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Single Structural GenePathway of Complement Are Encoded by athe Mannan-Binding Lectin Activation Two Constituents of the Initiation Complex of

Wilhelm J. SchwaebleLynch, Thomas Vorup-Jensen, Jens C. Jensenius and Cordula M. Stover, Steffen Thiel, Marcus Thelen, Nicholas J.

http://www.jimmunol.org/content/162/6/34811999; 162:3481-3490; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Two Constituents of the Initiation Complex of theMannan-Binding Lectin Activation Pathway of ComplementAre Encoded by a Single Structural Gene1,2,3

Cordula M. Stover,* Steffen Thiel,† Marcus Thelen,‡ Nicholas J. Lynch,§

Thomas Vorup-Jensen,† Jens C. Jensenius,† and Wilhelm J. Schwaeble4*§

Mannan-binding lectin (MBL) forms a multimolecular complex with at least two MBL-associated serine proteases, MASP-1 andMASP-2. This complex initiates the MBL pathway of complement activation by binding to carbohydrate structures present onbacteria, yeast, and viruses. MASP-1 and MASP-2 are composed of modular structural motifs similar to those of the C1q-associated serine proteases C1r and C1s. Another protein of 19 kDa with the same N-terminal sequence as the 76-kDa MASP-2protein is consistently detected as part of the MBL/MASP complex. In this study, we present the primary structure of this novelMBL-associated plasma protein of 19 kDa, MAp19, and demonstrate that MAp19 and MASP-2 are encoded by two differentmRNA species generated by alternative splicing/polyadenylation from one structural gene.The Journal of Immunology,1999,162: 3481–3490.

T he complement system may be activated via three differ-ent routes: the classical pathway is initiated by binding ofthe multimolecular C1 complex to Ag-Ab complexes, the

alternative pathway is initiated by certain structures on microor-ganisms, and the mannan-binding lectin (MBL)5 pathway is initi-ated by the binding of MBL to carbohydrates, e.g., on the surfaceof microorganisms.

The discovery of the MBL pathway began with the descriptionof a bactericidal factor in mouse plasma, Ra-reactive factor, which

was shown to activate complement upon binding to carbohydratesin Ra LPSs on Gram-negative bacteria (1). The molecule involvedin the recognition of carbohydrates by Ra-reactive factor in humanand mouse plasma was identified as plasma mannan-binding lectin(MBL) (2, 3). MBL-mediated activation leads to cleavage of thefourth and the second component of complement (C4 and C2, re-spectively) and subsequently to the formation of the C3 convertaseC4b2a (4, 5). In vitro experiments indicated that MBL may forma complex with C1r2C1s2 (6, 7).

In vivo, however, MBL associates with serine proteases sim-ilar to but distinct from C1r and C1s, termed MASP-1 (8 –11)and MASP-2 (12). Like the serine proteases C1r and C1s of theclassical activation pathway, MASP-1 and MASP-2 are com-posed of an N-terminal CUB domain (13), followed by an epi-dermal growth factor (EGF)-like domain, a second CUB do-main, two complement control protein (CCP) domains, and aserine protease domain. Upon activation, MASP-2 is cleavedinto two disulfide-linked chains, an N-terminal A chain com-posed of the first five domains and a C-terminal, catalyticallyactive B chain. As yet, very little is known about the stoichi-ometry, the composition, and activation sequence of the MBL/MASP complex. Previous reports indicated that MASP-1cleaves C2, C3, and C4 (5, 14). However, with the discovery ofthe second MBL-associated serine protease, it became evidentthat at least the cleavage of C4 is mediated by MASP-2 (12).Another constituent of the MBL/MASP complex, a plasma pro-tein of approximately 19 kDa with an N-terminal sequenceidentical 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 translationalproduct of an additional, abundantly expressed mRNA transcriptof 1 kb, processed together with the 2.6-kb MASP-2 mRNA fromthe same heterologous nuclear RNA transcript of a single struc-tural gene. MAp19 would be expected to be enzymatically inactiveas it lacks the serine protease domain. Its presence in the MBL/MASP complex implies a possible regulatory role and emphasizesthat the composition of the MBL pathway activation complex isdifferent from that of the classical pathway.

