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AMINO ACID SOURCES 177
have been thoroughly characterized. Leishmanolysin
or gp63 (63 kDa glycoprotein) is
bound to the promastigote membrane by a
GPI anchor, and is the most abundant protein
exposed at the promastigote membrane
(about 5 10 5 molecules per cell). A homologous
enzyme with an acidic optimal pH, found
as a soluble protein in lysosomes, is present
at lower levels in amastigotes. The membranebound
enzyme is a Zn 2 -dependent HEXXH
metalloproteinase, able to degrade components
of the extracellular matrix, like fibrinogen.
Leishmanolysin is N-glycosylated at its three
potential sites, and contains three domains;
two have novel folds, while the N-terminal
domain is similar to the catalytic domain of the
metzincin class of zinc proteinases (family M8).
Leishmanolysin is produced as a proenzyme
and appears to be activated by a Cys-switch
mechanism, as proposed for matrix metalloproteinases.
Recently, genes presumably
encoding leishmanolysin homologs have been
cloned and sequenced from C. fasciculata,
Herpetomonas samuelpessoai, T. brucei and
T. cruzi. Several membrane-bound metalloproteinases
have been detected in T. cruzi and several
other Trypanosomatids, but have not been
characterized. Two soluble metalloproteinases,
a carboxypeptidase and an aminopeptidase,
also have been identified in L. major.
P. falciparum contains a recently characterized
metalloproteinase, falcilysin, which has
homology with pitrilysin and other members
of the M16 family. Falcilysin is located in the
food vacuole, and seems to be involved in
the degradation of the peptides derived from
the action of the falcipains and plasmepsins.
A gene encoding an aminopeptidase belonging
to the M1 family of metallopeptidases
also has been cloned from P. falciparum.
Antibodies raised against a peptide predicted
from the sequence recognizes two schizont
proteins with M r values of 96 and 68 kDa.
Aspartic proteinases
These enzymes have been described and studied
in most detail in malarial parasites. In contrast,
no members of this class have yet been
identified in Trypanosomatids. P. falciparum
contains aspartic proteinases with molecular
masses ranging from 10 to 148 kDa. Plasmepsins
I and II have 73% amino acid identity,
but cleave hemoglobin with different specificities.
However, both enzymes cleave the Phe
33/Leu 34 bond in the -chain of native hemoglobin,
which destabilizes hemoglobin, leading
to its unraveling and further proteolysis. The
crystal structure of a complex of plasmepsin II
with the inhibitor pepstatin is similar to that of
mammalian and fungal aspartic proteinases.
The pro-plasmepsins are Type II integral membrane
proteins that are cleaved to yield the soluble
mature enzymes. The interaction of the
pro-domain of pro-plasmepsin II with the
catalytic moiety is unusual, since, instead of
blocking the active site cleft, the pro-domain
interacts with the C-terminal domain keeping
both domains apart and preventing the formation
of the active site. Genes encoding aspartic
proteinases have also been identified in other
Apicomplexa, including Toxoplasma, Eimeria
and Cryptosporidium.
Threonine proteinases
The 20S proteasome, belonging to the threonine
peptidase class, is a high molecular weight complex
which presents, in eukaryotes, three proteolytic
activities with different specificity, and is
the major proteolytic activity in the cytosol and
within the nucleus. 20S proteasomes have been
recently identified in E. histolytica, T. brucei,
T. cruzi, L. mexicana, P. falciparum and P. berghei,
G. lamblia and Entamoeba invadens. The 20S
proteasome has an apparent M r of about
670 kDa, and is made up of a number of subunits
with apparent M r values ranging, in different
parasites, from 22 to 35 kDa, and with pI
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA