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Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark;
Department of Microbiology and Immunology, University of Leicester, Leicester, United Kingdom;
Medical Research Council Immunochemistry Unit, University of Oxford, Oxford, United Kingdom; and
§
Molecular Sciences Division, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
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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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MBL is a calcium-dependent lectin that binds carbohydrates with 3- and 4-hydroxo groups oriented in the equatorial plane of the pyranose ring (3). Prominent ligands for MBL are thus D-mannose and N-acetyl-D-glucoseamine, while carbohydrates not fitting this steric requirement, e.g., D-galactose, have undetectable affinity for MBL. This selectivity, along with differences in the spatial organization of the ligands, allows for the specific recognition of carbohydrates on pathogenic microorganisms and viruses and avoids recognition of noninfectious self. The importance of this pathway is underlined by a number of clinical studies linking MBL deficiency with increased susceptibility to a variety of infectious diseases (4).
It has recently been suggested that the minimal ancestral components of the primordial complement system were MBL complexes and C3 (1). Several of the structural and functional properties of MBL and the C1 complexes are certainly consistent with the notion that the classical pathway evolved from the MBL pathway. Electron microscopy indicates that the quaternary structure of MBL is similar to that of C1q, the nonenzymatic part of the C1 complex (5). MBL is oligomeric and composed of up to 18 identical chains, each consisting of a C-terminal lectin domain and a coiled structure, termed the neck region, which is important for trimerization of the adjacent collagen-like region (6). Characterization of the proteases associated with MBL has provided the strongest evidence for an evolutionary relationship between MBL and C1, however. The two serine proteases associated with MBL, MBL-associated serine protease (MASP)-1 and MASP-2, share identical domain organizations with those of C1r and C1s, the enzymatic components of the C1 complex (2, 7, 8, 9). These domains include an N-terminal C1r/C1s/sea urchin Uegf/bone morphogenic protein (CUB) domain, an epidermal growth factor-like domain, a second CUB domain, a tandem of complement control protein domains, and a serine protease domain. A 19-kDa MBL-associated protein of unknown function, MAp19, which consists of the first CUB domain and the epidermal growth factor-like domain of MASP-2 and therefore lacks protease activity, has been described (10, 11). As in the C1 proteases, activation of MASP-1 occurs through cleavage of an Arg-Ile bond adjacent to the serine protease domain, which splits the enzyme into disulfide-linked A and B chains, the latter consisting of the serine protease domain (7). The Arg-Ile site is conserved in MASP-2, and a similar cleavage appears to occur in the course of MASP-2 activation (2). The MASP-1 and MASP-2 genes are located on chromosomes 3 and 1, respectively (12, 13). Genetic analyses suggest that the MASP genes and the C1r and C1s genes arose via a series of duplications from a common ancestor (14). The identification of MASPs in tunicate species and lampreys places MASP evolution on a time scale of 500–600 million years and points to the MBL pathway as an antecedent of the classical complement pathway (14, 15).
These considerations have led to the expectation that C1 activation will prove to be a useful model for complement activation through the MBL pathway. In the C1 complex, autoproteolytic cleavage of the Arg-Ile site of C1r is followed by C1r activation of C1s, which thereby acquires the ability to cleave C4 and C2 (16). The cleavage of C4 into two fragments, designated C4a and C4b, allows the C4b fragments to form covalent bonds with adjacent hydroxyl or amino groups and the subsequent generation of C3 convertase through noncovalent interaction with the C2b fragment of activated C2. By analogy, it would be predicted that MASP-1 activates MASP-2.
As an initial step toward understanding the role of MASP-2 in complement activation, we have generated a recombinant form of MASP-2 for reconstitution studies with MBL. We find, unexpectedly, that whereas C1 complex autoactivation is a two-step mechanism involving both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. Our results suggest that the roles of the serine protease diversified considerably in the course of vertebrate complement evolution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The coding region of human MASP-2 cDNA was amplified by PCR from
the cloned cDNA (2) by a sense primer (synthesized by
Genosys, Cambridge, U.K.) engineering rat CD4 5' leader sequence into
the construct: tccccatccgctcaagcaggccaccATGAGGCTGCTGACCCTCCTG (MASP-2
coding sequence in capitals). The antisense primer was constructed to
encode a C-terminal His6 tag:
ctagtgatggtgatggtgatgAAAATCACTAATTATGTTCTCGATC. PCR was conducted
with an annealing temperature of 55°C under conditions as recommended
by the enzyme supplier (BIOTAQ; Bioline, London, U.K.). The product was
TA cloned into the pCR2 plasmid vector according to the manufacturer’s
protocol (K2000-01; Invitrogen, Leek, The Netherlands). The insert was
excised from the vector by EcoRI restriction digest and
ligated into the SmaI site of the pDR2
EF1
expression
vector (17), according to standard procedures for
blunt-end ligation (18). The construct was selected and
propagated in Escherichia coli TG44.
Expression in HEK 293 cells
Plasmid DNA was recovered from bacterial culture by use of Qiagen’s Mega-prep procedure (12381; Qiagen, Hilden, Germany). Human embryonic kidney (HEK) 293 cells (CRL-1573; American Tissue Type collection, Manassas, VA) were grown in DMEM (01-055-1A; Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 4 mM L-glutamine, 10,000 U penicillin/ml, 0.1% (w/v) streptomycin, and 10% (v/v) FBS (10106-169; Life Technologies, Paisley, U.K.). Transfections were conducted in 150-cm2 flasks (TPP AG, Trasadingen, Switzerland) containing a 80% confluent monolayer of cells. Two hours before transfection, the medium in the flask was replaced with 35 ml of fresh medium. A calcium phosphate/plasmid DNA precipitate was made by mixing 1680 µl of 150 mM NaCl, 268 mM CaCl2,1 mM EDTA, 10 mM Tris-HCl (pH 7.1), and 100 µg plasmid DNA (in 150 µl of 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) with 1680 µl of 50 mM HEPES, 250 mM NaCl, 1.5 mM Na2HPO4/NaH2PO4 (pH 7.12). This precipitate was added to one flask. Cells were incubated with the precipitate for 16 h, then emptied and rinsed twice with PBS before adding 35 ml of RPMI 1640 (Life Technologies formulation) supplemented with glutamine and penicillin as above and a 1/100 vol of a solution of insulin, transferrin, and selenium (51300-44; Life Technologies).
