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INTRODUCTION |
Protease nexin 1 (PN1)1
is a member of the SERPIN super-family (1, 2), and an important
physiological regulator of thrombin (Th) and urinary plasminogen
activator (uPA) (2). PN1 forms stoichiometric complexes with both Th
and uPA (3) that ultimately results in their removal by cellular
endocytosis and degradation (4). The precise biochemical nature of the
complexes is still not completely clear, but they are extremely stable
and possibly covalent (5-7). When complexes are formed between the
SERPIN and its target protease, there is an accompanying conformational change in the SERPIN that either unmasks or causes the formation of a
new binding site in the complexed SERPIN that is not present in the
free SERPIN (8, 9). The cryptic nature of the LRP-binding site in the
free SERPIN makes sense biologically. It ensures that SERPINs will
remain extracellular, either in plasma or in tissues near cell
surfaces, until they have formed an irreversible complex with a protease.
The list of SERPINs dependent on the LRP for cellular internalization
includes, protease nexin 1 (PN1) (10, 11), heparin cofactor-II (12),
antithrombin III (ATIII) (12) and
-1-antitrypsin (12). It is
interesting to note that although the LRP acts as the internalization
receptor, other cellular components are most likely required for the
efficient catabolism of the SERPIN-Protease complexes (12-15). In the
case of the plasma SERPINs these components remain to be identified.
However PN1, which is primarily restricted to tissues, utilizes at
least two different cell surface molecules to assist LRP-mediated
internalization. When PN1 forms complexes with uPA, the uPA receptor is
required for efficient concentration at the cell surface of uPA-PN1
complexes and subsequent internalization via the LRP (11, 16). In
contrast, when PN1 is in complex with thrombin, heparin chains present
at the cell surface greatly facilitate the uptake and turnover of
thrombin-PN1 complexes (15), and uPA receptor is not involved (11, 16).
Thus, in the case of PN1, the nature of the target protease directly
plays a role in the clearance mechanism.
In a recent study using a synthetic peptide library strategy, a
putative LRP-binding site was identified in PN1 (10). The library
consisted of peptides 12 amino acids in length, and spanned nearly the
entire PN1 sequence. A single peptide in the library, 47PHDNIVISPHGI58 was identified as a potent
inhibitor of Th-PN1 internalization and degradation. Using
-1-antitrypsin structure and sequence alignments, this sequence is
predicted to be a transition sequence that occurs just after helix A
and continues to form the sixth strand of sheet B (1). Consequently,
this site meets the criteria of being at least partially buried in the
intact SERPIN, with the potential of becoming more exposed after
complex formation with thrombin. There is no direct evidence, however,
to distinguish this possibility from a simple conformational change in
which the exposure of this site remains constant.
In the present studies we further investigate the potential role of
this putative site in LRP-mediated internalization using two different
approaches. In the first approach, a polyclonal IgG was generated
against Pro47-Ile58 with a cysteine residue
added after the Ile to facilitate haptenization to ovalbumin. We
demonstrate that this antibody specifically and selectively inhibits
the binding of Th-PN1 complexes to the LRP, but does not affect the
interaction of the complexes with cell surface heparins. In the second
approach we utilize site-directed mutagenesis and baculovirus-driven
expression in insect cells. Two variant forms of PN1 were expressed;
one with an alanine substitution at the position of His 48 (H48A), and
another with alanine substitutions at the positions of His 48 and Asp
49 (H48A,D49A). Each PN1 variant was characterized biochemically by
determining the kassoc value for thrombin
inhibition and ability to form SDS-resistant complexes with thrombin.
Additionally, the PN1 variant-thrombin complexes and native
PN1-thrombin complexes were assayed for their capacity to bind to cell
surface heparins. While the PN1 variants were found to be very similar
to native PN1 in their ability to inactivate thrombin and bind to cell
surface heparins, complexes made with each of the PN1 variants showed
decreased rates of catabolism. These experiments define a critical role
for the structural determinant, Pro47-Ile58,
in the LRP-mediated internalization of Th-PN1 complexes. These data
also demonstrate that with the use of
anti-(Pro47-Ile58) antibody, cell surface
heparin binding and LRP-mediated internalization of Th-PN1 complexes
can be studied as independent events, even though they act
cooperatively to facilitate complex catabolism.