*Department of Microbiology and Immunology, University of Leicester, Leicester,United Kingdom;†Department of Medical Microbiology and Immunology, Univer-sity of Aarhus, Aarhus, Denmark;‡Theodor-Kocher-Institute, University of Bern,Bern, Switzerland; and§Institute for Anatomy and Cell Biology, University of Mar-burg, Marburg, Germany

Received for publication October 30, 1998. Accepted for publication December17, 1998.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by the Wellcome Trust, Deutsche Forschungsgemein-schaft SFB 297, the Danish Medical Research Council, and the Deutscher Akade-mischer Austauschdienst.2 The expression of an additional mRNA species of 1 kb generated by an alternativesplice mechanism from a single structural MASP-2 gene was first presented at theIVth International Workshop on C1 and Collectins, Mainz, Germany, Oct. 3–5, 1997(C. Stover, N. Lynch, B. Schutz, S. Thiel, J. Jensenius, and W. Schwaeble: A com-parative analysis of expression patterns for classical and lectin pathway componentsin vivo); similar data to those presented in this original publication were presented asa poster at the XVIIth International Complement Workshop, Rhodes, Greece, Oct.12–16, 1998 (M. Takahashi, M. Matsushita, Y. Endo, and T. Fujita: MBL-MASPcomplex is associated with a truncated protein derived from MASP-2 gene by alter-native RNA processing).3 Sequence data are available from EMBL/GenBank/DDBJ under the accession num-bers: 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 ofMicrobiology and Immunology, University of Leicester, University Road, LeicesterLE1 9HN, U.K. E-mail address: ws5@le.ac.uk5 Abbreviations used in this paper: MBL, mannan-binding lectin; CCP, complementcontrol protein; CUB, C1r/C1s/Uegf/bone morphogenetic protein 1; EGF, epidermalgrowth factor; MALDI-TOF, matrix-assisted laser desorption ionization-time offlight; MAp19, MBL-associated plasma protein of 19 kDa; MASP, MBL-associatedserine protease; UT region, untranslated region.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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Materials and MethodsMaterials

Restriction enzymes were purchased from Boehringer Mannheim (Mann-heim, Germany). The human liver cDNA library was purchased from Strat-agene (Cambridge, U.K.). Poly(A)Tract mRNA Isolation System IV wasfrom Promega (Madison, WI). RT-PCR kit (Superscript preamplificationsystem) was from Life Technologies (Paisley, U.K.). [a-32P]dCTP andblotting membranes were from Amersham-Pharmacia (Upsala, Sweden).PCRII vector and prokaryotic expression vector pTrxFus were purchasedfrom Invitrogen (Leek, The Netherlands). A kit (Easy DNA kit) to purifygenomic DNA was purchased from Invitrogen.

Isolation of MASP-2-related transcripts from a human livercDNA library

A 59-specificSmaI subfragment (450 bp) of clone phl-4 (12) was used toscreen approximately 107 plaques of a human liver oligo(dT)-primedcDNA library cloned in the phage vectorl ZAP (Stratagene). After rescueof the plasmid pBluescript SK2 by in vivo excision, each of the eightclones isolated was characterized by Southern blot hybridization followingrestriction analysis. Four of these 59-specific transcripts (phl-5, phl-6,phl-7, phl-8; 0.7–1.2 kb) were sequenced on both strands using the dideoxychain termination method of Sanger (T7 Sequencing Kit; Amersham-Phar-macia). A characteristic restriction site forSmaI was used to generate sub-clones in pBluescript KS1 for sequencing using reverse and universe M13primers.

