(Received for publication, November 6, 1995; and in revised form, December 21, 1995)
From the
A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which
lacks an amino-terminal lipoyl domain, p45, has been identified in the
pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited
significant similarity with internal E3-binding domains of
dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution
of a pyruvate dehydrogenase-depleted adult A. suum PDC in
guanidine hydrochloride resulted in two E3-depleted E2 core
preparations which were either enriched or substantially depleted of
p45. Following reconstitution, the p45-enriched E2 core exhibited
enhanced E3 binding, whereas, the p45-depleted E2 core exhibited
dramatically reduced E3 binding. Reconstitution of either the bovine
kidney or A. suum PDCs with the A. suum E3 suggested
that the ascarid E3 was more sensitive to NADH inhibition when bound to
the bovine kidney core. The expression of p45 was developmentally
regulated and p45 was most abundant in anaerobic muscle. In contrast,
E3s isolated from anaerobic muscle or aerobic second-stage larvae were
identical. These results suggest that during the transition to
anaerobic metabolism, E3 remains unchanged, but it appears that a novel
E3BP, p45, is expressed which may help to maintain the activity of the
PDC in the face of the elevated intramitochondrial NADH/NAD ratios associated with anaerobiosis.
The pyruvate dehydrogenase complex (PDC) ()plays a
key role in the unique mitochondrial metabolism of the parasitic
nematode, Ascaris suum(1, 2) . During larval
development, A. suum exhibits a marked aerobic-anaerobic
transition in energy metabolism. Early larval stages are aerobic, and
the PDC functions to supply acetyl-CoA for tricarboxylic acid cycle
oxidation(3) . In contrast, energy metabolism in adult ascarid
muscle is anaerobic. Adult muscle mitochondria lack both a functional
tricarboxylic acid cycle and cytochrome oxidase activity and use
unsaturated organic acids, such as fumarate and 2-methyl branched-chain
enoyl-CoAs, instead of oxygen, as terminal electron
acceptors(4, 5, 6) . The PDC is overexpressed
in adult muscle and is designed to function under the elevated
NADH/NAD
and acyl-CoA/CoA ratios present in the
microaerobic environment of the host gut(7, 8) .
The subunit composition of the adult ascarid muscle PDC differs
significantly from the PDCs isolated from other eukaryotic organisms (7, 9, 10) . In both yeast and mammalian
PDCs, incubation of the complex with
[2-C]pyruvate in the absence of CoA results in
the acetylation of two proteins, dihydrolipoyl transacetylase (E2) and
dihydrolipoyl dehydrogenase (E3)-binding protein
(BP)(11, 12) . E2 and E3BP are present in a ratio of
about 5:1, and both contain similar multidomain structures consisting
of an amino-terminal lipoyl domain, a subunit binding domain, and an
inner domain(13, 14, 15) . E2 preferentially
binds pyruvate dehydrogenase (E1) and exhibits transacetylase activity,
whereas E3BP contains a high-affinity binding site for E3(16) .
In contrast, incubation of the ascarid complex under similar conditions
results in the acetylation of only E2 and no protein corresponding to
E3BP has been identified(10) . In addition, the PDC from A.
suum and other closely related nematodes contains subunits of 43
kDa (p43) and 45 kDa (p45) of unknown function, which do not appear to
be present in PDCs from other organisms(17) . Whether the
E3-binding site in the ascarid PDC resides in E2, as has been observed
for PDCs from prokaryotes, or in one of these novel proteins has not
been determined(18) .
In the present study, we report that
sequence near the amino terminus of p45 exhibits significant similarity
to the putative E3-binding domains of E2 and E3BP and demonstrate that
E3 binding to the E2 core is dependent on the presence of p45. In
addition, we show that ascarid E3 bound to the bovine kidney core
(containing E3BP) appears to be more sensitive to NADH inhibition than
when it is bound to the ascarid core (containing p45). Therefore, it
appears that during the switch from aerobic to anaerobic energy
metabolism in ascarid development, E3 remains unchanged, but a novel
anaerobic E3BP, p45, is expressed to maintain PDC activity in the face
of elevated NADH/NAD ratios.
Figure 1:
Resolution of A. suum E1-depleted PDC with guanidine hydrochloride. E1-depleted A.
suum PDC (0.6 mg) was incubated in 50 mM MOPS, 1 M GdnHCl, 1 mM EDTA, 3 mM dithiothreitol, and 1
mg/ml Pluronic F68 (pH 7.4) as described under
``Experimental Procedures.'' The sample (0.2 ml) was applied
to a Superose 12 column equilibrated in the same buffer. The flow rate
was 0.4 ml/min and 0.4-ml fractions were collected. Inset,
Coomassie Brilliant Blue-stained 10% SDS-polyacrylamide gel of the
pooled peak fractions; *, adult A. suum PDC (12 µg); SC, E1-depleted A. suum PDC (10 µg); 1,
Peak 1, fractions 18-20 (
8 µg), (p45-enriched E2 core); 2, Peak 2, fractions 23-27 (8 µg) (p45-depleted A. suum E2 core); and 3, Peak 3, fractions
29-32, (4 µg). Molecular mass markers of 64, 50, and 36 kDa
are indicated at the right.