Purification of rMASP-2 on Ni-nitrilotriacetic acid (NTA) agarose
After 6 days of incubation, the medium from four flasks (
140
ml) was harvested and centrifuged at 3300 x g to
remove cellular debris, the pH was adjusted 8, and 0.1% (v/v)
NaN3 was added. The supernatant was incubated for
16 h at 4°C with 5 ml of Ni-NTA agarose (30230; Qiagen),
preequilibrated with 5 vol of RPMI 1640, under gentle agitation. The
beads were collected on a PolyPrep 10-ml column cartridge (731-1550;
Bio-Rad, Hercules, CA) and washed with 40 ml 10 mM Tris-HCl, 1.1 M
NaCl, 0.05% (v/v) polyoxyethylene 10-tridecyl ether, pH 7.4 (buffer
A). Elution was conducted in two steps, collecting 4 x 2.5-ml
fractions of protein eluting with buffer A, containing 24 mM imidazole,
and subsequently collecting 4 x 2.5-ml fractions of protein
eluting in buffer A, containing 24 mM imidazole and 10 mM EDTA. Three
fractions of the EDTA eluate containing rMASP-2, as judged by Western
blotting, were pooled and dialyzed against 10 mM Tris-HCl, 140 mM NaCl,
pH 7.4 (TBS) with 0.05% (v/v) polyoxyethylene 10-tridecyl ether
(referred to as purified rMASP-2). A negative control was made by
processing in parallel 140 ml of supernatant from untransfected HEK 293
cells prepared as the rMASP-2-containing supernatant.
SDS-PAGE, Western blotting, and silver staining
SDS-PAGE and Western blot analysis was conducted with 4% to 20% (w/v)) gradient acrylamide gels, as described by Jensen et al. (19). Detection of rMASP-2 was conducted with polyclonal rabbit anti-MASP-2 or mAb anti-MASP-2 Abs 1.29F11 and 1.3B7 (20), followed by HRP-conjugated anti IgG Ab (P0260 or P0448; Dako A/S, Glostrup, Denmark) and developed with chemiluminescent substrate (34080; Pierce, Rockford, IL). Relative molecular sizes were interpolated from curves constructed on the basis of colored protein markers (Rainbow, RPN756; Amersham-Pharmacia, Uppsala, Sweden), i.e., myosin (200 kDa), phosphorylase (98 kDa), BSA (77 kDa), OVA (51 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (19 kDa), and lysozyme (15 kDa). The electrophoretic mobility of these colored proteins differed significantly from those of nonstained proteins, and the molecular masses given above were estimated from calibration curves constructed using the unlabeled MARK12 molecular mass markers (NOVEX, San Diego, CA). To analyze the protein content of the pooled fractions of purified rMASP-2, 200 µl was precipitated by mixing with 300 µl of acetone and incubating at -20°C for 16 h. After redissolving precipitated protein in SDS-PAGE sample buffer, samples were boiled and analyzed by SDS-PAGE under reducing or nonreducing conditions. Silver staining of the gels was conducted essentially as described by Nesterenko et al. (21).
N-terminal sequencing
Purified rMASP-2 corresponding to a volume of 500 µl of EDTA/imidazole eluate was applied to SDS-PAGE. After electrophoresis, the gel was equilibrated for 10 min in 10 mM 3-cyclohexylamino-L-propanesulfonic acid, 10% (v/v) methanol, pH 11. Blotting of the proteins onto a Problot membrane (PE Applied Biosystems, Foster City, CA) was conducted in the same buffer at 7.5 V/cm for 10 h. Protein species were visualized with Coomassie staining, and the species appearing at 75 kDa was excised and sequenced on an Applied Biosystems 470/120A sequencer.
MBL/MASP-1/MASP-2/MAp19 and MBL preparations
The MBL concentration was determined by a time-resolved immunofluorometric assay (TRIFMA), as described earlier (22). Clinical grade MBL/MASP-1/MASP-2/MAp19 complexes (MO-04; State Serum Institute, Copenhagen, Denmark) were prepared from pooled plasma from human donors (23). Human serum albumin was removed from the pharmaceutical formulation by carbohydrate affinity chromatography of MBL. A total of 3 mg of MBL/MASP-1/MASP-2/MAp19 was diluted in TBS with 0.05% (v/v) Tween-20 (TBS/Tw) and 5 mM CaCl2. The diluted MBL/MASP-1/MASP-2/MAp19 was passed over mannose-derivatized Fractogel TSK HW-75 beads (14985; Merck KgaA, Darmstadt, Germany), prepared according to a procedure by Fornstedt and Porath (24). After washing in calcium-containing buffer, MBL/MASP-1/MASP-2/MAp19 was eluted in TBS/Tw with 15 mM EDTA. To free MBL of MASP and MAp19, fractionation was conducted at acid pH (25). Fractions from the affinity chromatography containing MBL/MASP-1/MASP-2/MAp19 complexes were pooled and diluted in 50 mM CH3COONa, 0.1% (w/v) NaN3, 0.01% (v/v) polyoxyethylene 10-tridecyl ether, 1 mM EDTA, pH 4.5, and passed over a 5 x 50-mm Mono S HR 5/5 ion-exchange column (17-0547-01; Amersham-Pharmacia). After washing and elution of bound material by a NaCl gradient (final concentration 1 M NaCl), MBL-containing fractions were analyzed by the capability to activate C4 (see below) and by Western blotting with anti-MASP-1 and anti-MASP-2 Abs. Fractions with undetectable capability to activate C4, that is, with the C4-cleaving activity reduced more than 6000-fold, and with no MASP as judged from Western blots, were pooled. This MASP-depleted MBL is referred to as natural MBL or simply MBL. MBL/MASP-1/MASP-2/MAp19 complexes with MASP in the proenzymatic state were prepared as described by Stover et al. (10). rMBL was produced under serum-free conditions in HEK 293EBNA cells and purified by carbohydrate affinity chromatography on mannose-derivatized Fractogel TSK beads (to be published).
