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*
Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark;
Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan; and
Department of Microbiology and Immunology, University of Leicester, Leicester, United Kingdom.
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There have been reports indicating that MBL can activate C1r and C1s in vitro (11, 12), and an electron microscopy study has shown C1r/C1s in association with MBL (13). These studies were performed with purified proteins. The studies characterizing the MASPs and MAp19 as MBL-associated proteins were also conducted on purified material, and in these studies neither C1r nor C1s was found to be associated with MBL. However, there is a risk that physiologically associated proteins may be eluted from MBL during the purification procedures, which include EDTA treatment and exposure to high salt concentrations. Thus, it appears prudent to examine whether, in whole serum, the MASPs and MAp19 are associated only with MBL and whether C1r and C1s are associated only with C1q. We have also compared the nature of the interaction of the proteases with their corresponding recognition molecules by exposing the complexes to buffers of different ionic strength and/or to calcium ions or calcium chelators. The molecular sizes of complexes formed by the MASPs and MAp19 in whole serum were examined by gel-permeation chromatography (GPC).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human sera from three donors were used, with 3, 0.3, and 0.01
µg/ml MBL concentrations, respectively. These sera had normal levels
of C1q (around 70 µg/ml). Serum of low MBL concentration was depleted
of C1q and complement factor D (14) by passage through
ion-exchange chromatography beads (BioRex-70; Bio-Rad, Hercules, CA)
and was in some cases further depleted of MBL by passage through a
column of mannan-Sepharose beads prepared by coupling purified mannan
(15) to CNBr-activated Sepharose (Amersham-Pharmacia,
Uppsala, Sweden) essentially as described by the manufacturers.
C1r-deficient serum with a C1s concentration 
30% of normal was from
a previously described individual (16) and was kindly made
available by Drs. A. Fay and V. Broomhead (Royal Victoria Infirmary,
Newcastle-upon-Tyne, U.K.). Serum completely deficient in functional
C1q was kindly provided by Dr. Schellens-Sanders (University Hospital,
Utrecht, The Netherlands).
C1q was prepared as described (14) by binding to BioRex-70 beads, which were eluted with a salt gradient to separate C1q and factor D. Human IgG and purified MBL/MASP/MAp19 (lot no. 002) used for reconstituting MBL-deficient serum were from Statens Serum Institut (Copenhagen, Denmark). Human serum albumin (HSA) was from Novo Nordisk (Bagsværd, Denmark).
Mouse monoclonal anti-C1q (clone 42, IgG1
)
was a gift from Dr. C. E. Hack (CLB, Amsterdam, The Netherlands)
(17). Mouse monoclonal anti-MBL (clone 131-1,
IgG1) was from Statens Serum Institut. Mouse
monoclonal anti-MASP-1 (clone 2B11, IgG1)
was as described (18). These Abs and nonspecific
monoclonal IgG1 (M 7894; Sigma, St. Louis, MO)
were purified from ascitic fluid by affinity chromatography on protein
A-Sepharose.
A mouse mAb to MASP-2, reacting with the A chain, was produced by
fusing spleen cells from a mouse immunized with recombinant MASP-2 with
mouse myeloma cells (Sp2/0). The expression construct was made by
cyclic amplification (using the BamHI modified sense primer
5'-GGG ATC CCT TAG GCC CGA AGT GGC C-3' and the XhoI
modified antisense primer 5'-CTC GAG ATC CAG GGA ATA TAG TTA ATA AC-3')
with a template from a previously described plasmid (phl-4) containing
a sequence corresponding to full-length MASP-2 (6). The
product obtained was subcloned in PCRII (Invitrogen, Leek, The
Netherlands). After excision with BamHI/XhoI, the
fragment was subcloned into a BamHI/SalI-cut
expression vector, pTrxFus (K350-01; Invitrogen). The construct,
lacking the codons for the first amino-acid residue at the N terminus
and the last five amino-acid residues at the C terminus of MASP-2 (as
analyzed by sequencing of the construct), was expressed in
Escherichia coli and purified on ThioBond resin beads
(R350-10; Invitrogen) according to the manufacturer’s protocol. Mice
were immunized five times at 2- to 4-wk intervals with 5 µg
recombinant MASP-2 per immunization. The first immunization was with
the Ag emulsified in Freund’s complete adjuvant, the next three with
the Ag in Freund’s incomplete adjuvant, and the last immunization was
given i.v. with the Ag dissolved in saline. Hybridomas were screened in
microtiter wells coated with MBL/MASP/MAp19 and subcloned twice. The
resulting mAbs were purified on protein G-Sepharose. Three clones
(1.3B7, 1.19C7, and 1.29F11) were established, all producing
IgG1. Ab from clone 1.3B7 was used in the
present assay.
Polyclonal rat anti-MASP-2 antiserum was produced by immunizing rats with recombinant MASP-2, as described for mice above.
