From the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
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ABSTRACT |
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The properties of recombinant staphylokinase
(SakSTAR) expressed in Pichia pastoris cells have been
determined. The single consensus N-linked oligosaccharide
linkage site in SakSTAR (at Asn28 of the mature protein)
was occupied in approximately 50% of the expressed protein with
high-mannose-type oligosaccharides. The majority of these glycans
ranged in polymerization state from Man8GlcNAc2
to Man14GlcNAc2, with the predominant species
being Man10GlcNAc2 and
Man11GlcNAc2. Glycosylated SakSTAR
(SakSTARg) did not differ from its aglycosyl form in its
aggregation state in solution, its thermal denaturation properties, its
ability to form a complex with human plasmin (hPm), the amidolytic
properties of the respective SakSTAR-hPm complexes, or its ability to
liberate the amino-terminal decapeptide required for formation of a
functional SakSTAR-hPm plasminogen activator complex. However, this
latter complex with SakSTARg showed a greatly reduced
ability to activate human plasminogen (hPg) as compared with the same
complex with the aglycosyl form of SakSTAR. We conclude that
glycosylation at Asn28 does not affect the structural
properties of SakSTAR or its ability to participate in the formation of
an active enzymatic complex with hPm, but it is detrimental to the
ability of the SakSTAR-hPm complex to serve as a hPg activator. This is
likely due to restricted access of hPg to the active site of the
SakSTARg-hPm complex.
Staphylokinase (Sak),1 a
protein secreted by certain strains of Staphylococcus
aureus, functions as a profibrinolytic agent by virtue of its
ability to convert the zymogen human plasminogen (hPg) to its active
serine protease form, plasmin (hPm). The Sak gene has been
cloned and sequenced from serotype B and F bacteriophages sak Sak does not possess proteolytic activity. Thus, as with streptokinase
(SK), the ability of Sak to convert hPg to hPm, a process that requires
cleavage of the Arg561-Val562 peptide bond in
hPg (9), is indirect. However, unlike SK, Sak requires catalytic
amounts of preformed hPm for this activation to occur (10-13), a
feature that illustrates important differences in the mechanisms by
which these two bacterial proteins activate hPg. Specifically, whereas
the complex between SK and hPg is capable of self-generation of the
enzymatic species SK-hPg' and SK-hPm, both of which serve as hPg
activators (for a review, see Ref. 14), the Sak-hPg complex must be
processed by another hPg activator to provide Sak-hPm, which then
functions as a hPg activator complex. During the conversion of hPg to
hPm by Sak, a decapeptide is cleaved from the amino terminus of Sak
(1). Although this peptide cleavage is not a rate-limiting step in hPg
activation (15), it has been concluded that this event is necessary for
this activation to occur (16).
Interest in Sak is based on its ability to enhance fibrinolysis in a
fibrin-dependent manner, thus holding promise for Sak as an
agent for thrombolytic therapy (17, 18). Therefore, elaboration of the
essential properties of Sak required for hPg activation represents a
timely area of investigation. In analysis of the amino acid sequence of
SakSTAR, only one consensus site for N-linked glycosylation
(Asn28-Val-Thr) is present. To test the importance of the
occupancy of Asn28 by carbohydrate, which becomes a
relevant issue for the expression of SakSTAR in systems other than
bacteria, we examined the ability of this locus to be processed in a
yeast-derived expression system and the effects of glycosylation on the
properties of this protein. This communication presents the results of
this study.
Insertion of SakSTAR cDNA into pPIC9K--
The starting
plasmid for this investigation was the E. coli expression
plasmid pMEX602sakB (a gift from H. R. Lijnen, Leuven, Belgium),
which contained the cDNA of SakSTAR isolated from a lysogenic S. aureus strain (4) that was cloned into the
expression vector pMEX6 (7). PCR primers were designed to introduce
restriction sites AvrII and NotI upstream and
downstream of the SakSTAR cDNA, respectively, to allow
insertion of this coding sequence cDNA into the Pichia
pastoris expression plasmid pPIC9K (Stratagene, La Jolla, CA). To
introduce the AvrII site (underlined), we used the primer
TAC/CTAGGTCAAGTTCATTCGACAAAG(+), which codes for the mature
amino-terminal sequence Ser1-Ser-Ser-Phe-Asp-Lys. The
primer for the NotI site was
ATGC/GGCCGCTTATTATTTCTTTTCTATAACAACC(
PCR was carried out using Pfu polymerase (5.0 units) and the
pMEX602sakB plasmid (100 ng). The primer annealing temperature was
47 °C. The PCR products were purified by electroelution and cloned
into pPIC9K via its AvrII and NotI restriction sites.
