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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), Grenoble, France; and
Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark
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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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MASP-1 and MASP-2 exhibit a modular structure homologous to that of C1r
and C1s, the proteases of the C1 complex of complement
(9), with an N-terminal module originally found in
complement subcomponents C1r/C1s, Uegf, and bone morphogenetic
protein-1 (CUB module) (10) followed by an epidermal
growth factor (EGF)-like module, a second CUB module, two contiguous
complement control protein modules (11), and a C-terminal
chymotrypsin-like serine protease domain (see Fig. 1
). MAp19 is a
nonenzymic protein comprising the N-terminal CUB-EGF segment of MASP-2
followed by four unique residues (7, 12). As yet, very
little is known about the stoichiometry, the assembly, and the
activation mechanisms of the MBL-MASP complex(es). An analysis of the
interaction between recombinant fragments of MASP-1 and MASP-2, and MBL
from the rat was published very recently, providing evidence that both
MASP-1 and MASP-2 associate with MBL in a
Ca2+-dependent manner through interactions
involving the N-terminal CUB-EGF-CUB moiety of the proteases
(13).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Diisopropyl phosphorofluoridate was obtained from Acros Organics (Noisy-le-Grand, France). The plasmids containing the full-length MASP-2 and MASP-1 cDNAs were obtained as described previously (3, 14). Oligonucleotides were obtained from Oligoexpress (Paris, France). VentR polymerase was purchased from New England Biolabs (Beverly, MA). The Ultralink-MBL column was prepared by coupling 0.5 mg of MBL purified from human plasma to 2 ml of Ultralink Biosupport medium according to the manufacturer’s instructions (Interchim, Montluçon, France). Peptide:N-glycosidase F was purified from cultures of Flavobacterium meningosepticum according to the method of Tarentino et al. (15), modified as described by Aude et al. (16).
Proteins
MBL was isolated from human plasma according to the procedure described by Tan et al. (17), with a further purification step using ion-exchange chromatography on a 1-ml Resource S column (Amersham Pharmacia Biotech, Piscataway, NJ). The MBL sample was loaded onto the column in 50 mM sodium acetate, 1 mM EDTA, 0.1% (w/v) Tween 20, pH 4.5, and eluted with a gradient to 1.0 M NaCl over 45 min at a flow rate of 0.5 ml/min. Proenzyme recombinant MASP-2 was expressed in human embryonic kidney 293 cells and purified to homogeneity as described recently (18). The concentrations of purified recombinant proteins were determined using absorption coefficients (A1%, 1 cm at 280 nm) calculated by the method of Edelhoch (19), and m.w. was calculated from the amino acid sequence or determined by mass spectrometry, as follows: MASP-1 CUB-EGF fragment, 10.0 and 21,000 (19 ; this study); MASP-2 CUB-EGF fragment, 11.7 and 18,861 (3 ; this study); MAp19, 11.6 and 19,086 (3 ; this study). Due to the low amounts of material recovered, estimation of the concentration of full-length MASP-1 and MASP-2 was based on Coomassie blue staining after SDS-PAGE analysis using appropriate internal standards and respective m.w. of 82,000 and 74,200 (3, 20).
Construction of expression plasmids containing MASP-2-derived fragments
The cDNA coding for MASP-2 was excised from the pBS-MASP-2 vector by digestion with XhoI and EcoRI and cloned into the corresponding restriction sites of the pFastBac1 baculovirus transfer vector (Life Technologies, Grand Island, NY). DNA fragments encoding the MASP-2 signal peptide followed either by aa 1–168 of the mature MASP-2 (corresponding to the N-terminal CUB-EGF module pair) or by aa 1–166 of the mature protein plus the EQSL residues corresponding to the MAp19 sequence (7) were amplified by PCR using VentR polymerase and pBS-MASP-2 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 MAp19 (5'-GGGGTACCCTAGAGGCTCTGCTCTGAGCAGGTGCGCTT-3') introduced the EQSL amino acid sequence (boldface and underlined) followed by a stop codon (boldface) and a KpnI site (underlined) at the 3' end of the PCR product. The antisense primer for the CUB-EGF module pair (5'-GGAATTCCTACAGGCGTGA-3') introduced a stop codon (boldface) followed by an EcoRI site (underlined) at the end of the PCR product. The amplified DNA fragments were digested with BamHI and KpnI, and BamHI and EcoRI for MAp19 and the CUB-EGF fragment, respectively, and cloned into the corresponding sites of the pFastBac1 vector. The resulting constructs were characterized by restriction mapping and checked by dsDNA sequencing (Genome Express, Grenoble, France).
