From the Lerner Research Institute, The Cleveland Clinic
Foundation, Cleveland, Ohio 44195, Institute of
Experimental Cardiology, Russian Cardiology Research Center, Moscow
121552, Russia, ¶ Department of Biochemistry, University of
Washington, Seattle, Washington 98195,
ZymoGenetics Inc.,
Seattle, Washington 98102, ** Behring Diagnostics GmbH, Marburg, Germany
D-35001, and
Department of
Medicine, East Carolina University School of Medicine,
Greenville, North Carolina 27853
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ABSTRACT |
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Coagulation factor XIIIa, plasma
transglutaminase (endo--glutamine:
-lysine transferase EC
2.3.2.13) catalyzes isopeptide bond formation between glutamine and
lysine residues and rapidly cross-links fibrin clots. A monoclonal
antibody (5A2) directed to a fibrinogen A
-chain segment 529-539 was
previously observed from analysis of end-stage plasma clots to block
fibrin
-chain cross-linking. This prompted the study of its effect
on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting
XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine
donor substrate dansylcadaverine into the glutamine acceptor
dimethylcasein in an uncompetitive manner with respect to
dimethylcasein utilization and competitively with respect to
dansylcadaverine. Uncompetitive inhibition was also observed with the
synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive
inhibition arises from preferential interaction of the inhibitor with
the enzyme-substrate complex but is also found to inhibit
-chain
cross-linking. The conjunction of the uncompetitive and competitive
modes of inhibition indicates in theory that this bireactant system
involves an ordered reaction in which docking of the glutamine
substrate precedes the amine exchange. The presence of substrate
enhanced binding of 5A2 to XIIIa, an interaction deemed to occur
through a C-terminal segment of the XIIIa A-chain (643-658,
GSDMTVTVQFTNPLKE), 55% of which
comprises sequences occurring in the fibrinogen epitope A
-(529-540)
(GSESGIFTNTKE). Removal of the
C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on
activity. Crystallographic studies on recombinant XIIIa place the
segment 643-658 in the region of the groove through which glutamine
substrates access the active site and have predicted that for
catalysis, a conformational change may accompany glutamine-substrate
binding. The uncompetitive inhibition and the
substrate-dependent binding of 5A2 provide evidence for the
conformational change.
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INTRODUCTION |
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Factor XIII (fXIII)1 is
a member of the class of enzymes known as transglutaminases, which
catalyze displacement of amide ammonia at the -position in glutamine
residues by replacing it with another amine, usually an
-amino group
from a suitable lysine residue (1-4). Formation of
-(
-glutamyl)lysine isopeptide bonds functions in both intra-
and intermolecular cross-linking of proteins. Among the many functions
of fXIII, the best known are 1) the cross-linking of fibrin, which
stabilizes clots against redissolution by fibrinogen (5) and plasmin
(6), 2) the linking of
2-plasmin inhibitor to fibrin for
further protection from plasmin (7, 8), and 3) the cross-linking of
matrix proteins, among them, fibronectin, collagen, and thrombospondin,
which facilitate wound healing (9). Platelet and placental fXIII are
homodimers composed of two identical A chains, but plasma fXIII is a
heterotetramer composed of two A and two B chains (10). Plasma fXIII is
activated by two steps: 1) thrombin cleavage of a 4 kDa fragment from
the N terminus of the A-chains and 2) Ca-dependent
dissociation of the two B chains from the tetramer, yielding
functionally active A-chain dimers (XIIIa) (11, 12). FXIII was
discovered in 1948 (13), and since that time has been extensively
studied. Its primary structure and its three-dimensional structure
including its active site are all known (14-17). But still unknown are
the determinants of substrate docking and specificity (18). Many of the
domains are highly conserved and do not yield antibodies to probe their function. In this study, we describe multiple types of inhibition of
XIIIa produced with a cross-reacting monoclonal antibody (5A2) directed
to fibrinogen A
-chains in the region of residues 529-539. The modes
of inhibition change from uncompetitive to competitive depending on
which substrate (amine donor or glutamine acceptor) is examined and
these changes give insight into the order of substrate usage. Enhanced
binding of 5A2 to XIIIa in the presence of substrate is viewed as
evidence that substrate binding induces conformational changes in
XIIIa. These observations support earlier inferences on the catalytic
mechanism gained from crystallographic studies on recombinant fXIII
A-chains (rXIIIA).
