(Received for publication, June 5, 1995; and in revised form, September 7, 1995 )
From the
Human C4b-binding protein (C4BP), which is a regulator of the
classical complement pathway C3 convertase, forms high affinity
complexes with anticoagulant protein S and with the pentraxin serum
amyloid P component (SAP). SAP is a plasma protein present in all
amyloid deposits. Recently, SAP was shown to inhibit the complement
regulatory functions of C4BP. In this investigation, we have studied
the structural requirements for the C4BP-SAP interaction. C4BP was
subjected to chymotrypsin digestion, which yielded two major fragments
corresponding to the central core (160 kDa) and to the cleaved-off
tentacles (48 kDa). SAP-Sepharose specifically bound the 160-kDa
fragment, suggesting that the central core of C4BP contains the binding
site for SAP. In a quantitative affinity chromatography assay, the
dissociation constants for binding of intact C4BP and of the 160-kDa
central core fragment to SAP were found to be 30 and 70 nM,
respectively. Recombinant C4BP composed of only -chains bound SAP
with similar affinity (K
= 22
nM), whereas nonglycosylated recombinant
-chain C4BP
(synthesized in the presence of tunicamycin) bound SAP with lower
affinity (K
= 126 nM).
This suggests that the carbohydrate moiety of the central core of C4BP
is important for binding of C4BP to SAP in contrast to the C4BP
-chain, which is not required. EDTA, heparin, and
phosphorylethanolamine as well as a peptide comprising amino acids
27-39 of SAP were found to completely displace C4BP from the SAP
matrix. Moreover, the immobilized SAP peptide bound C4BP in a reaction
that, in contrast to the C4BP-SAP interaction, was not dependent on
calcium.
C4b-binding protein (C4BP) ()is a regulator of the
complement system(1) . It binds multiple C4b molecules,
inhibits the assembly of the classical pathway C3 convertase (C4b2a),
dissociates C2a from the convertase, and functions as cofactor to
factor I in the degradation of C4b. In human plasma, C4BP also
interacts with the anticoagulant vitamin K-dependent protein S (2) and with serum amyloid P component
(SAP)(3, 4) . C4BP is a high molecular weight (M
= 570,000) plasma glycoprotein composed
of seven identical
-chains and one
-chain, the arrangement of
which gives C4BP an octopus-like structure(1, 5) . The
-chain (M
= 70,000) contains eight
tandemly repeated, internally homologous modules, designated short
consensus repeats (SCRs), and a C-terminal region that contains two
cysteines that are involved in interchain disulfide
bridging(6) . Each
-chain contains a binding site for C4b,
the detailed location of which is not known. The
-chain (45 kDa),
which is related structurally to the
-chain, contains three SCR
modules and a carboxyl-terminal region with two cysteines(7) .
The
-chain has three sites for N-linked glycosylation,
one in SCR-3 and two in SCR-8, whereas the
-chain contains five
sites for N-linked glycosylation, two in each of SCR-1 and
SCR-2 and one in SCR-3.
SAP is a serum glycoprotein composed of 10 identical, noncovalently linked 25-kDa subunits, which are arranged in two parallel cyclic pentagonal structures interacting face-to-face(8) . SAP is related in structure to C-reactive protein, and both proteins belong to the pentraxin protein family(9, 10) . Pentraxins have been highly conserved through evolution and are found in all vertebrate species. There are also pentraxins described in invertebrates(11) . Although the physiological function of SAP is largely unknown, SAP has received considerable medical interest as it associates with all types of amyloid deposits, including those of Alzheimer's disease(9, 12) . SAP has a high tendency to undergo calcium-dependent self-aggregation, which may be important for its deposition in amyloid. It has lectin-like properties and binds heparin, heparan and dermatan sulfate, and phosphorylethanolamine(13, 14, 15) . Another property of SAP is its ability to bind DNA and chromatin(16) . SAP displaces type H1 histones and renders solubility to the otherwise insoluble chromatin (17, 18) . SAP has also been reported to interact with fibronectin (19) and hirudin(13) . All these interactions are calcium-dependent and appear to involve a unique binding site in the SAP molecule and ligands containing phosphorylated or possibly sulfated residues(20, 21) .
