9 mol L−1 (Maingonnat et al., 1999), is of thesame order of magnitude as those for the non-specific complexesHA–BSA and HA–lysozyme, which are about 10−9 and10−8 mol L−1 respectively (Van Damme et al., 1994). Lysozymeis present in cartilage (Van Damme et al., 1994) and albumin is amajor protein of synovial fluid (Scott et al., 2000), two areas

which are also rich in HA. Knowing that the interactionsinvolved in the complex HA–BSA (Xu et al., 2000) and in thecomplex HA–lysozyme (Van Damme et al., 1994) are electro-static, and that a charge patch able to accommodate an HAdecamer exists at the surface of human serum albumin (asGrymonpré et al. (2001) have shown with integrated computermodeling), we suggest that the binding of HA to albumin andlysozyme might in fact be specific. Here, the specificity of thebinding would not be associated to 3D structural features orspecific sequences, but rather to the existence of such chargepatches on protein surfaces.

Non-specific (or maybe specific) interactions between poly-electrolytes and proteins may thus be responsible for enzymaticinhibition as well as activation behaviors. The inhibitionbehaviors may result from the formation of two types of com-plexes: i) polyanion–HAase complexes, which directly inacti-vate HAase, and ii) HA–polycation complexes, whichindirectly inactivate HAase by hindering HAase accessibilityto HA. Many polyanions, such as glycosaminoglycans (heparin,heparan sulfate, dermatan sulfate), HA derivatives (O-sulfo-nated HA) and synthetic polyanions (poly(styrene-4-sulfonate))are known to inhibit HAase (Mathews and Dorfman, 1955;Aronson and Davidson, 1967; Girish and Kemparaju, 2005;Toida et al., 1999; Isoyama et al., 2006). In the case of O-sulfonated HA, part of its inhibition activity could be of thecompetitive type (Toida et al., 1999). Indeed, such HA deri-vatives may behave as substrates analogues in inhibitingHAase. According to more recent studies, the inhibitory activityof all these compounds does not involve their binding to thecatalytic site of the enzyme. In good agreement with our findingabout the non-specific complexes HA–HAase, Girish andKemparaju (2005) suggest that inhibition by these polyanionsmight be due to the formation of non-specific electrostaticcomplexes between HAase and the polyanions. This phenom-enon is demonstrated in the case of heparin (Maksimenko et al.,2001). Such enzyme inhibition by formation of non-specificcomplexes between polyanion and enzyme has already beendocumented, for phospholipase A2 (Melo and Ownby, 1999;Diccianni et al., 1990), β-amylase and catalase, (Mathews andDorfman, 1955). Inhibition of HAase activity by formation ofHA–polycation complexes concerns, as mentioned above,proteins such as albumin, but also, polycationic polysaccharidessuch as chitosan. Denuziere et al. (2000) report that HA andchitosan form non-specific electrostatic complexes which makethe substrate unavailable for HAase. Considering the activationeffect of low protein content, it might also be interesting toinvestigate the possible activation effect of low chitosanconcentrations.

One of the more important questions, for which we canalready highlight some pertinent points for further research,concerns the extent of inhibition and activation effects withrespect to HAase within the ECM. The ECM is rich in poly-electrolytes such as proteins, glycosaminoglycans and otherpolysaccharides. The two essential conditions are thus pH andionic strength. The ability of a protein and a polysaccharide toform a non-specific complex is related to the pI and pKa valuesof the two species respectively. Considering the diversity in the


ECM, one might expect that HAase as well as HA could beinvolved in non-specific complexes at pH close to neutrality.For example, Van Damme et al. (1994) show that HA andlysozyme, a protein secreted into the ECM of cartilage, formnon-specific complexes at pH 7.5 and under 0.04 mol L−1 ionicstrength. Such a low ionic strength is likely to exist in cartilagebecause of the exclusion of small ions by its highly chargedproteoglycans (Van Damme et al., 1994). However, theinhibition and activation effects associated with non-specificcomplex formation probably exist under higher ionic strength.Indeed, the activation and inhibition of HAase by proteins isobserved by Maingonnat et al. (1999) when using an ionicstrength close to 0.15 mol L−1. Similarly, the activation ofHAase upon addition of BSA reported by Gacesa et al. (1981) ismeasured with reaction mixtures containing 0.1 mol L−1 NaCl.Moreover, while the spectrophotometric measurements withHA and BSA at pH 5 showed no turbidity, the strong activationeffect observed upon addition of BSA in the reaction mixture inour preliminary experiment at pH 4.85 suggested that solublenon-specific complexes may exist at pH close to 5 and have thesame effect as the complexes in suspension observed at pH 4.