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MATERIALS AND METHODS |
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Proteins and Reagents-- Initial studies on inhibitory effects of 5A2 were carried out directly in plasma. Kinetic studies used ZymoGenetics Inc. (Seattle, WA) (rXIIIA) (19). Synthetic substrates for XIIIa were used as supplied in Berichrom® XIIIa assay kits except when supplemented with the glutamine substrate LGPGQSKVIG from Behring Werke (Marburg, Germany). Bovine N,N-dimethylated casein, dansylcadaverine, phenylmethylsulfonyl fluoride, trypsin, dithiothreitol, Aprotinin®, and guinea pig liver tissue transglutaminase were obtained from Sigma. Bovine thrombin was from U. S. Biochemical Corp. Hirudin was from Pentapharm AG (Basel). Phenylprolylarginine chloromethyl ketone was from Chemica Alta Ltd (Edmonton, Alberta, Canada). Antifibrinogen monoclonal antibodies (mAb) 5A2 and 7F8 were made as described previously (20, 21), as was the anti-FPA (22). Anti-angiotensin-converting enzyme mAb 9B9 (23) was generously provided by Dr. S. M. Danilov (Russian Cardiology Research Center, Moscow). Rabbit anti-XIIIa antibodies were generated using rXIIIA as antigen and purified by elution (0.35 M citrate, pH 3.5) from protein A-Sepharose (Amersham Pharmacia Biotech). Except where indicated, all protein solutions were in Tris-buffered saline containing 0.1 mM EDTA (TBSE) at pH 7.4.
Human fibrinogen was purified from the donor plasma by sequential precipitations with ethanol, glycine, and ammonium sulfate/Effect of 5A2 on Cross-linking of Fibrinogen-- These studies simply involved adding calcium (5 mM) and dithiothreitol (2.5 mM) in the presence and absence of added 5A2 (4 mg/ml) to the fibrinogen (2 mg/ml) preparation before removing the contaminating XIIIa from the fibrinogen. The observed cross-linking was verified as due to the presence of active XIIIa in the fibrinogen preparation, because it occurred without thrombin activation and in presence of hirudin, it did not change during a parallel incubation (6 h, 22 °C) with 8 mg/ml rXIIIA added, and it did not occur at all after passing the fibrinogen through the anti-fXIII affinity gel.
Cross-linked products were separated by SDS-polyacrylamide gel electrophoresis of thiol-reduced specimens and identified after Coomassie staining by comparison with previous electropherograms identifying the polypeptide chain compositions of the products (26).Activation of rXIIIA-- To preactivate the rXIIIA (2 mg/ml), it was incubated (15 min, 37 °C) with thrombin (0.5 units/ml) at pH 7.4 in the presence of CaCl2 (2 mM) and a trace amount of fibrinogen (2 mg/ml). The activation was then terminated, and thrombin was inactivated with hirudin (10 units/ml). Activation of rXIIIA that was precoated on microtiter wells was performed with 0.07 units of thrombin with 5 mM calcium for 30 min at 37 °C, and the thrombin was subsequently inactivated with 0.3 units of hirudin before washing and blocking for enzyme-linked immunosorbent assay measurements.
Assays of XIIIa Activities Using Dimethylcasein As the Glutamine
Substrate and Dansylcadaverine As the Amine Substrate--
These
assays followed continuous changes in fluorescence arising from the
incorporation of dansylcadaverine into casein by the method of Lorand
et al. (27). The fluorescence was measured in 96-well
microplates using a Perkin-Elmer luminescence spectrometer LS50, with
excitation elicited at ex = 360 nm and emission followed at
em = 510 nm. The buffer consisted of 0.1 M sodium borate containing 10 mM
CaCl2 and 5 mM dithiothreitol at pH 9.0. The
concentrations of preactivated XIIIa in most of the experiments was
0.03 mg/ml. Dimethylcasein concentrations were varied from 0.06 to 0.38 mg/ml in the presence of a saturating concentration of dansylcadaverine (200 mM), and dansylcadaverine concentrations were varied
1-5 mM in the presence of a saturating concentration of
dimethylcasein (5 mg/ml). Reaction rates were assessed during initial
stages when fluorescence varied linearly with time.
