From the Protease inhibition by secretory leukocyte
protease inhibitor (SLPI) is accelerated by the sulfated
polysaccharides. The nature of the SLPI-polysaccharide interaction,
explored with affinity chromatography, indicated that this interaction
was sensitive to the charge and type of polysaccharide. Dextran and
chondroitin had the lowest affinity for SLPI, followed by dermatan,
heparan, and dextran sulfates. While heparin bound SLPI tightly, the
highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin
oligosaccharides, prepared using heparin lyase I were SLPI-affinity
fractionated. Surprisingly, undersulfated heparin oligosaccharides
bound SLPI with the highest affinity, suggesting the importance of free
hydroxyl groups for high affinity interaction. Isothermal titration
calorimetry was used to determine the thermodynamics of SLPI
interaction with a low molecular weight heparin, an undersulfated
decasaccharide and a tetrasaccharide. The studies showed 12-14
saccharide units, corresponding to molecular weight of ~4,800, were
required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore,
an undersulfated decasaccharide was able to bind SLPI tightly
(Kd ~13 nM), resulting in its
activation and the inhibition of neutrophil elastase and pancreatic
chymotrypsin. The in vitro assessment of heparin and the
decasaccharide and tetrasaccharide using stopped-flow kinetics
suggested that heparin was the optimal choice to study SLPI-based
in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway
hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in
combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.
Secretory leukocyte protease inhibitor
(SLPI)1 is a nonglycosylated
serine antiprotease with a molecular weight of 11,700 (29). It has been
isolated from human bronchial secretions, seminal fluid, cervix uteri
secretions, parotid saliva, articular cartilage chondrocytes, and
intervertebral disc fibrochondrocyte (2, 3). The biological function of
SLPI is believed to be the inhibition of elastase, cathepsin G, and
other proteases, thereby protecting tissue from self-degradation by
these enzymes (2, 4). SLPI is composed of two homologous domains and
contains many positively charged amino acid residues (15 lysine and 5 arginine residues) (2). It is within clusters of these positively
charged amino acid residues that the heparin polyanion probably
interacts (5, 14).
Heparin is biosynthesized in mast cells as a proteoglycan consisting of
a central core protein from which multiple glycosaminoglycan (GAG)
chains extend (6-8). On isolation and purification from tissue, such
as lung, heparin is released from its protein core and isolated as a
GAG. GAG heparin is a polydisperse, highly sulfated, linear
polysaccharide comprised of repeating 1 SLPI binds heparin and LMW heparin tightly (Kd of
6 ± 2 nM and 50 ± 9 nM,
respectively) resulting in an increase in SLPI's rate of elastase
inhibition (1). Heparin also increases inhibition of chymotrypsin by
SLPI (14). Recent studies by Ying et al. (15) showed that
heparin could restore the elastase inhibition by SLPI after the
inactivation of SLPI through the oxidation of a critical methionine
residue. Faller et al. (16) have shown that SLPI interacts
with heparin through seven ion pair interactions and have inferred that
85% of the binding energy is electrostatic in nature.
We report the further characterization of the interaction between SLPI
and heparin by using affinity chromatography. Heparin-derived oligosaccharides with high affinity for SLPI were isolated and their
structure identified. The interaction of heparin and heparin-derived oligosaccharides with SLPI was assessed using isothermal titration calorimetry (ITC) (17) as was their activation of SLPI's inhibition of
various proteases, using stopped-flow kinetics (18). Finally, the
in vivo activation of SLPI by heparin was studied using a sheep bronchoprovocation model for late asthmatic response (19).