Construction of expression plasmids containing MASP-1 and its CUB-EGF segment
DNA fragments encoding the MASP-1 signal peptide plus the mature
protein (aa 1–680) or the N-terminal CUB-EGF segment of MASP-1 (aa
1–165 of the mature protein) were amplified by PCR using
VentR polymerase and
MASP-1b/pDR2
EF1
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 both
PCR products. The antisense primers
(5'-GGAATTCTCAGTTCCTCACTCC-3' for MASP-1 and
5'-GGAATTCTCACTCCACTCGGCAGGT-3' for the
CUB-EGF fragment) introduced a stop codon (boldface) followed by an
EcoRI site (underlined) at the 3' end of both fragments. The
PCR products were digested with BglII and EcoRI,
purified, and cloned into the BamHI/EcoRI sites
of the pFastBac1 vector. The final constructs were analyzed by dsDNA
sequencing.
Cells and viruses
The Spodoptera frugiperda insect cells (Ready-Plaque Sf9 cells obtained from Novagen, Madison, WI) were routinely grown and maintained in Sf900II serum-free medium (Life Technologies) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin (Life Technologies). The Trichoplusia ni (High Five) insect cells (provided by Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France) were maintained in TC100 medium (Life Technologies) containing 10% FCS (Dominique Dutscher, Brumath, France) supplemented with 50 IU/ml penicillin and 50 mg/ml streptomycin. Recombinant baculoviruses were generated using the Bac-to-Bac system (Life Technologies). The bacmid DNA was purified using the Qiagen midiprep purification system (Qiagen, Courtaboeuf, France) and used to transfect Sf9 insect cells using cellfectin in Sf900 II SFM medium (Life Technologies) 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 (21).
Protein production and purification
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 for intact MASP-1, and 96 h for the fragments. The supernatants were collected by centrifugation and diisopropyl phosphorofluoridate was added to a final concentration of 1 mM in the case of MAp19 and both CUB-EGF fragments.
The culture supernatants containing MAp19 or the MASP-2 CUB-EGF fragment were dialyzed against 50 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 8.1, and loaded at 1.5 ml/min onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 12 cm) equilibrated in the same buffer. Elution was conducted by applying a 1.2-liter linear gradient to 350 mM NaCl in the same buffer. Fractions containing the recombinant fragments were identified by Western blot analysis, precipitated by addition of (NH4)2SO4 to 60% (w/v), and left overnight at 4°C. The pellets were resuspended in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4, and applied onto a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas, Montgomeryville, PA) equilibrated in the same buffer. The purified fragments were concentrated to 0.3 mg/ml by ultrafiltration on Microsep microconcentrators (m.w. cut-off = 10,000) (Filtron, Karlstein, Germany).
The supernatant containing the CUB-EGF fragment of MASP-1 was dialyzed against 75 mM NaCl, 10 mM imidazole, pH 6.1, and loaded at 1.5 ml/min onto a Q-Sepharose Fast Flow column (Amersham Pharmacia Biotech) (2.8 x 10 cm) equilibrated in the same buffer. Elution was conducted by applying a 1-liter linear gradient to 500 mM NaCl in the same buffer. Fractions containing the recombinant fragment were identified by Western blot analysis, and the protein was precipitated by the addition of (NH4)2SO4 to 60% (w/v). Final purification was achieved by high-pressure gel permeation on a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas) equilibrated in 145 mM NaCl, 1 mM EDTA, 50 mM triethanolamine hydrochloride, pH 7.4, and the purified fragment was concentrated to 0.5 mg/ml by ultrafiltration.
The MASP-1 containing supernatant was dialyzed against 50 mM NaCl, 50 mM triethanolamine hydrochloride, pH 8.1, and loaded at 1 ml/min onto a DE 52 column (Whatman, Tewksbury, MA) (2.8 x 10 cm) equilibrated in the same buffer. Elution was conducted by applying a 1-liter linear gradient to 500 mM NaCl in the same buffer. Fractions containing the recombinant fragment were identified by Western blot analysis and dialyzed against 145 mM NaCl, 2 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4. Further purification was achieved by affinity chromatography on an UltraLink-MBL column equilibrated in the same buffer. Elution was conducted by applying the same buffer containing 5 mM EDTA instead of calcium.