Polyclonal rabbit Abs, anti-A'MASP-2 (against the A chain of MASP-2), anti-B'MASP-2 (against the B chain of MASP-2), and anti-B'MASP-1 (against the B chain of MASP-1) were produced by immunizing rabbits previously primed with bacillus Calmette-Guérin vaccine with synthetic peptides coupled to purified protein derivative of tuberculin (Statens Serum Institut). The peptides represented amino-acid sequences from the N terminus of MASP-2 and internal regions of the serine protease domains of MASP-2 and MASP-1, respectively (6).
Rabbit anti-C4 Ab (A065; Dako, Glostrup, Denmark) was digested with pepsin, and the F(ab')2 fragment was purified by GPC. This fragment, as well as rabbit anti-C1r (AHC-002; Serotec, Kidlington, U.K.), rabbit anti-C1s (AHC-003; Serotec), and normal rabbit IgG (purified from rabbit serum on protein A-Sepharose), was biotinylated with 166 µg of biotin-N-hydroxysuccinimide per 1 mg of Ab in 1 ml PBS/bicarbonate (pH 8.5) (19).
Labeling of Abs with europium for use in time-resolved immunofluo-rometric assay was conducted with the N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid-europium labeling reagent (Wallac, Turku, Finland) as described by the manufacturers.
Goat anti-human C1r (80297) and goat anti-human C1s (80275) were from Bio-Rad. HRP-labeled goat anti-rabbit IgG (PO448), HRP-rabbit anti-goat IgG (PO449), HRP-rabbit anti-rat IgG (PO450), HRP-rabbit anti-mouse IgG (PO260), and rabbit anti-C1q (A136) were from Dako.
SDS-PAGE and Western blotting
SDS-PAGE in a discontinuous buffer system on 15-cm-long 4–20% polyacrylamide gels with subsequent electrophoretic transfer of proteins to polyvinylidene difluoride membranes (Immobilon-P; 0.45-µm pore size; Millipore, Bedford, MA) was performed as described (20). Relative molecular sizes were interpolated from curves constructed on the basis of colored marker proteins (Full-range Rainbow; NK9768; Amersham Life Science, Buckinghamshire, U.K.). The apparent molecular sizes of these dye-conjugated proteins were determined from calibration curves constructed from the unlabeled MARK 12 m.w. markers (Novex, San Diego, CA).
Assay for C4 deposition onto mannan
C4 activation was estimated by measuring the deposition of C4b onto mannan-coated microtiter wells. MaxiSorp (Nunc, Kamstrup, Denmark) wells were coated with mannan by overnight incubation at room temperature with 1 µg mannan in 100 µl 0.1 M sodium carbonate buffer (pH 9.6). Uncoated binding sites were blocked with 0.2 mg HSA in 200 µl TBS (10 mM Tris-HCl buffer (pH 7.4) containing 145 mM NaCl and 15 mM NaN3). After 1 h at room temperature, the microwell plate was washed with TBS/Tween (TBS containing 0.05% (v/v) polyoxyethylenesorbitan monolaurate, Tween 20) and kept at 4°C with TBS until use. Serum samples (100 µl) were diluted 1:90 in 4 mM barbital sodium buffer (pH 7.4) containing 0.14 M NaCl, 2 mM CaCl2, 1 mM MgCl2, and 7.5 mM NaN3 and were incubated in the wells at 37°C for 45 min. The wells were washed, and 100 ng biotinylated anti-C4 F(ab')2 was added to each well in 100 µl TBS/Tween containing 5 mM CaCl2 (TBS/Tween/Ca). Incubation was continued at room temperature for 1 h, after which the wells were washed. Alkaline-phosphatase-conjugated avidin (A2527; Sigma) diluted 1:2500 in TBS/Tween/Ca was then added at 100 µl/well and incubated for 1 h. After further washing, colorigenic substrate (p-nitrophenyl phosphate) was added, and the OD at 405 nm was read on an ELISA reader after suitable color development. MBL/MASPs/MAp19 was added to some sera to a concentration of 1 µg/ml MBL before the sample was diluted for assay.
Assay for deposition of C4 onto IgG
Microtiter wells were incubated with 1 µg human IgG in 100 µl 0.1 M sodium carbonate buffer (pH 9.6). Thereafter, the assay was conducted as described above for the assay for C4 deposition on mannan. C1 activity was reconstituted in C1q-deficient serum by adding C1q to 70 µg/ml.
