From the
To test the hypothesis that a large portion of the bait region
of human
Human
To compare the properties of the thiol esters in the
two variants with those of the thiol esters in normal plasma
We also
examined the kinetics of the small methylamine-induced protein
conformational change by monitoring TNS fluorescence changes and found
a major difference between the variants and plasma
The TNS fluorescence
spectra also showed different changes for the
In this study, we tested the hypothesis that portions of the
bait region of
If noncovalent
contacts between the C-terminal portion of the bait region of a monomer
of one dimer and regions on a second dimer are critical for
tetramerization, it might be expected that major differences in this
C-terminal region might parallel different oligomerization states in
other
Another
consideration in the failure of the variants to trap proteinase is that
the rate of proteinase reaction is very much slower than for wild-type
Uncoupling of bait region cleavage and thiol ester activation has
been reported in human
The values reported are based on
the measured change in absorbance. All measurements are the average of
three separate determinations. The accuracy of the measurements is
estimated to be ±0.1 SH/monomer.
We thank Dr. Assaf Steinschneider for carrying out the
N-terminal peptide sequencing, Dr. Esper Boel for the generous gift of
the wild-type
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-macroglobulin (
M) can be
removed without adversely affecting the protein's structural and
functional properties, we expressed two human
M
variants with truncated bait regions and examined whether these
variants folded normally and functioned as proteinase inhibitors. Each
variant contains sites that are normal bait region cleavage sites in
wild-type
M, including the primary trypsin cleavage
site. The truncated bait regions are shorter by 23 and 27 residues,
respectively, and lack the C-terminal portion as well as different
parts of the N-terminal section of the bait region. We found that such
bait region truncation permitted normal folding of the monomers as well
as formation of the thiol ester and dimerization by disulfide
cross-linking, although the resulting species bound
6-(p-toluidino)-2-naphthalenesulfonic acid in a manner more
like thiol ester-cleaved
M than native
M. The variants' thiol esters reacted with
nucleophiles at rates identical to wild-type
M.
Surprisingly, however, the truncations prevented the noncovalent
association of the covalent 360-kDa dimers that normally gives
tetrameric
M, decoupled bait region cleavage from
thiol ester activation, and resulted in the inability of the two
variants to ``trap'' proteinase. This was despite apparent
cleavage of the bait region by proteinase, albeit at very much reduced
rates relative to wild-type tetrameric
M. The kinetics
of thiol ester cleavage-dependent protein conformational changes also
changed from sigmoidal to exponential. These findings indicate that
residues in the bait region appear to be necessary for noncovalent
association of 360-kDa disulfide-linked dimers to give tetrameric
M and suggest a role for the bait region in normal
M in coupling bait region cleavage to the sequence of
conformational changes that result in thiol ester activation and
ultimately proteinase trapping.
-macroglobulin
(
M)
(
)
is a high molecular mass,
tetrameric, broad range plasma proteinase inhibitor
(1) that is
organized as a noncovalently associated dimer of disulfide-linked
dimers
(2) . It inhibits proteinases by the unusual mechanism of
physically sequestering the proteinase as a result of
proteinase-triggered large-scale conformational changes in
M and can inhibit up to 2 mol of proteinase/mol of
M tetramer, depending on the size of the proteinase
(3). The interaction between the proteinase and
M that
initiates the trapping sequence is a limited proteolytic cleavage of
M in a restricted region, termed the bait region. The
bait region occurs in all
-macroglobulins, but shows great
variation in length and amino acid sequence between different
-macroglobulin species
(1) (Fig. 1). In contrast, the
remainder of the polypeptide shows very high sequence similarity
between
-macroglobulins, including conservation of most of the
disulfides (1), suggesting very similar folding of the
-macroglobulin monomers.
Figure 1:
Bait region sequences of wild-type and
variant human M and, for comparison, of PZP, rat
M, and rat
I
using the
one-letter amino acid code. The bait region covers approximately
residues 666-706 in the human inhibitor. Within this region are
all of the primary proteinase cleavage sites (underlined),
most of the secondary cleavage sites, and very little sequence homology
with
M from other species. Outside of this region, the
primary structures of
M from different species show
very high sequence homology. Sites of cleavage in human
M by trypsin, chymotrypsin, and porcine pancreatic
elastase are indicated by downwardarrows and the
letters T, C, and P, respectively. The
positions corresponding to the DNA cleavage sites by the NsiI,
MluI, and BsiWI restriction endonucleases used in the
construction of the variants are indicated. Sequence data for human
M, PZP, rat
M, and rat
I
are taken from Sottrup-Jensen et al. (1).
