(Received for publication, September 5, 1995; and in revised form, December 27, 1995)
From the
To screen for high molecular weight proteases in Entamoeba
histolytica, we subjected a soluble amebal extract to density
gradient centrifugation and tested the fractions for activity against
the chymotryptic peptide substrate,
Suc-leucyl-leucyl-valyl-tyrosyl-4-methylcoumaryl-7-amide. Two peaks of
activity, of approximately 11 and 20 S, were clearly separated. Based
on SDS-electrophoretic pattern and immunoblot analysis, we ascribe the
20 S activity to proteasomes. The 11 S protein was purified from amebal
homogenates by a series of chromatographic steps. As determined by
molecular sieve chromatography and nondenaturing gel electrophoresis,
the native complex had an apparent M of 385,000
± 10%. On SDS gels, the purified enzyme exhibited a single band
of M
62,000 that yielded a single N-terminal
sequence, indicating that the preparation was homogeneous and that the
native complex consisted of six very similar or identical subunits. The
enzyme preferred peptides with aromatic residues at the P
position and had low but distinct activity toward azocasein. We
conclude that the 11 S enzyme is a novel high molecular weight protease
that is distinct from proteasomes.
Entamoeba histolytica is a parasitic protozoon that
resides in the human gut. It frequently occurs in developing countries,
causes amebic dysentery, and may lead to the formation of tumor-like
abscesses in liver and spleen(1) . As to its cell biology, E. histolytica is a low eukaryote that lacks mitochondria and
a well defined endoplasmatic reticulum/Golgi apparatus. Morphologically
conspicuous is the enormous amount of vacuoles in the amebae. These
occupy about 40% of the total cell volume and are functionally
equivalent to both the lysosomes and the cytotoxic vesicles of higher
eukaryotic cells(2, 3) . Besides its function in
cellular metabolism, protein degradation is essential for a range of
regulatory processes and for the elimination of unstable or abnormal
proteins(4) . In the last decade, new insights have emerged
into the mechanisms of intracellular protein metabolism. Generally,
there appear to be two major pathways for protein degradation: one is
lysosomal and employs the action of thiol- and aspartyl-dependent
cathepsins (5) , and the other is cytosolic and functions with
the aid of high molecular weight proteases. The latter are represented
by the Lon and ClpAP proteases in prokaryotes and the 20 S proteasome
or multicatalytic protease (MCP), ()which is the proteolytic
core of a 26 S protease, in eukaryotes(4, 6) . The 20
S proteasome has also been found in some Archaea(7) and in at least one Eubacterium(8) .
Protein degradation by Lon, ClpAP, and the 26 S protease is ATP
dependent, and protein substrates generally have to be ubiquitinated
prior to degradation by the 26 S protease(9) . By contrast,
small peptides are cleaved by Lon, ClpP, and the 20 S protease in the
absence of ATP and, as to the latter, without the need for ubiquitin
tagging(10) . Consequently, fluorogenic peptides have been used
for screening purposes and as model substrates to probe the specificity
of these high molecular weight proteases(11, 12) . For
proteasomes, the pattern of activity toward the different substrates is
variable. In particular, upon stimulation by
-interferon,
mammalian proteasomes exhibit an increase in chymotryptic activity,
which may serve to generate peptides for antigen presentation and
appears to be due both to the replacement of certain proteasomal
-subunits by others and to an enhanced response to activator
protein(6, 13, 14) . The three-dimensional
structure of an archaeal 20 S proteasome, which should be similar to
its eukaryotic counterpart, has recently been solved(7) . It
consists of four stacked rings with seven subunits each: two rings of
22.3-kDa
-subunits sandwiched between two rings of 25.8-kDa
-subunits. Remarkably, the bacterial ClpP seems to possess a
similar 7-fold symmetry(15) . The active site residue of the
proteasome has been identified as a
-chain threonine both by
site-directed mutagenesis (16) and by N-terminal
modification(17) .
In E. histolytica, a range of cysteine proteinases of the papain type has been extensively characterized both on the genomic and on the protein level(18, 19) . These enzymes are localized in the lysosome-like vacuoles mentioned above. By contrast, nothing is known yet about cytoplasmic protein degradation in this organism or about the proteases involved. In this study, we present evidence indicating that E. histolytica contains both proteasomes and a novel, unrelated high molecular weight protease.
Figure 1:
Purification of the 11 S protease.
Chromatographic runs are as follows: A, DEAE-cellulose; B, Sephacryl S-300; C, MonoQ; D, Superose 6
(second run). &cjs0822;, Suc-LLVY-MCA hydrolyzing activity; ,
Boc-LSTR-MCA hydrolyzing activity; - - -, NaCl
gradient; -, protein absorption. For further experimental
details, see text.
Figure 2: Effect of SDS on Suc-LLVY-MCA hydrolyzing activity in amebic extract. Samples were preincubated with the indicated concentrations of SDS, and enzyme activity was measured fluorimetrically as described under ``Experimental Procedures.'' Values are given as percent activity of a control without SDS.
