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*
Department of Microbiology and Immunology, University of Leicester, Leicester, United Kingdom;
Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark;
Theodor-Kocher-Institute, University of Bern, Bern, Switzerland; and
§
Institute for Anatomy and Cell Biology, University of Marburg, Marburg, Germany
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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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The discovery of the MBL pathway began with the description of a bactericidal factor in mouse plasma, Ra-reactive factor, which was shown to activate complement upon binding to carbohydrates in Ra LPSs on Gram-negative bacteria 1 . The molecule involved in the recognition of carbohydrates by Ra-reactive factor in human and mouse plasma was identified as plasma mannan-binding lectin (MBL) 2, 3 . MBL-mediated activation leads to cleavage of the fourth and the second component of complement (C4 and C2, respectively) and subsequently to the formation of the C3 convertase C4b2a 4, 5 . In vitro experiments indicated that MBL may form a complex with C1r2C1s2 6, 7 .
In vivo, however, MBL associates with serine proteases similar to but distinct from C1r and C1s, termed MASP-1 8, 9, 10, 11 and MASP-2 12 . Like the serine proteases C1r and C1s of the classical activation pathway, MASP-1 and MASP-2 are composed of an N-terminal CUB domain 13 , followed by an epidermal growth factor (EGF)-like domain, a second CUB domain, two complement control protein (CCP) domains, and a serine protease domain. Upon activation, MASP-2 is cleaved into two disulfide-linked chains, an N-terminal A chain composed of the first five domains and a C-terminal, catalytically active B chain. As yet, very little is known about the stoichiometry, the composition, and activation sequence of the MBL/MASP complex. Previous reports indicated that MASP-1 cleaves C2, C3, and C4 5, 14 . However, with the discovery of the second MBL-associated serine protease, it became evident that at least the cleavage of C4 is mediated by MASP-2 12 . Another constituent of the MBL/MASP complex, a plasma protein of approximately 19 kDa with an N-terminal sequence identical to MASP-2, was consistently detected 12 .
The present study defines the primary structure of this MBL-associated protein (MAp19) and shows that it is the translational product of an additional, abundantly expressed mRNA transcript of 1 kb, processed together with the 2.6-kb MASP-2 mRNA from the same heterologous nuclear RNA transcript of a single structural gene. MAp19 would be expected to be enzymatically inactive as it lacks the serine protease domain. Its presence in the MBL/MASP complex implies a possible regulatory role and emphasizes that the composition of the MBL pathway activation complex is different from that of the classical pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes were purchased from Boehringer Mannheim
(Mannheim, Germany). The human liver cDNA library was purchased from
Stratagene (Cambridge, U.K.). Poly(A)Tract mRNA Isolation System IV was
from Promega (Madison, WI). RT-PCR kit (Superscript preamplification
system) was from Life Technologies (Paisley, U.K.).
[
-32P]dCTP and blotting membranes were from
Amersham-Pharmacia (Upsala, Sweden). PCRII vector and prokaryotic
expression vector pTrxFus were purchased from Invitrogen (Leek, The
Netherlands). A kit (Easy DNA kit) to purify genomic DNA was purchased
from Invitrogen.
Isolation of MASP-2-related transcripts from a human liver cDNA library
A 5'-specific SmaI subfragment (450 bp) of clone
phl-4 12 was used to screen approximately 107 plaques of
a human liver oligo(dT)-primed cDNA library cloned in the phage vector
ZAP (Stratagene). After rescue of the plasmid pBluescript
SK- by in vivo excision, each of the eight clones isolated
was characterized by Southern blot hybridization following restriction
analysis. Four of these 5'-specific transcripts (phl-5, phl-6, phl-7,
phl-8; 0.7–1.2 kb) were sequenced on both strands using the dideoxy
chain termination method of Sanger (T7 Sequencing Kit;
Amersham-Pharmacia). A characteristic restriction site for
SmaI was used to generate subclones in pBluescript
KS+ for sequencing using reverse and universe M13 primers.