A puzzle regarding the functioning of
the trapping mechanism of -macroglobulins is that limited
proteolysis anywhere within the bait region results in triggering the
same conformational change. Similarly puzzling is that the same
mechanism appears to operate in different
-macroglobulins despite
the large differences in length, sequence, and composition of the bait
regions (Fig. 1).
H NMR studies on human
M have shown that at least parts of the bait region
are mobile and surface-accessible
(4, 5) . This has led
to a model for the organization of
M as a structurally
conserved scaffold formed by the majority of the polypeptide to which
is attached a less structured module composed of the bait region
residues
(6) . Since the structure of the flexible portion of the
bait region does not appear to depend on the conformation of the
remainder of the protein
(5, 7) , such a model could
readily accommodate the large differences between bait regions that
occur between different
-macroglobulins and suggested to us that
it might be possible to shorten a given bait region without loss of
inhibitory properties. To test this, we have expressed and
characterized two human
M variants in which a large
portion of the normal bait region sequence from residues 676 to 706 has
been removed and much shorter portions substituted, each of which
contains sites that are normally attacked by specific proteinases in
wild-type
M, including the primary trypsin cleavage
site
(8, 9) . These truncated bait regions are shorter by
23 and 27 residues, respectively.
Construction of Vectors for Expression of Bait Region
Variants of Human
The
vector for expression of variant 1, pA2M-BRC1, was constructed from the
wild-type expression plasmid p1167
(8) . In brief, a 1683-base
pair fragment of p1167, covering -Macroglobulin
M residues
146-707, was excised by digestion with the restriction
endonucleases BamHI and BsiWI and ligated into
M13mp18 containing a modified polylinker region in which the portion of
the polylinker between the PstI and SphI sites had
been removed and replaced by a synthetic oligonucleotide containing
AflII, BsiWI, and XhoI sites (coding strand,
5`-ACTTAAGCGTACGCTCGAGTCATG-3`; noncoding strand,
5`-ACTCGAGCGTACGCTTAAGTTGCA-3`). The phage construct was doubly
digested with NsiI and BsiWI to remove the portion of
DNA covering most of the bait region. A double-stranded linker (coding
strand, 5`-TGGCCTACGCGTGCAC-3`; noncoding strand,
5`-GTACGTGCACGCGTAGGCCATGCA-3`) containing a silent MluI site
was then inserted to create a new 1605-base pair
BamHI-BsiWI fragment with a modified bait region cDNA
sequence. The BamHI-BsiWI fragment was excised and
religated into p1167 that had been cut with BamHI and
BsiWI to create pA2M-BRC1, which was used for expression of
M variant 1. pA2M-BRC2, used for expression of
M variant 2, was derived directly from pA2M-BRC1.
pA2M-BRC1 was cut with MluI and BsiWI to remove a
portion of the bait region cDNA, and a longer double-stranded linker
(coding strand, 5`-CGCGTGGGCTTTTACGAGAGC-3`; noncoding strand,
5`-GTACGCTCTCGTAAAAGCCCA-3`) was inserted between the cut ends. The
success of the mutagenesis was confirmed by sequencing of the plasmid
pA2M species.
Stable Transfection of Baby Hamster Kidney
Cells
Stably transfected baby hamster kidney cells expressing
wild-type and bait region variants of human
-macroglobulin were prepared by cotransfection with
plasmids pRMH140 and pSV2dhfr and the appropriate
-macroglobulin expression plasmid, with selection for
high producers by resistance to methotrexate and neomycin, as
described
(9) . Typically, 5 µg of pRMH140 and pSV2dhfr and
20 µg of the
-macroglobulin plasmid were used.
Calcium phosphate precipitation was used for the transfection.
Isolation of
Plasma
-Macroglobulin
M was isolated from pooled outdated plasma obtained
from the Vanderbilt Blood Bank by chromatography on zinc chelate resin,
Cibacron blue gel, and AcA22 as described previously
(10) . All
forms of recombinant
M were isolated from serum-free
cycles of growth medium using only the zinc chelate resin
chromatography step, which yielded material sufficiently pure for
further characterization as judged by polyacrylamide gel
electrophoresis. Concentrations of all forms of
M were
determined spectrophotometrically using an extinction coefficient for
the plasma protein of 640,000 M
cm
(11) since the changes in the bait
region were expected to have a negligible effect on the absorbance at
280 nm.