Despite earlier reports showing
the ability of C4BP to bind immobilized SAP(19) , the presence
of a C4BPSAP complex in blood has only recently been
recognized(3, 4) . The latter studies demonstrate that
C4BP interacts with nonaggregated SAP in fluid phase and that the two
proteins form a 1:1 stoichiometric complex in plasma. Due to the
equimolar concentrations of the two proteins and the high affinity of
the interaction, essentially all SAP in blood is bound to
C4BP(3) , even under acute-phase conditions(22) .
Sucrose density gradient experiments have shown the complex between
SAP, C4BP, and protein S to have the potential to interact with
fluid-phase C4b, suggesting that the binding sites for the different
molecules on C4BP are distinct(3) . However, recent results
have shown SAP binding to C4BP to be associated with inhibition of the
factor I cofactor activity of C4BP and with inhibition of C4BP binding
to C4b-coated surfaces (23) .
In this study, we have localized the SAP-binding site to the 160-kDa chymotryptic fragment constituting the central core of C4BP and demonstrate that the binding is influenced by the presence of N-linked carbohydrates on C4BP. A SAP peptide previously reported to support cell attachment (24) is shown to be involved in the interaction with C4BP. The results provide basis for further understanding of the physiological role of SAP.
Figure 1:
Endo-N-glycosidase F treatment
of plasma-derived and recombinant C4BP. C4BP purified from plasma and
rC4BP expressed in eucaryotic cells in the presence or absence of
tunicamycin were analyzed before and after endo-N-glycosidase
F treatment. After glycosidase treatment, samples were reduced,
electrophoresed on 10% SDS-polyacrylamide gel, and silver-stained. The two arrows to the right point to the completely
nonglycosylated
-chain of C4BP (65 kDa) and to
endo-N-glycosidase F (PNGase). No effect of
endo-N-glycosidase F on nonglycosylated (non-glyc.)
rC4BP (from tunicamycin-treated cells) was observed, while the
molecular masses of both plasma C4BP and
rC4BP decreased from an
apparent molecular mass of
70 kDa to 65
kDa.
The K values for the interactions with the
SAP matrix (M) were estimated by fitting the data to ,
where the subscripts B, F, and T denote bound, free, and total
concentrations, respectively, and K and K
are the dissociation constants of C4BP to
the SAP matrix and SAP in solution, respectively.
and assume a 1:1 stoichiometry of the C4BP-SAP interaction (3) and [M]
[C4BP]
. Under the experimental conditions,
[SAP]
[C4BP]
, and
therefore, [SAP]
was a good approximation of
[SAP]
. The K
for the
interaction between the SAP matrix and the different C4BP fragments or
recombinant C4BP had the same form as the previous
equation(29) . This assumes a 1:1 stoichiometry of the
interaction and an excess of matrix over the C4BP tracer. The results
are expressed as fraction of C4BP bound as a function of the
concentration of C4BP
(C4BP fragments or recombinant
C4BP). As [C4BP
]
[C4BP]
or [M]
, the total
concentration of C4BP
is a good approximation of its free
concentration. Dissociation constants were calculated by nonlinear
regression with the STATISTICA program using mean values of at least
five determinations.
Figure 2:
Binding
of C4BP to SAP immobilized on matrix. A, measurement of C4BP
binding to a fixed concentration of SAP matrix as a function of the
C4BP concentration. The experiment was performed as described under
``Experimental Procedures'' with 50 µl of packed SAP
matrix dissolved in a total volume of 300 µl. The solid line was drawn using K = 30 nM and a maximal concentration of C4BP-binding sites on the SAP
matrix of 60 nM. The variance of the curve was r
= 0.97. B, measurement of C4BP
binding to varying amounts of SAP matrix at a fixed C4BP concentration.
The solid line (r
= 0.95) was
drawn using K
= 38 nM and
assuming a maximal fraction of C4BP of 1 at infinite concentration of
SAP matrix. C, measurement of the fraction of C4BP bound to
the SAP matrix as a function of varying concentrations of free SAP. The
experiment was performed with a fixed C4BP concentration and 50 µl
of SAP matrix. The solid line represents the best fit of data
to , with [M]/K
= 1.80, as calculated from the previous measurements, and K
= 15 nM (r
= 0.89). Data in A and C are the means
± S.E. of at least five experiments. Those in B are the
mean of two measurements.
The purified 160-kDa central core fragment of C4BP
competitively inhibited binding of C4BP to SAP-Sepharose, whereas the
48-kDa fragment was without effect (Fig. 3A). The
calculated K for the interaction between the
160-kDa central core fragment and the SAP matrix (K
= 70 nM) was
2-fold lower than the K
for the C4BP-SAP matrix interaction (K
). To further characterize binding of the
central core fragment to the SAP matrix, a purified 160-kDa core
preparation was radiolabeled and directly tested for binding to
SAP-Sepharose (Fig. 3B). The calculated K
(K
) was 58
nM. The concentration of core-binding sites on the SAP matrix
(105 nM) was distinctly higher than that measured for intact
C4BP (60 nM), indicating that the number of SAP molecules
accessible to the central core is higher than those accessible for
intact C4BP. The lower accessibility of C4BP for SAP as compared with
the central core fragment was presumably the result of steric hindrance
by the extended C4BP tentacles.
Figure 3:
Binding of C4BP chymotryptic fragments to
SAP immobilized on matrix. A, measurement of C4BP bound to the
SAP matrix as a function of varying concentrations of C4BP fragments.
The solid lines represent the best fit of data to . [M]/K was fixed to
1.82, and the K
value was fixed to 70 nM for the C4BP core fraction (
; r
=
0.94) and 2970 nM for the C4BP tentacle fraction (
; r
= 0.26). Data are the means ± S.E.
of five (core) or three (tentacles) experiments. B,
measurement of 160-kDa C4BP core binding to a fixed concentration of
SAP matrix as a function of core concentration (
). The
experiment was performed as described for plasma-purified C4BP (see Fig. 2and ``Results'') in a total volume of 300
µl. The solid line was drawn using K
= 58 nM and a maximal concentration of
C4BP-binding sites of 105 nM. The variance of the curve was r
= 0.96. Data are the means ± S.E.
of three experiments.
Binding of rC4BP to SAP was
also characterized in the QAC system (Fig. 4).
rC4BP was
found to bind to the SAP matrix in a similar fashion as plasma-derived
C4BP (K
= 22 nM), which
demonstrates that the
-chain of C4BP is not involved in the
interaction between C4BP and SAP. Recombinant C4BP obtained from
tunicamycin-treated cells lacks N-linked carbohydrates and was
found to be less efficient than intact C4BP in displacing the C4BP
tracer from the SAP matrix (K
= 126 nM). This suggests involvement
of the N-linked carbohydrate moiety of C4BP in the C4BP-SAP
interaction. However, removal of neuramidate residues by neuraminidase
treatment of plasma-derived C4BP did not affect its ability to bind SAP (K
= 21 nM) (data not
shown).
Figure 4:
Binding of recombinant C4BP to SAP
immobilized on matrix. Shown is the measurement of C4BP bound to the
SAP matrix as a function of varying concentrations of recombinant C4BP.
The solid lines represent the best fit of data to . [M]/K was fixed to
1.82, and the K
Figure 5:
Displacement of C4BP from SAP-Sepharose by
SAP ligands and peptides. A, heparin (),
phosphorylethanolamine (
), phosphorylcholine (
), and
phosphorylserine (
) were added at increasing concentrations to
fixed amounts of C4BP and SAP matrix. The displacement of C4BP was
measured as percentage of bound C4BP to C4BP bound in the absence of
SAP ligands. B, Pep-1 (
,
), containing the
27-39-amino acid sequence of SAP, or a peptide containing the
same amino acids scrambled (Pep-2;
,
) was added in
increasing concentrations to a fixed amount of C4BP and SAP matrix. The
displacement of C4BP was measured as percentage of bound C4BP to C4BP
bound in the absence of peptides. The peptides were used either reduced
(
,
) or reduced and carboxymethylated (
,
).