Analysis of proteins bound to C1q and MBL
Sera were diluted with an equal volume of TBS/Tween, and
100-µl samples were incubated overnight in microwells coated with 0.5
µg mouse mAb (anti-C1q, anti-MBL, anti-MASP-1, or
nonspecific IgG1) in 100 µl PBS. Sera
diluted as above were also incubated in microwells that had previously
been coated with 0.5 µg streptavidin (S-4762; Sigma) and then with
0.5 µg biotin-labeled rabbit anti-C1r, biotin-labeled rabbit
anti-C1s, or biotin-labeled normal rabbit IgG. The wells were then
washed with TBS/Tween/Ca, and the bound proteins were eluted for
analysis by SDS-PAGE and Western blotting. Each sample to be analyzed
was added to 12 identically coated microwells. To elute the bound
proteins, the first well was incubated with 120 µl of SDS-PAGE sample
buffer (0.5 M Tris-HCl buffer (pH 6.7) containing 4 M urea, 10% (v/v)
glycerol, 1.5% weight to volume ratio (w/v) SDS, and 0.1% (w/v)
bromphenol blue) diluted 1:2 in TBS. After 10 min, the eluate was
transferred to the next well, incubated for 10 min, and transferred to
the next well, and so on. The final eluate from the 11th well thus
contained the sum of material eluted from 11 identical wells. All 12
wells were then developed with the relevant Ab to ascertain the coating
efficiency (determined from the 12th well) and the efficiency of
elution from the other 11 wells. The eluted proteins were reduced by
adding DTT to 60 mM, and then they were boiled and subjected to
SDS-PAGE and Western blotting. Identical blots were developed with
monoclonal anti-MBL (1 µg/ml), rabbit anti-C1q (1:3000),
rabbit anti-B'MASP-1 (1:1500), rabbit anti-N'MASP-2
(1:3000), rat anti-MASP-2 (1:1000), goat anti-C1r (1:2000),
goat anti-C1s (1:2000), normal rabbit serum, normal goat serum, or
nonspecific monoclonal IgG1. All were diluted in TBS/Tween except for
the rat anti-MASP-2, which was diluted in TBS/Tween containing
0.1% (w/v) heat-aggregated human IgG (10 mg/ml aggregated at 63°C
for 30 min before removal of the precipitate at 10,000 x
g) and 0.1% (w/v) heat-aggregated HSA (10 mg/ml aggregated
at 75°C for 30 min). After incubation and washing with TBS/Tween
before a final wash with TBS/Tween without NaN3
(enhanced chemiluminescence (ECL) wash buffer), each membrane was
incubated for 2 h with the relevant HRP-labeled secondary Ab
(HRP-goat anti-rabbit Ig 1:3000, HRP-rabbit anti-goat 1:3000,
or HRP-labeled rabbit anti-mouse Ig 1:3000) in ECL wash buffer
containing 1 mM EDTA and normal human IgG 100 µg/ml. The membrane was
washed, impregnated with an ECL substrate system (SuperSignal CL-HRP,
34080; Pierce, Rockford, IL), and used to expose x-ray film (RX
Medical, Fuji, Japan).
Analysis of calcium ion dependency and influence of ionic strength on protease binding
Serum was diluted 1:2 in the following buffers: TBS/Tween/Ca, TBS/Tween/Ca containing a total of 0.5 M NaCl, TBS/Tween/Ca containing a total of 1 M NaCl, TBS/Tween containing 20 mM EDTA, TBS/Tween containing a total of 1 M NaCl and 20 mM EDTA, or TBS/Ca containing 2% (v/v) Tween 20. Serum was subsequently incubated in microwells coated with monoclonal anti-C1q or monoclonal anti-MBL Ab as described above. After washing the microwells with TBS/Tween/Ca, bound proteins were eluted with sample buffer and analyzed by SDS-PAGE and Western blotting as described above (using goat anti-C1r, rabbit anti-B'MASP-1, and monoclonal anti-MASP-2 primary Abs).
Sucrose density gradient centrifugation
Continuous 10–30% sucrose gradients (11 ml) were prepared in
either TBS containing 1 mM CaCl2 or TBS
containing 1 mM EDTA. Samples of 100 µl serum or 100 µl serum with
EDTA added to 10 mM were layered on top of the gradient. The tubes were
centrifuged in a swing-out rotor at 4°C for 24 h at 35,000 rpm
in a Beckman L8-70 M ultracentrifuge. The gradients were subsequently
fractionated by means of the Beckman Fraction Recovery System with 40%
sucrose as displacing fluid, with fractions of 270 µl being
collected.
The fractions were analyzed for C1q and MBL by time-resolved immunofluorometric assay (TRIFMA). For C1q assay, microtiter wells (FluoroNunc; Nunc) were coated with 100 ng F(ab')2 rabbit anti-C1q, incubated with HSA, and washed with TBS/Tween. Fractions diluted 1:200 in TBS/Tween containing 5 mM EDTA and HSA 100 µg/ml were added at 100 µl/well and incubated for 2 h. The wells were then washed with TBS/Tween and incubated with biotinylated rabbit anti-C1q diluted 1:4000 in TBS/Tween for 2 h. The wells were washed again, and europium-labeled streptavidin (1244-360; Wallac) diluted 1:1000 in TBS/Tween containing 25 µM EDTA was added and incubated for 1 h. The wells were washed with TBS/Tween, enhancer solution was added at 200 µl/well, and time-resolved fluorometry readings (Delfia; Wallac) were taken from the wells. For MBL assay, the microtiter wells were coated with mouse monoclonal anti-MBL Ab, and assay details were as described (21). Samples from the fractions were diluted 20-fold in TBS containing 0.5 M NaCl, 10 mM EDTA, and 0.1% (v/v) Tween 20. After incubation, the wells were washed, and europium-labeled monoclonal anti-MBL was added to the wells. The bound europium-labeled Ab was quantified as above.
IgG and IgM were determined in the fractions by sandwich ELISA and TRIFMA, respectively. For IgG analysis, microtiter wells were coated (at 1 µg/ml) with rabbit anti-human IgG (A107; Dako), and fraction samples diluted 1:7000 in TBS/Tween were incubated in the wells. The wells were washed, and alkaline phosphatase-conjugated anti-human IgG (D336; Dako) diluted 1:1500 was added. Bound conjugate was quantified with p-nitrophenyl phosphate substrate, and the OD was read at 405 nm. For IgM analysis, the microtiter wells were coated with monoclonal anti-human IgM (from hybridoma; No. HB 57; American Type Culture Collection, Manassas, VA) at 1 µg/ml and were incubated with fraction samples diluted 1:15 in TBS/Tween. The wells were washed and incubated with biotinylated anti-human IgM (HB 57) diluted 1:2000. Bound biotinylated Ab was quantified with europium-labeled streptavidin and time-resolved fluorometry. Sedimentation coefficients for MBL and C1q were estimated by assuming values of 7 S and 19 S for IgG and IgM, respectively.
GPC of serum
Serum was subjected to GPC in the FPLC system (Amersham-Pharmacia) on a 10 mm x 30 cm Superose 6 HR column (17-0537-01; Amersham-Pharmacia). The eluent was either TBS containing 5 mM EDTA and 0.01% (v/v) Tween 20 or TBS containing 2 mM CaCl2, 1 mM MgCl2, and 0.01% (v/v) Tween 20 at a flow rate of 0.45 ml/min. The column was loaded with 50 µl of undiluted sample (serum containing 2.5 µg/ml MBL), which had been centrifuged at 10,000 x g for 10 min. EDTA was added to the serum to 10 mM before fractionation in the EDTA-containing buffer. Fractions of 0.25 ml were collected in polystyrene microtiter plates previously blocked by incubation with TBS/Tween.
MASP-1, MASP-2, and MAp19 in the fractions from GPC were detected by SDS-PAGE and Western blotting. Samples of 30 µl from each of two consecutive fractions were pooled, and 23 µl SDS-PAGE sample buffer and 9 µl 0.06 M DTT were added. The samples were boiled and applied to the gel for SDS-PAGE and Western blotting. After blocking, the membrane was incubated with rabbit anti-B'MASP-1 1:2000, rabbit anti-B'MASP-2 1:500, or mouse monoclonal anti-MASP-2 1:1000 in TBS/Tween containing 0.01% (w/v) normal human IgG and 0.1% (w/v) HSA. After washing with TBS/Tween and ECL wash buffer, the membrane was incubated for 2 h with HRP-goat anti-rabbit IgG or HRP-rabbit anti-mouse IgG, diluted 1:3000 in ECL wash buffer containing 1 mM EDTA and human IgG 100 µg/ml. This was followed by washing in ECL wash buffer, the addition of chemiluminescence substrate, and exposure to film.
C1q, MBL, and IgM in GPC fractions were measured by TRIFMA as described above. The presence of MBL/MASP-1 complexes in MBL-containing fractions was analyzed by incubating fraction samples diluted 2-fold in TBS/Tween/Ca in microtiter wells previously coated with mannan. The wells were washed and incubated with monoclonal anti-MASP-1. Bound anti-MASP-1 was detected by means of europium-labeled rabbit anti-mouse IgG secondary Ab and time-resolved fluorometry.
Analysis of MASP-1 in MBL complexes was also performed by incubating 1-ml pools of fractions containing MBL in microtiter wells coated with monoclonal anti-MASP-1, using 9 wells per pool. After washing the wells with TBS/Tween, the bound complexes were eluted as above and were analyzed by SDS-PAGE and Western blotting, with the blots being developed with rabbit anti-B'MASP-1, monoclonal anti-MASP-2, or monoclonal anti-MBL Abs.
Quantification of MBL-associated and non-MBL-associated MASPs and MAp19
One milliliter of a serum containing MBL at 3 µg/ml was diluted 2-fold in TBS/Tween/Ca and incubated for 4 h at 4°C with 100 µl mannose-TSK beads (divinylsulfone-activated Fractogel TSK HW-75 beads (Merck, Darmstadt, Germany) coupled with mannose) (22). After centrifugation, the supernatant was taken, the beads were washed, and bound proteins were eluted with SDS-PAGE sample buffer diluted 3-fold in TBS/EDTA. Dilutions of the eluate (containing MBL-associated components), the supernatant (containing non-MBL-associated components), and the original serum were analyzed by SDS-PAGE and Western blotting, with the blots being developed with anti-B'MASP-1 or anti-MASP-2 before the appropriate HRP-labeled secondary Abs and ECL detection. Signals were quantified by means of the Flour-S MultiImager with Multianalyst software (Bio-Rad).
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Results |
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The activation of the complement system on a surface coated with a physiologically relevant carbohydrate ligand for MBL was compared with the complement activation on an Ig-coated surface.
The deposition of C4 fragments (C4b) onto mannan was found to depend on
the concentration of MBL in the serum (Fig. 1
A), whereas deposition onto
an IgG-coated surface did not correlate with the serum MBL
concentration (Fig. 1B). A serum sample was then
specifically depleted of C1q and MBL by ion-exchange chromatography on
BioRex 70 beads before affinity chromatography on mannan-Sepharose.
This removed over 99% of C1q and MBL as measured TRIFMA. The
C1q/MBL-depleted serum did not produce complement deposition onto
mannan- or IgG-coated microtiter wells (Fig. 1). Addition of MBL to the
depleted serum restored C4 deposition onto the mannan surface, and
addition of C1q restored C4 deposition onto the IgG surface (Fig. 1),
but not vice versa (data not shown). The dependence of complement
activation on the C1rC1s complex was subsequently studied on the two
surfaces. When a serum deficient in C1r was incubated in the
mannan-coated wells, C4 was deposited. The MBL concentration in the
C1r-deficient serum was 0.4 µg/ml, which was similar to that of the
serum containing a medium level of MBL (0.3 µg/ml), which produced a
similar deposition of C4. Addition of MBL to the C1r-deficient serum
increased C4 deposition. In contrast, when the C1r-deficient serum was
incubated in the IgG-coated wells, no C4 deposition was seen. As
expected, the addition of extra C1q did not induce C4 deposition
because the C1r-deficient serum already had a normal level of C1q (data
not shown). No C4 deposition was found on the IgG surface when a serum
deficient in C1q was used (Fig. 1B), whereas C4 deposition
occurred on the mannan surface (Fig. 1A). Addition of C1q to
the C1q-deficient serum restored C4 deposition on the IgG surface.
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The molecular composition of complexes containing MBL or C1q was studied, as well as the composition of complexes containing MASP-1, C1r, or C1s. Serum was incubated in microtiter wells coated with Ab against the respective molecules, and the bound proteins were eluted and analyzed by SDS-PAGE and Western blotting. No C1q was bound to monoclonal anti-MBL or monoclonal anti-MASP-1 Abs, as analyzed by developing the Western blots with anti-C1q Ab (data not shown). Biotinylated anti-C1r and anti-C1s Abs bound to streptavidin-coated microwells were used to catch complexes containing C1r or C1s. This approach was found to be superior to coating the Abs directly onto the wells, in that some of the directly coated IgG could be eluted and could give rise to signals on the Western blots as a result of the presence of anti-Ig reactivity in the developing Abs (data not shown). The efficiency of elution was assessed by developing the wells with anti-MBL and anti-C1q Abs (data not shown), and further blots were also developed with anti-MBL or anti-C1q Ab to detect the presence of any eluted MBL or C1q (data not shown).
A band at 100 kDa representing nonactivated MASP-1 was found in eluates
from anti-MBL- and anti-MASP-1-coated wells (Fig. 2, blot A). A band at 76 kDa
representing MASP-2 was found in eluates from anti-MBL-coated wells
(Fig. 2, blots B and C). A strong band
at 20 kDa representing MAp19 was found in eluates from anti-MBL-
and anti-MASP-1-coated wells (Fig. 2, blot C). Bands
representing nonactivated C1r were found in eluates from anti-C1q-,
anti-C1r-, and anti-C1s-coated wells (Fig. 2, blot
D), as were bands representing nonactivated C1s (Fig. 2, blot E).
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, blot C).
Activated forms (bands corresponding to the respective protease A
chains) were also seen for C1r and C1s (in eluates from anti-C1q,
anti-C1r, and anti-C1s) and for MASP-1 (in eluates from
anti-MBL and anti-MASP-1) when the x-ray film exposure time was
increased 10-fold (data not shown).