The possible existence of HA–protein and/or polyelectro-lyte–HAase complexes, including HA–HAase non-specificcomplexes, in ECM tissue may play an important role in theregulation of the HAase activity under normal and pathologicalconditions. Indeed, such complexes would allow for HAase tobe present but inactive in ECM. This situation would corres-pond to that suggested by Stern (2005), in which HAase must bedeposited in ECM together with its inhibitor in the same way asmatrix metalloproteinases (MMP) are deposited together withtissue inhibitors of MMP (TIMP). If HAase inhibition is due tothe existence of non-specific complexes, HAase activity mightthus be restored by any processes able to dissociate thosecomplexes. Such processes might be a slight pH or ionicstrength variation associated with ion exchange or, in the case ofnon-specific HA–HAase complexes, the presence of a protein,which might be a hyaladherin, capable of forming an HA–protein complex that would be more stable than the complexformed with HAase.

The existence of HA–protein and/or polyelectrolyte–HAasecomplexes in ECM tissue might also account for some of thedifficulties encountered in attempts to purify HAase. Indeed, itis observed that for highly purified HAase preparations noenzymatic activity is detectable in the absence of added proteins(Maingonnat et al., 1999; Mathews and Dorfman, 1955).According to our results, we may suggest that with thesepurified HAase preparations, HAase undergoes non-specificcomplex formation with HA and is thus inactivated. Of course,it has been observed (Stern, 2005) that, in addition to beingpresent at exceedingly low concentration, when HAase ispurified, it requires the presence of detergents and proteaseinhibitors to maintain its catalytic activity. These species areneeded to avoid enzyme denaturation. Thus, added BSA couldact by preventing HAase denaturation. However, our spectro-photometric and kinetic results show that to be catalyticallyactive HAase should not undergo non-specific complexformation with HA. Therefore, even though proteins such as

BSA could prevent enzyme denaturation by interacting withHAase, their interaction with HA allows non-specific HA–HAase complex formation to be avoided. Delpech (personalcommunication) observes that preparations of HAase, which donot show any enzymatic activity when they become highlypurified, have the same significant activity whether Triton X100or albumin is added to the reaction mixture. On the basis of ourresults, we may suggest that by interacting with HAase, TritonX100 allows for HAase to be catalytically active by preventingboth HAase denaturation and HA–HAase complex formation.Moreover, the ability of HA and HAase to form non-catalyticcomplexes with each other and with various polyelectrolytesmight also explain why no enzymatic activity has yet beendetected in HYAL3 and HYAL4 gene products. Consideringthese phenomena of complex formation might be useful indesigning an appropriate HAase activity assay method fordetecting new HAase activity.

Finally, although biochemists, in an attempt to identify asingle function for biomacromolecules such as enzymes aremaking great efforts to purify them to homogeneity, the presentstudy shows that biomacromolecules may not have just onebiological function. In addition, interactions between the bioma-cromolecules themselves may modulate their functions. Thisoften neglected fact should be kept in mind when attempting toelucidate the biological functions of biomacromolecules andalso when therapeutic uses of these molecules are to beconsidered.

4. Experimental procedures

4.1. Preparation and characterization of the HA solution

Sodium HA from human umbilical cord was obtained fromSigma (H 1876, lot 127H0482). An HA stock solution wasprepared by dissolving HA in 5 mmol L−1 phosphate (Prolabo28 015.294) buffer, then it was divided into fractions and storedat −20 °C before use. The HA average molar mass (

PMn ) and its

polydispersity index (Ip) were determined by steric exclusionHPLC equipped with both multi angle laser light scatterring andrefractive index detectors (Tranchepain et al., 2006): (

PMn ) was

equal to 1.45 106 g mol−1 and Ip equal to 1.8. HA being highlyhygroscopic, we used the uronic acid assay method (Dische,1947; Bitter and Muir, 1962) to measure the actual HA contentof the HA mother solution. The experimental procedure was asdescribed previously (Vincent et al., 2003). The method wascalibrated using sodium glucuronate (Sigma G 8645). The HAstock solution contained 7.3 g of HA per litre.

4.2. Preparation of the HAase and BSA solutions

Bovine testicular HAase (EC 3.2.1.35) and BSA were fromSigma and were used without further purification. HAase(Sigma H 3884, lot 76H8025) had a specific activity of 990 units(US Pharmacopeia, 1990) per mg. Since the enzymatic activityof frozen HAase solutions did not remain constant, solutionsfreshly prepared by dissolving HAase in 5 mmol L−1 phosphatebuffer were used. Similarly, concentrated BSA solutions were


freshly prepared by dissolving BSA (Sigma A 3675, lot78H1399) in 5 mmol L−1 phosphate buffer.