GPC analysis
The apparent size of MASP-2 under nondenaturing conditions was examined by subjecting 200 µl of purified rMASP-2 to GPC analysis on a 10 mm x 30-cm Superose 6 HR column (17-0537-01; Amersham-Pharmacia) in calcium- and magnesium-containing (10 mM Tris-HCl, 140 mM NaCl, 0.01% (v/v) Tween-20, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) or EDTA-containing (10 mM Tris-HCl, 140 mM NaCl, 0.01% (v/v) Tween-20, 5 mM EDTA, pH 7.4) buffer. Complex formation between rMASP-2 and MBL was tested by incubating 200 µl of the purified rMASP-2 with 10 µg of MBL for 2 h in the presence of 2 mM Ca2+ and 1 mM Mg2+, followed by fractionating in calcium- and magnesium-containing buffer as above. Also, 200 µl of the purified rMASP-2 was incubated with 10 µg of MBL for 2 h in the presence of 5 mM EDTA, followed by fractionating in EDTA-containing buffer as above. To test the ability of EDTA to dissolve MBL/rMASP-2 complexes, complexes were allowed to form by incubating for 2 h 200 µl of the purified rMASP-2 with 10 µg of MBL in the presence of 2 mM Ca2+ and 1 mM Mg2+. EDTA was then added to a final concentration of 8 mM, followed by incubation of the sample for 2 h. The sample was thereafter subjected to GPC in the EDTA-containing buffer described above. To test the ability of EDTA and NaCl to dissolve MBL/rMASP-2 complexes, complexes were allowed to form as before, and EDTA and NaCl were added to final concentrations of 8 mM and 1 M, respectively, followed by incubation of the sample for 2 h. The sample was subjected to GPC in 10 mM Tris-HCl, 140 mM NaCl, 0.01% (v/v) Tween-20, 5 mM EDTA, 0.35 M NaCl, pH 7.4. Fractions of 450 µl were collected and analyzed in reduced state by SDS-PAGE/Western blotting with mAb anti-MASP-2 Ab, as described above, using 60 µl of the fractions per lane. Chemiluminescent signals were detected by use of a charge-coupled device camera (FlourS; Bio-Rad).
MASP-2 proteolysis
The activation of rMASP-2 was examined by incubation in mannan-coated wells (prepared as for the C4 deposition assay). Three different solutions were prepared: 1) purified rMASP-2 diluted 20-fold in buffer containing 500 ng MBL/ml, and as controls: 2) purified rMASP-2 diluted 20-fold and 3) MBL diluted to 500 ng/ml. All dilutions were conducted with 4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4. After incubation for 5 h at 4°C, the solutions were applied to the mannan-coated wells, 4 x 11 wells receiving each 100 µl of rMASP-2 and MBL, 11 wells receiving each 100 µl of rMASP-2, and 11 wells receiving each 100 µl of MBL. The wells were incubated for 16 h at 4°C, followed by incubation for 2 h at 37°C. The supernatants from incubations with rMASP-2/MBL and with rMASP-2 were collected. Bound protein was eluted by adding 100 µl of 5% (v/v) glycerol, 1.5% (w/v) SDS, 24% (w/v) urea, 0.0005% (w/v) bromophenol blue, 63 mM Tris-HCl, pH 6.7, to a microtiter well, incubating for 10 min at ambient temperature, and then transferring the eluate to the next well in the row, in total collecting bound protein from 11 wells. Eluates were subjected to SDS-PAGE, either in reduced state and boiled for 3 min or in nonreduced state without boiling. One-third of the eluate from each row was analyzed by SDS-PAGE/Western blotting. The remaining two-thirds of eluate from each row were analyzed by SDS-PAGE/blotting, followed by incubation with C4 under conditions suitable for C4 activation and detection of C4b deposited on the blotting membrane with labeled anti-C4, as reported earlier (2). Western blotting was conducted as described above with mAb anti-MASP-2 Abs. Chemiluminescence signals were detected by light-sensitive films (Super RX; Fuji, Tokyo, Japan). For comparison, plasma-derived MBL/MASP-1/MASP-2/MAp19 complexes (MO-04; State Serum Institute) were applied to the SDS-PAGE in reduced and nonreduced state (corresponding to 200 ng of MBL per lane for analysis with anti-MASP-2 Ab, and 500 ng of MBL for analysis of C4b deposition). To identify the position, proenzymatic MASP-2, MBL/MASP-1/MASP-2/MAp19 complexes containing proenzymatic MASP were applied to the SDS-PAGE (corresponding to 344 ng of MBL, reduced, for analysis with anti-MASP-2 Ab, and 550 ng of MBL, reduced, for analysis of C4b deposition). The activation of natural MASP-2 in MBL/MASP-1/MASP-2/MAp19 complexes was compared with the activation of rMASP-2 in rMASP-2/MBL complexes in a time-course study: MBL-deficient serum was diluted 8-fold and incubated together with MBL in microtiter well strips at 4°C for 16 h, as described above. Similarly, purified rMASP-2 was diluted 40-fold and incubated with MBL in microtiter well strips (1244-550; Nunc, Kampstrup, Denmark). After washing in calcium-containing buffer, the wells were incubated between 10 min and 5 h at 37°C. Following each incubation, the wells were emptied and filled with buffer containing calcium and 10 mM PefaBlock (84900321; Boehringer Mannheim, Indianapolis, IN), which previously had been found to inhibit the enzymatic activity of rMASP-2. Elution from the wells was conducted at 4°C, as described above. The eluates were analyzed in the reduced state by SDS-PAGE/Western blotting, as described above.
C4 activation
C4 was prepared as described by Dodds (26). FlouroNunc microtiter wells (437958; Nunc) were coated with 1 µg mannan (prepared as described in Ref. 27) in 100 µl 0.1 M bicarbonate (pH 9.6) for 16 h at room temperature and subsequently blocked with 1 mg human serum albumin/ml (440511; State Serum Institute) for 2 h at room temperature. After washing with TBS, 0.05% (v/v) Tween-20 (TBS/Tw), wells received 10 ng MBL (natural MBL or rMBL) in 100 µl 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl2, 0.05% (v/v) Triton X-100, 1 mg human serum albumin/ml, pH 7.4 (TRIFMA buffer). After incubating for 16 h at 4°C, the wells were washed with TBS/Tw with 5 mM CaCl2 (TBS/Tw/Ca2+) and then received 100 µl of MBL-deficient serum, purified rMASP-2, or rMASP-2-containing culture supernatant diluted 50-fold or more in TRIFMA buffer. As control, HEK 293 cell serum-free culture supernatant or culture supernatant fractionated on Ni-NTA agarose was used. After 16-h incubation at 4°C and washing with TBS/Tw/Ca2+, wells were incubated at 37°C for 1.5 h with 0.5 µg C4 in 100 µl 4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4. Detection of deposited C4b in the wells was conducted according to procedures for TRIFMA (28) with europium-labeled anti-C4 mAbs (Hyb 162-1 and 162-2, kindly supplied by Dr. Claus Koch, The State Serum Institute) at 0.5 µg/ml TBS/Tw with 25 µM EDTA added. The Abs were labeled with Eu according to Wallac’s protocol (Wallac Oy, Turku, Finland).