Chemical characterization of the recombinant proteins
N-terminal sequence analyses were performed after SDS-PAGE and electrotransfer, using an Applied Biosystems model 477 A protein sequencer as described previously (22). 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 (23).
Enzymic deglycosylation of the MASP-1 CUB-EGF fragment
The CUB-EGF fragment of MASP-1 (0.65 mg/ml) was incubated in the presence of 10% (w/w) peptide: N-glycosidase F for 3 h at 30°C, in 50 mM triethanolamine hydrochloride, 145 mM NaCl (pH 7.4) containing either 1 mM CaCl2 or 5 mM EDTA. Deglycosylation was monitored by SDS-PAGE analysis.
PAGE and immunoblotting
SDS-PAGE analysis was performed as described previously (24). Western blot analysis and immunodetection of the recombinant proteins were conducted as described by Rossi et al. (25), using the mouse monoclonal anti-MASP-2 Ab 1.3B7 (26) or rabbit anti-peptide Abs directed against either the N-terminal end or the serine protease domain of MASP-1 (3).
Gel permeation chromatography
The recombinant fragments used in this study were analyzed by high pressure gel permeation chromatography on a TSK G3000 SWG column (7.5 x 600 mm) (Tosohaas) equilibrated in 145 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, containing either EDTA (2 mM) or CaCl2 (1 mM). Elution was performed at 1 ml/min, and proteins were detected from their absorbance at 280 nm.
Analytical ultracentrifugation
Sedimentation velocity analysis was performed at 20°C using a Beckman XL-I analytical ultracentrifuge. Experiments were conducted at 60,000 rpm using 12-mm path-length double-sector cells containing 400 µl of samples in 145 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, containing either 1 mM CaCl2 or 2 mM EDTA. Sedimentation coefficients (S) were calculated by using the time derivative method described by Stafford (27), or the SVEDBERG program as described by Philo (28). Translational diffusion constants (D) were obtained using the same program and the molecular masses were determined from S and D. The solvent density was taken at 1.006 g/ml at 20°C, and the partial specific volume of the proteins was estimated from the amino acid composition at 0.71 ml/g (MASP-1 CUB-EGF fragment) and 0.72 ml/g (MASP-2 CUB-EGF fragment and MAp19) using the SEDNTERP program. The same program was used to evaluate the maximal axial ratio of the proteins, assuming a prolate ellipsoid shape and a hydration coefficient of 0.4 g H2O/g protein (29).
Real-time surface plasmon resonance spectroscopy and data evaluation
Surface plasmon resonance measurements were performed using an upgraded BIAcore instrument (BIAcore AB, Uppsala, Sweden). The running buffer for protein immobilization was 145 mM NaCl, 5 mM EDTA, 10 mM HEPES, pH 7.4. Protein ligands were diluted to 10–20 µg/ml in 10 mM formate, pH 3.0 (MASP-1 CUB-EGF fragment) or in 10 mM acetate, pH 4.0 (MAp19 and MBL) and immobilized onto the carboxymethylated dextran surface of a CM5 sensor chip (BIAcore AB) using the amine-coupling chemistry (BIAcore AB amine coupling kit) according to the manufacturer’s instructions. Binding of the intact proteins (MASP-1, MASP-2) and of the fragments (MAp19 or the CUB-EGF fragments of MASP-1 and MASP-2) was measured over 2500 and 8700 resonance units (RU) of immobilized MBL, respectively, at a flow rate of 10 µl/min in 145 mM NaCl, 1 mM CaCl2, 50 mM triethanolamine hydrochloride, pH 7.4. Cross-interaction between MASP-1 and MASP-2 was analyzed under the same conditions as above, over immobilized MAp19 (1500 RU) or MASP-1 CUB-EGF (1350 RU). Equivalent volumes of each protein sample were injected over a surface with immobilized BSA to serve as blank sensorgrams for subtraction of the bulk refractive index background. Regeneration of the surfaces was achieved by injection of 10 µl of 5 mM EDTA.
