330
P. M. RUDD et al.
6
Inhibitors of the Complement Pathway, DAF (CD55)
and CD59 Protect Cells from Complement Mediated Cell Lysis
The complement system can be activated by either of two pathways. The classical
pathway is triggered by antigen-antibody complexes, specifically those of IgM
and IgG. The alternative pathway is activated by the surfaces of foreign bodies
such as viruses, fungi and bacteria. Activation by either pathway leads to the production of the bimolecular complex, C3 convertase. The C3 convertase enzyme
cleaves C3 into C3b and C3a. C3b can covalently attach to the particle or cell with
the resultant opsonisation leading to clearance by phagocytic cells with C3b
receptors. DAF and CD59 are glycosylphosphatidylinositol (GPI) anchored glycoproteins which protect cells from complement-mediated damage. The active site
of CD59 is located close to the cell surface at 3.5 nm from the membrane while
that of DAF is further away, at 16 nm (Fig. 23.7). While CD59 binds the C5b-8
component of the MAC, DAF both dissociates the classical and the alternative
pathway C3/C5 convertases and inhibits their assembly.
(i) The O-Glycosylated Region of DAF Serves as a Protease Resistant Spacer
to Position the Active Site
DAF is composed offour complement control protein (CCP) domains suspended
above the membrane by a heavily O-glycosylated Ser/Thr rich region of approximately 70 amino acids which is linked to the GPI anchor (Fig. 23.7). The structures of the O-glycans have been determined by glycan sequencing of the sugars
released by hydrazine under conditions optimised for O-glycan release (Rudd,
P.M., Morgan, B.P., Harris, c., Wormald, M.R. and Dwek, R.A.- unpublished
data). The regulatory activity against C4b2a is localised in CCP domains 2-4
while its activity against C3bBb also involves CCP domain 4. There is oneNglycosylation site which is located between CCP1 and CCP2. The N-glycan site
has been deleted without any associated loss of activity (Coyne et aI., 1992). However, deletion of the Ser/Thr rich region eliminated DAF function. This was
restored in a fusion construct in which the four CCP domains were added to the
HLA B44 molecule suggesting that the O-glycosylated region serves as an important protease resistant spacer which projects the DAF functional domains above
the plasma membrane (Coyne et al., 1992).
(ii) Potential Roles for the Glycans Attached to CD 59
CD 59 (Fig. 23.7) belongs to the Ly-6 superfamily and is present on a wide variety
of cell types, including leukocytes, platelets, epithelial and endothelial cells, placental cells and erythrocytes. CD59 is attached to the surfaces of these cells by
means of a GPI anchor containing three lipid chains (Davies et al., 1989, Rudd et
aI., 1997). Human erythrocyte CD59 consists of a heterogeneous mixture of more
than 120 glycoforms (Fig. 23.8) of which the major single sugar is a complex glycan containing both a bisecting GlcNAc residue and a core fucose (Rudd et aI.,
1997). A population of sialylated O-glycans was recovered from human erythrocyte CD59; the major species which were identified were two mono-sialylated
forms of the disaccharide Gal~1,3GalNAc. 90 % of the GPI anchor glycans contain
the tri-mannose sugar common to all mammalian anchors analysed to date.
P. M. RUDD et al.
6
Inhibitors of the Complement Pathway, DAF (CD55)
and CD59 Protect Cells from Complement Mediated Cell Lysis
The complement system can be activated by either of two pathways. The classical
pathway is triggered by antigen-antibody complexes, specifically those of IgM
and IgG. The alternative pathway is activated by the surfaces of foreign bodies
such as viruses, fungi and bacteria. Activation by either pathway leads to the production of the bimolecular complex, C3 convertase. The C3 convertase enzyme
cleaves C3 into C3b and C3a. C3b can covalently attach to the particle or cell with
the resultant opsonisation leading to clearance by phagocytic cells with C3b
receptors. DAF and CD59 are glycosylphosphatidylinositol (GPI) anchored glycoproteins which protect cells from complement-mediated damage. The active site
of CD59 is located close to the cell surface at 3.5 nm from the membrane while
that of DAF is further away, at 16 nm (Fig. 23.7). While CD59 binds the C5b-8
component of the MAC, DAF both dissociates the classical and the alternative
pathway C3/C5 convertases and inhibits their assembly.
(i) The O-Glycosylated Region of DAF Serves as a Protease Resistant Spacer
to Position the Active Site
DAF is composed offour complement control protein (CCP) domains suspended
above the membrane by a heavily O-glycosylated Ser/Thr rich region of approximately 70 amino acids which is linked to the GPI anchor (Fig. 23.7). The structures of the O-glycans have been determined by glycan sequencing of the sugars
released by hydrazine under conditions optimised for O-glycan release (Rudd,
P.M., Morgan, B.P., Harris, c., Wormald, M.R. and Dwek, R.A.- unpublished
data). The regulatory activity against C4b2a is localised in CCP domains 2-4
while its activity against C3bBb also involves CCP domain 4. There is oneNglycosylation site which is located between CCP1 and CCP2. The N-glycan site
has been deleted without any associated loss of activity (Coyne et aI., 1992). However, deletion of the Ser/Thr rich region eliminated DAF function. This was
restored in a fusion construct in which the four CCP domains were added to the
HLA B44 molecule suggesting that the O-glycosylated region serves as an important protease resistant spacer which projects the DAF functional domains above
the plasma membrane (Coyne et al., 1992).
(ii) Potential Roles for the Glycans Attached to CD 59
CD 59 (Fig. 23.7) belongs to the Ly-6 superfamily and is present on a wide variety
of cell types, including leukocytes, platelets, epithelial and endothelial cells, placental cells and erythrocytes. CD59 is attached to the surfaces of these cells by
means of a GPI anchor containing three lipid chains (Davies et al., 1989, Rudd et
aI., 1997). Human erythrocyte CD59 consists of a heterogeneous mixture of more
than 120 glycoforms (Fig. 23.8) of which the major single sugar is a complex glycan containing both a bisecting GlcNAc residue and a core fucose (Rudd et aI.,
1997). A population of sialylated O-glycans was recovered from human erythrocyte CD59; the major species which were identified were two mono-sialylated
forms of the disaccharide Gal~1,3GalNAc. 90 % of the GPI anchor glycans contain
the tri-mannose sugar common to all mammalian anchors analysed to date.