Altered Processing of Fibronectin in Mice Lacking Heparin
A ROLE FOR HEPARIN-DEPENDENT MAST CELL CHYMASE IN FIBRONECTIN
DEGRADATION*
Elena
Tchougounova
,
Erik
Forsberg§,
Gustaf
Angelborg
,
Lena
Kjellén
, and
Gunnar
Pejler
¶
From the
Swedish University of Agricultural Sciences,
Department of Veterinary Medical Chemistry, The Biomedical Center, 751 23 Uppsala, Sweden and the § Uppsala University, Department
of Medical Biochemistry and Microbiology, The Biomedical Center,
751 24 Uppsala, Sweden
Received for publication, September 14, 2000, and in revised form, October 13, 2000
 |
ABSTRACT |
We have previously generated a mouse strain with
a defect in its heparin biosynthesis by targeting the gene for
N-deacetylase/N-sulfotransferase-2 (NDST-2).
The NDST-2
/
mice show reduced levels of various mast
cell mediators such as histamine and various heparin-binding mast cell
proteases, including chymases, tryptases, and carboxypeptidase A. In
this work we have addressed the possible functional consequences of the
lack of sulfated heparin. Peritoneal cells were harvested from normal
and NDST-2
/
mice. After culturing the cells,
conditioned media were collected and were subjected to
SDS-polyacrylamide gel electrophoresis under reducing
conditions. Several differences in the protein patterns were observed,
including the presence of large amounts of a ~250-kDa protein in
medium from NDST-2
/
mice that was absent in normal
controls. Peptide microsequencing revealed identity of this protein
with fibronectin. Western blot analysis showed the presence of
fibronectin degradation products in cell cultures from normal mice,
which were absent in cultures from NDST-2
/
animals.
Further experiments showed that the degradation of fibronectin observed
in cell cultures from NDST-2+/+ mice was catalyzed by mast
cell chymase in a strongly heparin-dependent manner. This
report thus indicates a biological function for chymase/heparin proteoglycan complexes in fibronectin turnover.
 |
INTRODUCTION |
Heparin is a highly sulfated glycosaminoglycan with well known
anticoagulant properties commonly utilized in antithrombotic therapy.
However, its exclusive synthesis by mast cells and its extravascular
location within the secretory granules of mast cells strongly argues
against a major role for heparin as a physiological anticoagulant.
Heparin is synthesized as a (GlcA-GlcNAc)n backbone that
undergoes several polymer modification reactions, including
N-deacetylation, N-sulfation, C5 epimerization of
selected GlcA residues to IdoA, 2-O-sulfation of IdoA/GlcA
residues, and O-sulfation of glucosamine residues at the 6- and 3-positions (1). In its proteoglycan form, the heparin chains are
attached through a Xyl-Gal-Gal linkage region to a serglycin protein
core (2). To study the biological function of heparin proteoglycan, we
recently developed a heparin-deficient mouse strain by targeting the
gene for NDST-2,1 a
biosynthetic enzyme involved in the initial stages of the heparin modification process (3). In the absence of functional NDST-2, heparin
remains as the unmodified nonsulfated precursor. The
NDST-2
/
mice develop normally and are fertile. However,
their mast cells are severely affected (4, 5). The mast cells show
drastically reduced levels of inflammatory mediators such as histamine
and various mast cell proteases. In addition, the mast cell granules display an altered morphology as compared with the normal cells, with
large apparently "empty" vacuoles instead of the normal
electron-dense granule. Because the actual expression of mast cell
proteases was not affected by the inactivation of NDST-2 it appears
that, in the absence of mature heparin, the storage of these
inflammatory mediators is impaired (4).
Large amounts of various proteases are stored in the mast cell
secretory granules, constituting up to ~25% of the total cell protein in mast cells. The mast cell proteases are classified as either
(i) tryptases, tetrameric serine proteases with trypsin-like substrate
specificities, (ii) chymases, serine proteases with chymotrypsin-like
substrate specificities, or (iii) carboxypeptidase A (6). In both the
chymase and tryptase families, several related enzymes are expressed
within the same species. In mouse, the connective tissue type of mast
cells express the tryptases mouse mast cell protease 6 (mMCP-6) and
mMCP-7 as well as the chymases mMCP-4 and mMCP-5 (7-12). Both the
chymases and tryptases bind strongly to heparin proteoglycan within the
secretory granules. In addition, both tryptases and chymases are
dependent on heparin for their activity. The tryptases require binding
to heparin for gaining its active tetrameric form (13), and the
continuous presence of heparin is needed to maintain enzymatic
stability (14). The chymases, on the other hand, are active in the
absence of heparin. However, the presence of heparin leads to
potentiation of chymase activity toward some substrates (15) and to
protection from various macromolecular protease inhibitors (16).
