tains two Kunitz-type trypsin inhibitors
[133,219]. In the haemolymph of the horseshoe
crab, Limulus polyphemus, a novel trypsin inhibitor has been detected and constitutes a new class
of protease inhibitors [58]. The best-known invertebrate proteinase inhibitors are the hirudins, the
inhibitors of blood-clotting found in the saliva of
the leech Hirudo medicinalis. They inhibit both
thrombin and trypsin and are present in more
than ten isoforms, all of which are 65 amino acids
long and differ in just a few positions [243]. In the
anterior gut, but not in the saliva or the mid-gut
of H. medicinalis, are two further types of proteinase inhibitors which perhaps have antibacterial
functions: the bdellins belong to the Kazal group
and appear as smaller (5-7 kDa) and larger
(20 kDa) variants. The eglins have a length of 70
amino acids and belong to their own super-family
[9]. In the saliva of the North American bloodsucking leech, Haementaria officinalis, is an
inhibitor of the blood-clotting factor Xa; this is a
protein of 119 amino acids, including 20 cysteine
residues, belongs to a new super-family and has
been named "antistasin". The inhibitor known as
"ghilanten" from the closely related species
H. ghilinaii is very similar [16, 62]. A highly specific Xa inhibitor has also been detected in the tick
Ornithodorus moubata; this is a peptide of 60
amino acids of the Kunitz type [291].
The roundworm Ascaris lumbricoides is equipped with inhibitors against all proteinases present
in the host gut lumen: in each case there are several inhibitors of low molecular weight
(7-20 kDa) for pepsin, chymotrypsin and elastase, and trypsin. On the basis oftheir sequences,
the trypsin and chymotrypsin inhibitors have
been allocated to a new super-family. The horse
roundworm, Parascaris equorum, also produces
inhibitors, about which little is as yet known
[170]. A strictly elastase-specific inhibitor from
the sea anemone Anemonia sulcata has been
identified as an atypical Kazal type on the basis of
its sequence [280]. An inhibitor that inhibits trypsin, but not chymotrypsin or thrombin, has been
isolated from the coelom fluid of the starfish Asterias forbesi [167].
3.4.2 Cysteine-Proteinase Inhibitors
The inhibitors of the vertebrate cysteine-,
aspartate- and metalloproteinases have been
much less investigated than those of the serine
proteinases, and comparative biochemical data
are almost completely absent. Cystatin from
3.4.2 Cysteine-Proteinase Inhibitors
101
chicken egg albumin was the first representative
of a super-family of cysteine-proteinase inhibitors
of the same name, many more examples of which
have since been found in man and other mammals. Three, or perhaps four, families of closely
related proteins can be distinguished within the
cystatin super-family [38, 220]. Cystatins of
type 1 (stefins) are molecules of about 100 amino
acids (11 kDa) without disulphide bridges. They
include, for example, mammalian cystatins A and
B, of which B is found in many cell types and A is
restricted to the epithelium and leukocytes. The
function of cystatin A is probably to inhibit the
cysteine proteinases of invading parasitic Protozoa [12]. The type-2 cystatins, which include the
chicken egg cystatin, consist of about 115 amino
acids (13 kDa) with two disulphide bridges close
to the C-terminus. The cystatin family includes
not only various intra- and extracellular cystatins
from mammals and birds but also an inhibitor in
the poison of the puff adder, Bitis arientans
[12,224]. The cystatins of type 3, the kininogens,
have the most complicated structure of all. Their
polypeptide chain of about 355 amino acids
makes up three domains, similar in structure to
the type-2 cystatins but with different inhibitory
activities and specificities. It appears that two
gene duplications occurred during the evolution
of the kininogens. The C-terminal region of the
kininogen chain contains the sequence of the
blood-pressure reducing peptide hormone bradykinin, which is released through the activity of the
serine proteinase kallikrein (see Fig. 8.5, p. 302).
The kininogens also contain additional disulphide
bridges and are glycosylated [12].
