240
6 Immunoproteins
cells of a sponge are separated mechanically or
chemically, completely functional small sponges
form in the cell suspension within a matter of
days. Because of the great mobility of the sponge
cells and the dynamic changeability of the structures they form, even this primitive multicellular
organism requires highly developed mechanisms
for cell-cell recognition; these may be effectively
investigated by such reaggregation experiments.
Two aggregation systems can be distinguished
in most sponge species: a non-species-specific primary system and a species-specific secondary system, which is lacking only in the calcareous sponges (Calcarea). Both require Ca 2 + and therefore
treatment with ethylenediaminetetraacetic acid
(EDT A) leads to cell separation. In the primary
process, aggregates of about 400-500 !-tm in diameter are formed; this also applies to mixtures of
cells from different species, e.g. Cliona celata +
Geodia cydonium. The responsible factor here is
a primary aggregation factor (pAF) on the cell
surface and this has been purified from G. cydonium. It is a glycoprotein with 50 % carbohydrate
and consists of three polypeptides of 16.5, 15.5
and 13.5 kDa [118, 119].
In the secondary process, for example in
G. cydonium, large aggregates with a diameter of
more than 5 mm are formed, and can differentiate into small functional sponges. The secondary
aggregation factors (sAP) from Microciona prolifera and G. cydonium are large glycoproteins
of complex structure. The aggregation factor
from M. prolifera is a proteoglycan of 2 . 10 7 Da
containing 60-70 % carbohydrate [115]. The
aggregation factor from Geodia is a bivalent molecule with the capacity to link the aggregation
receptors (AR) of different cells; in addition, it
has both glucuronyl transferase and galactosyl
transferase activity. sAF-AR binding initiates a
process of signal transduction across the cell
membrane, in the course of which particular nuclear proteins are phosphorylated with the
involvement of inositol trisphosphate and diacylglycerol as secondary messengers [156]. The
aggregation factor of G. cydonium is associated
with a 32-kDa protein, calpactin, which binds
Ca 2 + ions in cooperation with a membranebound phospholipid. The sequence of this protein, derived from its cDNA, agrees 80 % with
that of vertebrate calpactin II [149]. The
membrane-bound AR from M. prolifera has been
purified and characterized; it is a glycoprotein of
15-18 kDa with 81 % carbohydrate. Binding of
sAP and AR is via a lysine residue of the aggregation factor and the terminal glucoronic acid of
the receptor. Cleavage at the glucuronyl residue
by a membrane-bound glucuronidase results in
cell separation; conversely, glucuronyl transfer
by the sAF restores the aggregation capacity. In
parallel, there is a further regulatory mechanism
in which an anti-aggregation receptor on the cell
surface interacts with the sAF-AR complex and
cleaves off a terminal galactose residue. The antiaggregation receptor of Geodia is a glycolipoprotein of about 180 kDa. It can be inactivated by a
galactose-specific lectin, and it may be that the
cell's own galactosidase and the galactosyl transferase activity of the sAP are involved in inactivation and reactivation. The species specificity of
cell aggregation is determined largely by the specific structure of the aggregation factor and its
receptor. In addition, transplant rejection may
occur through the action of an "inhibiting
aggregation factor", a glycoprotein of 26 kDa
with 60 % carbohydrate [118].
6.9.3 The Variable Surface Glycoproteins
of the Trypanosomes
The cell surface of the pathogenic African Trypanosoma species that infest mammals, e.g. T. brucei and T. congolense, which cause the nagana disease of cattle, or T. equiperdum, which is the
causative agent of horse murrain, is completely
covered by a 12- to 15-nm-thick layer of identical
glycoprotein molecules which constitute 7-10 %
of the total cell protein. The variability of these
surface proteins is unique; one single clone, i.e.
the progeny of a single cell, of T. equiperdum was
found to possess more than 100 different variant
proteins with no common immunological determinants. Alterations in these "variant-specific
surface glycoproteins" (VSGs) at a frequency of
about 10-6 per cell division enable the parasite to
avoid the immune defences of the host. The VSGs
are made up of polypeptide chains with a length
of 450-500 amino acids and these chains form a
compact aggregate of homodimers on the cell surface. The outwardly directed N-terminal domains
have several variable antigen determinants along
a length of about 400 amino acids. The Cterminal domain carries several oligosaccharides,
giving a carbohydrate content of 7-17 % [139].