Sensorgrams were analyzed with BIAevaluation 2.1 software (BIAcore AB) using a single-site binding model as previously described (30). Briefly, the equation Rt = R0 exp (-koff (t - t0)) was used for the dissociation phase, where Rt is the amount of ligand (in RU) remaining bound at time t and t0 is the beginning of the dissociation phase. The final dissociation rate constant koff was calculated from the mean of the values obtained from a series of injections. To analyze the association phase, the equation Rt = Req (1 - exp (-ks(t - t0))) was used, where Req is the amount of bound ligand (in RU) at equilibrium, t0 is the starting time of injection, and ks = kon x C + koff, where C is the concentration of the injected analyte. The association rate constant kon was determined from the slope of a plot of ks vs C, from a series of at least five analyte concentrations. The data were also analyzed by global fitting to a 1:1 Langmuir binding model of both the association and dissociation phases for several concentrations simultaneously, using the BIAevaluation 3.0 software (BIAcore AB). This analysis yielded kon and koff values similar to those calculated as described above, except in the case of the CUB-EGF fragment of MASP-1 for which global fitting was less satisfactory. The apparent equilibrium dissociation constants (KD) were calculated from the ratio of the dissociation and association rate constants (koff/kon).
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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 of the various
recombinant fragments used in this study are depicted in Fig. 1
. The recombinant baculoviruses for
expression of each protein and fragment were generated as described in
Materials and Methods and used to infect High Five insect
cells for various periods at 28°C. The amount of recombinant protein
secreted into the culture supernatants was estimated by SDS-PAGE and
Western blot analysis and found to be 
1, 5, 8, and 4 µg/ml for
MASP-1, MAp19, and the CUB-EGF segments of MASP-1 and MASP-2,
respectively. In each case, a significant portion of the recombinant
material ranging from 30 (MAp19) to 90% (MASP-1) was found in the
insoluble cell pellet fraction. Protein purification was performed as
described in Materials and Methods, using an initial
ion-exchange fractionation step in all cases. MASP-1, MAp19, and the
CUB-EGF segments of MASP-1 and MASP-2 were each purified to
homogeneity. It should be mentioned that the purification yield of
MASP-1 was very low, as only 20 µg of purified protein was
obtained from 500 ml of culture supernatant. Therefore, this protein
was detected routinely using Western blot analysis rather than
Coomassie blue staining.
Analysis of the recombinant MASP-1 by SDS-PAGE under reducing
conditions (Fig. 2) indicated that
40–50% of the protease migrated as a 90-kDa single-chain proenzyme
species reactive with both Abs directed to the N- and C-terminal ends
of the molecule and yielding a single N-terminal sequence
His-Thr-Val-Glu-Leu-Asn-Asn-Met-Phe-Gly... identical with that of
the mature protein (Fig. 2). The recombinant protease was found to be
partially activated, as shown by the presence of a 58-kDa band
corresponding to the N-terminal A chain (Fig. 2, lane 1),
and of a doublet at 30 and 32 kDa (Fig. 2, lane 2)
corresponding to the serine protease domain, with the expected
N-terminal sequence Ile-Phe-Asn-Gly-Arg-Pro-Ala-Gln-Lys-Gly... ,
indicating that cleavage had occurred at the susceptible site
Arg448-Ile449
(20). A further species of 28 kDa, exhibiting an
N-terminal sequence identical with that of the A chain, and
corresponding to a fragment derived from the N-terminal end of MASP-1,
was also detected (Fig. 2, lane 1). Using protein staining
with Coomassie blue (data not shown), it was checked that no other
major fragment or contaminant was present.
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The MASP-1 CUB-EGF fragment migrated as a diffuse band containing at
least three distinct populations: a major species of apparent molecular
mass 23–25 kDa (77%), surrounded by two minor species of 26–28 kDa
(22%) and 21–23 kDa (<2%) (Fig. 3, lanes 1 and
4). Treatment of the fragment with
peptide:N-glycosidase F in the presence of EDTA led to the
disappearance of the upper 26- to 28-kDa band, with a concomitant
decrease of the major 23- to 25-kDa band (51%), and a dramatic
increase of the lower 21- to 23-kDa species (49%). Given the
presence of two N-linked carbohydrates in the CUB-EGF region
of MASP-1 (20 ; see Fig. 1), it became clear that the three
populations described above corresponded to species with either two
carbohydrates, a single carbohydrate, or no carbohydrate.
Interestingly, performing the treatment with
peptide:N-glycosidase F in the presence of
Ca2+ ions almost totally prevented
deglycosylation, with only a slight increase in the 21- to 23-kDa
aglycosylated species, providing indirect evidence that
Ca2+ bound to the CUB-EGF fragment induced
protection against enzymic deglycosylation.