Measurements of XIIIa Activity Using Berichrom® fXIII Synthetic
Substrates--
This assay uses a synthetic decapeptide (LGPGQSKVIG)
as glutamine substrate and glycine ethyl ester as the amine substrate and measures transglutaminase activity from the rate of ammonia release
from the peptide using an auxiliary enzyme system (28). The ammonia
release is followed colorimetrically from consumption of NADH as the
ammonia is driven into reaction with -ketoglutarate by glutamate
dehydrogenase, all of these components apart from NADH being packaged
in the "reporting" solution of the Berichrom® fXIII kit. The
assays were carried out according to the recommended procedure of the
supplier except for two modifications made to characterize the kinetics
of 5A2 inhibition in relation to LGPGQSKVIG concentrations. We
substituted rXIIIA/5A2 mixtures for the plasma specified for measuring
plasma fXIII in the kit. Secondly, since the reporting solution
contained a saturating level of LGPGQSKVIG, we diluted it in half and
varied the concentration of LGPGQSKVIG by supplementing the diluted
solution with added peptide. As determined with the peptide
reconstituted to its original level, apparent reaction velocities, both
in the presence and absence of 5A2 reported with the diluted solutions,
were consistently one-half those reported with the undiluted solution
due to the lower concentration of reporting enzyme (glutamate
dehydrogenase) in the diluted system.
Preparation of C-terminal-truncated 51-kDa Fragment of rXIIIA-- This tryptic derivative of rXIIIA was prepared essentially as described by Greenberg et al. (29). The digest contained rXIIIA (2 mg/ml), trypsin (1 mg/ml), and CaCl2 (10 mM) at 37 °C. SDS-polyacrylamide gel electrophoresis showed that full conversion of rXIIIA to the 51-kDa fragment required 2.5 h of incubation under these conditions. The digestion was terminated by adding Aprotinin® (0.14 mg/ml). The electropherograms showed that none of the 80-kDa parent rXIIIA remained unconverted. A shorter incubation of 1.5 h left 75% converted. The 51-kDa fragment precipitated out of solution during the incubation, was centrifuged, and resuspended in TBSE, and the suspension was divided into 0.1-ml aliquots, which were stored frozen. Just before assay, the suspension was thawed, and admixed with an equal volume of 6 M urea (3 M final) to dissolve the protein immediately before measuring its activity.
Enzyme-linked Immunosorbent Assay for Binding-- Nunc immuno plates (Vanguard Intl., Neptune, NJ) were coated with rXIIIA in TBS (10 µg/ml, 100 µl/well, 18 h, 4 °C), washed with TBS, and blocked with 3% bovine serum albumin in TBS containing 0.1% Tween-20 (TBSTw). Primary antibodies, either 5A2 or control anti-FPA mAbs (100 µg/ml, 100 µl/well), were added in 0.1% bovine serum albumin in TBSTw supplemented with either Behrichrom substrate or blank (10 µl/well). After 18 h of equilibration at 4 °C followed by a TBSTw wash, the retained 1° antibody was fixed in place with cold 0.25% glutaraldehyde in phosphate-buffered saline (100 µl/well, 4 °C, 10 min), and the plates were washed (TBSTw) and quenched 0.1% bovine serum albumin in TBSTw (200 µl/well, 37 °C, 10 min). After further washing, secondary reporter antibody (horseradish peroxidase-goat-anti-mouse Ig at 1:1000 dilution, 100 µl/well) was added, and the excess was washed away after 2 h at 23 °C. Retained 2° antibody was assessed from peroxidase activity toward orthophenylenediamine.
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RESULTS |
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Inhibitory Effects of mAb 5A2 on Fibrinogen and
-Chain
Cross-linking--
In the course of studies on the effect of 5A2 on
the cross-linking of fibrinogen we observed (Fig.
1) that
-dimer formation was
inhibited to a degree comparable to the subsequent (26) incorporation
of fibrinogen
-chains2into
hybrid
-trimers. The inhibition of
-chain cross-linking was
not detected in our preceding study, which characterized only the
effect of 5A2 on end-stage plasma clots (20). The inhibition of
-chain cross-linking together with searches indicating that there
was no homology between the 5A2 epitope on fibrinogen (20) and the
amino acid sequences in the fibrinogen
-chains, prompted us to
investigate whether the inhibitory effect of 5A2 was arising from a
direct interaction with XIIIa. We then proceeded to examine the effects
of 5A2 on substrates other than fibrinogen.
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Kinetics of Dansylcadaverine Incorporation into
Dimethylcasein--
This reaction was inhibited by 5A2 in a
concentration-dependent manner and was investigated further
in relation to the utilization of the dimethylcasein (Fig.
2) and the dansylcadaverine (Fig. 3) at varying concentrations. When
dimethylcasein concentrations were varied, we obtained parallel,
nonintersecting Lineweaver-Burk plots of 1/ versus
1/[dimethlycasein] at 5A2 concentrations ranging to 0.6 mg/ml (Fig.