Anchorage of VSGs in the membrane is brought
about by binding of the C-terminus to a complex
glycosyl-phosphatidyl-inositol via a covalent
bond between the carboxyl group and an ethanolamine residue. The precursor of this structure in
T. brucei is the free glycolipid ethanolamine-
6 Immunoproteins
cells of a sponge are separated mechanically or
chemically, completely functional small sponges
form in the cell suspension within a matter of
days. Because of the great mobility of the sponge
cells and the dynamic changeability of the structures they form, even this primitive multicellular
organism requires highly developed mechanisms
for cell-cell recognition; these may be effectively
investigated by such reaggregation experiments.
Two aggregation systems can be distinguished
in most sponge species: a non-species-specific primary system and a species-specific secondary system, which is lacking only in the calcareous sponges (Calcarea). Both require Ca 2 + and therefore
treatment with ethylenediaminetetraacetic acid
(EDT A) leads to cell separation. In the primary
process, aggregates of about 400-500 !-tm in diameter are formed; this also applies to mixtures of
cells from different species, e.g. Cliona celata +
Geodia cydonium. The responsible factor here is
a primary aggregation factor (pAF) on the cell
surface and this has been purified from G. cydonium. It is a glycoprotein with 50 % carbohydrate
and consists of three polypeptides of 16.5, 15.5
and 13.5 kDa [118, 119].
In the secondary process, for example in
G. cydonium, large aggregates with a diameter of
more than 5 mm are formed, and can differentiate into small functional sponges. The secondary
aggregation factors (sAP) from Microciona prolifera and G. cydonium are large glycoproteins
of complex structure. The aggregation factor
from M. prolifera is a proteoglycan of 2 . 10 7 Da
containing 60-70 % carbohydrate [115]. The
aggregation factor from Geodia is a bivalent molecule with the capacity to link the aggregation
receptors (AR) of different cells; in addition, it
has both glucuronyl transferase and galactosyl
transferase activity. sAF-AR binding initiates a
process of signal transduction across the cell
membrane, in the course of which particular nuclear proteins are phosphorylated with the
involvement of inositol trisphosphate and diacylglycerol as secondary messengers [156]. The
aggregation factor of G. cydonium is associated
with a 32-kDa protein, calpactin, which binds
Ca 2 + ions in cooperation with a membranebound phospholipid. The sequence of this protein, derived from its cDNA, agrees 80 % with
that of vertebrate calpactin II [149]. The
membrane-bound AR from M. prolifera has been
purified and characterized; it is a glycoprotein of
15-18 kDa with 81 % carbohydrate. Binding of
sAP and AR is via a lysine residue of the aggregation factor and the terminal glucoronic acid of
the receptor. Cleavage at the glucuronyl residue
by a membrane-bound glucuronidase results in
cell separation; conversely, glucuronyl transfer
by the sAF restores the aggregation capacity. In
parallel, there is a further regulatory mechanism
in which an anti-aggregation receptor on the cell
surface interacts with the sAF-AR complex and
cleaves off a terminal galactose residue. The antiaggregation receptor of Geodia is a glycolipoprotein of about 180 kDa. It can be inactivated by a
galactose-specific lectin, and it may be that the
cell's own galactosidase and the galactosyl transferase activity of the sAP are involved in inactivation and reactivation. The species specificity of
cell aggregation is determined largely by the specific structure of the aggregation factor and its
receptor. In addition, transplant rejection may
occur through the action of an "inhibiting
aggregation factor", a glycoprotein of 26 kDa
with 60 % carbohydrate [118].
6.9.3 The Variable Surface Glycoproteins
of the Trypanosomes
The cell surface of the pathogenic African Trypanosoma species that infest mammals, e.g. T. brucei and T. congolense, which cause the nagana disease of cattle, or T. equiperdum, which is the
causative agent of horse murrain, is completely
covered by a 12- to 15-nm-thick layer of identical
glycoprotein molecules which constitute 7-10 %
of the total cell protein. The variability of these
surface proteins is unique; one single clone, i.e.
the progeny of a single cell, of T. equiperdum was
found to possess more than 100 different variant
proteins with no common immunological determinants. Alterations in these "variant-specific
surface glycoproteins" (VSGs) at a frequency of
about 10-6 per cell division enable the parasite to
avoid the immune defences of the host. The VSGs
are made up of polypeptide chains with a length
of 450-500 amino acids and these chains form a
compact aggregate of homodimers on the cell surface. The outwardly directed N-terminal domains
have several variable antigen determinants along
a length of about 400 amino acids. The Cterminal domain carries several oligosaccharides,
giving a carbohydrate content of 7-17 % [139].
Anchorage of VSGs in the membrane is brought
about by binding of the C-terminus to a complex
glycosyl-phosphatidyl-inositol via a covalent
bond between the carboxyl group and an ethanolamine residue. The precursor of this structure in
T. brucei is the free glycolipid ethanolamine-