Polyacrylamide Gel Electrophoresis
Polyacrylamide
gels were run under nondenaturing conditions in 5% acrylamide slabs
according to the procedure of Davis
(12) or under denaturing
conditions on 7.5% gels according to the procedure of
Laemmli
(13) . Unless otherwise noted, samples for SDS-PAGE were
denatured and reduced for 45 min at 37 °C (to prevent heat
cleavage
(14, 15) ) in buffer containing 1% SDS and 0.8%
dithiothreitol. 1 mM phenylmethanesulfonyl fluoride was added
to M samples containing proteinase to inactivate the
proteinase and thus prevent additional cleavage of
M
during denaturation. Molecular mass standards were myosin (200 kDa),
-galactosidase (116.3 kDa), phosphorylase b (97.4 kDa),
serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31
kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa), and aprotinin
(6.5 kDa).
N-terminal Sequencing
Samples for N-terminal
sequence analysis were prepared by electroblot transfer of trypsin
cleavage fragments from a 5% polyacrylamide gel run under
denaturing/reducing conditions to a polyvinylidene difluoride membrane
using published procedures
(16) . The sequences were determined
on an ABI Model 477A protein sequencer in the Protein
Sequencing/Synthesis Laboratory at the University of Illinois at
Chicago.
Assays
Trypsin trapping assays were carried out as
described previously
(17) and involved measurement of residual
trypsin activity following addition of excess soybean trypsin inhibitor
to complex any nontrapped trypsin. Trypsin activity was measured
spectrophotometrically with the substrate tosylarginine methyl ester.
Assays of protected trypsin were carried out after a 5-min reaction of
trypsin for plasma M and after up to a 60-min reaction
with trypsin for the variants. Free sulfhydryl groups were determined
by spectrophotometric assay on a Shimadzu 2100PC apparatus using
5,5`-dithiobis(2-nitrobenzoic acid)
(17) .
Fluorescence Measurements
Wavelength and kinetic
measurements of TNS fluorescence were made on an SLM-AMINCO 8000
spectrofluorometer using samples of 1.2 ml in acrylic cuvettes. A TNS
concentration of 50 µM was used for all measurements.
Excitation was at 316 nm, with slits of 4 nm. Emission slit settings of
4 nm for emission spectra and of 16 nm for monitoring emission at a
constant wavelength were used.
Data Analysis
Kinetic data were fitted by
nonlinear least squares using the program MINSQ II (MicroMath
Scientific Software, Salt Lake City, UT).
Electrophoretic Behavior of Bait Region
Variants
To compare the properties of the two variants with
those of wild-type M, we examined their
electrophoretic behavior on polyacrylamide gels run under nondenaturing
conditions. Both variants migrated much faster than wild-type
recombinant or plasma
M, at a position consistent with
their being dimeric species, as judged by comparison with Limulus
M, which occurs naturally as a dimer, and with
SDS-dissociated wild-type
M (Fig. 2, lanesa, c, e, h, and j).
When run under denaturing conditions in the presence of dithiothreitol,
the differences between the normal and variant forms of
M disappeared (Fig. 3), reflecting similar size
of the monomers. Plasma and wild-type recombinant
M
gave a single band corresponding to the
M monomer
(Fig. 3, lanesb and c). The two
M variants also gave single bands at the same position
as wild-type recombinant
M (Fig. 3, lanesd and e). In the absence of dithiothreitol, all
species gave a single band at higher molecular mass, corresponding to
the disulfide-linked 360-kDa dimer (data not shown).
Figure 2:
Electrophoretic mobilities of wild-type
and variant human M and of Limulus
M on a 5% polyacrylamide gel run under
nondenaturing conditions in the absence and presence of trypsin.
pA2M, rA2M, LA2M, BR1, and BR2 refer to plasma
M, recombinant wild-type
M, Limulus
M, variant 1, and
variant 2, respectively. The designations ``-'' and
``+'' signify the absence and presence of trypsin.
Laneg contains wild-type recombinant
M in the presence of 1% SDS to promote dissociation
into dimers. Trypsin treatment involved incubation at room temperature
for 10 min with 1.1 eq of trypsin/
M dimer at an
M concentration of 1 mg/ml. The positions of dimer and
tetramer are indicated by arrows. Phenylmethanesulfonyl
fluoride to a concentration of 2 mM was added to terminate the
proteinase reactions.
Figure 3:
SDS-PAGE (7.5% gel) of wild-type and
variant M run under reducing conditions. Samples were
prepared as described under ``Materials and Methods.''
Lanea, molecular mass standards; laneb, plasma
M; lanec,
wild-type recombinant
M; laned,
variant 1; lanee, variant
2.
Presence and Properties of Internal Thiol
Esters
Although the thiol ester is an intrinsically reactive
group, it is kinetically stabilized in the native M
structure by being located in a hydrophobic cleft with limited solvent
accessibility
(6, 18, 19, 20) . The
presence of an intact thiol ester in an
M variant is
thus a sensitive indicator of the correct structure of the variant in
the vicinity of the thiol ester, needed both to generate and to
stabilize the thiol ester. Both bait region variants were found to
contain intact thiol esters as judged by the ability to generate free
sulfhydryl groups from them upon incubation with methylamine
().