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EXPERIMENTAL PROCEDURES |
Materials--
Cell culture media and reagents were purchased
from Irvine Scientific and JRH Scientific. Cell culture plastics were
from Corning. Thrombin, 3,000 NIH units/mg, was purchased from
Calbiochem, and Na125I was from Amersham Pharmacia Biotech.
High-trap heparin-Sepharose and Cibacron blue-Sepharose were from
Amersham Pharmacia Biotech. Recombinant enzymes were from Boehringer
Mannheim. All other common shelf reagents were either from Sigma or
Calbiochem. The synthesis of Pro47-Ile58-Cys
has been described previously (10). Antibodies against this peptide
were raised in rabbits, using Pro47-Ile58-Cys
coupled to ovalbumin as described previously (17). The LRP agonist,
receptor-associated protein, was expressed as a receptor-associated protein-glutathione S-transferase fusion protein (RAP-GST)
consisting of an amino-terminal glutathione S-transferase
sequence followed by the rat RAP sequence. The fusion protein was
affinity purified on a glutathione-Sepharose column as described
(15).
Cell Culture--
Human foreskin fibroblasts (HF) were grown and
maintained as described previously (8). Experimental cultures were
seeded at 1 × 105 cells/well into 24-well plates.
When the cells reached confluence, they were changed to serum-free
medium and used 48 h later.
Preparation and Expression of Recombinant Proteins--
The
methods used to prepare and express recombinant forms of PN1 in a
baculovirus-driven expression system have been described in detail
elsewhere (18) and are briefly summarized here. Recombinant cDNA
constructs of PN1 in pBluescript with Ala substitutions at His48 and Asp49 were prepared using overlapping
polymerase chain reaction to introduce the desired nucleotide
substitutions as described previously (19). The constructs were
sequenced and cloned into the pVL baculovirus shuttle vector and
co-transfected into Sf9 insect cells along with BaculoGold
baculovirus. Recombinant viruses were purified by a single round of
plaque purification. For protein expression, Sf9 cells grown in
T175 flasks were infected at a multiplicity of infection of 10:1. Five
to seven days later the media were harvested, and the recombinant forms
of PN1 were purified by affinity chromatography on Cibacron
blue-Sepharose (20).
Determination of PN1 Activity and kassoc
Constants--
Purified samples of PN1 and the recombinant variants of
known protein concentration (21) were titrated with active thrombin to
determine the percentage of activity. 30 ng of thrombin were added to
various amounts of the PN1 samples in a final volume of 100 µl of
PBS, pH 7.2, containing 0.1% bovine serum albumin. At the end of a
30-min incubation, the reactions were chilled on ice, and a 200-fold
molar excess of Chromozym-Th was added. The reactions were returned to
room temperature for 30 min to allow for color development as a measure
of residual thrombin activity. Absorbance measurements were taken at
405 nm to quantify color development. kassoc
values were determined as described previously (2).
Protein Radioiodination--
125I-Thrombin was
prepared as described previously, using the Iodogen method (22).
Specific activities ranged from 8,000 to 15,000 cpm/ng of protein.
Th-PN1 Complex Formation and Analysis--
600 ng of
125I-thrombin was added to amounts of PN1, PN1(H48A), and
PN1(H48A,D49A) required to achieve complete thrombin inhibition as
determined in the titration assay described above. Reactions were
carried out in 300 µl of PBS containing 0.1% bovine serum albumin.
At the end of a 30-min incubation, the reactions were diluted with
binding medium to a final concentration of 200 ng/ml. Prior to
dilution, 5-µl aliquots were removed and added to 15 µl of SDS-PAGE
sample buffer, and analyzed by SDS-PAGE on 10% polyacrylamide gels
(23).
Cell Binding, Internalization, and Degradation
Assays--
Binding and internalization experiments were done in
binding medium that consisted of serum-free, bicarbonate-free
Dulbecco's modified Eagle's medium, containing 20 mM
Hepes buffer, pH 7.2, and 0.1% bovine serum albumin. When binding
assays were done at 4 °C, all reagents were pre-chilled, and the
cells were placed on ice in a 4 °C cold room. Competing ligands were
added simultaneously with radiolabeled ligands. Concentrations of
ligands are indicated in the text and figure legends. At the completion
of the incubations, unbound ligand was removed, and the cells were
washed four times with 1 ml of PBS and finally lysed with 10% SDS.