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SDS-PAGE analysis of the MASP-2 CUB-EGF fragment and of MAp19 indicated
that both migrated as a homogeneous band of apparent molecular mass
19–21 kDa (Fig. 3, lanes 2, 3, 5, and 6). Both
proteins showed a single N-terminal sequence
Thr-Pro-Leu-Gly-Pro-Lys-Trp-Pro-Glu-Pro... identical with that of
the mature MASP-2, and mass spectrometry analysis yielded mass values
of 18,861 ± 20 (MASP-2 CUB-EGF fragment) and 19,086 ± 20
(MAp19) consistent with the calculated values (18,807 and 19,080,
respectively), indicating that both recombinant proteins had the
expected sequences.
MAp19 and the CUB-EGF fragments of MASP-1 and MASP-2 form homodimers
The three recombinant segments produced in this study were
submitted to sedimentation velocity analysis, and the data were
analyzed as described in Materials and Methods. Analysis of
MAp19 in the presence of 1 mM Ca2+ (Fig. 4A) indicated that the protein
population was homogeneous, with S20,w = 3.0, and
a molecular mass calculated from S and D of 30 ± 3 kDa,
consistent with the occurrence of a dimer, although slightly lower than
the expected value (38.16 kDa). Performing the analysis in the presence
of 2 mM EDTA resulted both in a broadening of the sedimentation peak
and an overall decrease of the signal (Fig. 4B), although
the mean S value was comparable to that determined in the presence of
Ca2+ (Table I).
Experiments using size-exclusion chromatography indicated that EDTA
significantly modified the elution position of MAp19 and also induced
formation of a significant amount of high-m.w. aggregates. Similar
results were obtained in the case of the MASP-2 CUB-EGF fragment, which
in the presence of Ca2+ also exhibited an S value
and a calculated mass consistent with a homodimer (Table I). EDTA had
the same effect on the sedimentation profile of this fragment as
observed in the case of MAp19.
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C) or EDTA (Fig. 4D), with S values and calculated molecular mass values
consistent with the occurrence of stable dimers in both cases (Table I). In contrast to MAp19 and the CUB-EGF fragment of MASP-2, EDTA had
no significant effect on the sedimentation profile of the MASP-1
CUB-EGF fragment. Analysis by size-exclusion chromatography indicated
that EDTA induced no detectable aggregation of the protein, but
significantly increased its elution position from 17.3 min in the
presence of Ca2+ to 18.7 min. There is no interaction between MASP-1 and MASP-2
The ability of MASP-1 and MASP-2 to interact with each other was
investigated by surface plasmon resonance spectroscopy using the
recombinant full-length proteins and their N-terminal fragments
expressed in this study. As illustrated in Fig. 5, both recombinant MASP-1 and its
CUB-EGF fragment showed no ability to bind to MAp19 covalently
immobilized onto the surface of a sensor chip, whether in the presence
or absence of Ca2+ ions. In another series of
experiments (data not shown), the CUB-EGF fragment of MASP-1 was
immobilized, and both MAp19 and MASP-2 were used as soluble analytes.
Again, no significant interaction could be detected in the presence or
absence of Ca2+ ions.
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MASP-1, MASP-2, and MAp19 each interact individually with MBL in the presence of Ca2+
The ability of MASP-1, MASP-2, and MAp19 to bind to MBL was
studied using surface plasmon resonance spectroscopy. As shown in Fig. 6A, recombinant MASP-1 readily
bound to immobilized MBL in the presence of 1 mM
CaCl2, as shown by the increase in the RU during
the association phase of the sensorgram, and bound MASP-1 could be
quantitatively eluted at the end of the dissociation phase by a pulse
injection of EDTA. No interaction occurred when EDTA was substituted
for Ca2+ in the running buffer (Fig. 6B). The CUB-EGF fragment of MASP-1 also bound to MBL in a
Ca2+-dependent manner (Fig. 6C), but
the dissociation in this case was much faster than that observed for
full-length MASP-1 (Fig. 6B).
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A). The CUB-EGF fragment of
MASP-2 (Fig. 7B) and the parent protein MAp19 (Fig. 7C) also bound MBL in the presence of
Ca2+, yielding virtually identical curves. Again,
as observed above in the case of MASP-1, both the MASP-2 CUB-EGF
fragment and MAp19 dissociated faster from MBL than full-length MASP-2.
