(Received for publication, August 19, 1994; and in revised form, June 19, 1995)
From the
A library of monoclonal antibodies to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate
decarboxylating (EC 1.2.4.1; E1) subunit is reported. 21 monoclonal
antibodies were generated, and 20 were investigated, of which 9 were
elicited to PDHc and 11 to pure E1 subunit; 19 were of the IgG1 isotype
and one of the IgG3 isotype. According to an enzyme immunoassay, all 20
of the monoclonal antibodies bound the PDHc, and 17 bound the E1
subunit. According to Western blot analysis, 14 of the 19 monoclonal
antibodies bound to the E1 subunit. The monoclonal antibodies inhibited
PDHc from 0 to >98%. The six monoclonal antibodies that displayed
greater than 30% inhibition of E. coli PDHc were unable to
inhibit porcine heart PDHc nor did they bind porcine heart PDHc
according to dot blot analysis. Radiolabeling gave binding constants
ranging from 5 to 10 10
M
on these six monoclonal antibodies, with greater than 80% of
maximal inhibition achieved in less than 1 min. One of the six, 18A9,
gave >98% inhibition, required two antibodies/E1 subunit for maximum
inhibition, and was shown to be a non-competitive inhibitor. Monoclonal
antibody 15A9 was shown to counteract GTP-induced inhibition, while 1F2
influenced the conformation of E1, allowing two antibodies, which did
not previously bind E1, to bind to it. A new mechanism-based kinetic
assay is presented that is specific for the E1 component of 2-keto acid
dehydrogenases. This assay confirmed that the three most strongly
inhibitory monoclonal antibodies specifically inhibited the E1 function
while antibody 1F2 led to enhanced activity, suggesting an induced
conformational change in PDHc or in E1.
Pyruvate dehydrogenase (PDHc) ()is a multienzyme
complex that is found in both prokaryotic and eukaryotic cells. The
enzyme complex catalyzes the oxidative decarboxylation of pyruvate in
the following overall reaction ((1) )
In Escherichia coli, three different enzyme components are involved in the above reaction: pyruvate dehydrogenase, utilizing thiamin diphosphate (ThDP) as a cofactor (EC 1.2.4.1; E1); dihydrolipoamide transacetylase, which contains covalently linked lipoic acid residues (EC 2.3.1.12; E2); and dihydrolipoamide dehydrogenase, containing tightly bound FAD (EC 1.8.1.4; E3). The multienzyme complex performs the following series of reactions ((2, 3, 4) )
The complex consists of multiple copies of each subunit: 24 E1,
molecular weight 99,474 ((5) ); 24 E2, molecular weight 65,959 ((6) ); and 12 of E3, molecular weight 50,554 ((7) )
for a total calculated molecular weight of 4.57 10
daltons.
Metabolic inhibitors of PDHc include nicotinamide
adenine dinucleotide (NADH), acetyl-CoA, and guanosine triphosphate
(GTP)(8, 9, 10, 11) . Mammalian PDHc
is regulated in a more complex fashion by phosphorylation and
dephosphorylation of the subunit of E1(4) . Other studies
were directed at the investigation of various substrate analogues and
their ability to inhibit PDHc. Small molecule inhibitors of PDHc
include bromopyruvate(12) ,
fluoropyruvate(13, 14) , phosphonate analogues of
pyruvate(15, 16) , mono- and bifunctional
arsenoxides(17, 18, 19) , branched chain keto
acids(20) , and tetrahydrothiamin pyrophoshate (21) .
Within the last decade, there has been significant interest in the role of PDHc in various clinical disorders. In some of these studies, there were reported polyclonal rabbit antibodies being made against various species of PDHc and its subunits. Two studies reported that these rabbit antibodies have the ability to inhibit various species of PDHc activity to some extent(22, 23) . Although immunochemical inhibition of PDHc was not the major emphasis of these reports, they do constitute the first reported immununochemical inhibition of PDHc. Interest in another clinical disorder, primary biliary cirrhosis, has resulted in monoclonal antibodies being generated to the human E2 component of PDHc (24, 25) . These antibodies have been reported to inhibit the PDHc but are directed specifically at the E2 subunit.
