From the
Because proteases free in the body are damaging to the tissues,
animals have evolved various agents for their inactivation and
clearance. Mammals, for instance, have a diverse array of active site
protease inhibitors in the plasma. In addition, mammals have
Proteases are involved in the invasion and pathogenesis of
bacteria, molds, and multicellular parasites (Breton et al.,
1992; Hotez et al., 1990; McKerrow et al., 1993).
Metazoans have evolved a variety of agents for the inactivation of
proteases that help limit the destructive potential of these pathogens.
The most important of these regulatory molecules are the polypeptide
protease inhibitors, which can be classified into two groups, the
active site-inhibitors and the
Homologues of
In mammals, the binding of
The
definitive characterization of the essential functions of human
All experiments were conducted on adult male Limulus
polyphemus (380-460 g) obtained from the Marine Resources
Center of the Marine Biological Laboratory in Woods Hole, MA. Animals
were used within a few days of their initial capture from the wild and
were returned to the ocean unharmed at the completion of the
experiment. No animal was used for more than one experiment.
Labeled proteins were
dissolved in endotoxin-free 3% NaCl (Travenol). The total blood volume
was estimated as 30% of the total body volume (Armstrong, 1985).
Solutions of labeled proteins as 1% of the total blood volume were
introduced into a 50-ml syringe fitted with a 16-gauge needle, which
was inserted through the cephalo-thoracic joint directly into the lumen
of the heart. 30-40 ml of blood was aspirated into the syringe to
mix with the sample of labeled protein, and then the entire syringe
content was slowly injected back into the heart. At various times
following injection, blood samples (2 ml volume) were taken from the
blood spaces of the joints of the fourth pair of legs. Cells were
removed from the blood samples by centrifugation at 1000
The molecular mass of trypsin is
23,800 Da (Desnuelle, 1960). When FITC-trypsin was applied to Centricon
filters with 10- and 100-kDa molecular exclusions, less than 5% of the
fluorescence appeared in the flow-through of the Centricon 10 kDa, but
more than 97% of the fluorescence was found in the flow-through of the
Centricon 100 kDa. The trypsin-associated fluorescence of plasma from
preclearance stages (10 min after injection) was associated with a
molecular complex larger than 100 kDa, because 84 and 82%,
respectively, of the fluorescence was retained by filters with 10- and
100-kDa molecular exclusion limits (). However, the
fluorescence that reappears in the plasma after clearance (40-45
min) is associated with small molecules since 82% of the fluorescence
of plasma collected 40 min after injection and 89% of 45 min samples
passed through the 10-kDa filter (). This indicates a
degradation of the labeled trypsin following its clearance from the
plasma. When samples of plasma recovered 10 min after injection of
FITC-trypsin were applied to a calibrated 100
It will
be interesting to determine the processing pathway of
-macroglobulin (
M), which binds
active proteases, and the
M-protease complex is then
cleared from the plasma by a receptor-mediated endocytotic process.
M is also present in the plasma of many invertebrates,
and in the American horseshoe crab, Limulus polyphemus, it is
the only protease inhibitor in the plasma. To search for a clearance
process for proteases in Limulus, fluorescein isothiocyanate
(FITC)-labeled proteins were injected into the blood, and the
fluorescence in the plasma and associated with the blood cells was
determined. FITC-labeled trypsin was cleared with an initial mixing
period (0-10 min) and a rapid clearance period (10-30 min),
followed by the reappearance of FITC in the plasma (45-90 min).
Before and during the clearance process, the labeled trypsin was
associated with a complex having a molecular mass identical to that of
Limulus
M, and that was precipitated by
antibodies directed against Limulus
M. The
fluoresceinated material that reappeared in the plasma after 45 min was
of low molecular mass (<10 kDa) and thus appears to have experienced
degradation. The clearance of trypsin requires its protease activity,
since phenylmethylsulfonyl fluoride-inactivated, FITC-labeled trypsin
was cleared only very slowly if at all (t > 180 min).