Materials
Porcine heparin (Mr(avg) 14,000), low
molecular weight heparin (Mr(avg) 4,800), and
heparan sulfate (Mr(avg) 11,000) were obtained
from Celsus Laboratories (Cincinnati, OH). Chondroitin (Mr(avg) 25,000) and dermatan sulfate
(Mr(avg) 11,000-25,000), sodium salts, and
heparin/heparan sulfate disaccharide standards for capillary
electrophoresis were purchased from Seikagaku America, Inc. (Rockville,
MD). Dextran (Mr(avg) 1,000), dextran sulfate (Mr(avg) 5,000), heparin-Sepharose 4B prepacked
columns, CNBr-activated Sepharose 4B
N-Suc-Ala-Ala-Pro-Phe-pNA and MeO-Suc-Ala-Ala-Pro-Val-pNA were purchased from Sigma. Heparin lyase I (heparinase I, EC 4.2.2.7), heparin lyase II, (heparinase II, no EC number), and heparin lyase III
(heparinase III, EC 4.2.2.8) were a gift from IBEX (Montreal, Canada).
Recombinant human SLPI was expressed and purified as described
previously (20). Recombinant protein was >99% pure as assessed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high
performance liquid chromatography and contained <0.72 enzyme unit
lipopolysaccharide/mg of protein. Bovine pancreatic chymotrypsin was
purchased from Boehringer Mannheim. Human neutrophil elastase and human
neutrophil cathepsin G were from Calbiochem-Novabiochem, San Diego, CA.
Dialysis tubing was purchased from Spectrum Laboratory Products
(Houston, TX).
Methods
Affinity Chromatography Studies
Heparin-Sepharose Affinity Chromatography of SLPI--
SLPI (100 µg in 10 µl buffer) was loaded onto a 2.5-ml heparin-Sepharose
column (1 mg/ml) was first washed with 20 mM sodium phosphate, pH 7.2, buffer and a 50-ml linear salt gradient of 0-1.5
M NaCl in 5 mM sodium phosphate buffer, pH 7.4, was used to elute the column. The eluent was monitored at 280 nm, and
the salt concentration was determined using a conductivity meter and fractions were collected.
Preparation of SLPI-Sepharose--
CNBr-activated Sepharose (300 mg) was washed with 75 ml of 1 mM hydrochloric acid and
swollen for 15 min at room temperature. The swollen beads were
suspended in coupling buffer (100 mM sodium bicarbonate,
100 mM NaCl, pH 8.4) containing 10 mg of
N-acetylated heparin (21) (to protect SLPI's interacting
lysine residues), SLPI (1 mg) was added slowly to avoid precipitation,
and the slurry was mixed at 4 °C for 12 h. The gel was then
washed with coupling buffer, 100 mM sodium acetate buffer,
pH 4.5, resuspended in 1 M ethanolamine, pH 8.9, to block
remaining reactive sites on the beads, and the slurry was again mixed
at 4 °C for 12 h. The gel was subsequently poured into a column
(0.5 × 2 cm) and thoroughly washed with 5 mM sodium
phosphate buffer, pH 7.4, containing 2 M NaCl and stored at
4 °C until used. A control column was also prepared using the same
method but without adding the SLPI-N-acetylated heparin
mixture. All affinity chromatography was performed at 4 °C.
SLPI-Sepharose Affinity Chromatography of
Polysaccharides--
The SLPI-Sepharose column was equilibrated in 5 mM, pH 7.4, sodium phosphate buffer. In separate
experiments, heparin (500 µg), heparan sulfate (500 µg), dermatan
sulfate (500 µg), chondroitin (600 µg), dextran (600 µg), or
dextran sulfate (1.2 mg) were affinity fractionated on this column.
After loading the polysaccharide, the column was washed with 5 mM sodium phosphate buffer, pH 7.4, and a 20-ml linear salt
gradient (from 0 to 1.75 M NaC1) in the same buffer was
used to elute the column and fractions were collected. GAGs were
detected by carbazole assay (22), dextran and dextran sulfate by
phenol-sulfuric acid assay (23), and salt concentrations were
determined by measuring the conductivity.