Reported here is a description of a library of monoclonal antibodies (MAb) recently generated to aerobic E. coli PDHc and its E1 subunit. 21 MAbs were cloned to 100% homogeneity. 20 of the 21 MAbs were studied for their ability to bind and inhibit PDHc. In addition, further biochemical characterization was done on six that displayed high levels of inhibition. A new chromophoric assay has been developed for measuring E1 activity in the PDHc, and its application provides complementary support for the conclusions. These findings 1) represent the first reported MAbs to aerobic K-12 E. coli pyruvate dehydrogenase and its E1 subunit, 2) represent the first MAbs to bind and inhibit the E1 subunit, and 3) provide relevant information for future studies that will continue to define the E1 active center and the thiamin diphosphate binding site, since no high resolution x-ray studies have yet been reported for this enzyme.
Figure 1:
Best
fit linearity for the E1 assay. , 2 mg/ml PDHc;
, 1 mg/ml
PDHc;
, 0.5 mg/ml PDHc;
, 0.25 mg/ml PDHc;
, 0.125
mg/ml PDHc; +, BSA. This assay was run at 20 °C with a 20-s
lag time. Other components in the assay were 5.0 mM pyruvate,
12 mM MgCl
, 0.1 mM ThDP, 1.25 mM 4,4`-dithiodipyridine, and 1.25 mM 4-mercaptopyridine in
50 mM Tris, pH 7.7. See ``Experimental Procedures''
for details.
Figure SI: Scheme IMechanism of new E1 assay.
Two separate fusions, one using a mouse injected with the PDHc antigen and the other with the E1 antigen, were performed. Out of these two fusions, 21 MAbs were selected and cloned to homogeneity. 11 antibody clones resulted from the E1 fusion and were assigned an E1 suffix. The remaining 10 resulted from the PDHc fusion and were assigned the suffix PDHc. Isotyping studies done on tissue culture supernatants showed 20 of the monoclonal antibodies to be IgG1 and 1 to be an IgG3.
Experiments were conducted to determine the maximum
inhibition produced by each MAb. All 21 ascites fluids were first
titered in a microtiter plate with PDHc bound to the wells, and titers
were expressed as one-half of the antibodies' maximum binding
capacity. Each ascites had to have a titer of at least 2.5
10
to be used (one MAb, 12A9PDHc, failed to meet this
criterion and was rejected). The remaining 20 ascites fluids were
diluted, and the inhibition was determined as described under
``Experimental Procedures.'' Although it is known that the
concentration of MAbs in ascites fluid can vary from one ascites to the
next, the inhibition and radiolabel data were obtained under saturating
conditions, as demonstrated by the stability of the results subsequent
to more than one dilution. In addition, six of the MAbs (18A9, 21C3,
15A9, 1F2, 7C9, and 13A8) that exhibited greater than 30% inhibition
were purified, and the maximum inhibition data summarized in Table 1were verified by using greater than 700-fold molar excess
of MAb to enzyme. To ensure that the inhibition was specific, ascites
fluid from a non-relevant MAb was titered and demonstrated to give no
inhibition. The extent of inhibition of PDHc by the remaining 20 MAbs
is in the range from 0 to 99% (Table 1). Those monoclonal
antibodies, which gave greater than 60% inhibition, were all generated
from the PDHc immunogen, whereas those generated from the E1 antigen
gave a maximum of 50% inhibition.
After all of the characterizations of the MAbs summarized in Table 1were completed, 19 of the 20 monoclonal antibody ascites fluids remained. The remaining 19 antibodies were further evaluated by Western blot analysis. 14 of these MAbs were observed to bind the E1 subunit of the PDHc antigen of E. coli (data not shown). However, five of the MAbs (23H4, 8G10, 7C9, 8C9, and 13A8) did not show binding in Western blot analysis with PDHc or E1 from E. coli. Those five MAbs were reblotted at a higher concentration of ascites fluid and still did not demonstrate binding to E. coli PDHc or E1.
Six MAbs (18A9, 21C3, 15A9, 1F2, 7C9, and 13A8) that exhibited greater than 30% inhibition were also tested against porcine heart PDHc purchased from Sigma (specific activity, 0.45 IU/mg). Because of the low specific activity, 100 IU/liter of enzyme activity for both E. coli and porcine heart PDHc was used with 100 µg/ml of purified MAb. The data show no cross-species inhibition with porcine heart PDHc.