Cloning of a partial cDNA transcript of rat MASP-2 by reverse-transcriptional amplification
To generate a species-specific MASP-2 cDNA probe for use in Northern blot analysis, rat liver RNA was reverse transcribed using the Superscript preamplification system (Life Technologies). The obtained oligo(dT)-primed cDNA was used in cyclic amplification with randomized primers derived from the human MASP-2 amino acid sequences ATLCGQES (position: 72–79 of the mature protein, 12) and TGWKIHYT (position: 272–279). A standard PCR program was used (95°C, 5 min; 35 cycles, denaturation at 95°C for 30 s, annealing at 50°C for 1 min, and extension at 72°C for 1 min; final extension step at 72°C for 10 min). A 623-bp product was obtained, subcloned in PCRII (TA cloning kit; Invitrogen), and sequenced on both strands. The nucleotide sequence of RT-PCR rl-1 revealed an overall identity of 81.1% with the corresponding human MASP-2 cDNA; the two deduced translational products show 80.2% identity.
Northern blot analysis
Total RNA was extracted from human, mouse, rat, and guinea pig liver tissues according to standard protocols 15 , and mRNA was purified using Poly(A)Tract mRNA Isolation System IV (Promega). Approximately 2 µg of poly(A)+ RNA was separated per lane on a denaturing 0.8% (w/v) agarose gel and transferred to Hybond N membrane (Amersham-Pharmacia). The blots were hybridized according to standard protocols 16 with random primed 32P-dCTP-labeled (Random Primed Labeling Kit; Boehringer Mannheim) cDNA probes generated from our full-length human MASP-2 cDNA transcript phl-4, the full-length cDNA transcript of the MASP-2-related mRNA species of 1 kb, phl-5, and the partial rat-specific MASP-2 cDNA clone RT-PCR rl-1.
Prokaryotic expression of rMASP-2
rMASP-2 was expressed in Escherichia coli using the
Thiofusion Expression System (Invitrogen). phl-4 cDNA was used as a
template for cyclic amplification (for primers, see Table I
). The expected 1989-bp PCR
product was obtained, subcloned in PCRII (Invitrogen), excised with
BamHI/XhoI, then cloned in-frame into pTrxFus
(Invitrogen) linearized with BamHI and SalI. The
construct was confirmed by restriction mapping and sequencing.
Expression and purification were performed according to the
manufacturer’s protocol.
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N-acetyl-glucosamine was coupled to TSK-75 beads (Merck, Darmstadt, Germany) following the procedures of Fornstedt and Porath 17 . Plasma was isolated by drawing blood in a vial containing heparin (final concentration, 25 mM), iodoacetamide (5 mM), cyclocaprone (20 mM) (Amersham-Pharmacia), leupeptin (2 mg/ml) (Boehringer Mannheim), pepstatin A (4 mg/ml), benzamidine (100 mM) (Sigma-Aldrich Company, Dorset, U.K.), o-phenanthroline (10 mM) (Sigma-Aldrich), trazylol (200 U/ml) (Bayer, Leverkusen, Germany), soybean trypsin inhibitor (25 mg/ml) (Sigma-Aldrich), and 5 mM CaCl2. The plasma was then diluted in an equal volume of buffer A (10 mM barbital, 140 mM NaCl, 13 mM NaN3, 0.05% (w/v) Emulphogene (Sigma-Aldrich), 0.5 mM iodoacetamide, 2 mM cyclocaprone, 0.2 mg/ml leupeptin, 0.4 mg/ml pepstatin A, 10 mM benzamidine, 1 mM o-phenanthroline, and 2 mM CaCl2) and passed through N-acetyl-glucosamine-TSK beads. The beads were washed with buffer A, and calcium-dependent proteins, including the MBL/MASP complex, were eluted with buffer A containing 5 mM EDTA instead of 2 mM CaCl2.
