(Received for publication, October 23, 1995; and in revised form, February 21, 1996)
From the
Inter--trypsin inhibitor (ITI), a human serum protease
inhibitor of molecular mass 240 kDa which may release physiological
derivatives, has been shown to interact with hyaluronic acid (HA),
resulting in pericellular matrix stabilization (Chen, L., Mao, S. J.
T., McLean, L. R., Powers, R. W., and Larsen, W. J.(1994) J. Biol.
Chem. 269, 28282-28287). The purpose of this study is to
determine whether ITI binding to tumor cell surface is mediated by
urinary trypsin inhibitor (UTI) receptor or cell-associated hyaluronic
acid (HA). We demonstrated specific complex formation of the heavy (H)
chains of ITI with HA. Binding of the H-chains of ITI to immobilized HA
was detected and quantified using colorimetric immunoassays. Binding
was time-, temperature-, and concentration-dependent. However, UTI and
HI-8 (the carboxyl terminus of UTI) failed to bind to immobilized HA.
ITI bound to HA remained functional protease inhibiting activity. After
incubation of SMT-cc1 cells with purified biotinylated ITI,
biotinylated ITI is bound to the cells, dissociated, and gives rise to
the H-chains and UTI on the cell surface. The cell surface
receptor-bound UTI derived from ITI may be the result of the limited
proteolysis on the cell surface. In the cells treated with
hyaluronidase, bound H-chains disappeared from the surface of the
cells, while most of the cell surface ITI derivatives was present in
deglycosylated UTI (28 kDa). It is suggested that the binding of ITI to
the cell surface is mediated by HA on the cells. This was confirmed by
the fact that the hyaluronidase-treated cells can abolish the ITI
binding. The cell surface UTI formation was inhibited by diisopropyl
fluorophosphate, phenylmethylsulfonyl fluoride, and eglin C, suggesting
that elastase-like enzyme(s) may be responsible for the UTI formation.
Preincubation of the cells with UTI did not decrease in exogenously
added ITI on the cell surface. A model for cell surface UTI formation
is proposed in which ITI binding to cells from serum used for the
culture is followed by the limited proteolysis by trace amounts of
active serine proteases, to form cell-surface receptor-bound UTI and
the H-chains intercalated into cell surface HA. This process is subject
to regulation of cell-associated UTI and of stabilization of
pericellular matrix.
Inter--trypsin inhibitor (ITI) (
)is the
precursor of urinary trypsin inhibitor (UTI), which is one of the
Kunitz-type protease inhibitors present in human serum and urine (1, 2, 3, 4) . ITI was found to act
as a stabilizer of pericellular matrix. The serum factor, identified as
an ITI, is a structural component of the matrix. ITI is required to
stabilize the fully expanded cumulus cell-oocyte complexes matrix, thus
supporting the process of ovulation (5) . Huang et al.(6) showed previously that hyaluronic acid (HA)
synthesized by cultured fibroblasts firmly bound 85-kDa proteins, which
were derived from serum used for the culture and appeared to be
covalently linked to HA. The serum-derived HA-associated protein was
confirmed to be the heavy (H)-chains of ITI, suggesting that
cooperative binding to HA of the H-chains of ITI is required to
stabilize the pericellular matrix(7) .
Hyaluronic acid is a
glycosaminoglycan of the extracellular matrix in most mammalian
tissues. It consists of a linear polysaccharide chain with repeating
glucuronic
acid-(1-3)-N-acetylglucosamine-(
1-4)
structure. It is bound by cell surface receptors and extracellular
matrix proteins (8) . HA is also believed to play an important
role in controlling tumor cell growth, migration, invasion, and
differentiation. Synthesis and degradation of HA are significantly
related to these cellular functions such as cell proliferation. Yoneda et al.(9, 10) have shown that HA added
exogenously or supplied endogenously by increased synthesis may act as
a modulator of fibroblast proliferation.
We showed previously that highly purified human UTI efficiently inhibits soluble and tumor cell-associated plasmin and subsequently prevents tumor cell invasion and metastasis(11) . Inhibition of cell-bound plasmin by UTI is associated with significantly reduced tumor cell invasiveness in vitro and a decreased number of metastasis in vivo(11) .
Recently we found that some tumor cells have specific binding sites for UTI on the cell surface(12) . Exogenously applied UTI may be bound to specific binding sites on the surface of tumor cells. This potentially leads to the build up of a substantial amount of UTI at the surface of the tumor cells(12) . There is good evidence that UTI may play an important role in prevention of tumor cell invasion and metastasis(13, 14) .