Expression and Purification of Recombinant SakSTAR from E. coli--
DH5 Expression and Purification of Recombinant SakSTAR from P. pastoris Cells--
The cDNA encoding SakSTAR was also expressed
in the P. pastoris yeast system using the GS-115 strain.
Detailed procedures used in this laboratory to select appropriate yeast
transformants have been published previously (19). After transformation
and selection of the high producing clones, the cells were grown to a
cell density of 250 g/liter in a 5-liter fermentor using glycerol as
the primary carbon source, after which methanol was added, and the
cells were allowed to ferment for 8-12 h to induce extracellular production of SakSTAR. The medium was then collected and dialyzed against two changes of H2O to remove the high quantity of
salt contained in the fermentor medium and, finally, against two
changes of 0.01 M sodium phosphate, pH 6.0. SakSTAR was
then purified in a two-step procedure as described above for the
E. coli expression system. Fractions contained both
SakSTARu and SakSTARg. To separate these
unglycosylated and glycosylated forms of the protein, the solution was
dialyzed against 10 mM sodium phosphate, 5 mM
MgCl2, and 400 mM NaCl, pH 7.0, and purified by
affinity chromatography over a 10-ml bed volume of concanavalin
A-Sepharose (Sigma Chemical Co.) at room temperature.
SakSTARu was collected in the flow-through and wash.
SakSTARg was then eluted from the column with 500 mM
When SakSTARg was to be used for carbohydrate analysis,
elution from the concanavalin A-Sepharose column was accomplished using
1 M acetic acid to avoid any possible contamination with mannose. However, in this case, further purification was necessary. The
SakSTARg preparation was dialyzed against H2O
containing 0.1% trifluoroacetic acid and injected onto a 4.6 × 150-mm C8 reverse phase HPLC column (Vydac, Hesperia, CA) that was
preequilibrated with a 75:25 (v:v) H2O:CH3CN
solution containing 0.1% trifluoroacetic acid. Elution was
accomplished by increasing the CH3CN to 75% over 40 min at
a flow rate of 1 ml/min. The major peak that eluted at 19.5 min
contained highly purified SakSTARg.
Activation of hPg by SakSTAR--
To determine the plasminogen
activator activity of SakSTAR, SakSTARu, and
SakSTARg, the conversion of hPg to hPm was monitored using
a coupled assay. An amount of 5 nM SakSTAR,
SakSTARu, or SakSTARg was added to 5 nM hPg that contained endogenous trace amounts of hPm. The
chromogenic substrate, S2251, was added to a final concentration of 0.9 mM. Release of p-nitroanilide was monitored
continually at 405 nm for 1 h at 37 °C. The rate of activation
of hPg by the SakSTAR variants was determined by the increase in
absorbance at 405 nm as a function of time. The buffer used was 10 mM Na-Hepes and 150 mM NaOAc, pH 7.4.
To examine the activity of the preformed SakSTAR-hPm complexes, the
appropriate SakSTAR samples were added to hPm at a 1:1 molar
stoichiometry. Urokinase was used to activate hPg before the addition
of SakSTAR (0.5 nM urokinase and 13.5 µM hPg
for 1 h at 37 °C). The activated hPm was then incubated with
SakSTAR, SakSTARu, or SakSTARg at 37 °C to
form the corresponding complex, respectively. Catalytic amounts (1:30,
m:m) of these enzyme complexes were then added to 25 nM hPg
in activation buffer containing the substrate. The continuous
activation assay was carried out as indicated above. In separate
experiments to determine the amidolytic activities of these same
complexes, substrate S2251 was used without the addition of hPg.
Determination of Protein Concentrations and Spectrophotometric
Absorptivity by Analytical Ultracentrifugation--
SakSTAR samples
were dialyzed against 10 mM sodium phosphate buffer, pH
6.0, and adjusted to an adsorption range of 0.75-1.2 at 280 nm. The
absorption spectra and the interference fringe pattern were measured on
a Beckman XL-I analytical ultracentrifuge with integrated absorbance
and Rayleigh interference optics. A synthetic boundary cell was used
with a path length of 1.2 cm. The volumes of the buffer reference and
SakSTAR samples were 400 and 190 µl, respectively. Measurements were
made at a rotor speed of 20,000 rpm at 20 °C with a detection
wavelength of 280 nm for absorbance and 675 nm for fringe displacement.
