(Received for publication, May 10, 1995)
From the
CD59 antigen is a membrane glycoprotein that inhibits the
activity of the C5b-9 membrane attack complex (MAC), thereby protecting
human cells from lysis by human complement. The inhibitory function of
CD59 derives from its capacity to interact with both the C8 and C9
components of MAC, preventing assembly of membrane-inserted C9 polymer.
MAC-inhibitory activity of CD59 is species-selective and is most
effective when both C8 and C9 derive from human or other primate
plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in
MAC, mediate virtually unrestricted lysis of human cells expressing
CD59. In order to identify the segment of human C8 that is recognized
by CD59, recombinant peptides containing human or rabbit C8 sequence
were expressed in Escherichia coli and purified. CD59 was
found to specifically bind to a peptide corresponding to residues
334-385 of the human C8 -subunit, and to require a disulfide
bond between Cys
and Cys
. No specific
binding was observed to the corresponding sequence from rabbit C8
(residues 334-386). To obtain functional evidence that this
segment of human C8
is selectively recognized by CD59, recombinant
C8 proteins were prepared by co-transfecting COS-7 cells with
human/rabbit chimeras of the C8
cDNA, and cDNAs encoding the
C8
and C8
chains. Hemolytic activity of MAC formed with
chimeric C8 was analyzed using target cells reconstituted with CD59.
These experiments confirmed that CD59 recognizes a conformationally
sensitive epitope that is within a segment of human C8
internal to
residues 320-415. Our data also suggest that optimal interaction
of CD59 with this segment of human C8
is influenced by N-terminal
flanking sequence in C8
and by human C8
, but is unaffected by
C8
.
Human CD59 antigen is a 18-21-kDa plasma membrane protein
that functions as an inhibitor of the C5b-9 membrane attack complex
(MAC) ()of human (hu) complement(1) . CD59 interacts
with both the C8 and C9 components of MAC during its assembly at the
cell surface, thereby inhibiting formation of the membrane-inserted C9
homopolymer responsible for MAC cytolytic activity (2, 3) . This serves to protect hu blood and vascular
cells from injury arising through activation of complement in plasma.
CD59's inhibitory activity is dependent upon the species of
origin of C8 and C9, with greatest inhibitory activity observed when C8
and C9 are from hu or other primates. By contrast, CD59 exerts little
or no inhibitory activity toward C8 or C9 of most other species,
including rabbit (rb) (4, 5, 6) .
Human C9
is a single-chain 72-kDa polypeptide, whereas C8 consists of a
heterotrimer of polypeptides, arranged as a disulfide-linked
-
subunit that is non-covalently associated with a
-chain(7, 8) . C8
and C8
chains exhibit
extensive sequence homology with C9. Analysis of the physical
association of CD59 with components of MAC suggested that separate
binding sites for CD59 are contained within the
-chain of hu C8
and within hu C9.(9) Consistent with the evidence for a CD59 binding
site in hu C8
, C8 hybrids formed by combining rb C8
-
with hu C8
displayed unrestricted hemolytic activity toward hu
erythrocytes (huE)(10) . Whereas the CD59 recognition site in
hu C9 has been localized to a segment of the polypeptide spanning
residues 359-415, the corresponding site within hu C8
remains to be identified(11, 12) . Comparison of the
aligned sequences of hu and rb C8
-chains revealed maximal
divergence of sequence between residues 349-385, suggesting that
this segment of the polypeptide might contain the site within hu C8
that is selectively recognized by CD59(10) . In this study, we
use recombinant C8
peptides and hemolytically active C8 hybrids
and chimeras containing hu and rb polypeptide sequence to determine the
contribution of this sequence to the species-selective interaction of
CD59 with the C8 component of hu MAC.
Fusion proteins were purified
from periplasmic extracts by amylose affinity chromatography, according
to the manufacturer's recommendation. Digestions with factor Xa
were performed in 20 mM Tris, 20 mM NaCl, pH 7.4 (pH
7.0 for rb C8), after which the sample was applied directly to a
Q-Sepharose (Sigma) column equilibrated in the same buffer. Released
peptide was collected in the wash and determined to be >95% pure
when analyzed on 15% acrylamide/Tricine SDS-polyacrylamide gel
electrophoresis gels. Electrophoretic mobility differences observed
upon reduction of the isolated peptide suggest that the single cysteine
pair (corresponding to Cys
and Cys
in hu
C8
) spontaneously forms an intrachain disulfide bond. Identity of
purified peptides was confirmed by amino acid analysis.