Division of Medicinal and Natural Products
Chemistry,
Amgen Inc., Boulder, Colorado 80301, and the ** University of
Miami School of Medicine, Miami Beach, Florida 33140
ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results & Discussion
References
4 linked uronic acid and
glucosamine residues, with a molecular weight range of 5,000-40,000
and an average molecular weight (Mr(avg)) of
14,000 (9, 10). While it has as many as 10 different saccharide residues that comprise its sequence, not all sequences are
biosynthetically favored. The major sequence in heparin is
4)-
-L-IdoAp2S(1
4)-
-D-GlcNpS6S(1
(where IdoAp is idopyranosyluronic acid, GlcNp is
2-amino-2-deoxyglucopyranose and S is sulfate) comprising from 75 to
90% of its structure (Fig. 1) (9, 10). Low molecular weight (LMW)
heparin has an Mr(avg) ~5,000 and is prepared
from heparin by chemical or enzymatic treatment as a clinical
antithrombotic agent (11). Homogeneous heparin oligosaccharides,
ranging in molecular weight from 665 to 4655, have also been prepared
from heparin using heparin lyase I (12, 13). These oligosaccharides
have been purified to homogeneity and structurally characterized using
high-field multidimensional NMR spectroscopy (13).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results & Discussion
References
SLPI-Sepharose Affinity Chromatography of Partially Depolymerized Oligosaccharides-- Heparin was digested to 30% reaction completion using heparin lyase I and fractionated by Sephadex G50 gel exclusion chromatography into sized oligosaccharide mixtures as previously reported (13). Two size-fractionated oligosaccharide mixtures were chosen for these studies, one in which the average oligosaccharide size was a hexasaccharide and the other in which the average oligosaccharide size was a hexadecasaccharide. The hexasaccharide mixture (50 and 500 µg) was loaded onto the SLPI-Sepharose column equilibrated with 5 mM sodium phosphate buffer, pH 7.4, the column was washed with the same buffer and then eluted in the same buffer with 20 ml of linear 0-1.5 M NaCl. The eluent was monitored by conductivity and at 232 nm. Fractions corresponding to the interacting peak were collected, exhaustively dialyzed (500 molecular weight cutoff membranes) and lyophilized. The hexadecasaccharide mixture was similarly fractionated. Fractions from the affinity chromatography experiments were subjected to gel electrophoresis (PAGE) (25). Sample (5-10 µg) were dissolved in 10-15 µl of distilled water and 15 µl of a 50% (w/v) sucrose solution and subjected to electrophoresis on a 20-cm-long linear gradient (12-22% total acrylamide) gel for 5 h at 400 V or through a 32-cm-long gradient gel for 18 h at 400 V. The gels were stained with Alcian blue (0.5% in 2% acetic acid) for 30 min, destained by washing with distilled water for 24 h, and silver-strained as described previously (25).
Binding, Inhibition, and Rate Studies
ITC-- ITC of SLPI with LMW heparin and the homogeneous heparin-derived tetrasaccharide and decasaccharide (13) were performed as described previously (17). Titrations of SLPI (1 ml at 100-200 µM) with tetrasaccharide (10 10-µl injections at 8-9 mM) were in 50 mM sodium phosphate buffer, pH 7.4, containing 10-150 mM NaC1 to vary ionic strength. Titrations of LMW heparin (1 ml at 100 µM) or decasaccharide (1 ml at 142 µM) with SLPI (20 5-µl injections at 853 µM) were in 50 mM sodium phosphate buffer, pH 7.1, containing 100 mM NaC1.
Effect of Polysaccharides on Proteinase Inhibition by SLPI-- The effect of heparin and other polysaccharides on the inhibition of three proteases by SLPI was determined by reacting a fixed concentration of protease and substrate with a series of SLPI concentrations in the presence or absence of 10 µg/ml polysaccharide. After 15 min of incubation at 37 °C, residual enzymatic activity was measured as the rate of change in absorbance at 405 nm caused by formation of pNA on a SpectraMAX 340 plate reader (Molecular Devices, Sunnyvale, CA). Human neutrophil elastase was assayed in a reaction mixture with 7.5 nM enzyme and 0.3 mM MeO-Suc-Ala-Ala-Pro-Val-pNA (26) in 100 mM Tris-HCl, pH 8.3, 0.1 M NaCl, 1% bovine serum albumin. Human cathepsin G was assayed in a reaction mixture with 16 nM enzyme and 0.4 mM N-Suc-Ala-Ala-Pro-Phe-pNA (27) in 625 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.125% Brij 35. Bovine pancreatic chymotrypsin was assayed using 13 nM enzyme and 100 µM N-Suc-Ala-Ala-Pro-Phe-pNA (28) in 100 mM Tris-HCl, pH 7.8, 10 mM CaCl2. The Km for each substrate was determined by iteratively fitting the initial velocities to the Michaelis-Menten equation using Sigma Plot (Jandel Scientific, San Rafael, CA). Data were visualized by plotting according to the Lineweaver-Burk transformation. The concentration of elastase, cathepsin G, and chymotrypsin were determined by active site titration with human SLPI (29). The inhibition constant (Ki) of recombinant human SLPI for each protease was determined as described previously (30).