4.3. Spectrophotometric measurements

The HA and protein solutions were diluted with 5 mmol L−1

phosphate buffer. When necessary, before pH adjustment withphosphoric acid (Sigma P 6560), an appropriate volume of aconcentrated sodium nitrate (Prolabo 27 950.298) solution wasadded to the HA and protein solutions to adjust ionic strength. Forionic strength adjustment of the solutions used to perform spectra,potassium chloride was substituted for sodium nitrate because ofthe absorbance of the latter between 250 and 350 nm. All thesolutions were preincubated at 37 °C. When the HA/proteinmixtures were analyzed, the HA solution was first placed in thecuvette and the addition of the protein solution corresponded totime zero for data recording. Quartz cuvettes of 1 cm pathlengthwere used to obtain the spectra of the HA, BSA andHA plus BSAsolutions. For measurements of the absorbance at 400 nm overtime, the HA/protein mixtures were placed in a 1 cm pathlengthplastic cuvette. In all cases, the cuvette was placed in the spec-trophotometer (Uvikon 860, Kontron) equipped with a magneticstirrer and maintained at 37 °C.

4.4. Kinetics of the HA hydrolysis

The HA stock solution was placed in a reactor, diluted to thedesired concentration with 5 mmol L−1 phosphate buffer, ad-justed to pH 4 with phosphoric acid, stirred and maintained at37 °C. An appropriate volume of a concentrated BSA solutionadjusted to pH 4 was then added to the HA solution. For theexperiments performed in the absence of BSA, the BSA wasreplaced by phosphate buffer at pH 4. The reaction was started2 min after the addition of BSA, by adding an adequate volumeof a concentrated HAase solution previously adjusted to pH 4.The BSA and HAase solutions were preincubated at 37 °C.

According to Gacesa et al. (1981) and Gold (1982), the factthat the enzyme is preincubated with or without the non-catalytic protein (serum, serum proteins or albumin) for severalhours before mixing with HA does not significantly modifyenzyme activity means that the non-catalytic protein does notact by preventing enzyme inactivation. By taking into accountour hypothesis, we chose to preincubate BSA with HA, thusallowing for BSA to complex with HA and under the expe-rimental conditions used, for the maximum HAase to be freeand catalytically active. We have tested the influence of thepreincubation time of BSA with HA: no significant differencesin the initial hydrolysis rate were observed for preincubationtimes ranging from 0 to 20 min and a preincubation time of2 min was retained.

Throughout the reaction, 200 μL aliquots of the reactionmedium were removed from the reactor and assayed with the N-acetyl-D-glucosamine reducing ends assay. The concentration ofN-acetyl-D-glucosamine reducing ends, generated from HAhydrolysis, was measured by using our improved version(Astériou et al., 2001) of the Reissig method (Reissig et al.,1955) according to the experimental procedure detailed in

Vincent et al. (2003). This improved version allows the turbi-dimetric component, due to the presence of proteins in thereaction medium samples, to be deduced from the total absor-bance measured at 585 nm, thus giving the colorimetric com-ponent of the absorbance. This color part of the absorbance at585 nm (Abs585) is related to the molar concentration of N-acetyl-D-glucosamine reducing ends, which also corresponds tothe molar concentration of HA reducing ends ([HA reducingends]), according to the following equation:

HA reducing ends½ � ¼ Abs585= 1:8103 � 1:63� �

where 1.8 ·103 corresponds to the slope of the calibration curveobtained by using N-acetyl-D-glucosamine (Sigma A 8625) and1.63 is a corrective factor due to the fact that in HA, N-acetyl-glucosamine is substituted in position 3 (Shimada andMatsumura, 1980; Vincent et al., 2003).

For each kinetic experiment, the hydrolysis reaction wasfollowed for 3 h and the concentration of the HA reducing endswas plotted against time. The experimental points obtained werefitted by the bi-exponential model described in Vincent et al.(2003) and the initial hydrolysis rate was calculated as beingequal to the value of the first derivative of that function at timezero.

Acknowledgements

We thank Dilys Moscato for reading the manuscript. We aregrateful to the “Conseil Régional de Haute-Normandie” for thefellowship granted to Trias Astériou, to the “Matériaux Poly-mères, Plasturgie” network for the fellowship granted to FrédéricTranchepain and to the FrenchResearch and TechnologyMinistryfor the fellowship granted to Hélène Lenormand.

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hyaluronidase activity is modulated by complex ing

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