To prepare
Cys Gly mutants of hu C8
-derived peptide 334-385,
PCR-mediated site-directed mutagenesis was performed using overlapping
primers to change the two Cys codons(18) . The product was
cloned into pMAL-p2 and expressed as a fusion protein. The peptide was
released with factor Xa and purified as described above. For certain
experiments, cysteine residues of the purified peptides were reduced
and alkylated by incubation (37 °C, 60 min) with 10 mM dithiothreitol, followed by 30 mM iodoacetamide.
Figure 5:
Inhibitory function of CD59 requires hu
C8 residues 320-415. Bar graph (right panel)
summarizes combined results of all experiments performed under
conditions of Fig. 1with recombinant hu C8 containing chimeric
-subunits. From each C8 titration, the inhibitory activity of CD59
(expressed as the percentage of inhibition of hemolysis due to CD59, ordinate) was calculated at the concentration of C8 resulting
in 50% hemolysis in the absence of CD59. Error bars denote mean
± S.D. (n = 5); asterisks indicate
significance (p < 0.01) compared to recombinant rb C8. To
the left of each data bar, the protein tested is depicted so
as to designate those portions containing hu (open) or rb (shaded) C8
sequence. Human C8 and rabbit C8 denote C8 purified from hu and rb plasma, respectively.
Recombinant C8 proteins contain hu (H) or rb (R)
-chain sequence numbered according to the deduced primary
structure of the hu or rb mature polypeptide, respectively. In some
chimeric constructs, numbering appears discontinuous because of a gap
in the alignment of the rb and hu C8
sequences. In all cases,
chimeric
-chains were expressed with hu C8
and hu C8
chains. Domains depicted in the proposed structure of C8 include
thrombospondin type 1 (TS), low density lipoprotein receptor (LDLR), hinge (Hinge), membrane binding (MB), and epidermal growth factor precursor (EGFP).
Figure is adapted from (8) .
Figure 1:
Effect of CD59 on hemolytic activity of
recombinant C8. Functional assay of hu, rb, and hu/rb hybrids of
complement C8 measured in the absence () or presence (
)
of cell-surface CD59. Hemolytic activity of each protein (ordinate) was determined by titration of each C8 construct in
the presence of 50 ng/ml rb C9, using hu C5b-7, chE target cells
reconstituted with hu CD59 (see ``Experimental Procedures'').
Results shown are for plasma-derived hu C8 (panel A),
plasma-derived rb C8 (panel B), recombinant hu C8 (panelC), and recombinant hybrid C8 containing rb C8
, hu
C8
, and hu C8
(panel D).
Figure 2:
The rb/hu C8 hybrids reveal role of the
hu C8 chain in recognition by CD59. C8 hybrids composed of either
rb or hu
,
, and
subunits were expressed in COS-7 and
assayed for functional inhibition by CD59 under conditions described
for Fig. 1. From each C8 titration performed as in Fig. 1, the inhibitory activity of CD59 (expressed as the
percentage of inhibition of hemolysis due to CD59, ordinate)
was calculated at the concentration of C8 that resulted in 50%
hemolysis in the absence of CD59. HumanC8 and rabbitC8 represent the plasma-derived proteins; constructs 1-8 represent recombinant C8 containing the
indicated subunits. Bargraph summarizes combined
results of all experiments, normalized to results obtained in each
experiment for recombinant hu C8 (100%). Errorbars denote mean ± S.D.; parentheses indicate number of
independent experiments; asterisks indicate significance (p < 0.01) compared to recombinant rb C8. , significance (p < 0.06)
Figure 3:
CD59 binding to C8-chain peptide.