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 Northernblot analysis, rat liver RNA was reverse transcribed using the Superscriptpreamplification system (Life Technologies). The obtained oligo(dT)-primed cDNA was used in cyclic amplification with randomized primersderived from the human MASP-2 amino acid sequences ATLCGQES (po-sition: 72–79 of the mature protein, 12) and TGWKIHYT (position: 272–279). A standard PCR program was used (95°C, 5 min; 35 cycles, dena-turation at 95°C for 30 s, annealing at 50°C for 1 min, and extension at72°C for 1 min; final extension step at 72°C for 10 min). A 623-bp productwas obtained, subcloned in PCRII (TA cloning kit; Invitrogen), and se-quenced on both strands. The nucleotide sequence of RT-PCR rl-1 revealed

an overall identity of 81.1% with the corresponding human MASP-2cDNA; the two deduced translational products show 80.2% identity.

Northern blot analysis

Total RNA was extracted from human, mouse, rat, and guinea pig livertissues according to standard protocols (15), and mRNA was purified usingPoly(A)Tract mRNA Isolation System IV (Promega). Approximately 2mgof poly(A)1 RNA was separated per lane on a denaturing 0.8% (w/v)agarose gel and transferred to Hybond N membrane (Amersham-Pharma-cia). The blots were hybridized according to standard protocols (16) withrandom primed32P-dCTP-labeled (Random Primed Labeling Kit; Boehr-inger Mannheim) cDNA probes generated from our full-length humanMASP-2 cDNA transcript phl-4, the full-length cDNA transcript of theMASP-2-related mRNA species of 1 kb, phl-5, and the partial rat-specificMASP-2 cDNA clone RT-PCR rl-1.

Prokaryotic expression of rMASP-2

rMASP-2 was expressed inEscherichia coliusing the Thiofusion Expres-sion System (Invitrogen). phl-4 cDNA was used as a template for cyclicamplification (for primers, see Table I). The expected 1989-bp PCR prod-uct was obtained, subcloned in PCRII (Invitrogen), excised withBamHI/XhoI, then cloned in-frame into pTrxFus (Invitrogen) linearized withBamHI andSalI. The construct was confirmed by restriction mapping andsequencing. Expression and purification were performed according to themanufacturer’s protocol.

Detection of MAp19 in the MBL/MASP complex

N-acetyl-glucosamine was coupled to TSK-75 beads (Merck, Darmstadt,Germany) following the procedures of Fornstedt and Porath (17). Plasmawas isolated by drawing blood in a vial containing heparin (final concen-tration, 25 mM), iodoacetamide (5 mM), cyclocaprone (20 mM) (Amer-sham-Pharmacia), leupeptin (2 mg/ml) (Boehringer Mannheim), pepstatinA (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) (Sig-ma-Aldrich), and 5 mM CaCl2. The plasma was then diluted in an equalvolume 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 cy-clocaprone, 0.2 mg/ml leupeptin, 0.4 mg/ml pepstatin A, 10 mM benza-midine, 1 mM o-phenanthroline, and 2 mM CaCl2) and passed through

Table I. Primers and oligonucleotides used in this study

Nucleotide position Sequence

Primers derived from cDNA clone phl-4a

65-84 bp (mature start) 59-GGG ATC CCT TAG GCC CGA AGT GGC C-39(sense,BamHI modified)

2030-2053 bp (ser. prot.domain) 59-CTC GAG ATC CAG GGA ATA TAG TTA ATA AC-39(antisense,XhoI modified)

Primers derived from cDNA clone phl-4b

1372-1390 (serine prot.domain) 59-ACC TGG TGA TTT TCC TTG G-39 (sense)2278-2296 (39UT clone phl-4) 59-GTC ACT CTC GTG GTT TAT G-39 (antisense)

Primers derived from cDNA clone phl-4c

2-16 (59UT) 59-CTGGACGGGCACACC-39(sense)529-549 (EGF-like domain) 59-CG CTT GTT ACG GTG CAG GAC G-39 (antisense)461-481 (EGF-like domain) 59-GCG CCC ACC TGC GAC CAC CAC-39 (sense)846-868 (CUB II) 59-TCC TGA TTC ATC TGT GAC AAA GG-39 (antisense)846-868 (CUB II) 59-CCT TTG TCA CAG ATG AAT CAG GA-39 (sense)1063-1083 (CCP I) 59-GAA AGA TGG ATC TTG GGA CCG-39 (sense)1243-1263 (CCP I-II) 59-CCA TCA GCC TCA CAC ACA TAT-39 (antisense)1213-1233 (CCP II) 59-TTC ACT TTC ATT GTG TAG AAG-39 (antisense)1255-1275 (CCP II) 59-GGC TGA TGG ATT CTG GAC GAG-39 (sense)1372-1390 (serine prot.domain) 59-ACC TGG TGA TTT TCC TTG G-39 (sense)2278-2296 (39UT clone phl-4) 59-GTC ACT CTC GTG GTT TAT G-39 (antisense)