In the present study, we investigated whether ITI binding to tumor cell surface is mediated by UTI receptors or by cell-associated HA. Available information on the interaction of ITI derivatives with cells or with immobilized HA is mainly based on the use of biotinylated compounds as ligands. We show that binding of ITI to the cell-associated HA, but not to the UTI receptor, may be essential to the formation of the aggregates comprising the H-chains of ITI and HA which constitutes pericellular matrix.
SMT-cc1 cell monolayers or U937 cell
suspensions (1 10
cells/ml) were grown in the above
medium, then in complete medium without serum for 16 h. The cells were
then incubated with increasing concentrations of biotinylated ITI
(0-2 µM) in RPMI 1640 medium containing ITI-depleted
FCS for 16 h at 37 °C. Then, the cells were washed twice with PBS
containing 0.1% bovine serum albumin (BSA) (washing buffer). The cells
were homogenized in washing buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 µM each
aprotinin, pepstatin, and cystatin. 50 µl of anti-ITI
antibody-coupled CNBr-activated Sepharose 4B were incubated with the
supernatants of cell lysates (16 h, 4 °C) to detect the
cell-associated ITI and its derivatives. The samples were washed
several times with 50 mM PBS, 0.5 M NaCl, and 0.1%
Triton X-100, and the final pellet (immobilized immunocomplex) was
resuspended in electrophoresis sample buffer, heated, and analyzed by
SDS-PAGE followed by Western blot using avidin-peroxidase.
ITI was biotinylated according to the method of Guesdon et al.(19) , using N-hydroxysuccinimidylbiotinamide caproate (Sigma). ITI purified from human serum was treated with hyaluronidase (hyaluronidase/ITI = 1/100 molar ratio; 37 °C, 6 h). Hyaluronidase-treated ITI was subjected to preparative gel electrophoresis. Eluted materials were concentrated by ultrafiltration (Amicon) for analysis on reverse phase high performance liquid chromatography. The C18 silica columns were packed for high performance and equilibrated with 5% acetonitrile, 0.1% trifluoroacetic acid before loading. Fractions containing H-chains were pooled.
K was calculated using the
experimentally determined association (k
) and
dissociation (k
) rate constants. k
was monitored by allowing proteases and ITI
bound to immobilized HA to react for given periods of time before
addition of substrate which stopped the association process and then
measuring the remaining enzyme activity. Final concentrations: trypsin,
plasmin, and elastase, 10 nM; Substrates, S-2222 (1
mM), S-2251 (200 µM), and S2484 (200
µM). k
was monitored by adding
-macroglobulin to a mixture of enzyme and inhibitor.
The
-macroglobulin-protease complexes are
enzymatically active on synthetic substrates. Dissociation kinetics
could be monitored by measuring the time-dependent appearance of
hydrolytic activity. Final concentrations: enzymes, 10 nM at
time 0;
-macroglobulin, 3 µM; substrate,
S-2222 (1 mM), S-2251 (200 µM), and S-2484 (200
µM). The inhibition equilibrium constant (K
; nM) was
calculated(20, 21) .
In addition, 96-well microtiter plates were coated with ITI or its derivatives (2 µg/ml, 16 h, 4 °C) that subsequently was blocked with 2% BSA. HA (0-0.1 mg/ml) was applied into the 96-well plates coated either with ITI derivatives or BSA (as a control) and incubated (2 h, 23 °C). HA specifically bound to ITI derivatives was detected with peroxidase-conjugated hyaluronic acid-binding protein (HRP-HABP, 0.5 µg/ml; this compound was kindly supplied by Dr. Takashi Kondo (Chugai Pharmaceutical Co., Tokyo, Japan))(22, 23, 24) . Wells were allowed to react with tetramethylbenzidine as described above.
We made an
additional experiment to determine the equilibrium dissociation
constant of ITI and HA. ITI was radioiodinated using the chloramine-T
method(25) . The specific activity was adjusted to 1.5
10
cpm/µg ITI by adding unlabeled ITI. HA was
conjugated to AH-Sepharose 4B using the carbodiimide
method(25) . Various amounts of
I-labeled ITI
were added to HA-Sepharose and incubated (16 h, 23 °C). Bound and
free
I-labeled ITI were separated by centrifugation and
counted with a
-counter. Nonspecific binding was measured in the
presence of an excess amount of unlabeled ITI. The data were analyzed
by a Scatchard plot.