In the present study, the aim was to study the consequences of the lack
of functional heparin. Peritoneal cells, a cellular population
containing both macrophages and lymphocytes as well as mast cells of
the connective tissue subtype were cultured, and the proteins secreted
by normal and NDST-2
/
cells were compared. Although
several differences were noticed, the most striking finding was the
presence of a ~250-kDa protein band in conditioned media from
NDST-2
/
cells, that was absent in media from normal
cells. The protein was identified as fibronectin. Subsequent analyses
showed that, in normal cells, fibronectin is degraded in a
heparin-dependent manner by mast cell chymase, whereas very
little degradation is seen in the NDST-2
/
cells. This
report thus indicates that heparin in complex with chymase may be
involved in the turnover of fibronectin.
 |
MATERIALS AND METHODS |
Reagents--
Protamine (Mr ~4500)
grade IV, cycloheximide, pepstatin, N-ethylmaleimide were
purchased from Sigma. PefablocSC, a serine protease inhibitor, was
purchased from Pentapharm Ltd. (Basel, Switzerland). Rat chymase
(rMCP-1) and heparin proteoglycan were purified from peritoneal mast
cells as described previously (17).
1-Antichymotrypsin
and
1-antitrypsin were purchased from Calbiochem (La
Jolla, CA), and donkey anti-rabbit Ig conjugated to horseradish peroxidase was from Amersham Pharmacia Biotech. Fibronectin purified from human plasma and rabbit anti-human fibronectin antiserum were
kindly provided by Staffan Johansson (Dept. of Medical Biochemistry and
Microbiology, Uppsala University).
Cells--
Peritoneal cells from NDST-2
/
mice and
corresponding NDST-2+/+ mice (4) (females, 12-15 weeks
old) were collected by peritoneal washing with 10 ml of cold
phosphate-buffered saline (PBS), pH 7.4. Cells were centrifuged
(300 × g, 4 °C, 10 min) and cultured in serum-free Dulbecco's
modified Eagle's medium/Ham's F-12 medium (Life Technologies, Inc.).
The Dulbecco's modified Eagle's medium was supplemented with 2 mM L-glutamine (Life Technologies, Inc.) and
penicillin-streptomycin (100 IU/ml, 100 mg/ml; Life Technologies, Inc.). The cells were distributed in 24-well plates (Nunc; ~2 × 106 cells in 0.4 ml/well). Cells were incubated at 37 °C
overnight in a humidified atmosphere of 5% CO2.
Protein Microsequencing--
Samples from conditioned media were
recovered from peritoneal cell cultures and were subjected to SDS-PAGE
on 10% polyacrylamide gels under reducing conditions. Gels were
silver-stained according to Morrissey (18). Bands of interest were
subsequently excised, trypsin-digested and subjected to microsequencing
using a Q-tof (Micromass, Manchester, UK) electrospray tandem mass spectrometer.
Western Blot Analysis--
After culturing peritoneal cells for
~20 h, conditioned media were collected. To 400 µl of
conditioned medium, 3× SDS-PAGE sample buffer (200 µl) was added.
Cell extracts were prepared by adding 300 µl of 3× SDS-PAGE sample
buffer to the culture dishes after removal of conditioned media.
Samples (40 µl) of these mixtures were subjected to SDS-PAGE on 6%
polyacrylamide gels under reducing conditions in a Laemmli system.
Proteins were subsequently blotted onto nitrocellulose membranes,
followed by blocking with 5% milk powder in PBS for 2 h at
20 °C. Next, the membranes were incubated with rabbit anti-human
fibronectin, diluted (1: 200) in 5% milk powder/TBS/0.1% Tween 20, at
4 °C for 20 h. After washing the membranes extensively with
TBS/0.1% Tween 20, the membranes were incubated with donkey
anti-rabbit Ig conjugated to horseradish peroxidase (Amersham Pharmacia
Biotech; 1:3000 dilution in TBS/0.1% Tween 20). After 45 min of
incubation at 20 °C, the membranes were washed extensively with
TBS/0.1% Tween 20, followed by washing with TBS without detergent. The
membranes were developed with the ECL system (Amersham Pharmacia
Biotech) according to the protocol provided by the manufacturer.
Purification of Mast Cells--
Peritoneal cells from normal
mice (females, 12-15 weeks old) were collected by peritoneal washing
with 10 ml of cold PBS, pH 7.4. Mast cells (connective tissue type) of
~95% purity (the majority of the contaminating cells were red blood
cells), as judged by staining with toluidine blue, were prepared by
density gradient centrifugation on metrizamide (19).
Inhibition of Fibronectin Degradation--
Peritoneal cells from
normal mice were collected and cultured as described above. Different
inhibitors were added to the cells during the culture period: protamine
(0.25 µg/ml, 2.5 µg/ml, 25 µg/ml); cycloheximide (5 µg/ml);
1-antichymotrypsin (5 µg/ml, 45 µg/ml);
1-antitrypsin (5 µg/ml, 45 µg/ml);
N-ethylmaleimide (1 mM); EDTA (10 mM); pepstatin (10 µg/ml); and PefablocSC (2 mM).