Microtiter plate wells were coated with 10 µg/ml of hu C8 or the
C8
peptide indicated (abscissa) and CD59 binding
performed as described under ``Experimental Procedures.'' Solid bars represent total binding; open bars represent nonspecific binding obtained in presence of 50-fold
excess unlabeled CD59. All absorbances (405 nm) are normalized to that
obtained in each separate assay for wells coated with 10 µg/ml hu
C8 (OD = 1.0), after correction for nonspecific binding to wells
coated with BSA. Error bars denote mean ± S.D. of all
results obtained from six separate experiments so performed. Results
shown are for plasma-derived hu C8, hu C8
peptide 334-385,
rb C8
peptide 334-386, and hu C8
peptide 334-385
containing Cys
Gly mutations at residues 345 and
369.
Figure 4:
Role of
hu C8 Cys
/Cys
disulfide in expression
of CD59 binding site. Microtiter plate binding of biotin-CD59 was
performed for wells coated with 10 µg/ml MBP-C8
334-385
or MBP-C8
334-385 containing Cys
Gly mutations at
Cys
and Cys
. hu C8 refers to wells
coated with plasma-derived hu C8. Solid bars denote specific
binding to each protein that was plate-coated without prior disulfide
reduction; hatched bars denote proteins that were reduced and
alkylated prior to plate coating (see ``Experimental
Procedures''). MBP itself contains no Cys residues, and therefore
MBP fusion proteins were employed for these experiments to circumvent
potential changes in plate-coating efficiency of free peptides after
reduction and alkylation. Absorbances (OD
; ordinate) are normalized to data obtained in each assay for hu
C8 (OD = 1), after correction for nonspecific binding to
MBP-lacZ
. Error bars denote mean ± S.D. of all
results obtained from six separate experiments so
performed.
The data of the present study identify the sequence between
residues 320-415 of the hu C8 -chain as critical to the
selective recognition of hu C8 by cell-surface CD59. Our experiments
also suggest that a disulfide-bonded loop contained within residues
334-385 (Cys
-Cys
) is required for
CD59 binding, providing an initial clue to the structural motif through
which this inhibitor selectively regulates the lytic activity of hu
MAC. These data further suggest that the capacity of CD59 to interact
with this segment of C8
is influenced by other portions of C8. In
particular, we note that replacing hu C8
with rb C8
decreased
the inhibitory activity of CD59 toward hu C8 (see constructs 7 and 8 in Fig. 2). Similarly, retention of N-terminal flanking rb sequence
in chimeric C8
constructs containing hu residues 320-415
partially decreases the inhibitory activity of CD59 toward an otherwise
hu C8 (see constructs 5 and 6 in Fig. 5). By contrast to the
partial loss of CD59 inhibitory function observed for the above C8
constructs, mere substitution of C8
residues 320-415 in hu
C8 with corresponding rb sequence (construct 4 in Fig. 5) is
sufficient to completely abrogate CD59's selective inhibition of
the lytic activity of hu C8, resulting in a protein that is
functionally indistinguishable from rb C8. Taken together, these data
support the interpretation that C8
residues 320-415 contain
the CD59 binding site in hu C8, but that the conformation of this site
is allosterically influenced by the hu C8
subunit as well as by
C8
sequence flanking this site. Our interpretation that
CD59's selective inhibitory activity toward hu C8 reflects its
capacity to bind hu C8
residues 320-415 is consistent with
the observed species-selective binding of CD59 to both hu C8
and
to the hu C8
-derived peptide 334-385 (Fig. 3), and by
the lack of detectable CD59 binding to either hu C8
or C8
subunits (9) . Nevertheless, we cannot exclude the possibility
that CD59 also interacts with these other regions of C8 that are
potentially exposed during MAC assembly.
Our conclusion that CD59
recognizes sequence unique to hu C8 as derived from the present
analysis of the interaction of recombinant C8 hybrids with purified and
membrane-reconstituted CD59 is consistent with results of earlier
experiments in which the lytic activity of plasma-derived forms of C8
toward huE were evaluated. In particular, it was observed that hu C8
and a derivative of hu C8 that lacked C8
were equally restricted
in their capacity to lyse huE, suggesting that the C8
subunit does
not contribute to the mechanism by which CD59 or other huE complement
regulatory factors restrict MAC assembly(21) . Furthermore, a
C8 hybrid formed by combining the isolated rb C8
-
and hu
C8
subunits exhibited the same level of unrestricted lytic
activity toward huE as was observed for rb C8, implicating C8
as
the determinant of species-selective restriction of C8 lytic
activity(10) . As discussed above, measurement of the physical
association of CD59 with isolated hu C8 subunits also revealed that
whereas CD59 binds specifically to C8
-
or C8
, such
binding is not detected for either C8
or C8
(9) .