An unexpected finding was that, whereas MAp19 was present in eluates
from both anti-MBL- and anti-MASP-1-coated wells (Fig. 2, blot C), MASP-2 was eluted only from anti-MBL-coated
wells. This is seen both from the blot developed with rabbit
anti-B'MASP-2 Ab (which only reacts with MASP-2) and from that
developed with monoclonal anti-MASP-2 (which reacts with both
MASP-2 and MAp19). The fact that equal amounts of MAp19 were present in
eluates from anti-MBL- and anti-MASP-1-coated wells (Fig. 2, blot C) shows that this phenomenon is not due to a
difference in the amount of MAp19-containing complexes caught in the
wells. As discussed below, this result probably results from the fact
that most MASP-1/MAp19 complexes in serum are not associated with
MBL.
The signal obtained from the band representing MAp19 was always
stronger than the signal from MASP-2 (Fig. 2, blot C).
Influence of calcium and ionic strength on the composition of complexes caught by anti-C1q or anti-MBL Abs
It has previously been reported that the C1 complex is disrupted
by high NaCl concentrations and by removing calcium ions. Therefore, we
studied the composition of MBL- or C1q-containing complexes in serum
diluted in buffers of different composition. Only traces of C1r and C1s
could be found in association with C1q in serum diluted with buffer
containing 0.5 M or 1 M NaCl before application to the anti-C1q
Ab-coated wells (Fig. 3). Under the same
conditions, MASP-1, MASP-2, and MAp19 were still bound to MBL (Fig. 3).
When the serum was diluted in EDTA-containing buffer, no C1r or C1s was
associated with C1q, whereas most of MASP-1, MASP-2, and MAp19 remained
in association with MBL (Fig. 3). Full dissociation of MASP-1, MASP-2,
and MAp19 from MBL was only observed when the serum was diluted in a
buffer containing both EDTA and a high (1 M) NaCl concentration (Fig. 3). Developing the wells after elution showed that none of these
buffers influenced the binding of MBL to the anti-MBL Ab or of C1q
to the anti-C1q Ab.
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The MBL/MASPs/MAp19 and C1 complexes were studied by sucrose
density gradient centrifugation in the presence of either calcium or
EDTA. Undiluted serum samples were layered on the gradients because it
has been observed that the C1 complex has a tendency to dissociate at
higher dilutions of serum (23). MBL sedimented to a
position corresponding to 10 S at its peak concentration in both
calcium- and EDTA-containing media (Fig. 4). On the other hand, C1q sedimented at
about 11 S in the presence of calcium (Fig. 4, top panel)
and at about 10 S in the presence of EDTA (Fig. 4, bottom
panel). Sedimentation of the C1 complex was thus reduced by the
addition of EDTA, whereas that of the MBL complex was essentially
unaltered.
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Calcium dependence of the integrity of the MBL/MASPs/MAp19 complex
was further studied by GPC and analysis of fractions by SDS-PAGE and
Western blotting. When a serum of MBL concentration 2.5 µg/ml was
fractionated in the presence of calcium, MASP-1 immunoreactivity
emerged at volumes corresponding to 650 kDa and to a broader range
around 300 kDa (Fig. 5A2). The
MASP-1 emerging at 650 kDa on GPC migrated as a 100-kDa band on
SDS-PAGE in the reduced state, whereas that emerging around 300 kDa
gave rise to both 70-kDa and 100-kDa bands. The 70-kDa form was found
in the later fractions emerging from GPC in this region. When
chromatography was performed in the EDTA-containing buffer, no 100-kDa
MASP-1 immunoreactivity was seen at the elution volume corresponding to
650-kDa forms, whereas the pattern of 70- and 100-kDa forms emerging at
around 300 kDa was similar to that seen in the presence of calcium
(Fig. 5B2). A 50-kDa form of MASP-1 immunoreactivity was
seen in both calcium- and EDTA-containing buffers at an elution volume
corresponding to molecules smaller than HSA (Fig. 5, A2 and
B2). Nonactivated MASP-2 (running as 76-kDa immunoreactive
band on SDS-PAGE) emerged in fractions corresponding to 650 kDa on GPC
in calcium-containing buffer (Fig. 5A3), whereas it emerged
at around 300 kDa in EDTA-containing buffer (Fig. 5B3). The
majority of MAp19 (detected as a 19-kDa band with anti-MASP-2 Ab)
emerged at 650 kDa on GPC in calcium-containing buffer (Fig. 5A3) and at around 300 kDa in EDTA-containing buffer (Fig. 5B3). Thus, even in the EDTA-containing buffer most of the
MAp19 emerges considerably earlier than expected for a 19-kDa molecule.
Also, MAp19 was seen at an elution volume corresponding to molecules
smaller than HSA in both calcium- and EDTA-containing buffers (Fig. 5, A3 and B3). As analyzed by TRIFMA, MBL emerged at
volumes corresponding to 720 kDa in the calcium-containing buffer and
850 kDa in the EDTA-containing buffer, in both cases before the
fractions containing the majority of the MBL-associated proteins. C1q
emerged at volumes corresponding to 850 kDa in the
calcium-containing buffer and 800 kDa in the EDTA-containing buffer.