FITC-labeled, trypsin-reacted Limulus
M was
cleared rapidly from the plasma of Limulus, whereas
FITC-labeled, native Limulus
M persisted
undiminished in excess of 400 min. The blood cells of Limulus bound FITC-labeled trypsin-reacted Limulus
M, and the peak of recovery from the blood cells
coincided with the minimum concentration of FITC-labeled protein in the
plasma, suggesting that the blood cells participate in the clearance of
M-protease complex from the plasma. Thus, we have
demonstrated the existence of a clearance pathway in Limulus that operates selectively on enzymatically active proteases and
have shown that Limulus
M is the probable
agent for protease clearance. This is the first documentation of a
protease clearance pathway in invertebrates and represents the first
identified physiological function for
M in
invertebrates.
-macroglobulin-
(
M)
(
)
-like inhibitors (Laskowski
and Kato, 1980; Travis and Salvesen, 1983). The active site inhibitors
bind to and inactivate the active sites of target proteases (Laskowski
and Kato, 1980; Travis and Salvesen, 1983), whereas
M
enfolds the target protease molecule in a molecular cage that shields
it from protein substrates (Sottrup-Jensen, 1987; Barrett and Starkey,
1973; Barrett, 1981). In marked contrast to the active site inhibitors,
M-bound proteases retain the enzymatic activity of the
active site and can still hydrolyze amide or ester substrates small
enough to diffuse into the
M cage (Bieth et
al., 1978; Beatty et al., 1982; Berthillier et
al., 1968). Protease binding begins with the proteolytic cleavage
of the
M polypeptide at a region known as the
``bait'' region (Barrett and Starkey, 1973; Barrett et
al., 1979; Hall and Roberts, 1978; Sottrup-Jensen et al.,
1981) and is immediately followed by a physical folding of
M around the target protease to ``trap'' it
within the
M cage (Barrett et al., 1979;
Cummings et al., 1984; Nelles et al., 1980; Hall and
Roberts, 1978). The bait region contains peptide bonds susceptible to
almost all endopeptidases, thereby conferring the broad reactivity of
M and enabling it to react with proteases from
endogenous sources involved in coagulation, fibrinolysis, and
inflammation as well as with foreign proteases introduced by invading
pathogens (Barrett and Starkey, 1973; Howell et al., 1983).
M does not interact with enzymatically inactive
proteases.
M have recently been
described from a variety of invertebrates, including mollusks and
chelicerate and mandibulate arthropods (Armstrong and Quigley, 1995).
Most of the unique structural and functional characteristics of
M have been conserved over the 0.5-0.6 billion
years of evolutionary divergence of the arthropod and vertebrate
lineages, including significant sequence identity (Sottrup-Jensen
et al., 1990), the internal thiol ester (Armstrong and
Quigley, 1987; Spycher et al., 1987), the unique protease
trapping mechanism (Armstrong and Quigley, 1985; Quigley et
al., 1991), and the reactivity against a broad spectrum of
proteases of diverse catalytic mechanisms (Quigley and Armstrong,
1985).
M to
endopeptidases is followed by the clearance of the
M-protease complex from the circulation by hepatocytes
(Van Leuven, 1984; Gliemann et al., 1983; Davidsen et
al., 1985; Feldman et al., 1985). The conformational
change of
M that accompanies protease binding both
entraps the protease and exposes a previously buried domain close to
the carboxyl terminus (Holtet et al., 1994) that is then
recognized by a cell surface receptor system that mediates the binding
and endocytosis of the complex (Marynen et al., 1981, 1982;
Van Leuven et al., 1983). The mammalian
M
receptor has been identified as being identical to the low density
lipoprotein receptor-related protein and is specific for the
protease-reacted form of
M, lacking binding activity
for unreacted
M, which remains free in the plasma
(Kristensen et al., 1990; Strickland et al., 1990). A
conformational change in
M similar to that produced by
reaction with proteases can be produced by treatment with small primary
amines such as methylamine and ammonium (Barrett et al.,
1979). Like protease-reacted
M, methylamine-reacted
M is bound by cells (Kaplan and Nielsen, 1979; Kaplan
et al., 1981; Debanne et al., 1976). Once
internalized, the
M-protease and
M-methylamine complexes are degraded in secondary
lysosomes (Sottrup-Jensen, 1987). Thus, in contrast to other protease
inhibitors that bind to the active site of the protease resulting in
immediate enzymatic inactivation, the receptor-mediated endocytotic
clearance of
M-protease complex from the circulation
must also be regarded as part of its inactivation mechanism.