In all cases, complete elution of the bound material could be achieved
by a pulse injection of EDTA (data not shown).
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A shows the association and
dissociation curves from a representative series of experiments
performed with six different concentrations of the MASP-2 CUB-EGF
fragment (10–100 nM). The association phase was analyzed as described
in Materials and Methods, to yield
ks values at each concentration, and a
plot of ks vs the analyte
concentrations gave a straight line (Fig. 8B) with a slope
corresponding to the association rate constant
kon. The dissociation phase was also
analyzed as described Materials and Methods to yield the
dissociation rate constant koff. The
values of kon,
koff, and of the resulting apparent
equilibrium dissociation constant KD
determined for the various proteins and fragments are shown in Table II. Full-length MASP-1 and its N-terminal
CUB-EGF fragment showed very similar
kon values (2.1–2.8 x
105 M-1
s-1). In contrast, in agreement with previous
observations (see Fig. 6, B and C), the MASP-1
CUB-EGF fragment showed a large increase in the
koff value, resulting in a 15-fold
increase in the apparent KD.
Similarly, full-length MASP-2, its CUB-EGF fragment, and MAp19 all
exhibited comparable kon values
ranging from 3.5 to 3.7 x 105
M-1 s-1, whereas the
latter two fragments showed significantly increased
koff values, resulting in a 16-fold
increase in the apparent KD. It was
concluded that both MASP-1 and MASP-2 specifically bound to MBL in the
presence of Ca2+, with
KD values in the nanomolar range. This
property is shared by, and therefore clearly arises from, their
N-terminal CUB-EGF segments, which nevertheless exhibit higher
dissociation rates than the full-length proteins.
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Discussion |
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Both the recombinant MASP-2 CUB-EGF fragment and MAp19 had the expected
amino acid sequence as judged from N-terminal sequence and mass
spectrometry analyses, whereas, as expected from the occurrence of two
N-glycosylation sites at Asn30 and
Asn159 (20), the MASP-1 CUB-EGF
fragment was produced in a glycosylated form. As previously observed
for the homologous CUB-EGF fragment of C1r produced in a baculovirus
system (30), the latter fragment underwent heterogeneous
glycosylation, with the occurrence of a major species (75%)
containing a single carbohydrate, and a minor species (25%) bearing
two oligosaccharides. In keeping with previous data (25, 30), both carbohydrate chains were short high-mannose
oligosaccharides. It is not known whether such heterogeneity also
occurs in the case of the intact recombinant MASP-1, which exhibited an
apparent molecular mass of 90 kDa, a value that appears consistent with
the occurrence of four oligosaccharides at the predicted four
N-glycosylation sites of the protein (Ref. 20 ;
see Fig. 1).
Our data obtained by sedimentation velocity analysis and surface plasmon resonance spectroscopy provide strong evidence that the CUB-EGF fragments of human MASP-1 and MASP-2, as well as MAp19, each form homodimers in solution, and that there is no cross-interaction between MASP-1 and MASP-2, or between MASP-1 and MAp19. Thus, sedimentation velocity analyses show unambiguously that the above three fragments have S20,w and molecular mass values derived from S and D consistent with the occurrence of homodimers. For comparison, the recombinant CUB-EGF fragment of C1s associates as a Ca2+-dependent dimer and has an S value of 3.0–3.1 S (35), a value that is close to those determined here for the three fragments, which range from 2.8 to 3.3 S. The axial ratios long semi-axis/short semi-axis derived from the S values assuming a hydration value of 0.4 g H2O/g protein were similar for all fragments, with values of 4.4–4.8 for MAp19 and the MASP-2 CUB-EGF fragment and 3.8 for the MASP-1 CUB-EGF fragment, indicating that these have comparable, rather elongated overall shapes. Further evidence for the absence of cross-interaction between MASP-1, MASP-2, and MAp19 was obtained by surface plasmon resonance spectroscopy and size-exclusion studies, which showed no interaction between the N-terminal regions of MASP-1 and MASP-2. Our finding that recombinant MASP-1 and MASP-2 do not associate to each other is consistent with recent studies performed on human serum (36, 37) and on recombinant fragments of rat MASP-1 and MASP-2 (13). Thus, unlike C1r and C1s (which naturally associate to each other in the presence of Ca2+ ions), MASP-1 and MASP-2 appear to be designed to form homodimers only. This property is not unexpected, because C1s also forms homodimers in the absence of C1r (38).
