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* Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat à l’Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble, France; and
Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan
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Abstract |
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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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Collectins are another family of oligomeric lectins involved in the first line of defense against pathogens that comprise a collagenous region and a C-type lectin carbohydrate recognition domain (9). Mannan-binding lectin (MBL) is a member of this family that binds to mannose and GlcNAc groups present on the surface of various pathogens and is able to initiate activation of the lectin pathway of complement (10). MBL circulates in association with four structurally related proteins, the MBL-associated serine proteases (MASP)-1, -2, and -3 (10, 11, 12) and a truncated form of MASP-2 called MAp19 (19-kDa MBL-associated protein) or small MBL-associated protein (13, 14). Although the precise roles of the different complexes have not yet been elucidated, it is clear that the MBL-MASP-2 complex is sufficient to trigger complement activation through cleavage of C4 and C2 (15, 16, 17).
The MASPs are modular proteins homologous to the C1r and C1s proteases of the C1 complex that trigger the classical pathway of complement (18). They comprise, from the N-terminus, two CUB modules (module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1) (19) surrounding an epidermal growth factor (EGF)-like module with a consensus motif for Ca2+ binding (20), two contiguous complement control protein modules, and a serine protease domain. Studies using recombinant human MASPs and modular fragments derived from their N-terminal part have demonstrated that MASP-1, MASP-2, and MAp19 each individually form Ca2+-dependent complexes with MBL through their N-terminal CUB1-EGF moieties (21). Using recombinant fragments from the rat, it was shown that the interaction of MASP-1 and MASP-2 with rat MBL involves their three N-terminal CUB1-EGF-CUB2 modules (22).
It has been demonstrated recently that, like MBL, human L-ficolin/P35 associates with the MASPs and MAp19 in serum and forms complexes able to activate complement (23). The objective of the present study was to investigate the interaction properties of these proteins by surface plasmon resonance spectroscopy, using L-ficolin/P35 derived from human serum and recombinant MASPs and MAp19 expressed in a baculovirus/insect cells system. Our data demonstrate that these proteins bind individually to L-ficolin/P35 in a Ca2+-dependent fashion, with affinities in the nanomolar range, comparable to those determined previously for their interaction with MBL. Evidence is also provided that MBL and L-ficolin/P35 compete with each other for binding to the MASPs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The plasmids containing the full-length MASP-1 and MASP-2 cDNAs were obtained as described previously (11, 24). Oligonucleotides were purchased from Oligoexpress (Paris, France). VentR polymerase was from New England Biolabs (Beverly, MA). Asialofetuin-Sepharose was prepared by coupling asialofetuin (Sigma-Aldrich, St. Louis, MO) to cyanogen bromide-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ).
Proteins
MBL was isolated from human plasma according to the procedure described by Tan et al. (25), modified as described by Thielens et al. (21). The MBL molar concentration was estimated assuming a hexameric structure of 450 kDa (18 polypeptide chains of 25 kDa each). Recombinant MASP-1, MAp19, and the CUB1-EGF segments of MASP-1 and MASP-2 were expressed using a baculovirus/insect cells system and purified as described previously (21). The concentrations of purified recombinant proteins were determined using absorption coefficients (A1%, 1cm at 280 nm) calculated by the method of Edelhoch (26) and an m.w. calculated from the amino acid sequence or determined by mass spectrometry, as follows: MASP-1 CUB1-EGF-CUB2 segment, 10.0 and 34,300 (this study); MASP-1 CUB1-EGF fragment, 10.0 and 21,000 (21); MASP-2 CUB1-EGF fragment, 11.7 and 18,861 (21); and Map19, 11.6 and 19,086 (21). Due to the low amount of material available, estimation of the concentrations of full-length MASP-1, MASP-2, and of MASP-2 CUB1-EGF-CUB2 was based on Coomassie blue staining after SDS-PAGE analysis using appropriate internal standards and m.w. of 82,000 (27), 75,100, and 31,600, respectively.