M, we examined the kinetics of their reaction with
methylamine. The rate of reaction of the thiol esters in both
M variants, determined by the rate of appearance of
free SH groups, was indistinguishable from that in plasma
M (Fig. 4). Since the reactivity of the thiol
ester in plasma
M is determined not only by the
nucleophilicity of the attacking base, but also by the accessibility to
the attacking nucleophile, this identity between the variants and
plasma
M suggests very similar or identical local
environments.
Figure 4:
Time
course of free thiol release from plasma and variant M
by reaction with 0.2 M methylamine at pH 8. Free thiol
concentration was determined by continuous spectrophotometric
monitoring of the change in absorbance caused by reaction of the
liberated thiol with 5,5`-dithiobis(2-nitrobenzoic acid).
,
variant 1;
, variant 2;
, wild-type
M. The
solidlines represent the least-squares best fits to
the data.
Effects of Variants on TNS Fluorescence
TNS
fluorescence has been used extensively to follow the protein
conformational changes that result from bait region and/or thiol ester
cleavage in
M
(21, 22, 23, 24) . We
initially examined the ability of the
M variants to
bind to and alter TNS fluorescence and compared the effects with those
of the well studied human plasma
M. In contrast to the
relatively modest enhancement produced by plasma
M
(Fig. 5A), the two variants produced
5-8-fold
greater enhancement of TNS fluorescence (Fig. 5, B and
C) and a larger blue shift, with
at
430 nm, indicative of a TNS-binding site that more closely
resembles that of conformationally altered plasma
M
than that of native
M.
Figure 5:
Comparison of the changes in TNS
fluorescence produced by plasma and variant M before
and after reaction with methylamine. A, plasma
M; B, variant 1; C, variant 2. In
each panel, spectruma is of unreacted
M, and spectrumb is of
methylamine-reacted
M. The TNS concentration was 50
µM in each case, and the concentration of dimeric
M was 0.4 µM.
Conformational Changes following Reaction of Variants
with Methylamine
The mobility of tetrameric human plasma
M or dimeric Limulus
M on
polyacrylamide gels run under nondenaturing conditions can be used to
distinguish between native and methylamine-treated or
proteinase-complexed
M
(14, 25) . The
increase in mobility between native and methylamine-treated or
proteinase-reacted forms has led to the designation of native
M as the ``slow'' form and reacted
M as the ``fast'' form (14) and has been
correlated with a compaction of the molecule
(14, 26) .
The two dimeric variants showed imperceptible changes in mobility
following reaction with methylamine, compared both with tetrameric
human
M and dimeric Limulus
M (Fig. 6), suggesting that any
conformational change resulting from thiol ester cleavage is different
both in magnitude and kind from that which occurs in the normal
tetramer or even in a fully functional dimer.
Figure 6:
Effect of thiol ester cleavage on
electrophoretic mobilities of plasma and variant human
M and Limulus
M run under
nondenaturing conditions on a 5% polyacrylamide gel. The designations
``-'' and ``+'' signify that
M was untreated or reacted with methylamine,
respectively. ``S'' and ``F''
indicate positions of slow and fast conformers, respectively. For
plasma
M and for the two variants, methylamine-treated
samples were prepared by reaction with 0.2 M methylamine at pH
8.0 for 1 h, which is sufficient to ensure complete cleavage of the
thiol esters. For Limulus
M, which reacts
with methylamine more slowly, reaction was with 0.4 M
methylamine at pH 8.0 overnight.
As a second way of
comparing the thiol ester cleavage-induced conformational change in the
two variants with that in plasma M, we examined the
effect that these species had on the fluorescence emission spectrum of
TNS. Both variants gave a small increase in fluorescence intensity,
although without an alteration in the position of the maximum, whereas
plasma
M gave a 4-fold increase in intensity and a
shift in the emission maximum of
40 nm (Fig. 5).
M.
In plasma
M, cleavage of two thiol esters, presumably
within the dimer that constitutes a proteinase-binding site, is
required before any large-scale conformational change occurs in that
dimer
(21) . This cooperativity leads to sigmoidal kinetics of
methylamine-induced conformational change that can be well fitted to a
model of initial reaction of the thiol esters within a half-molecule
(S
and S
) (with rate constant k`) and
subsequent slow conformational change (with rate constant
k
) once both thiol esters within a given
half of the tetramer have been cleaved (S
and
S
) (Fig. S1). (Note, however, that more
complex schemes could be envisaged involving additional steps. Indeed,
a more complex scheme is necessary to explain sigmoidal kinetics for
the monomeric thiol ester-containing protein C3 upon thiol ester
cleavage
(27) . Also note that the two subunits in tetrameric
plasma
M that constitute a proteinase-binding site and
that are presumably the ones that change conformation cooperatively
upon thiol ester cleavage of both subunits may well be from
different disulfide-linked half-molecules rather than the same
one.)
