From the Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India
Received for publication, February 24, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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Hyaluronidases constitute a special group of polysaccharide-degrading enzymes that have the ability to degrade primarily hyaluronan (HA),1 yielding various lengths of oligosaccharides as the end product (5). The bacterial hyaluronidases are called hyaluronate lyase. Various Gram-positive microorganisms including species of Streptococcus, Clostridium, Propionibacterium, Staphylococcus, and Peptostreptococcus produce hyaluronate lyase (6). This enzyme is either cell-associated or released outside of cell. It primarily degrades hyaluronan and certain chondroitin sulfates, the major components of host extracellular matrix of tissue (5, 7, 8). The HA degradation directly contributes to bacterial invasion by allowing greater microbial access to, or migration between, host tissues for colonization (8, 9). Because of this reason hyaluronate lyase is also being called "spreading factor." The protein sequence of four bacterial hyaluronate lyase from S. pneumoniae (10), Streptococcus agalactiae (11), Staphylococcus aureus (12), and Propionibacterium acnes (13) have been reported so far. Their sequence homologies range from 28 to 68% suggesting their structural, functional, and evolutionary similarities.
The crystal structure of S. pneumoniae hyaluronate lyase (SpnHL)
shows that the enzyme is built up of two domains, an N-terminal domain or
catalytic domain, which is predominantly -helical, and a C-terminal
domain or the supportive domain, which is predominantly
-sheet
(14). The two domains are
connected by a short linker polypeptide. The interface between the N- and
C-terminal domains is composed of several residues from the two domains
forming several interactions. The cleft in between the
- and
-domains is the place where the HA binding and degradation occurs and is
wide at the two ends and narrower in the middle. The crystal structure of
S. agalactiae hyaluronate lyase is similar to SpnHL in whole
structure architecture and active site geometry except that it has an
additional
-sheet domain before the N-terminal
-domain present in
the SpnHL (15).
Although crystal structure of hyaluronate lyase and S. agalactiae in complex with HA has been reported (1416), detailed insight into important structural features like existence of co-operative interactions between C- and N-terminal domains and the specific role of C-terminal domain in functional activity of SpnHL have not been provided so far. Besides this, despite the importance of SpnHL in pneumococcal infection no sincere efforts to search suitable inhibitors for this enzyme have been made (5, 17).
We report detailed studies on the structural changes associated with the thermal denaturation and guanidine hydrochloride-, calcium chloride-, and magnesium chloride-induced unfolding of SpnHL using optical spectroscopic techniques, limited proteolysis, and enzymatic activity measurements. The N-terminal domain of SpnHL was isolated and purified after limited proteolysis. Comparative analysis of enzymatic activity and stability of the isolated N-terminal domain of SpnHL with that when present along with C-terminal in the native enzyme have also been reported. Furthermore, efficiency of guanidine hydrochloride (GdnHCl), L-arginine, L-arginine methyl ester (L-NAME), and guanidine isothiocyanate (GdnHSCN) as inhibitors of SpnHL has also been studied.
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EXPERIMENTAL PROCEDURES |
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Overexpression and PurificationOverexpression and purification for histidine-tagged protein was carried out as described earlier (18). Purification of non histidine-tagged protein was carried out using various chromatographic techniques. Briefly, an overnight culture was harvested, and the cell pellet was resuspended in 20 mM Tris-Cl, pH 7.4, containing 2 mM EDTA. After sonication the cell lysate was loaded onto Mono-Q pre-equilibrated with the same buffer. The column was washed with the same buffer containing 100 mM NaCl, and the protein was eluted with the linear gradient of buffer containing 100500 mM NaCl. The fractions containing SpnHL was pooled, and ammonium sulfate was added to a final concentration of 1.7 M. The protein solution was loaded onto phenyl-Sepharose (high substituted) pre-equilibrated with the same buffer containing 1.7 M ammonium sulfate. The protein was eluted using a linear gradient of 1.7 to 0 M ammonium sulfate. The active fractions were pooled, concentrated, and finally purified on a Superdex 200 HR 10/30 column on AKTA fast protein liquid chromatography equilibrated with the same buffer without ammonium sulfate. Eluted protein was finally concentrated, dialyzed against the 20 mM Tris-HCl, pH 7.4, and stored as a 10 mg/ml stock solution at 4 °C. The purity of purified enzyme was checked by electrospray ionization mass spectrometry and found to be 99% pure. The final purified protein sample was also subjected to the N-terminal protein sequencing.