Stimulation of Chymase-catalyzed Fibronectin Degradation by
Heparin--
Fibronectin (0.2, 2, and 20 µg) purified from human
plasma was incubated with 25 ng of purified rMCP-1 and 50 ng of rat
heparin proteoglycan in PBS (total volume, 100 µl). Samples (30 µl)
were taken after 15 and 40 min of incubation, mixed with 15 µl of 3× SDS-PAGE sample buffer and subjected to SDS-PAGE under reducing conditions in a Laemmli system, utilizing 6% polyacrylamide gels, followed by Western blot analysis as described above. To equalize the
amount of fibronectin in each lane, samples containing 20 µg of
fibronectin were diluted 100-fold in SDS-PAGE sample buffer before
electrophoresis, and samples containing 2 µg of fibronectin were
diluted 10 × in SDS-PAGE sample buffer before analysis (samples containing 0.2 µg of fibronectin were analyzed without dilution).
Determination of Cleavage Sites in Fibronectin--
Fibronectin
(40 µg) purified from human plasma was incubated with 100 ng of
purified rMCP-1 and 250 ng of rat heparin proteoglycan in PBS (total
volume 50 µl). Samples (25 µl) were taken after 90 min of
incubation, mixed with 12.5 µl of 3× SDS-PAGE sample buffer, and
subjected to SDS-PAGE under reducing conditions in a Laemmli system,
using 6% polyacrylamide gels. After electrophoresis, degradation
products were transferred to a polyvinylidene difluoride membrane
(Bio-Rad; equilibrated ~10 min in methanol) by using a Mini
Trans-Blot Cell (Bio-Rad). After transfer (~1 h, 90 mA), the membrane
was stained with Coomassie Brilliant Blue for ~5 min and destained in
50% methanol/10% acetic acid. The membrane was dried during 1 h
on Whatman paper. N-terminal sequence analysis of recovered fragments
was carried out using an ABI 476A (Applied Biosystems, Foster City, CA)
automatic protein sequenator.
 |
RESULTS |
Total peritoneal cell populations from both NDST-2+/+
and NDST-2
/
mice were cultured overnight. To elucidate
any differences among the proteins expressed by these cells,
conditioned media as well as cell solubilisates were subjected to
SDS-PAGE analysis followed by silver staining (Fig.
1). The cell fractions from both normal and NDST-2
/
mice displayed highly similar protein
patterns. In contrast, several differences were seen among the proteins
present in the conditioned media (Fig. 1). In particular, high amounts
of a ~250-kDa band are seen in media from NDST-2
/
cells but are largely absent in media from normal cells. Material corresponding to this band was recovered and subjected to
microsequencing after trypsin digestion. Several tryptic peptides were
obtained, and their sequences are displayed in Table
I. The sequences of all these peptides
matched the sequence of mouse fibronectin. Thus, large amounts of
soluble fibronectin are present in conditioned media from
NDST-2
/
cells but are largely absent in media from
normal cells.

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Fig. 1.
SDS-PAGE analysis of cell fractions and
conditioned media from peritoneal cell cultures. Peritoneal cells
from NDST-2+/+ and NDST-2 / mice were
incubated for ~20 h. Cell fractions (after removal of conditioned
media) and conditioned media were subjected to SDS-PAGE under reducing
conditions in a Laemmli system, utilizing gels containing 10%
polyacrylamide followed by silver staining. The protein band indicated
by an arrow was identified as fibronectin.
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Table I
Tryptic peptide sequences obtained by microsequencing of ~250-kDa
band present in conditioned media from NDST-2 / peritoneal
cells
|
|
Western blot analysis, utilizing a polyclonal anti-fibronectin
antibody, confirmed the presence of fibronectin in media from NDST-2
/
cells. Again, very little intact fibronectin
antigen was found in normal cell culture media. However, several bands
of lower molecular weight that reacted with the anti-fibronectin
antiserum were observed, indicating that fibronectin was degraded in
cell cultures from normal cells (Fig.
2A). Also cellular extracts
displayed reduced amounts of fibronectin in the normal as compared with NDST-2-deficient cells (Fig. 2B). When cycloheximide was
present during the culture period, the fibronectin band was markedly
reduced, indicating that the fibronectin was synthesized by the cells
and not carried over from the peritoneal wash fluid (results not
shown). Most likely, macrophages are the main source of fibronectin in this cellular system because macrophages are known producers of fibronectin (20).

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Fig. 2.
Degradation of fibronectin by
NDST-2+/+ peritoneal cells. Peritoneal cells from
NDST-2+/+ and NDST-2 / mice were cultured
~20 h. A, after culture, the conditioned media
(A) and cell fractions (B) were subjected to
Western blot analysis using an anti-fibronectin antiserum.
|
|
Further studies were conducted to determine the mechanism of
fibronectin degradation in the normal cell cultures. When peritoneal cells were depleted from mast cells, the degradation of fibronectin was
markedly reduced, indicating that mast cells were responsible for the
proteolytic activity (Fig. 3). Further,
reconstitution of the mast cell-depleted peritoneal cells with purified
mast cells resulted in increased fibronectin degradation in a
dose-dependent fashion (Fig. 3). Purified mast cells did
not show any detectable expression of fibronectin (Fig. 3).

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Fig. 3.