After dialysis against 10 mM barbital, 140 mM NaCl, pH 7.4, to remove enzyme inhibitors, the MASPs in the MBL/MASP preparation were partially activated by incubation with mannan 18 for 2 h at 37°C. The preparation was analyzed using reduced (with DTT) or nonreduced samples by SDS-PAGE and Western blotting using a 4–20% (w/v) gradient gel, followed by blotting onto a polyvinylidene difluoride membrane (Hybond-P; Amersham-Pharmacia), as described 19 . After blotting, the membranes were incubated in TBS (10 mM Tris, 140 mM NaCl, pH 7.4) with 0.1% (v/v) Tween-20 and subsequently incubated with rabbit anti-MASP-2 antiserum diluted 1000-fold in TBS (13 mM NaN3) with 0.05% (v/v) Tween-20 (TBS/Tween), followed by washing with TBS/Tween and TBS/Tween without NaN3.
The anti-human MASP-2 antiserum was produced by immunizing rabbits with rMASP-2 (S. Petersen et al., unpublished data) expressed in E. coli using the ThioFusion Expression System (Invitrogen), as described above. After exposure to the polyclonal MASP-2 antiserum for 2 h at room temperature, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (DAKOpatts, Glostrup, Denmark), followed by addition of enhanced chemoluminescence (ECL) substrate (Pierce, Rockford, IL), and then exposed to x-ray film. Colored m.w. marker proteins were used as standards (Full Range Rainbow; Amersham-Pharmacia).
Mass spectrometry of plasma MAp19
The MBL/MASP/MAp19 complex was purified from a therapeutic MBL preparation from Statens Serum Institute, Copenhagen, Denmark. In the presence of calcium, the MBL preparation was passed through mannose-conjugated TSK-75 beads (Merck), and, after washing, the MBL/MASP/MAp19 complex was eluted from the beads with buffer containing EDTA. An amount equivalent to 500 µg MBL was fractionated by SDS-PAGE on a 4-20% gradient gel. The gel was stained with Coomassie blue, and the band corresponding to MAp19 excised. The gel slice was decolored with 30% (w/v) CH3CN/50 mM NH4HCO3, washed with water, and dried in a speed vac. The gel was then incubated in 100 mM DTT and heated to 55°C for 30 min and washed with water. Free sulphydryl groups were alkylated with 250 mM iodoacetamide for 30 min at room temperature. After washing with water, the gel was dried in a speed vac. In-gel digestion with endopeptidase Lys-C or trypsin and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) were performed as described 20 . Alternatively, tandem mass spectrometry (MS/MS) was employed.
Internal calibration of MALDI-TOF measurements was accomplished by adding desArg-1 bradykinin (Mass 904.4681) (Sigma-Aldrich) and adrenocorticotropic hormone fragment 18-39 (Mass 2465.1989) (Sigma-Aldrich) to the samples. For the protein-fragment search, monoisotopic peptide masses were used and a mass tolerance of 0.006% was allowed.
Characterization of the human gene for MASP-2
Human genomic DNA, isolated from peripheral blood leukocytes
using Easy DNA Kit (Invitrogen), was digested overnight with
EcoRI, separated on a 0.8% (w/v) agarose gel, and alkali
blotted to Hybond N+ nitrocellulose membrane
(Amersham-Pharmacia). MASP-2 cDNA transcript phl-4
(EcoRI/KpnI excised) and an amplification product
representing the coding region for the serine protease domain and the
3'-untranslated (UT) region of the 2.6-kb mRNA species
(EcoRI excised from PCRII; for oligonucleotides, see Table I
) were used as probes. Further analysis of the gene structure for
MASP-2 was performed by cyclic amplification of genomic DNA from two
donors using oligonucleotides derived from the cDNA sequence of human
MASP-2 clone phl-4 12 and of clone phl-5 (see Table I). PCR
amplification was confirmed by Southern blot analysis using cDNA clone
phl-4, and hybridizing fragments were subcloned into PCRII vector
(Invitrogen) and sequenced on both strands (T7 Sequencing Kit;
Amersham-Pharmacia). In addition, the 2.8-kb genomic amplification
products, obtained from each of the two genomic DNA preparations, were
excised from PCRII by EcoRI and PstI restriction
digest, subcloned in pBluescript KS+, and sequenced on both
strands.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight cDNA clones representing a novel MASP-2-related mRNA species
of approximately 1 kb were isolated from a human liver cDNA library.