Isolation of L-ficolin/P35
Cohn fraction III from human plasma was fractionated with polyethylene glycol 4000 at a concentration of 8%. The precipitate was dissolved in 50 mM Tris, 200 mM NaCl, and 20 mM CaCl2, pH 7.8 (starting buffer), and applied to a GlcNAc-agarose column (Sigma). MBL-MASPs and L-ficolin/P35-MASPs complexes were eluted sequentially using the starting buffer containing 0.3 M mannose and 0.3 M GlcNAc, respectively. Fractions containing L-ficolin/P35-MASPs were dialyzed against the starting buffer and applied to an asialofetuin-Sepharose column. L-ficolin/P35-MASP complexes were eluted with the starting buffer containing 0.3 M GlcNAc; dialyzed against 50 mM Tris, 1 M NaCl, and 20 mM EDTA, pH 7.8; and applied again to the asialofetuin-Sepharose column. The MASPs and small MBL-associated protein/MAp19 passed through the column, whereas L-ficolin/P35 was retained on the column and eluted with a buffer containing 0.3 M GlcNAc. L-ficolin/P35 was finally passed through anti-MBL (3E7)-Sepharose (10) and anti-H-ficolin (4H5)-Sepharose (28) columns. The molar concentration of L-ficolin/P35 was estimated assuming a tetrameric structure of 420 kDa (12 polypeptide chains of 35 kDa each).
Construction of expression plasmids containing full-length MASP-2 and its CUB1-EGF-CUB2 segment
DNA fragments encoding the MASP-2 signal peptide, the mature protein (aa 1–671), and an additional C-terminal Arg-(His)6 sequence, or the MASP-2 signal peptide followed by residues 1–281 of the mature protein (corresponding to the CUB1-EGF-CUB2 segment) were amplified by PCR using VentR polymerase and pFastBac-MASP-2 (17) as a template, according to established procedures. The sequence of the sense primer (5'-CGGGATCCATGAGGCTGCTGACCCTC-3') introduced a BamHI restriction site (underlined) at the 5' end of both PCR products. The antisense primer for full-length MASP-2 (5'-GGAATTCCTAGTGATGGTGATGGTGATGTCTAAAATCACTAATTATGTTCTCGATCCAGGGAAT-3') introduced the Arg-(His)6 sequence (bold and underlined) followed by a stop codon (bold) and an EcoRI site (underlined) at the 3' end of the fragment. The antisense primer for the CUB1-EGF-CUB2 segment (5'-GGAATTCCTATGTGCTCGTGTAGTGG-3') introduced a stop codon (bold) followed by an EcoRI site (underlined) at the end of the PCR product. The amplified DNA fragments were digested with BamHI and EcoRI, purified, and cloned into the corresponding sites of the pFastBac1 baculovirus transfer vector (Invitrogen, San Diego, CA). The final constructs were characterized by restriction mapping and were checked by dsDNA sequencing (Genome Express, Grenoble, France).
Construction of the expression plasmid containing the MASP-1 CUB1-EGF-CUB2 segment
A DNA fragment encoding the signal peptide and the N-terminal
CUB1-EGF-CUB2 segment of MASP-1 (aa 1–278 of the mature protein) was
amplified by PCR using VentR polymerase and
MASP-1b/pDR2
EF1
(24) as a
template, according to established procedures. The sequence of the
sense primer (5'-GAAGATCTATGAGGTGGCTGCTTCT-3') introduced a
BglII restriction site (underlined) at the 5' end of the PCR
product, and that of the antisense primer
(5'-GGAATTCCTATGCAGCCCTGTATGAG-3')
introduced a stop codon (bold) followed by an EcoRI site
(underlined) at the 3' end. The PCR fragment was digested with
BglII and EcoRI, purified, and cloned into the
BamHI/EcoRI sites of the pFastBac1 vector. The
final construct was verified by dsDNA sequencing.
Cells and viruses
The insect cells Spodoptera frugiperda (Ready-Plaque Sf9 cells; Novagen, Madison, WI) and Trichoplusia ni (High Five cells; Invitrogen) were routinely grown and maintained as described previously (21). Recombinant baculoviruses were generated using the Bac-to-Bac system (Invitrogen). The bacmid DNA was purified using the Qiagen midiprep purification system (Qiagen, Courtaboeuf, France) and was used to transfect Sf9 insect cells with Cellfectin in Sf900 II SFM medium (Invitrogen) as described in the manufacturer’s protocol. Recombinant virus particles were collected 4 days later, titrated by virus plaque assay, and amplified as described by King and Possee (29).