Comparison of MASP-2 captured in wells incubated with MBL-deficient serum and purified rMASP-2
Mannan-coated microtiter plates were prepared and incubated with natural MBL as described for the C4 activation assay. MBL-deficient serum was diluted 2-, 8-, 32-, 128-, and 512-fold in TRIFMA buffer. Each dilution was applied to 11 wells using 100 µl of dilution per well. The Ni-NTA-purified rMASP-2 was diluted 10-, 20-, 160-, and 640-fold, applied to the wells as for the dilutions of MBL-deficient serum. After incubation for 16 h at 4°C and washing three times with cold calcium-containing buffer, the bound protein was eluted from the wells by incubation with denaturing buffer, as described above, but carrying out the elution procedure at 4°C. Eluates were subjected to SDS-PAGE in nonreduced state after boiling for 3 min. Western blot analysis was conducted as described above with polyclonal rabbit anti-MASP-2 Ab. Chemiluminescence signals were detected by light-sensitive films (Fuji). Controls were included by analyzing the eluate from mannan-coated wells that had not received MBL before the incubation with MBL-deficient serum (diluted 2-fold) or Ni-NTA-purified rMASP-2 (diluted 10-fold).
C3 activation
C3 was purified from serum and kindly provided by Dr. Alister Dodds (26). C2 was purified according to a method described by Kerr and Gagnon (29). Microtiter wells were coated with mannan, followed by 20 ng natural MBL or rMBL in 100 µl TRIFMA buffer. Wells were then incubated for 16 h at 4°C with 100 µl of purified rMASP-2 diluted 50- or 200-fold or MBL-deficient serum diluted 200- or 1000-fold in TRIFMA buffer. After washing with TBS/Tw/Ca2+, complement components were added to the wells in 100 µl of 4 mM barbital, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4, with 2.5 µg C4/ml, 0.3 µg C2/ml, and 1 µg C3/ml. Controls were prepared by adding buffer with C2 and C3, or C4 and C3, or only C3. The wells were incubated at 37°C for 1.5 h and subsequently washed in TBS/Tw/Ca2+. Rabbit anti-C3c Ab (A 062; Dako A/S), biotinylated according to a procedure described by Guesdon et al. (30), was diluted to 1 µg/ml in TBS/Tw/Ca2+ and added to the wells, each well receiving 100 µl. After washing in calcium-containing buffer, bound Ab was detected with europium-labeled streptavidin (1244-360; Wallac) diluted 1000-fold in TBS/Tw with 25 µM EDTA added, followed by flourometry as for the C4 deposition assay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of rMASP-2
A construct suitable for expression of MASP-2 in mammalian cell
lines (rMASP-2) was generated by cloning the human MASP-2 cDNA sequence
into the pDR2EF1 expression vector (17).
Simultaneously, the cDNA was modified to encode a 5' Kozak sequence and
a COOH-terminal His6 tag. Transcription from the
pDR2EF1 vector is controlled by the human elongation factor 1
promoter. rMASP-2 was produced by transient expression in HEK 293 cells
maintained in serum-free medium. The production of rMASP-2 was
monitored by Western blotting with anti-MASP-2 mAbs raised against
rMASP-2 expressed in E. coli (20). On Western
blots, the Abs recognized a 75-kDa species in the culture supernatant
from transfected cells, whereas no signal was detected in control
supernatant. Normal murine IgG, when used instead of mAbs, did not give
rise to any signals when used to analyze the supernatant from
transfected cells.
Purification of rMASP-2
rMASP-2 was captured by incubating the supernatant with
Ni-NTA-derivatized agarose under nondenaturing conditions. Western blot
analysis of the supernatant following the incubation with the
Ni-NTA-agarose suggested a nearly complete binding of the rMASP-2. By
passing one column volume of imidazole-containing buffer over the
affinity matrix, about 50% of the bound rMASP-2 was eluted (as judged
by Western blotting); however, the rMASP-2 contributed only a minor
part of the eluted protein. Subsequent elution with buffer containing
both imidazole and EDTA released the remaining rMASP-2 in a fraction
that migrated as a single species with an apparent
Mr of 75 kDa on silver-stained
SDS-PAGE gels (Fig. 1
). Fractions of the
control supernatant, processed in parallel with the rMASP-2-containing
supernatant, did not give rise to any signals on Western blots, nor
were any proteins with a similar Mr
detected by silver staining (data not shown). Amino acid sequencing of
the 75-kDa species yielded the 10 NH2-terminal
amino acids of MASP-2. Comparison with the NOVEX MARK12 protein weight
marker by densitometry of the silver-stained gel suggested that the
concentration of rMASP-2 in the purified fraction was 8 µg/ml. The
concentration of rMASP-2 in the culture supernatant before
fractionation is therefore estimated to be about 1 µg/ml.
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The ability of rMASP-2 to form stable complexes with MBL was
examined by GPC analysis in the presence of Ca2+
and Mg2+ or in EDTA-containing buffer. GPC
fractions were analyzed by Western blotting. In the absence of MBL,
rMASP-2 elutes either in the presence or absence of
Ca2+/Mg2+ in the same
fractions as monomeric C1s (Fig. 2). A
major change in elution volume was observed when rMASP-2 was incubated
with MBL in the presence of Ca2+ and
Mg2+. Under these conditions, most of the rMASP-2
eluted just before free MBL, consistent with the larger size of the
MBL/rMASP-2 complex compared with that of free MBL. In the presence of
EDTA, a much smaller fraction of the rMASP-2 formed complexes with MBL.
The MBL/rMASP-2 complexes thus formed were very stable since the
complexes dissociated in buffers containing 1 M NaCl and 8 mM EDTA, but
not in those containing 8 mM EDTA only (data not shown). According to
Western blot analysis of the rMASP-2/MBL complexes, the rMASP-2
remained in the proenzymatic state, as judged by its appearance as a
single species of Mr 75 kDa.