Determination of Rates of Inhibition-- The association rate constant for inhibition of proteases by SLPI was determined using the progress curve method (31). Enzyme containing heparin or heparin-derived oligosaccharide (120 µl) was added to a mixture (120 µl) of SLPI and substrate, and the concentration of product (absorbance at 402 nm) was measured as a function of time at 25 °C using stopped-flow apparatus (Biologic SFM-3, Molecular Kinetics, Pullman, WA). Human neutrophil elastase (10 nM) containing heparin or heparin-derived oligosaccharide (10 µg/ml) was added to a mixture of SLPI (50 to 100 nM) and substrate MeO-Suc-Ala-Ala-Pro-Val-pNA (1.0 mM) in 50 mM Hepes, pH 7.4, 100 mM NaCl. Human neutrophil cathepsin G (30 nM) containing heparin or heparin-derived oligosaccharide (10 µg/ml) was added to a mixture of SLPI (150-350 nM) and substrate N-Suc-Ala-Ala-Pro-Phe-pNA (1.0 mM) in 50 mM Hepes, pH 7.4, 100 mM NaCl. Bovine pancreatic chymotrypsin (40 nM) containing heparin or heparin-derived oligosaccharide (10 µg/ml) was added to a mixture of SLPI (250 to 500 nM) and substrate N-Suc-Ala-Ala-Pro-Phe-pNA (500 µM) in 50 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM CaCl2. The observed pseudo-first order rate constant (kobs) for the approach to the steady state was obtained by nonlinear regression analysis of the progress curves using Biokine software (Biologic Instruments de Laboratories). The apparent second-order association rate constant (ka) for proteinase-SLPI complex formation was calculated from kobs using the following relationship (14),
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(Eq. 1) |
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(Eq. 2) |
Antigen-induced Airway Responses in Sheep
Airway Mechanics--
Adult ewes (median weight 30 kg)
were instrumented as described previously (32). Mean pulmonary flow
resistance (RL) was calculated from an analysis
of 5-10 breaths by dividing the change in transpulmonary pressure by
the change in flow at midtidal volume. Immediately after
RL determination, thoracic gas volume
(Vtg) was measured in a constant volume body
plethysmograph to calculate specific lung resistance
(SRL) by the equation SRL = RL × Vtg).
Airway Hyperresponsiveness-- Base-line airway responsiveness was determined by measuring the SRL immediately after saline inhalation and consecutive administration of 10 breaths of increasing concentrations of carbachol (0.25, 0.5, 1.0, 2.0, and 4.0%, w/v). Airway responsiveness was estimated by determining the cumulative carbachol breath units required to increase SRL by 400% over the post-saline value (PC400). One breath unit was defined as 1 breath of an aerosol containing 1% w/v carbachol (32). Antigen-induced airway hyperresponsiveness was determined by repeating the carbachol dose response 24 h after antigen challenge. A paired t test (one-tailed) was used to evaluate hyperresponsiveness studies.