Production and purification of MASP-2 and the CUB1-EGF-CUB2 segments of MASP-1 and MASP-2
High Five cells (1.75 x 107 cells/175-cm2 tissue culture flask) were infected with the recombinant viruses at a multiplicity of infection of 2 in Sf900 II SFM medium at 28°C for 72 h. Culture supernatants were collected by centrifugation.
The culture supernatant containing MASP-2 (530 ml) was concentrated to 35 ml by ultrafiltration and dialyzed against 500 mM NaCl and 20 mM Na2HPO4, pH 7.4 (buffer A), containing 5 mM imidazole. The concentrated supernatant was incubated for 1 h at room temperature under gentle agitation with 4 ml of Chelating-Sepharose Fast Flow resin (Amersham Pharmacia Biotech) loaded with Ni2+ ions according to the manufacturer’s protocol and equilibrated in the dialysis buffer. The gel was collected in a column cartridge and was washed three times with 20 ml of the same buffer and three times with 20 ml of buffer A containing 25 mM imidazole. Elution was conducted in two steps, with three washes with 8 ml of buffer A containing 100 mM imidazole and then three washes with 8 ml of buffer A containing 200 mM imidazole. The fractions containing full-length MASP-2 were identified by Western blot analysis; concentrated 20-fold by ultrafiltration; dialyzed against 145 mM NaCl, 1 mM CaCl2, and 50 mM triethanolamine hydrochloride, pH 7.4; and stored at -20°C.
The CUB1-EGF-CUB2 segments of MASP-1 and MASP-2 were purified according to the protocol used for the CUB1-EGF segments of MASP-1 and MASP-2, respectively (21).
Chemical characterization of the recombinant proteins
N-terminal sequence analyses were performed after SDS-PAGE and electrotransfer, using an Applied Biosystems model 477A protein sequencer (Foster City, CA) as described previously (30). Mass spectrometry analyses were performed using the matrix-assisted laser desorption ionization technique on a Voyager Elite XL instrument (Perseptive Biosystems, Cambridge, MA), under conditions described previously (31).
PAGE and immunoblotting
SDS-PAGE analysis was performed as described previously (32). Western blot analysis and immunodetection of the recombinant proteins were conducted as described by Rossi et al. (33), using mouse anti-MASP-2 mAb 1.3B7 (34) or rabbit anti-peptide Abs directed against the N- and C-terminal ends of MASP-1 (11).
Real-time surface plasmon resonance spectroscopy and data evaluation
Surface plasmon resonance measurements were performed using an upgraded BIAcore 1000 or a BIAcore 3000 instrument (BIAcore, Uppsala, Sweden). The running buffer for protein immobilization was 145 mM NaCl, 5 mM EDTA, and 10 mM HEPES, pH 7.4. Protein ligands were diluted to 30 µg/ml in 10 mM sodium acetate, pH 4.0 (MBL) or pH 5.0 (L-ficolin/P35), and immobilized onto the carboxymethylated dextran surface of a CM5 sensor chip (BIAcore) using amine coupling chemistry (BIAcore amine coupling kit). Binding of the recombinant proteins to immobilized L-ficolin/P35 or MBL was measured at a flow rate of 20 µl/min in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4, and 0.005% surfactant P20. Equivalent volumes of each analyte were injected over a surface with immobilized BSA to serve as blank sensorgrams for subtraction of the bulk refractive index background. Regeneration of the MBL and L-ficolin/P35 surfaces between analyses was achieved by injection of 10 µl of 5 mM EDTA or 1 M NaCl/20 mM EDTA, respectively. The Ca2+ dependence of the interaction between the MASPs or their N-terminal fragments and MBL or L-ficolin/P35 was studied in 145 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, and 0.005% surfactant P20 containing 1 mM EGTA and various amounts of calcium calculated to give the desired free calcium concentrations as previously described (35).
The data were analyzed by global fitting to a 1:1 Langmuir binding model of both the association and dissociation phases for several concentrations simultaneously, using BIAevaluation 3.1 software (BIAcore). The apparent equilibrium dissociation constants (Kd) were calculated from the ratio of the dissociation and association rate constants (koff/kon).