Additional studies were conducted to determine if these six MAbs (18A9, 21C3, 15A9, 1F2, 7C9, and 13A8) bound the mammalian PDHc antigen. Dot blot analysis was performed as described under ``Experimental Procedures.'' The control antigen, E. coli PDHc, was matched to the mammalian total IU of enzyme added. The qualitative results illustrated that these six MAbs show binding to as low as 29 ng of E. coli PDHc but show no binding to mammalian PDHc when accounting for nonspecific binding signals.
These six MAbs were also chosen for further study, three because they exhibited the greatest inhibition (18A9, 21C3, 15A9) and three because they were generated from an E1 immunogen and displayed relatively high inhibition (7C9, 1F2, 13A8). All six MAbs were purified to homogeneity by HPLC protein A and were shown to be distinct and separate entities by their different migration patterns (data not shown).
The binding constants of these six MAbs to the PDHc were determined (39) by a competitive radioimmunoassay (Table 2). The binding constants for these MAbs are similar, but MAb 18A9, which exhibits greatest inhibition, also exhibited the strongest binding constant.
The time course for attaining maximum inhibition by the six MAbs was also determined as described earlier. Greater than 85% of the maximum inhibition was achieved after 1 min, and by 10 min, maximum inhibition was essentially achieved (data not shown).
The stoichiometry of the interaction of MAb 18A9 with PDHc
was also determined. Antibody concentrations ranging from 6.25
10
M to 6.25
10
M were added to 1000 IU/liter of enzyme, the mixtures
were incubated for 1 h, and then the activity was assayed and a
titration curve was drawn. Fig. 2A shows the titration
curve for inhibition of the PDHc by MAb 18A9, and Fig. 2B illustrates the determination of the stoichiometry. Initial
velocity was determined in the presence of MAb 18A9 (Fig. 3);
the double reciprocal plot was suggestive of linear non-competitive
inhibition with a K
of 2.50
10
M. This behavior would be consistent
with the MAb binding near or over but not within the active center
pocket. Alternatively, it could be binding in a region distant from the
active center and changing the conformation of the enzyme, thus
changing its turnover number.
Figure 2:
A, Inhibition of PDHc with purified
monoclonal antibody 18A9. Errorbars represent
± 3 standard deviations. Antibody concentrations ranging from
6.25 10
M to 6.25
10
M were added to 2000 IU/liter of PDHc
having a specific activity of 25 IU/mg and incubated for 1 h. The
activity was assayed, and a titration curve was drawn. B,
stoichiometry of interaction of monoclonal antibody 18A9 with PDHc.
2000 IU/liter total PDHc activity was used having a specific activity
of 25 IU/mg.
Figure 3:
Lineweaver-Burk kinetic plot for
monoclonal antibody 18A9 interacting with PDHc. the K was determined to be 2.50
10
M.
, no 18A9 inhibitor;
, 7.5
µg/ml 18A9 inhibitor;
, 30 µg/ml 18A9 inhibitor;
, 15 µg/ml 18A9 inhibitor. The PDHc 500 IU/liter enzyme and
monoclonal antibody were diluted in 20 mM potassium P
buffer, pH 7.0, with 0.1% BSA to the above stated final inhibitor
concentrations. The mixtures were allowed to incubate 1 h at 4 °C.
The various inhibitor concentrations were then run in the NADH assay.
The various substrate concentrations were 5.0
10
M, 1.0
10
M, 2.0
10
M, 4.0
10
M, 8.0
10
M, and 1.6
10
M. The complete reaction mixture
also consisted of 50 mM Bicine buffer, pH 8.1, 1.0
10
M CoASH, 3.0
10
M cysteine, 2.33
10
M NAD
, 2.0
10
M ThDP, and 1.0
10
M MgSO
. The production of NADH was continuously
monitored at 340 nm at 27 °C. 1 unit of activity produced 1
µmol of NADH per min under these
conditions.