Figure S1:
Scheme 1.
Using the change in TNS fluorescence that accompanies this
change in protein conformation, we were able to reproduce the sigmoidal
behavior for plasma M (Fig. 7C) and to
obtain a rate constant for the protein conformational change (9
10
s
) very similar to the
published value (10
10
s
)
(21) . The behavior of the variants
was different and simpler. No sigmoidal kinetics were observed.
Instead, the TNS-monitored conformational change was well described by
a single exponential, giving a pseudo first-order rate constant (1.9
10
s
) similar to that of
thiol ester cleavage (Fig. 7, A and B). Thus,
the protein conformational change in the two variants, which is a
different change from wild-type
M, did not depend on
cleavage of both thiol esters within the same disulfide-linked
half-molecule.
Figure 7:
Time course of protein conformational
changes resulting from reaction of 0.4 µM M species with 0.2 M methylamine at pH
8.0 followed by changes in TNS fluorescence. A, variant 1;
B, variant 2; C, plasma
M. The
solidlines represent the least-squares fits to a
pseudo first-order reaction for variants 1 and 2 and to a two-step
conformational change described in Scheme 1 for plasma
M (21). Using the sigmoidal fit for the variants gave
a very much poorer fit than using the single
exponential.
The ability of each of the two variants to protect
trypsin from inhibition by soybean trypsin inhibitor through
sequestration of the proteinase was determined by reaction of the
variant with trypsin, addition of excess soybean trypsin inhibitor, and
assaying for residual trypsin activity. Although plasma
M was capable of protecting proteinase in this assay,
neither of the variants showed any significant trypsin protection
ability after incubation for either 5 or 60 min with trypsin.
Proteolytic Reaction of Bait Region Variants
To
determine whether the M variants retained an exposed
flexible bait region that remained the preferred site of proteolytic
cleavage, we examined the reaction of each of the variants with
proteinase. Reaction with trypsin resulted in cleavage of both variants
as judged by the disappearance of the 180-kDa monomer band and the
appearance of cleavage products on SDS-PAGE (Fig. 8). Although
there was evidence for cleavage in the bait region, cleavages at
additional sites made interpretation of the degradation patterns
problematical. The rate of cleavage of the bait region of each variant
by trypsin was very much less than for plasma
M, and
the reaction was catalytic rather than stoichiometric. For variant 2,
bait region cleavage bands were the first formed, but prolonged
incubation resulted in the appearance of smaller fragments with the
consequent disappearance of the higher molecular mass bait region
cleavage bands (data not shown). For variant 1, bait region cleavage
occurred, but another major cleavage site resulted in only a small
reduction in size of the 180-kDa band and the appearance of a slightly
smaller intense band. These findings can be understood in terms of
cleavage within the bait region still being possible in both variants,
but at such a reduced rate that secondary cleavages outside of the bait
region also become important. As a result, further degradation of the
variants can occur, particularly with longer incubations. The reduced
rate of reaction of proteinase with the shortened bait regions must
reflect reduced accessibility or flexibility of the bait region peptide
as a result of removal of flanking residues on the N- and/or C-terminal
sides.
Figure 8:
Effect of incubation of plasma and variant
M with trypsin for 5 min as analyzed by SDS-PAGE
(7.5%). pA2M, BR1, and BR2 refer to plasma
M, variant 1, and variant 2, respectively. The
designations ``-'' and ``+'' signify the
absence or presence of trypsin. Trypsin treatment involved incubation
of 2.8 µM
M dimer with 3.0
µM trypsin at room temperature for 5 min. The positions of
the bait region cleavage bands (95-85 kDa) and intact monomer
bands (180 kDa) are indicated by arrows. Phenylmethanesulfonyl
fluoride to a concentration of 2 mM was added to terminate the
proteinase reactions.