Denaturation/Unfolding of SpnHLSpnHL (1 µM) was incubated in the presence/absence of increasing concentration of GdnHCl or CaCl2 or MgCl2 and incubated for 1 h at 25 °C before taking the measurements. Initial studies were carried out with both the histidine-tagged and non-histidine-tagged enzyme. Similar results were obtained with both enzymes, so only histidine-tagged enzyme was used for further studies, as it was easy to purify.
Assay of Enzymatic ActivityThe activity of SpnHL is defined as its ability to breakdown HA to unsaturated disaccharide units (20). In a 1-ml solution of 0.2 mg/ml HA in 50 mM sodium acetate buffer, 10 mM CaCl2, pH 6.0 (or the different salt concentration was maintained in the HA solution), 10 µl of enzyme sample (4 ng/µl, diluted just before taking measurement or stated otherwise) was added. The reaction mixture was incubated for 5 min during which the measurements were carried out by monitoring the increase in absorbance at 232 nm at 25 °C. Percent inhibition was determined as described previously (17).
Fluorescence SpectroscopyFluorescence spectra were recorded with a PerkinElmer Life Sciences LS 50B spectroluminesencemeter in a 5-mm path length quartz cell at 25 °C. For monitoring tryptophan fluorescence, excitation wavelength of 290 was used, and the spectra were recorded between 300 and 400 nm.
Circular Dichroism MeasurementsCD measurements were made
with a Jasco J800 spectropolarimeter calibrated with ammonium (+)-
10-camphorsulfonate. The results are expressed as the mean residual
ellipticity [], which is defined as [
] = 100 x
obs/(lc), where
obs is the
observed ellipticity in degrees, c is the concentration in moles of
residue per liter, and l is the length of the light path in
centimeters. The values obtained were normalized by subtracting the base line
recorded for the buffer having the same concentration of denaturant or salt
under similar conditions.
Limited ProteolysisSpnHL (0.2 mg/ml) incubated for 1 h under specified conditions was subjected to limited proteolysis with carboxypeptidase B under similar conditions like at 55 °C or in the presence of 0.75 M GdnHCl or 1.5 M CaCl2 at 25 °C for 15 min. A protease to enzyme ratio of 1:100 (w/w) was used for the study. The protease reaction was stopped by adding 2 mM EDTA to reaction mixture, and the samples were analyzed on 10% SDS-PAGE.
Size-exclusion ChromatographyGel filtration experiments were carried out on a Superdex 200 HR 10/30 column and Sephadex 75 HR 10/30 column on AKTA fast protein liquid chromatography (Amersham Biosciences) for the purification of native SpnHL and the N-terminal domain, respectively. Column was calibrated with various molecular weight standard markers (Amersham Biosciences) before use. Column was equilibrated and run with 20 mM Tris-HCl, pH 7.4, at 25 °C. 200 µl of the sample was loaded on the column and run at 25 °C, flow rate of 0.3 ml/min, with detection at 280 nm.
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RESULTS AND DISCUSSION |
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The Two Structural Domains of SpnHL Have Significantly Different
Stabilities
Thermal DenaturationThe thermal unfolding of SpnHL was
characterized by monitoring the loss of secondary structure of enzyme with
temperature. Fig. 1 summarizes
the changes in ellipticity at 222 nm of SpnHL at increasing temperature, where
two distinct transitions were observed between 20 and 100 °C. These
transitions were in the temperature regions from 35 to 60 °C and 65 to 100
°C and centered at about 50 and 82 °C, respectively. Furthermore, the
transitions were found to be reversible as they were also observed on cooling
the SpnHL samples from 100 to 20 °C after the heating scan (data not
shown). These observations suggest that the SpnHL molecule contains two
distinct structural domains of significantly different thermal
stabilities.