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RESULTS AND DISCUSSION |
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Affinity chromatography experiments were performed on
SLPI-Sepharose using various polysaccharides (Table
I). Affinity for SLPI increased with the
increasing negative charge of the polysaccharides studied. Dextran, an
uncharged 16-linked polymer of
-D-glucopyranose, failed to bind. Chondroitin, containing one carboxylate group and
~0.15 sulfate groups/disaccharide unit, bound weakly to the column,
eluting as a sharp peak at 0.04 M NaCl. Heparan sulfate and
dermatan sulfate, both containing one carboxylate group and ~1
sulfate per disaccharide unit, bound with moderate affinity eluting
with 0.12 and 0.24 M NaCl, respectively. Heparin and
dextran sulfate (Fig. 1) bound with high
affinity to SLPI both eluting from the SLPI-Sepharose column with peaks
at 0.26 M NaCl. Heparin contained chains having a higher
affinity for SLPI (eluting at 0.74 M NaC1) than the highest
affinity chains in dextran sulfate (eluting at 0.65 M
NaC1). This is surprising as heparin has a lower negative charge
density (
3.7/disaccharide, one carboxylate and ~2.7 sulfate groups)
than dextran sulfate (
4.5/ disaccharide). These results suggest that
while SLPI-polysaccharide interaction increases with increased anionic
character of the polysaccharide, this interaction is not strictly
dependent on charge density. The glycosidic linkage, type of saccharide
present, and molecular weight may also be important in displaying the
interacting groups of the polysaccharide in a productive manner.
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Heparin eluting from SLPI-Sepharose gives a broad peak with significant trailing (up to 0.74 M) (Table I). However, when SLPI is fractionated on heparin-Sepharose, it gives a sharp and symmetric peak at 0.26 M (not shown). These data suggest that affinity fractionation of heparin is taking place on SLPI-Sepharose and that tightly binding chains might contain structurally unique SLPI-binding sites. In contrast, dextran sulfate fractionated on a SLPI-Sepharose column gave a much sharper symmetrical peak, suggesting that fractionation was the result of ion-exchange with the most highly charged chains binding with the highest avidity. Previous studies report that SLPI elutes from heparin-Sepharose at 0.7 M (16) and 0.5 M (14) NaC1, while the current study shows a lower affinity (0.26 M NaC1) between heparin and SLPI. These previous studies used Hitrap columns (Amersham Pharmacia Biotech) having a significantly higher concentration of heparin (10 mg/ml of beads) compared with a 0.75-1 mg/ml ligand density in the column used in the current study. The increased ligand density may cause an increase in both binding strength and nonspecific interactions (33).
To study the structural differences of heparin affinity-fractionated on
SLPI-Sepharose, the disaccharide composition of selected heparin
fractions was depolymerized using a mixture of heparin lyases I, II,
and III, and analyzed by capillary electrophoresis. The disaccharide
composition of heparin eluting at 0.26 M NaC1 (the peak
fraction) was compared with heparin eluting at 0.58 M NaC1
(the highest affinity fraction). The disaccharide analysis of both
fractions afforded a predominant peak at 7 min assigned to the
trisulfated disaccharide (Fig. 2,
peak 1), UA2S(1
4)-
-D-GlcNpS6S (where
UA is
4-deoxy-
-L-threo-hexa-4-enopyranosyluronic
acid) that arises from heparin's major sequence (Fig. 1). The heparin fraction with highest affinity for SLPI contained an increased amount
of undersulfated disaccharides of the structure
UAp(1
4)-
-D-GlcNpS,
UAp(1
4)-
-D-GlcNpAc6S, and
UAp(1
4)-
-D-GlcNpAc (Fig. 2, peaks 2-4,
respectively) with the
UAp arising from unsulfated iduronic or
glucuronic acid residues present in heparin. The enrichment of
undersulfated sequences in the heparin fraction with the highest affinity toward SLPI-Sepharose is surprising, since highly sulfated chains in heparin most often exhibit the greatest affinity for heparin
binding proteins (5). The enrichment of chains containing undersulfated
sequences suggests some degree of specificity in the SLPI-heparin
interaction. It is also possible that the less highly sulfated
glycosaminoglycan heparan sulfate may be an endogenously important
ligand for SLPI. Furthermore, the additional hydroxyl groups present in
undersulfated iduronic or glucuronic acid residues enriched in the
heparin, having the highest affinity for SLPI, suggest that the
SLPI-heparin interaction may involve a hydrogen-bonding component. This
is supported by the observation of Faller et al. (16). that
the SLPI-heparin interaction is not purely ionic.