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To determine whether or not the rMASP-2/MBL complexes could be activated by mannan binding, rMASP-2 was incubated with MBL, added to mannan-coated microtiter wells, and then analyzed after reduction by Western blotting to test for cleavage of the proenzyme into disulfide-linked A and B chains. The C4-activating capability of activated and nonactivated rMASP-2 was initially tested qualitatively in a C4 deposition assay in which the nonreduced, unboiled samples were electroblotted onto nitrocellulose filters after SDS-PAGE analysis and then incubated with C4, as described previously (2).
Under reducing conditions, Western blotting with an anti-MASP-2 Ab
(reacting with the A chain only) revealed that a control
MBL/MASP-1/MASP-2/MAp19 preparation contained only a single MASP-2
species of 50 kDa corresponding to the A chain (Fig. 3, blot A, lane 11),
indicating that the native MASP-2 was fully activated. After
electrophoresis and electroblotting under nonreducing conditions, this
preparation also induced C4 deposition on the nitrocellulose filters
(Fig. 3, blot B, lane 12). In contrast, purified rMASP-2
analyzed in an equivalent manner in the absence of MBL (Fig. 3, blot A,
lanes 8 and 9, respectively) appeared as an
uncleaved species of 75 kDa that was incapable of inducing C4b
deposition (Fig. 3, blot B, lane 9). The apparent molecular
mass of rMASP-2 matches closely that of the proenzyme purified from
plasma in the presence of protease inhibitors.
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, blot A, lane 6). Remnants of proenzyme as well as
some cleaved rMASP-2 were found in the supernatant from this incubation
(Fig. 3, blot A, lane 2). The activated rMASP-2 was also
able to elicit C4b deposition on the membrane to a degree similar to
that observed for plasma-derived MASP-2 (Fig. 3, blot B, lane
5). In the absence of MBL, rMASP-2 did not show any sign of
proteolytic cleavage (Fig. 3, blot A, lane 3). rMASP-2 added
to mannan-coated wells with MBL at 4°C underwent only partial
cleavage (Fig. 3, blot A, lane 4). A time-course study confirmed that complete cleavage of rMASP-2 in complex with MBL required about 2–3 h of incubation at 37°C. In contrast, natural MASP-2 in MBL-MASP complexes, generated by incubation of MBL with MBL-deficient serum, appeared to be all activated after 16 h of incubation at 4°C, as judged by SDS-PAGE/Western blotting (data not shown).
C4 activation
Quantitative comparisons of C4 activation by rMASP-2 were
undertaken using a C4b deposition assay. Samples of purified or
unpurified rMASP-2 were incubated in microtiter wells coated with
mannan and MBL in the presence of C4. The rMASP-2 was compared with
MBL-deficient serum as a MASP source. As shown in Fig. 4A, C4b deposition took place
in the presence of rMASP-2 and MBL, while control culture supernatants
that were unfractionated or had been fractionated in parallel with
rMASP-2 did not give rise to any signal. The C4b deposition was MBL
dependent, as no signal was detected in wells without MBL. Fig. 4B indicates that rMASP-2 mediates C4 activation when bound
to either rMBL or natural MBL. To rule out contamination of the C4
preparation with MASP-1, the C4 was preincubated with anti-MASP-1
mAb-coated beads. Incubation with the beads had no effect on subsequent
C4 deposition (data not shown). Similarly, no MASP-1 could be detected
in the C4 preparation by a time-resolved immunofluorometric assay for
MASP-1 Ag, suggesting that the level of such contaminating MASP-1, if
it is present at all, is at least 3000-fold lower than the MASP-1
concentration in normal human serum.
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4 x
104 counts, i.e., half-maximal C4b deposition by
the MBL-deficient serum, 10-fold more rMASP-2 preparation was required
than MBL-deficient serum (Fig. 4A). To relate the actual
mass of bound MASP-2, the bound MASP-2 was eluted from the respective
wells and quantified by Western blotting with a polyclonal
anti-MASP-2 Ab. On this basis, it was estimated that for a given
dilution of the respective preparations, 5-fold more rMASP-2 than
native MASP-2 bound to the wells to which MBL was immobilized (Fig. 4C). With regard to the ability to activate C4, therefore,
these data suggest that the activity of rMASP-2/MBL complexes is
50-fold lower than that of natural MASP-2/MBL complexes generated by
incubating MBL with MBL-deficient serum. Concerning the capacity of MBL
for binding of native MASP-2 and rMASP-2, saturation was observed after
incubating 8-fold dilutions of each preparation on immobilized MBL
(Fig. 4C; data not shown). Together with the estimated
5-fold difference in the amounts of MASP-2 protein present in each
preparation, this indicates that MBL at the conditions of the
experiment binds 5-fold more rMASP-2 than natural MASP-2, which
presumably will be integrated to form complexes consisting of
MBL/MASP-1/MASP-2/MAp19. C3 activation
The ability of MBL/rMASP-2 to also elicit C3b deposition in the
microtiter wells was tested by mixing C2 and C4 with C3 in a manner
similar to that for the C4 activation assay. As indicated in Fig. 5, C3 deposition required C4 and C2, and
was also completely dependent on a MASP source (MBL-deficient serum or
rMASP-2). As observed for C4, generation of the C3 convertase occurred
on surfaces coated with either rMBL or natural MBL. The possibility of
contamination of the C2 and C3 preparation with MASP-1 was excluded, as
described for the C4 preparation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and ionic strength dependent (data
not shown) suggests the interactions to be similar to the native
complexes. Taken altogether, these observations suggest that the
rMASP-2 that we have produced is active and correctly folded. An
equivalent recombinant form of MASP-1 produced in a similar manner
nevertheless had no enzymatic activity according to the complement
activation assays when tested alone or in combination with rMASP-2
(data not shown). The secreted protein differed from natural MASP-1
with regard to its molecular mass, however, so we are reluctant to draw
any conclusions regarding the biological properties of this
material. The discovery of the two MASP proteins each with a domain organization identical with that of the C1 proteases, combined with the striking overall structural similarity between C1q and MBL, has led to the expectation that complement activation via the MBL pathway involves the coordinated activity of two MASP proteins in a similar fashion to that already well established for C1r and C1s (16, 32). According to this proposal, MASP-1 should be required for MASP-2 activation. Somewhat surprisingly, the results of the present study contradict such a model insofar as we find that C4 and C2 can be activated by reconstituted MBL/rMASP-2 complexes, indicating that the activation of these complement components may occur independently of the presence of all other components of natural MBL/MASP-1/MASP-2/MAp19 complexes. Thus, a system consisting of only MBL and rMASP-2 immobilized on a mannan-coated surface is sufficient for inducing the autoactivation of the MASP-2 proenzyme, its cleavage into disulfide-linked A and B chains, and its ability to generate the C3 convertase, C4bC2b, through proteolysis of C4 and C2. Others have reported that fluid phase-activated MBL complexes directly cleave C3 via the enzymatic activity of MASP (33). We did not detect any direct conversion of C3 in the absence of C4 and C2 when using either rMASP-2 or MBL-deficient serum as a MASP source. Thus, we were unable to confirm these observations using our experimental system.