Taken together with the results of the present study, these data
provide compelling evidence that the primary CD59 binding site in hu C8
is within the C8
subunit, centered on residues 334-385.
In addition to interacting with the C8 component of hu MAC, CD59 is
also known to interact with hu C9, a polypeptide that exhibits sequence
similarity to hu C8 and to several other MAC
components(9, 11, 12) . The CD59 recognition
domain in hu C9, as deduced from analysis of the lytic activity of
hu/rb C9 chimeras, was localized to residues 334-415, whereas
analysis of CD59 binding to hu C9-derived peptides suggested that C9
residues 359-411 were required for specific binding (11, 12) . It is of interest to note that from the
aligned sequences of hu C8
and hu C9, the site in C8
that we
now identify as conferring recognition by CD59 (C8
320-415)
overlaps the same region of C9 that was shown to contain the CD59
recognition domain (Fig. 6). Surprisingly, inspection of the
aligned amino acid sequences of hu C8
and C9 that span the
respective CD59 recognition sites of these two proteins reveals very
limited sequence identity and unremarkable sequence homology. This
suggests that the CD59 binding sites expressed by these two MAC
components are not structurally identical, but comprise distinct motifs
that are individually recognized by the inhibitor. In this context, it
is of interest to note that by contrast to the apparent conformational
sensitivity of the CD59 recognition site in hu C8
(see
above), the segment of hu C9 recognized by CD59 shows virtually no
dependence on flanking C9 sequence, nor does it require formation of
the intrachain disulfide (between C9
Cys
-Cys
, corresponding to C8
Cys
-Cys
) that is normally present in hu
C9(11, 19, 20, 22, 23) .
Figure 6:
Alignment of sequence within the CD59
recognition sites in hu C8 and C9. The segment of hu C8
identified to contain the site recognized by CD59 is aligned to the
corresponding segment of hu C9. Identity of this segment in C8
(residues 320-415) is from data of Fig. 5. Alignment is
based upon the entire amino acid sequence of the mature hu C8
and
C9 polypeptides (overall identity = 32%). Arrows depict
the peptide sequence from C8
(residues 334-385; see Fig. 3and Fig. 4) and from C9 (residues 359-411; from (11) ) shown to specifically bind CD59. Asterisks denote sequence identity; dashed lines denote the putative intrachain disulfide bond contained in this
segment of each polypeptide (see ``Discussion'').
Domain structure of the polypeptides is depicted as described in Fig. 5, except that C9 lacks the C-terminal thrombospondin
domain contained in C8
.
It remains unresolved how the interaction of CD59 with its binding
site in C8 serves to inhibit MAC assembly. In membranes expressing
CD59, there is reduced incorporation of C9 and reduced lytic activity
of MAC, suggesting that CD59 inhibits C9 binding to C5b-8 or subsequent
conformational changes required for membrane insertion and C9 polymer
formation(2, 3, 5) . The capacity of CD59 to
restrict C9 binding to C5b-8 as well as to inhibit MAC lytic activity
after C9 is initially bound implies potential inhibition at both steps
of MAC assembly(3, 5, 24) . In addition to
providing a binding site for CD59, the
-chain of C8 also has the
capacity to bind C9, and therefore this subunit of the C5b-8 complex is
thought to mediate binding and incorporation of C9 into
MAC(7, 9, 25) . This raises the possibility
that the segment of C8
to which CD59 binds either overlaps or is
closely apposed to a segment of C8
that interacts with C9,
accounting for CD59's inhibitory effect on MAC assembly. Of note,
whereas CD59 binding to C8 is highly species-selective, such species
selectivity is not observed for the interaction of C9 with C5b-8, in
the absence of CD59(5, 12) . This suggests that the C9
and CD59 binding sites in C8
that are both exposed when C8
incorporates into MAC are not identical.