The fringe displacement ( Differential Scanning Calorimetry--
Samples of SakSTAR,
SakSTARu, or SakSTARg were dialyzed overnight
against PBS buffer (0.01 M sodium phosphate and 0.14 M NaCl, pH 7.4). Protein concentrations of 0.75-1.3 mg/ml
of SakSTAR, SakSTARu, or SakSTARg were
determined at 280 nm using the experimentally determined molar
extinction coefficient of 9,800. Protein samples and buffers were
degassed for 30 min. All SakSTAR samples were heated at 1 °C/min,
and the denaturation temperatures (Tm) and calorimetric enthalpies
( Circular Dichroism (CD)--
CD spectra were recorded at 222 nm
on an AVIV model 62DS spectrophotometer. SakSTAR, SakSTARu,
or SakSTARg was dialyzed against PBS, pH 7.4, and
concentrated to 0.2 mg/ml. A 0.2-cm path length cell was used. The data
were collected at a 1.0-nm bandwidth, and each represents the average
of three measurements. The SakSTAR samples were heated at 1 °C/min,
and changes in ellipticity were monitored at 222 nm. Mean residue
ellipticities were calculated using a mean residue molecular weight of
114.6 for SakSTAR (20). Points were fitted to a Boltzman curve, and
midpoints were established as Tm values.
Enzyme-linked Immunosorbent Assays--
A constant concentration
of hPm (50 nM) was placed in individual wells of a 96-well
microtiter plate, and the plates were washed with PBS buffer after an
overnight incubation at 4 °C. A 2-h incubation with blocking buffer
(6% milk in PBS buffer) was followed by washings with PBS buffer.
SakSTAR, SakSTARu, or SakSTARg was then added
to the wells at a series of concentrations between 0 and 100 nM. After a 2-h incubation at room temperature, the wells
were washed once again as described above. A mouse anti-SakSTAR IgG
monoclonal antibody (5 µg/ml; obtained from H. R. Lijnen) was
then incubated in the wells, and the wells were washed after 90 min.
Alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad, Richmond,
CA) was then added to the wells and allowed to incubate for 90 min.
After washing, the substrate Sigma-104 (Sigma Chemical Co.) was added,
and the absorbancies of the wells were determined at 405 nm in a
microtiter plate reader to detect the amount of alkaline phosphatase
present. After subtraction of the values determined for light scatter
(absorbancy at 490 nm), plots were fitted to a hyperbolic curve, and
C50 values were determined from the midpoints using Origin
software. Control experiments demonstrated that the monoclonal antibody
reacted with equal affinity to the three forms of SakSTAR.
Sedimentation Equilibrium Experiments using Analytical
Ultracentrifugation--
Sedimentation equilibrium experiments were
performed on a Beckman XL-I analytical ultracentrifuge at speeds of
18,000, 20,000, and 25,0000 rpm with protein concentrations of 0.33, 0.5, and 1.0 mg/ml. Before centrifugation, the protein solutions were
equilibrated in 10 mM sodium phosphate and 100 mM NaCl, pH 7.3, at 20 °C. Dialysates were loaded into
the reference chambers for all experiments. The apparent molecular
weights and deviations from a single fit as functions of the radius
from the center of rotation were determined using the Beckman XL-I
software that accompanied the instrument.
Analytical Techniques--
Oligonucleotides were synthesized
using phosphoramidite-based methodology on the Beckman Oligo 1000M DNA
synthesizer. For DNA sequencing, the dideoxy chain termination method
(21) using the sequenase version 2.0 DNA sequencing kit (U.S.
Biochemical Corp., Cleveland, OH) was used with the
ALFexpress sequencer (Pharmacia Biotech, Piscataway, NJ).
Fluorescence labeling was accomplished with Cyp5'-dATP. All
reagents for this procedure were purchased from Pharmacia. The
sequencing gel was run at 1500 V, 60 mA, and 25 W at 55 °C. The
laser power was stabilized between 700 and 800. The sequence was
processed using software supplied by Pharmacia.
