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Interaction Properties of Human Mannan-Binding Lectin (MBL)-Associated Serine Proteases-1 and -2, MBL-Associated Protein 19, and MBL^{1 ,2}

Nicole M. Thielens^3,*, Sándor Cseh^*, Steffen Thiel, Thomas Vorup-Jensen, Véronique Rossi^*, Jens C. Jensenius and Gérard J. Arlaud^*

^* 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

	Abstract

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The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8–3.2 S) in the presence of Ca²⁺. Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K_D values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca²⁺-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca²⁺-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

	Introduction

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Studies performed over the past decade have led to the discovery of a novel complement activation pathway named the mannan-binding lectin (MBL)⁴ or lectin pathway. This pathway may play an important role in innate immunity, especially against encapsulated microorganisms that have the ability to evade activation of the classical and alternative pathways of complement (2, 3, 4). The recognition component of this pathway is MBL, a member of the collectin family (5) that binds through C-type lectin domains to neutral carbohydrates on the surface of various pathogenic microorganisms (6). Two modular MBL-associated serine proteases, MASP-1 (2) and MASP-2 (3), were initially found to be associated to MBL. A third protein component MBL-associated protein 19 (MAp19), generated by alternative splicing of the MASP-2 gene (7) and, more recently, a further protease MASP-3 (8) were also shown to be associated with MBL.

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).

View larger version (22K): [in this window] [in a new window]   FIGURE 1. Modular structures of the various recombinant proteins and fragments used in this study. The nomenclature and symbols used for protein modules are those defined by Bork and Bairoch (1 ). Ser Pr, Serine protease domain. The arrow indicates the Arg-Ile bond cleaved upon activation of MASP-1. The only disulfide bridge shown is that connecting the activation peptide to the serine protease domain. , N-linked oligosaccharides. , Specific EQSL sequence at the C-terminal end of MAp19.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

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). Vent_R 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 Vent_R 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 Vent_R polymerase and MASP-1b/pDR₂EF1 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 R_t = R₀ exp (-k_off (t - t₀)) was used for the dissociation phase, where R_t 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 k_off was calculated from the mean of the values obtained from a series of injections. To analyze the association phase, the equation R_t = R_eq (1 - exp (-k_s(t - t₀))) was used, where R_eq is the amount of bound ligand (in RU) at equilibrium, t₀ is the starting time of injection, and k_s = k_on x C + k_off, where C is the concentration of the injected analyte. The association rate constant k_on was determined from the slope of a plot of k_s 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 k_on and k_off 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 (K_D) were calculated from the ratio of the dissociation and association rate constants (k_off/k_on).

	Results

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Production and chemical characterization of recombinant MASP-1, MAp19, and the CUB-EGF segments of MASP-1 and MASP-2

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.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. SDS-PAGE analysis of recombinant MASP-1. Lane 1, MASP-1 (reduced) revealed by Western blot analysis using an Ab recognizing the N-terminal end of the protein; lane 2, MASP-1 (reduced) revealed using an Ab recognizing the serine protease domain of the protein. Molecular masses of standard proteins (expressed in kDa) are shown on the left side of the blot.

View larger version (125K): [in this window] [in a new window]   FIGURE 3. SDS-PAGE analysis of the recombinant fragments used in this study. Lanes 1 and 4, MASP-1 CUB-EGF fragment; lanes 2 and 5, MASP-2 CUB-EGF fragment; lanes 3 and 6, MAp19; lanes 1–3 contain unreduced samples and lanes 4–6 contain reduced samples. Molecular masses of unreduced and reduced standard proteins (expressed in kDa) are shown on the left and right sides of the gel, respectively.