M in vivo has proven difficult because
mammalian plasma contains a diverse array of active site protease
inhibitors that, in the aggregate, are able to bind most exogenous and
endogenous proteases (Laskowski and Kato, 1980; Travis and Salvesen,
1983). In contrast,
M is the only trypsin inhibitor in
Limulus plasma (Quigley and Armstrong, 1983), making the
horseshoe crab a much simpler system for its functional
characterization. In general, the diversity of the plasma proteins in
Limulus is relatively low, and the blood contains but a single
cell type, the granular amebocyte, in the general circulation
(Armstrong, 1985; 1991). Our aim is to investigate the fate of
exogenous proteases introduced into the blood of Limulus and
to characterize the molecules involved in protease clearance. In this
report, we document a protease clearance pathway and demonstrate that
M is the mediator for clearance. Furthermore, we show
that the blood cells are capable of binding the
M-protease complex.
Assay of Protein Clearance
For angiography the animals
were restrained upside down on an x-ray table. 10 ml of Angiovist-282
(diatrizoate meglumine, 28% organically bound iodine) (Berlex
Laboratories Inc., Wayne, NJ) was injected either into the blood space
of one of the leg joints or into the heart at the cephalo-thoracic
joint. The injection and distribution of the dye was observed by
fluoroscopy and recorded onto video tape.
g for 5 min, and the fluorescence of the plasma was measured with a
fluorometer (Turner Model 112; filters: emission: 47B, 2A, excitation
2A-12). Blood cell-associated fluorescence was measured after lysis of
the pelleted blood cells in 10% sodium dodecyl sulfate and
centrifugation (10,000
g, 20 min) to remove cell
debris.
Fluoresceination of Proteins
Limulus M was purified as described previously (Armstrong
et al., 1991). The trypsin-binding activities of the
M preparations were determined by the soybean trypsin
inhibitor protection assay (Armstrong et al., 1985). The thiol
ester of Limulus
M was inactivated by
reaction with methylamine as described previously (Quigley and
Armstrong, 1985), resulting in a loss of 96% of the trypsin binding
activity. Limulus hemocyanin and Limulus
M were labeled with fluorescein isothiocyanate
(FITC) (Harlow and Lane, 1988). Trypsin (Sigma, catalog no. T-8003) was
fluoresceinated with FITC in carbonate/bicarbonate buffer (pH 9.1)
containing 20 mM benzamidine followed by dialysis against 1
mM HCl. To purify proteolytically active FITC-labeled trypsin,
the pH of the sample was adjusted to 8.0 with Tris buffer, and the
sample was applied to a column of benzamidine-Sepharose, which was then
washed with Tris-buffered 0.1 M NaCl (pH 8.0) and eluted with
10 mM HCl (Hixson and Nishikawa, 1973). The labeling
efficiency for all proteins, as measured by the ratio of optical
absorbance (495/280 nm), ranged from 0.357 to 0.507. Biotinylated
trypsin was purchased from Sigma (catalog no. T6640). To form the
protease-Limulus
M complex, a 2-fold excess
of trypsin was exposed to Limulus
M in 50
mM Tris (pH 8.0) for 3 min at room temperature, and the
Limulus
M was then separated from the unbound
trypsin by gel filtration on a Sephadex G-50 column under
lipopolysaccharide (LPS)-reduced conditions.