PDHc from E. coli, unlike its mammalian counterparts, is allosterically inhibited by GTP(10, 11) . Experiments were conducted, as described under ``Experimental Procedures,'' to determine if any of the monoclonal antibodies could interfere with the inhibition of PDHc by GTP. The mixture was assayed in the presence and absence of GTP. A ratio of activity in the absence of GTP to that in the presence of GTP was then calculated for each MAb. A nonspecific MAb was also included to show the effect of ascites on the ratio. If a MAb altered the regulatory binding site of the GTP, one would expect the regulating ability of GTP on the PDHc enzyme to be affected. When the ratio of activities in the absence to that in the presence of GTP approaches unity, inhibition due to GTP is blocked. Several MAbs may have some limited effect (data not shown), but purified MAb 15A9 at a concentration of 6.25 µM totally shuts down the regulation of the enzyme by GTP (Fig. 4).
Figure 4: Effect of increasing monoclonal antibody 15A9 concentration on GTP-induced inhibition of PDHc. With greater than two antibodies per E1 subunit (0.78 µM 15A9), the effect of GTP inhibition is blocked. At 6.25 µM, concentration of 15A9 the effect of 0.5 mM GTP is completely blocked. 600 IU/liter of PDHc enzyme was used. The error of measure is similar to that in Fig. 5. The fractional enhancement of the activity in the absence of GTP compared to the activity in the presence of GTP is plotted for the indicated levels of MAb 15A9.
Figure 5:
An
induced conformational change of radiolabeled E1 antigen by monoclonal
antibody 1F2. , monoclonal antibody 7C9;
, monoclonal
antibody 13A8;
, monoclonal antibody 1F2. B/B
binding
of the E1 radiolabel in the presence of 7C9 or 13A8/binding of E1 label
in the absence of 7C9 or 13A8. Monoclonal antibody 1F2 was bound to the
microtiter plate and was incubated in the presence of radiolabeled E1
and 7C9 or 13A8. This figure demonstrates that once 1F2 binds
radiolabeled E1, 7C9 and 13A8 are then able to bind, increasing uptake
of E1 antigen.
Radiolabeling experiments were
also conducted with MAbs 18A9, 21C3, 15A9, 1F2, 7C9, and 13A8. It was
observed that MAbs 7C9 and 13A8, although elicited to the E1 antigen,
did not bind the radiolabeled E1. This was in agreement with the plate
binding E1 assay (see Table 1) and Western blot analysis. A
cooperative binding experiment was done, as described under
``Experimental Procedures.'' The data in Fig. 5suggest a conformational change induced in the E1 antigen
when MAb 1F2 is bound to the radiolabeled E1, a conformational change
that enables MAbs 7C9 and 13A8 to bind the E1. This could be deduced
from Fig. 5based on the increasing
[B]/[B] ratio for the
binding of the radiolabeled E1 to the solid phase MAb 1F2 in the
presence of 7C9 and 13A8. The control for this experiment was the
unbound 1F2 in the absence of the other two MAbs competing for
radiolabeled E1 at increasing concentrations of MAb 1F2.
We hypothesized that if one of the MAbs could inhibit the reaction monitored by the E1-specific assay, this would provide further evidence that the MAbs were specifically bound to the E1 subunit and blocked the transfer of the acetyl group to the nitrogen of the 4-mercaptopyridine. Fig. 6demonstrates that according to the E1-specific assay, both PDHc and E1 (latter data not shown) were indeed inhibited by MAbs 18A9, 21C3, and 15A9, the best inhibitors. Next, purified MAb 18A9 was titrated against a constant concentration of purified E1 or PDHc at 1 mg/ml. Fig. 7illustrates that the inhibition can be reversed when less than saturating amounts of antibody are used as demonstrated by the increased E1 activity, thus suggesting it to be specific. When titering the MAb 18A9 against PDHc and E1, at 0.25 mg/ml MAb 18A9 concentration, there is more activity observed with E1 than with PDHc. This is simply explained by an increased ratio of E1 to antibody with the purified E1, resulting in unbound E1 giving rise to enzyme activity.
Figure 6: Inhibition of the E1 of PDHc enzyme with monoclonal antibody ascites diluted 1:5, monitored by the E1-specific assay. TOTL refers to total PDHc activity without monoclonal antibody inhibition. ASC refers to a nonspecific ascites fluid diluted 1:5. All other bars represent specific monoclonal antibodies in the presence of the enzyme. See ``Experimental Procedures'' for details.