Data are the mean of two experiments.
A peptide comprising amino acids 27-39 of SAP (Pep-1) has been shown to sustain cell binding when coupled to surfaces (24) and to displace SAP from heparin and other ligands(21) . The effect of Pep-1 on C4BP binding to the SAP matrix was tested in the QAC system (Fig. 5B). The presence of 20 µM Pep-1 completely displaced C4BP from the SAP matrix. Carboxymethylated Pep-1 was even more efficient in displacing C4BP from SAP-Sepharose. A peptide with a scrambled sequence (Pep-2) gave only a 20% displacement at 120 µM.
Figure 6:
Binding of plasma and recombinant C4BP to
immobilized Pep-1. A radioactive C4BP tracer was incubated with
increasing concentrations of Pep-1 (,
) and Pep-2 (
,
) in microtiter wells with immobilized Pep-1. The peptides were
used either reduced (
,
) or reduced and carboxymethylated
(
,
). Data are the mean of at least two experiments. Inset, measurement of C4BP binding to Pep-1 with different
concentrations of calcium or EDTA in the buffer. Data are the means
± S.E. of three experiments.
The interaction between SAP and C4BP that occurs in the blood stream (3, 4) affects the functions of both proteins. Thus, C4BP bound to SAP has reduced capacity to regulate the classical C3 convertase of complement(23) . Moreover, calcium-dependent self-aggregation of SAP is inhibited by binding of C4BP to SAP(3) , which may be important to keep SAP soluble in blood. We have now characterized the C4BP-SAP interaction using a quantitative affinity chromatography method and elucidated some of the structural requirements for this protein-protein interaction.
The -chain
of C4BP, which is imperative for the interaction between C4BP and
protein S, was found to be dispensable for binding of C4BP to SAP.
Thus, the multiple carbohydrate site chains of the
-chain do not
affect binding of C4BP to SAP. We have recently shown that C4BP lacking N-linked carbohydrate side chains is able to bind
SAP-Sepharose(27) , which is surprising because SAP is known to
recognize carbohydrate chains(21) . However, the carbohydrate
side chains still appear to affect the C4BP-SAP interaction because N-nonglycosylated C4BP bound SAP with 4-fold lower affinity
than intact C4BP. Treatment of C4BP with neuraminidase did not impede
binding of C4BP to SAP-Sepharose, indicating that terminal sialic acids
are not involved in the interaction.
Digestion of C4BP with
chymotrypsin results in two major fragments, the 160-kDa central core
and the 48-kDa cleaved-off tentacles(32) . From a chymotryptic
digest, the core fraction was retained on a SAP-Sepharose column,
suggesting that this portion of the molecule contains the SAP-binding
site. This was further shown by the specific displacement of C4BP from
SAP-Sepharose by the central core fragment. However, it cannot be
excluded that the cleaved-off tentacle fragments, to some extent,
affect the C4BP-SAP interaction because the affinity of the binding of
the central core to SAP was consistently found to be 2-fold lower than
that of the binding of intact C4BP to SAP. It is noteworthy that the
eight SCRs and part of the C-terminal non-repeat region of the C4BP
-chain belong to the best preserved regions of the molecule in
different species(33, 34) . This part of C4BP is
presumably involved in the polymerization of the chains that occurs
during its biosynthesis. It might also be speculated that a SAP-binding
motif is preserved in this part of the molecule.
Both the
interaction between C4BP and immobilized C4b and the factor I cofactor
activity of C4BP are modified by binding of SAP to C4BP(23) .
SAP probably affects C4b binding by an allosteric mechanism rather than
by direct steric hindrance because the binding site on C4BP for C4b
appears to be located within the three most N-terminal SCRs of the C4BP
-chain. Thus, these SCRs of mouse C4BP were found to support
binding of C4b(35) . Moreover, results of electron microscopy
of the C4BP
C4b complex (36) and comparisons of C4BP
sequences of different species (34) suggested that the
C4b-binding site is located within the first three SCRs. SAP may bind
to multiple
-chains (to SCR-8) of the same C4BP molecule, thus
acting as a cross-linker. This may lock the C4BP molecule in a
``closed'' conformation similar to that suggested by Perkins et al.(28) (Fig. 7). This conformation is
quite different from the ``open'' conformation observed in
electron microscopy(36) , and this difference suggests that the
C4BP molecule is highly flexible. The locked closed conformation may
reduce the ability of C4BP to interact with multiple C4b molecules on a
surface and impede the factor I cofactor activity of C4BP(23) .
This model is compatible with the observed ability of C4BP to form a
ternary complex with SAP and C4b in fluid phase and the ability of this
complex to bind protein S(4) . The model may also explain how a
monoclonal antibody reacting with SCR-6 is able to inhibit the
interaction between C4b and C4BP(37) . The monoclonal antibody
may act as SAP acts, locking C4BP in a closed conformation.
Figure 7:
Schematic model of C4BP-SAP interaction. A, the seven 70-kDa -subunits (white or shaded) are arranged as suggested by Perkins et
al.(28) . SAP is represented by the two black
ovals. SAP binds to the central portion of C4BP (SCR-8 and the
C-terminal domain of C4BP). This interaction may lock C4BP in a closed
structure, as illustrated. C4BP interacts with protein S via its
-chain, which is not involved in the interaction with SAP. B, shown is a schematic representation of the 160-kDa central
core of C4BP and the SAP pentamer. The approximate position of Pep-1 in
the SAP monomer is indicated in white(39) . The size
of the SCRs is drawn to scale according to the schematic representation
of Barlow et al.(44) . The overall size of the central
core is taken from results of electron microscopy (36) .
Several
compounds known to interfere with C4BPSAP complex formation were
tested in the QAC method, and the obtained results were in accordance
with those on record(4, 15, 21) .
Phosphorylethanolamine efficiently disrupted the C4BP-SAP interaction;
phosphorylcholine had only a minor effect; and phosphorylserine was
without effect. A recent report demonstrated that the SAP decamer
contains only one binding site for phosphorylcholine, whereas
phosphorylethanolamine probably binds each SAP monomer(38) .
This may help explain the potent effect of phosphorylethanolamine as
each of the SAP monomers is likely to participate in C4BP binding (Fig. 7). A peptide comprising amino acids 27-39 of SAP
(Pep-1) was able to displace C4BP from the SAP matrix and also showed
binding properties by itself. It was noteworthy that calcium was not
required for binding of C4BP to Pep-1, in contrast to the C4BP-SAP
interaction, which is dependent on the presence of calcium (3) . The recent resolution of the three-dimensional structure
of SAP demonstrated that both the amino acid residues that bind calcium
(distinct from those of Pep-1) and calcium ions are directly involved
in binding acidic groups of SAP ligands (phosphate in
phosphorylethanolamine and acetate in the 4,6-cyclic pyruvate acetal of
-D-galactose)(39) . In contrast, binding of SAP
to C-reactive protein appears to be mediated by the C-terminal region
of SAP(40) . In addition, binding of SAP to immune complexes is
not dependent on calcium(41) . Thus, these interactions do not
seem to involve the same binding site on SAP as that involved in
binding of phosphorylethanolamine. Pep-1 has binding properties that
are independent of calcium, and the corresponding part of SAP may play
a role in SAP binding both to immune complexes and to C4BP. The
C4BP-SAP interaction presumably involves multiple contact points in
each SAP monomer (Fig. 7). If so, the sequence of Pep-1 might
contribute to binding of SAP to C4BP even though it may not be the
whole binding site.
Several reports suggest that SAP is involved in
the opsonization process(17) . Thus, SAP interacts with immune
complexes(41) , macrophage receptors(42) , and
neutrophils(43) . The C4BPSAP complex may represent inert
forms of both C4BP and SAP in blood that are converted to their active
states when dissociating from the complex.