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Heparin-derived oligosaccharide mixtures, prepared through the partial
digestion of heparin with heparin lyase I, were next used to better
understand the structural requirements for SLPI binding to heparin. An
oligosaccharide mixture, comprised primarily of hexasaccharides
(~80%) but containing small amounts of di-, tetra-, octa-, and
decasaccharides, was initially used for these experiments. This mixture
was fractionated on SLPI-Sepharose (Fig. 3, A and B) and
analyzed by gradient polyacrylamide gel electrophoresis (PAGE) (Fig.
3C), a method that fractionates oligosaccharides based on
their molecular size (34). In the first affinity fractionation experiment (Fig. 3A), the SLPI-Sepharose column was
deliberately underloaded, resulting in the binding of the entire sample
and its release with NaC1 (from 0.05 to 0.96 M, peak at
0.26 M). Fractions eluting in the early, middle, and late
portions of the peak were analyzed by gradient PAGE (Fig.
3C, lanes a-c, respectively). Oligosaccharides
ranging in size from disaccharide through octasaccharide bound to
SLPI-Sepharose. The sample eluting at the highest salt concentration
(Fig. 3C, lane c) was enriched in larger oligosaccharides and disenriched in disaccharide (degree of polymerization (dp) 2) and
tetrasaccharide (dp 4). Of particular note is the enrichment of an
undersulfated hexasaccharide in the higher affinity fractions (Fig.
3C, lanes b and c) migrating just
below the fully saturated hexasaccharide standard. Next, the
SLPI-Sepharose column was overloaded with the same oligosaccharide
mixture (Fig. 3B). The affinity chromatogram showed two
peaks, unbound oligosaccharides eluting in the column wash (1-11 ml)
and SLPI-bound oligosaccharides eluting again at 0.26 M
NaC1. Gradient PAGE analysis of the nonbinding oligosaccharides eluting
in the wash (not shown) contained all of the oligosaccharides present
in the original sample. Oligosaccharides eluting in the early middle
and late portion of the peak were collected and analyzed (Fig.
3D, lanes d-f). The interacting fractions contained oligosaccharides of dp 6, with both di- and
tetrasaccharides conspicuously absent in the PAGE analyses. The larger
oligosaccharides (dp
6) apparently displace the weaker binding
smaller oligosaccharides (dp 2-4) from the SLPI-Sepharose column. It
was also clear that largest oligosaccharides in the mixture (dp 8 and
10) are enriched in the later fractions (Fig. 3D, lanes e
and f). Finally, it is interesting that the
octasaccharide and decasaccharide most enriched in the high affinity
fractions (Fig. 3D, lanes e and f) migrate just
below the fully sulfated octa- and decasaccharide standards, suggesting
that they contain a slightly lower level of sulfation.
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A higher molecular weight, heparin-derived oligosaccharide mixture (average dp 16, containing dp 12-26) was next loaded on the SLPI-Sepharose column. The affinity chromatogram again showed two peaks, one in the column wash (the result of overloading the column) and a second peak (from 0.05 to 1 M, peak at 0.32 M NaC1) of high affinity oligosaccharides (Fig. 4A). Gradient PAGE analysis again showed a trend with the larger oligosaccharides eluting in fractions collected at higher salt concentrations (Fig. 4B, lanes a-c). No interaction was observed below dp 14, suggesting that this represents the minimum sequence within heparin for optimal interaction with SLPI. Again, the most interesting observation is that heparin oligosaccharides binding the tightest to SLPI migrate just below the intense bands corresponding to the fully sulfated oligosaccharides in the standard heparin oligosaccharide mixture. The enriched oligosaccharides are again undersulfated, thus supporting the data obtained from the disaccharide analysis showing that the heparin fractions binding most tightly to SLPI are enriched in unsulfated iduronic and glucuronic acid residues (Fig. 2).