Association of Trypsin with
To determine the association of
FITC-labeled trypsin with blood proteins, the isolated plasma samples
recovered from trypsin-injected animals were treated with 3%
polyethylene glycol 8000 followed by centrifugation at 8000 M
g for 20 min to remove a majority of the hemocyanin, which
interfered with molecular filtration. The molecular mass of the
FITC-labeled material was estimated by centrifugation through molecular
filters with defined size exclusion characteristics and by gel
filtration chromatography. Aliquots of the supernatants were subjected
to centrifugation for 4 h through Centricon filters (Amicon) with 10 or
100-kDa molecular mass retentions. The fluorescence of the flow-through
and retained fractions was measured with the fluorometer. Gel
filtration chromatography employed a calibrated 100
1.6-cm
column of Sephacryl S-300 HR (Pharmacia Biotech Inc.) resin that was
eluted at 1 ml/min. Pore-limit gel electrophoresis was conducted with
4-15% gradient gels (Mini-Protean II ready gels, Bio-Rad catalog
no. 161-0902) with a Tris borate buffer system (84 mM Tris, 75
mM boric acid (pH 8.9)) for the upper and bottom chamber at
100 V for 3 h. Immunoblotting was conducted as described by Towbin
et al. (1979). Immunoprecipitation was performed using an
affinity-purified rabbit anti Limulus
M
antibody following the method of Harlow and Lane(1988). The antiserum
was affinity purified on Limulus
M-conjugated
Sepharose with elution with 0.1 M glycine (pH 2.5).
Angiogram of Limulus
The circulation of
Limulus is very different from that of mammals, with a
dorsally situated tubular heart that beats very slowly (30
beats/min at 23 °C). Blood is expelled into the frontal artery, one
pair of anterior arteries, and four pairs of lateral arteries (Redmond
et al., 1982; Shuster, 1978). From the arterial system, the
blood flows into open sinuses. In addition to the heart pump, the gills
participate in the venous system as accessory pumps (Freadman and
Watson, 1989). The relatively lethargic circulation resulted in slow
mixing with the blood of samples injected into the peripheral
circulation (the leg joints; data not shown). Instead, good mixing of
injected samples was obtained for tracer dye injected directly into the
heart, with significant transfer of label to the peripheral circulation
within 1 min and dissemination of the injected material by 5 min
(Fig. 1, A-D).
Figure 1:
Angiogram of adult
male Limulus polyphemus. A, fluorography before
injection of Angiovist. H, Limulus heart; N,
needle introduced into the heart with connecting tubing to syringe
containing 10 ml of Angiovist. B, distribution of the contrast
medium during injection. The heart and the proximal arteries appear in
much higher contrast. C, 1 min after injection of
Angiovist-282. Contrast medium has reached the distal arteries but
almost none has been transported to the blood sinuses peripheral to the
heart. D, 5 min after injection of Angiovist. Contrast medium
is still present in the heart and arteries, but it has also been
transported from the heart to the vascular sinuses and the
gills.
Stability of Endogenous Proteins in the Circulation of
Limulus
Hemocyanin, which is the main oxygen transport protein
of the blood and, at a concentration of 20-60 mg/ml, is the most
abundant protein in the plasma (Volbeda and Hol, 1988; Van Holde and
Miller, 1982), was selected for the determination of the behavior of an
inert protein. Hemocyanin was collected from Limulus plasma by
precipitation with 3% polyethylene glycol 8000 and was labeled with
FITC. The protein was separated from the unbound FITC by gel filtration
on Sephadex G-50 under LPS-reduced conditions and was then injected
into the heart at 6 µg/g of body weight. To obtain a rapid mixing
of the injected protein, 30-40 ml of blood was aspirated into the
injection syringe to mix with the sample destined for injection, and
the mixture was immediately reinjected into the heart. The Limulus blood cells react to mechanical stress and exposure to LPS by
degranulation and clotting of the blood (Armstrong, 1979, 1980;
Armstrong and Rickles, 1982; Armstrong, 1980). The LPS-reduced
conditions ensured that no clotting of the blood cells occurred in the
syringe during aspiration and reinjection. After 5 min, 90% mixing of
the labeled protein with the peripheral blood was achieved, and a
steady-state concentration was established by 10 min (Fig. 2).
Over the observation period of 180 min, there was no significant
clearance of FITC-labeled hemocyanin from the circulation
(Fig. 2).
Figure 2:
Distribution of FITC-labeled hemocyanin in
the Limulus circulation following intracardiac injection.