To confirm that the bait region remained a major site of
proteolysis, N-terminal sequencing was carried out on the dominant
cleavage bands of variant 2 after reaction with
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (see Fig. 8for appearance of the gel). Since individual
bands were not resolved in this region, the diffuse stained region
around 90 kDa was cut into two parts (upper and lower) and separately
sequenced. The normal N-terminal sequence SVSGK was found to be a major
component of the lower and a minor component of the upper part of the
band. A new N terminus, with the sequence VGFYE and corresponding to
cleavage at the expected bait region trypsin cleavage site, was found
to be a major component of the upper and a minor component of the lower
part of the band. In both the upper and lower parts, another N-terminal
sequence, AFQPF, was also found. This corresponds to secondary cleavage
near the bait region at residue 765 (normal M
numbering) and may reflect greater accessibility of this region in the
absence of dimer-dimer interactions.
Proteinase-induced Conformational Change
We
examined the nature of the proteinase-induced conformational changes in
the two variants both by PAGE under nondenaturing conditions and by TNS
fluorescence changes. Trypsin reaction with both variants resulted in a
different change in electrophoretic mobility from that with both
wild-type tetrameric human M and dimeric Limulus
M. Whereas plasma and wild-type recombinant human
M as well as Limulus
M
showed the characteristic increase in mobility upon reaction with
proteinase (Fig. 2, lanesb, d, and
f), both variants showed a slight decrease in mobility
(lanesi and k).
M
variants upon reaction with proteinase compared with plasma
M. Thus, both variants produced a decrease in TNS
fluorescence intensity with little change in
upon
reaction with proteinase, whereas plasma
M gave a
large increase in intensity and a blue shift of
40 nm in
(data not shown).
Coupling between Bait Region Cleavage and Thiol Ester
Activation
In plasma M, bait region cleavage by
proteinase results in limited conformational change that alters the
environment of the thiol ester and makes it much more reactive to
nucleophilic attack, generating the so-called ``nascent
state''
(28) . Cleavage of both thiol esters within a
half-molecule is then the trigger for the large-scale conformational
change that results in proteinase trapping
(21) . There is thus a
necessary coupling of bait region cleavage and thiol ester activation
for the proteinase trapping mechanism to occur in plasma
M. To determine whether such coupling still occurred
in the dimeric
M variants, we assayed
proteinase-treated
M for free SH release. Even after 1
h of incubation with 1.1 eq of trypsin/half-molecule, no free SH groups
were detectable by continuous 5,5`-dithiobis(2-nitrobenzoic acid)
assay, in contrast to the behavior of plasma
M
(). That the thiol esters were still present and accessible
to nucleophile was demonstrated by the subsequent ability of
methylamine to release free SH groups (), at a rate
6
times faster than in non-proteinase-treated variants.
M can be removed without adversely
affecting the properties of
M by examining the
properties of two bait region-truncated human
M
variants to see whether these truncations affected the correct folding
of the protein and the ability of
M to function as a
proteinase inhibitor. The findings proved to be both more complex and
more interesting than anticipated due to unexpected effects of the bait
region truncation on tetramer formation. Thus, removal of 23 or 27 of
the
40 bait region residues apparently still resulted in correct
folding of the
M monomer and normal dimerization via
intermolecular disulfide formation, but prevented noncovalent
association of the disulfide-linked half-molecules to give tetrameric
M. The dimers were incapable of protecting trypsin
from soybean trypsin inhibitor by trapping or of significantly
activating the thiol esters toward nucleophilic attack and reacted much
more slowly than the wild type with proteinase. This inability to trap
proteinase may be due to one of several causes: (i) the uncoupling of
bait region cleavage from thiol ester activation; (ii) the failure to
form tetramer; or (iii) the interrelationship of both of these
processes, namely the requirement of parts of the bait region for
tetramer formation, which in turn is needed for the coupling of bait
region cleavage to thiol ester activation and of thiol ester cleavage
to proteinase-trapping conformational change. These unexpected findings
should provide new insights into the mechanism of activation of the
thiol ester by bait region proteolysis.
Correct Folding of Monomers
Correct folding of the
monomers was indicated by several properties of the variants. Thus, (i)
the thiol ester bond formed in each variant was stable in the absence
of nucleophile and reacted with methylamine at a rate comparable to
that of wild-type M to give close to the expected
number of free SH groups. Our understanding of thiol ester formation in
M to give functional slow form
M is
that it occurs after the correct folding of the
M
polypeptide to give a form resembling fast form
M in
which the cysteine and glutamine residues (positions 949 and 952,
respectively) are correctly positioned and in a suitable environment
for formation of a stable thiol ester by elimination of NH
(9, 29). Thus, both
M variants must provide
favorable folded structures for formation of the thiol ester linkage
similar to those in wild-type
M. However, it should be
noted that the effect of these variants on TNS fluorescence was more
like that of conformationally altered
M rather than
native
M, so that the overall conformation of
correctly folded domains may not be identical to that in wild-type
M. (ii) Wild-type
M is composed of
pairs of disulfide-linked dimers
(2) . Formation of
disulfide-linked dimer rather than higher molecular mass multimers must
require correct folding of the appropriate domains such that these two
cross-links can form. Both on native gel electrophoresis and SDS-PAGE
under nonreducing conditions, the two variants migrated as covalently
linked dimers, which dissociated into 180-kDa monomers upon reduction.