Radioactivity in the samples was quantified by
counting.
Internalization assays were done in the same binding medium in a
37 °C water bath. 125I-Th-PN1 complexes were added to
the wells at a concentration of 200 ng/ml, and at the indicated times,
triplicate samples were rapidly chilled to 4 °C. Cell surface bound
complexes and internalized complexes were determined as described
previously (8).
Degradation assays were also done in the same binding medium in a
37 °C water bath. 125I-Th-PN1 complexes were added to
each well at a final concentration of 200 ng/ml in a volume of 250 µl. At the indicated times, 100-µl aliquots from triplicate samples
were each added to 1 ml of ice-cold 12% trichloroacetic acid. After a
minimum incubation of 2 h on ice, trichloroacetic acid
precipitable material was removed by centrifugation at 10,000 × g for 10 min in a refrigerated microcentrifuge. Aliquots of
the supernatants were quantified by
counting to measure
nonprecipitable radioactivity. Samples from control incubations performed in the absence of cells were processed identically to determine background levels of trichloroacetic acid nonprecipitable radioactivity and were subtracted from the values determined in parallel samples.
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RESULTS |
Anti-(Pro47-Ile58) Antibody Selectively
Blocks the LRP-mediated Internalization of Th-PN1 Complexes but Does
Not Affect Heparin-mediated Cell Surface Binding--
A rabbit
polyclonal IgG was generated against
Pro47-Ile58, and the total IgG fraction was
purified on protein G-Sepharose and brought to a final concentration of
1.0 mg/ml in PBS. To test the effect of this IgG on Th-PN1 complex
degradation, 125I-Th-PN1 complexes were formed and
pre-incubated with various dilutions of the antibody for 30 min at
37 °C. The complexes were then added to cells for 3 h at
37 °C, and release of trichloroacetic acid soluble radioactivity
into the incubation medium was measured. At a 1:10 dilution of the IgG,
complex degradation was inhibited approximately 70% (data not shown).
Because the peptide sequence, Pro47-Ile58,
lies in close proximity to the heparin-binding site, additional experiments were done to determine whether the decrease in complex degradation was due to inhibiting the LRP-binding site in the complexes
or to a steric hindrance of heparin binding. Inhibition of heparin
binding could also account for the lower degradation rate in the
presence of the antibody, because we have previously shown that a
heparin binding deficient variant of PN1 in complex with thrombin is
degraded very slowly when compared with native PN1 (15). Shown in Fig.
1 are parallel experiments done at 4 and
37 °C, where we simultaneously measured the effect of
anti-(Pro47-Ile58) IgG on both the degradation
and cell association of Th-PN1 complexes. Relative to cultures that
received only 125I-Th-PN1 complexes or a control pre-immune
IgG, experimental cultures that received
anti-(Pro47-Ile58) IgG degraded significantly
lower amounts of complexes during a 3-h incubation at 37 °C;
approximately 70% less when compared with the complex only controls
(Fig. 1A). As expected, degradation was reduced by 80% in
the presence of the LRP agonist, RAP-GST. Insignificant quantities of
trichloroacetic acid soluble radioactivity were generated at 4 °C,
demonstrating that complex degradation is dependent on endocytosis and
occurs intracellularly. In contrast to the marked effect that
anti-(Pro47-Ile58) IgG had on complex
degradation, it had no effect on total cell surface binding of the
Th-PN1 complexes at 4 °C or on total cell association of the
complexes at 37 °C (Fig. 1B). These data indicate that
the inhibitory effect of anti-(Pro47-Ile58)
IgG on Th-PN1 complex degradation is due to the specific inhibition of
binding of the complexes to the LRP and that the binding of complexes
to cell surface heparins occurs via a structural determinant that is
distinct from the heparin-binding site.

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Fig. 1.