Fluorescence was measured in cell-free plasma samples collected from
the blood spaces of the leg joints over a period of 180 min after
injection. Break and scale change at 45 min. , fluorescence of
plasma samples from an individual animal;
, sample fluorescence
(mean ± standard deviation) as fraction of the fluorescence at
10 min postinjection, with subtraction of background fluorescence of
the t = 0 sample. Number of animals = 4. The
introduced hemocyanin becomes uniformly mixed with the blood by 10 min
following injection and then remains at a constant concentration during
the observation period.
Clearance of Trypsin from the Plasma of
Limulus
FITC-labeled, purified trypsin was introduced into the
blood of Limulus as described above at 14.5 µg/g of body
weight. The concentration of trypsin in the peripheral blood increased
for 5-10 min and was then rapidly cleared from the plasma by
30-35 min after injection with a t for this clearance of
26.5 min. Clearance was followed by the reappearance of fluorescence in
the plasma between 45-90 min to 52% of the amount of 10 min
(Fig. 3). Clearance requires the proteolytic activity of trypsin.
FITC-labeled, purified trypsin was inactivated by three sequential 1
mM additions of the irreversible active site inhibitor
phenylmethylsulfonyl fluoride (PMSF) over a period of 90 min at room
temperature (remaining activity < 4%) followed by dialysis against 1
mM HCl to remove free inhibitor. In marked contrast to the
efficient clearance of active trypsin, inactivated enzyme was not
removed from the circulation over the observation period of 180 min
(Fig. 3).
Figure 3:
Clearance of trypsin from Limulus plasma after intracardiac injection. FITC-trypsin () is
removed from the plasma after 35 min (t = 26.5 min,
n = 5, mean ± standard deviation). After 45 min,
52% of the fluorescence has reappeared in the plasma.
,
FITC-trypsin, inactivated by three sequential treatments with 1
mM PMSF. There is no detectable clearance of the inactivated
protease over the 180-min observation period (t > 180 min,
n = 4, mean ± standard deviation).
,
fluorescence associated with the blood cells following injection of
FITC-trypsin into the Limulus heart. The maximum of the
injected fluorescence associated with the blood cells, 51% of the
concentration at 10 min, coincides approximately with the time of
minimal fluorescence in the plasma (n = 3, mean
± standard deviation).
Clearance of Reacted and Unreacted
Limulus-
Limulus M from the Limulus
Plasma
M was labeled with
FITC as described above. In excess of 85% of the FITC-labeled
Limulus
M was reactive with trypsin, as
determined by the soybean trypsin inhibitor protection assay using
trypsin whose activity was determined by active site titration with
p-nitrophenyl p`-guanidinobenzoate hydrochloride
(Chase and Shaw, 1967). Unreacted FITC-labeled Limulus
M was injected at 6.6 µg/g of body weight and
became uniformly mixed with the blood by 10 min after injection and was
not significantly cleared from the plasma over a period of 400 min
(Fig. 4; data shown to 200 min after injection). Methylamine
(MA)-reacted Limulus
M was prepared by
incubation with 0.1 M MA for 24 h at 4 °C followed by
dialysis against 3% NaCl. FITC-labeled, MA-reacted Limulus
M was injected at 6.4 µg/g of body weight and
was cleared to 23% of the starting concentration by 24 min after
injection. Clearance was followed by a reappearance of FITC in the
plasma after 45 min (Fig. 4). FITC-labeled trypsin was reacted
with Limulus
M at pH 8.0 for 5 min followed
by gel filtration to remove unbound trypsin from the complex. The
trypsin-Limulus
M complex experienced a
clearance to 0.6% of the starting amount by 20 min after injection,
followed by a reappearance in the plasma of 28% of the FITC at
30-90 min). Thus, the efficient clearance of the
trypsin-
M complex was similar to the clearance of
MA-reacted
M and distinct from the persistence in the
plasma of native, unreacted
M.
Figure 4:
Clearance of Limulus -macroglobulin
(Limulus-
M) from the plasma of Limulus after intracardiac injection.