Figure 1:
Immunoprecipitation of ITI in
cells saturated with biotinylated ITI. SMT-cc1 cells were cultured in
RPMI 1640 supplemented with 10% FCS. We confirmed that endogenous UTI
is localized on the cell surface or to the pericellular matrix by
immunohistochemical staining(12) . This is the significance of
the usage of the biotinylated ITI. After the cells were washed twice
with PBS, 0.1% BSA, the cell monolayers were incubated in serum-free
medium for 16 h at 37 °C. Then, the cells were treated with RPMI
1640 medium containing ITI-depleted serum plus biotinylated ITI (2
µM) and incubated for 3 h (lane 1), 12 h (lane 2), and 16 h (lane 3) at 37 °C. Then, the
cells were washed and homogenized. Anti-ITI antibody coupled to
Sepharose (50 µl) was incubated with cell lysate supernatants (from
5 10
cells). Immobilized immunocomplex was analyzed
by 12% SDS-PAGE, followed by Western blot using avidin-peroxidase. a, ITI; b, H-chains (HC-1, HC-2, and HC-3); c, UTI. Lane M, molecular masses of standards are
indicated.
SMT-cc1 cell monolayers were treated with ITI-depleted serum containing 2 µM biotinylated ITI and incubated for 16 h at 37 °C. After the cells were washed twice with washing buffer, the cells were incubated in the presence or absence of hyaluronidase (20 µg/ml, 30 min, 37 °C). After washing, the cells were homogenized. Anti-ITI antibody coupled to CNBr-activated Sepharose 4B were incubated with the supernatant of SMT-cc1 cell lysate (16 h, 4 °C). Immobilized immunocomplexes were analyzed by SDS-PAGE, followed by Western blot. Fig. 2shows that in the absence of hyaluronidase, sharp bands of >200 kDa, 120 kDa, 90-100 kDa, and 80 kDa as well as a polydisperse one of 40 kDa were obtained in the cell lysate sample. In the hyaluronidase-treated samples, the staining intensity of >200-kDa, 120-kDa, 90-100-kDa and 80-kDa bands disappeared, and a new band (deglycosylated UTI, molecular mass = 28 kDa) appeared. It has been reported that the 28-kDa form is the deglycosylated UTI. UTI contains a low-sulfated chondroitin 4-sulfate chain, and its apparent molecular mass upon SDS-PAGE shifts from 40 kDa to 28 kDa upon chondroitinase ABC or hyaluronidase treatment(26) . SDS-PAGE of UTI yields a molecular mass of 40 kDa, whereas the amino acid sequences yield values of 15-16 kDa. The discrepancy between these value is due to the chondroitin sulfate chain, whose removal results in a shift of the UTI band to 28 kDa. Hyaluronidase will not digest the whole glycosaminoglycan chain but will leave the innermost disaccharide repeat and the linkage region intact.
Figure 2:
Immunoprecipitation of ITI in cells
saturated with biotinylated ITI in the presence or absence of
hyaluronidase. SMT-cc1 cell monolayers were treated with 2 µM biotinylated ITI and incubated for 16 h at 37 °C. The cells
were washed twice with washing buffer and were incubated in the absence (lane 3) or presence (lane 4) of hyaluronidase (20
µg/ml, 30 min, 37 °C). Cell-associated biotinylated compounds
(from 1 10
cells) were immunoprecipitated with
anti-ITI antibody coupled to Sepharose and analyzed by 12% SDS-PAGE,
followed by Western blot using avidin-peroxidase. Lane 1, ITI (a); lane 2, UTI (c); lane 3,
H-chains (b); lane 4, deglycosylated UTI (d).
Although deglycosylated UTI could be recovered from the cell lysate, the H-chains of ITI could not be detected in the immunoprecipitable proteins (Fig. 2). It is likely that the serum-derived HA-associated proteins may be the H-chains of ITI, but without the light chain.