After restriction analysis, three of these (clones phl-5, phl-6, phl-7;
Fig. 1A)
were sequenced in full. Clones phl-5 (736 bp) and phl-6 (729 bp)
comprise an open reading frame of 555 bp, preceded by a 5' UT sequence
identical to the 5' UT region of the 2.6-kb MASP-2 mRNA species (clone
phl-4 12 ; Fig. 1, A and B). Clone phl-7 (684
bp) is a partial transcript of the same mRNA that gives rise to phl-5
and phl-6. This novel mRNA species shares complete identity over 540 bp
with the 5' coding sequence of the 2.6-kb MASP-2 mRNA (Fig. 1B). This stretch of identity is followed by a coding
sequence of 12 nucleotides, and the open reading frame terminates with
a stop codon (TAG) 553 bp downstream of the translation initiation
codon (ATG). The 3' UT region differs from the 3' UT region of the
2.6-kb MASP-2 mRNA species (Fig. 1B). This novel mRNA
species therefore codes for the same signal peptide as determined for
MASP-2 and two of the six structural motifs of MASP-2, the N-terminal
CUB domain and the EGF-like domain (Fig. 1C). The deduced
translation product of this mRNA has a unique C-terminal sequence of
four amino acids (EQSL) that are not contained in MASP-2 (Fig. 1C). The calculated molecular mass of the deduced
amino acid sequence of the mature translation product is 19,075 Da.
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A, two hybridization signals
(one at 2.6 kb, one at 1 kb) were detected consistently when probing
human liver mRNA with the full-length cDNA transcripts, represented by
clones phl-4 and phl-5 (see Figs. 1A and 2A). A
cDNA probe specific for the sequence encoding the serine protease
domain and the 3' UT region (see above) hybridizes to the 2.6-kb mRNA
species only (not shown). The 1-kb MASP-2-related mRNA species is more
abundant than the 2.6-kb mRNA species (Fig. 2A). In
addition, rat-specific MASP-2 cDNA (RT-PCR rl-1), representing the
coding sequence for the N-terminal CUB domain, the EGF-like domain, and
the second CUB domain, was also probed on mRNA preparations of rat,
mouse, and guinea pig liver tissue. As depicted in Fig. 2B,
the presence of both MASP-2 and a more abundant 1-kb mRNA transcript
was detected consistently, showing that the coexpression of both mRNA
species is conserved.
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Analysis of two different plasma proteins with MASP-2
immunoreactivity was performed by SDS-PAGE and Western blotting. The
purified MBL-MASP complex was dialyzed to remove protease inhibitors
and then incubated in presence of mannan to obtain approximately 50%
cleavage of MASP-2 contained in the preparation (see Materials
and Methods). When this preparation was analyzed in its nonreduced
form, the Ab stained three bands, representing uncleaved (nonactivated)
MASP-2 (at 76 kDa), a slightly smaller and less abundant band (the
disulfide-linked A and B chains of cleaved MASP-2), and a small
MBL-associated protein at 17 kDa (Fig. 3A, lane N). When
analyzed after reduction, several bands are seen (Fig. 3A,
lane R). The upper 76-kDa band represents nonactivated
MASP-2, in which no cleavage of the polypeptide chain has occurred. The
band at 52 kDa represents the A chain of cleaved MASP-2, whereas the
band at 31 kDa represents the B chain of MASP-2, and the lower band at
19 kDa represents MAp19.
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B) and
subjected to mass spectrometry analysis. Purified MAp19 was in-gel
digested with endopeptidase Lys-C or trypsin, and the petides were
analyzed by MALDI-TOF or by tandem mass spectrometry (MS/MS). Hereby,
nearly complete coverage (96%) of the deduced amino acid sequence
encoded by the open reading frame of the 1-kb MASP-2-related mRNA
species was obtained (Table II).