(iii) Other evidence for correct folding is the immunological
cross-reactivity of the variants with the polyclonal antibody used in
the radial immunodiffusion assay and the normal behavior of the
variants during the zinc chelate affinity chromatography step that is
used for isolation of tetrameric human plasma
M.
Involvement of the Bait Region in Tetramer
Formation
Although we had expected correct folding of the
monomers and intersubunit disulfide bonding to give dimers, we had not
anticipated that these dimers would fail to form tetramers by
noncovalent association. A common feature of the variants was that a
large part of the C-terminal region was missing in each case, including
the portion that does not contain principal proteolysis sites in the
wild-type inhibitor (residues 701-705). It thus appears that some
or all of the C-terminal portion may be involved in the noncovalent
association of M dimers to give tetramers. Perhaps
relevant to the role of the bait region in interdimer interactions is
that it is much harder to disrupt the noncovalent interactions in
proteinase-reacted
M than in native
M, suggesting a major alteration in the interdimer
contacts
(30, 31) . Although this could be another
consequence of the global conformational changes in
M
that occur upon bait region and thiol ester cleavage, it should be
noted that fluorescence studies have localized both the thiol ester and
the bait region to the central region of
M
(32, 33) , which is the region where
the noncovalent interdimer contacts appear to occur
(34) . This
localization of the thiol ester has more recently been confirmed by
electron microscopy studies
(35, 36) .
-macroglobulins. This appears to be the case. Thus, human
PZP, which has a much longer bait region than human
M
(1, 37) , is a predominantly
disulfide-linked dimer in the native state and has the sequence SSGPVP
immediately preceding the conserved ETXR sequence, whereas a
very different sequence, VEEPHT, is found in human
M
(Fig. 1). Upon reaction with proteinase, but not methylamine, the
equilibrium between dimer and tetramer shifts markedly toward
tetramer
(38) , indicating an alteration in the nature of the
noncovalent dimer-dimer interactions following bait region cleavage and
thus possibly involving the bait region directly. The bait regions of
the two rat
I
variants are even more
different from human
M than is PZP, having a large
number of prolines in the C-terminal region of the bait region (8 out
of 18 residues) (Fig. 1). These
I
proteins also differ in oligomerization state from human
M in being monomeric.
What Constitutes a Proteinase Trap?
Tetrameric
human M can inhibit up to 2 mol of proteinase.
However, it has still not been definitively shown whether two subunits
from the same or different covalent dimers form a proteinase-binding
site (Fig. 9). Analysis of the pattern of
proteinase-
M covalent cross-links favors a
side-to-side mode of noncovalent association of covalent dimers
(Fig. 9B), and electron microscopy studies of the
difference in density between methylamine- and proteinase-treated
M indicate that the upper and lower halves of the
M ``H'' represent proteinase-binding
sites
(39) . Thus, the schematic representation in
Fig. 9B most probably represents the mode of proteinase
inhibition in human
M. The simplest explanation for
the failure of either of the variants to trap proteinase is therefore
that a trap could only exist in the
M tetramer as a
result of the bringing together of the separate halves of the trap from
different covalent dimers.
Figure 9:
Schematic representation of two ways in
which the two disulfide-linked dimers of plasma M
might interact noncovalently to give tetrameric
M and
to form the two proteinase-binding sites of the tetramer. A,
formation of a proteinase-binding site by a disulfide-linked dimer;
B, formation of a proteinase-binding site by two monomers, one
from each of two noncovalently associated disulfide-linked dimers. In
A and B, one of the disulfide-linked dimers is shown
hatched. No attempt has been made to indicate the position of
the intersubunit disulfide bonds or the position of the bait regions.
However, we propose that the bait regions lie in the contact region
between a hatched and a nonhatched dimer. C and D represent the type of half-molecules that would
result from failure of the dimers to associate noncovalently for cases
A and B, respectively. Note how proteinase
sequestration (i.e. trapping) is only possible in C.
The circles labeled P represent trapped proteinase
that is not free to dissociate from complex with
M.