, unreacted FITC-labeled
Limulus
M. Unreacted Limulus
M was not cleared from the plasma during the
observation period (t > 200 min, n = 5,
mean ± standard deviation).
, MA-treated Limulus
M. MA-treated Limulus
M
was cleared from the plasma to 80% of its initial concentration by 25
min after injection (t
= 20 min, n = 5,
mean ± standard deviation). 47% of the fluorescence reappeared
in the plasma by 45 min.
, cell-associated fluorescence
associated with the injection of unreacted FITC-Limulus
M (n = 5, mean ± standard
deviation). Very little unreacted Limulus
M
became associated with the blood cells.
The clearance of
trypsin and trypsin-M complex resembles the patterns
of clearance of proteases from mammals, where enzymatically active
proteases are selectively eliminated. Protease-reacted
M is cleared rapidly from the circulation of dogs
(Ohlsson, 1971a), rabbits (Debanne et al., 1973), rats
(Katayama and Fujita, 1974a 1974b), and mice (Imber and Pizzo, 1981)
(t = 5-8 min). Trypsin-reacted Limulus
M was injected at 6.3 µg/g of body weight.
The clearance half-times for Limulus ranged from 18 min for
trypsin-Limulus
M complex (Fig. 5) to
26.5 min for trypsin (Fig. 3). Although the times for clearance
are longer for Limulus than for mammals, this is largely
attributable to the slower transport of the injected samples from the
heart to the periphery in Limulus. The actual durations of
clearance itself for mammals and for Limulus are similar after
the lengthened mixing times in the latter are subtracted. The
requirement for active enzyme (Fig. 3) is consistent with an
involvement of protease inhibitors in clearance and not with the
activity of a pathway that nonselectively clears any and all foreign
proteins. For example, because proteolytic cleavage of the bait region
is required for entrapment and binding (Hall and Roberts, 1978; Harpel,
1973; Sottrup-Jensen et al., 1989),
M
interacts only with active proteases and ignores inactive enzyme
(Lanchantin et al., 1966; Starkey and Barrett, 1973).
Figure 5:
Clearance of trypsin-reacted Limulus M from the Limulus plasma after
intracardiac injection.
, unreacted FITC-Limulus
M persisted in the plasma for the duration of the
observation period (for details, see Fig. 4).
, FITC-labeled
trypsin-reacted Limulus
M was eliminated to
99.4% of the injected fluorescence after 20 min (t = 18
min, n = 5, mean ± standard deviation).
,
cell-associated fluorescence after injection of FITC-labeled
trypsin-reacted Limulus
M. The maximal peak
of 48% of the injected fluorescence coincided approximately with the
time of minimal fluorescence in the plasma (n = 5, mean
± standard deviation).
Blood
cells from injected animals were solubilized with 10% SDS, and after
centrifugation the cell-associated fluorescence of the solution was
measured with the fluorometer. Whereas neither inactivated trypsin nor
unreacted Limulus M associated with the blood
cells at any time after injection (Fig. 4), 51% of the
fluorescence of trypsin (Fig. 3) and 48% of the
trypsin-Limulus
M complex (Fig. 5)
could be recovered in the cell-associated fraction at 30 and 20 min
postinjection, respectively. The time course of the association of FITC
with the blood cells was the inverse of the amount of fluorescence in
the plasma ( Fig. 3and Fig. 5).
Association of Trypsin with Limulus
Microfiltration, gel
filtration chromatography, and immunoprecipitation were used to
characterize the association of the injected trypsin with plasma
proteins. Postinjection plasma samples were precipitated with 3%
polyethylene glycol 8000 and, after centrifugation to remove the
precipitated hemocyanin, the supernatant was analyzed by
microfiltration through Centricon filters (Amicon) with various
molecular retention capabilities. After a 4-h centrifugation, the
fluorescence of the flow-through and of the retained solution were
measured with the fluorometer.
M in Vivo
1.6-cm Sephacryl
S-300 HR column, over 80% of the fluorescence was recovered in a high
molecular mass complex that eluted at the same position on the column
as purified Limulus
M, indicating that
trypsin binds to a molecule of the Limulus-plasma with a
molecular mass identical to that of
M (Fig. 6).