2). A replot of the 1/
intercepts versus the 5A2
concentrations was linear, with a negative intercept at 5A2 concentrations corresponding to a Ki of 0.8 mM (Fig. 2, inset). The parallel Lineweaver-Burk
plots and linear replot conformed with a pattern of inhibition termed
"uncompetitive," where the inhibitor is directed principally toward
the enzyme-substrate complex (30), the glutamine substrate in this
instance. These effects were specifically caused by 5A2 because no
inhibition was observed with 7F8 and were also specifically directed to
XIIIa because no inhibitory effects of 5A2 were observed toward
transamination of these substrates by tissue transglutaminase (negative
results not shown).
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Transamination of Berichrom® Decapeptide LGPGQSKVIG with Glycine
Ethyl Ester--
As illustrated (Fig.
4), 5A2 exhibited a
concentration-dependent inhibition of XIIIa-catalyzed
release of ammonia from the decapeptide in a pattern consistent with
the uncompetitive mode of inhibition observed with the other
nonfibrinogen-related glutamine substrate, dimethylcasein. The 1/
intercepts (corresponding to values for 1/Vmax)
at 1/S = 0 on the Lineweaver-Burk plots varied linearly
with 5A2 concentrations (Fig. 4, inset). Extrapolating this
replot to the base-line 1/Vmax = 0 yielded an
estimate of 0.9 µM for the value of
Ki, which could be off by a factor of two (95%
confidence interval) because of the length of the extrapolation.
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Homology Searches--
The inhibitory effects of 5A2 on
utilization of nonfibrinogen substrates and the absence of effect on
tissue transglutaminase suggested that 5A2 was interacting with a
segment of XIIIa with a sequence similar to that of the fibrinogen
epitope. The fibrinogen epitope was identified from CNBr and tryptic
fragments to be located in the region of A 529-539, the only
fragment from the combined digests that substantially inhibited (18%)
binding of 5A2 to fibrinogen A
-chains (20). Searches for
similarities between the sequence of this peptide and the amino acid
sequences (16, 31) in XIIIA were made using both the PROPHET (Bolt
Beranek and Newman, Inc., Cambridge MA) and the BLAST network service
(NCBI). Both searches found a segment near the C terminus of the
A
-chain 643-658 of XIIIA that consists in part (55%) of sequences
occurring in the region of the fibrinogen A
-chain epitope, and no
other regions were found (>3% homology) in either XIIIA, fibrinogen,
or casein. XIIIA:
643GSDMTVTVQFTNPLKE;
Fgn A
:
529GS
GSGIFTN-TKE540.
No Effect on an Enzymically Truncated XIIIA-- To test the importance of the C-terminal domain for the inhibitory effects of 5A2, we examined its effect on cross-linking of a tryptic fragment of 5A2 lacking the putative cross-reacting domain. Greenberg et al. (29) observed that a tryptic fragment of XIIIA comprising residues Gly38-Lys513 retained 20% of its initial activity. On trypsinizing rXIIIA we obtained a pure 51-kDa fragment that precipitated during the digestion. The fragment exhibited transglutaminase activity in the Berichrom® fXIII assay when it was admixed (0.015 mg/ml) with the substrates immediately after dissolving it in 3 M urea. Unlike intact XIIIa, activity of the fragment was unaffected by 5A2 (Fig. 5). This result supported the hypothesis that 5A2 was inhibiting intact XIIIa by interacting with the segment in the XIIIa C-terminal domain, residues 643-658 with sequence similar to the 5A2 epitope.
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Binding of 5A2 with rXIIIA-- Attempts to detect binding of 5A2 to plated rXIIIA by enzyme-linked immunosorbent assay methods failed until we resorted to fixing the initially bound 5A2 with glutaraldehyde to prevent dissociative losses during exposure (30 min) to secondary, reporter antibody. This approach detected binding of 5A2 to the rXIIIA zymogen substantially above that of a control mAb, anti-FPA (Fig. 6). Activating the rXIIIA with thrombin promoted binding of both the 5A2 and anti-FPA mAbs (5A2 more so), whether the activation was performed before plating the rXIIIA (columns labeled XIIIa) or after plating (columns labeled XIIIA*). We suspect that the enhanced binding of the anti-FPA was due to an effect of thrombin, because plating thrombin caused some retention of the anti-FPA not observed with 5A2. Unlike the binding of anti-FPA, the 5A2 binding became substantially enhanced further when the synthetic substrate from the Berichrom® fXIII kit was added to the medium during incubation. The added substrate also caused a slight increase in binding to the zymogen, which could conceivably be due to the normal development of low levels of activity by zymogen without thrombin treatment. Essentially the same enhancing effects of added substrate were reproducibly obtained in repeated experiments. The enhanced binding of 5A2 after activation with thrombin and the further enhancement after adding substrate were viewed as indications that substrate binding was inducing a conformational change leading to greater exposure of the epitope for 5A2 binding.