Although functional dimers exist in other
species, e.g.Limulus, there is no requirement that
these dimers be equivalent to the disulfide-linked dimer within the
human M tetramer. Rather, the Limulus dimer
may resemble the two noncovalently associated monomeric units of the
M tetramer that we propose form a trap, with the
difference that in Limulus these are the subunits that are
covalently linked. In keeping with a structural difference between
Limulus covalent dimers and human
M covalent
dimers (represented by the present bait region variants) is that the
Limulus dimer undergoes an increase in electrophoretic
mobility upon reaction with proteinase (analogous to tetrameric human
M), whereas the
M variant dimers
showed a small decrease in electrophoretic mobility (Fig. 2). In
addition, the dimeric
M variants behave quite
differently toward TNS binding than would be expected from them simply
acting as one-half of an
M tetramer.
M. However, it is not the rate of reaction that is
important, but the rate of conformational change following bait region cleavage that determines the efficiency of proteinase
sequestration. Thus, rapid reaction followed by a very slow trapping
conformational change could result in less trapping than a slow
proteolysis followed by a rapid conformational change.
Uncoupling of Bait Region Cleavage and Thiol Ester
Activation and Hydrolysis
An alternative explanation for the
failure of the variants to trap proteinase is that bait region cleavage
did not result in significant activation of the thiol ester toward
nucleophilic attack. Thus, no free SH groups were generated after
reaction of the variants with trypsin, although there was a small
increase in the rate of methylamine cleavage of the thiol esters in
trypsin-treated M variants (
6-fold faster than in
the native proteins). This, however, is a very much smaller enhancement
in reactivity than is normally found in plasma or wild-type recombinant
M, where cleavage of the thiol ester is 90% complete
within 15 s after reaction with proteinase
(40) . The absence of
the appropriate conformational change after proteolytic cleavage is,
however, more likely to be due to the absence of cooperative
interactions between noncovalently associated subunits than to the
uncoupling of thiol ester activation and bait region cleavage. Thus,
direct cleavage of the thiol esters with methylamine, although
proceeding at a normal rate, failed to bring about the normal
conformational change and did not show the cooperativity that is found
in tetrameric
M (Fig. 7). It is therefore more
likely that the uncoupling of thiol ester activation from bait region
cleavage and the absence of cooperative thiol ester cleavage-dependent
protein conformational change in the variants result from the same
cause, namely the absence of noncovalent dimer-dimer interactions.
M that had been cross-linked
between dimeric subunits by cis-platinum. Reaction of this
cross-linked species with trypsin gave bait region cleavage without
thiol ester activation or conformational change. Consequently, no
trapping of proteinase occurred until the cross-links were removed
(41). Reaction with methylamine did, however, result in a
conformational change since the dimer-dimer interface was
present
(42) . There may thus be a parallel between such
cross-linked
M species and the dimeric
M variants of the present study in that particular
dimer-dimer interactions may be needed both for thiol ester activation
and for the conformational change that follows thiol ester cleavage. In
the cross-linked tetramer, the bait region-thiol ester linkage may be
disrupted by covalently locking the dimer-dimer interface, whereas in
the dimeric variants, the interactions are not achievable because
tetramers do not form. Thus, the need for tetramer formation may be
2-fold: first, to physically constitute a potential trap by bringing
together the two halves of the trap located in different
disulfide-linked half-molecules; and second, to provide the changes in
dimer-dimer interactions consequent to bait region cleavage that are
necessary for thiol ester activation and for linking thiol ester
cleavage in both halves of the trap to conformational change. Either of
these on its own is, however, sufficient to account for the failure of
the present variants to trap proteinase.
Choice of Bait Region Variants
Our choice of bait
region variants was originally guided by the expectation that the whole
of the bait region outside of the conserved regions is a discrete
domain that is not involved in interdimer interactions and that major
deletions could be made that would only affect proteinase
specificity. The variants that we constructed were thus appropriate for
testing bait region requirements for accessibility to, and specificity
for, proteinase, but not appropriate for delineating the function of
the bait region in maintaining the tetrameric, and therefore
functional, state of the inhibitor. Consequently, we plan to make
further bait region variants in which (i) selected portions of the
C-terminal region of the bait region will be either added back to the
present variants or deleted from the wild type and (ii) the remainder
of the deleted N-terminal portion of the bait region will be restored.
This will allow us to determine whether there are variants that remain
dimers but are more rapidly cleaved in the bait region compared with
the present variants and also to determine which residues are needed
for tetramer formation.
Table:
Thiol ester cleavage of recombinant
M as a result of treatment with methylamine, trypsin,
or trypsin followed by methylamine
M,
-macroglobulin(s); PAGE, polyacrylamide gel
electrophoresis; TNS, 6-(p-toluidino)-2-naphthalenesulfonic
acid; PZP, pregnancy zone protein.
M expression plasmid p1167, and Dr.
Steven T. Olson for critical comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.