Fibronectin degradation is mast
cell-dependent. Nonfractionated peritoneal cells
(Control), mast cell-depleted cells, and mast cell-depleted
cells reconstituted with different numbers of mast cells (5 × 103, 20 × 103, and 100 × 103) and different numbers of purified
NDST-2+/+ mast cells (20 × 103 and
100 × 103) were incubated ~20 h. After incubation,
the conditioned media were subjected to Western blot analysis using an
anti-fibronectin antiserum. MC, mast cells.
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|
Fibronectin degradation was not inhibited by EDTA,
N-ethylmaleimide, nor pepstatin, inhibitors of
metalloproteases, thiol proteases, and aspartyl proteases,
respectively. In contrast, complete inhibition of fibronectin
proteolysis was obtained by PefablocSC, a specific inhibitor of serine
proteases (Fig. 4A). These
results strongly suggest that fibronectin is degraded by mast cell
serine proteases. To distinguish between tryptases and chymases, the
effect of the macromolecular weight protease inhibitors
1-antichymotrypsin and
1-antitrypsin
was investigated. Tryptase is known to be insensitive to both of these
inhibitors, whereas chymase is inhibited to some extent by
1-antichymotrypsin but slowly by
1-antitrypsin (16). Results presented in Fig.
4B show that the degradation of fibronectin was completely
inhibited by
1-antichymotrypsin but not by
1-antitrypsin, indicating that mast cell chymase is the
enzyme mainly responsible for fibronectin degradation in the normal
cells. Because chymase is essentially absent in NDST-2-deficient mast
cells (4), the lack of fibronectin degradation in the NDST-2-deficient
cells is thus in agreement with this notion.

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Fig. 4.
Inhibition of fibronectin degradation.
Peritoneal cells from NDST-2+/+ mice were incubated for
~20 h. A, different protease inhibitors were present
during the culture period: N-ethylmaleimide
(NEM;1 mM), EDTA (10 mM), pepstatin
(10 µg/ml), and PefablocSC (2 mM). As a control,
peritoneal cells from NDST-2+/+ mice were cultured without
protease inhibitors. B, different amounts of
1-antichymotrypsin (5 and 45 µg/ml) or
1-antitrypsin (5 and 45 µg/ml) were present during the
culture period. As a control, peritoneal cells from
NDST-2+/+ mice were cultured without inhibitors.
C, different concentrations of the polycationic heparin
antagonist protamine (0.25, 2.5, and 25 µg/ml) were present during
the culture period. As a control, peritoneal cells from
NDST-2+/+ mice were cultured in the absence of protamine.
For all panels, samples from cell culture media were subjected to
Western blot analysis using an anti-fibronectin antiserum.
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Chymases have previously been shown to be dependent on heparin for
optimal proteolysis of certain substrates (17, 21, 22). To investigate
whether the degradation of fibronectin is dependent on heparin, we
tested the effect of protamine, a heparin antagonist, on the
degradation of fibronectin in cell cultures from normal mice. Addition
of protamine to the cell cultures caused inhibition of fibronectin
proteolysis in a dose-dependent fashion, with essentially
complete inhibition obtained after addition of 10 µg of protamine to
the cells (Fig. 4C). This indicates that chymases are highly
dependent on heparin for optimal degradation of fibronectin in the cell cultures.
Further studies were performed in a purified system to study the effect
of heparin on chymase-catalyzed fibronectin degradation and to
determine major cleavage sites in fibronectin. Chymase (rMCP-1; rat
homologue to the major chymase in mouse peritoneal mast cells, mMCP-4;
Ref. 23) was incubated with different concentrations of human
fibronectin, in either the absence or the presence of heparin. Rapid
degradation of fibronectin was observed, and the rate of
chymase-catalyzed fibronectin proteolysis was enhanced by heparin
proteoglycan (Fig. 5). The enhancing
effect of heparin was most prominent at low fibronectin concentrations
(Fig. 5). Fibronectin fragments were blotted onto nitrocellulose (Fig.
6) and subjected to N-terminal sequence
analysis. N-terminal sequences were obtained for fragments 1-4 (Fig.
6) and are listed in Table II. The
N-terminal of fragment 5 (Fig. 6) was blocked. Because the N-terminal
of human fibronectin is known to be blocked (24), fragment 5 may
correspond to the original N-terminal portion of fibronectin.
Comparison of the obtained N-terminal sequences with that of intact
fibronectin revealed three major cleavage sites for chymase (Table
III). Fragments 2 and 4 contained the
same N-terminal sequence, indicating that fragment 4 had undergone
further processing in the C-terminal portion. In accordance with the
known substrate specificities of chymotrypsin-like enzymes, all
cleavage sites contain an aromatic amino acid residue at the P1
position and show no obvious homologies in the P2-P5 or P1'-P5'
regions. The cleavage sites are all located in the first three type III
repeats of fibronectin, on the C-terminal side of the
collagen/gelatin-binding domain (Fig.
7).

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Fig. 5.
Stimulation of chymase-catalyzed fibronectin
degradation by heparin proteoglycan. Fibronectin (0.2, 2, and 20 µg) was incubated with 25 ng of purified chymase in the presence or
absence of 50 ng of heparin proteoglycan (hep PG; total
volume 100 µl, in PBS). Samples (30 µl) were taken after 15 and 40 min of incubation and subjected to SDS-PAGE under reducing conditions
followed by Western blot analysis using an anti-fibronectin antiserum.