After being treated with hyaluronidase, cell-associated ITI and the H-chains disappeared. This means that H-chains were released into the medium by hyaluronidase treatment. In a parallel experiment, soluble ITI purified from human serum was incubated with hyaluronidase at the same conditions as described above (20 µg/ml, 30 min, 37 °C). This treatment did not cause the complete disappearance of the 240-kDa band and the appearance of several new bands, major ones of 80 kDa and 28 kDa as well as minor bands of 70 kDa, 60 kDa, and 50 kDa (Fig. 3). In addition, soluble UTI purified from human urine was incubated with hyaluronidase at the same conditions. This treatment caused the incomplete disappearance of the 40-kDa band and the appearance of new bands (molecular mass of 60 kDa and 28 kDa). Broad staining with several bands shown in Fig. 3(lane 2, d) may represent the difference in the extent of deglycosylation.
Figure 3: Specific cleavage of ITI or UTI by hyaluronidase. ITI (1 µg) purified from human serum was incubated for 30 min at 37 °C in the absence (lane 1) or presence (lane 2) of hyaluronidase (ITI/hyaluronidase molar ratio = 10/1) (10% SDS-PAGE and Western blot using anti-ITI antibody). Lane 3, UTI 0.5 µg. UTI (5 µg) purified from human serum was incubated in the absence (lane 4) or presence (lane 5) of hyaluronidase (UTI/hyaluronidase molar ratio = 10/1) (18% SDS-PAGE and Western blot using anti-UTI antibody (see (11, 12, 13, 14) )). a, ITI; b, H-chains; c, UTI; d, deglycosylated UTI (28 kDa, black arrow). White arrow, UTI degradation/hyaluronidase complex. SDS-PAGE and Western blot were carried out using anti-ITI antibody, followed by biotin-anti-rabbit IgG, avidin-peroxidase, and by tetramethylbenzidine substrate solution.
We speculated that the cell surface UTI might have been derived from ITI bound on the cell surface, but not from UTI formed in the medium and subsequently bound to the cells. It was confirmed by Western blot analysis that biotinylated ITI does not contain UTI fragments. In a parallel experiment, the cells were treated with the biotinylated ITI (0.2 µM) in the presence of an excess of unlabeled ITI (2 µM). Under this condition, H-chains and UTI could not be detected in the immunoprecipitable proteins (Fig. 4).
Figure 4: Immunoprecipitation of ITI in cells saturated with biotinylated ITI in the presence of an excess of unlabeled ITI. The experiment was performed as described in the legends to Fig. 1and Fig. 2. SMT-cc1 cell monolayers were incubated with biotinylated ITI (0.2 µM) in the presence of unlabeled ITI (0, 0.5, and 2.0 µM) in RPMI 1640 medium containing ITI-depleted FCS for 16 h at 37 °C. Anti-ITI antibody-coupled Sepharose 4B was incubated with the supernatants of cell lysates (16 h, 4 °C) and immunoprecipitated.
To test which enzymes were responsible for the cell surface ITI cleavage, we added biotinylated ITI to the serum-free culture medium of SMT-cc1 cells in the presence or absence of several protease inhibitors including diisopropyl fluorophosphate, PMSF, aprotinin, leupeptin, pepstatin, cystatin, eglin C, and E64. Several ITI derivatives were found even in the presence of aprotinin, leupeptin, pepstatin, cystatin, and E64. However, virtually no UTI was detected on the cell surface when PMSF or eglin C was added to the medium, while there was a considerable dose-dependent and time-dependent UTI formation in the absence of PMSF or eglin C (Fig. 5). These findings indicated that the cell-bound UTI was formed by cleavage of ITI on the surface of the cells. In a parallel experiment, soluble ITI was incubated with elastase. This treatment caused the complete disappearance of ITI and the appearance of H-chains and UTI. Cell-associated elastase-like enzyme(s) may be responsible for the cell-bound UTI formation (Fig. 6).
Figure 5: Immunoprecipitation of ITI in SMT-cc1 cell layers saturated with biotinylated ITI in the presence of protease inhibitors. SMT-cc1 cell monolayers were treated with 2 µM biotinylated ITI and incubated for 16 h at 37 °C in the presence or absence of diisopropyl fluorophosphate (1 mM), PMSF (1 mM), aprotinin (1 µM), leupeptin (10 µM), pepstatin (1 µM), cystatin (1 µM), eglin C (5 µg/ml), or E64 (10 µM). a, ITI; b, H-chains; c, UTI. SDS-PAGE and Western blot were carried out using avidin-peroxidase, followed by enzyme substrate.
Figure 6: Specific cleavage of ITI by elastase. ITI (5 µg) was treated with leukocyte elastase (0-2 µg) for 30 min at 37 °C. a, ITI; b, H-chains; c, UTI; d, 22-kDa UTI fragment. The protein bands were detected as described in Fig. 3. Although lanes 2 and 3 appear to have much more protein compared with lane 1, the same amount of ITI (5 µg) was used for each lane.