Anti-(Pro47-Ile58)
antibody inhibits the degradation but not the cell surface binding of
125I-Th-PN1 complexes by HF cells. A,
triplicate confluent cultures of HF cells in 24-well plates were
incubated with 125I-Th-PN1 complexes at a concentration of
100 ng/ml in binding medium, either alone or in the presence of
RAP-GST, anti-(Pro47-Ile58) antibody, or
control antibody (pre-immune IgG antibody). After a 2-h incubation at
4 °C (closed bars) and 37 °C (open bars),
cell surface binding and turnover of the 125I-Th-PN1
complexes were measured. 100-µl aliquots of the culture supernatants
were removed and precipitated in 12% trichloroacetic acid to measure
the changes in trichloroacetic acid nonprecipitable radioactivity.
Background trichloroacetic acid nonprecipitable radioactivity was
determined as described under "Experimental Procedures" and
subtracted from the mean of triplicate samples. B, remaining
culture supernatants were removed by aspiration, and the cells were
then washed four times with PBS. The cell monolayers were solubilized
in 1 ml of 10% SDS, and radioactivity was quantified by counting.
Error bars indicate the standard deviation from the mean of
the triplicate samples in both panels.
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To rule out the possibility that these data might be an artifact of
antibody bivalency or the possibility that these complexes may be
associated with another cell surface component in the presence of the
antibody, we next directly demonstrated that the complexes are indeed
bound to cell surface heparins in the presence of
anti-(Pro47-Ile58) IgG. The 4 °C binding
experiment shown in Fig. 1B was repeated, this time using
complexes in the presence and absence of a 1:10 dilution of
anti-(Pro47-Ile58) IgG. At the end of the 3-h
incubation, the cells were rapidly rinsed four times with ice-cold PBS,
and 1 ml of binding medium was added back with or without 200 nM soluble heparin. The dissociation and release of the
complexes in the medium was then followed over a 12-min time course. In
the absence of soluble heparin, dissociation was not detectable either
in the presence or absence of
anti-(Pro47-Ile58) IgG (Fig.
2). In contrast, dissociation in the
presence of soluble heparin was extremely rapid, greater than 70% in 3 min. The addition of anti-(Pro47-Ile58) IgG,
at the same concentration that effectively inhibited complex degradation, had no effect on the dissociation rate. These data clearly
demonstrate that anti-(Pro47-Ile58) IgG does
not inhibit binding of Th-PN1 complexes to the cell surface and that
complex binding is indeed to cell surface heparins, because
dissociation was accelerated by the addition of soluble heparin. The
simplest interpretation of the data in Figs. 1 and 2 is that cell
surface heparin binding and LRP binding are mediated by distinct
structural determinants in PN1.

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Fig. 2.
Anti-(Pro47-Ile58)
antibody does not inhibit the binding of 125I-Th-PN1
complexes to cell surface heparins on HF cells.
125I-Th-PN1 complexes were incubated at 37 °C for 1 h in the presence or absence of
anti-(Pro47-Ile58) antibody. Confluent
cultures of HF cells in 24-well plates were incubated at 4 °C for
4 h with 100 ng/ml of either 125I-Th-PN1 complexes
alone ( , ) or 125I-Th-PN1 complexes in the presence
of anti-(Pro47-Ile58) ( , ). At the end
of the incubation, the cells were washed four times with ice-cold PBS.
Binding medium alone ( , ) or with soluble heparin (200 nM) ( , ) was then added to the cells. Culture medium
was removed at the specified times, and radioactivity was quantified by
counting. Error bars indicate the standard deviation
from the mean of duplicate samples.
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Characterization of PN1 Variants with Alanine Substitutions at
His48 and Asp49--
Both of the PN1 variants,
PN1(H48A) and PN1(H48A,D49A), were expressed in Sf9 insect cells
under control of the polyhedron promoter in baculovirus and purified
using Cibacron blue-Sepharose as described previously (20). Native PN1
was purified from the serum-free conditioned medium harvested from HF
cells using heparin-Sepharose affinity chromatography (8). Active site
titrations using thrombin in a chromogenic substrate assay were
performed on both native PN1 and each of the variants. Based on the
active site titrations and the actual protein concentration, the
percentage of active protein was calculated (Table
I). Very little difference in the activity of the protein preparations was measured. In fact, PN1(H48A) and PN1(H48A,D49A) were 74 and 76% active, respectively, which was
slightly higher than native human fibroblast PN1 that displayed 70%
activity. Using the activity measurements, the concentrations of each
of the PN1 variants and native PN1 were then adjusted to equivalent
active concentrations, and the kassoc were
determined for each of the forms of PN1 as described previously (24).