The identification of the unique carboxyl-terminal sequence (EQSL) in
plasma MAp19 finally revealed that this protein is the translational
product of the 1-kb MASP-2-related mRNA species.
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The Southern blot analysis shown in Fig. 4A revealed two bands of 10
and 12 kb, which hybridized with the cDNA probe phl-4 (representing the
entire MASP-2 2.6-kb mRNA), whereas only the 10-kb band was detected
when the same filter was hybridized with a subfragment representing the
coding sequence for the serine protease domain (Fig. 4B).
This provided evidence for the existence of only one structural MASP-2
gene, with exons being spread over a distance of less than 22 kb.
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; Fig. 5, B
and C). The genomic sequence obtained comprises 4464
nucleotides (pgM-2 A; pgM-2 B, 4465 nucleotides) and starts with the
exon for the 5' UT region shared by both types of mRNA species and
spans up to the exon for the 3' UT region of the 2.6-kb MASP-2 mRNA
species (see Figs. 5A and 6).
The sequences obtained from the genomic DNA of two different donors
were in full agreement with the cDNA sequences of the previously
published MASP-2 cDNA clone (phl-4, 12) and the cDNA sequences obtained
from the 1-kb MASP-2-related mRNA species shown in this study.
Polymorphic sequences were obtained for the sequence representing the
3' UT regions of the 1- and 2.6-kb mRNA species, respectively, and are
listed in Table III. The 5' UT region and
the coding sequence for the signal peptide, the N-terminal CUB domain
(CUB-I), and the EGF-like domain of MASP-2 are located on one single
exon (exon a), separated by an intron sequence of 443 bp from an exon
containing the coding sequence for the unique C-terminal sequence
glutamic acid, glutamine, serine, and leucine (EQSL), the stop codon
(TAG), and the 3' UT sequence (including the polyadenylation initiation
signal AATAAA) of the 1-kb MASP-2 mRNA species (exon b; see Fig. 6).
Approximately 1.3 kb further downstream follows an exon encoding the
N-terminal 65 amino acids of the second CUB domain (CUB-II) (exon c).
This exon is separated by an intron sequence of 315 bp from the coding
sequences for the C-terminal portion of CUB-II, the two CCP domains,
and the serine protease domain as well as the 3' UT region of the
2.6-kb MASP-2 mRNA species (exon d). As illustrated in Fig. 5D, classical splice sites (according to the GTAG rule,
including pyrimidine-rich tracts and branchpoint sequences located 5'
thereof; Refs. 21–24; Fig. 6, Table IV) allow joining of exon a to exon b.
Usage of the polyadenylation initiation signal in exon b results in the
mRNA transcript of 1 kb. Alternatively, ligation of the 5' splice site
of exon a with the 3' splice site of exon c results in the loss of the
alternative splice exon and leads to the mRNA transcript of 2.6 kb.
Furthermore, the obtained primary structure of the human MASP-2 gene
redefines the C-terminal boundary of the EGF-like domain (see Fig. 6).
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A
and 6). We regard it as more than likely that this clone is an artifact
resulting from incomplete processing (see below), as we were unable to
detect a corresponding mRNA signal by Northern blot analysis using a
probe (an internal SmaI/Sau3A subfragment of 386 bp)
specific for the divergent sequence contained in phl-8 (data not
shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we show that two distinct gene products, MASP-2, a functionally active serine protease, and MAp19, a novel MASP-2-related protein devoid of the serine protease domain, are encoded by one structural gene. The generation of two mRNA species leading to two different gene products appears not to be restricted to human MASP-2, as Northern blot analyses of guinea pig, mouse, and rat liver RNA also revealed the presence of an abundant, smaller transcript of approximately 1 kb. The striking degree in which the presence of an abundant, small MASP-2 gene product is conserved implies a physiologic requirement for MAp19 and MAp19 homologues. Likewise, the conservation of the genomic mechanism that generates the small MASP-2 gene product is supported by results of Southern blot analyses of rat genomic DNA indicating that the MASP-2 gene in the rat is similarly organized as the human gene described in this work (C. Stover, S. Thiel, N. Lynch, and W. Schwaeble, manuscript in preparation), thus favoring the view of evolutionary stability 25 .