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Guanidine Hydrochloride UnfoldingThe guanidine hydrochloride-induced changes in the structural properties of SpnHL were studied using optical spectroscopic techniques. Time-dependent changes in structural parameters of SpnHL at increasing concentrations of GdnHCl showed maximum changes within1hof incubation and no further alteration up to the next 12 h (data not shown) suggesting that incubation time of 1 h is sufficient for achieving equilibrium under any GdnHCl concentration studied.
The GdnHCl-induced alterations in the tertiary structure of SpnHL were monitored by studying the changes in the tryptophan fluorescence of enzyme at increasing concentration of GdnHCl. If a significant difference exists between the fluorescence emission maxima of native and denatured protein, corelation between the GdnHCl concentration and the functional changes in wavelength at which fluorescence emission is maximal provides useful information on the stabilization of intermediates during protein unfolding (21). For SpnHL, the emission wavelength maximum in the absence of GdnHCl (fo) and at GdnHCl concentration greater than 2.5 M (fd) were 334 and 355 nm, respectively. The fractional change in wavelength at which the tryptophan fluorescence emission spectrum of SpnHL is maximal is plotted against the concentration of GdnHCl in Fig. 2A. Two well separated transitions were observed on increasing the GdnHCl concentration between 0 and 2.5 M. The first transition was very sharp and occurred between 0.4 and 0.7 M GdnHCl. A plateau region existed between 0.7 and 0.9 M and was followed by a broad second transition between 0.9 and 2 M GdnHCl. Furthermore, the changes in emission wavelength associated with the first transition was only from 334 to 341 nm, suggesting that only partial unfolding of the SpnHL molecule is associated with the first transition. At 2 M GdnHCl, an emission wavelength of 355 nm was observed indicating that all the tryptophan moieties present in the SpnHL are exposed to solvent under these conditions (22). Such a situation can arise only when the protein is completely unfolded.
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The effect of GdnHCl on the secondary structure of SpnHL was studied by
monitoring changes in the far-UV CD profiles of the enzyme. In the far-UV
region, the CD spectrum of native SpnHL shows the presence of substantial
-helical and
-sheet conformation.
Fig. 2B summarizes the
effect of increasing GdnHCl concentration on loss of ellipticity at 222 nm for
SpnHL. Two well separated transitions, a sharp transition between 0.4 and 0.75
M GdnHCl, and a broad transition between 0.9 and 2.2 M
GdnHCl were observed. Furthermore, only about 30% loss of secondary structure
was found to be associated with the first transition indicating only partial
unfolding of SpnHL molecules under these conditions. However, at GdnHCl
concentrations greater than 2 M, a complete loss of far-UV CD
signal was observed suggesting complete unfolding of SpnHL molecules at higher
GdnHCl concentrations.
As only partial unfolding of SpnHL molecules was found to be associated with the first transition observed at low GdnHCl concentrations, it seems that probably only one of the structural domains of enzyme unfolds under these conditions whereas the other domain remains intact. The thermal denaturation profile of 0.75 M GdnHCl-treated SpnHL further strengthened this possibility as only a single transition with mid-point of thermal transition (Tm) of about 40 °C was observed for this sample (Fig. 2C).
CaCl2 Interaction with SpnHL Results in Unfolding of Only
One Domain
Calcium ions are essential for the normal enzymatic activity of SpnHL
(18,
23). It has been speculated
that the calcium ions most likely bind to the C-terminal domain of SpnHL and
induce structural changes in the loop of the catalytic cleft
(24). We studied the effect of
CaCl2 and MgCl2 on the domain structure of SpnHL.