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To further understand the structural requirements for oligosaccharide
interaction with SLPI, selected members of a small collection of
purified heparin oligosaccharides previously prepared in our laboratory
were next examined. A fully sulfated tetrasaccharide UAp2S(1
4)-
-D-GlcNpS6S(1
4)-
-L-IdoAp2S(1
4)-
-D-GlcNpS6S
(Fig. 1) was the smallest interacting oligosaccharide (eluting with 0.15 M NaC1). An undersulfated decasaccharide, having the
structure
UAp2S(1
4)-
-D-GlcNpS6S(1
4)-
-L-IdoAp2S(1
4)-
-D-GlcNpS6S(1
4)-
-L-IdoAp2S(1
4)-
-D-GlcNpS6S(1
4)-
-L-IdoAp2S(1
4)-
-D-GlcNpS6S(1
4)-
-D-GlcAp(1
4)-
-D-GlcNpS6S (where GlcAp is glucopyranosyluronic acid) (Fig. 1), was identified that bound tightly (~0.3 M) to SLPI. The unsulfated
glucuronic acid residue, found in this decasaccharide, is probably
the same residue enriched in SLPI affinity fractionated heparin giving rise to the
UAp containing disaccharides (Fig. 2).
ITC affords useful data on the thermodynamics of binding including
H, Ka and n (the number of
ligand interactions per mol of macromolecule) (17). The interaction of
the fully sulfated, heparin-derived tetrasaccharide (Fig. 1) with SLPI
was first examined by ITC. This tetrasaccharide bound weakly, affording a Kd of 197 µM,
H of
2.7 kcal/mole and an n of 3 (3 mol of tetrasaccharide/mol
of SLPI). These data suggested that the heparin binding domain might be
a dodeca- to tetradecasaccharide (dp 12-14) in size. SLPI interaction
with LMW heparin (Mr(avg) 5,000, corresponding
to dp ~15) had been previously examined using intrinsic fluorescence
spectroscopy and a Kd of 0.5 µM in
similar ionic strength buffer (50 mM Hepes, 150 mM NaC1, pH 7.4) (16), and a 1:1 binding stoichiometry was
reported for this interaction (1). ITC was used to explore this
interaction in more detail. Titrations of LMW heparin
(Mr(avg) 4,800) into SLPI was not possible, due
to precipitation of SLPI. In the reverse experiment, titrating SLPI
into LMW heparin, no precipitation was observed. The fitted heats of
interaction yielded a Kd of 2.4 nM,
H of -6.9 kcal/mol and an n of 1. The binding
of the undersulfated decasaccharide (Fig. 1) to SLPI was next examined by ITC. This decasaccharide showed tight binding with a
Kd of 13 nM and displayed an
n of 1.4. The ITC data suggest that a tetradecasaccharide
(dp 14) represents the minimum SLPI binding domain on heparin,
consistent with the minimum high affinity oligosaccharide observed in
the SLPI affinity chromatography experiment (Fig. 4B).
Record analysis was performed using the weakly binding tetrasaccharide,
which was available in sufficient quantities to perform multiple
titrations. The contribution of non-ionic interaction to binding was
determined using NaC1 concentrations ranging from 10 to 150 mM in 50 mM sodium phosphate buffer, pH 7.4. These data show a 40% ionic component when comparing the extrapolated
G (Kd 573 µM) at the y
intercept (1 M NaC1) in 50 mM sodium phosphate
buffer, pH 7.4, with the
G (Kd 4.3 µM) under physiologic conditions (150 mM NaC1
in the same buffer). The remaining non-ionic component (~60%) of the
interaction probably results from hydrogen bonding of the unsubstituted
hydroxyl groups present in this tetrasaccharide (Fig. 1).
Heparin is found in the granules of mast cells present in substantial quantities in the human lung tissue (6-9) where SLPI is found. Thus, the observed binding affinity between heparin and SLPI probably plays a biologically important role. Other negatively charged glycoconjugates, prevalent in the lung include heparan sulfate and mucin (35, 36). Both heparan sulfate (Table I) and mucin (35) bind to SLPI and, thus, might also be of physiologic importance. While porcine intestinal heparin binds with substantially higher affinity for SLPI than does porcine intestinal heparan sulfate (Table I), a highly sulfated heparan sulfate (10) might bind with comparable affinity.