Primers derived from cDNA clone phl-5c

621-644 (39UT clone phl-5) 59-GGA GCT GAG GCC AGC AGG CTG GCT-39 (antisense)Antisense primer specific for the intron separating exons a and bc

59-cca ggc cct cac ccc tgg-3 9

a Oligonucleotides used for cyclic amplification of human MASP-2 cDNA to generate prokaryotic expression constructs.b Oligonucleotides used for cyclic amplification of human MASP-2 cDNA to generate a specific probe representing the

coding region for the serine protease domain and the 39 UT region of the 2.6-kb MASP-2 mRNA species.c Oligonucleotides used for cyclic amplification of human genomic DNA from two donors.

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N-acetyl-glucosamine-TSK beads. The beads were washed with buffer A,and calcium-dependent proteins, including the MBL/MASP complex, wereeluted 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 removeenzyme inhibitors, the MASPs in the MBL/MASP preparation were par-tially activated by incubation with mannan (18) for 2 h at37°C. The prep-aration was analyzed using reduced (with DTT) or nonreduced samples bySDS-PAGE and Western blotting using a 4–20% (w/v) gradient gel, fol-lowed by blotting onto a polyvinylidene difluoride membrane (Hybond-P;Amersham-Pharmacia), as described (19). After blotting, the membraneswere 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 an-tiserum 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 rabbitswith rMASP-2 (S. Petersen et al., unpublished data) expressed inE. coli usingthe ThioFusion Expression System (Invitrogen), as described above. After ex-posure to the polyclonal MASP-2 antiserum for 2 h at room temperature, themembranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (DAKOpatts, Glostrup, Denmark), followed by addition of en-hanced chemoluminescence (ECL) substrate (Pierce, Rockford, IL), and thenexposed 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 MBLpreparation from Statens Serum Institute, Copenhagen, Denmark. In thepresence 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 500mg MBL was fractionated by SDS-PAGE ona 4-20% gradient gel. The gel was stained with Coomassie blue, and theband corresponding to MAp19 excised. The gel slice was decolored with30% (w/v) CH3CN/50 mM NH4HCO3, washed with water, and dried in aspeed vac. The gel was then incubated in 100 mM DTT and heated to 55°Cfor 30 min and washed with water. Free sulphydryl groups were alkylatedwith 250 mM iodoacetamide for 30 min at room temperature. After wash-ing with water, the gel was dried in a speed vac. In-gel digestion withendopeptidase Lys-C or trypsin and matrix-assisted laser desorption ion-ization-time of flight mass spectrometry (MALDI-TOF) were performed asdescribed (20). Alternatively, tandem mass spectrometry (MS/MS) wasemployed.

Internal calibration of MALDI-TOF measurements was accomplishedby adding desArg-1 bradykinin (Mass 904.4681) (Sigma-Aldrich) and ad-renocorticotropic hormone fragment 18-39 (Mass 2465.1989) (Sigma-Al-drich) to the samples. For the protein-fragment search, monoisotopic pep-tide 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 usingEasy DNA Kit (Invitrogen), was digested overnight withEcoRI, separatedon a 0.8% (w/v) agarose gel, and alkali blotted to Hybond N1 nitrocellu-lose membrane (Amersham-Pharmacia). MASP-2 cDNA transcript phl-4(EcoRI/KpnI excised) and an amplification product representing the codingregion for the serine protease domain and the 39-untranslated (UT) regionof the 2.6-kb mRNA species (EcoRI excised from PCRII; for oligonucle-otides, see Table I) were used as probes. Further analysis of the genestructure for MASP-2 was performed by cyclic amplification of genomicDNA from two donors using oligonucleotides derived from the cDNAsequence of human MASP-2 clone phl-4 (12) and of clone phl-5 (see TableI). PCR amplification was confirmed by Southern blot analysis usingcDNA clone phl-4, and hybridizing fragments were subcloned into PCRIIvector (Invitrogen) and sequenced on both strands (T7 Sequencing Kit;Amersham-Pharmacia). In addition, the 2.8-kb genomic amplificationproducts, obtained from each of the two genomic DNA preparations, wereexcised from PCRII byEcoRI andPstI restriction digest, subcloned inpBluescript KS1, and sequenced on both strands.

