Download Structural and functional studies on C4b

Bioscience Reports 5, 855-865 {1985)
Printed in Great Britain
855
Structural and functional studies on C4b-binding protein,
a regulatory c o m p o n e n t of the human complement system
L. Ping CHUNG and Kenneth B. M. REID
M.R.C. Im munoehemistry Unit, Department of Biochemistry, South Parks Road,
Oxford OX1 3QU; U.K.
{Received 22 August 1985)
The binding and cofactor activities of C4b-binding protein
were examined before and after limited proteolysis by pepsin,
trypsin and chymotrypsin. The major fragments generated
were characterized by amino acid sequencing, thus establishing the precise points of limited proteolysis. These studies
allow a tentative assignment of the cofactor activity site to
the residues 177-322 of the 549 amino acid long chain of
C4b-binding protein but indicated that residues in the region
332-395 are important in the binding activity.
Human C4b-binding protein (C4BP) is an important regulatory component
of the serum complement system, being involved in the control of the classical pathway enzyme complex C4bC2a. The C4BP displays binding activity
by interacting with C4b and thus displacing C2a from the complex (Gigli
et al., 1979) and it is also a cofactor in the degradation of C4b by the
enzyme factor I (Fujita & Nussenzweig, 1979). Human C4BP is a glycoprotein of approx. 5 5 0 0 0 0 Mr in dissociating and non-dissociating conditions and is composed of probably seven identical, disulphide-linked chains
each of 70 000 Mr and 549 amino acids long (Dahlb~ick & M/iller-Eberhard,
1984; Chung et al., 1985a). In the electron microscope the C4BP has a
spider-like structure with seven flexible tentacles (each 3 nm • 33 nm)
joined at one end to a small central body (Dahlb/ick et al., 1983). However,
X-ray solution scattering experiments by synchroton radiation indicated that
in solution the molecule is more compact, the tentacles being relatively close
together in distinction to the splayed out images seen in the electron micrographs (Perkins et al., 1985). In this paper some correlation between the
structure and function of the C4BP molecule has been established by
examining the binding and cofactor activities of C4BP before and after
limited proteolysis by various enzymes.
Materials and Methods
Preparation and assay o f c o m p l e m e n t c o m p o n e n t s
Cls, C4, factor I and C4BP were prepared as described by Gigli et al.
(1976), Bolotin et al. (1977), Hsiung et al. ( 1 9 8 2 ) a n d Chung et al. (1985b)
856
CHUNG & REID
respectively. C4i was prepared by the repeated freezing (at --70~
and
thawing of native C4 or by treatment of the native C4 with 50 mM methylamine at 37~ for 1 h. The complete inactivation of the purified C4 was
demonstrated by the cleavage of the C4i c~i chain by factor I in the presence
of C4BP and the fact that the C4i was insensitive to Cls. The C4 haemolytic
assays were performed as described by Kerr (19-80).
Radioiodination of proteins. The iodination procedure used was adapted
from the method of Fraker and Speck (1978).
Assay of C4BP cofactor activity. Protease-treated or untreated C4BP
was incubated with C4i (0.4 rag/m1) and factor I (0.01-0.02 mg/ml) at 37~
for 1-3 h, followed by SDS-polyacrylamide gel electrophoresis analysis.
Prefiminary experiments had shown that it was preferable to follow the
degradation of C4i rather than C4b because of the clearer pattern of the
degraded fragments on SDS-polyacrylamide gel. The cofactor activity o f
the C4BP sample, determined by the degree of degradation of the od chain
of C4i, was estimated from the staining intensities of the 80000 M, call
fragment (generated after the first cleavage) and the 45 000 Mr C4d fragment
(generated after the second cleavage).
