Download Monoclonal antibodies to human plasma Protein X alias

Bioscience Reports 5, 343-352 (1985)
Printed in Great Britain
343
M o n o c l o n a l a n t i b o d i e s to human plasma P r o t e i n X
alias c o m p l e m e n t S - p r o t e i n
Dieter 3ENNE, Ferdinand HUGO and Sucharit BHAKDI
Institute of Medical Microbiology, University of Giessen,
Schubertstrasse i, D-6300 Giessen, Federal Republic of Germany
(Received 15 April 1985)
Protein X alias complement S-protein was isolated by
dissociation from purified XCSb-9 (fluid-phase terminal
C5b-9) c o m p l e x e s with 250 mM d e o x y c h o l a t e and
subsequent sucrose density gradient centrifugation and
SephacryJ gel chromatography.
Polyclonal rabbit and
monoclonal mouse antibodies were used to preliminarily
characterize the protein in human serum and plasma.
In plasma, Protein X yielded a symmetrical immunoprecipitate of ~2-mobility in a crossed immunoelectrophoresis assay.
However, a second immunoprecipitate
of Oh-mobility was observed when serum was analysed;
this precipitate represented Protein X in complex with
antithrombin-III. The co-precipitation of Protein X with
serum antithrombin-III was exploited for establishing a
simple screening test for unequivocal identification of
monocJonal a n t i - P r o t e i n X a n t i b o d i e s .
SDS-PAGE
immunoblotting with monoclonal antibodies showed that
Protein X exhibits pronounced microheterogeneity,
migrating as a diffuse moiety of approx. Mr 80-90 000.
Additionally, a small amount of polymeric aggregates
appear to be present in plasma. Reduction of disulfide
bonds led to liberation of a polypeptide of approx. 15 K
as d i s c e r n e d by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular
weight entities during the process of blood coagulation
or during formation of fluid-phase terminal complement
complexes. The plasma concentrations in healthy adults
were in the range of 500-700 pg/ml.
The availability
of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the
dual role of this protein in the terminal complement
and coagulation cascades.
A plasma protein, originally termed complement 'S-protein' (Podack
& MiJller-Eberhard, 1979), appears to inactivate nascent, fluid-phase
C5b-9 complexes by firm binding to these molecules with generation of
cytolytically inactive complement complexes (Kolb & Mtlller-Eberhard,
1975).
This process may represent the last regulatory step in the
complement cascade (Bhakdi & Tranum-3ensen, 1983). Further studies
3##
3ENNE ET AL.
on the biochemistry and function of this plasma protein have been
impeded by the difficulties encountered in a t t e m p t s to isolate it from
plasma.
Following the development of a simple method for isolating
the fluid-phase complement complex (Bhakdi & Roth, 19gl), we have
t h e r e f o r e r e s o r t e d to an a l t e r n a t i v e procedure for purifying the
'S-protein' which involves dissociation of the protein from C5b-9 by
high concentrations of deoxycholate (Podack & Mi~ller-Eberhard, 1980).
The purified protein has successfully been used as an immunogen and
we report some unusual features disclosed by application of poly- and
monoclonal antibodies in electroimmunoassays. Because the protein has
been found to fulfil a second function in the coagulation cascade, we
have proposed that it provisionally be re-named plasma Protein X
(3enne et al., 1985).
Materials
and Methods
Isolation
of protein
X f r o m XC5b-9
XCSb-9 was i s o l a t e d from inulin-activated serum as described
elsewhere (Bhakdi & Roth, 1981). Solutions containing 0.6-1.2 mg/ml
protein were made 250 mM in deoxycholate (DOC) through addition of
solid detergent, and incubated in the presence of 2 mM PMSF for 60
min, 37~
Four ml aliquots were applied to linear sucrose density
gradients containing 6.25 mM DOC (10-#0% w/w sucrose in 10 mM
Tris, 50 mM NaCI, 7.5 mM NAN,, pH 8.i) and centrifugation performed at 250 000 g x 3 h at #~ in a Beckmann v e r t i c a l rotor type
VTi-50 (#0 ml total gradient volume).
Twenty equal fractions were
c o l l e c t e d from t h e b o t t o m of the tubes and aliquots applied in
SDS-PAGE analyses.
Fractions containing Protein X were pooled,
diluted with # vols. 10 mM Tris, 50 mM NaCI, pH 8.1, and concentrated to approx, one-fifth of the original XCSb-9 volume (Amicon PM
10 membranes).