For each variant of PN1, the kassoc constants
for thrombin were found to be similar to the
kassoc constant of native PN1 for thrombin. The
PN1(H48A) variant had the highest measured
kassoc (6.8 × 10
5
M
1 s
1), but the PN1(H48A,D49A)
variant and native PN1 displayed very similar
kassoc values (5.7 × 10
5
M
1 s
1 and 6.3 × 10
5 M
1 s
1,
respectively).
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Table I
Biochemical characterization of PN1 (native), PN1(H48A), and
PN1(H48A,D49A)
Native PN1, PN1(H48A), and PN1(H48A,D49A) were purified as described
under "Experimental Procedures." The protein concentration of each
was determined, and active site titration assays were performed against
a known amount of thrombin to determine the percentage of activity of
each form of PN1. kassoc values were determined
using the colorimetric substrate, Chromozym-Th (2).
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In addition to the kassoc constant and activity
analyses presented above, which argue strongly that the introduced
substitutions do not significantly affect the biochemical activities of
PN1, we also examined the ability of the variant forms of PN1 to form complexes stable to SDS-PAGE with 125I-thrombin. The
characterization of the complex formation was critical, because any
variation in the capacity of the PN1 variants to form stable complexes
with thrombin due to the introduced substitutions could have a major
effect on their LRP-mediated catabolism. Human recombinant native PN1,
purified from Sf9 cells by the same method as each variant of
PN1, was used for comparison. Shown in Fig. 3 is a digitized image of complexes
formed between each form of PN1 and 125I-thrombin. In each
lane where recombinant native PN1 or a variant form of PN1 was present,
greater than 90% of the thrombin appeared in complex with the PN1 or
PN1 variant, demonstrating that each of the variants formed stable
complexes with 125I-thrombin at levels comparable with the
recombinant native PN1. Note that some 125I-thrombin
becomes inactivated and unable to form complexes, probably due to
oxidation during the radioiodination procedure, but all lanes showed a
similar low level of this nonreactive thrombin. Cumulatively, the close
similarity in activity, kassoc constants for
thrombin, and ability to form SDS-resistant high molecular weight
complexes with 125I-thrombin, argue that the alanine
substitutions introduced at the positions of His48 and
Asp49 do not affect the stability nor thrombin inhibitory
activity of either of the PN1 variants.

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Fig. 3.
Recombinant native PN1 and the variants
PN1(H48A) and PN1(H48A,D49A) form complexes with
125I-thrombin that are stable to SDS-PAGE. 600 ng of
125I-thrombin were incubated with amounts of native PN1 and
each of the recombinant variants required to achieve complete thrombin
inactivation within 30 min at 37 °C in a final volume of 300 µl.
At the end of the incubation, 5-µl aliquots of the reactions were
removed and added to 15 µl of SDS-PAGE sample buffer. The reactions
were analyzed by SDS-PAGE on 10% polyacrylamide gels to resolve free
125I-Th from 125I-Th-PN1 complexes. After
drying, the gel was exposed to a Bio-Rad Phospho-Imager screen for 30 min. The digitized image was developed on a Bio-Rad GS-250 Molecular
Imager. Lane 1, 125I-Th; lane 2,
125I-Th-PN1 (native); lane 3,
125I-Th-PN1(H48A); lane 4,
125I-Th-PN1(H48A,D49A).