Our observation that there is no interaction between recombinant MASP-1
and MAp19 appears consistent, considering that MAp19 contains the
CUB-EGF pair of MASP-2 and is therefore expected to share its binding
properties, including the inability to bind to MASP-1. However, this
finding is intriguing because recent studies (36, 37)
indicate that MASP-1 and MAp19 are associated in serum. A possible
explanation for this apparent discrepancy is that the recombinant
proteins used in our studies lack a particular posttranslational
modification essential for this particular function. In this respect,
it is noteworthy that, unlike human C1s, recombinant C1s expressed in a
baculovirus/insect cells system was found to lack 
-hydroxylation of
Asn134 but, nevertheless, retained its binding
and catalytic activities (32). Further, the data obtained
by Thiel et al. (36) and Matsushita et al.
(37) are based on the use of serum or serum-derived
fractions; therefore, the involvement of a yet unidentified serum
factor, which would act as a linker between MASP-1 and MAp19, cannot be
excluded. A third possibility is that a MASP-1/MAp19 complex can be
formed only between newly synthesized monomers in the hepatocyte, and
not when dimers of the two proteins are mixed. Obviously, additional
experiments are required to elucidate this point.
Another important question deals with the involvement of
Ca2+ ions in the observed homodimerization
processes. In the case of the MASP-2 CUB-EGF fragment and MAp19, the
interaction is obviously sensitive to EDTA, as shown by the observation
that the chelating agent induces significant peak broadening and loss
of material through aggregation (see Fig. 4), providing indirect
evidence that Ca2+ is involved, at least to some
extent, in the dimerization process. The observation that the elution
position of MAp19 on size-exclusion chromatography is different when
the analysis is performed in Ca2+ or EDTA also
supports this hypothesis. Although EDTA has no significant effect on
the sedimentation behavior of the MASP-1 CUB-EGF fragment, it
nevertheless modifies its elution position on size-exclusion
chromatography. In addition, whereas efficient deglycosylation of the
MASP-1 CUB-EGF fragment by peptide:N-glycosidase F occurs in
the presence of EDTA, Ca2+ almost totally
prevents enzymic deglycosylation, again providing indirect evidence
that Ca2+ ions bind to the CUB-EGF pair and
thereby contribute to maintain a compact structure of the assembly.
It should also be emphasized that Ca2+-dependent
association of CUB-EGF pairs was demonstrated in the case of both C1r
and C1s (30, 35), and that evidence was provided that
Ca2+ binds poorly to the C1r EGF module, and more
strongly to the corresponding CUB-EGF pair (30, 39, 40).
In this respect, it should be pointed out that the N-terminal CUB
modules of C1r, C1s, MASP-1, MASP-2, and MAp19 all belong to the same
subfamily of CUB modules (10). In the same way, the EGF
modules of these proteins all exhibit the particular consensus motif
(... Asp/Asn-Asp/Asn-Asp*/Asn*-Tyr/Phe... , in which * indicates
a -hydroxylated residue) that characterizes the subset of EGF
modules known to bind Ca2+ (41).
Finally, MASP-1, MASP-2, and MAp19 all contain the five residues
predicted from the three-dimensional structure of the C1r EGF module to
act as Ca2+ ligands (40).
Therefore, a likely hypothesis is that the homodimerization process observed in the case of the MASP-1 and MASP-2 CUB-EGF fragments and MAp19 also involves Ca2+-dependent interaction between the CUB and EGF modules in each monomer, as demonstrated in the case of the homologous part of C1r (30). A major difference between these closely related CUB-EGF pairs likely lies in the accessibility of the Ca2+ ion with respect to EDTA. It was shown that Ca2+ is well accessible in the case of C1r (30) and C1s, the CUB-EGF fragment of the latter sedimenting in the presence of EDTA as a monomer with an S20,w value of 1.7 S (N.M.T., unpublished data). In contrast, the Ca2+ ion would be only poorly accessible in the case of MASP-2, and probably not accessible at all in the case of MASP-1. A possible explanation is that, due to particular structural features in this protein, the Ca2+ ion would be sequestered within the CUB-EGF assembly or located in a slightly hydrophobic environment, and would not be accessible to EDTA. In contrast with the interpretation given by Wallis and Dodd (13) who conclude that dimer formation in rat MASP-1 and MASP-2 is not Ca2+-dependent, we suggest that this interaction, at least in humans, is most likely Ca2+-dependent, but poorly sensitive, or not sensitive at all to EDTA.