Splice junction sequences in the human MASP-2 gene suggest that the
generation of two mRNA transcripts encoding two different proteins by
the same single structural gene is due to an alternative
splicing/polyadenylation mechanism. Yet, as neither the 5' nor the 3'
splice sites match the consensus sequences 26 , these sites may be
suboptimal for recognition by the nuclear spliceosome 27 . Thus, it is
possible that the intron separating exon a from exon b is retained and
that this unspliced transcript is transported to the cytoplasm. Indeed,
isolation of cDNA clone phl-8 showed the presence of such an
intermediate transcript. However, splice sites may be strengthened by
certain nucleotides surrounding the splice junctions: these criteria of
primary structure are found in the human MASP-2 pre-mRNA and comprise
cytosines at positions -9, +14, +15 of the first 5' splice site, a
branchpoint sequence at position -72 of the first 3' acceptor site,
and polypyrimidine tracts preceding all three characterized acceptor
sites (Fig. 6). Candidates for so-called exonic splicing enhancers 27
are found in purine-rich sequences in exons b and d, downstream of the
respective 3' splice junctions. Similarly, the high content of cytidine
nucleotides in exon b may qualify certain stretches as oligo (C)
tracts, which were shown by others to support splicing 28 . Taken
together, it appears possible that additive strengtheners of splice
sites concentrate around the first 5' and 3' splice junctions that
might make these, together with the polyadenylation signal (AATAAA) at
position +133 of the first 3' acceptor site, more efficient in the
splicing process of exons a and b and overrule further downstream
splicing signals. By comparison, the polyadenylation signal used in the
generation of the poly(A) tail of the 2.6-kb MASP-2 mRNA species,
CATAAA, is rather weak 29 . In in vitro systems, this signal sequence
has been shown to significantly decrease polyadenylation and cleavage
efficiencies 30 , which may engender low levels of mRNA expression due
to impairments in stability 31 . Thus, these structural features
within the MASP-2 gene may be the molecular basis for the observation
that the 1-kb MASP-2-related mRNA species is abundantly expressed,
approximately eightfold more than the 2.6-kb MASP-2 mRNA species. When
analyzing MBL/MASP preparations by protein staining of SDS-PAGE gels
and by Western blotting, it seems that MAp19 is present at a higher
concentration than MASP-2 (not shown), indicating that the relative
amounts of the two mRNA species are also reflected by the relative
abundance of the respective proteins in plasma.
Our data suggest that the major translational product of the MASP-2 gene is a plasma protein, which is devoid of the serine protease domain and consists solely of the N-terminal CUB domain and the EGF-like module with four unique amino acids at the C terminus. Its presence in the MBL-MASP complex implies an architecture of the MBL/MASP complex that is different from the initiation complex of the classical complement activation pathway (C1qC1r2C1s2).
The function of MAp19 remains to be elucidated. A tryptic derivative of C1s consisting mainly of N-terminal regulatory domains was shown to bind to C1q, compete with the binding of native C1s, and thus inhibit hemolysis of Ab-coated erythrocytes 32 . A CUB domain motif present in calreticulin and expressed as a recombinant protein was shown in an in vitro assay to bind to C1q and to MBL 33 . Thus, MAp19 may have a modulating role in the activation of complement via the MBL pathway.
The primary structure of the human gene of MASP-2 presented in this
study indicates that both MASP-2 and MAp19 are encoded by two
differentially processed mRNA species encoded by one single structural
gene. In its genomic organization, the MASP-2 gene is quite distinct
from those of MASP-1 and the classical pathway serine proteases. The
human MASP-1 gene is located on chromosome 3q27-q28 34 . Its size was
estimated at approximately 50 kb. Sequence analysis showed that there
are 16 exons, 6 of which code for the serine protease domain 35, 36, 37 .