Stimulation of peptidolytic activity by SDS has typically been found
for proteasomes(27) . We therefore set out to investigate
whether these complexes were present in E. histolytica. To
this end, a soluble amebal extract was subjected to sucrose density
gradient centrifugation, and the fractions were tested for chymotryptic
activity. As shown in Fig. 3(closed circles), two
Suc-LLVY hydrolyzing peaks with sedimentation velocities of
approximately 11 and 20 S were clearly separated. Only the second peak
exhibited significant activity toward the tryptic substrate,
BOC-LSTR-MCA (Fig. 3, open squares). The
SDS-electrophoretic pattern of the 20 S peak (Fig. 3, inset
A) was restricted to a series of bands between 25 and 30 kDa and
roughly corresponded to that found for proteasomes from a number of
organisms(21) , whereas the 11 S fraction exhibited bands over
a much wider range (data not shown) and obviously contained a crude
mixture of proteins. In immunoblot experiments, we tested the reactions
of a cell homogenate and of the 11 and 20 S peaks with an antibody
(MCP231) against a sequence motif common to -chains of
proteasomes(26) . As shown in Fig. 3, inset B,
the 20 S peak (lane 3) exhibited a series of cross-reacting
bands around 30 kDa. By contrast, neither the 11 S fraction (lane
2) nor the purified 11 S protease (lane 1; see below)
cross-reacted with the antibody. These findings indicated that E.
histolytica contained both 20 S proteasomes and an 11 S peptidase.
Figure 3:
Density gradient centrifugation of a
soluble amebic extract. , Suc-LLVY-MCA hydrolyzing activity;
, Boc-LSTR-MCA hydrolyzing activity. Positions of the marker
proteins catalase and thyroglobulin are indicated. Inset A,
SDS-PAGE of the 20 S peak and protein staining. Inset B,
immunoblot probed with MCP231. Lane 1, purified 11 S protease; lane 2, 11 S peak; lane 3, 20 S peak. The positions
of two marker proteins (size in kDa) are indicated with arrows. For further details, see ``Experimental
Procedures.''
Figure 4: Gel electrophoresis of the purified 11 S protease and interpretation of the data. A, gel electrophoresis. 1, native gel electrophoresis through 5% polyacrylamide; 2, SDS-PAGE under weakly denaturing conditions (no boiling, no mercaptoethanol, 1 mg/ml SDS) through 10% polyacrylamide; 3, SDS-PAGE under strongly denaturing conditions (boiling, mercaptoethanol, 4 mg/ml SDS) through 15% polyacrylamide. Activity staining was performed with Suc-LLVY-MCA as substrate and protein staining with Coomassie Brilliant Blue G-250. Positions of molecular mass protein markers (in kDa) are indicated. For further details, see ``Experimental Procedures.'' B, inferred structures of the complexes visualized in the respective gel systems; see text for details.
Figure 5: Electron micrograph of the 11 S protease. Scale bar, 100 nm. For details, see ``Experimental Procedures.''
In this study, we have identified two high molecular weight
(11 and 20 S) proteases in E. histolytica. The heavier enzyme
appears proteasome-like both from its subunit composition and from its
cross-reactivity with an antibody against proteasomal -subunits.
By contrast, the lighter enzyme exhibited several novel
characteristics. Clearly, its activity cannot be ascribed to the
vesicular cysteine proteinases described in a series of earlier reports (18, 19) because it was not inhibited by E-64, a
specific inhibitor of these enzymes. In addition, its high molecular
weight and its substrate specificity (preferred cleavage of the peptide
bond of hydrophobic residues at the P
position) distinguish
this enzyme from the known amebic proteases histolysain and amebapain,
which favor arginine in P
and P
(28) .
Also, although peptidolytic activity was effectively inhibited by a
calpain inhibitor and stimulated by Ca
(Table 5), we obviously were not dealing with a calpain, as
these enzymes have a very different subunit composition and are blocked
by EDTA and E-64(29) . We originally thought the 11 S enzyme
might correspond to part (specifically, to the
-core) of the
proteasome. Indeed, apart from its complete lack of peptidylglutamyl
activity, the substrate specificity, kinetics, and effector profile of
the 11 S enzyme (Table 2-V) were not that different from
those found for proteasomes; SDS at low concentrations stimulated
activity to a comparable degree, and in electron-optical images the 11
S complex had a similar diameter (Fig. 5). However, a very
strong argument against the 11 S enzyme being part of the proteasome is
its radically different subunit composition (identical or very similar
62-kDa subunits rather than a range of bands between 25 and 30 kDa). Of
course, we cannot at present exclude a distant relationship between the
two complexes. For instance, the 62-kDa subunits might be the product
of natural gene duplications of sequences coding for far relatives
(with deviating N-terminal ends) of proteasomal
-chains. However,
the inferred symmetry of the 11 S particle (3-fold rather than 7-fold,
see Fig. 4B) would seem to argue even against a distant
relationship. Evidence has recently been reported (30) that Trypanosoma brucei, another parasitic protozoon that branched
off relatively early from the main eukaryotic line(31) ,
contains a high molecular weight protease that migrates faster than
mammalian 20 S proteasomes and that exhibits a deviating substrate
specificity. This enzyme could possibly be related to the E.
histolytica 11 S protease.