It is important to note that the activity of MBL reconstituted with rMASP-2 is lower than the activity of MBL reconstituted with MBL-deficient serum. In particular, it appears that the activation kinetics of natural MASP-2 differs from that of rMASP-2 in complex with MBL since all natural MASP-2 was activated after 16-h incubation at 4°C, while an equivalent amount of rMASP-2 required a further 2–3-h incubation at 37°C for all of it to become fully activated. It would seem, therefore, that the rMASP-2/MBL complexes cannot account for all properties of natural MBL complexes. The modification of the rMASP-2 COOH terminus with a His6 tag had no apparent qualitative effect on the properties of the recombinant protein with regard to C3 and C4 activation when compared with natural MASP-2, although it may still be possible that this alteration from the natural protein lowers the activity of the recombinant enzyme. It seems more likely, however, that other components, such as MASP-1 or MAp19, confer additional catalytic activity to the native complexes or affect the efficiency of MASP-2-mediated complement fixation, or both. In this regard it is of interest that Dobó et al. (34) have found that the addition of a mutated form of C1r that could not autoactivate nevertheless increased the hemolytic activity of C1 complexes formed from this mutant and wild-type C1r, C1q, and C1s. By analogy, this suggests that noncatalytic components, e.g., MAp19, may contribute to the activity of MBL/MASP-1/MASP-2/MAp19 complexes, possibly through interaction with MASP-2. To saturate MBL 50% with MASP-2, as estimated from the amounts captured in mannan-coated wells, it appears that a somewhat higher concentration of rMASP-2 was required in comparison with the concentration of MASP-2 in diluted MBL-deficient serum saturating at the same level (T. Vorup-Jensen, unpublished data). Thus, the concentration saturating 50% taken as an approximate measure of the dissociation constant, it may be the case that other proteins in MBL-deficient serum, e.g., MAp19 and MASP-1, can increase the binding stability of the MBL/MASP-2 interaction, allowing for a more potent complement activation. The influence of other proteins in serum was clearly observed on the binding capacity of MBL, which could bind about 5-fold more rMASP-2 than natural MASP-2 captured from MBL-deficient serum, suggesting that rMASP-2 in the absence of MAp19 and MASP-1 may fill in vacant binding sites.
It has been reported that C1 activation can occur through interaction with proteases of the blood-clotting cascade (35, 36). Although the physiological significance of this mechanism is unclear, it may be speculated also to contribute to the observed difference in the activation of rMASP-2 and natural MASP-2 presented in the form of MBL-deficient serum. Stringent evaluation of the physiologically relevant mechanism of C4 and C2 activation by the MBL pathway will require careful analysis of the identity and stoichiometry of all the components involved, and at present it remains unclear whether rMASP-2 is intrinsically less active than native MASP-2, or whether the complexes it forms with MBL are less active than the native complexes, or both. Such analyses await the purification of native MASP-2 to homogeneity for direct comparisons with the recombinant protein. Importantly, our results already indicate that the MASP/MBL pathway of complement activation is qualitatively distinct from the C1 pathway, which is entirely dependent on the presence of both proteases (37, 38).
Previous work has highlighted the relatively close phylogenetic
relationship between the C1 proteases and MASP-2, while the
evolutionary relatedness between MASP-1 and the other serine proteases
seems more distant (14, 15, 39). The MBL pathway appears
to have originated earlier than the classical pathway of complement
activation since MASP and an MBL-like lectin have been identified in
protochordates (40), while the C1 proteins have only been
described in vertebrates (41). These considerations
suggest the following scenario for the evolution of these molecules: in
the course of the evolution of the classical complement-activation
pathway, the two catalytic activities of MASP-2, i.e., the
autoproteolytic and C4 and C2 cleavage activities, were split between
the two proteases of the C1 complex, perhaps after the duplication of
the MASP-2-like primordial gene likely to have given rise to C1r and
C1s. According to this proposal, the separation of these activities
would have generated enzymes each with more restricted proteolytic
activities than the ancestral protease. Such a process may have
conferred the benefit of tighter control of complement activation via a
pathway that could be regulated in the manner of the classical
activation pathway in contrast to that of an MBL-like pathway. However,
the factors controlling complement activation through the MBL pathway
are not fully elucidated; interactions between MASP and
2-macroglobulin have been reported (42, 43), but the regulatory features of the MBL pathway remain very
poorly characterized. The next important question to be addressed
concerns the roles of MAp19 and MASP-1 in complement activation by MBL
complexes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Vorup-Jensen, Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark.
3 Abbreviations used in this paper: MBL, mannan-binding lectin; CUB, C1r/C1s/sea urchin Uegf/bone morphogenic protein; GPC, gel permeation chromatography; HEK, human embryonic kidney; MAp, MBL-associated protein; MASP, MBL-associated serine protease; NTA, nitrilotriacetic acid; TRIFMA, time-resolved immunofluorometric assay.