To equalize the amount of fibronectin in each lane, samples containing
20 µg of fibronectin were diluted 100-fold in SDS-PAGE sample buffer
before electrophoresis, and samples containing 2 µg of fibronectin
were diluted 10× in SDS-PAGE sample buffer before analysis.
FN, fibronectin.
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Fig. 6.
Determination of cleavage sites in
fibronectin. Fibronectin (FN, 40 µg) was incubated
with 100 ng of purified chymase and 250 ng of heparin proteoglycan
(total volume, 50 µl, in PBS). Samples (25 µl) were taken after 90 min of incubation and subjected to SDS-PAGE under reducing conditions
followed by transfer of proteins to a polyvinylidene difluoride
membrane and N-terminal sequence analysis. N-terminal sequences were
obtained for fragments 1-4 (Table II). The N-terminal of fragment 5 was blocked.
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Table II
N-terminal sequences of various fibronectin fragments generated after
cleavage with rMCP-1 in the presence of heparin proteoglycan
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Fig. 7.
Schematic view of fibronectin. The
following symbols are used: , type I repeats; , type II repeats;
shaded oval, type III repeats; , V segment (24). Binding
regions for various ligands are shown. Arrows indicate the
identified cleavage sites for chymase.
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 |
DISCUSSION |
The recently developed heparin-deficient mouse strain provides an
excellent tool to elucidate the biological function of this polysaccharide. In addition, because the heparin-deficient mice also
are essentially completely devoid of the various heparin-binding proteases that are expressed by the connective tissue subtype of mast
cells, these mice may also be valuable for studying functional aspects
of the mast cell proteases (4).
The biological function(s) of the mast cell proteases is not
understood. However, the secretion of these enzymes during mast cell
degranulation suggests involvement in inflammatory reactions. Indeed,
several studies have pointed to important pro-inflammatory properties
of both tryptases (25-28) and chymases (29, 30) in vivo.
Although their involvement in inflammatory responses appears clear, the
exact mechanism by which they act is not certain. It has been proposed
that tryptases can modulate inflammation by activating protease
activated receptor-2 on the surface of various cell types, leading to
cellular activation (31). In addition, various other proteins of
potential importance in the inflammatory response, including high
molecular weight kininogen (32), fibrinogen (33, 34), and complement
factor C3 (35), have been shown to be substrates for tryptase. Several
substrates for various chymases have been identified (22, 36-44),
although it is not clear whether any of these proteins are substrates
for chymase in vivo. In particular, it is worth noting that
several known substrates for both tryptases and chymases are connective tissue components or are involved in connective tissue turnover (21,
40, 45-48). One possible biological function of the mast cell
proteases, although not proven in vivo, may thus be to carry out or assist in connective tissue turnover.
Mast cell chymases have rather broad substrate specificities. Thus, it
is likely that most proteins contain peptide sequences that potentially
could be recognized as substrates by mast cell chymase provided that
they are exposed. Accordingly, chymase may have a general degradative
role in the tissue after release from the mast cells. However, the
cleavage preferences of the chymases may not solely be related to the
structure of the actual active site but could also be influenced by
other factors. One such factor may be the association of chymases with
the strongly negatively charged heparin proteoglycan (49). Because
chymases are present as tight complexes with heparin proteoglycan both
within the mast cell granules and after exocytosis, it is relevant to
consider the complex of chymase with heparin as the physiological form of chymase. Therefore, effects on substrate cleavage properties imposed
by heparin proteoglycan are likely to reflect the situation in
vivo. We demonstrated recently that the chymase-catalyzed cleavage of thrombin, a heparin-binding protein, was strongly potentiated by
heparin proteoglycan (15). Blocking of the heparin-binding site of
thrombin abolished the stimulatory effect of heparin on thrombin
degradation. These results led to a model of chymase action where the
association of chymase with heparin proteoglycan was suggested to
direct the substrate specificity preferentially toward heparin-binding
proteins. Various proteins containing a heparin-binding region may be
"captured" by the heparin chains in the chymase/heparin complex,
thereby facilitating contact between chymase and susceptible regions in
its potential heparin-binding substrates. The present report suggests
that fibronectin is a substrate for chymase/heparin proteoglycan
complexes. Because fibronectin is a known heparin-binding protein (24),
the degradation of fibronectin by chymase in complex with heparin
proteoglycan is thus consistent with the suggested model.
The degradation of fibronectin in peritoneal cell cultures from
NDST-2+/+ mice was essentially completely inhibited when
protamine, a heparin antagonist, was present. Protamine is a
polycationic protein that has been shown to compete with chymase for
binding to heparin proteoglycan (50). The addition of protamine would
therefore result in release of chymase from heparin proteoglycan,
without affecting the actual active site of chymase. Our results
suggest that free chymase is less effective in degrading fibronectin
than when bound to heparin proteoglycan, supporting a vital importance of the association of chymase with heparin proteoglycan for optimal rate of chymase-catalyzed fibronectin proteolysis.