ResultsCharacterization of a novel MASP-2-related mRNA transcript

Eight cDNA clones representing a novel MASP-2-related mRNAspecies of approximately 1 kb were isolated from a human livercDNA library. After restriction analysis, three of these (clonesphl-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 of555 bp, preceded by a 59 UT sequence identical to the 59 UT regionof the 2.6-kb MASP-2 mRNA species (clone phl-4 (12); Fig. 1,Aand B). Clone phl-7 (684 bp) is a partial transcript of the samemRNA that gives rise to phl-5 and phl-6. This novel mRNA spe-cies shares complete identity over 540 bp with the 59 coding se-quence of the 2.6-kb MASP-2 mRNA (Fig. 1B). This stretch ofidentity is followed by a coding sequence of 12 nucleotides, andthe open reading frame terminates with a stop codon (TAG) 553 bpdownstream of the translation initiation codon (ATG). The 39 UTregion differs from the 39 UT region of the 2.6-kb MASP-2 mRNAspecies (Fig. 1B). This novel mRNA species therefore codes forthe same signal peptide as determined for MASP-2 and two of thesix structural motifs of MASP-2, the N-terminal CUB domain andthe EGF-like domain (Fig. 1C). The deduced translation product ofthis mRNA has a unique C-terminal sequence of four amino acids(EQSL) that are not contained in MASP-2 (Fig. 1C). The calcu-lated molecular mass of the deduced amino acid sequence of themature translation product is 19,075 Da.

The presence of an additional, MASP-2-related mRNA speciesis supported by Northern blot results. As shown in Fig. 2A, twohybridization signals (one at 2.6 kb, one at 1 kb) were detectedconsistently when probing human liver mRNA with the full-lengthcDNA transcripts, represented by clones phl-4 and phl-5 (see Figs.1A and 2A). A cDNA probe specific for the sequence encoding theserine protease domain and the 39 UT region (see above) hybrid-izes to the 2.6-kb mRNA species only (not shown). The 1-kbMASP-2-related mRNA species is more abundant than the 2.6-kbmRNA species (Fig. 2A). In addition, rat-specific MASP-2 cDNA(RT-PCR rl-1), representing the coding sequence for the N-termi-nal CUB domain, the EGF-like domain, and the second CUB do-main, was also probed on mRNA preparations of rat, mouse, andguinea pig liver tissue. As depicted in Fig. 2B, the presence of bothMASP-2 and a more abundant 1-kb mRNA transcript was detectedconsistently, showing that the coexpression of both mRNA speciesis conserved.

Characterization of MAp19

Analysis of two different plasma proteins with MASP-2 immuno-reactivity was performed by SDS-PAGE and Western blotting.The purified MBL-MASP complex was dialyzed to remove pro-tease inhibitors and then incubated in presence of mannan to obtainapproximately 50% cleavage of MASP-2 contained in the prepa-ration (seeMaterials and Methods). When this preparation wasanalyzed in its nonreduced form, the Ab stained three bands, rep-resenting uncleaved (nonactivated) MASP-2 (at 76 kDa), a slightlysmaller and less abundant band (the disulfide-linked A and Bchains of cleaved MASP-2), and a small MBL-associated proteinat 17 kDa (Fig. 3A,lane N). When analyzed after reduction, sev-eral bands are seen (Fig. 3A, lane R). The upper 76-kDa bandrepresents nonactivated MASP-2, in which no cleavage of thepolypeptide chain has occurred. The band at 52 kDa represents theA chain of cleaved MASP-2, whereas the band at 31 kDa repre-sents the B chain of MASP-2, and the lower band at 19 kDa rep-resents MAp19.