The protein samples were then chromatographed at
#~ over a Sephacryl S-300 column (1 x 60 cm, Pharmacia, Uppsala)
equilibrated in the same buffer. The flow rate was # ml/h, and 2 ml
fractions were collected.
This step removed the detergent from the
protein and was essential for immunological renaturation.
Purified
P r o t e i n X was finally concentrated to approx. #00-600 pg/ml and
stored frozen at -20~
Rabbit antisera and monoclonal antibodies
Polyclonal antisera were raised in rabbits following the immunization schedule of Harboe and Ingild (1973). Initial injections were with
100 IJg of protein per animal, and booster injections performed with
30-50 pg of protein.
Monoclonal antibodies
Female balb/c mice were given 5 x 100 pl Protein X in complete
F r e u n d ' s a d j u v a n t ( l : l v / v ) subcutaneously at different sites for
primary immunization.
They were b o o s t e r e d 5 weeks later by
subcutaneous injection of 500 pl of antigen mixed 1:1 with incomplete
Freund's adjuvant. A third booster series was given intraperitoneally 5
weeks later without adjuvant, and the mice sacrificed 3-# d t h e r e a f t e r .
CHARACTERIZATION OF PLASMA PROTEIN X
3t~5
Myeloma cells were cultured in RPMI 1640 medium supplemented
with 10% i n a c t i v a t e d f e t a l c a l f s e r u m ( B i o c h r o m , Berlin), 1%
glutamine, 5000 U/ml penicillin and 5 mg/ml st rept om yci n and 0.02
mM 2 - m e r c a p t o e t h a n o l in a 7% CO 2 atmosphere at 37~
Myeloma
cells from 12 petri dishes (9 cm d i a m e t e r ) grown to half confluence
were admixed with cells from one spleen in s e r u m - f r e e medium and
p e l l e t e d by c e n t r i f u g a t i o n at 12000 r.p.m.
The fusion solution
consisted of 50% PEG 4000 (Merck, D a r m s t a d t ) , sterilized in RPMI
and kept at 37~
in the CO 2 incubator for one day.
2 ml of this
solution was added to the cell pellets, which were suspended by gentle
pipetting for 2 rain.
Four ml of prewarmed medium was then added
and the agglutinating ceils continuously swirled for tt rain. The fusion
solution was then diluted by addition of 16 ml of RPMI.
Cells were
pelleted and resuspended in 20 ml of myeloma medium.
They were
distributed over 4 x 96 cups (0.32 cm 2 culture area) of microtrays
containing macrophages from two mice (obtained by peritoneal lavages
with 0.34 M sucrose solution). On the next day and every second or
third day t h e r e a f t e r ) half of the medium was replaced by HAT-medium
(0.04 x 10-2 mM aminopterin, 1.6 x 10 -2 mM thymidine, 0.1 x 10 -2
mM hypoxanthine (in myeloma m e d i u m ) ) .
Hybridoma ceils were cloned thrice by limiting dilution on a feeder
layer of macrophages in microculture trays.
Ceils that were to be
seeded were viewed and numbered under the microscope in a Terasaki
plate (Nunc GmbH, Wiesbaden). Positive clones were cultured in 157
ml bottles and culture supernatants were collected.
Hybridoma cells
from two densely grown culture flasks were intraperitoneally injected
in balb/c mice p r e - t r e a t e d with 0.5 ml of pristane, and ascites fluid
co llected 2-5 weeks t h e r e a f t e r .
Screening assays.
R o u t i n e s c r e e n i n g was p e r f o r m e d using
m i c r o - E L I S A a s s a y s with p l a t e s c o a t e d with purified Protein X;
development was with peroxidase-labelled second antibodies. Definitive
i d e n t i f i c a t i o n of a n t i b o d y specificities was p e r f o r m e d by e l e c t r o immunoassay-immunoblotting (EIA-IB) as described e l s e w h e r e - ( B h a k d i
et al., 1985). In the later stages of this work, a simple line-immunoe l e c t r o p h o r e s i s - i m m u n o b l o t t i n g screening assay was used.
Human
s e r u m was a p p l i e d in line i m m u n o e l e c t r o p h o r e s i s against specific
antibodies to antithrombin-III (AT-III; Atlantic Antibodies, Scarborough,
Memphis).
The r e s u l t i n g line immunoprecipitate, which has been
shown to contain not only antithrombin-III, but also complexed Protein
X, was solubilized in SDS and e l e c t r o b l o t t e d onto nitrocellulose. The
filter paper containing the blotted proteins was then sectioned into 0.5
cm wide strips and used to test the r e a c t i v i t y of individual antibody
clones for protein X - r e a c t i v i t y as described elsewhere (Bhakdi et al.,
1985).