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The Heparin-mediated Binding of 125I-Th-PN1 Variant
Complexes to the Cell Surface Is Identical to Native
125I-Th-PN1 Complexes--
In recent studies (15), we have
shown that the binding of Th-PN1 complexes to cell surface heparins
acts synergistically to enhance LRP-mediated internalization. Heparin
binding plays a similar role in the catabolism of thrombospondin 1 (25)
and lipoprotein lipase (26). The aim of our next experiments was to
measure the ability of the PN1 variant complexes to bind to cell
surface heparins, because this could also significantly affect catabolism rates. Confluent cultures of HF cells in 24-well plates were
incubated with native or variant 125I-Th-PN1 complexes for
4 h at 4 °C, in the presence and absence of the indicated
concentrations of heparin (Fig. 4). At
the end of the incubation, unbound ligand was removed, and the cultures were assayed for bound complexes by
counting. The absolute binding of native 125I-Th-PN1 complexes,
125I-Th-PN1(H48A,D49A) complexes, and
125I-Th-PN1(H48A) complexes were all virtually identical,
as were the competition curves with soluble heparin. In all three
cases, binding was inhibited by 50% at a heparin concentration of
around 5 nM. Importantly, the concentration of complexes
used in this experiment (200 ng/ml) is the same concentration used in
the internalization and degradation studies presented below. From these
results we determined that any change observed in the rate of
catabolism was not due to a difference in the initial binding
interaction of these PN1 variant complexes to cell surface
heparins.

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Fig. 4.
Substitutions of Ala at His48 and
Asp49 do not affect the cell surface heparin binding of
125I-Th-PN1 complexes. Confluent cell cultures in
24-well plates were incubated with 200 ng/ml of 125I-Th-PN1
(native) complexes ( ), Th-PN1(H48A) complexes ( ), or
125I-Th-PN1(H48A,D49A) complexes ( ) in the presence and
absence of the indicated concentrations of soluble heparin. The
incubations were allowed to reach equilibrium at 4 °C (4 h), at
which time the unbound complexes were removed, and wells were washed
four times with cold PBS. The cell monolayers were lysed with 10% SDS
and quantified by counting.
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Alanine Substitutions at His48 and Asp49
Markedly Impair Th-PN1 Complex Catabolism--
Based on our recent
studies, which identified the peptide sequence
47PHDNIVISPHGI58 as a putative binding site in
PN1 required for LRP-mediated internalization and catabolism of
125I-Th-PN1 complexes, we next evaluated the effect of
alanine substitutions as His48 and Asp49 on
125I-Th-PN1 catabolism. 125I-Thrombin-PN1
complexes were formed from each variant of PN1 and from native PN1 and
used at a final concentration of 200 ng/ml in binding medium as
described under "Experimental Procedures." Complexes were added to
confluent HF cell cultures in 24-well plates in triplicate. At the
indicated time points, trichloroacetic acid nonprecipitable
radioactivity in the media, which corresponds to
125I-tyrosine released from lysosomal degradation of the
radioiodinated thrombin (22) was determined (Fig.
5). Relative to native
125I-Th-PN1 complexes, the 125I-Th-PN1(H48A)
variant complexes were degraded at about a 50% slower rate.
125I-Th-PN1(H48A,D49A) variant complexes, in which the PN1
has an additional replacement of Asp48 with Ala, reduced
the rate of degradation to about 15% of native 125I-Th-PN1
complexes. The control experiments presented in Figs. 3 and 4 and in
Table I demonstrate that this differential rate of degradation is not
due to a difference in PN1 inhibitory activity, complex stability, or a
differential capacity to bind to cell surface heparins. Therefore,
these data would strongly suggest that there is an altered interaction
of the PN1 variant complexes with their receptor, LRP, which is
required for the internalization of the complexes prior to
intracellular degradation.

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Fig. 5.
The degradation of
125I-Th-PN1(H48A) and 125I-Th-PN1(H48A,D49A)
complexes by HF cells is markedly reduced. HF cells were incubated
with 200 ng/ml of 125I-Th-PN1 (native) complexes ( ),
Th-PN1(H48A) complexes ( ), or 125I-Th-PN1(H48A,D49A)
complexes ( ) at 37 °C in binding medium. At the indicated time
points, aliquots of triplicate samples were analyzed for the appearance
of trichloroacetic acid nonprecipitable radioactivity. Error
bars represent the standard deviation from the mean.
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The LRP-mediated Internalization of 125I-Th-PN1(H48A)
and 125I-Th-PN1(H48A,D49A) Complexes Is Sharply
Lowered--
The results shown in Fig. 5 measure the intracellular
degradation of native and variant 125I-Th-PN1 complexes as
judged by an increase in trichloroacetic acid soluble radioactivity.