The binding curves recorded for each analyte at varying Ca2+ concentrations were analyzed in the same way to determine the equilibrium level of analyte binding to the surface (Req). Ca2+ concentrations corresponding to half-maximum binding were determined by nonlinear regression analyses of the Req vs log Ca2+ concentration curves using Sigmaplot 5.0 software (Sigma-Aldrich).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The modular structures of MASP-1, MASP-2, and the recombinant
fragments used in this study are represented in Fig. 1
. The recombinant baculoviruses for
production of MASP-2 and the CUB1-EGF-CUB2 segments of MASP-1 and -2
were generated as described in Materials and Methods and
used to infect High Five insect cells for 72 h at 28°C. The
amount of recombinant protein secreted into the culture supernatants,
as estimated by SDS-PAGE and Western blot analysis, ranged from 0.15
µg/ml (MASP-2 and its CUB1-EGF-CUB2 segment) to 10 µg/ml (MASP-1
CUB1-EGF-CUB2 segment). In each case a significant part of the
recombinant material, ranging from 30% (MASP-1 CUB1-EGF-CUB2) to
>90% (MASP-2 and its CUB1-EGF-CUB2 segment), was found in the cell
pellet fraction. In the case of MASP-2, two bands reacting with a
specific Ab were obtained, one corresponding to the full-length
protease and the other to a 45-kDa truncated fragment derived from the
N-terminal end of the protein, as observed previously for production in
the same expression system of the recombinant protease without a
six-histidine tag (17). The presence of the six-histidine
tag at the C-terminal end of the protease allowed separation of
full-length MASP-2 from the truncated fragment using immobilized nickel
ion affinity chromatography. Although the fragment unexpectedly
remained bound to the column at a low imidazole concentration (25 mM),
it was totally eluted at 100 mM imidazole, whereas most of the
full-length protein eluted at 200 mM imidazole. The purification yield
of MASP-2 was very low, as only 
20 µg of purified protein was
recovered from 500 ml of culture supernatant. The recombinant protease
was therefore detected routinely by Western blot analysis using an mAb
specific for the N-terminal end of MASP-2 rather than by Coomassie Blue
staining. Analysis of the purified MASP-2 by SDS-PAGE under
nonreducing conditions (Fig. 2A, lane 1)
indicated that the protease migrated as a single 80-kDa species that
yielded two sequences upon Edman degradation:
Thr-Pro-Leu-Gly-Pro-Lys-Trp-Pro-Glu-Pro... and
Ile-Tyr-Gly-Gly-Gln-Lys-Ala-Lys-Pro-Gly..., corresponding to the
N-terminal ends of the mature protein and the serine protease
domain, respectively. Analysis under reducing conditions showed the
presence of a 45-kDa band reactive with Abs directed to the N-terminal
end of the molecule and corresponding to the A chain and of a 28-kDa
band corresponding to the serine protease domain that was revealed by
Coomassie blue staining (not shown) but did not react with the Ab (Fig. 2A, lane 2). These results indicate that the
protein was fully activated at the end of the purification
procedure.
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20% of the total protein contents.
It migrated under nonreducing and reducing conditions as a single band
with an apparent molecular mass of 32 kDa (Fig. 2
B) that
yielded a single N-terminal sequence identical with that of the MASP-2
A chain.
The CUB1-EGF-CUB2 fragment of MASP-1 could be purified to homogeneity
using ion exchange and gel permeation chromatography, as described in
Materials and Methods. SDS-PAGE analysis of the recombinant
protein indicated that it migrated as a species with an apparent
molecular mass of 35 kDa (Fig. 3),
yielding a single N-terminal sequence
(His-Thr-Val-Glu-Leu-Asn-Asn-Met-Phe-Gly...) identical with that of
the mature MASP-1. The diffuse character of the band can be accounted
for by the carbohydrate content of the protein, as it contains two
potential N-linked oligosaccharides (27) (see
Fig. 1). Analysis by matrix-assisted laser desorption ionization mass
spectrometry yielded a heterogeneous peak centered on a mass value of
34,296 ± 30 Da. Given the predicted mass of the polypeptide
moiety of the protein (31,973 Da), this yields a deduced mass
value of 1,161 ± 30 Da for each carbohydrate component,
consistent with the occurrence of two oligosaccharide chains comprising
two N-acetylglucosamine and four or five mannose residues
(calculated masses, 1,055–1,217 Da).