Heparin and the heparin-derived tetrasaccharide and decasaccharide were examined for their effect on SLPI inhibition of human lung elastase as well as cathepsin and chymotrypsin (Table II). In the absence of heparin or heparin-derived oligosaccharide, SLPI is a potent (Ki ~200 pM) inhibitor of both human neutrophil elastase and bovine pancreatic chymotrypsin but considerable less inhibitory of neutrophil cathepsin G activity (Ki ~9 nM). The addition of heparin, decasaccharide or tetrasaccharide had no significant effect on the Ki of SLPI for any of these three proteases. Stopped-flow kinetics permits the measurement of the association rate (ka) of SLPI and proteases (18). In the absence of heparin or heparin-derived oligosaccharide, the association rate of SLPI for human neutrophil elastase was five times faster than SLPI association with human neturophil cathepsin G and bovine pancreatic chymotrypsin. Taken together with the low Ki of SLPI for neutrophil elastase, this fast association rate suggests neutrophil elastase is an important protease target for this inhibitor. The ka of SLPI for all three enzymes increased by 3-5-fold on the addition of heparin. The undersulfated decasaccharide also showed a 5-fold enhancement for the ka of SLPI with both human neutrophil elastase and bovine pancreatic chymotrypsin but not for human neutrophil cathepsin G. The tetrasaccharide had no effect on the ka of SLPI for any of the three proteases when compared with control. These data demonstrate that heparin, and to a lesser extent the undersulfated decasaccharide, enhance SLPI inhibition of protease by enhancing the ka of SLPI with protease without effecting a change in the Ki. Furthermore, the effect of heparin on the ka of the SLPI-protease interaction, particularly human lung elastase, suggests its therapeutic application to elastase based diseases of the lung such as asthma.
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The effect of SLPI-heparin combination therapy against antigen-induced airway hyperresponsiveness was next evaluated in a sheep bronchoprovocation model. In saline-treated animals, antigen challenge resulted in the development of airway hyperresponsiveness, as demonstrated by the decreased amount of carbachol required to induce a 4-fold increase in specific lung resistance (Fig. 5). Aerosol treatment with SLPI (10 mg) or heparin (16.7 mg of corresponding to 2,500 units) alone had no effect on the development of hyperresponsiveness. In contrast, combination therapy with SLPI (10 mg) and heparin (16.7 mg of corresponding to 2,500 IU) provided protection against the development of hyperresponsiveness (p < 0.05 versus antigen-stimulated response). In comparison, the individual treatments had no inhibitory effect on peak early and late phase bronchoconstriction following antigen challenge, while the combination therapy inhibited the peak responses by 37 (p < 0.1) and 48% (p < 0.05), respectively. These results demonstrate that heparin increases the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness. Recombinant human-SLPI has been shown to inhibit ovine elastase (37). The identification of other ovine protease(s) inhibited by recombinant human-SLPI to promote this pharmacologic effect requires further investigation.
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The sheep bronchoprovocation model has been used extensively to study the pathobiology of asthma (19). Epidemiological data suggests that patients who suffer from more severe asthma are the same ones that develop late antigen-induced response in the laboratory. Thus, the effect of SLPI-heparin combination therapy, which largely eliminates late phase bronchoconstriction in sheep, may represent a useful therapeutic approach to the treatment of severe asthma in humans.
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FOOTNOTES |
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* 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: University of Iowa, PHAR-S328, Iowa City, IA 52242. Tel.: 319-335-8834; Fax: 319-335-6634; E-mail: robert-linhardt{at}uiowa.edu.
1 The abbreviations used are: SLPI, secretory leukocyte protease inhibitor; GAG, glycosaminoglycan; LMW, low molecular weight; ITC, isothermal titration calorimetry; PAGE, polyacrylamide gel electrophoresis; pNA, p-nitroanaline; PBS, phosphate-buffered saline; dp, degree of polymerization.
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