102
3 The Structural Variety and Metabolism of Proteins
10 kDa, which is produced in the fat bodies and
released in increasing amounts into the haemolymph during pupae development. Its function
would appear to be to protect tissues from attack
by proteinases, released during metamorphosis
from degraded larval cells [263].
3.4.3 Clz-Macroglobulins
Cl2-Macroglobulins are large glycoproteins present
in the blood of vertebrates and invertebrates and
in the egg albumin of birds and reptiles. The Cl2M
of mammalian plasma consists of four subunits
connected in pairs by disulphide bridges to form
dimers. The human subunits are made up of 1451
amino acids which agree at 73 % of positions with
those of the rat. This super-family also includes
the human pregnancy zone protein, the Clzmacroglobulin and the Clrinhibitor 3 of the rat.
Most members of the family are tetramers, but
some are dimers (frog Cl2M, human pregnancy
zone protein) or monomers (rat Cll-inhibitor 3).
Ovostatin, a proteinase inhibitor from chicken
egg albumin, is similar in sequence to the blood
plasma Cl2M at least at the N-terminus. There is
significant agreement between Cl2M and partial
sequences of the complement components C3
and C4 ofthe mouse; the latter, however, contain
regions that are not present in Cl2M. The evolution
of the complement components possibly included
a gene fusion (exon shuffling). The complement
components, like Cl2M, contain an internal thioester bond that is formed by reaction of a cysteine
thiol group with the y-carboxyl group of a neighbouring glutamate [256, 357].
The inhibitory action of azM is basically different from that of typical proteinase inhibitors;
Cl2M inhibits proteinases of all classes and the
formation of the Cl2M/enzyme is irreversible.
Only proteolytic activity against macromolecular
substrates is inhibited; the proteinases retain
their activity against low molecular weight substrates, even when complexed with Cl2M.· The
explanation for this behaviour lies in the mechanism of inhibition. According to the trap hypothesis, the proteolytic cleavage of at least one subunit brings about a conformational change in the
azM which allows it to envelope the proteinase
and prevent interaction with macromolecular
substrates. The establishment of covalent bonds
binds the proteinase irreversibly to the Cl2M; the
complex is then rapidly eliminated by uptake into
macrophages or other cells [181]. The active
centre of the proteinase is involved in the initial
cleavage of the Cl2M but not in complex formation, and therefore retains it activity against low
molecular weight substrates. The Cl2M chain is
cleaved in a region in the last third of the molecule before the C-terminus; this contains peptide bonds that correspond to the different specificities of the individual proteinases [257]. It is
characteristic of Cl2M that it only inhibits that
activity of the bound proteinase that is directed
against protein substrates protecting it from other
macromolecular proteinase inhibitors; furthermore, the activity of Cl2M is itself inhibited by
methylamine. Based on this property, azM can be
detected in the blood plasma of all classes of vertebrates from the agnathans Petromyzon and
Myxine to the mammals [235]. The Cl2M forms of
the chicken and the frog Rana pipiens are apparently very similar to those of the mammals in
structure and mode of action [70]. In contrast, the
fish plasma protein (ClMh) that is homologous to
Cl2M is only half the size of the mammalian protein and consists of two heterodimers with 105and 90-kDa subunits (I and II) linked by a disulphide bridge. The evolution of ClMh to Cl2M either
involved the fusion of two genes (I and II) or, if I
and II are the products of one gene and arose by
post-translational modification, the cleavage site
has been lost in the evolution of Cl2M [259]. In the
pig, dog, rat and rabbit there are two structurally
different azMs whose relative proportions change
during development, pregnancy and acute
inflammation, and which apparently have different biological functions.
Cl2M-like proteinase inhibitors are also detectable in the xiphosuran Limulus polyphemus and
in several crustaceans. Sequence comparisons
between the thioester-containing regions or comparisons of the peptide cleavage patterns reveal
similarities between the inhibitors of L. polyphemus, Homarus americanus and Pacifastacus
leniusculus and the Cl2M of mammalian plasma.
Like the mammalian Cl2M, the inhibitors of the
above-mentioned arthropods are inactivated by
methylamine; an exception to this is found in
Cancer borealis [6,65]. The Cl2M-like protein
from Astacus haeniolymph is different to the proteinase inhibitor found in the haemocytes and
cuticle; the latter inhibitor is highly specific for
the proteases of the fungus Aphanomyces astaci,
the causative organism of the crayfish pest.