To determine the origin of the above-described 19-kDa MASP-2-related protein, plasma MAp19 was purified by SDS-PAGE(Fig. 3B) and subjected to mass spectrometry analysis. PurifiedMAp19 was in-gel digested with endopeptidase Lys-C or trypsin,and the petides were analyzed by MALDI-TOF or by tandem massspectrometry (MS/MS). Hereby, nearly complete coverage (96%)of the deduced amino acid sequence encoded by the open readingframe of the 1-kb MASP-2-related mRNA species was obtained

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FIGURE 1. Primary structure of theMASP-2-related mRNA species of 1 kb andits deduced translational product in compar-ison with the 2.6-kb MASP-2 mRNA and itsdeduced translational product.A, Schematicalignment of three cDNA transcripts (phl-5,phl-6, phl-7) that represent the novel 1-kbMASP-2-related mRNA species isolatedfrom a human liver cDNA library. The in-dicated restriction sites were used to estab-lish subclones for sequence analysis. Thecomparison includes phl-4, the previouslydescribed MASP-2 mRNA species of 2.6 kb(12). These two species share the same 59UT region and differ in their 39 UT regions.B, Nucleotide sequences of phl-5, phl-6, andphl-7. A comparison between the 1-kbMASP-2-related and the 2.6-kb MASP-2mRNA species is given by aligning phl-5,phl-6, and phl-7 with the previously pub-lished primary structure of phl-4 (12). Se-quence identity is indicated by dots. The de-rived amino acid sequences are givenbeneath. At the 39end, the 1-kb mRNA spe-cies terminates with five triplets not con-tained in the 2.6-kb mRNA species. Aster-isks mark the signal peptide, and numbersindicate amino acid positions from the startof the mature protein (12). Polyadenylationinitiation signals within the distinct 39 UTregions of these two mRNA species are un-derlined (aataaa, cataaa).C, Schematic rep-resentation of the modular composition ofthe deduced translational products of the2.6-kb MASP-2 mRNA species (12) and the1-kb MASP-2-related mRNA species. Atthe C terminus of the latter there are fourunique amino acids, glutamic acid, glu-tamine, serine, and leucine.

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(Table II). The identification of the unique carboxyl-terminal se-quence (EQSL) in plasma MAp19 finally revealed that this proteinis the translational product of the 1-kb MASP-2-related mRNAspecies.

Organization of the human gene encoding MASP-2 and MAp19

The Southern blot analysis shown in Fig. 4A revealed two bands of10 and 12 kb, which hybridized with the cDNA probe phl-4 (rep-resenting the entire MASP-2 2.6-kb mRNA), whereas only the10-kb band was detected when the same filter was hybridized witha subfragment representing the coding sequence for the serine pro-tease domain (Fig. 4B). This provided evidence for the existence ofonly one structural MASP-2 gene, with exons being spread over adistance of less than 22 kb.

FIGURE 2. Northern blot analysis of liver mRNA using human- andrat-specific cDNA probes for MASP-2.A, Approximately 2 mg ofpoly(A)1-selected RNA of human liver (lane 1) was hybridized with ra-diolabeled phl-4, showing both the 1-kb MASP-2-related and 2.6-kbMASP-2 mRNA species.B, Approximately 2mg of poly(A)1-selectedRNA of guinea pig liver (lane 1), mouse liver (lane 2), and rat liver (lane3) was hybridized with radiolabeled RT-PCR rl-1, showing both the 1-kbMASP-2-related and the 2.6-kb MASP-2 mRNA species. Additional bandsat approximately 2 and 3 kb are observed in mouse and guinea pig RNApreparations (lanes 1and2) and may be alternatively polyadenylated tran-scripts for MASP-2 and/or incompletely spliced intermediary products ofheteronuclear MASP-2 RNA.