C4BP binding assay. To test for the binding of intact C4BP (or treated
C4BP) to C4i, 50 tal of C4i (0.4 mg/ml) in 50 mM Tris HCI/5 mM EDTA/
150 mM NaC1, pH 7.4, was incubated in each well of a microtitre plate for
2 h at 37~ After this incubation, a solution of bovine serum albumin (2.5
mg/ml) in the Tris HC1/EDTA/NaC1 buffer was used to wash the wells (three
times) and to saturate any non-specific binding sites by incubation at 37~
for 30 min. The wells were then emptied and the C4BP sample (0-10 tag) of
untreated or protease-treated C4BP, '2sI-C4BP (approx. 3 0 0 0 0 c.p.m, in
less than 0.1 tag of protein) and bovine serum albumin (2.5 mg/ml) in a total
vol of 35 tal was added and incubated for 90 rain at 37~ After washing the
wells three times with BSA/Tris HC1/EDTA/NaC1 the plate was dried, and
the wells were cut out and placed in tubes for gamma counting. The binding
activity of t h e C4BP sample was estimated by its inhibitory effect on the
binding of 12sI-C4BP to the C4i coated microtitre wells.
Limited proteolysis o f C4BP. Trypsin treatment. The trypsin-treated
C4BP was prepared by incubating purified C4BP (0.4-0.6 mg/ml) in 50 mM
TrisHC1/0.15 M NaC1, pH 7.4, with trypsin at a substrate/enzyme ratio of
250/1 (w/w) at 37~ for 4 h. The digestion was stopped by the addition of
1 mM DFP or a three-fold excess of soy bean trypsin inhibitor and the
sample stored at 4~ before use. For amino acid sequencing the tryptic fragments (from a digest designed to yield only T1 and T3) were dissolved in
an SDS/urea/Tris buffer and separated on a BRL preparative SDS-polyacrylamide gel apparatus. After extensive dialysis and freeze drying, the
purified fragments were then subjected to automated N-terminal amino acid
sequencing.
Chymotrypsin treatment. The 4 8 0 0 0 Mr and 1 6 0 0 0 0 M r chymotryptic
fragments used in the eofactor assay were prepared fi'om chymotrypsindigested C4BP samples by gel filtration on a Sephacryl S-300 column (2.5
cm • 80 cm) equilibrated with 25 mM Tris HC1/2 mM EDTA/0.15 M NaC1
pH 7.4 at room temperature. C4BP (20 ml of 0.5 mg/ml) was incubated with
chymotrypsin (at a substrate/enzyme ratio of 100/1, w/w) at 37~ for 3.5 h
C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY
857
to give a good yield of the 48 000 Mr fragment or for 6 h to obtain a good
yield of the 160 000 Mr fragment. In both cases the chymotryptic activity
was stopped by 1 mM DFP and the sample applied to Sephacryl S-300. For
sequencing studies, native C4BP (10 mg) was incubated with chymotrypsin
(at a substrate to enzyme ratio of 400/I, w/w) at 37~ for 75 min in 50 mM
Tris HC1/100 mM NaC1, pH 7.4. DFP (1 mM) was added to stop the reaction
and the sample was dialysed at 4~ against 0.2 M NH4HCO 3. The dialysate
was then freeze dried, reduced and alkylated (Chung et al., 1985b). The
fragments were separated on a Sephacryl $300 column (1.5 cm X 90 cm)
equilibrated in 6 M guanidine HC1/20 mM Tris HC1, pH 7.4.
Pepsin treatment. Pepsin-digested C4BP used in the cofactor and binding
activity assays was prepared by incubating C4BP (0.3 mg/ml) in 0.1 M
sodium acetate/0.1 M NaC1, pH 4.8, with pepsin (at a substrate/enzyme ratio
of 25/1, w/w) at 37~ for 2.5 h. The pH of the digest was adjusted to 7.1
and the solution made 1 mM with respect to DFP. In all the experiments
performed on the pepsin-treated C4BP, a C4BP sample dialysed in the pH
4.8 buffer and subsequently adjusted to pH 7.1 was used as an extra control.
To prepare pepsin-treated C4BP for amino acid sequence analysis, 12 mg of
C4BP (0.5 mg/ml) in 0.1 M sodium acetate/0.1 M NaC1, pH 4.2, was incubated with pepsin (at a substrate/enzyme ratio of 75/1, w/w) at 37~ for 4 h.