3 The Structural Variety and Metabolism of Proteins
10 kDa, which is produced in the fat bodies and
released in increasing amounts into the haemolymph during pupae development. Its function
would appear to be to protect tissues from attack
by proteinases, released during metamorphosis
from degraded larval cells [263].
3.4.3 Clz-Macroglobulins
Cl2-Macroglobulins are large glycoproteins present
in the blood of vertebrates and invertebrates and
in the egg albumin of birds and reptiles. The Cl2M
of mammalian plasma consists of four subunits
connected in pairs by disulphide bridges to form
dimers. The human subunits are made up of 1451
amino acids which agree at 73 % of positions with
those of the rat. This super-family also includes
the human pregnancy zone protein, the Clzmacroglobulin and the Clrinhibitor 3 of the rat.
Most members of the family are tetramers, but
some are dimers (frog Cl2M, human pregnancy
zone protein) or monomers (rat Cll-inhibitor 3).
Ovostatin, a proteinase inhibitor from chicken
egg albumin, is similar in sequence to the blood
plasma Cl2M at least at the N-terminus. There is
significant agreement between Cl2M and partial
sequences of the complement components C3
and C4 ofthe mouse; the latter, however, contain
regions that are not present in Cl2M. The evolution
of the complement components possibly included
a gene fusion (exon shuffling). The complement
components, like Cl2M, contain an internal thioester bond that is formed by reaction of a cysteine
thiol group with the y-carboxyl group of a neighbouring glutamate [256, 357].
The inhibitory action of azM is basically different from that of typical proteinase inhibitors;
Cl2M inhibits proteinases of all classes and the
formation of the Cl2M/enzyme is irreversible.
Only proteolytic activity against macromolecular
substrates is inhibited; the proteinases retain
their activity against low molecular weight substrates, even when complexed with Cl2M.· The
explanation for this behaviour lies in the mechanism of inhibition. According to the trap hypothesis, the proteolytic cleavage of at least one subunit brings about a conformational change in the
azM which allows it to envelope the proteinase
and prevent interaction with macromolecular
substrates. The establishment of covalent bonds
binds the proteinase irreversibly to the Cl2M; the
complex is then rapidly eliminated by uptake into
macrophages or other cells [181]. The active
centre of the proteinase is involved in the initial
cleavage of the Cl2M but not in complex formation, and therefore retains it activity against low
molecular weight substrates. The Cl2M chain is
cleaved in a region in the last third of the molecule before the C-terminus; this contains peptide bonds that correspond to the different specificities of the individual proteinases [257]. It is
characteristic of Cl2M that it only inhibits that
activity of the bound proteinase that is directed
against protein substrates protecting it from other
macromolecular proteinase inhibitors; furthermore, the activity of Cl2M is itself inhibited by
methylamine. Based on this property, azM can be
detected in the blood plasma of all classes of vertebrates from the agnathans Petromyzon and
Myxine to the mammals [235]. The Cl2M forms of
the chicken and the frog Rana pipiens are apparently very similar to those of the mammals in
structure and mode of action [70]. In contrast, the
fish plasma protein (ClMh) that is homologous to
Cl2M is only half the size of the mammalian protein and consists of two heterodimers with 105and 90-kDa subunits (I and II) linked by a disulphide bridge. The evolution of ClMh to Cl2M either
involved the fusion of two genes (I and II) or, if I
and II are the products of one gene and arose by
post-translational modification, the cleavage site
has been lost in the evolution of Cl2M [259]. In the
pig, dog, rat and rabbit there are two structurally
different azMs whose relative proportions change
during development, pregnancy and acute
inflammation, and which apparently have different biological functions.
Cl2M-like proteinase inhibitors are also detectable in the xiphosuran Limulus polyphemus and
in several crustaceans. Sequence comparisons
between the thioester-containing regions or comparisons of the peptide cleavage patterns reveal
similarities between the inhibitors of L. polyphemus, Homarus americanus and Pacifastacus
leniusculus and the Cl2M of mammalian plasma.
Like the mammalian Cl2M, the inhibitors of the
above-mentioned arthropods are inactivated by
methylamine; an exception to this is found in
Cancer borealis [6,65]. The Cl2M-like protein
from Astacus haeniolymph is different to the proteinase inhibitor found in the haemocytes and
cuticle; the latter inhibitor is highly specific for
the proteases of the fungus Aphanomyces astaci,
the causative organism of the crayfish pest.