The suggested mechanism for the enhancement of fibronectin degradation
by heparin implies that heparin increases the likelihood that
fibronectin molecules reach contact with chymase. Thus, heparin enhances the apparent affinity between chymase and fibronectin, which
would be reflected by a decreased Km for the rate of
proteolysis. Hence, if the rate of fibronectin proteolysis follows
Michaelis-Menten kinetics, the stimulatory effect of heparin would be
largest at low fibronectin concentrations. When increasing amounts of
fibronectin is available, the rates of fibronectin proteolysis would
reach the same Vmax both in the absence or
presence of heparin. Our results show that the stimulatory effect of
heparin is largest at lower fibronectin concentrations, in agreement
with an effect of heparin on the Km for the rate of
fibronectin proteolysis.
Peritoneal mast cells are known to express at least two different
chymases, mMCP-4 and -5 (4). Because both mMCP-4 and -5 are highly
basic and thus capable of interaction with heparin, either or both of
these chymases may be responsible for the observed degradation of
fibronectin. However, because no reagents that specifically block
either mMCP-4 or -5 activity are available, we can at present not
distinguish whether fibronectin is primarily degraded by mMCP-4 or
mMCP-5.
Fibronectin is a multi-functional protein, built up of several distinct
domains capable of interacting with various ligands, including
collagens, integrins, heparin/heparan sulfate, and fibrin (Ref. 24 and
Fig. 7). Accordingly, fibronectin is known to play a role in
diverse biological processes, such as cell binding, cell migration,
matrix assembly, embryonic development, and connective tissue
remodeling (24, 51). Altered fibronectin processing has been noted in
numerous pathological conditions, including cancer, arthritis,
thrombosis, and wound healing. For instance, inflammatory conditions
are often associated with the generation of fibronectin degradation
products that are not found under normal conditions (52). Such
fibronectin fragments may reflect tissue degradation because of the
release of proteolytic enzymes during disease. The present report
raises the possibility that mast cell chymase may be involved in the
formation of such fibronectin degradation products. Degradation of
fibronectin could, for example, affect the ability of inflammatory
cells to attach to matrix and may disrupt the organization of the
extracellular matrix, thereby facilitating movement of cells within the
tissue. Importantly, it has been suggested that some of the fibronectin
fragments display activities that are cryptic in the intact molecule
(53-55). Thus, proteolysis of fibronectin by chymase could unleash
fibronectin activities that potentially may be important components in
the regulation of e.g. inflammatory responses.
 |
ACKNOWLEDGEMENT |
We are grateful to Staffan Johansson for
helpful discussions and for providing the anti-fibronectin antiserum.
 |
FOOTNOTES |
*
This work was supported by grants from Polysackaridforskning
AB, the Swedish Medical Research Council, the Swedish Natural Science
Research Council, Magnus Bergvalls Stiftelse, Wibergs Stiftelse, and
from King Gustaf V's 80th Anniversary Fund.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: Swedish University
of Agricultural Sciences, Dept. of Veterinary Medical Chemistry, The
Biomedical Center, Box 575, 751 23 Uppsala, Sweden. Tel.: 46-18-4714090; Fax: 46-18-550762; E-mail:
Gunnar.Pejler@vmk.slu.se.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008434200
 |
ABBREVIATIONS |
The abbreviations used are:
NDST-2, N-deacetylase/N-sulfotransferase;
PBS, phosphate-buffered saline;
mMCP, mouse mast cell protease;
rMCP, rat
mast cell protease;
PAGE, polyacrylamide gel
electrophoresis.
 |
REFERENCES |
1.
|
Lindahl, U.,
Kusche-Gullberg, M.,
and Kjellén, L.
(1998)
J. Biol. Chem.
273,
24979-24982[Free Full Text]
|
2.
|
Kjellén, L.,
Pettersson, I.,
Lillhager, P.,
Steen, M. L.,
Pettersson, U.,
Lehtonen, P.,
Karlsson, T.,
Ruoslahti, E.,
and Hellman, L.
(1989)
Biochem. J.
263,
105-113[Medline]
[Order article via Infotrieve]
|
3.
|
Eriksson, I.,
Sandbäck, D.,
Ek, B.,
Lindahl, U.,
and Kjellén, L.
(1994)
J. Biol. Chem.
269,
10438-10443[Abstract/Free Full Text]
|
4.
|
Forsberg, E.,
Pejler, G.,
Ringvall, M.,
Lunderius, C.,
Tomasini-Johansson, B.,
Kusche-Gullberg, M.,
Eriksson, I.,
Ledin, J.,
Hellman, L.,
and Kjellén, L.
(1999)
Nature
400,
773-776[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Humphries, D. E.,
Wong, G. W.,
Friend, D. S.,
Gurish, M. F.,
Qiu, W. T.,
Huang, C.,
Sharpe, A. H.,
and Stevens, R. L.
(1999)
Nature
400,
769-772[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Huang, C.,
Sali, A.,
and Stevens, R. L.