The SakSTAR variants were identified in part based on their molecular
weight by TOF-MALDI-DE on a Voyager-DE Spectrometer (PerSeptive
Biosystems, Framington, MA). The samples were dialyzed against
H2O to remove any residual salts, after which they were concentrated to 0.1-1.0 mg/ml. A volume of 0.5 µl of sample was added to 0.5 µl of 10 mg/ml sinapinic acid in a mixture of 50:50 (v:v) H2O:CH3CN containing 0.1%
trifluoroacetic acid on a 100-well sample plate. The drops were air
dried. The dried samples were irradiated with a N2 laser
(337 nm; pulse time, 4 ns). Linear mode positive ionizations were used.
Signal transients were recorded at a time resolution of 5 ns.
For determination of the molecular weights of SakSTAR variants in the
stoichiometric complex with hPm, the SakSTAR preparation was incubated
at a 1:1 (m/m) complex with hPm for 40 min in a buffer containing 10 mM sodium phosphate, 5 mM MgCl2,
and 400 mM NaCl, pH 7.0. The reaction mixtures were then
dialyzed overnight against H2O at 4 °C. The molecular
weights of the SakSTAR samples in the presence and absence of hPm were
determined by TOF-MALDI-DE in the positive ion mode as described above.
Oligosaccharides were released from SakSTARg by digestion
with PNGase F. The conditions of digestion were 1 unit PNGase F/200 µg SakSTARg in a total volume of 50 µl of a buffer
containing 100 mM sodium phosphate, pH 7.2, for 18 h
at 37 °C. Hydrazinolysis was also employed using Oxford GlycoSystems
(Rosedale, NY) Glycoprep 1000 for automated performance with 1 mg of
SakSTARg in the cell. The released oligosaccharides were
then labeled with 2-aminobenzamide (22, 23). Methodology used for
characterization of the fluorescence-labeled oligosaccharides by HPLC
and by gel filtration using the Oxford GlycoSystems RAM 2000 GlycoSequencer has been described previously (22).
Recombinant SakSTAR was purified from conditioned culture media
obtained from an E. coli-based expression system using a
combination of SP-Sepharose (Fig.
1A) and phenyl-Sepharose (Fig.
1B) chromatography in quantities of approximately 300-400
mg/liter cell culture. As determined by TOF-MALDI-DE, the final
material possessed a molecular weight of 15498.9 (calculated molecular
weight, 15494.6), and the amino-terminal amino acid sequence was found
to be Ser-Ser-Ser-Phe-Asp-Lys. Recombinant SakSTAR, purified as
described in the Fig. 1 legend from P. pastoris cells,
contained both the glycosylated and nonglycosylated forms, which were
readily separated on a column of concanavalin A (Fig. 1C).
The final yields were approximately 60 mg/liter SakSTARu and 50 mg/liter SakSTARg. The molecular weight of
SakSTARu, which was 15,497.3 (calculated molecular weight,
15,494.6), was very similar to that of SakSTAR, whereas that of
SakSTARg was displayed as a heterogeneous broad peak
approximately 3000 higher. Both forms of SakSTAR resulting from yeast
expression possessed the same amino-terminal amino acid sequence as the
material obtained from E. coli.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
C (1) and sak42D (2), respectively, and
from the genome of a lysogenic strain of S. aureus
(SakSTAR) (3). The protein was initially expressed in
relatively low yield and with variable amino-terminal processing in
Escherichia coli (1, 4) and Bacillus subtilis (2,
5), but later improvements in the expression plasmid provided much
larger quantities of intact Sak (6, 7). Some differences exist in the
coding regions of the Sak genes in sak
C,
sak42D, and sakSTAR. Nonetheless, in each case,
Sak is synthesized as a 163-amino acid protein that contains a 28-amino
acid residue signal sequence. The x-ray crystallographic structure of
SakSTAR has been determined, thus allowing a three-dimensional modeling
of this protein (8).