For immunoblotting,
samples were transferred to nitrocellulose (Schleicher and Schuell)
overnight at 40 V in 25 mM Tris-HCl, 192 mM glycine,
and 20% (v/v) methanol. To detect bands of interest, each specific
antiserum was used in conjunction with the Promega kit,
Protoblot Western blot AP system (Promega Corp.), using
goat anti-rabbit IgG secondary antibody conjugated to alkaline
phosphatase. The procedure was performed as described by the
manufacturer. Affinity purified antisera against p45, E1
, and
E1
were prepared as described previously (10, 25, 26) . E2 antiserum was
affinity-purified in a manner previously
described(25, 27) .
Figure 2:
Binding of A. suum peripheral
components (E1 and E3) by two E2 core populations. A. suum p45-enriched E2 core (Peak 1) was reconstituted with excess A.
suum E3, and p45-depleted A. suum E2 core (Peak 2) was
reconstituted with excess A. suum E1 and E3 as described under
``Experimental Procedures.'' Samples were separated by
SDS-PAGE on 10% gels and stained with Coomassie Brilliant Blue. Lane 1, adult A. suum PDC (12 µg); Lane
2, E1-depleted A. suum PDC (10 µg); Lane 3, p45-enriched E2 core (Peak 1) (8 µg); Lane 4, reconstituted Peak 1 pellet (
12 µg); Lane 5,
reconstituted Peak 1 supernatant (4 µg); Lane 6,
p45-depleted A. suum core (Peak 2) (8 µg); Lane
7, reconstituted Peak 2 pellet (10 µg); and Lane 8,
reconstituted Peak 2 supernatant (5
µg).
Figure 3: Association of p45 with the core of the complex after selective proteolysis. E1-depleted A. suum PDC was treated with V8 protease and the proteolytic products were separated as described under ``Experimental Procedures.'' Samples (9 µg) were separated by SDS-PAGE on 12% gels and stained with Coomassie Brilliant Blue. The identity of E2 domain fragments was determined by immunoblotting and amino-terminal sequencing. Lane 1, E1-depleted PDC; Lane 2, E1-depleted PDC following proteolysis; Lane 3, pellet; Lane 4, supernatant.
Figure 4:
NADH sensitivity of A. suum,
bovine kidney, and hybrid pyruvate dehydrogenase complexes. Hybrid
complexes were constructed as described under ``Experimental
Procedures.'' A, PDC activity is expressed as a
percentage of the activity measured in the absence of NADH using a
fixed NAD concentration with varying NADH
concentrations. B, PDC activity is expressed as a percentage
of the activity measured in the absence of NADH using conditions of a
fixed nicotinamide pool (200 µM). Closed circles, A. suum PDC; squares, bovine kidney PDC; and open
circles, bovine kidney E2
E3BP
E1/A. suum E3
hybrid complex.
Figure 5:
Immunoblot of A. suum larval
homogenates with antisera against the adult A. suum E2, p45,
E1, and E1
. Larval homogenates and the affinity purified
antisera were prepared as described under ``Experimental
Procedures.'' E2 and p45 antisera were used at dilutions of 1:5000
and 1:2000, respectively. E1
and E1
antisera were both used
at dilutions of 1:2000. UE, unembryonated eggs (150 µg); L1 (150 µg); L2 (150 µg); L3 (80
µg); and M, adult muscle PDC (1 µg). *, rabbit IgG: L3
are recovered from rabbit lungs and homogenates often contain rabbit
IgG.
Perhaps surprisingly, significant p45 staining
was observed in homogenates of third-stage larvae (L3), even though
they are aerobic and cyanide-sensitive. However, other key enzymes of
anaerobic metabolism, such as the 2-methyl branched-chain enoyl-CoA
reductase also have been identified in this transitional larval stage.
Their expression may represent an adaptation to the switch to
anaerobiosis that accompanies the impending molt to the
fourth-stage(30) . Taken together, these results suggest that
p45 is found predominantly in anaerobic muscle. In addition, antisera
against the adult muscle E1 and E1
recognize proteins of
different mobilities in the aerobic larval stages. This confirms the
presence of stage-specific E1
isozymes and further suggests that
stage-specific forms of E1
also may be present.
To determine if
E3s also were different in aerobic and anaerobic stages, E3 was
purified to homogeneity from aerobic L2 (Table 2). E3 from L2
behaved similarly to the E3 from adult muscle during purification and
had identical kinetic properties and subunit molecular mass (55 kDa,
data not shown). More important, amino-terminal sequence analysis of
both E3s indicated that the first 53 amino acids were identical,
suggesting that stage-specific isoforms of E3 were not present in the A. suum PDC (Table 3). Therefore, it appears that during
the switch from aerobic to anaerobic energy metabolism in ascarid
development, E3 remains unchanged, but a novel anaerobic E3BP, p45, is
expressed to maintain PDC activity in the face of elevated
NADH/NAD ratios observed in adult body wall muscle.