Received for publication February 11, 2000. Accepted for publication May 25, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-helical bundle at the nucleation site of collagen triple-helix formation. FEBS Lett. 344:191.[Medline]
p36.2 by in situ hybridization and somatic cell hybrid analysis. Cytogenet. Cell Genet. 84:148.[Medline]
-2-macroglobulin: a protein model for the interaction. Scand. J. Immunol. 42:373.[Medline]
2-Macroglobulin binds to and inhibits mannose-binding protein-associated serine protease. Int. Immunol. 7:1579.This article has been cited by other articles:
|
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 |
|
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|
|
|
|
 |
|
 R. Ramasawmy, G. S. Spina, K. C. Fae, A. C. Pereira, R. Nisihara, I. J. Messias Reason, M. Grinberg, F. Tarasoutchi, J. Kalil, and L. Guilherme Association of Mannose-Binding Lectin Gene Polymorphism but Not of Mannose-Binding Serine Protease 2 with Chronic Severe Aortic Regurgitation of Rheumatic Etiology Clin. Vaccine Immunol., June 1, 2008; 15(6): 932 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Takahashi, D. Iwaki, K. Kanno, Y. Ishida, J. Xiong, M. Matsushita, Y. Endo, S. Miura, N. Ishii, K. Sugamura, et al. Mannose-Binding Lectin (MBL)-Associated Serine Protease (MASP)-1 Contributes to Activation of the Lectin Complement Pathway J. Immunol., May 1, 2008; 180(9): 6132 - 6138. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Rawal, R. Rajagopalan, and V. P. Salvi Activation of Complement Component C5: COMPARISON OF C5 CONVERTASES OF THE LECTIN PATHWAY AND THE CLASSICAL PATHWAY OF COMPLEMENT J. Biol. Chem., March 21, 2008; 283(12): 7853 - 7863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Teillet, M. Lacroix, S. Thiel, D. Weilguny, T. Agger, G. J. Arlaud, and N. M. Thielens Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55 J. Immunol., May 1, 2007; 178(9): 5710 - 5716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Moller-Kristensen, S. Thiel, A. Sjoholm, M. Matsushita, and J. C. Jensenius Cooperation between MASP-1 and MASP-2 in the generation of C3 convertase through the MBL pathway Int. Immunol., February 1, 2007; 19(2): 141 - 149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zhang, K. Takahashi, E. M. Alicot, T. Vorup-Jensen, B. Kessler, S. Thiel, J. C. Jensenius, R. A. B. Ezekowitz, F. D. Moore, and M. C. Carroll Activation of the Lectin Pathway by Natural IgM in a Model of Ischemia/Reperfusion Injury J. Immunol., October 1, 2006; 177(7): 4727 - 4734. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
J. N. Arnold, R. Wallis, A. C. Willis, D. J. Harvey, L. Royle, R. A. Dwek, P. M. Rudd, and R. B. Sim Interaction of Mannan Binding Lectin with {alpha}2 Macroglobulin via Exposed Oligomannose Glycans: A CONSERVED FEATURE OF THE THIOL ESTER PROTEIN FAMILY? J. Biol. Chem., March 17, 2006; 281(11): 6955 - 6963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 J. Seyfarth, P. Garred, and H. O. Madsen The 'involution' of mannose-binding lectin Hum. Mol. Genet., October 1, 2005; 14(19): 2859 - 2869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Arnold, M. R. Wormald, D. M. Suter, C. M. Radcliffe, D. J. Harvey, R. A. Dwek, P. M. Rudd, and R. B. Sim Human Serum IgM Glycosylation: IDENTIFICATION OF GLYCOFORMS THAT CAN BIND TO MANNAN-BINDING LECTIN J. Biol. Chem., August 12, 2005; 280(32): 29080 - 29087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Lynch, S.-u.-H. Khan, C. M. Stover, S. M. Sandrini, D. Marston, J. S. Presanis, and W. J. Schwaeble Composition of the Lectin Pathway of Complement in Gallus gallus: Absence of Mannan-Binding Lectin-Associated Serine Protease-1 in Birds J. Immunol., April 15, 2005; 174(8): 4998 - 5006. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Teillet, B. Dublet, J.-P. Andrieu, C. Gaboriaud, G. J. Arlaud, and N. M. Thielens The Two Major Oligomeric Forms of Human Mannan-Binding Lectin: Chemical Characterization, Carbohydrate-Binding Properties, and Interaction with MBL-Associated Serine Proteases J. Immunol., March 1, 2005; 174(5): 2870 - 2877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Iwaki and T. Fujita Production and purification of recombinants of mouse MASP-2 and sMAP Innate Immunity, February 1, 2005; 11(1): 47 - 50. [Abstract] [PDF]
|
|
|
|
|
|
Y. Aoyagi, E. E. Adderson, J. G. Min, M. Matsushita, T. Fujita, S. Takahashi, Y. Okuwaki, and J. F. Bohnsack Role of L-Ficolin/Mannose-Binding Lectin-Associated Serine Protease Complexes in the Opsonophagocytosis of Type III Group B Streptococci J. Immunol., January 1, 2005; 174(1): 418 - 425. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Windbichler, B. Echtenacher, T. Hehlgans, J. C. Jensenius, W. Schwaeble, and D. N. Mannel Involvement of the Lectin Pathway of Complement Activation in Antimicrobial Immune Defense during Experimental Septic Peritonitis Infect. Immun., September 1, 2004; 72(9): 5247 - 5252. [Abstract] [Full Text] [PDF]
|
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|
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L. A. Gregory, N. M. Thielens, M. Matsushita, R. Sorensen, G. J. Arlaud, J. C. Fontecilla-Camps, and C. Gaboriaud The X-ray Structure of Human Mannan-binding Lectin-associated Protein 19 (MAp19) and Its Interaction Site with Mannan-binding Lectin and L-ficolin J. Biol. Chem., July 9, 2004; 279(28): 29391 - 29397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-B. Chen and R. Wallis Two Mechanisms for Mannose-binding Protein Modulation of the Activity of Its Associated Serine Proteases J. Biol. Chem., June 18, 2004; 279(25): 26058 - 26065. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Wallis, J. M. Shaw, J. Uitdehaag, C.-B. Chen, D. Torgersen, and K. Drickamer Localization of the Serine Protease-binding Sites in the Collagen-like Domain of Mannose-binding Protein: INDIRECT EFFECTS OF NATURALLY OCCURRING MUTATIONS ON PROTEASE BINDING AND ACTIVATION J. Biol. Chem., April 2, 2004; 279(14): 14065 - 14073. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zundel, S. Cseh, M. Lacroix, M. R. Dahl, M. Matsushita, J.