EXPERIMENTAL PROCEDURES
), a sequence that
encodes the carboxyl-terminal region of the protein Val-Val-Ile-Glu-Lys-Lys136-Stop-Stop.
cells containing the pMEX602 intracellular expression
plasmid were grown in 100 ml of Terrific Broth (Sigma Chemical Co.) at
37 °C with vigorous shaking in 500-ml culture flasks. Induction was
accomplished during the late log phase with 200 mM
isopropyl-
-thio-D-galactopyranoside for 4-8 h. Cells
were centrifuged, and pellets were resuspended in 0.25 the volume of culture medium in 0.04 M sodium phosphate buffer, pH 6.0, and disrupted by pulsed sonication for 5 min at 4 °C. Cell debris was separated by centrifugation at 45,000 × g for 30 min. The cleared lysates were diluted with H2O to 0.01 M sodium phosphate, pH 6.0. SakSTAR was then purified using
minor modifications of a previously described method (7). First, the
solution was purified by cation exchange chromatography over a 10-ml
bed volume of SP-Sepharose at room temperature. The column was washed
with 0.01 M sodium phosphate buffer, pH 6.0, and eluted
with a 200-ml (total volume) linear gradient from 0 to 2.0 M NaCl. The pooled fractions containing SakSTAR, as
identified by an S2251 chromogenic substrate assay, SDS-polyacrylamide
gel electrophoresis, and TOF-MALDI-DE, were adjusted to 2.5 M NaCl and applied to a 10-ml bed volume of
phenyl-Sepharose. The column was washed with 0.01 M sodium phosphate and 2.5 M NaCl, pH 6.0, and eluted with 0.01 M sodium phosphate, pH 6.0. Fractions containing SakSTAR
were identified as described above.
-D-mannopyranoside in 10 mM
sodium phosphate, 5 mM MgCl2, and 400 mM NaCl, pH 7.0.
J) was determined by subtracting the mean
value of the baseline from the plateau. The concentration (C) of the
protein sample was then related to the fringe displacement by C =
J/k, where k is the specific fringe
displacement for a cell optical path length (L), defined as
k = (dn/dc)L/
. The specific
refractive increment, dn/dc, was 0.187 (g/ml)
1
at the wavelength (
) of the laser light source (675 nm), a value that is generally accepted to be independent of the amino acid composition of the protein.
H) for each protein were determined from computer analysis (with
manufacturer-designed software) of the thermograms.
RESULTS
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Fig. 1.
Elution profiles of SakSTAR from E. coli (A and B) and P. pastoris (A-C) systems. A, SakSTAR from
E. coli. A profile of cation exchange chromatography using
SP-Sepharose column chromatography (10-ml bed volume) is shown. A
volume of 100 ml of the cytosolic fraction in 10 mM sodium
phosphate, pH 6.0, was loaded onto the column, which was equilibrated
and washed with 10 mM sodium phosphate, pH 6.0. The protein
was eluted with a gradient containing 0 (start solution)-2
M NaCl (limit solution) in the same buffer. The elution
began at fraction 31, and fractions 37-41 were collected.
B, fractions 37-41 from A were adjusted to 2.5 M NaCl in 50 ml and loaded onto a phenyl-Sepharose column
(10-ml bed volume). The bound protein was eluted with 10 mM
sodium phosphate, pH 6.0, applied at fraction 9. Fractions 16-25 were
collected. This material was pooled and dialyzed against a solution
containing 10 mM sodium phosphate, 5 mM
MgCl2, and 400 mM NaCl, pH 7.0. C,
concanavalin A column chromatography of recombinant SakSTAR from
P. pastoris (after steps described in A and
B). The collected fractions from B were placed
over a concanavalin A-Sepharose column. SakSTARu was
collected in the flow-through and wash in fractions 11-16. After this,
the column was washed with a solution of 10 mM sodium
phosphate, 5 mM MgCl2, and 400 mM
NaCl, pH 7.0. SakSTARg was then eluted with 10 mM sodium phosphate, 5 mM MgCl2,
400 mM NaCl, and 50 mM
-D-mannopyranoside, pH 7.0. Fractions 37-41 were
collected. All chromatography was performed at room temperature at a
flow rate of 1.6 ml/min.
A determination was made of the nature of the N-linked
glycosylation at the lone consensus Asn site contained on
Asn28 of SakSTARg. After release of the
N-linked oligosaccharides by PNGase F, the glycan pool was
fluorescence-labeled, and the components were identified in terms of
their equivalent glucose units based on their elution positions from a
calibrated BioGel P4 column (Fig. 2). The
elution profile of the sample shows the presence of
Man8-14GlcNAc2.