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 S_20,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.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Sedimentation velocity analysis of MAp19 and the CUB-EGF segment of MASP-1. Analysis was performed as described in Materials and Methods using the time derivative method of Stafford (26 ). The solid curve is the least square fit of the data corresponding to 10 absorbance scans. A, MAp19 in the presence of 1 mM CaCl2; B, MAp19 in the presence of 2 mM EDTA; C, MASP-1 CUB-EGF segment in the presence of 1 mM CaCl2; D, MASP-1 CUB-EGF segment in the presence of 2 mM EDTA.

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.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. Analysis by surface plasmon resonance spectroscopy of the interaction of MASP-1 or its CUB-EGF fragment with immobilized MAp19. MAp19 was immobilized on the sensor chip as described in Materials and Methods. Fifty microliters of either MASP-1 (100 nM) or its CUB-EGF fragment (144 nM) were injected in the running buffer containing 1 mM CaCl2 at a flow rate of 10 µl/min. The specific signal shown was obtained by subtracting the background signal as described in Materials and Methods.

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).

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Analysis by surface plasmon resonance spectroscopy of the interaction of MASP-1 with immobilized MBL. MBL was immobilized on the sensor chip as described in Materials and Methods. Fifty microliters of each analyte were injected in the running buffer containing either 1 mM CaCl2 or 1 mM EDTA, at a flow rate of 10 µl/min. A, MASP-1 (100 nM) injected in the presence of Ca2+, followed by the running buffer alone, and then by the running buffer plus EDTA; B, MASP-1 (60 nM) in the presence of Ca2+ (top) and in the presence of EDTA (bottom); C, MASP-1 CUB-EGF fragment (150 nM) in the presence of Ca2+ (top) and in the presence of EDTA (bottom). The specific signal shown in panels B and C was obtained by subtracting the background signal as described in Materials and Methods. No correction was done in panel A.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. Analysis by surface plasmon resonance spectroscopy of the interaction of MASP-2 or MAp19 with immobilized MBL. MBL was immobilized on the sensor chip as described in Materials and Methods. Fifty microliters of each analyte was injected in the running buffer containing either 1 mM CaCl2 or 1 mM EDTA, at a flow rate of 10 µl/min. A, MASP-2 (15.6 nM); B, MASP-2 CUB-EGF fragment (80 nM); C, MAp19 (80 nM). The specific binding signal shown was obtained by subtracting the background signal as described in Materials and Methods.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Kinetics of the binding of the MASP-2 CUB-EGF fragment to immobilized MBL. A, Representative sensorgrams (after background subtraction) illustrating the binding of the MASP-2 CUB-EGF fragment (bottom to top curves: 10, 20, 40, 60, 80, and 100 nM) to immobilized MBL (8700 RU) in the running buffer containing 1 mM CaCl2; B, concentration dependence of ks values determined by analysis of the data from A.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 S_20,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 S_20,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 K_D 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 k_on and k_off 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 K_D 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 K_D 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.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. Summary of the data obtained in this study illustrating the association of MASP-1, MASP-2, and MAp19 into homodimers, and their interaction with MBL. Each protein is proposed to bind Ca2+ through its N-terminal CUB-EGF module pair, inducing formation of a Ca2+-dependent CUB-EGF domain that mediates homodimerization. MASP-1, MAp19, and MASP-2 each individually associate to MBL through their CUB-EGF homodimeric regions. It cannot be excluded that the second CUB module plays an accessory role in the interaction with MBL. C indicates the C-terminal catalytic regions of MASP-1 and MASP-2, comprising complement control protein modules 1 and 2 and the serine protease domain. LECT, C-type lectin modules.

	Acknowledgments

 
We thank C. Ebel for her help and advice with analytical ultracentrifugation, M. Jaquinod for performing mass spectrometry analyses, and J.-P. Andrieu and J. Gagnon for performing N-terminal sequence analyses.

	Footnotes

 
¹ This work was supported in part by the Commissariat à l’Energie Atomique, the Center National de la Recherche Scientifique, and the Danish Medical Research Council. S.C. is the recipient of a FEBS long-term fellowship.

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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