O n e - and t w o - d i m e n s i o n a l SDS-PAGE/immunoblotting were performed as described elsewhere (Bhakdi & Tranum-3ensen, 1982; Bhakdi
et al., 198/4).
Blots were ceveloped with peroxidase-labelled rabbit anti-mouse IgG
(Dakopatts, Copenhagen).
Plasma concentrations of Protein X in 10
h e a l t h y d o n o r s w e r e d e t e r m i n e d by r o c k e t - i m m u n o e l e c t r o p h o r e s i s immunoblotting using purified protein as standard,
Protein c o n c e n t r a tions in the standard were determined by amino acid analyses.
3/46
3ENNE ET AL.
R e s u l t s and D i s c u s s i o n
Isolation of Protein X from XC5b-9
XCSb-9 p r e p a r a t i o n s were given 230 mM DOC and centrifuged
through sucrose density gradients.
SDS-PAGE analyses of individual
gradient fractions showed that Protein X was selectively and partially
dissociated from the complex. The upper gradient fractions containing
the protein (Fig. IA) were chromatographed over Sephacryl S-300 to
remove detergent.
This step led to recovery of the protein (Fig. 1B)
in immunologically intact form in overall yields of 23-30%.
The
amino acid composition of Protein X is given in Table 1. The results
a r e in good a g r e e m e n t with data of Podack and Mi~ller-Eberhard
(1979) for Protein X purified from plasma.
The absorbance at 280
nm of a 1 mg/ml protein solution (A280, 1 cm) was 1.7.
Electroimmunoassays with polyclonal rabbit antibodies
P u r i f i e d P r o t e i n X y i e l d e d a single immunoprecipitate of c~e l e c t r o p h o r e t i c mobility upon crossed immunoelectrophoresis against
specific rabbit antiserum (Fig. 2A). Surprisingly, however, we always
observed the development of 3-10 immunoprecipitates upon analysis of
whole human serum with this antiserum (Fig. 2B). Thus, the Protein
X preparations still contained strongly immunogenic contaminants.
In
Fig. I. Purification of Protein X from fluid-phase
XC5b-9 (formerly SC5b-9) complement complexes.
XCSb-9 (A: left gel) was treated with 250 mM DOC.
The sample was centrifuged through a linear sucrose
density gradient, twenty fractions were collected,
and aliquots applied to SDS-PAGE (A: lanes 1-20).
Protein X was selectively and partially dissociated
from the XCSb-9 complex to be recovered in the upper
fractions (15-19) of the gradient.
Fractions 16-19
were pooled and chromatographed over Sephacryl S-300
to remove detergent.
Purified Protein X recovered
from the column (gel B) exhibited the typical
diffuse staining pattern in SDS-PAGE.
CHARACTERIZATION
Table I.
OF PLASMA
PROTEIN
X
Amino acid composition of Protein X
Amino acid
Residues/100 residues
Asp
Thr
Ser
Gln
Pro
Gly
Ala
Cys
Val
Met
Ile
Leu
Tyr
Phe
Lys
His
Arg
12.1
5.4
7.6
13.4
6.5
7.0
6.1
2.1
5.0
i. 7
2.6
8.2
3.1
5.2
5.9
2.2
5.9
Fig. 2.
Crossed immunoelectrophoresis of isolated
Protein X (plate A) and human serum (plates B, C)
developed with a polyclonal rabbit antiserum to
Protein X.
The isolated protein (A) yielded a
symmetrical immunoprecipitate of e-electrophoretic
mobility
with this antiserum.
However,
8-10
precipitates were observed when serum was applied
(B, C).
In plate C, purified Protein X was
incorporated
into a 1 cm wide intermediate gel;
plate B (control) contained a blank gel.
The line
immunoprecipitate (curved arrow, plate C) representing Protein X fused with a major, double-peaked
immunoprecipitate (arrows), provisionally identifying the latter as serum Protein X.
347
348
3ENNE ET AL.
order to provisionally identify Protein X, a crossed-line immunoelectrophoresis (Kr~ll, 1973) was performed with incorporation of purified
protein in an intermediate gel (Fig. 2C). This resulted in fusion and
elevation of a single, double-peaked precipitate corresponding to serum
Protein X onto the line precipitate (plate 2C).
EIA-IB with monoclonal antibodies
Five monoclonal antibodies were obtained against Protein X, three
of subclass IgG-1, one of subclass IgG-2b, and one of not clearly
identifiable subclass.