The diminished degradation rates of 125I-Th-PN1(H48A) and
125I-Th-PN1(H48A,D49A) suggest an impaired interaction with
the LRP but do not demonstrate this directly. To address this, we
measured the internalization rates of 125I-Th-PN1(H48A) and
125I-Th-PN1(H48A,D49A) relative to native
125I-Th-PN1 complexes (Fig.
6). Confluent cultures of HF cells in 24-well plates were incubated with native 125I-Th-PN1
complexes, 125I-Th-PN1(H48A) and
125I-Th-PN1(H48A,D49A), each at a concentration of 200 ng/ml at 37 °C. At the indicated times, ranging from 5 to 30 min,
triplicates wells were rapidly chilled to 4 °C and processed to
remove cell surface bound complexes as described previously (8). Not
surprisingly, the internalization data directly mirror the results of
the degradation studies. Relative to native 125I-Th-PN1
complexes, 125I-PN1(H48A) were internalized at a 50%
slower rate. 125I-Th-PN1(H48A,D49A) complexes were
internalized at only about 15% the rate of native
125I-Th-PN1 complexes.

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Fig. 6.
Reduced LRP-mediated internalization of
125I-Th-PN1(H48A) and 125I-Th-PN1(H48A,D49A)
complexes accounts for the decreased rates of degradation.
Confluent cultures of HF cells were incubated with 200 ng/ml of
125I-Th-PN1 complexes ( ), Th-PN1(H48A) complexes ( ),
or 125I-Th-PN1(H48A,D49A) complexes ( ) at 37 °C. At
the indicated times triplicate wells were rapidly chilled to 4 °C
and rinsed four times with PBS to remove unbound complexes. Cell
surface bound complexes were stripped at 4 °C with a solution of
EDTA and heparin as described previously (8). The remaining monolayers
were solubilized in 10% SDS to determine the amount of internalized
complexes. In both cases the radioactivity was quantified by counting. Error bars represent the standard deviation from
the mean.
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DISCUSSION |
The present studies were undertaken to further probe the potential
role of the PN1 peptide sequence,
47PHDNIVISPHGI58, in the LRP-mediated clearance
of Th-PN1 complexes and to determine what structural features of this
sequence might be important in this process. The potential importance
of this sequence was discovered using a synthetic peptide library of
PN1 sequences and screening for peptides that inhibited the catabolism
of complexes by HF cells (10). The requirement for heparin-mediated
binding of the complexes to the cell surface to promote an efficient
interaction between the complexes and the LRP (15) precluded more
detailed studies of this sequence using a synthetic peptide approach,
because the sites in PN1 that interact with heparin and the LRP are
distinct. In the present studies, two different approaches have been
used to more clearly define the role of the structural determinant, Pro47-Ile58, in Th-PN1 complex catabolism and
to identify critical residues within this determinant.
To independently confirm and more narrowly define the role of this
structural determinant in Th-PN1 catabolism, an
anti-(Pro47-Ile58) polyclonal IgG was
generated. This antibody specifically inhibited the internalization and
subsequent degradation of Th-PN1 complexes but had no effect on the
binding of complexes to cell surface heparins. These data independently
confirm the role of Pro47-Ile58 in
LRP-mediated internalization, which had previously been based solely on
synthetic peptide competition studies (10). In addition, they strongly
suggest that the LRP and heparin-binding determinants in PN1 are
structurally distinct, despite the fact that they act cooperatively to
promote efficient complex catabolism (15).
In addition to the antibody studies, a genetic approach was used in
which alanine substitutions were introduced at positions His48 and Asp49 of the intact PN1 protein. The
rationale for these choices is derived from the predicted structural
location of 47PHDNIVISPHGI58 in PN1 based on
its homology to
-1-antitrypsin (1). This sequence is predicted to be
at least partially interior to the protein surface. Pro47
most likely represents the beginning of the transition sequence between
helix A and strand 6B(1). His48 and Asp49 are
transition amino acids, and Asn50 through Pro55
become strand 6 of sheet B. Given this structural information, we
hypothesized that the charged residues, His48 and
Asp49, might be important for either direct interaction
with the LRP receptor or assisting in the attainment of the active
conformation of this structural determinant when fully exposed to the
hydrophilic exterior of the molecule.