The elution of the MASPs and MAp19 on GPC of a serum containing
only 10 ng/ml MBL exhibited the same pattern as the serum containing
2.5 µg/ml MBL.
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Non-MBL-associated and MBL-associated MASPs and MAp19
Non-MBL-associated and MBL-associated MASPs and MAp19 in serum
were estimated semiquantitatively by fractionating serum with
mannose-coupled TSK beads to adsorb MBL-containing complexes. Western
blots from bound and supernatant fractions were analyzed by ECL
quantified by a digital camera to count the light emission.
Approximately 91% of total MASP-1, 95% of total MASP-2, and 98% of
total MAp19 were found in the supernatant fraction, i.e., they were not
associated with MBL (Fig. 6).
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Discussion |
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The overall structural similarity between MBL and C1q has been emphasized (25). Electron microscopy reveals both proteins to be multimers with long arms, protruding from a central hub in the case of MBL and from a stalk in the case of C1q (11). It has been contentious whether MBL can utilize both the MASPs and the C1r/C1s complex and, inversely, whether C1q can utilize the MASPs. MBL has been found to initiate cleavage of C1r and C1s in vitro. One study reported that the binding of radiolabeled C1r/C1s complexes to E. coli depended on the presence of MBL (12), and another study found that radiolabeled C1s, complexed with C1r, was activated when mixed with MBL and zymosan (11). A recent report indicates that C1r and C1s may be associated with MBL, as analyzed by electron microscopy (13).
The above findings have all been based on studies with purified proteins. In the present report, we have studied the possible associations of these components in serum. To study the initiation of the complement cascade by MBL and C1q, we employed an assay reflecting the opsonization of microorganisms by these two molecules. Microtiter wells were coated with ligand for MBL or C1q, and complement deposition was estimated after incubation with different sources of complement.
Activation of the complement system on mannan was found to depend on the presence of MBL in the sera, whereas activation on IgG depended on the presence of C1q. This accords with other studies describing a correlation between serum levels of MBL (and not anti-mannan Ab) and the deposition of complement factors onto mannan (26). When we employed a serum deficient in C1r, we observed no activation on IgG, whereas activation comparable to that seen with normal serum occurred on mannan surfaces. Addition of further MBL to the C1r-deficient serum enhanced complement deposition on mannan. Thus, the MBL complement-activating pathway was fully functional in the absence of C1rC1s complexes, whereas the classical pathway was nonfunctional.
We subsequently examined the molecular composition of MBL- or C1q-containing complexes. One approach was to catch MBL or C1q and their associated proteins on Ab-coated microtiter wells before analysis of the bound proteins by SDS-PAGE and Western blotting. This strategy revealed that C1r and C1s are only found in association with C1q, whereas MASP-1, MASP-2, and MAp19 are associated with MBL and not with C1q. Wells coated with anti-MASP-1 Ab revealed complexes between MASP-1 and MAp19 but no complexes between MASP-1 and MASP-2. The expected complexes between C1r and C1s were found when employing anti-C1r or anti-C1s as catching Abs. We did not observe any complexes between the MASPs/MAp19 and C1r or C1s.
The integrity of the C1 complex depends on calcium, but there is controversy as to the requirement of calcium for maintaining the MBL/MASP/MAp19 complex. The isolation of MASP-1 by EDTA treatment of MBL/MASP complex bound to anti-MBL-coated beads has been reported (5). On the other hand, a study of the behavior of purified MBL/MASP on GPC in different buffers indicated that the MASPs are bound to MBL independently of calcium (27). In the present report we studied the dependency of the assembly of MBL/MASP/MAp19 on divalent cations in whole serum by several methods and found only a very modest influence of adding EDTA, a chelator of divalent metal ions, at physiological ionic strength.
On sucrose density gradient centrifugation, MBL-containing complexes
sediment at the same rate, corresponding to about 10 S for the peak
concentration, whether in the presence of calcium or EDTA. However,
C1q-containing complexes suffer a shift in sedimentation from a peak
corresponding to about 11 S in the presence of calcium to a peak
corresponding to 10 S in the presence of EDTA. The latter agrees with
the 10.2 S value found for purified C1q (28). This
phenomenon is strongly suggestive of an EDTA-induced dissociation of
the C1 complex. It previously has been found that the MBL in sera from
individuals with the wild-type genotype of the MBL gene separate into
several forms corresponding to 10.3, 11.9, 13.6, and 14.6 S, with
70% (based on analysis by Western blotting) in the fractions at or
above 11.9 S (29). The largest form was assumed to
represent pentamers and the smallest form to represent dimers of the
structural subunit of three polypeptide chains. The data in the present
report are based on a quantitative assay, and we see a symmetrical peak
around 10 S.