After raising the pH to 7.1 and adding DFP (to 1 mM) the sample was
dialysed, freeze dried, reduced and alkylated. The peptic fragments P1 and
P2 were fractionated on a Sephacryl S-300 column run in 6M guanidine
HC1/20 mM Tris HC1, pH 7.4, and then prepared for automated amino acid
sequencing.
Results and Discussion
Trypsin-treated C4BP
The present results confirm and extend those of Reid and Gagnon (1982),
which showed that trypsin-treated C4BP retained its cofactor activity and
behaved in an identical fashion to the untreated molecule in non-dissociating
conditions on Sephacryl S-300 and in non-reducing conditions on SDSPAGE electrophoresis (Fig. 1a). Under reducing conditions on SDS-PAGE,
each of the 70 000 Mr chains was shown to be split initially into two major
fragments, T1 of 40 000 M r and T3 of 30 000 Mr (Fig. lb), and with longer
digestion times, or a higher trypsin concentration, the 40 000 Mr T 1 fragment
was further digested to give a 36 000 Mr T2 fragment. Both forms of C4BP,
the C4BP-H and C4BP-L (Fig. la), were split by trypsin to the same extent.
Similar fragments to T1, T2 and T3 can be generated by the use of plasmin
and are observed during the purification of C4BP if a plasmin inhibitor is not
used during the purification procedure.
Trypsin-treated C4BP still retained its cofactor activity although its
efficiency was approx. 5 times lower than that of untreated C4BP (Fig. 2).
However, as shown in Fig. 3, the binding of I~sI-C4BP to the C4i coated on
microtitre wells was not inhibited significantly on the addition of trypsintreated C4BP while untreated C4BP gave good inhibition. The difference
858
CHUNG & REID
Fig. 1. L i m i t e d p r o t e o l y s i s of C4BP by t r y p s i n . C4BP (0.4 m g / m l ) was i n c u b a t e d w i t h
v a r y i n g c o n c e n t r a t i o n s of t r y p s i n at 37~ f o r 15 m i n t o 4 h. T h e e n z y m e was i n a c t i v a t e d
b y a d d i t i o n o f a 3-fold excess of soy b e a n t r y p s i n i n h i b i t o r f o l l o w e d b y an e q u a l v o l u m e
o f gel l o a d i n g b u f f e r ( S D S / u r e a / T r i s ) a n d h e a t i n g at 100 ~ for 10 m i n . T h e samples were
e l e c t r o p h o r e s e d o n (a) 3.5% ( w / v ) S D S - P A G E u n d e r n o n - r e d u c i n g c o n d i t i o n s a n d (b) on
15% ( w / v ) S D S - P A G E u n d e r r e d u c i n g c o n d i t i o n s . T h e s u b s t r a t e / e n z y m e ratios a n d t i m e
of digestion f o r e a c h t r a c k are 1 - n o e n z y m e , 4 h ; 2 - 2 0 0 / 1 , 4 h ; 3 - 3 0 0 / 1 , 4 h ; 4 4 0 0 / 1 , 4 h ; 5 - 2 0 0 / 1 , 1 h ; 6 - 3 0 0 / 1 , 1 h ; 7 - 4 0 0 / 1 , 1 h ; 8 - 2 0 0 / 1 , 15 m i n ; 9 - 3 0 0 / 1 ,
15 r a i n ; 10 - 4 0 0 / 1 , 15 m i n .
C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY
859
Fig. 2. Cofactor activity assay of trypsin-treated C4BP. Samples were run on an 8.5%
(w/v) SDS-PAGE in reducing conditions. Tracks A, C4i, B and I refer to untreated C4BP,
C4i, trypsin-treated C4BP and factor I respectively. C4i (0.4 mg/ml), factor I (0.01 mg/
ml) and C4BP (untreated or trypsin-digested) at 250 to 1 #g/ml (as indicated by the
number above each track) were incubated at 37 ~ for 2 h. The positions of the c~i (uncleared), ~il (generated after first cleavage) and C4d (generated after second cleavage)
chains are shown. The intensities of the Coomassie Blue stained ~il and C4d fragments
were used as a measure of cofactor activity.
b e t w e e n the binding activities o f the t r e a t e d and u n t r e a t e d C4BP was over
100-fold (Fig. 3).
T h e N - t e r m i n a l a m i n o acid s e q u e n c e o b t a i n e d for T1 m a t c h e d t h a t o f the
N - t e r m i n u s o f the i n t a c t C4BP, indicating t h a t the 40 000 M r t r y p t i c fragm e n t was derived f r o m t h e N-terminal end o f the 70 000 M r chain o f C4BP.
A d o u b l e a m i n o acid s e q u e n c e was o b t a i n e d for T3 as detailed below:
Derived a m i n o acid
sequence from
c D N A studies
Fragment
T3
Sequence 1
Sequence2
325
335
345
G E I T Q H R K S R P H N H CV Y F Y G D E
KSRPHNHCVYFAGDE
SRPP
NHCVYFAGDE
B o t h s e q u e n c e s 1 and 2 m a t c h e d w i t h the s e q u e n c e derived f r o m the c D N A
studies ( C h u n g et al., 1985a) b u t the yields o f P T H a m i n o acids f r o m
s e q u e n c e 1 were a p p r o x i m a t e l y twice t h a t o f s e q u e n c e 2, indicating t h a t the
g e n e r a t i o n o f the t r y p t i c f r a g m e n t s T1 and T3 was due to splitting b y
860
CHUNG & REID
[]
"
-0
C
D
0
-Q
"
Ik
9 Non-trypsindigestedC4BP
i TrypsindigestedCt,BP
~ ~ .
1
iili
o.lo's i
.....
. . . . . .
,ug CLBPadded
lb
Fig. 3. Binding activity of trypsin-treated C4BP. The binding activity
of trypsin-treated C4BP was assayed by comparing its ability to inhibit
the binding of 12SI-C4BP to C4i coated microtitre plates with that of
untreated C4BP. The amount of radioactivity bound (mean of two
determinations) was plotted against the amount of C4BP untreated (o)
or trypsin-treated (u), added to each well.
trypsin at arginine 331 and partial splitting at lysine 332. An interesting
feature which emerged from this sequencing study was that the cDNA
sequence studies predicted that residue 343 is tyrosine yet the amino acid
sequencing showed that the major amino acid present at that position was
alanine. Thus this is evidence for a third polymorphic site in the C4BP
molecule in addition to those observed at positions 44 and 309 (Chung
et al., 1985a).
Chymotrypsin-treated C4BP
Nagasawa et al. ( t 9 8 2 , 1983) reported that C4BP could be split by mild
chymotrypsin treatment to a 4 8 0 0 0 M r fragment which contained the
cofactor active site and a 160 000 Mr fragment. After reduction of disulphide
bonds the 160 000 M~ fragment was shown to be composed o f probably 7
disulphide-linked chains of approx. 25 000 Mr. It was established by amino
acid sequence studies (Nagasawa et al., 1983; Dahlbgck & M(iller-Eberhard,
1984; Chung et al., 1 9 8 5 a ) t h a t the 4 8 0 0 0 M r and 2 5 0 0 0 M r fragments
were derived from the N-terminal and C-terminal portions of the 70 000 Mr
C4BP chain respectively, and thus the 25 000 M r fragments form a disulphidelinked C-terminal core o f 160 000 M r .
In this study a more detailed time course o f chymotrypsin treatment with
low enzyme concentrations showed that a lightly digested form o f the
molecule in which the 48 000 Mr fragment was disulphide-bonded to the
C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY
Fig. 4. Limited proteolysis of C4BP by chymotrypsin and pepsin.
C4BP (0.7 mg/ml) was incubated with chymotrypsin (substrate/enzyme
ratio 50/1, 100/1, 150/1,200/1, w/w in tracks 1, 2, 3 and 4 respectively)
at 30~ for 12 h and electrophoresed on 4% (w/v) non-reduced (a) and
8% (w/v) reduced (b) SDS-PAGE. Tracks in 5(a) and 5(b) are nondigested C4BP.
Pepsin digestion of C4BP is shown for samples of C4BP incubated
with pepsin for 3 h at 37 ~ at pH 4.2 at substrate/enzyme ratios of 50/1,
75/1, 100/1 and 150/1 and then electrophoresed under (c) non-reducing
conditions on 4.5% (w/v) SDS-PAGE and (d) reducing conditions on
8.5% (w/v) SDS-PAGE. Track T contained trypsin-digested C4BP.
861
862
CHUNG & REID
25 000 Mr fragment was produced after the initial cleavage by chymotrypsin. As shown in Fig. 4 (a and b), the liberation of the 48 000 Mr fragment from the disulphide-linked core fragments correlated with the further
spfitting of the 48 000 Mr and 25 000 Mr fragments. Since chymotryptic
digestion progressively removes an N-terminal 48 000 Mr fragment from each
of the 70 000 Mr C4BP chains, analysis of the chymotrypsin treated C4BP
on non-reduced SDS-PAGE gives an estimate of the number of chains. The
value obtained was seven, which is consistent with that reported by Dahlb~ick
and Miiller-Eberhard (1984), who had further shown directly by electronmicroscopy studies that the 48 000 Mr and 160 000 M r fragments were the
'tentacles' and 'core' respectively of the unusual C4BP molecule. Cofactor
assays performed on the purified chymotryptic fragments confirmed the
findings of Nagasawa et al. (1982) that the 4 8 0 0 0 Mr fragment retained
the cofactor activity of C4BP while none could be detected in the 160 000 Mr
fragment. No binding assays were performed using the chymotryptic fragments; however, Fujita et al. (1985) have shown quite clearly that both the
48 000 Mr and the 160 000 Mr fragment had lost the ability to bind to C4b.
N-terminal sequence analysis of the 25 000 Mr chymotrypsin fragment
showed that chymotrypsin initially splits the 70 000 Mr chain principally
at the Tyr-Ser bond at position 395-396. This point of splitting is 30 amino
acids upstream from the Trp-Ser bond at position 425-426 which was the
major site of splitting reported by Dahlbgck and Miiller-Eberhard (1984)in
their study of the limited proteolysis of C4BP by chymotrypsin. The difference between the two studies is probably due to the use of different digestion
conditions.
Limited proteolysis by pepsin
When C4BP was treated with pepsin and examined in reducing conditions
on SDS-PAGE the 70 000 Mr chain was shown to be split into a 55 000 Mr
(P1) fragment which was further rapidly degraded into a 50000Mr (P2)
fragment (Fig. 4c and d). No fragment of 15-20 000 Mr, which might have
been expected to be present after the cleavage of the 70 000 Mr C4BP chain
into the 50-55 000 Mr fragment, was observed on SDS-PAGE or gel filtration, and it was concluded that the 'missing' 15-20000 Mr fragment had
been extensively digested by pepsin. Under non-reducing conditions the
pepsin-treated C4BP behaved as a diffuse band with an Mr of approx. 400 000.
The conversion of the 550 000Mr C4BP doublet into the 400 000 Mr band
observed on the non-reduced gel correlated with the conversion of the
7 0 0 0 0 Mr chain into P1/P2 fragments on the reduced gel, which showed
that the peptic fragments are disulphide-linked to each other and therefore
likely to represent the C-terminal portion of the chain (as was confirmed by
the sequencing studies). The cofactor and binding assays performed using
pepsin-digested C4BP (containing approximately equal amounts of the P1
and P2 fragments) showed that the protease-treated C4BP had an identical
activity to that of the untreated C4BP.
The P1 and P2 fragments were isolated from the reduced and alkylated
pepsin-treated C4BP by fractionation on a Sephacryl S-300 column run in
6 M guanidine HC1. Although P1 and P2 were not completely separated,
863
C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY
NH2~.