To evaluate the role of these specific amino acids we generated two
different PN1 variants: one in which only His48 was
replaced by Ala and another where both His48 and
Asp49 were replaced by Ala. Replacement of the first
charged residue, His48 by Ala, had a significant effect on
the catabolism of PN1(H48A) complexes, reducing it by 50%. The
additional substitution of Ala for Asp49, reduced complex
catabolism by 85%. In both cases this was demonstrated to be due to a
decreased rate of LRP-mediated internalization. Control studies
revealed that the substitutions had no effect on the heparin-mediated
binding of the complexes to the cell surface nor on the biochemical
characteristics of the PN1 variant complexes with thrombin. Taken
together, it is clear that both His48 and Asp49
play very important roles in the interaction of the PN1 structural determinant, 47PHDNIVISPHGI58, with the LRP.
Whether this is due to a direct interaction of these residues with the
LRP or because these residues are required to establish a particular
structural conformation of this determinant is presently unknown. Given
the overall hydrophobic character of
Pro47-Ile58, (PHDNIVISPHGI), it is likely that
removal of the charged residues affects the solubility of the sequence.
The genetic evidence using PN1 variants with point substitutions and
the anti-(Pro47-Ile58) antibody experiments
represent two important and independent lines of evidence that support
and extend the original observation that the sequence
Pro47-Ile58 in PN1 is required for the
LRP-mediated clearance of Th-PN1 complexes.
A common pathway for many LRP internalized ligands seems to be emerging
that involves cell surface proteoglycans and perhaps other accessory
proteins in many cases. Several of the ligands first bind to cell
surface heparin sulfate proteoglycans and are subsequently internalized
by the LRP (15, 25-27). Even Th-ATIII complexes, which display a
negligible affinity for heparin, use hepatic heparin sulfate
proteoglycans as a part of their clearance mechanism but do so by an
association with vitronectin (Vn) (14). Th-ATIII complexes first bind
to Vn and then utilize the heparin-binding site in Vn, which is exposed
only after it binds to Th-ATIII complexes (14). Recently, another
accessory molecule, cytokeratin 18, has been shown to play an important
role in the clearance of Th-ATIII-Vn ternary complexes (13). Antibodies
specific for cytokeratin 18 were shown to markedly reduce the
LRP-mediated internalization of Th-ATIII-Vn ternary complexes. Although
the heparin-mediated pathway appears to be common for many LRP
internalized ligands, there is at least one example where the
involvement of heparin has not yet been documented (27). The uPA
receptor binds complexes between high molecular weight urokinase and
plasminogen activator inhibitor 1 or PN1 prior to LRP-mediated
internalization (27). There are data suggesting that the uPA receptor,
as well as its bound ligand, is co-internalized along with the LRP
(16). Because plasminogen activator inhibitor 1 does contain a
heparin-binding site, however, the potential involvement of heparin in
this pathway should be examined more carefully.
Structural information on binding sites in ligands that interact with
the LRP is, however, limited. The highest resolution studies have been
done on the LRP-binding site in activated
2-macroglobulin, which identified lysine residues 1370 and 1374 as essential for binding to the LRP (28). The LRP-binding site
in thrombospondin 1 has been localized to the same amino-terminal
fragment that contains the heparin-binding domain (25), and the
carboxyl-terminal folding domain of lipoprotein lipase, which also
contains the heparin-binding domain, has been implicated in binding to
the LRP (26). Most recently, studies on the binding of plasminogen activator inhibitor 1-protease complexes to the LRP have implicated two
residues located in the heparin-binding site in LRP binding (27). These
data may be subject to alternate interpretations, because the opposite
charge nature of the amino acid substitutions in plasminogen activator
inhibitor 1 variants that resulted in a decreased LRP affinity may
impart structural changes in the general region that are not
necessarily part of the heparin-binding site (27). In addition, data
supporting any type of a universal structural motif shared by all LRP
ligands are not very compelling, because the ligands are diverse, and
many of the ligands do not cross-compete for binding (28). Although the
binding of several LRP ligands to cell surface heparins has been shown
to play an important role in their clearance by the LRP, it remains to
be determined whether the heparin and the LRP-binding sites overlap in
some cases. The data in the present report, along with previously published studies using synthetic peptides strongly argue against this
in the case of PN1.