Figure 7:
Titration of monoclonal antibody 18A9 in
the E1 assay. TOTAL refers to the PDHc or E1 subunit activity
in the absence of monoclonal antibody 18A9. , PDHc enzyme,
E1 subunit (both the PDHc and E1 were at 1 mg/ml). See
``Experimental Procedures'' for
details.
According to Fig. 6, there is an apparent enhancement of E1 activity in the presence of MAb 1F2. This stimulation or enhancement by MAb 1F2 was shown to be statistically significant.
20 of the 21 MAbs generated to PDHc and its E1 subunit have been studied. Western blot analysis revealed that 14 of 19 MAbs analyzed bound to the E1 subunit of PDHc. The observation that two of the five remaining MAbs, 23H4 and 8G10, bind E1 on the microtiter plate assay (Table 1) but not according to Western blot analysis is thought to be due to the fact that Western blotting conditions destroy (while plate coating conditions preserve) these epitopes. It is implied, therefore, that these epitopes are non-continuous in nature.
The question of possible cross-species inhibition by selected MAbs was addressed, and no binding or inhibition of these MAbs to mammalian PDHc was observed. In view of the structural dissimilarity shown by genetic mapping, the lack of cross-reactivity of these MAbs to mammalian PDHc is not surprising.
Of the six MAbs exhibiting greater
than 30% inhibition of PDHc, four (18A9, 21C3, 15A9, and 1F2) bind the
E1 subunit of PDHc. Surprisingly, the other two, 7C9 and 13A8, although
elicited to the E1 immunogen, appeared not to bind E1 at all. The
possibility that the E1 antigen was contaminated with E2-E3 can never
be totally ruled out; however, SDS-electrophoresis of the E1 antigen
showed no other bands (data not shown), nor did Western blot analysis
show any evidence of blotting to the E1 or to the PDHc antigen. If
E2-E3 contaminant proteins did exist, since they would be present in
such small percentages, they would have had to have been extremely
immunodominant antigens. The possibility of other contaminating protein
was also remote because these MAbs inhibited the enzyme quite
effectively. We hypothesize that these MAbs are directed toward
epitopes at a protein domain region where E1 contacts E2 or within some
other domain within E1. Once the E1 antigen is resolved from PDHc,
these regions may become distorted or are hidden from antibody
recognition. During the process of producing these MAbs, it is assumed
that peptide fragments that represented or conformed to the antigenic
domain of the native E1 protein on the PDHc were exposed. This
hypothesis is supported by the evidence that short peptide fragments
from protein digests can represent domain region epitopes or
discontinuous epitopes(40, 41) . It is also believed
that these MAbs are expressed to a conformational epitope that is only
available on the holo-PDHc and not on the purified E1 subunit or on the
SDS-denatured PDHc antigen. In the purified E1, the epitope must be
buried according to the data in Fig. 5. MAbs 7C9 and 13A8 did
not bind radiolabeled E1 by themselves, yet in the presence of
monoclonal antibody 1F2 they bound E1. This suggests that 1F2 exposes
the epitope that MAbs 7C9 and 13A8 can then bind. It is believed that
1F2 binds to the radiolabeled E1, causing a conformational change to
occur. This then allows 7C9 or 13A8 to bind, which in turn stabilizes
the complex. The 1F2-E1-7C9 complex has a higher binding constant
than the 1F2-E1 complex, thus exhibiting higher B/B values.
Enhancement of the E1 activity according to the E1-specific assay was observed in the presence of MAb 1F2, further supporting a conformational change induced in the E1 subunit when MAb 1F2 was bound. Such a conformational change would allow for a more efficient transfer of the acetyl group to the 4-mercaptopyridine. When looking at the overall reaction, MAb 1F2 inhibited the formation of NADH by decreasing the rate of transfer of the acetyl group to E2.
Due to the highest level of inhibition
exhibited by MAb 18A9, one would expect a very high level of
specificity for this MAb to a distinctly defined epitope.
Theoretically, only one MAb should bind each E1 subunit to obtain
maximal inhibition. The structural evidence appears to show that E1
binds along the 12 edges of an octahedral E2 core, with E3 binding or
attaching on the six faces(42, 43, 44) . The
inhibitor, rather than being a classical low molecular weight substrate
analog, is an antibody with an M of about 150,000.