A. suum undergoes a marked aerobic/anaerobic
transition during its development and energy metabolism in adult muscle
mitochondria is anaerobic and characterized by elevated
NADH/NAD ratios(3) . The results of the
present study suggest that E3 is identical in both aerobic and
anaerobic stages, but that the PDC from anaerobic mitochondria has a
novel E3BP, p45. In contrast to E3BPs identified in either yeast or
mammalian PDCs, p45 is not acetylated during incubation in
[2-
C]pyruvate and does not contain the highly
conserved amino-terminal PS/ALSPTM sequence characteristic of lipoyl
domains(10, 32) . In contrast, the amino terminus of
p45 does contain sequence which exhibits significant similarity to
E3-binding domains found internally in E2s, E3BPs, and
succinyltransferases isolated from a number of organisms. However, it
should be noted that the E3-binding domain of p45 also exhibits
significant differences from these putative E3-binding domains.
Supporting the proposed role of p45 as an E3-binding protein is the
observation that E2 core substantially depleted of p45 still retains
its ability to fully bind E1 and function catalytically, but does not
bind additional E3, while E2 core enriched with p45 binds additional
E3. Whether the PDC from early larval stages contains an E3BP similar
to that observed in yeast and mammals must await the purification of
the PDC from these stages. Unfortunately, purification is complicated
by the limited amounts of available tissue and the high protease
activity found in the hatching fluids of those early larval stages.
Although the function of a novel E3BP in anaerobic ascarid
mitochondria is still not completely clear, it appears that the binding
of E3 to p45 may decrease the sensitivity of the complex to NADH
inhibition. When a hybrid complex was constructed using the bovine
kidney core, bovine kidney E1, and the ascarid E3, the hybrid complex
had a significantly higher apparent K for
NAD
and was more sensitive to NADH inhibition than the
native ascarid complex. PDCs exhibit a wide range of sensitivities to
NADH inhibition. For example, PDCs from facultative anaerobes, such as Escherichia coli, are very sensitive to NADH inhibition. As
NADH accumulates during anaerobiosis, PDC activity is inhibited, which
in turn, diverts pyruvate from the TCA cycle to fermentative
pathways(33) . In contrast, PDCs from anaerobes, such as Enterococcus faecalis, are much less sensitive to NADH
inhibition(29) . PDCs from higher eukaryotes also are less
sensitive to NADH inhibition. In these organisms, PDC activity is
regulated primarily by covalent modification and not end product
inhibition. In addition, elevated NADH/NAD
levels have
a marked stimulatory effect on pyruvate dehydrogenase kinase activity
which catalyzes the phosphorylation and inactivation of the
complex(34, 35) . The PDC from adult A. suum muscle and other gut dwelling parasitic helminths is the most
insensitive to NADH inhibition, and pyruvate dehydrogenase kinase
activity in these complexes is also less sensitive to activation by
NADH(7, 17) . Interestingly, when the amino acid
sequence of the conserved redox active disulfide site of E3s from
anaerobic parasitic helminths is compared to that of other E3s, a
consistent sequence difference is observed. Phenylalanine is
substituted for leucine at position 40 and a proline is located at
position 38. These sequence alterations are conserved in E3s from
anaerobic helminths representing two distinct helminth phyla, but not
in the E3 from the aerobic free-living nematode, Caenorhabditis
elegans. The significance of this sequence difference remains to
be determined.
The function of E3BP has been elucidated most directly in the S. cerevisiae PDC. Disruption of the gene encoding E3BP yields a catalytically inactive complex with a properly assembled E2 core and attached E1, but no E3, and suggests that while E3BP is required for E3 binding, it is not an integral part of the core or required for assembly(36) . More important, deletion of most of the lipoyl domain of E3BP had no effect on PDC activity, suggesting that the lipoyl domain was not essential for E3 binding or E3BP function(36) . While E3BP has a domain structure similar to E2, it has no catalytic activity, and lacks the HXXKG sequence near the carboxyl terminus proposed as part of the active site of all dihydrolipoamide acyltransferases (37) . However, after the removal of the lipoyl domains of E2 by direct proteolysis or site-directed mutagenesis, it does appear that the lipoyl domain of E3BP can support the overall reaction of the complex, albeit at a greatly reduced rate(38, 39) . Indeed, the final specific activity of the PDC purified from A. suum muscle is significantly lower than that reported for PDCs from either yeast or mammals(7) . Whether this reduced specific activity is related to the absence of a lipoyl domain on E3BP is not clear. Recently, an E3BP in the PDC of the insect trypanosomatid, Crithidia fasciculata, has been identified which appears to contain, not one, but multiple lipoyl domains(40) . A similar situation has been described previously for E2s, where 1, 2, or 3 lipoyl domains have been identified, depending on the species, and there appears to be no physiological significance to the number of lipoyl domains in E2(41) . For example, site-directed mutagenesis of the three lipoyl domains of the E. coli E2 suggests that the inactivation of two of the three lipoyl domains does not affect catalysis(42) . Whether more subtle regulatory properties are affected by these alterations has not been determined.