FIGURE 3. Detection of MASP-2 and MAp19 in a MBL/MASP prep-aration and purification of MAp19.A, Western blot analysis of a humanMBL/MASP preparation using rabbit anti-MASP-2 antiserum. The MBL/MASP preparation was separated by SDS-PAGE under nonreducing (N)and reducing (R) conditions. Indicated are the positions of uncleaved (non-activated) MASP-2, the A chain of MASP-2, the B chain of MASP-2, andMAp19. The m.w. of marker proteins are given on theleft. B, SDS-PAGEanalysis and Western blotting of purified MAp19. A fraction of the MBL/MASP preparation was separated on a 15% SDS-PAGE under reducingconditions and stained with Coomassie blue or blotted and stained usingpolyclonal anti-MASP-2 Ab. Indicated is the position of the band excisedfor MALDI-TOF analysis.

Table II. Sequence of plasma MAp19 as determined by tandem mass spectrometry (MS/MS) andMALDI-TOF

Position PeptideMALDI-TOFa

(endopeptidase Lys-C digested)MS/MS

(trypsin digested)

1 1–6 TPLGPK 1 12 7–14 WPEPVFGR 1 13 15–30 LASPGFPGEYANDQER 2 14 31 R 2 25 32–41 WTLTAPPGYR 2 16 42–43 LR 2 27 44–63 LYFTHFDLELSHLCEYDFVK 1 18 64–69 LSSGAK 1 29 70–84 VLATLCGQESTDTER 2 1

10 85–88 APGK 2 211 89–103 DTFYSLGSSLDITFR 2 112 104–110 SDYSNEK 1 213 111–162 PFTGFEAFYAAEDIDECQ

VAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNK 1 2

14 163–170 RTCSEQSL 1 2

a Accuracy. 0.006%.

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The exon-intron boundaries of this gene were characterized bynucleotide sequence analysis of overlapping regions of genomicDNA isolated by PCR amplification using MASP-2-specific oli-

gonucleotide primers (Table I; Fig. 5,B and C). The genomicsequence obtained comprises 4464 nucleotides (pgM-2 A; pgM-2B, 4465 nucleotides) and starts with the exon for the 59 UT regionshared by both types of mRNA species and spans up to the exonfor the 39UT region of the 2.6-kb MASP-2 mRNA species (seeFigs. 5Aand 6). The sequences obtained from the genomic DNAof two different donors were in full agreement with the cDNAsequences of the previously published MASP-2 cDNA clone(phl-4, 12) and the cDNA sequences obtained from the 1-kbMASP-2-related mRNA species shown in this study. Polymorphicsequences were obtained for the sequence representing the 39 UTregions of the 1- and 2.6-kb mRNA species, respectively, and arelisted in Table III. The 59 UT region and the coding sequence forthe signal peptide, the N-terminal CUB domain (CUB-I), and theEGF-like domain of MASP-2 are located on one single exon (exona), separated by an intron sequence of 443 bp from an exon con-taining the coding sequence for the unique C-terminal sequenceglutamic acid, glutamine, serine, and leucine (EQSL), the stopcodon (TAG), and the 39 UT sequence (including the polyadenyl-ation initiation signal AATAAA) of the 1-kb MASP-2 mRNA spe-cies (exon b; see Fig. 6). Approximately 1.3 kb further downstreamfollows an exon encoding the N-terminal 65 amino acids of thesecond CUB domain (CUB-II) (exon c). This exon is separated byan intron sequence of 315 bp from the coding sequences for theC-terminal portion of CUB-II, the two CCP domains, and theserine protease domain as well as the 39 UT region of the 2.6-kbMASP-2 mRNA species (exon d). As illustrated in Fig. 5D, clas-sical splice sites (according to the GTAG rule, including pyrimi-dine-rich tracts and branchpoint sequences located 59 thereof;Refs. 21–24; Fig. 6, Table IV) allow joining of exon a to exon b.