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DISCUSSION |
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The C-terminal domain of XIIIA had been shown by Greenberg
et al. (29) to have some influence on the transglutaminase
activity of the enzyme, as judged by the substantial (80%) loss of
activity after removing the domain with trypsin. Attempts to probe its function immunochemically have proven difficult, because it is poorly
immunogenic (29). As described here, we serendipitously found that an
antibody (5A2) directed to a C-terminal domain of the fibrinogen
A-chain (529-539) inhibits XIIIa in a manner dependent on the
C-terminal domain of the enzyme. This inhibition is deemed to involve a
cross-reactivity with residues 643-658 in the XIIIA chain, which share
homology with the fibrinogen epitope, and further appears to be
partially dependent on interaction with XIIIa complexed with substrate.
The modes of inhibition have important implications for the structure
and activity of XIIIa.
The crystallographic structure of XIIIA was recently elucidated (17,
32, 33). As illustrated (Fig. 7), the
cross-reacting segment 643-658 forms a strand in the outer sheet
of the C terminus of the barrel 2 domain of XIIIA. The lysine 657 residue is located at the gap between the barrel 1 and barrel 2 domains, which has been identified as one of two possible loci through
which glutamine substrates gain access to the catalytic site of the
enzyme (32). The inhibition of utilization of glutamine substrates
(both the dimethylcasein and the synthetic peptide LGPGQSKVIG) was
uncompetitive, an indication (30) that the principal mode of that
inhibition was directed to the enzyme-glutamine complex. Any other mode
of enzyme-inhibitor interaction is incidental. In essence, the
glutamine binding acts as an activator of the catalytic conformational
change that enables utilization of the amine substrate. Utilization of the amine substrate was inhibited competitively, probably not because
5A2 acts as an analog of the amine substrate, but, more likely, because
the amine substrate cannot bind to the ternary glutamine
donor·XIIIa·5A2 complex. The independent observation of increased
5A2 binding to XIIIa in the presence of glutamine substrate is
consistent with that explanation. Thus, these two modes of inhibition
indicate that the transamination reaction is an ordered event dependent
on docking of the glutamine substrate to trigger reaction with
subsequent binding of the amine substrate.
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Binding of 5A2 to XIIIa was enhanced by co-incubation with the Berichrome® fXIII substrate system, an indication that substrate binding enhances exposure of the 5A2 epitope on XIIIa. It had been predicted that a conformational change accompanying the glutamine binding functions in the catalytic activity of the enzyme (32). Crystal structures of rXIIIA both before and after activation with either thrombin or high concentrations of calcium (34, 35) led to the inference that the conformational change that must accompany catalysis arises from the docking of the glutamine substrate. Our findings provide evidence for a substrate-induced conformational change and primacy of glutamine binding in the catalytic reaction.
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FOOTNOTES |
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* This work was supported in part by NHLBI, National Institutes of Health, Grants HL-16361 and HL-50355 and by the Russian Fund for Fundamental Investigation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Research Institute FF4, The Cleveland Clinic Foundation, Cleveland, OH 44195. Tel.: 216-444-5851; Fax: 216-445-5480; E-mail: shainoj{at}cesmtp.ccf.org.
1
The abbreviations used are: fXIII, factor XIII;
XIIIa, the enzymically active form of blood coagulation fXIII; XIIIA,
A-chains of fXIII; rXIIIA, recombinant fXIII A-chains; 5A2,
anti-fibrin(ogen) 529-539; mAb, monoclonal antibody; FPA,
fibrinopeptide A (fibrinogen A
1-16); TBSE, Tris-buffered saline
with EDTA; TBSTw, Tris-buffered saline with Tween.
2
Fibrinogen -chains (64 kDa) differ from
fibrin
-chains by containing the N-terminal fibrinopeptide A (1.5 kDa) that blocks the fibrin aggregation site located just penultimate
to the fibrinopeptide. The fibrinogen
-chains are normally
designated as A
-chains except when qualified as fibrinogen rather
fibrin chains. We defer the A
convention here to simplify the
multimeric structure of the cross-linked chains as
trimers
rather than A
trimers.
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REFERENCES |
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