trans

AMINO ACID SOURCES 175
from T. rangeli and C. fasciculata retain the
C-terminal extension in the mature enzyme,
and thus have a molecular weight appreciably
higher than those of the papain-like
cysteine proteinases. The longest C-terminal
extension described so far, that of cruzipain
(130 amino acids) undergoes a number of
post-translational modifications, including
N-glycosylation. Cruzipain and congopain
are strongly antigenic. In the case of cruzipain,
the C-terminal extension is responsible for
antigenicity.
A truncated recombinant form of cruzipain
(cruzainc), lacking the C-terminal domain, has
been crystallized and its three-dimensional
structure, complexed with several synthetic
peptidic inhibitors, has been determined. The
single polypeptide chain of 215 amino acids
folds into two distinct domains which define
the active site cleft, as well as seven substratebinding
subsites, a structure similar to that
found in other members of the C1 family. The
active site cleft contains the catalytic triad,
formed by Cys25 and His159, which form a
thiolate–imidazolium ion pair essential for the
enzymatic activity, and Asn175. The major
structural differences between cruzipain and
papain are found in the regions of the loops and
turns. Processing of the pro-enzymes to their
mature forms may be due to self-activation,
but at least in the case of the CPb enzymes
from L. mexicana, it appears that CPa can
process the CPbs. The CPb enzymes are specific
to the amastigote stage, and their expression
is induced in promastigotes when the pH
drops to about 4, accompanying differentiation
to the amastigote stage.
Type II and Type III cysteine proteinases
detected in Leishmania (CPa and CPc) are
encoded by single copy genes (lmcpa and
lmcpc), and lack gelatinolytic activity. Mature
CPa has a characteristic three-amino acid
insertion close to the N-terminus, and a very
short C-terminal extension. CPc completely
lacks a C-terminal extension, is amphiphilic
and possibly associated with membranes.
Trypanosomatid Type I and II cysteine proteinases
are lysosomal enzymes, and recently
a role for the pro-domain in their targeting to
the lysosome has been proposed. At least in
the case of L. mexicana, trafficking to the lysosomes
seems to occur via the flagellar pocket.
In addition to the lysosomal forms, which
certainly account for most of the activity,
membrane-associated forms of Type I and
Type III enzymes also appear to be present.
P. falciparum and other malarial parasites
contain a complex array of proteolytic enzymes,
representing all catalytic classes. Plasmodial
proteinases are thought to be involved in red
blood cell invasion and rupture, and are responsible
for the digestion of hemoglobin inside the
food vacuole. Several cysteine proteinases have
been described in P. falciparum and other
Plasmodium species, specifically falcipains
1 and 2. Falcipain 1 is a cathepsin L-like enzyme.
The gene sequence predicts a 67 kDa protein
which contains a signal peptide and a large proenzyme
domain, about 280 amino acids long,
and thus larger than the mature cysteine proteinase,
which has an apparent M r of 26.8 kDa.
Falcipain 2 is the major cysteine proteinase in
the trophozoite.
E. histolytica secretes a number of cysteine
proteinases into the medium. Those best characterized
have apparent molecular masses of
22–27 kDa (amoebapain), 26–29 kDa (histolysain),
16 kDa (cathepsin B) and 56 kDa (neutral
cysteine proteinase). At least seven genes
encoding enzymes from this class have been
identifed from E. histolytica; Eh-CPp1 encodes
amoebapain whereas Eh-CPp2 encodes histolysain.
The mature proteins are 86.5% identical
and are cathepsin B-like. As in most
other parasitic protozoa, these enzymes are
found in organelles similar to lysosomes,
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA