(Received for publication, November 29, 1994)
From the
The immunoregulatory plasma protein
-microglobulin (
-m) and the
proteinase inhibitor
-inhibitor-3
(
I
) form a complex in rat plasma. In the
present work, it was demonstrated that the
I
-m complex has no
inhibitory activity, the bait region was not cleaved by low amounts of
proteinases, and it was unable to covalently incorporate proteinases.
The results also indicated that the thiolester bond of the
I
-m complex was
broken. The
I
-m
complex was cleared from the circulation much faster than native
I
, with a half-life of approximately 7
min. Structurally, however, the
I
-m complex was
similar to native
I
rather than
I
cleaved by proteinases. It is speculated
that the role of
-m is to destroy the function of
I
by blocking the bait region and breaking
the thiolester and causing its physical elimination by rapid clearing
from the blood circulation. It is also possible that the formation of
complexes between
-m and
I
may serve as a mean to regulate the function of
-m since its complex with
I
is taken up rapidly by cellular receptors for
-macroglobulins.
-microglobulin (
-m), (
)also known as protein HC, is a low molecular weight plasma
glycoprotein with various immunoregulatory properties (reviewed in
Åkerström and Lögdberg,
1990 and Åkerström, 1992).
-m is translated in the liver together with bikunin
(Kaumeyer et al., 1986; Salier, 1990) as a precursor protein,
which is cleaved late in the trans-Golgi network previous to secretion
of free
-m (Bratt et al., 1993). Part of the
secreted
-m is linked covalently to other plasma
proteins, and circulating complexes with human IgA (Grubb et
al., 1986), rat
-inhibitor-3
(
I
) (Falkenberg et al., 1990),
and rat fibronectin (Falkenberg et al., 1994) have been
isolated and characterized.
I
-m is a 220-kDa
1:1 covalent complex circulating at a concentration of approximately 40
µg/ml (Falkenberg et al., 1994). Both
-m
and
I
are produced by rat hepatocytes, but
the site of formation of the complex is not known (Pierzchalski et
al., 1992).
I
is a 180-kDa proteinase
inhibitor belonging to the same superfamily as human
-macroglobulin (
M), the
-macroglobulins (Sottrup-Jensen, 1987). Its binding and inhibition
of proteinases is dependent on the ability to form covalent cross-links
to the proteinases (Enghild et al., 1989a). During the
reaction,
I
is cleaved in the bait region
by a variety of proteinases, leading to a change in conformation and a
subsequent activation of an internal thiolester bond. A highly reactive
glutamyl residue of the thiolester forms a covalent cross-link with a
lysyl residue in the attacking proteinase. The thiolester, which is a
common property of many of the
-macroglobulins, can also be broken
by a small nucleophilic reagent such as methylamine (Barrett and
Starkey, 1973). The conformational shift of
I
leads to exposure of a domain with high affinity for receptors on
hepatocytes and macrophages, resulting in a rapid clearance of the
I
-proteinase complex, a property shared by
many
-macroglobulins (Debanne et al., 1976; Van Leuven et al., 1979; Gliemann and Sottrup-Jensen, 1987).
The
complex formation between -m and
I
raised the question whether
-m could influence the proteinase inhibitory activity
or receptor recognition of
I
. In this
work, the complex was isolated and compared with the native proteinase
inhibitor,
I
.
Figure 3:
Incorporation of
[C]methylamine into
I
and
I
-m.
[
C]methylamine was allowed to react with 10
µg of
I
or
I
-m for 2 h at pH
8.2, followed by incubation with unlabeled methylamine for 2 h.
SDS-PAGE was performed on a 5-15% gradient gel of reduced
I
(lane1) and
I
-m (lane2), and non-reduced
I
(lane3) and
I
-m (lane4). Proteins are shown stained (A) and
autoradiographed (B).
Figure 2:
Bait region cleavage of
I
and
I
-m.
I
and
I
-m were incubated
with PPE or papain at a molar ratio of 10:1 for 5 min. PPE incubations
were terminated by adding DCI to a 250-µM final
concentration and papain by adding E-64 to a 50-µM final
concentration. Samples were run reduced in SDS-PAGE as follows: lane1,
I
or
I
-m; lane2,
I
or
I
-m + PPE; lane3,
I
or
I
-m +
papain.
Figure 1:
Incorporation of I-labeled proteinases into
I
and
I
-m. 2
µg of
I
or
I
-m were incubated
with different amounts (3-50 pmol) but with the same amount of
radioactivity of
I-labeled proteinases. After 5 min, the
reactions were terminated by the addition of proteinase inhibitors.
Non-reduced samples were separated by SDS-PAGE on a 5-15%
gradient gel. Gels were dried and autoradiographed. Lane 1 of
both gels represents incubation with 3 pmol of papain; lane 2, 35 pmol of cathepsin G; lane 3, 33 pmol of chymotrypsin; lane 4, 3 pmol of S. aureus V8 proteinase; lane
5, 8 pmol of PPE; lane 6, 50 pmol of trypsin. The
position of uncleaved
I
-m as determined
by staining of the gel is marked. Molecular mass markers are shown in
kilodaltons.