Figure 7: Quantitative assessment of specific binding of ITI derivatives to hyaluronic acid immobilized to microtiter plate wells. 96-well microtiter plates were coated with hyaluronic acid or BSA by incubation of 100-µl aliquots of each solution (0.1 mg/ml hyaluronic acid or 2% (w/v) BSA). After washing the wells, nonspecific binding sites were blocked with 200-µl aliquots of PBS containing 2% BSA, pH 7.4. ITI and its derivatives (0-100 nM) diluted in 100 µl of PBS, 2% (w/v) BSA was allowed to bind for 2 h at 23 °C. Then the plates were washed three times with PBS. Wells were allowed to react with anti-ITI antibody (1 µg/ml, 1 h, 23 °C). This polyclonal antibody reacts with ITI, H-chains, UTI, and HI-8. Specific binding of ITI derivatives (total binding (HA-coated wells) - nonspecific binding (BSA-coated wells)) approached saturation at approximately 100 nM ITI or H-chains. Bar, S.D. The experiments were performed at least three times.
Figure 8: Quantitative assessment of specific binding of hyaluronic acid to immobilized ITI derivatives. 96-well plates were coated with ITI derivatives (2 µg/ml, 16 h, 4 °C). HA specifically bound to ITI derivatives was detected with HRP-HABP (0.5 µg/ml; 1 h, 23 °C). It was confirmed previously that HRP-HABP can bind to HA (see (19, 20, 21) ).
Figure 9:
Time- and temperature-dependent binding of
ITI to immobilized HA. 96-well plates were coated with HA (0.1 mg/ml,
16 h, 4 °C). Biotinylated ITI (0.1 µM, 100
µl/well) was added to HA-coated wells and incubated at 37 °C
(), 23 °C (
), or 4 °C (
) for the various times
indicated. Biotinylated ITI specifically bound to HA was detected with
avidin-peroxidase.
, 2 µM ITI was added to one
sample prior to incubation (37 °C, 48
h).
In the next
experiment, we investigated whether ITI bound to HA has functional
protease inhibiting activity. The relative enzyme activity obtained
when trypsin, plasmin, or elastase was titrated with ITI bound to
immobilized HA was measured (Table 1). ITI bound to HA also
strongly inhibited trypsin, plasmin, and elastase activities.
Inhibition constant (K), a measure of the
stability of protease inhibition, varied with a difference of more than
10 between trypsin and plasmin. Trypsin exhibited the most stable
inhibition as well as the greatest association rate (Table 1).
Recently, the heavy chain 2 of ITI (HC2) has been shown to be easily degraded by HLE whereas HC1 is more resistant(27) . The molecular mass of fragments derived by limited proteolysis of UTI with HLE was 22 kDa (13) . The 22-kDa protein does not appear to be degraded further in lower molecular mass forms, despite prolongation of the incubation time or increased protease concentrations, indicating that the 22-kDa protein is resistant to further degradation(13) . The 22-kDa protein inhibited HLE with essentially the same affinity as native UTI. ITI-enzyme complexes can dissociate, which allows for a cleavage of ITI by formerly inhibited enzyme molecules. It has been observed in vitro that such a cleavage releases UTI-based by-products whose enzyme inhibitory capacity remains intact. We consider that HLE is inhibited by ITI bound to HA, although the same enzyme (2 ng) may digest ITI (5 µg) (see Fig. 6).
Figure 10:
Specific binding of ITI to the surface of
tumor cells (cell ELISA). A, in cell ELISA, SMT-cc1 cells
() cultured in 96-well microtiter plates were incubated with
varying concentrations of biotinylated ITI (0-200 nM, 1
h, 23 °C). SMT-cc1 cells preincubated with hyaluronidase (
)
failed to bind to ITI. The assays were carried out in triplicate. Bar, S.D. B, in competitive inhibition assay, cell
monolayers were incubated with 200 nM biotinylated ITI in the
presence of increasing concentrations of UTI (
) or ITI
(
).