Trypsin IChymo- /
COox/"
S,S
A - important in cofactor activity
B -important in binding activity
Cofactor
Activity
Binding
Activity
Major Sites
of Limited
Proteolysis
Treatment
SDS-PAGE
Non-reduced
Reduced
Untreated
550000
70000
+
+
Trypsin
550000
400001
30000
+
-
Arg 331
Lys 332
Chymotrypsin
160000
48000
25000
48000
+
-22
-
Tyr 395
Trp 425
Pepsin
400000
550003
+
+
Leu 161
Gly 177
i.
Further cleaved to 36000
2.
F u j i t a et al (1985)
3.
Further cleaved to 50000
Fig. 5. Summary of the major sites of limited proteolysis by trypsin,
pepsin and chymotrypsin on C4BP along with the cofactor and binding
activities of the protease-treated C4BP.
864
CHUNG & REID
pools enriched in each fragment were obtained. The amino acid sequence
data from each pool showed that the major pepsin cleavage points, which
yield P1 and P2, are at the Leu-Leu bond (position 161-162) and the
Gly-Val bond (position 177-178) respectively, i.e.
155
CDPRF
160
165
170
SLLGHASISCTVENETIGVWR
t
P1
175
180
P2
The asparagine residue at position 173 was not detected in the identification
of the PTH-amino acids in the automated sequencing, indicating that it may
be attached to N-linked carbohydrate. This would be consistent with the fact
that the P1 fragment contains only 17 amino acids more than P2 and yet
there was approx. 5000 Mr difference in their apparent mol.wts, on SDSPAGE, and they could be partially separated by gel filtration.
The properties of the fragments generated by limited proteolysis along
with the location of the major cleavage sites are given in Fig. 5. The results
obtained using trypsin-treated C4BP are similar to the finding that trypsintreated factor H retained its activity on fluid phase C3b but lost the ability
to bind or to act as a cofactor in the degradation of C3b on the cell surface
(Hong et al., 1982). Both the chymotrypsin- and pepsin-treated C4BP
showed cofactor activity at concentrations 10 times lower than the normal
plasma C4BP concentration, although the protease-treated C4BP was estimated to be approximately 5 times less efficient on a molar basis than the
untreated C4BP in the cofactor assay. The absence of a 15-20 000 Mr peptic
fragment after the cleavage of the 70000 Mr C4BP chain into the P1/P2
fragments indicates that there may be multiple pepsin-sensitive sites in the
region between residues 1-160. Since the pepsin-treated C4BP still retained
its cofactor and binding activities it can be concluded that residues 1-160 are
not of primary importance in these functions. This observation is consistent
with the finding by Reid and Gagnon (1982) that the N-terminal 40 amino
acids of C4BP are probably not important for the cofactor activity. The
limited proteotysis results indicate that the cofactor active region of C4BP
may lie between the cleavage sites of pepsin and chymotrypsin. However,
the electron-microscopy studies of Dahlb~ick et al. (1983) showed that fluid
phase C4b appeared to be attached close to the tips of the C4BP tentacles
which are about 29 nm from the chymotryptic cleavage site (Dahlbgck &
Miiller-Eberhard, 1984) and thus near to the N-terminal portions of the
C4BP chains, i.e. apparently N-terminal to the tryptic cleavage site. It is
possible, however, that the N-terminal regions of the C4BP chains fold
inwards towards the core leaving the putative cofactor active sites at the
extremities of the molecule. The present observations allow a tentative
assignment of the cofactor site to the residues 177-332 of the 549 amino
acid long chains of C4BP but that residues in the region 332-395 are important in the binding activity (Fig. 5).
C4b-BINDING PROTEIN STRUCTURE AND ACTIVITY
865
Dedication
We dedicate this paper in memory of Professor R. R. Porter in appreciation of his friendship, encouragement and advice over the past sixteen years
(K.B.M.R.) and four years (L.P.C.).
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