All bound to the protein in crossed-immunoe l e c t r o p h o r e s i s / i m m u n o b l o t t i n g analyses.
In these assays purified
P r o t e i n X was first immunoprecipitated in crossed immunoelectrophoresis with the rabbit antiserum (as in Fig. 2A).
The precipitate
was dissolved in SDS, etectroblotted, and reacted with the monoclonal
antibodies.
The blots were developed with peroxidase-labelled second
a n t i b o d i e s to mouse immunoglobulins.
Fig. 3A shows a positive
immunoblot obtained with a monoclonal anti-Protein X antibody.
We next used the monoclonal antibodies to analyse the immunoprecipitation behaviour of Protein X in serum (Fig. 3C) as opposed to
plasma (Fig. 3B).
These analyses showed that whereas the protein
yielded a symmetrical immunoprecipitate of ~-electrophoretic mobility
when analysed in plasma, a double-peaked precipitate formed when
serum was applied as antigen.
These results confirmed the observations of Fig. 2 and demonstrated a partial change in the physical state
of the protein that occurred during blood coagulation. As reported in
a separate communication, the slowly moving immunoprecipitate has
been i d e n t i f i e d as a complex between Protein X and serum antithrombin III (3enne et al., 1985). Here the co-precipitation of Protein
Fig. 3. Crossed immunoelectrophoresis combined with
e l e c t r o b l o t t i n g with monoclonal anti-Protein X
antibodies.
A: Purified Protein X. B: EDTA-plasma
(3 ~I).
C: Whole serum (5 ~i).
In the first
stage,
the samples were i m m u n o e l e c t r o p h o r e s e d
against
the polyclonal rabbit anti-Protein X
antiserum
of Fig. 2.
The precipitates were
subsequently dissolved in SDS and electroblotted
o n t o nitrocellulose.
The blots were finally
developed with monoclonal anti-Protein X antibodies.
Note the presence of a symmetrical Protein X
immunoprecipitate
in plate B (EDTA-plasma)
as
opposed to the double-peaked precipitate in plate C
(serum).
CHARACTERIZATION OF PLASMA PROTEIN X
3t+9
X with serum antithrombin-III is demonstrated in another manner.
Isolated AT-III (obtained from Behringwerke, Marburg) or whole serum
was s u b j e c t e d to crossed immunoelectrophoresis against anti-AT-III
antibodies.
C o n d i t i o n s were adjusted such that similar immunoprecipitates developed in both plates.
The precipitates were then
dissolved in SDS and electro-immunoblotted using monoclonal antiP r o t e i n X antibodies.
Fig. 4 depicts the results showing positive
staining for Protein X in the AT-III precipitate derived from serum,
but entire absence in the precipitate derived from purified plasmaderived AT-III, which contained no complexed Protein X.
The above observation was exploited for development of a simple
screening assay which permitted definitive identification of monoclonal
anti-Protein X antibodies.
For this purpose, serum was subjected to
line i m m u n o e l e c t r o p h o r e s i s (Krgll, 1973) against anti-AT-III.
The
ensuing line immunoprecipitate (Fig. 4C) containing the co-precipitated
Protein X was electroblotted, and the filter paper then sectioned into
0.5 cm wide strips.
Cell culture supernatants or ascites fluids were
applied to individual strips, and specific anti-Protein X clones were
identified through their positive reactions (Fig. 4C).
Plasma levels of Protein X in i0 healthy adults were determined by
rocket immunoelectrophoresis-immunoblotting, using purified protein as
the standard, and found to be of the order of 500-700 tJg/ml. This
e s t i m a t e agrees with the values given by Podack and Mflller-Eberhard
(1979).
SDS-PAGE immunoblotting
SDS-PAGE immunoblots developed with a mixture of 4 monoclonal
anti-Protein X antibodies consistently led to the generation of multiple
diffuse protein bands in the approx. Mr region of 80-90 000 (Fig. 5).
A d d i t i o n a l , faint staining was observed in higher molecular weight
regions.
No differences were noted between plasma and serum, and
the pattern also remained unaltered a f t e r inulin activation of serum
with formation of XC5b-9
(Fig. 5A).
Thus, Protein X does not
appear to be cleaved to lower molecular weight entities during the
process of blood coagulation or during XC5b-9 formation. Reduction
with 10 mM d i t h i o t h r e i t o l led to sharpening and more intensive
staining of the protein in the 80 K region, and concomitant generation
of a lower molecular weight polypeptide of 15 K. These results were
confirmed and extended by two-dimensional SDS-PAGE/immunoblotting
(Fig. 5B,C).