Figure 6:
Gel filtration on a calibrated Sephacryl
S-300 HR 100 1.6-cm column of a hemocyanin-depleted plasma
sample 10 min after intracardiac injection of FITC-trypsin.
,
fluorescence of the sample;
, optical absorbance at 280 nm of
the sample. 80% of the fluorescence was associated with a high
molecular mass complex that eluted at the same position as purified
Limulus
M.
Pore-limit polyacrylamide gel electrophoresis with Western blotting
and immunoprecipitation further implicated M as the
plasma protein that binds the injected trypsin prior to clearance.
Biotinylated trypsin (active or inactivated by three sequential
incubations with 1 mM PMSF) was injected into the animal, and
plasma samples were taken 10 min after injection, a time of complete
mixing but of minimal clearance. The samples were subjected to pore
limit gel electrophoresis followed by electrophoretic transfer to
nitrocellulose. One half of the nitrocellulose sheet was stained with
affinity-purified rabbit anti-Limulus
M and
horseradish peroxidase-labeled anti-rabbit-IgG to localize
M, and the other half was stained with horseradish
peroxidase-labeled avidin to localize trypsin. The biotinylated trypsin
(Fig. 7, lanes8 and 10) occupied the
same position on the gel as did Limulus
M
(Fig. 7, lanes1-5). Lanes containing
plasma of animals injected with PMSF-inactivated biotinylated trypsin
did not contain trypsin (Fig. 7, lane9).
Trypsin is a basic protein (pI = 10.5) (Desnuelle, 1960), which
would be expected to migrate toward the cathode under the conditions of
pore limit electrophoreses (electrophoresis buffering at pH 8.9) unless
bound to an acidic protein. The migration into the pore limit gel only
of enzymatically active trypsin and its localization at a position
coincident with Limulus
M is consistent with
the suggestion that active trypsin introduced into the plasma becomes
bound to
M. The association of both active and
PMSF-inactivated biotinylated trypsin with
M in 10-min
postinjection plasma samples was probed further by immunoprecipitation
of
M with an affinity-purified anti-Limulus
M antiserum. Biotinylated enzymatically active
trypsin co-immunoprecipitated with Limulus
M
(Fig. 8, lane8), whereas biotinylated
PMSF-inactivated trypsin did not (Fig. 8, lane9). Taken together, these observations indicate that a
majority of the injected active trypsin becomes associated with a
molecule with the properties of Limulus
M.
Furthermore, it appears that the trypsin-
M complex is
degraded to small peptides following clearance. The blood cells appear
to contribute to the clearance of the trypsin-
M
complex, since
50% of the cleared fluorescence of trypsin or
M associates with the blood cells ( Fig. 3and
Fig. 5
).
Figure 7:
Western blot of pore limit gel
electropherogram of plasma sample of animal injected 10 min previously
with biotinylated trypsin (pH 8.9 on a 4-15% polyacrylamide
gradient gel). Lanes1-5, blot probed with
rabbit anti-Limulus M antibody; lanes7-10, blot probed with horseradish
peroxidase-avidin. Lane1, methylamine-reacted
Limulus
M; lane2,
Limulus
M reacted with excess biotinylated
trypsin; lane3, hemocyanin-depleted plasma from an
animal injected 10 min previously with biotinylated trypsin; lane4, hemocyanin-depleted plasma sample from an animal
injected 10 min previously with PMSF-inactivated biotinylated trypsin;
lane5, unreacted Limulus
M; lane6, blank lane where the
nitrocellulose blot was cut in two; lane7,
biotinylated trypsin; lane8, hemocyanin-depleted
plasma from an animal injected 10 min previously with biotinylated
trypsin; lane9, hemocyanin-depleted plasma sample
from an animal injected 10 min previously with PMSF-inactivated
biotinylated trypsin; lane10, Limulus
M reacted with biotinylated trypsin. Biotinylated
trypsin is associated with a molecule that electrophoreses at the same
position as Limulus
M only in lanes8 and 10.