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The ability of the full-length MASPs, their N-terminal
CUB1-EGF-CUB2 and CUB1-EGF segments, and MAp19 to associate with
L-ficolin/P35 was studied using surface plasmon resonance spectroscopy.
As shown in Fig. 4, MASP-1 and its
CUB1-EGF-CUB2 and CUB1-EGF segments bound to immobilized L-ficolin/P35
in the presence of 1 mM CaCl2. Whereas the
association and dissociation phases of the binding curves exhibited
comparable shapes for full-length MASP-1 and its CUB1-EGF-CUB2 fragment
(Fig. 4, A and B), the dissociation was much
faster in the case of the shorter CUB1-EGF fragment (Fig. 4C). In each case binding of the proteins to L-ficolin/P35
was inhibited when EDTA was substituted for Ca2+
in the running buffer, and residual binding at the end of the
association phase accounted for <10% of the value observed in the
presence of CaCl2. Binding of MASP-2 was studied
in the same way, providing evidence for a
Ca2+-dependent interaction between L-ficolin/P35
and the full-length protease, its N-terminal fragments, and MAp19.
Again, comparable binding curves were obtained for MASP-2 and its
CUB1-EGF-CUB2 fragment (Fig. 5, A and B), whereas MASP-2 CUB1-EGF (not shown) and
MAp19 (Fig. 5C) dissociated faster from L-ficolin/P35.
Binding of MASP-2 CUB1-EGF and MAp19 to L-ficolin/P35 was not
detectable in the presence of EDTA. In contrast, residual binding
was still observed in EDTA for MASP-2 and its CUB1-EGF-CUB2 fragment,
although to relative extents of only 30 and 10%, respectively,
compared with binding achieved in the presence of
CaCl2.
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. Full-length MASP-1 and its N-terminal
fragments showed comparable kon values
(1.1–1.8 x 105 M-1
s-1), whereas koff
values were similar for MASP-1 and its CUB1-EGF-CUB2 fragment
(1.0–1.2 x 10-3
s-1) and significantly higher for the CUB1-EGF
fragment (4.8 x 10-3
s-1), in accordance with the shape of the
binding curves (Fig. 4). This resulted in a 3-fold increase in the
apparent Kd for the binding of the shorter
CUB1-EGF fragment. Similarly, MASP-2, its N-terminal fragments, and
MAp19 exhibited comparable kon values,
ranging from 2.2 to 2.7 x 105
M-1 s-1, whereas MAp19
and the CUB1-EGF segment both showed increased
koff values, also resulting in a 3-fold
increase in their apparent Kd.
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As previously observed in the case of full-length MASP-1 and
MASP-2 and their CUB1-EGF fragments (21), the
CUB1-EGF-CUB2 fragments of MASP-1 and MASP-2 bound to immobilized MBL
in the presence of calcium, and the interaction was totally prevented
in the presence of EDTA (Fig. 6). The
kinetic parameters for interaction of the CUB1-EGF-CUB2 fragments and
the full-length proteases with MBL were determined and compared with
the values previously obtained for MAp19 and the CUB1-EGF fragments
(Table II). The
kon values were of the same order for
MASP-1 and its N-terminal fragments (1.9–2.1 x
105 M-1
s-1) and for MASP-2, its N-terminal fragments,
and MAp19 (2.3–3.7 x 105
M-1 s-1). The
koff values increased from 5.9–6.8 x
10-4 s-1 for the
full-length proteases to 4.6–5.4 x 10-3
s-1 for the CUB1-EGF segments and MAp19, with
intermediate values for the CUB1-EGF-CUB2 segments (1.3–1.5 x
10-3 s-1), resulting in
similar variations in the Kd values.
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As the binding constants for the interaction of the recombinant
proteins with either L-ficolin/P35 or MBL were in the same range (see
Tables I and II), we addressed the question of whether MBL and ficolin
compete with each other for binding to MASP-1 and MASP-2. Preincubation
of the MASPs and each of their fragments with equimolar amounts of
soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35
and vice versa. This is illustrated in Fig. 7 in the case of full-length MASP-2
(A and A'), MASP-1 CUB1-EGF-CUB2 (B
and B'), and MAp19 (C and
C'). Preincubation of the various analytes with soluble MBL
also inhibited subsequent binding to immobilized MBL (data not shown).