(1998)
J. Clin. Immunol.
18,
169-183[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Serafin, W. E.,
Reynolds, D. S.,
Rogelj, S.,
Lane, W. S.,
Conder, G. A.,
Johnson, S. S.,
Austen, K. F.,
and Stevens, R. L.
(1990)
J. Biol. Chem.
265,
423-429[Abstract/Free Full Text]
|
8.
|
Huang, R. Y.,
Blom, T.,
and Hellman, L.
(1991)
Eur. J. Immunol.
21,
1611-1621[Medline]
[Order article via Infotrieve]
|
9.
|
Serafin, W. E.,
Sullivan, T. P.,
Conder, G. A.,
Ebrahimi, A.,
Marcham, P.,
Johnson, S. S.,
Austen, K. F.,
and Reynolds, D. S.
(1991)
J. Biol. Chem.
266,
1934-1941[Abstract/Free Full Text]
|
10.
|
McNeil, H. P.,
Austen, K. F.,
Somerville, L. L.,
Gurish, M. F.,
and Stevens, R. L.
(1991)
J. Biol. Chem.
266,
20316-20322[Abstract/Free Full Text]
|
11.
|
Reynolds, D. S.,
Gurley, D. S.,
Austen, K. F.,
and Serafin, W. E.
(1991)
J. Biol. Chem.
266,
3847-3853[Abstract/Free Full Text]
|
12.
|
Stevens, R. L.,
Friend, D. S.,
McNeil, H. P.,
Schiller, V.,
Ghildyal, N.,
and Austen, K. F.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
128-132[Abstract]
|
13.
|
Hallgren, J.,
Karlson, U.,
Poorafshar, M.,
Hellman, L.,
and Pejler, G.
(2000)
Biochemistry
39,
13068-13077[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Schwartz, L. B.,
and Bradford, T. R.
(1986)
J. Biol. Chem.
261,
7372-7379[Abstract/Free Full Text]
|
15.
|
Pejler, G.,
and Sadler, J. E.
(1999)
Biochemistry
38,
12187-12195[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Pejler, G.,
and Berg, L.
(1995)
Eur. J. Biochem.
233,
192-199[Abstract]
|
17.
|
Pejler, G.,
Söderström, K.,
and Karlström, A.
(1994)
Biochem. J.
299,
507-513[Medline]
[Order article via Infotrieve]
|
18.
|
Morrissey, J. H.
(1981)
Anal. Biochem.
117,
307-310[Medline]
[Order article via Infotrieve]
|
19.
|
Sterk, A. R.,
and Ishizaka, T.
(1982)
J. Immunol.
128,
838-843[Abstract/Free Full Text]
|
20.
|
Alitalo, K.,
Hovi, T.,
and Vaheri, A.
(1980)
J. Exp. Med.
151,
602-613[Abstract]
|
21.
|
Saarinen, J.,
Kalkkinen, N.,
Welgus, H. G.,
and Kovanen, P. T.
(1994)
J. Biol. Chem.
269,
18134-18140[Abstract/Free Full Text]
|
22.
|
Gervasoni, J. E., Jr.,
Conrad, D. H.,
Hugli, T. E.,
Schwartz, L. B.,
and Ruddy, S.
(1986)
J. Immunol.
136,
285-292[Abstract/Free Full Text]
|
23.
|
Lützelschwab, C.,
Pejler, G.,
Aveskogh, M.,
and Hellman, L.
(1997)
J. Exp. Med.
185,
13-29[Abstract/Free Full Text]
|
24.
|
Hynes, R. O.
(1990)
in
Fibronectins: Springer Series in Molecular Biology
(Rich, A., ed)
, Springer Verlag, New York
|
25.
|
Compton, S. J.,
Cairns, J. A.,
Holgate, S. T.,
and Walls, A. F.
(1999)
Int. Arch. Allergy Immunol.
118,
204-205[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
He, S.,
Peng, Q.,
and Walls, A. F.
(1997)
J. Immunol.
159,
6216-6225[Abstract]
|
27.
|
Huang, C.,
Friend, D. S.,
Qiu, W. T.,
Wong, G. W.,
Morales, G.,
Hunt, J.,
and Stevens, R. L.
(1998)
J. Immunol.
160,
1910-1919[Abstract/Free Full Text]
|
28.
|
Molinari, J. F.,
Moore, W. R.,
Clark, J.,
Tanaka, R.,
Butterfield, J. H.,
and Abraham, W. M.
(1995)
J. Appl. Physiol.
79,
1966-1970[Abstract/Free Full Text]
|
29.
|
He, S.,
and Walls, A. F.
(1998)
Eur. J. Pharmacol.
352,
91-98[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
He, S.,
and Walls, A. F.
(1998)
Br. J. Pharmacol.
125,
1491-1500[Abstract]
|
31.
|
Steinhoff, M.,
Vergnolle, N.,
Young, S. H.,
Tognetto, M.,
Amadesi, S.,
Ennes, H. S.,
Trevisani, M.,
Hollenberg, M. D.,
Wallace, J. L.,
Caughey, G. H.,
Mitchell, S. E.,
Williams, L. M.,
Geppetti, P.,
Mayer, E. A.,
and Bunnett, N. W.
(2000)
Nat. Med.