In agreement with the findings by Wallis and Dodd (13) in
the rat system, our data clearly show that human MASP-1 and MASP-2 each
individually bind to MBL in a Ca2+-dependent
fashion, with KD values of 1.7 and 0.8
nM, respectively, indicating strong affinity in both cases. As shown by
the ability of the corresponding fragments to bind to MBL, this
specific property is mediated, for the most part, by the N-terminal
CUB-EGF moiety of each protein. In this respect, it should be
emphasized that MAp19 has the same behavior as the CUB-EGF fragment of
MASP-2, including strikingly similar
kon and
koff values (see Table II), indicating
that the extra four residues at the C-terminal end of MAp19 have no
influence on its binding ability and specificity.
The fact that the CUB-EGF fragments exhibit higher dissociation rate constants than the corresponding full-length proteins suggests that additional contacts are provided by other regions of the proteins. In this respect, it is noteworthy that the three N-terminal modules CUB-EGF-CUB of rat MASP-1 were found to be required for efficient binding to MBL (13). A further explanation lies in the fact that, in contrast with their CUB-EGF fragments that are dimers, full-length MASP-1 and MASP-2 may self-associate through other regions of the proteins and thereby form complexes providing multivalent interaction with MBL, which may explain the fact that they dissociate more slowly.
The interaction with MBL is clearly Ca2+-dependent in all cases, including MASP-1, and, in contrast with the homodimerization process, appears to be fully EDTA-sensitive. A plausible explanation is that, as a result of the interaction with MBL, the CUB-EGF assembly may undergo a conformational change that makes the Ca2+ ion accessible to the solvent and therefore to chelation by EDTA. However, it cannot be excluded that the interaction between MBL and the MASPs, itself, may involve a Ca2+ bridge that would be fully accessible to EDTA.
The fact that MASP-1, MASP-2, and MAp19 each associate with high
affinity with MBL suggests that each individual protein may form a
complex with MBL, as summarized in Fig. 9. In the case of MASP-1 and MBL, the
KD values for the interaction (1.4 nM)
and the estimated average concentrations of the two proteins in serum
(70 and 2 nM, respectively) (42, 43) appear consistent
with the occurrence of a stable MBL-MASP-1 complex in vivo. Provided
that the MASP-2 and MAp-19 serum concentrations are also in the
nanomolar range, the same reasoning would apply in the case of
MBL-MASP-2 and MBL-MAp19 complexes. In this respect, it should be
emphasized that, as the KD for MAp19
is found to be 9- to 16-fold higher than that for MASP-1 and MASP-2
(see Table II), if MAp19 binds to the same MBL population as MASP-1 or
MASP-2, then the MAp19 concentration required for effective binding
needs to be significantly higher than that of the MASPs. In this
hypothesis, depending on the relative concentrations of MAp19 and
MASP-2 in serum, MAp19 may act as a regulator of activation of the
lectin pathway. However, it cannot be excluded that MAp19, MASP-1, and
MASP-2 each bind to distinct MBL populations. In the same way, it
cannot be excluded that two of the three MBL ligands (e.g., MASP-1 and
MAp19) may bind simultaneously to the same MBL molecule, as this type
of experiment involving simultaneous binding of two analytes to the
same immobilized ligand could not be readily performed using surface
plasmon resonance spectroscopy.
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Acknowledgments |
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Footnotes |
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2 A preliminary report of this study was presented at the XVIIIth International Complement Workshop in Salt Lake City, Utah, July 23–27, 2000.
3 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.
4 Abbreviations used in this paper: MBL, mannan-binding lectin; the nomenclature of protein modules defined by Bork and Bairoch (1 ); CUB module, module originally found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1; EGF, epidermal growth factor; MAp19, 19-kDa MBL-associated protein; MASP, MBL-associated serine protease; RU, resonance unit(s); S, sedimentation coefficient; D, diffusion constant.
Received for publication November 22, 2000. Accepted for publication February 14, 2001.
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fragments of human complement proteases C1r and C1s. J. Biol. Chem. 265:14469.-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex. Biochemistry 31:4254.[Medline]
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,
N-linked oligosaccharides. 
, Specific EQSL sequence
at the C-terminal end of MAp19.