Cystatins inactivate the plant cysteine proteinase papain and the lysosomal cathepsins B, H
and L; calpain II, in contrast, is only inhibited by
particular kininogens. The mechanism by which
the cystatins inhibit enzymes is not yet completely
understood [17]. A family tree can be constructed
using the known sequences of cystatins from
mammals and birds. Assuming that the rate of
evolution of the cystatins has been constant, then
the common ancestral form of the present cystatins must have existed before the eukaryotes. In
fact, a cysteine-proteinase inhibitor with clear
homology to the animal cystatins has been found
in rice plants [1, 52]. A proteinase inhibitor specific for cathepsin L has been isolated from pig
leukocytes; the sequence of 96 amino acids shows
no significant homology with cystatin and apparently belongs to a new super-family [225]. In the
fly Sarcophaga peregrina, there is a cysteineproteinase inhibitor (sarcostatin A), of about
[133,219]. In the haemolymph of the horseshoe
crab, Limulus polyphemus, a novel trypsin inhibitor has been detected and constitutes a new class
of protease inhibitors [58]. The best-known invertebrate proteinase inhibitors are the hirudins, the
inhibitors of blood-clotting found in the saliva of
the leech Hirudo medicinalis. They inhibit both
thrombin and trypsin and are present in more
than ten isoforms, all of which are 65 amino acids
long and differ in just a few positions [243]. In the
anterior gut, but not in the saliva or the mid-gut
of H. medicinalis, are two further types of proteinase inhibitors which perhaps have antibacterial
functions: the bdellins belong to the Kazal group
and appear as smaller (5-7 kDa) and larger
(20 kDa) variants. The eglins have a length of 70
amino acids and belong to their own super-family
[9]. In the saliva of the North American bloodsucking leech, Haementaria officinalis, is an
inhibitor of the blood-clotting factor Xa; this is a
protein of 119 amino acids, including 20 cysteine
residues, belongs to a new super-family and has
been named "antistasin". The inhibitor known as
"ghilanten" from the closely related species
H. ghilinaii is very similar [16, 62]. A highly specific Xa inhibitor has also been detected in the tick
Ornithodorus moubata; this is a peptide of 60
amino acids of the Kunitz type [291].
The roundworm Ascaris lumbricoides is equipped with inhibitors against all proteinases present
in the host gut lumen: in each case there are several inhibitors of low molecular weight
(7-20 kDa) for pepsin, chymotrypsin and elastase, and trypsin. On the basis oftheir sequences,
the trypsin and chymotrypsin inhibitors have
been allocated to a new super-family. The horse
roundworm, Parascaris equorum, also produces
inhibitors, about which little is as yet known
[170]. A strictly elastase-specific inhibitor from
the sea anemone Anemonia sulcata has been
identified as an atypical Kazal type on the basis of
its sequence [280]. An inhibitor that inhibits trypsin, but not chymotrypsin or thrombin, has been
isolated from the coelom fluid of the starfish Asterias forbesi [167].
3.4.2 Cysteine-Proteinase Inhibitors
The inhibitors of the vertebrate cysteine-,
aspartate- and metalloproteinases have been
much less investigated than those of the serine
proteinases, and comparative biochemical data
are almost completely absent. Cystatin from
3.4.2 Cysteine-Proteinase Inhibitors
101
chicken egg albumin was the first representative
of a super-family of cysteine-proteinase inhibitors
of the same name, many more examples of which
have since been found in man and other mammals. Three, or perhaps four, families of closely
related proteins can be distinguished within the
cystatin super-family [38, 220]. Cystatins of
type 1 (stefins) are molecules of about 100 amino
acids (11 kDa) without disulphide bridges. They
include, for example, mammalian cystatins A and
B, of which B is found in many cell types and A is
restricted to the epithelium and leukocytes. The
function of cystatin A is probably to inhibit the
cysteine proteinases of invading parasitic Protozoa [12]. The type-2 cystatins, which include the
chicken egg cystatin, consist of about 115 amino
acids (13 kDa) with two disulphide bridges close
to the C-terminus. The cystatin family includes
not only various intra- and extracellular cystatins
from mammals and birds but also an inhibitor in
the poison of the puff adder, Bitis arientans
[12,224]. The cystatins of type 3, the kininogens,
have the most complicated structure of all. Their
polypeptide chain of about 355 amino acids
makes up three domains, similar in structure to
the type-2 cystatins but with different inhibitory
activities and specificities. It appears that two
gene duplications occurred during the evolution
of the kininogens. The C-terminal region of the
kininogen chain contains the sequence of the
blood-pressure reducing peptide hormone bradykinin, which is released through the activity of the
serine proteinase kallikrein (see Fig. 8.5, p. 302).
The kininogens also contain additional disulphide
bridges and are glycosylated [12].
Cystatins inactivate the plant cysteine proteinase papain and the lysosomal cathepsins B, H
and L; calpain II, in contrast, is only inhibited by
particular kininogens. The mechanism by which
the cystatins inhibit enzymes is not yet completely
understood [17]. A family tree can be constructed
using the known sequences of cystatins from
mammals and birds. Assuming that the rate of
evolution of the cystatins has been constant, then
the common ancestral form of the present cystatins must have existed before the eukaryotes. In
fact, a cysteine-proteinase inhibitor with clear
homology to the animal cystatins has been found
in rice plants [1, 52]. A proteinase inhibitor specific for cathepsin L has been isolated from pig
leukocytes; the sequence of 96 amino acids shows
no significant homology with cystatin and apparently belongs to a new super-family [225]. In the
fly Sarcophaga peregrina, there is a cysteineproteinase inhibitor (sarcostatin A), of about