We demonstrated that cell-associated HA binds to the H-chains
of ITI, but not to UTI or HI-8. They are consistent with the reports in
which it has been demonstrated that HA synthesized by cultured
fibroblasts firmly bound 85-kDa protein and the serum-derived
HA-associated protein is confirmed to be the H-chains of the ITI. The
H-chains could be involved in the formation of HA-rich extracellular
matrix through their calcium-dependent HA binding activity (5, 6, 7, 28, 29) . Our
results are also supported by a recent study showing that
pre--inhibitor stabilizes the expanding cumulus extracellular
matrix by binding directly with HA and that pre-
-inhibitor may
serve as a structural protein to organize the function of the cumulus
extracellular matrix(28, 29) . It was also shown that
binding of pre-
-inhibitor and HA is thought a stereospecific
charge interaction. The stabilizing action of pre-
-inhibitor may
not be indicated through protease inhibiting
activity(28, 29) .
It was reported that
stabilization of the pericellular matrix may require more than a simple
binding interaction between pre--inhibitor (or ITI) and
HA(22, 23, 24) . We found that ITI and UTI
interact strongly with hyaluronic acid-binding protein (HABP) (data not
shown). This binding interaction was relatively fast. We propose that
HABP may further strengthen or stabilize an interaction between ITI and
HA.
More recently, it has been reported that the carboxyl-terminal Asp of H-chains was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of hyaluronic acid chain, demonstrating the covalent binding of proteins to hyaluronic acid(30) .
ITI is a unique protease inhibitor that can be proteolyzed by the same enzymes that are inhibited to generate smaller inhibitors. Complete digestion of ITI by each protease was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme.
Expression of CD44 in lymphocytes or tumor cells and HAITI
complex in the matrix may mediate cell-matrix interactions in
inflammation and tumor invasion(22) . HA has been found in
tissues and body fluids, as part of larger molecular structures as in
aggregates with proteoglycans and as a coat attached to cell
surfaces(22) . Some blood proteins such as fibronectin,
collagen type IV, fibrinogen, IgG, or IgM have been shown to possess HA
binding ability. Lymphocyte CD44 may recognize HA linked to
ITI/H-chains located on the cell surface. Our results strongly support
the hypothesis that serum factor, which has been identified recently as
a protein belonging to the ITI family, acts as a structural component
of the close environment of cell surface and showed that specific
binding of ITI to HA is essential for successful organization of the
matrix around the cell surface.
We demonstrated that specific receptors for UTI have been determined on the surface of certain tumor cells(12) . However, ITI can bind to HA strongly but not to the UTI receptor. The receptor binding domain within the UTI has been localized to the amino acid sequences 1-79 (domain I) of UTI. Domain I and the domain II (79-143, HI-8) are the result of extensive treatment of UTI with trypsin-like enzyme(31) . HI-8, lacking the domain I, does not bind to the cells(31) . We speculate that, in a complete ITI molecule, domain I of UTI may be masked by the H-chains of ITI. The HA-bound ITI may be cleaved into the active UTI and the H-chains of ITI by elastase-like protease(s) on the cell surface(27) . It is likely that the UTI moiety binds to the UTI receptor after ITI is processed on the cell surface.
Recently, possible implications for the physiological function of the ITI family were discussed. Mast cell protease inhibitor, trypstatin, was found to be a fragment of ITI light chain(32) . Plasma UTI fragment is taken up into mast cells and stored in their cytoplasmic granules, since UTI fragment may not be generated by mast cells themselves (32) .
In addition, UTI was found to
inhibit interleukin 8 gene expression induced by lipopolysaccharide in
HL60 cells(33) . UTI inhibited increase of cytosolic
Ca stimulated by lipopolysaccharide but not by the
calcium ionophore A23187. The cell membrane may be the site of action
of UTI, and the effect of UTI may be due to inhibition of
Ca
influx or mobilization.
We can postulate the pathways of specific binding of H-chains to HA as follows. ITI is produced in the liver and immediately secreted into the serum(26, 34) . ITI is captured on the cell surface through the covalent linkage to HA, thereby mediating the binding of HA to the cell surface and stabilizing pericellular matrix. Then, cell-associated ITI may be cleaved into UTI and H-chains on the cell surface. The H-chains may act as structural linkers through stabilization of pericellular matrix. On the other hand, receptor-bound UTI may act as a membrane-associated protease inhibitor(12) . Besides protease inhibiting activity, UTI has additional functions including prevention of cytokine production or inhibition of cellular calcium influx(33) . This attractive hypothesis will be clarified by the demonstration of ITI-cleaving enzyme(s) as well as of UTI receptors on the cell surface.