Man10GlcNAc2 and
Man11GlcNAc2 were present in approximately
equal amounts and represented >80% of the oligosaccharides that were
released from SakSTARg. When hydrazinolysis was used as the
method for release of N-linked and O-linked saccharides, an
elution profile very similar to that seen in Fig. 2 was obtained. Further treatment of the entire neutral oligosaccharide pool of Fig. 1
with jack bean -mannosidase followed by rechromatography on the
BioGel P4 column resulted in the identification of a single fraction of
the Man(
1,4)GlcNAc(
1,4)GlcNAc core.
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To assess the comparative properties of SakSTARu and SakSTARg, their thermal stabilities were determined. Differential scanning calorimetry analysis (Fig. 3) yielded Tm values of 71.7 °C for SakSTARu and 71.8 °C for SakSTARg, with calorimetric enthalpies of 80.3 and 84.8 kcal/mol, respectively. These are similar to a Tm value of 70.0 °C and an enthalpy of 83.1 kcal/mol obtained for SakSTAR (data not shown). Comparable Tm values for SakSTAR, SakSTARu, and SakSTARg of 72.3 °C, 73.2 °C, and 76.0 °C, respectively, were obtained by monitoring the temperature dependence of their molar ellipticities at 222 nm (data not shown).
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Sedimentation equilibrium centrifugation analyses of each of the SakSTAR samples were undertaken to examine whether aggregation of the polypeptide chain occurred. The graphs provided (Fig. 4) show that the data for both proteins could be fit to single molecular weights over a wide concentration range in the cell. This is supported by the randomness of the residuals also provided in Fig. 4. The apparent molecular weights determined from this approach were 15,994 ± 214 for SakSTARu and 18,241 ± 347 for SakSTARg. These compare very closely with the calculated molecular weights for the monomeric species. Similar data were collected for bacterially expressed SakSTAR.
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The overall abilities of the various forms of SakSTAR to activate hPg were assessed in an assay wherein the various forms of SakSTAR were added to hPg in a continuous assay of plasmin formation. Formation of the SakSTAR-hPm activator complex occurred due to the presence of small amounts of hPm that are present in the hPg preparations. Under identical activation conditions, the results illustrated in Fig. 5A demonstrated that whereas SakSTARu displayed a high level of hPg activator activity under these conditions, SakSTARg did not generate an effective hPg activator complex with hPm. On the other hand, the data in Fig. 5B demonstrate that the addition of equimolar levels to preformed hPm did not inhibit the amidolytic activity of hPm toward S2251. However, Fig. 5C shows that the ability of a preformed SakSTARg-hPm complex to activate hPg is significantly diminished.
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The ability of the SakSTAR proteins to associate with hPm was determined from the enzyme-linked immunosorbent assay-based binding experiments shown in Fig. 6. The data demonstrate that all three forms of the protein interact in a nearly identical manner to hPm, with C50 values of 16, 21, and 22 nM for SakSTAR, SakSTARu, and SakSTARg, respectively. These experiments were performed by titration of hPm with the SakSTAR variants. When similar titrations of insolubilized SakSTAR preparations were conducted with hPm (the complex was detected with a hPg-derived monoclonal antibody), the C50 values were nearly the same (~1.5-fold higher in each case) as those reported above.
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Lastly, because amino-terminal proteolysis (at
Lys10-Lys11) of Sak has been shown to be
necessary for hPg activation to occur (3, 6), the amino-terminal amino
acid sequences of these forms of SakSTAR were determined after
complexation with hPm. After incubation of equimolar amounts of each
form of SakSTAR with hPm for 20 min at 37 °C, the samples were
subjected to SDS gel electrophoresis under reducing conditions. The
band containing the relevant SakSTAR was excised, and the
amino-terminal amino acid sequence analysis was performed. For all
three samples, the sequence (>90%) obtained was
Lys11-Gly-Asp-Asp-Ala-Ser. Further confirmation that this
critical cleavage of SakSTAR occurs in the SakSTAR-hPm complex was
achieved using TOF-MALDI-DE analysis. The SakSTARu and
SakSTARg complexes displayed molecular mass differences of
1164 and 1203, respectively, from their parent proteins in the presence
of hPm. These values (or their Na+ adducts) are consistent
with the loss of a decameric peptide from the amino terminus of SakSTAR
(calculated molecular weight, 1170.6).