If the antibody's effector antigen is E1, theoretically 24 E1
subunits must be bound by 24 MAbs to achieve maximum inhibition. The
first antibodies binding the E1 subunits have binding constants similar
in magnitude to the constants measured in Table 2. Depending on
the accessibility of the particular epitope, binding of each additional
18A9 antibody to the other E1 subunits within the complex could become
increasingly more difficult due to increasing steric problems. This
means that the binding constants for the last antibodies to be bound
would be lower than the initial binding constants, resulting in ratios
of greater than one antibody per E1 subunit needed to achieve maximal
inhibition at 1 h. In viewing the stoichiometry of inhibition by 18A9 (Fig. 2B), greater than 90% inhibition is achieved with
a ratio of [18A9]/[E1] of 1:1 (24 copies of E1 per
PDHc), but to achieve that last 5-8% inhibition, a ratio of 2:1
is required. Observation of such a high level of inhibition for such a
low ratio of antibody to enzyme antigen is nearly unprecedented. This
can be explained by the allosteric behavior described above, as can the
sigmoidal kinetic curve observed in the inhibition by 18A9 (Fig. 2A).
It is very likely that MAb 18A9 directly affects the E1 component only, since isolated E1 component is also totally inhibited by it according to the E1-specific assay (data not shown). This would be in contrast to a polyclonal antibody elicited to an interlipoyl domain linker peptide, which interfered with the overall reaction without affecting the activities of the resolved components and therefore probably blocked the interaction between the E2 and E3 subunits(45) .
The extent of maximal inhibition achieved by any MAb can be viewed analogously to the inhibitory efficacy of a small molecule inhibitor; the closer the inhibitor to the catalytic site, the greater the extent of inhibition. The linear non-competitive inhibition exhibited by MAb 18A9 is not surprising in view of this multisubunit and multicopy complex. This MAb clearly does not resemble the pyruvate substrate and hence should not exhibit competitive inhibition. It could act as a reversible ``cap'' over the active center, physically blocking entry to it but capable of interacting with PDHc both in the absence and in the presence of the substrate.
With some of the other purified MAbs (7C9 in particular), maximum inhibition is only achieved with molar ratios of MAb:PDHc greater than or equal to 700:1. In our earlier publication (46) we demonstrated that the the polyclonal antibodies generated to PDHc were to the three distinct subunits. In the immunoelectrophoresis we observed only three lines, suggesting three populations of antibodies (E1, E2, and E3). This evidence would suggest that the monoclonal antibodies should behave the same way.
Those MAbs (18A9, 21C3, and 15A9) displaying the greatest inhibition could be predicted to be directed at areas of the E1 subunit that are vitally important for enzyme activity. The E1 active site with its ThDP binding structural motif (47) is a good candidate. This binding region would likely constitute a discontinuous epitope (48) due to the nature of coenzyme or substrate binding sites in enzymes forming pockets. The motif is comprised of 30-40 amino acids that have common structural similarities among ThDP-dependent enzymes(47) , is located on the E1 subunit, and is responsible for anchoring the Mg(II) ion and the diphosphate tail of the coenzyme. These three MAbs would likely bind over or near this active center. Epitope mapping studies (49) clearly demonstrate that the three MAbs have the same binding regions, whereas those MAbs derived from the E1 antigen have different binding regions, strongly supporting epitope dissimilarity.
Finally, it has been demonstrated that MAb 15A9 (Fig. 4) can block the allosteric inhibitory effect of GTP. This MAb could be bound at or near the GTP binding locus, or it could have caused a conformational change that distorted the binding locus for GTP, thus rendering it ineffective as an allosteric regulator. The behavior of MAb 15A9 suggests it to be a concentration-dependent inhibitory (negative heterotropic) effector molecule.
In summary, a MAb library has been presented that contains antibodies that bind as well as inhibit the E. coli PDHc and its decarboxylating E1 subunit. Of the MAbs characterized, 18A9 inhibits the enzyme to greater than 98%, and 15A9 can block the effect of GTP regulation of E. coli PDHc. This MAb should provide an excellent tool in studying the GTP binding site or its effector site. Lastly, conformation-dependent epitopes on the E1 subunit have been identified. MAbs 7C9 and 13A8, which could not bind the purified E1 subunit and which were elicited to this purified E1 antigen, were shown to bind E1 antigen in the presence of MAb 1F2.