The CaCl2- and MgCl2-induced alterations in the tertiary structure of SpnHL were studied by monitoring the changes in the tryptophan fluorescence of enzyme. Fig. 3A summarizes the changes in the emission wavelength of tryptophan fluorescence of SpnHL at increasing concentrations of CaCl2 and MgCl2. A sigmoidal dependence of enhancement of emission wavelength (from 334 to 341 nm) with increasing concentration of CaCl2 or MgCl2 was observed. However, even at 1.5 M CaCl2 or MgCl2 an emission wavelength of about 341 nm was observed suggesting only partial unfolding of enzyme under these conditions (as discussed under "GdnHCl Denaturation"). The far-UV CD studies on SpnHL with increasing concentrations of CaCl2 or MgCl2 also support the suggestion of partial unfolding of enzyme by these salts. Fig. 3B shows the loss of CD ellipticity at 222 nm of SpnHL with increasing concentration of CaCl2 or MgCl2. A maximum of only about 30% loss of secondary structure was observed for CaCl2- or MgCl2-induced transition. As CaCl2 or MgCl2 induces only partial unfolding of SpnHL molecules it seems that these salts probably induce unfolding of only one of the structural domains of enzyme whereas the other domain remains intact.
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As GdnHCl (at low concentration), CaCl2, or MgCl2, which are all electrolytes, induced partial unfolding of SpnHL, the possibility of a nonspecific ion-induced effect on the enzyme was also explored. For this, the effect of NaCl on structural parameters of SpnHL was studied and is summarized in Fig. 2, A and B. NaCl even up to 2 M concentration showed no significant change in the tryptophan fluorescence or to the secondary structure of SpnHL indicating that NaCl does not induce any change in enzyme structure probably because of lack of its interaction with the enzyme. These observations collectively suggest that GdnHCl, CaCl2, and MgCl2 have specific interaction/binding sites with/in SpnHL.
Identification of Structural Domain Susceptible to Low
Temperature or Low GdnHCl Concentration or CaCl2
The factors determining the vulnerability for proteolysis of a protein by
protease depend on the conformational parameters such as accessibility,
segmental motion, and protrusions. For this reason limited proteolysis has
been effectively used to monitor structural domains in proteins,
ligand-induced conformational changes, and protein folding/unfolding
(25).
To characterize the structural domains corresponding to the transitions observed during thermal-, GdnHCl-, and CaCl2-induced unfolding of SpnHL (see Figs. 1,2,3) limited proteolysis studies were carried out under conditions where only one transition was complete. Fig. 4A summarizes the SDS-PAGE profile of the protein fragments obtained on limited proteolysis with carboxypeptidase B of the SpnHL molecule heated at 55 °C or treated with 0.75 M GdnHCl or 1.5 M CaCl2. For all three samples, a single protein band corresponding to a molecular mass of about 40 kDa was observed on proteolysis. There are about 96 cleavage sites of Carboxypeptidase B spread throughout the SpnHL molecule, including one in the linker region. However, limited proteolysis of SpnHL by the protease in all the conditions studied resulted in only a single fragment of about 40 kDa. This suggests that the obtained fragment is a folded one in which the proteolytic sites are buried in the protein interior and not accessible to protease for cleavage. The N-terminal amino acid sequencing of the 40-kDa fragments were carried out following Western blotting of protein bands on polyvinylidene difluoride membranes. In all the three cases the nine amino acids obtained corresponded to the first nine residues (ASVKDTYTD) of the N-terminal domain of SpnHL. These observations suggest that heating of the SpnHL molecule to 55 °C or treating it with 0.75 M GdnHCl or 1.5 M CaCl2 results in unfolding of only the C-terminal domain. The N-terminal domain of SpnHL obtained on limited proteolysis was purified by size exclusion chromatography (Fig. 4B). For characterization of the carboxypeptidase B cleavage site in SpnHL that generates the N-terminal fragment on proteolysis, electrospray ionization mass spectrometry analysis of the N-terminal fragments obtained were carried out. Only two peaks corresponding to molecular massed of 40501 and 42414.5 Da with relative intensity ratios of 3:1, respectively, were observed (data not shown). As the major peak corresponded to a molecular mass of 40501 Da, it suggests that the carboxypeptidase B cleavage site in the SpnHL is between Tyr520 and Lys521. This site is located at the end of N-terminal domain just before the polypeptide linkers analyzed from the crystal structure.
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Characterization of N-terminal Domain
The size exclusion chromatographically purified N-terminal domains of SpnHL
(Fig. 4B) were
analyzed for thermal stability, GdnHCl denaturation, and enzymatic
activity.