Each of the regions coding for the CUB-I, CUB-II, CCP-I, and CCP-II
structural domains of MASP-1 is encoded by two exons each, while the
region coding for the EGF-like domain is encoded by one exon only 37 .
C1r and C1s are closely linked on chromosome 12p13 and thought to have
arisen by gene duplication from a common ancestor 38 . The gene for
C1s spans about 13 kb, comprises 12 exons, and compares with the MASP-1
gene in exon/intron structure 37 , except for the region coding for
the serine protease domain: it is encoded by one exon only, as is also
the case for C1r and haptoglobin 37, 39 . By comparative analysis of
cDNA and genomic DNA amplification products for the coding
sequence of the serine protease domain of human MASP-2, it was
suggested that it is encoded by one exon 37 . This work assigns the
regions coding for the CUB-I domain and the EGF-like domain to one exon
(exon a), the sequence coding for the C terminus of MAp19 to exon b,
that for the N-terminal portion of CUB-II domain to exon c, and assigns
the sequence coding for the C-terminal part of the CUB-II domain, the
two CCP domains, and the serine protease domain to one exon (exon d).
Interestingly, the position of the intron within the region coding for
the CUB-II domain is conserved between the genes for C1s, MASP-1 37 ,
and MASP-2 (Fig. 6). The generation of two functionally distinct gene
products, the structural features of the translational product of the
2.6-kb mRNA species 12 , the exon-intron structure of the gene (see
above), and the localization on human chromosome 1p36.2-3 40 may
imply a distinct branch for MASP-2 on the evolutionary tree of
complement serine proteases.
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Acknowledgments |
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Footnotes |
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2 The expression of an additional mRNA species of 1 kb generated by an alternative splice mechanism from a single structural MASP-2 gene was first presented at the IVth International Workshop on C1 and Collectins, Mainz, Germany, Oct. 3–5, 1997 (C. Stover, N. Lynch, B. Schütz, S. Thiel, J. Jensenius, and W. Schwaeble: A comparative analysis of expression patterns for classical and lectin pathway components in vivo); similar data to those presented in this original publication were presented as a poster at the XVIIth International Complement Workshop, Rhodes, Greece, Oct. 12–16, 1998 (M. Takahashi, M. Matsushita, Y. Endo, and T. Fujita: MBL-MASP complex is associated with a truncated protein derived from MASP-2 gene by alternative RNA processing).
3 Sequence data are available from EMBL/GenBank/DDBJ under the accession numbers: Y18281 (clone phl-5), Y18282 (clone phl-7), Y18283 (clone phl-6), Y18284 (clone phl-8), Y18285 (clone RT-PCR rl-1), Y18286 (clone pgM-2 A), and Y18287 (clone pgM-2 B).
4 Address correspondence and reprint requests to Dr. W. Schwaeble, Department of Microbiology and Immunology, University of Leicester, University Road, Leicester LE1 9HN, U.K. E-mail address:
5 Abbreviations used in this paper: MBL, mannan-binding lectin; CCP, complement control protein; CUB, C1r/C1s/Uegf/bone morphogenetic protein 1; EGF, epidermal growth factor; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; MAp19, MBL-associated plasma protein of 19 kDa; MASP, MBL-associated serine protease; UT region, untranslated region.