Analysis of complexes bound to anti-C1q Abs in different buffers showed that the C1 complex was dissociated by high salt concentrations, as previously reported (30). However, high salt concentrations did not dissociate the MBL/MASP/MAp19 complexes. This observation is supported by the fact that in certain MBL/MASP purification procedures, e.g., affinity chromatography on mannose- or mannan-Sepharose, the MASPs are eluted together with MBL after washing the affinity matrix with buffers containing NaCl at concentrations above 1 M.
The MBL/MASPs/MAp19 complexes could be dissociated by diluting serum in buffers containing both a high salt concentration and EDTA. This observation is supported by the fact that MASP-1 was eluted with EDTA at high salt concentration from MBL bound to anti-MBL beads (5).
Increasing the ionic strength usually promotes hydrophobic interactions and inhibits ionic interactions, indicating that the interaction between MASPs/MAp19 and MBL is primarily hydrophobic. This differs from the interaction of C1r2C1s2 with C1q, which is strictly calcium-dependent and in which electrostatic interactions apparently play a significant role. The interaction of C1r and C1s with C1q has been proposed to be mediated by the first CUB domain and by the EGF domain (31, 32). Thus, it would be of interest to examine the involvement of these two domains of MASP-1, MASP-2, and MAp19 in the interaction with MBL and to investigate the calcium dependence of such an interaction. The simple approach of comparing the sequences of these domains in the different proteins does not reveal any obvious physicochemical difference among C1r, C1s, MASP-1, and MASP-2.
Another difference between the complexes is that the C1 complex is reported to be stable at low pH (pH 5.5) in the presence of calcium (33), whereas the MASPs are dissociated from MBL at low pH (27).
Over 95% of the total MAp19, MASP-1, and MASP-2 in serum are not associated with MBL. In addition, GPC showed that part of the non-MBL-associated MASP and MAp19 in serum occurs as large calcium-dependent complexes. It has previously been suggested that most of the MASP-1 in serum is not associated with MBL (18). This contrasts with the components of the C1 complex, where only relatively small amounts of non-C1q-associated C1r and C1s have been reported in serum (34).
We propose that most MASP-1 and MAp19 are found together in serum as a non-MBL-associated complex and that MASP-2 forms non-MBL-associated complexes with itself. Some of the non-MBL-associated MASPs/MAp19 could be associated with ficolins (35). However, we have not ruled out the presence of uncharacterized proteins in complex with the non-MBL-associated MASPs. By analogy with the C1sC1rC1rC1s complex, the interaction between the different MASP and MAp19 components may occur via the first CUB and the EGF domain (9). On the other hand, the specificities of the serine proteases are probably determined by the two CCP domains in conjunction with the serine protease domain (9).
It should be noted that MBL emerged later from the Superose GPC column in calcium-containing buffer than it did in EDTA-containing buffer. This is probably because of a calcium-dependent interaction of MBL with the carbohydrate of the agarose beads and does not necessarily reflect a change in conformation or oligomeric state. Binding of MBL to Sepharose has previously been used as a step in purifying MBL (27, 36).
As discussed above, the MBL-associated MASPs and MAp19 are not dissociated at physiological ionic strength in the presence of EDTA. The EDTA-mediated dissociation occurs only at a high salt concentration. However, EDTA does mediate the dissociation of the high molecular weight non-MBL-associated MASP-1/MAp19 complexes and non-MBL-associated MASP-2 complexes at physiological ionic strength. The elution positions on GPC of the MASPs and MAp19 in EDTA-containing buffer are still significantly different from those expected for monomers, indicating the existence of non-calcium-dependent dimers, which might be analogous to the dimers of C1r (37). Some MAp19 is found at the expected position of monomers of this protein, in both calcium-containing and EDTA-containing buffers. The stoichiometric composition of the complexes remains to be determined.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steffen Thiel, Department of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, DK-8000 Aarhus, Denmark.
3 Abbreviations used in this paper: MBL, mannan-binding lectin; MASP, MBL-associated serine protease; MAp19, 19-kDa MBL-associated protein; EGF, epidermal growth factor; CCP, complement control protein; GPC, gel-permeation chromatography; CUB, acronym of complement subcomponent C1r/C1s, Uegf, and Bmp 1; w/v, weight to volume ratio; ECL, enhanced chemiluminescence; TRIFMA, time-resolved immunofluorometric assay.
Received for publication September 13, 1999. Accepted for publication May 4, 2000.
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-C1q), monoclonal anti-MBL (
) and C1q (left axis,
) concentrations by TRIFMA and for IgG and IgM (peak positions
marked) by ELISA and TRIFMA, respectively. The fractionation volume is
shown on the x-axis.