Thermal StabilityThe thermal denaturation profiles of the isolated N-terminal fragments were obtained by monitoring loss of CD ellipticity at 222 nm with increasing temperature. Fig. 5 summarize the Tm observed for the isolated N-terminal domain obtained on limited proteolysis of SpnHL at 55 °C and on treatment with 0.75 M GdnHCl. A single transition with the Tm of about 76 and 75 °C was observed for the two samples, which is very close to the Tm of 82 °C (Fig. 1) observed for N-terminal domain in the native enzyme. This observation demonstrates that the fragments obtained on proteolysis of SpnHL (Fig. 4B) are intact N-terminal domains. Furthermore, it also demonstrates that the stability of N-terminal domain was not affected by removal of C-terminal domain suggesting the absence of cooperative interactions between the two domains of SpnHL.
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GdnHCl DenaturationThe GdnHCl denaturation profile of the purified N-terminal domain obtained on limited proteolysis of SpnHL at 55 °C and on treatment with 0.75 M GdnHCl was studied using tryptophan fluorescence and far-UV CD spectroscopies and is summarized in Fig. 6. The alterations in the tertiary structure of N-terminal fragment of SpnHL were monitored by studying the changes in the tryptophan fluorescence at increasing concentration of GdnHCl. When fractional change in emission wavelength at which the fluorescence intensity of N-terminal fragment of SpnHL is maximal is plotted against the increasing concentration of GdnHCl, a sigmoidal transition in the GdnHCl concentration range between 0.5 and 2 M was observed (Fig. 6A). A Cm (denaturant concentration where 50% denaturation of protein is observed) of about 1.25 M GdnHCl was found to be associated with this transition.
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The effect of GdnHCl on the secondary structure of N-terminal fragment of
SpnHL was studied by monitoring changes in the far-UV CD profiles of the
enzyme. The CD spectrum of N-terminal fragment of SpnHL in the far-UV region
shows the presence of substantial -helical conformation.
Fig. 6B summarizes the
effect of increasing GdnHCl concentration on the loss of ellipticity at 222 nm
for N-terminal fragment of SpnHL. A single transition in the GdnHCl
concentration range between 0.5 and 2 M was observed. A
Cm of about 1.25 M GdnHCl was found to
be associated with this transition. As for both tryptophan fluorescence and
far-UV CD, a single transition between 0.5 and 2 M GdnHCl was
observed, which suggests that the N-terminal fragment of SpnHL undergoes a
cooperative unfolding with simultaneous loss of both the secondary and the
tertiary structure.
The thermal denaturation profile of N-terminal fragment of SpnHL obtained as described above, treated with 0.75 M GdnHCl, was studied. A single transition with a Tm of about 40 °C (Fig. 6C) was observed, which is similar to that observed for the 0.75 M GdnHCl-treated SpnHL (Fig. 2C).
In native SpnHL, for the second transition observed at higher concentrations of GdnHCl, a Cm of about 1.25 M was observed by both tryptophan fluorescence and far-UV CD (Fig. 2, A and B), which is similar to that reported above for the purified N-terminal fragment of SpnHL. These observations lend further support to the contention that the N-terminal fragments obtained on limited proteolysis of SpnHL treated under specified conditions is in folded conformation.
N-terminal Domain Is the Functional Domain Capable of Carrying Out
Enzymatic Reaction on Its Own
The reported structure of SpnHL in complex with hyaluronic acid
(16) showed that the three
residues, Tyr408, His399, and Asn349, from
N-terminal domain and HA are perfectly placed to support the mechanism of HA
degradation (14,
16). This indicates that
probably the C-terminal domain does not play any significant role in the
enzymatic activity of SpnHL. This possibility is further supported by the
evidence from some structurally related enzyme like glucoamylase
(26) and alginate lyase
(27) in which only the
-domain is present.
As we were able to obtain an intact N-terminal domain of SpnHL we carried out enzymatic activity measurements of this domain. Fig. 7 summarizes the comparison of enzymatic activity of isolated N-terminal domain with that of the native enzyme. The N-terminal domain showed about 12% enzymatic activities compared with the native enzyme. This demonstrates that the N-terminal domain is the functional domain of SpnHL and is itself sufficient for carrying out the enzymatic reaction. The C-terminal domain seems to modulate the enzymatic activity probably by holding the substrate, hyaluronic acid, in a suitable conformation for enzymatic reaction as speculated earlier (14).