Unexpectedly, preincubation of the MASPs with soluble L-ficolin/P35
resulted in increased binding of the injected material to immobilized
L-ficolin/P35. In a control experiment, injection of soluble
L-ficolin/P35 on immobilized L-ficolin/P35 also resulted in binding to
a level comparable to that obtained when using soluble P35/MASPs
complexes. No significant binding of isolated L-ficolin/P35 to
immobilized MBL or of soluble MBL to immobilized L-ficolin/P35 or MBL
was observed in the concentration range used (<50 nM).
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We next studied the Ca2+ dependence
of the binding of MASP-1 and MASP-2 or their fragments to immobilized
L-ficolin/P35 or MBL. Sensorgrams were recorded at different
Ca2+ concentrations, and resonance units at
equilibrium (Req) were determined for each
analyte as described in Materials and Methods. Binding of
the MASPs and their N-terminal fragments to the immobilized ligands
increased with Ca2+ concentration to reach a
plateau at Ca2+ concentrations ranging from
3–10 µM for binding to L-ficolin/P35 (Fig. 8, A and B), and
from 10–30 µM for binding to MBL (Fig. 8, A' and
B'). No binding of any protein to MBL or of the N-terminal
fragments of MASP-1 to L-ficolin/P35 was observed at calcium
concentrations <100 nM. Residual binding to L-ficolin/P35 was observed
at 10 nM calcium or in the absence of calcium for MASP-1, MASP-2, and
its CUB1-EGF-CUB2 and CUB1-EGF fragments, accounting for 8.5, 33, 13.5,
and 2% of the maximal binding observed at the plateau, respectively,
in accordance with previous observations (see Fig. 4A and
Fig. 5, A and B). To determine the
Ca2+ concentrations that yielded half-maximal
binding, Req values were normalized for
each recombinant protein to the maximal value obtained at the plateau,
and the values obtained are reported in Table III. Half-maximal binding to
L-ficolin/P35 was found to occur at comparable
Ca2+ concentrations for MASP-1 and its fragments
(0.47–0.79 µM) as well as for MASP-2 and its fragments (0.31–0.45
µM). Half-maximal binding to MBL occurred at comparable
Ca2+ concentrations for the MASP-1 and MASP-2
fragments (0.63–1.3 µM), but at significantly higher concentrations
for the full-length proteases (2.7–2.9 µM).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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70%), and the
purified protein was totally recovered in a two-chain activated form,
resulting from cleavage at the expected Arg-Ile bond at position
444.
The CUB1-EGF-CUB2 segment of MASP-2 was also produced at a low yield
(0.1 µg/ml) comparable to that of the full-length protease, in
contrast to the shorter CUB1-EGF segment and the related MAp19 protein,
which were produced at higher yields (50- to 80-fold) using the same
expression system (21). It should be mentioned that the
low amount of secreted protein correlated with the high amount of
recombinant material present inside the cells, suggesting that the
secretion rate, rather than the expression level, is responsible for
the low production yield. In contrast, the CUB1-EGF-CUB2 fragment of
MASP-1 was produced at a satisfactory yield (10 µg/ml), comparable
to that obtained previously with the CUB1-EGF fragment of MASP-1
(21). As expected from the occurrence of two
N-glycosylation sites at Asn30 and
Asn159 (27), the CUB1-EGF-CUB2
fragment of MASP-1 was produced in a glycosylated form. The data
obtained by mass spectrometry analysis were consistent with the
presence of two short high mannose oligosaccharides and provided no
evidence for distinct species bearing either one or two carbohydrates,
as observed previously in the case of the CUB1-EGF fragment
(21).