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DISCUSSION |
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SakSTAR contains a single N-linked oligosaccharide consensus site at Asn28. This investigation was initiated to ascertain the effects on the activity of this protein consequent to glycan occupancy of that site. In its natural state in S. aureus, the relevant Asn28 is not glycosylated, but in expression systems other than bacteria, this residue is potentially capable of assembling oligosaccharide. A full understanding of whether this residue can be modified in the conformational environment of the intact protein and how processing at this site in other host cells affects its properties is relevant to the elucidation of the structure-function relationships of SakSTAR. An additional consideration concerns the extent to which SakSTAR activity is altered by glycosylation in expression systems that could be effectively utilized for its preparation. This is especially pertinent if large amounts of material would be required for use as a therapeutic fibrinolytic agent.
We used yeast cells of the strain P. pastoris as an
expression system to express SakSTAR. The choice of this expression
vehicle was governed by the potentially large amounts of secreted
protein that could be obtained in a simple and inexpensive culture
medium under simple fermentation conditions with methanol/glycerol as the only carbon sources. Furthermore, when compared with the much more
extensively characterized Saccharomyces cerevisiae system, the glycosylation machinery of P. pastoris cells only
permits assembly of relatively short N-linked high-mannose
saccharides (22), a subpopulation of which may be phosphorylated (23). O-linked glycans with a small number of mannose residues consisting of
mannose and (1,2)-linked dimer to pentamer saccharides of mannose
have also been observed (24).
Purification of SakSTAR from 3 liters of fermentation medium of P. pastoris cells using an expression plasmid that allows for secretion of the protein results in total final yields of approximately 100-150 mg of the recombinant material. The relative amount of protein that contains N-linked glycans ranges from 50% to 75% of the total, which varies in different fermentations. We have not performed a systematic study of the routine variables in the fermentation protocol that affect the extent of protein modification.
The purified glycosylated and aglycosylated proteins were subjected to molecular weight analysis by TOF-MALDI-DE and amino-terminal acid sequence analysis. Both analyses indicated that signal peptide processing was identical for SakSTARu and SakSTARg and yielded the same mature protein as that obtained for bacterially expressed SakSTAR. Neither SakSTARu nor SakSTARg displays association or concentration-dependent aggregation in solution as demonstrated by sedimentation equilibrium centrifugation. Thus, both yeast-expressed proteins exist as single chain molecules in solution.
The nature of the Asn28-linked oligosaccharides assembled
on SakSTARg by P. pastoris cells was similar to
that found earlier for another peptide expressed in this system (22).
Man10GlcNAc2 and
Man11GlcNAc2 represented approximately 80% of
the total glycans released from Asn28.
Exoglycosidase-catalyzed digestion of the entire oligosaccharide pool
with an -mannose-specific jack bean mannosidase showed that all
mannose residues except the single core mannose possessed
-anomeric
linkages. Digestion of this same oligosaccharide pool with
(
1,2)-specific mannosidase yielded a single product,
Man6GlcNAc2. This conclusion is consistent with
previous results showing that the P. pastoris-derived core
glycan structure contains an additional (
1,6)-mannose on the arm of
the (
1,3)-mannose of the mammalian-type core oligosaccharide
Man5GlcNAc2, with mannose extensions from this
latter yeast-based core structure of the (
1,2)-type (22). Based on
the HPLC profiles of N-linked oligosaccharides and on the
lack of additional saccharide release by hydrazinolysis from SakSTARg after liberation of N-linked glycans by
PNGase F, no evidence was obtained for charged N-linked
oligosaccharides or O-linked saccharides, respectively, on
SakSTARg. Thus, all carbohydrate chains assembled on this
protein were Asn28-linked neutral high-mannose
oligosaccharides with yeast-type mannose extensions from the
mammalian-like core.
To assess the conformational stability of the protein, thermal stability measurements were carried out by two independent techniques. From both differential scanning calorimetry analysis and CD-monitored temperature scans, Tm values between 70 °C and 76 °C were obtained for SakSTAR, SakSTARu, and SakSTARg. These high Tm values suggest that the native conformation is highly resistant to thermal denaturation. This property is displayed despite the lack of added stability that would be present if the protein contained disulfide bonds. Furthermore, the presence of the glycan does not significantly alter the conformational stability of SakSTAR. This conclusion also suggests that interactions between the protein and the oligosaccharide chain, if any, have minimal impact on the maintenance of stability in SakSTARg.