To exclude
the possibility that the purification procedure of the
I
-m complex caused
the loss of the capability of the molecule to interact with various
proteinases,
I-labeled PPE was added directly to rat
plasma. After stopping the reaction by adding the low molecular weight
inhibitor DCI, half of the plasma volume was applied to
anti-
-m-Sepharose to isolate a pool including the
I
-m complex, and
the other half was added to
anti-
I
-Sepharose to isolate
I
Equal amounts from each column were
then separated by SDS-PAGE under non-reducing circumstances (not
shown). Radioactive PPE had been incorporated to
I
but not to
I
-m. This agrees
well with the former experiment.
The ability of
I
and
I
-m to inhibit
trypsin was also compared by using the high molecular weight substrate
blue hide powder azure. As expected,
I
inhibits the trypsinmediated degradation of the substrate, but
I
-m could not
inhibit trypsin even at high concentrations (not shown).
I
and
I
-m were incubated
with two proteinases of different specificities, PPE and papain, in a
molar ratio of 10:1. The reaction products were separated by reduced
SDS-PAGE (Fig. 2). The cleavage of
I
resulted in doublet bands migrating around 100 kDa, which
previously have been shown to result from
I
bait region cleavage (Enghild et. al., 1989a).
I
-m, on the other
hand, was not cleaved by the proteinases using these conditions, as
judged by SDS-PAGE (Fig. 2) and N-terminal amino acid sequence
analysis (not shown), suggesting that the bait region was not
accessible on the complex.
To
exclude the possibility that the thiolester reactivity of
I
-m had been
destroyed as a result of purification,
[
C]methylamine was also incubated with rat
plasma. The plasma was then applied to either anti-
-m
or anti-
I
-Sepharose. Equal amounts of
proteins eluted from the two columns were then separated by
non-reducing SDS-PAGE. Only very faint radioactivity could be seen in
the
I
-m complex as
compared with
I
after elution from the
anti-
-m column (not shown). These experiments thus
suggest that the thiolester of
I
is intact
and reactive with methylamine, whereas the thiolester of
I
-m is broken.
Figure 4:
Clearance of I-labeled
I
and
I
-m from mouse
blood circulation. Native
I
,
trypsin-treated
I
(A), or native
I
-m,
trypsin-treated
I
-m, and native
I
-m in competition
with methylamine-treated
M (B) were injected
into the lateral vein of a mouse. The clearance of the radioactive
proteins was determined by removing 25-µl aliquots of blood and
counting them in a
-counter.
Figure 5:
Transverse urea gradient-polyacrylamide
gel electrophoresis of I
and
I
-m. A single
sample was loaded along the top of each polyacrylamide (5%) gel and
allowed to migrate toward the bottom, as indicated by the arrow. The gel was cast with a continuous urea gradient
(0-8 M urea) running from left to right.
I
,
I-labeled
I
-m, and
I-labeled
I
-m incubated with
PPE migrated identically, and only one of these samples,
I
, is shown in A. The migration
of
I
incubated with PPE is shown in B. Further details are described under ``Experimental
Procedures.''
Finally, no major
difference between I
and the
I
-m complex could
be seen by electron microscopy. Both appeared as rounded molecules
containing granular structures, suggesting the presence of
intramolecular domains in both
I
and the
I
-m complex.
In this work, the
I
-m complex was
purified from rat plasma, and its proteinase inhibitory activities and
some structural properties were compared with those of free
I
, the rat
-macroglobulin
homologue. The results demonstrate that the proteinase inhibitory
activity of
I
-m is
lost and its thiolester bond broken, and it was cleared from the
circulation almost as quickly as proteinase-cleaved
I
. However, the structure of the
I
-m complex, both
before and after proteinase treatment, is similar to native
I
rather than proteinase-cleaved
I
.
The
I
-m complex lacked
the proteinase inhibitory activity associated with
I
. Two observations can explain this
finding. First, proteinases were unable to cleave the peptide backbone
at the bait region of
I
-m (Fig. 2). As a result, there was no conformational shift of the
I
-m molecule (Fig. 5), which has previously been demonstrated to be the
result of proteinase treatment of
I
(Enghild et al., 1989a). It can be speculated that
-m is bound closely to the bait region of the
I
peptide, sterically blocking the attack
of the proteinase. Second, methylamine was not incorporated into
I
-m as it was into
I
(Fig. 3). This indicates that the
thiolester bond of
I
-m is cleaved,
making the complex unable to bind proteinases covalently via the
glutamyl residue of the thiolester (see also Fig. 1). As a
result, there can be no covalent binding of the attacking proteinase by
I
-m, which has been
shown to be prerequisite for the efficient proteinase inhibition of
I
(Enghild et al., 1989a).