Figure 8:
Immunoprecipitation with affinity-purified
anti-Limulus M, followed by
SDS-polyacrylamide gel electrophoresis and Western blotting, probed
with horseradish peroxidase-avidin. Lanes1-5,
samples were immunoprecipitated with preimmune serum; lanes7-9, samples were immunoprecipitated with rabbit
anti-Limulus
M antibody that had been
affinity purified on Limulus
M-conjugated
Sepharose. Lane1, sample was methylamine-reacted
Limulus
M; lane2,
biotinylated trypsin-reacted Limulus
M;
lane3, plasma collected 10 min after intracardiac
injection of biotinylated trypsin; lane4, plasma
collected 10 min after intracardiac injection of PMSF-inactivated
biotinylated trypsin; lane5, unreacted Limulus
M; lane6, biotinylated trypsin
loaded directly onto the gel without immunoprecipitation; lane7, biotinylated trypsin-reacted Limulus
M; lane8, plasma sample
collected 10 min after intracardiac injection of biotinylated trypsin;
lane9, plasma sample collected 10 min after
intracardiac injection of PMSF-inactivated biotinylated trypsin.
Biotinylated trypsin is present only in lanes6,
7, and 8. Unlike mammalian
M,
Limulus
M fails to establish
N
(
-glutamyl)-lysine bonds between the
thiol ester glutamyl residue of
M and lysine residues
of the reacting protease (Quigley et al., 1991), so
biotinylated trypsin bound to Limulus
M
migrates at the position of unreacted trypsin in the presence of
SDS.
The mammalian receptor for the
M-protease complex has been shown to be identical to
the low density lipoprotein-related protein and belongs to the family
of low density lipoprotein receptors (Zheng et al., 1994;
Strickland et al., 1990). Low density lipoprotein
receptor-related protein binds a variety of ligands in addition to the
M-protease complex, including plasminogen
activator-plasminogen activator inhibitor-1 complex (Kounnas et
al., 1993), apoE-enriched very low density lipoprotein (Rebeck
et al., 1993), lipoprotein lipase (Williams et al.,
1994; Chappell et al., 1992), Pseudomonas exotoxin A
(Kounnas et al., 1992), and the 40-kDa receptor-associated
protein (Herz et al., 1991). We have not yet identified a
specific cell surface receptor for Limulus
M-protease complex from the blood cells, but
detergent extracts of Limulus blood cells contain a protein
that specifically binds mammalian receptor-associated protein and that
may be a molecular homologue of low density lipoprotein
receptor-related protein of mammals.
(
)
M-protease complex in Limulus. After binding
to its receptor, mammalian
M is internalized via
coated pits and sequestered in secondary lysosomes (Goldstein et
al., 1979; Sottrup-Jensen, 1987), with the
M-protease complex subsequently being degraded to low
molecular weight products (Ohlsson, 1971b; Katayama and Fujita, 1974a,
1974b). The mechanism of endocytosis and the fate of the endocytosed
protein seems to be similar to that of low density lipoprotein (Pastan
et al., 1977; Brown and Goldstein, 1979; Goldstein et
al., 1979) and of peptide hormones and other proteins that bind to
specific cell surface receptors (Pastan and Willingham, 1981; Steinman
et al., 1983; Besterman and Low, 1983). In Limulus,
40-50% of the fluorescence reappeared in the plasma after
clearance from the plasma. The soluble postclearance fluorescence was
associated with molecules smaller than 10 kDa, based on the free
passage of fluorescence through molecular filters with a 10 kDa cutoff.
This is consistent with the possibility of proteolytic degradation of
the endocytosed complex in secondary lysosomes and with the suggestion
that the entire pathway of inactivation of proteases by
M has been conserved during the evolution of lineages
as diverse as the vertebrates and the arthropods.
Table:
Estimation of sizes of molecular complexes
formed by FITC-trypsin with molecules of the plasma at different stages
of the clearance cycle
M,
-macroglobulin; FITC, fluorescein isothiocyanate; LPS,
lipopolysaccharide; MA, methylamine; PMSF, phenylmethylsulfonyl
fluoride.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.