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Inhibitors of SpnHL
The degradation of hyaluronan in the connective tissues by SpnHL is an
important step in the pneumococcal invasion. The bacterial strains producing
more hyaluronate lyase are more virulent than those strains producing less
enzyme (28). Furthermore,
S. pneumoniae strains with SpnHL and cell toxin pneumolysin double
mutations showed significant additive attenuation in virulence
(29). Therefore, the
inhibition of SpnHL is expected to reduce the spreading of S.
pneumoniae pathogen in the early stages of the pneumococcal invasion.
Furthermore, hyaluronate lyase from bacteria and Streptomyces differ
from hyaluronidases from other sources, including mammals, by their mode of
action. They catalyze an elimination reaction, rather than hydrolysis of the
1,4-glycosidic linkage between
N-acetyl-
-D-glucosamine and D-glucuronic
acid residues in hyaluronan. Because of these reasons SpnHL becomes a
potential target for developing antimicrobial agents.
Salicylate, vitamin C, heparin, dicumarene, and flavonoids have been reported to have certain inhibitory effects on the enzyme activity of hyaluronidase (5). However, recent studies by Li et al. (17) have reported that of the above mentioned molecules only vitamin C inhibits SpnHL activity. They also reported the crystal structure of SpnHL·vitamin C complex (17), which shows that only one vitamin C molecule binds to SpnHL at the active site and forms about 25 interactions with seven residues (mainly from N-terminal domain and only one from C-terminal domain) of SpnHL.
The structural studies on SpnHL presented in this paper demonstrated that
GdnHCl interacts with enzyme and induces significant structural changes in it.
As the active site of an enzyme is very susceptible to subtle adjustments or
conformational variations in the enzyme we thought it worthwhile to study the
effect of GdnHCl and some related molecules (having guanidino moiety) on the
enzyme activity of SpnHL so that their efficiency as inhibitors can be
explored. Fig. 8 summarizes the
effect of increasing concentrations of GdnHCl, L-arginine,
L-NAME, and GdnHSCN on the inhibition of enzymatic activity of
SpnHL. All four molecules inhibited the SpnHL activity in a dose-dependent
manner. The IC50 of enzyme activity by GdnHCl,
L-arginine, L-NAME, and GdnHSCN was found to be 150,
8.0, 0.85, and 0.14 mM, respectively. The interesting observation
is that at the concentration of GdnHCl at which there is complete inhibition
of SpnHL activity (about 350 mM), no significant effect on
structural properties of enzyme was observed
(Fig. 2). Similar observations
were obtained for GdnHSCN, L-NAME, and L-arginine (data
not shown). These observations suggest that GdnHCl or L-arginine or
L-NAME and GdnHSCN affect the active site of enzyme at a
concentration much lower than that required for bringing out any significant
structural change in enzyme. This is possible only if these molecules bind
directly to the active site first, followed by binding to other sites in the
enzyme, which results in structural changes. For vitamin C, which has been
reported as an inhibitor of SpnHL, an IC50 of 5.8
mM was observed
(17). In the present study an
IC50 of
0.85 and 0.14 mM were observed for
L-NAME and GdnHSCN, respectively, which are significantly lower
(about seven and 40 times) than vitamin C. These studies present a new
chemical entity that can be exploited for better designing of inhibitors for
SpnHL.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 212411-18 (ext. 4282); Fax:
91-522-223405; E-mail:
bhakuniv{at}rediffmail.com.
1 The abbreviations used are: HA, hyaluronan; SpnHL, S. pneumoniae
hyaluronate lyase; GdnHCl, guanidine hydrochloride;
Cm, denaturant concentration where 50%
denaturation of protein is observed; GdnHSCN, guanidine isothiocyanate;
L-NAME, Nw-nitro-L-arginine methyl
ester; Tm, mid-point of thermal denaturation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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