-P. Andrieu, W. J. Schwaeble, J. C. Jensenius, T. Fujita, G. J. Arlaud, et al. Characterization of Recombinant Mannan-Binding Lectin-Associated Serine Protease (MASP)-3 Suggests an Activation Mechanism Different from That of MASP-1 and MASP-2 J. Immunol., April 1, 2004; 172(7): 4342 - 4350. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Lynch, S. Roscher, T. Hartung, S. Morath, M. Matsushita, D. N. Maennel, M. Kuraya, T. Fujita, and W. J. Schwaeble L-Ficolin Specifically Binds to Lipoteichoic Acid, a Cell Wall Constituent of Gram-Positive Bacteria, and Activates the Lectin Pathway of Complement J. Immunol., January 15, 2004; 172(2): 1198 - 1202. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Stengaard-Pedersen, S. Thiel, M. Gadjeva, M. Moller-Kristensen, R. Sorensen, L. T. Jensen, A. G. Sjoholm, L. Fugger, and J. C. Jensenius Inherited Deficiency of Mannan-Binding Lectin-Associated Serine Protease 2 N. Engl. J. Med., August 7, 2003; 349(6): 554 - 560. [Full Text] [PDF]
|
|
|
|
|
|
Y. Endo, M. Nonaka, H. Saiga, Y. Kakinuma, A. Matsushita, M. Takahashi, M. Matsushita, and T. Fujita Origin of Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and MASP-3 Involved in the Lectin Complement Pathway Traced Back to the Invertebrate, Amphioxus J. Immunol., May 1, 2003; 170(9): 4701 - 4707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Ambrus, P. Gal, M. Kojima, K. Szilagyi, J. Balczer, J. Antal, L. Graf, A. Laich, B. E. Moffatt, W. Schwaeble, et al. Natural Substrates and Inhibitors of Mannan-Binding Lectin-Associated Serine Protease-1 and -2: A Study on Recombinant Catalytic Fragments J. Immunol., February 1, 2003; 170(3): 1374 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Uemura, M. Saka, T. Nakagawa, N. Kawasaki, S. Thiel, J. C. Jensenius, and T. Kawasaki L-MBP Is Expressed in Epithelial Cells of Mouse Small Intestine J. Immunol., December 15, 2002; 169(12): 6945 - 6950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Collin, M. D. Svensson, A. G. Sjoholm, J. C. Jensenius, U. Sjobring, and A. Olsen EndoS and SpeB from Streptococcus pyogenes Inhibit Immunoglobulin-Mediated Opsonophagocytosis Infect. Immun., December 1, 2002; 70(12): 6646 - 6651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cseh, L. Vera, M. Matsushita, T. Fujita, G. J. Arlaud, and N. M. Thielens Characterization of the Interaction Between L-Ficolin/P35 and Mannan-Binding Lectin-Associated Serine Proteases-1 and -2 J. Immunol., November 15, 2002; 169(10): 5735 - 5743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Endo, M. Takahashi, M. Kuraya, M. Matsushita, C. M. Stover, W. J. Schwaeble, and T. Fujita Functional characterization of human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2 promoters, and comparison with the C1s promoter Int. Immunol., October 1, 2002; 14(10): 1193 - 1201. [Abstract] [Full Text] [PDF]
|
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|
|
|
S. Gulati, K. Sastry, J. C. Jensenius, P. A. Rice, and S. Ram Regulation of the Mannan-Binding Lectin Pathway of Complement on Neisseria gonorrhoeae by C1-Inhibitor and {alpha}2-Macroglobulin J. Immunol., April 15, 2002; 168(8): 4078 - 4086. [Abstract] [Full Text] [PDF]
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|
|
|
M. Matsushita, M. Kuraya, N. Hamasaki, M. Tsujimura, H. Shiraki, and T. Fujita Activation of the Lectin Complement Pathway by H-Ficolin (Hakata Antigen) J. Immunol., April 1, 2002; 168(7): 3502 - 3506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Celik, C. Stover, M. Botto, S. Thiel, S. Tzima, D. Kunkel, M. Walport, W. Lorenz, and W. Schwaeble Role of the Classical Pathway of Complement Activation in Experimentally Induced Polymicrobial Peritonitis Infect. Immun., December 1, 2001; 69(12): 7304 - 7309. [Abstract] [Full Text] [PDF]
|
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|
|
|
|
A. Roos, L. H. Bouwman, D. J. van Gijlswijk-Janssen, M. C. Faber-Krol, G. L. Stahl, and M. R. Daha Human IgA Activates the Complement System Via the Mannan-Binding Lectin Pathway J. Immunol., September 1, 2001; 167(5): 2861 - 2868. [Abstract] [Full Text] [PDF]
|
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|
|
|
N. M. Thielens, S. Cseh, S. Thiel, T. Vorup-Jensen, V. Rossi, J. C. Jensenius, and G. J. Arlaud Interaction Properties of Human Mannan-Binding Lectin (MBL)-Associated Serine Proteases-1 and -2, MBL-Associated Protein 19, and MBL J. Immunol., April 15, 2001; 166(8): 5068 - 5077. [Abstract] [Full Text] [PDF]
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V. Rossi, S. Cseh, I. Bally, N. M. Thielens, J. C. Jensenius, and G. J. Arlaud Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2 J. Biol. Chem., October 26, 2001; 276(44): 40880 - 40887. [Abstract] [Full Text] [PDF]
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) purified rMASP-2 (Ni-NTA agarose-purified rMASP-2), and
(
) MBL-deficient serum. The following negative controls were used:
(
) culture supernatant from untransfected cells (culture
supernatant); (
) fractionated culture supernatant from untransfected
cells (Ni-NTA frac. control sup.). A control (
) was also prepared by
no addition of MBL to the wells (Without MBL), but with the addition of
rMASP-2 (A only). Error bars are SDs on measurements in
duplicate wells. In C, the MASP-2 captured in wells
incubated with MBL-deficient serum and purified rMASP-2 was compared.
MBL-deficient serum was diluted 2-, 32-, 128-, and 512-fold in TRIFMA
buffer and applied to wells in microtiter plates coated with mannan and
natural MBL. The Ni-NTA-purified rMASP-2 was diluted 10-, 20-, 160-,
and 640-fold, and similarly applied to microtiter wells. Eluates from
the wells were analyzed in nonreduced state and after 3 min of boiling
by SDS-PAGE/Western blotting with anti-MASP-2 Ab. Controls were
prepared by incubating either MBL-deficient serum or purified rMASP-2
in mannan-coated wells without MBL (W/o MBL). The positions of MAp19
(in lanes loaded with eluate from wells incubated with MBL-deficient
serum) and full-length MASP-2 are indicated.