The
thiolester of native -macroglobulins is believed to be hidden in a
pocket that is too small for large molecules to enter (Barrett et
al., 1979). The conformational shift induced by proteinase
cleavage of the bait region leads to exposure of the thiolester,
followed by a nucleophilic attack on the thiolester by a lysyl
-amino group on the nearby located proteinase, and a subsequent
Glu-Lys cross-linking of
I
and the
proteinase. However, the thiolester of the
I
-m complex is
apparently cleaved, but since the conformation of the
I
-m complex is
similar to native
I
, it is not likely that
the thiolester has reacted with a proteinase. Instead, the thiolester
could have reacted with a small nucleophilic molecule, such as
methylamine, or with a structure on the
-m molecule
that somehow can reach the thiolester bond without disrupting the
conformation of the native
I
.
Consequently, it is possible that
-m is cross-linked
to
I
via the glutamyl or thiol group
involved in the thiolester bond.
-m is not dissociated
from the
I
-m
complex by extensive boiling in the presence of reducing agents. (
)This suggests that if the two proteins are cross-linked by
a disulfide bond, it is most likely of an unusual type as in the
IgA
-m complex in human plasma, which is also
reduction resistant (Calero et al., 1994). The nature of the
bond between
-m and
I
is
the subject of present investigations at our laboratories.
Cleavage
of I
by proteinases ultimately leads to an
exposure of receptor-binding domains, and a rapid clearance of the
proteinase-
I
complex from the circulation
(Enghild et al., 1989a). In agreement with this, the
half-life of trypsin-treated
I
was
approximately 2 min (Fig. 4). Surprisingly, the
I
-m complex was
also rapidly cleared, with a half-life of approximately 7 min in the
mouse blood circulation. Trypsin treatment of
I
-m did not, as
expected from the inability of the proteinase to induce conformational
changes in the complex, further increase the clearance rate. Thus,
despite the intact peptide backbone of
I
-m, its
insusceptibility to bait region proteinase attacks, and the structural
similarity between
I
-m and native
I
, it is possible that the
receptor-binding domain of the
I
-m complex is
exposed, resulting in a rapid clearing of the complex. The clearance of
I
-m could be
inhibited by an excess of
M, suggesting that it is in
fact cleared by the same receptors that normally bind
proteinase-cleaved
I
and not by receptors
with affinity for
-m or new epitopes formed by the
complex binding of
-m and
I
. (
)
The
I
-m complex is
found at a relatively high and stable concentration in rat plasma.
Thus, to match the rapid elimination from the circulation, the complex
must be synthesized at a comparable rate. It has been shown previously
that, although rat hepatocytes synthesize both
-m and
I
, the complex is not synthesized at all
or at a very low rate by rat hepatocytes (Pierzchalski et al.,
1992). Moreover, a simple mixing and incubation of
I-labeled
-m with rat plasma or
injection of
I-labeled
-m into living
rats do not lead to the formation of
I-labeled
I
-m (Falkenberg et al., 1994). Thus, despite a high rate of synthesis of the
I
-m complex, the
site of formation of the complex is still unknown.
The consequences
of complex formation between I
and
-m are at present unknown. Complex formation may serve
to regulate
-m immunoregulatory activity. Recently, we
have shown that covalent complexes of rat or human
-macroglobulins
with lysozyme seem to augment antigen presentation of lysozyme by
macrophages to T-cells (Chu and Pizzo, 1993; Chu et al.,
1994). The
-macroglobulin-lysozyme covalent complexes were taken
up very efficiently by macrophages via the low density
lipoprotein-related protein-
M receptor and then
processed to present lysozyme peptides in complex with Ia antigen to
T-cells that recognized the epitope. Whether uptake of
-m via the low density lipoprotein-related
protein-
M receptor serves a functional role in
macrophage regulation will require further study.
It has been shown
in this work that I
, the most abundant
proteinase inhibitor in the rat, has lost its proteinase inhibitory
activity when it is complex-bound to
-m. Furthermore,
I
-m is cleared from
the blood circulation at an unexpectedly high rate. A function of
-m could be to ``kill''
I
by blocking the inhibitory activity and
to cause its physical elimination from the circulation. Indeed, it can
be speculated that
-m ``kills'' its various
complex partners, i.e. albumin, fibronectin, and IgA, by
blocking one or more of their effector functions. It has, for example,
been shown that
-m is linked to IgA via the C-terminal
part of the
-chain (Grubb et al., 1986), which is also
involved in the binding to the secretory component (Mestecky and
McGhee, 1987), potentially blocking the transport of IgA from blood to
secretions. Thus, a scenario linking these observations would be for
-m to rapidly down-regulate the activity of plasma
proteins that are already in circulation before their concentration can
be lowered by transcriptional down-regulation.