MASP-1 and MASP-2 each bound individually to L-ficolin/P35 in the
presence of Ca2+, with
Kd values of 9.2 and 4.6 nM, respectively,
indicating high affinity in both cases. Each CUB1-EGF-CUB2 segment had
the same behavior as its corresponding full-length protein, including
comparable kon and
koff values. In contrast, the shorter
CUB1-EGF segments exhibited similar kon
values, but significantly higher dissociation constants. These results
are reminiscent of those obtained using rat proteins (22, 36), indicating that the three N-terminal CUB1-EGF-CUB2 modules
of MASP-1 and MASP-2 are required for efficient binding to MBL. Our
current data show that this also applies to the human proteins, since
human MASP-1 and MASP-2 dissociate from MBL with
koff values comparable to those of their
respective CUB1-EGF-CUB2 segment, but significantly lower than those of
their CUB1-EGF segment (see Table II). Comparable
Kd values were obtained in the present
study for the binding of MASP-1 and MASP-2 to MBL (3.2 and 2.6 nM,
respectively). The fact that these values differ slightly from those
determined previously (1.4 and 0.8 nM, respectively) probably results
from an underestimation of the MASP-2 concentration in the former study
(21). Overall, the apparent Kd
values of MASP-1 and MASP-2 for L-ficolin/P35 and MBL are in the same
range, although slightly higher values were obtained in the case of
L-ficolin/P35, mainly because of higher dissociation rates (see Tables I and II). Taken together, the above data indicate that MASP-1 and
MASP-2 associate with L-ficolin/P35 and MBL in similar ways. In both
cases the interaction involves a major contribution of the CUB1-EGF
module pair of the proteases, but is strengthened by the following CUB2
module. The latter may either stabilize the structure of the preceding
CUB1-EGF module pair or contribute additional contacts and hence
tighten the interaction. As previously observed in the case of MBL
(21), MAp19 and the CUB1-EGF segment of MASP-2 exhibited
virtually identical Kd values for
L-ficolin/P35, providing further evidence that the extra four residues
at the C-terminal end of MAp19 have no influence on its interaction
properties.
Interaction of the MASPs with L-ficolin/P35 showed a clear Ca2+ dependence, but was only partly sensitive to EDTA. Thus, significant binding of MASP-1, MASP-2, and the MASP-2 CUB1-EGF-CUB2 segment was still observed in the absence of Ca2+ ions. In the same way, complete removal of the bound proteins from immobilized L-ficolin/P35 required both EDTA and high salt concentration (1 M NaCl). The Ca2+ concentrations yielding half-maximal binding to L-ficolin/P35 were close for the full-length proteins and their CUB1-EGF or CUB1-EGF-CUB2 segments (0.47–0.79 µM for MASP-1 and 0.31–0.45 µM for MASP-2), indicating that all species bind Ca2+ with comparable affinities. It may therefore be concluded that the CUB1-EGF module pair contains all the ligands involved in Ca2+ binding and that the observed increased Kd of this fragment for L-ficolin/P35 does not result from a decreased affinity for Ca2+ ions.
Significant differences were observed between L-ficolin/P35 and MBL with respect to the Ca2+ dependence of their interaction with the MASPs. First, binding of the recombinant MASPs to MBL was totally prevented in the presence of EDTA, and complete elution of the bound proteins could be achieved by treatment with 5 mM EDTA (21). In addition, half-maximal binding of full-length MASP-1 and MASP-2 consistently occurred at lower Ca2+ concentrations in the case of L-ficolin/P35 (0.47 and 0.45 µM, respectively) than in the case of MBL (2.7 and 2.9 µM, respectively). Indeed, identical values would have been expected if these would account only for the affinity of the MASPs for Ca2+ ions. A plausible hypothesis may be the occurrence of a Ca2+ bridge at the interface between MBL and the MASPs, which would be consistent with the observed strict sensitivity of the MBL-MASP interaction to EDTA. Alternatively, Ca2+ ions could be necessary to maintain a biologically active conformation of MBL, which itself would be required for optimal binding to the MASPs. Interestingly, the Ca2+ concentrations yielding half-maximal binding of MASP-1 and MASP-2 to MBL or L-ficolin/P35 are in the low micromolar range, suggesting that the MASPs have a higher affinity for Ca2+ than that determined previously for C1r and C1s, the homologous proteases of the C1 complex of complement (Kd = 15–38 µM) (32, 35). The CUB1-EGF-CUB2 fragments of rat MASP-1 and MASP-2 were also shown to bind Ca2+ with significantly higher apparent Kd values (350 and 190 µM, respectively) (22). The discrepancy between these and our current data could reflect a difference between the Ca2+ affinities of the human and rat MASPs, although the sequences of the CUB1-EGF-CUB2 regions are well conserved between the two species (92 and 79% identity for MASP-1 and MASP-2, respectively). A more likely hypothesis lies in the nature of the techniques used, namely the Ca2+ dependence of the susceptibility to trypsin digestion, on the one hand (22), and the Ca2+ dependence of the interaction with L-ficolin/P35 or MBL, on the other hand (this study).