6,
151-158[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Maier, M.,
Spragg, J.,
and Schwartz, L. B.
(1983)
J. Immunol.
130,
2352-2356[Abstract/Free Full Text]
|
33.
|
Schwartz, L. B.,
Bradford, T. R.,
Littman, B. H.,
and Wintroub, B. U.
(1985)
J. Immunol.
135,
2762-2767[Abstract/Free Full Text]
|
34.
|
Huang, C.,
Wong, G. W.,
Ghildyal, N.,
Gurish, M. F.,
Sali, A.,
Matsumoto, R.,
Qiu, W. T.,
and Stevens, R. L.
(1997)
J. Biol. Chem.
272,
31885-31893[Abstract/Free Full Text]
|
35.
|
Schwartz, L. B.,
Kawahara, M. S.,
Hugli, T. E.,
Vik, D.,
Fearon, D. T.,
and Austen, K. F.
(1983)
J. Immunol.
130,
1891-1895[Abstract/Free Full Text]
|
36.
|
Nakano, A.,
Kishi, F.,
Minami, K.,
Wakabayashi, H.,
Nakaya, Y.,
and Kido, H.
(1997)
J. Immunol.
159,
1987-1992[Abstract]
|
37.
|
Mizutani, H.,
Schechter, N.,
Lazarus, G.,
Black, R. A.,
and Kupper, T. S.
(1991)
J. Exp. Med.
174,
821-825[Abstract]
|
38.
|
Pejler, G.,
and Karlström, A.
(1993)
J. Biol. Chem.
268,
11817-11822[Abstract/Free Full Text]
|
39.
|
Kokkonen, J. O.,
Vartiainen, M.,
and Kovanen, P. T.
(1986)
J. Biol. Chem.
261,
16067-16072[Abstract/Free Full Text]
|
40.
|
Suzuki, K.,
Lees, M.,
Newlands, G. F.,
Nagase, H.,
and Woolley, D. E.
(1995)
Biochem. J.
305,
301-306[Medline]
[Order article via Infotrieve]
|
41.
|
Taipale, J.,
Lohi, J.,
Saarinen, J.,
Kovanen, P. T.,
and Keski-Oja, J.
(1995)
J. Biol. Chem.
270,
4689-4696[Abstract/Free Full Text]
|
42.
|
Reilly, C. F.,
Schechter, N. B.,
and Travis, J.
(1985)
Biochem. Cell Biol. Commun.
127,
443-449
|
43.
|
Lindstedt, L.,
Lee, M.,
Castro, G. R.,
Fruchart, J. C.,
and Kovanen, P. T.
(1996)
J. Clin. Invest.
97,
2174-2182[Abstract/Free Full Text]
|
44.
|
Reilly, C. F.,
Tewksbury, D. A.,
Schechter, N. M.,
and Travis, J.
(1982)
J. Biol. Chem.
257,
8619-8622[Abstract/Free Full Text]
|
45.
|
Kofford, M. W.,
Schwartz, L. B.,
Schechter, N. M.,
Yager, D. R.,
Diegelmann, R. F.,
and Graham, M. F.
(1997)
J. Biol. Chem.
272,
7127-7131[Abstract/Free Full Text]
|
46.
|
Fang, K. C.,
Raymond, W. W.,
Blount, J. L.,
and Caughey, G. H.
(1997)
J. Biol. Chem.
272,
25628-25635[Abstract/Free Full Text]
|
47.
|
Banovac, K.,
Banovac, F.,
Yang, J.,
and Koren, E.
(1993)
Proc. Soc. Exp. Biol. Med.
203,
221-235[Abstract]
|
48.
|
Vartio, T.,
Seppa, H.,
and Vaheri, A.
(1981)
J. Biol. Chem.
256,
471-477[Free Full Text]
|
49.
|
Pejler, G.,
and Maccarana, M.
(1994)
J. Biol. Chem.
269,
14451-14456[Abstract/Free Full Text]
|
50.
|
Pejler, G.
(1996)
FEBS Lett.
383,
170-174[CrossRef][Medline]
[Order article via Infotrieve]
|
51.
|
George, E. L.,
Georges-Labouesse, E. N.,
Patel-King, R. S.,
Rayburn, H.,
and Hynes, R. O.
(1993)
Development
119,
1079-1091[Abstract/Free Full Text]
|
52.
|
Homandberg, G. A.
(1999)
Front. Biosci.
4,
D713-D730[Medline]
[Order article via Infotrieve]
|
53.
|
Werb, Z.,
Tremble, P. M.,
Behrendtsen, O.,
Crowley, E.,
and Damsky, C. H.
(1989)
J. Cell Biol.
109,
877-889[Abstract]
|
54.
|
Wachtfogel, Y. T.,
Abrams, W.,
Kucich, U.,
Weinbaum, G.,
Schapira, M.,
and Colman, R. W.
(1988)
J. Clin. Invest.
81,
1310-1316[Medline]
[Order article via Infotrieve]
|
55.
|
Xie, D. L.,
Meyers, R.,
and Homandberg, G. A.
(1993)
Blood
81,
186-192[Abstract]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.