First dimension samples were electrophoresed under
non-reducing conditions, followed by second dimension electrophoresis
with disulfide bond cleavage. We consistently observed the generation
of a major polypeptide of approx. 80 000 daltons also deriving from
high m o l e c u l a r
w e i g h t m a t e r i a l in the f i r s t d i m e n s i o n gels.
Additionally, we found faint but distinct staining of a low molecular
weight polypeptide (approx. 15 K, Fig. 5C) and microheterogeneity in
the 80-90 000 dalton region.
The collective results indicated that
Protein X exists in heterogeneous form in plasma, and that disulfide
bonds play a role in stabilizing its structure. A low molecular weight
polypeptide of 15 K is cleaved from the native protein by dithiothreitol; whether this cleavage product is generated as the result of
i n t r a m o l e c u l a r ' n i c k i n g ' of Protein X, or whether the protein is
actually primarily composed of two disulfide-bonded polypeptide chains
350
3ENNE ET AL.
Fig. 4.
A:
Crossed immunoelectrophoresis followed
by i m m u n o b l o t t i n g of purified antithrombin-III
developed with monoclonal anti-Protein X antibodies.
In the first stage, antithrombin-III was precipitated with anti-AI-III in conventional crossed
immunoelectrophoresis.
The
precipitate was
dissolved in SDS, electroblotted, and subsequent
incubation of the nitrocellulose blot with monoclonal anti-Protein X antibodies yielded an entirely
blank immunoblot.
Thus, AT-III isolated from human
plasma contained no Protein X.
B:
In contrast,
AT-III derived from human serum was precipitated
with anti-AT-III and the immunoprecipitate was shown
to contain Protein X.
C:
Line immunoelectrophoresis combined with immunoblotting for identification
of anti-Protein X antibody clones.
Human serum was
e l e c t r o p h o r e s e d in a line immunoelectrophoresis
against anti-AT-III.
The precipitate was electroblotted, the filter paper then sectioned into 0.5 cm
wide strips, and individual antibody clones tested
for their reactivity.
In this manner, anti-Protein
X clones (strips y, z) were easily distinguishable
from other clones (strip x), which yielded blank
immunoblots.
CHARACTERIZATION
OF
PLASMA
PROTEIN
X
Fig. 5.
A:
SDS-PAGE immunoblotting of (a)
EDTA-plasma; (b) serum; (c) inulin-activated serum
developed with monoclonal anti-Protein X antibodies.
Note the development of diffuse bands (arrows) in
the M r region 80-90 000 and additional faint
staining
in higher molecular weight regions.
Samples were not reduced prior to electrophoresis.
B and C:
Two-dimensional SDS-PAGE immunoblotting
of EDTA-plasma.
First dimension SDS-PAGE (left to
right) under non-reducing conditions.
Second
dimension electrophoresis on 12.5% gels (top to
bottom) under non-reducing (plate B) or reducing
conditions (plate C).
Note the generation of
s t r o n g l y staining spots exhibiting pronounced
m i c r o h e t e r o g e n e i t y and the presence of a weakly
staining moiety of approx. 15 K (arrow) in plate C.
The major 80 K moiety and the small 15 K polypeptide
were also generated from high molecular weight
material stemming from the origin of the first
dimension gel.
Thus, polymeric aggregates of
Protein X appear to be present in plasma; their weak
staining in plate B and gels a-c is probably due to
poor transferability from the SDS-gels by electroblotting in the absence of disulfide reducing
agents.
351
352
JENNE ET AL.
requires future clarification. It is noteworthy that Protein X present
in isolated XC5b-9 complexes was reported to be cleaved by dithiothreitol into a major #0 000 dalton subunit (Bhakdi & Tranum-3ensen,
1982).
The present data would indicate that this was due to secondary nicking of the protein occurring during the isolation procedure.
The m e t h o d s d e s c r i b e d in this paper a p p e a r well suited for
p u r i f i c a t i o n of Protein X in immunologically intact form.
In an
accompanying paper, we have shown that the isolated protein still
binds to AT-III/thrombin complexes to exert a net thrombin-protective
function (3enne et al., 1985).
The availability of poly- and monoclonal antibodies to the protein should facilitate and promote further
studies on this novel and interesting plasma component.
Acknowledgem ents
We thank Margit Pohl and Marion Muhly for outstanding technical
assistance and M. Wiesner (Max-Planck-Institute, Freiburg) for kindly
performing the amino acid analyses. This work was supported by the
D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (Bh 2/1-5) and the Fonds der
Chemischen Industrie.
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