From a biological point of view, a major finding is that MASP-1, MASP-2, and Map19 interact with both L-ficolin/P35 and MBL with high affinity at physiological Ca2+ concentrations and that L-ficolin/P35 and MBL compete with each other for binding to these proteins. In this respect it should be noted that the estimated concentration of L-ficolin/P35 in serum, although somewhat controversial (3.7–13.7 µg/ml; 9–30 nM) (8, 37, 38) is much higher than the average value for MBL (1 µg/ml; 2 nM) (39). Thus, it is likely that MBL and L-ficolin/P35 each form stable complexes with the MASPs in vivo, a hypothesis consistent with the observation that only part of MASP-1 and MASP-2 is associated with MBL in serum (40, 41). This is probably also true for MAp19, which is assumed to be present in serum in excess over MASP-2 (13). It has been shown recently that a third serine protease, MASP-3, associates with MBL and L-ficolin/P35 in serum (12, 28). This protease is generated through alternative splicing of the MASP1/3 gene and comprises the N-terminal A chain of MASP-1 connected to a different serine protease domain. The recombinant protein was shown to have interaction properties with MBL and L-ficolin/P35 identical with those of MASP-1, as expected from the presence of the same N-terminal CUB1-EGF-CUB2 interaction region in the two molecules (S. Cseh, N. M. Thielens, and G. J. Arlaud, unpublished observations). Whereas the roles of MASP-1, MASP-3, and MAp19 are not elucidated, it is clearly established that MASP-2 is the protease that triggers activation of the lectin pathway of complement through self-activation and subsequent cleavage of C4 and C2 (15, 17). Given the relative concentrations of MBL and L-ficolin/P35 in serum, our data relative to their affinity for MASP-2 provide support for the physiological relevance of L-ficolin/P35-MASP-2 complexes in initiation of the lectin pathway of complement. It has been shown recently that a second human serum ficolin, H-ficolin (or Hakata Ag), also associates with the MASPs and is able to trigger the lectin pathway of complement (28). Although the kinetic constants for its interaction with the MASPs remain to be determined, it may be expected from its plasma concentration (7–23 µg/ml) (3) that H-ficolin also forms stable complexes with these proteases. L- and H-ficolins have been suggested to possess differential specificities for GlcNAc, whereas MBL preferentially binds to mannose and GlcNAc residues (1). Thus, the coexistence of MBL, L-ficolin, and H-ficolin in serum is expected to enlarge the spectrum of pathogenic micro-organisms that can be recognized and eliminated through the lectin pathway of complement activation. These considerations emphasize the role of this pathway in the innate immune defense.
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2 Current address: Department of Physiology, University of Tennessee Health Sciences Center Memphis, 894 Union Avenue, Memphis, TN 38163.
3 Current address: Institute of Glycotechnology and Department of Applied Biochemistry, Tokai University, 1117-Kitakaneme, Hiratsuka, Kanagarua 259-1292, Japan.
4 Address correspondence and reprint requests to Dr. Nicole Thielens, Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. E-mail address: nicole.thielens{at}ibs.fr
5 Abbreviations used in this paper: GlcNAc, N-acetylglucosamine, the nomenclature of protein modules is that defined by Bork and Bairoch (6 ); CUB module, module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1; EGF, epidermal growth factor; MBL, mannan-binding lectin; MAp19, 19-kDa MBL-associated protein; MASP, MBL-associated serine protease.
Received for publication July 12, 2002. Accepted for publication September 18, 2002.
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fragments of human complement proteases C1r and C1s. J. Biol. Chem. 265:14469.This article has been cited by other articles:
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, N-linked oligosaccharides; 
, specific EQSL
sequence at the C-terminal end of MAp19.