Received for publication October 30, 1998. Accepted for publication December 17, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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]
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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]
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D. Iwaki, K. Kanno, M. Takahashi, Y. Endo, N. J. Lynch, W. J. Schwaeble, M. Matsushita, M. Okabe, and T. Fujita Small Mannose-Binding Lectin-Associated Protein Plays a Regulatory Role in the Lectin Complement Pathway J. Immunol., December 15, 2006; 177(12): 8626 - 8632. [Abstract] [Full Text] [PDF]
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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. 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]
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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]
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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]
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 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]
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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]
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F. Larsen, H. O. Madsen, R. B. Sim, C. Koch, and P. Garred Disease-associated Mutations in Human Mannose-binding Lectin Compromise Oligomerization and Activity of the Final Protein J. Biol. Chem., May 14, 2004; 279(20): 21302 - 21311. [Abstract] [Full Text] [PDF]
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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]
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M. M. Estabrook, D. L. Jack, N. J. Klein, and G. A. Jarvis Mannose-Binding Lectin Binds to Two Major Outer Membrane Proteins, Opacity Protein and Porin, of Neisseria meningitidis J. Immunol., March 15, 2004; 172(6): 3784 - 3792. [Abstract] [Full Text] [PDF]
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 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]
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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]
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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]
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 L. Zhao, Y. Ohtaki, K. Yamaguchi, M. Matsushita, T. Fujita, T. Yokochi, H. Takada, and Y. Endo LPS-induced platelet response and rapid shock in mice: contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system Blood, October 16, 2002; 100(9): 3233 - 3239. [Abstract] [Full Text] [PDF]
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O. Neth, D. L. Jack, M. Johnson, N. J. Klein, and M. W. Turner Enhancement of Complement Activation and Opsonophagocytosis by Complexes of Mannose-Binding Lectin with Mannose-Binding Lectin-Associated Serine Protease After Binding to Staphylococcus aureus J. Immunol., October 15, 2002; 169(8): 4430 - 4436. [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]
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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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M. Matsushita, S. Thiel, J. C. Jensenius, I. Terai, and T. Fujita Proteolytic Activities of Two Types of Mannose-Binding Lectin-Associated Serine Protease J. Immunol., September 1, 2000; 165(5): 2637 - 2642. [Abstract] [Full Text] [PDF]
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T. Vorup-Jensen, S. V. Petersen, A. G. Hansen, K. Poulsen, W. Schwaeble, R. B. Sim, K. B. M. Reid, S. J. Davis, S. Thiel, and J. C. Jensenius Distinct Pathways of Mannan-Binding Lectin (MBL)- and C1-Complex Autoactivation Revealed by Reconstitution of MBL with Recombinant MBL-Associated Serine Protease-2 J. Immunol., August 15, 2000; 165(4): 2093 - 2100. [Abstract] [Full Text] [PDF]
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S. Thiel, S. V. Petersen, T. Vorup-Jensen, M. Matsushita, T. Fujita, C. M. Stover, W. J. Schwaeble, and J. C. Jensenius Interaction of C1q and Mannan-Binding Lectin (MBL) with C1r, C1s, MBL-Associated Serine Proteases 1 and 2, and the MBL-Associated Protein MAp19 J. Immunol., July 15, 2000; 165(2): 878 - 887. [Abstract] [Full Text] [PDF]
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M. Matsushita, Y. Endo, and T. Fujita Cutting Edge: Complement-Activating Complex of Ficolin and Mannose-Binding Lectin-Associated Serine Protease J. Immunol., March 1, 2000; 164(5): 2281 - 2284. [Abstract] [Full Text] [PDF]
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C. M. Stover, S. Thiel, N. J. Lynch, and W. J. Schwaeble The Rat and Mouse Homologues of MASP-2 and MAp19, components of the Lectin Activation Pathway of Complement J. Immunol., December 15, 1999; 163(12): 6848 - 6859. [Abstract] [Full Text] [PDF]
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R. Wallis and R. B. Dodd Interaction of Mannose-binding Protein with Associated Serine Proteases. EFFECTS OF NATURALLY OCCURRING MUTATIONS J. Biol. Chem., September 29, 2000; 275(40): 30962 - 30969. [Abstract] [Full Text] [PDF]
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C.-B. Chen and R. Wallis Stoichiometry of Complexes between Mannose-binding Protein and Its Associated Serine Proteases. DEFINING FUNCTIONAL UNITS FOR COMPLEMENT ACTIVATION J. Biol. Chem., July 6, 2001; 276(28): 25894 - 25902. [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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10 and 12 kb. B, A radiolabeled 3'-specific cDNA
subfragment representing the coding sequence for the serine protease
domain hybridizes with a band of 