Measurements of the comparative abilities of the P. pastoris-derived SakSTAR preparations to activate hPg were made. When added to identical preparations of hPg that contained a small amount of hPm, it was found that whereas SakSTARu possessed the capacity to activate hPg, SakSTARg was nearly inactive under these conditions (Fig. 5A). This result then prompted additional experiments designed to address the basis for these differential effects. The first set of experiments was intended to address the question of whether the oligosaccharides present on SakSTARg could have precluded the formation of the SakSTAR-hPm activator complex. The data in Fig. 6 demonstrate that this is not the case, and complexes of nearly equal affinity were formed between hPm and each form of SakSTAR.
It has been found that release of a decapeptide from the amino terminus of SakSTAR, consequent to cleavage at Lys10-Lys11, is required for generation of an effective SakSTAR-hPm activator complex (3, 6). We then examined the possibility that the presence of the carbohydrate at Asn28 is affecting plasmin-mediated processing of SakSTAR to its fully active low molecular weight form. This was determined through incubation of both SakSTAR forms with hPm followed by electrophoretic separation. Amino-terminal sequence analysis of the excised gel band corresponding to SakSTARg showed a clear sequence, beginning at Lys10. This indicates that the requisite peptide bond in SakSTARg was cleaved in the complex, thus potentially rendering the complex capable of displaying hPg activation activity.
The possibility that the oligosaccharides present on SakSTARg could have blocked the active site of hPm, thus inhibiting the activity of the SakSTARg-hPm complex, was eliminated based on the experiments in Fig. 5B. Here, it is seen that the addition of an equimolar level of SakSTARg to hPm had little effect on the amidolytic activity of this enzyme toward the small substrate, S2251. However, upon activation of hPg with catalytic levels of preformed complexes of SakSTARu-hPm and SakSTARg-hPm (Fig. 6C), a clear diminution of SakSTARg-hPm activity, similar to that of hPm alone, is observed. Thus, we conclude that the basis of the poor hPg activator activity of SakSTARg results from the loss of specificity of the SakSTARg-hPm complex for hPg, perhaps due to a more restricted access of hPg to the active site of hPm in the activator complex.
Examination of a recently reported x-ray crystallographic structure of a ternary microplasmin-SakSTAR-microplasmin complex wherein an activating complex of SakSTAR and microplasmin is bound to a second substrate-like molecule of microplasmin (25) reveals that a Met residue at position 26 of SakSTAR is part of a hydrophobic network having surface complementarity to the carboxyl-terminal region of the microplasmin. This Met residue has been shown to be critical for the efficient activation of hPg by SakSTAR (26) and lies in close proximity to Asn28. The results displayed in Fig. 6 demonstrate that no appreciable decrease in hPm binding affinity is associated with glycosylation of SakSTAR. This observation strongly suggests that the presence of the oligosaccharide moiety on Asn28 is not interfering with the docking of the microplasmin moiety. However, a subtle change in orientation of hPm at the SakSTARg interface may be occurring, which can then restrict access of the activation loop of the hPg substrate to its requisite subsites in the active site cleft. The active site entry and proper orientation of a smaller substrate is not affected in the SakSTARg-hPm complex.
In conclusion, this investigation has provided clear evidence that
glycosylation of the lone N-linked consensus site in SakSTAR is detrimental to its hPg activator activity and has revealed the
mechanism of this effect. As such, this study provides valuable contributions to understanding the structure-function relationships of
this protein. In addition, whereas oligosaccharides assembled on
SakSTAR other than those identified herein may not produce these same
effects, this work should serve to heighten awareness that processing
events of this protein in other systems may not yield fully functional protein.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL-13423 (to F. J. C.) and the Kleiderer-Pezold family endowed professorship (to F. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame, IN 46556. Tel.:
219-631-6456; Fax: 219-631-8017; E-mail: castellino.1{at}nd.edu.
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ABBREVIATIONS |
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The abbreviations used are: Sak, staphylokinase; SakSTAR, recombinant staphylokinase; SakSTARg, glycosylated SakSTAR; SakSTARu, aglycosyl form of SAKSTAR; hPm, human plasmin; hPg, human plasminogen; SK, streptokinase; TOF-MALDI-DE, time-of-flight matrix-assisted-laser-desorption-ionization with delayed-extraction mass spectrometry; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline.
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REFERENCES |
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