FIGURE 4. Southern blot analysis ofEcoRI-digested human genomicDNA. A, Radiolabeled phl-4 hybridizes with two bands of;10 and 12 kb.B, A radiolabeled 39-specific cDNA subfragment representing the codingsequence for the serine protease domain hybridizes with a band of;12 kbonly.

FIGURE 5. Primary structure of the human MASP-2 gene.A, Schematic presentation of the split gene organization of the human MASP-2 gene. It iscomposed of four exons.B, Genomic fragments were amplified by PCR from genomic human DNA of two donors using MASP-2-specific oligonucleotides.The location of amplification products is schematically presented; the numbers indicate the nucleotide positions in the sequence of cDNA clones phl-4 andphl-5, to which the primers anneal (see Table I).C, Sequencing strategy of genomic MASP-2 after subcloning of amplification products and restrictionfragments thereof. Restriction map of genomic human MASP-2. Note the presence of aEcoRI restriction site in the intron separating exon b from exonc, consistent with hybridization results usingEcoRI-digested genomic DNA (Fig. 4).D, Schematic presentation of posttranscriptional constitutive splicingand alternative splicing/polyadenylation of the human MASP-2 gene. Splicing sites are indicated as well as polyadenylation initiation signals.

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Usage of the polyadenylation initiation signal in exon b results inthe mRNA transcript of 1 kb. Alternatively, ligation of the 59splice site of exon a with the 39 splice site of exon c results in theloss of the alternative splice exon and leads to the mRNA tran-script of 2.6 kb. Furthermore, the obtained primary structure of thehuman MASP-2 gene redefines the C-terminal boundary of theEGF-like domain (see Fig. 6).

Interestingly, one of the cDNA transcripts (clone phl-8, 1187bp) characterized retained the complete sequence (443 bp) for thedesignated intron separating exon a and exon b (see Figs. 5A and6). We regard it as more than likely that this clone is an artifactresulting from incomplete processing (see below), as we were un-able to detect a corresponding mRNA signal by Northern blot anal-ysis using a probe (an internalSmaI/Sau3A subfragment of 386

FIGURE 6. Primary structure of the human MASP-2 gene (pgM-2 A) that gives rise to two plasma proteins, MASP-2 and MAp19 (numbering:bp position). Intron sequences are in lower case; nucleotide sequences of the transcribed regions are shown in upper case.gt, ag, in-framedinucleotide consensus splice sites; 59, 39 splice junctions, branchpoint sequence, and polypyrimidine tracts are underlined.AATAAA, CATAAA arepolyadenylation signals used in the transcription of MASP-2 and MAp19 mRNA.A, Poly(A) addition site of MAP19 mRNA. Signal peptide:positions 16 – 60.

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bp) specific for the divergent sequence contained in phl-8 (data notshown).

DiscussionIn our original publication on MASP-2 (12), we noticed the pres-ence of a protein of approximately 20 kDa (now termed MAp19)with the same N-terminal sequence as MASP-2. With the devel-opment of purification methods that prevent activation of theMBL/MASP complex, we have been able to study the appearanceof MASP-2 and MAp19 in their activated and nonactivated forms.Upon activation, MASP-2 is cleaved at the beginning of the serineprotease domain (12). This domain is attached to the remainder ofthe molecule by a disulfide bond. The mobility of MAp19 on SDS-PAGE is unaffected by activation of the MBL/MASP complex.MALDI-TOF analysis of purified plasma MAp19 revealed identityof MAp19 with the translational product encoded by the 1-kbmRNA transcript of the MASP-2 gene. The association of MAp19with the MBL/MASP complex has also been shown by employingAbs toward the different components of the complex (S. Thiel,S. V. Petersen, T. Vorup-Jensen, M. Matsushita, T. Fujita, C. Sto-ver, W. Schwaeble, and J. C. Jensenius, manuscript in prepara-tion). Thus, it was found, for example, that anti-MBL or anti-MASP-1 Abs will also bind MAp19, as part of a complex withMBL and/or MASP-1.

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 encodedby one structural gene. The generation of two mRNA species lead-ing to two different gene products appears not to be restricted tohuman 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 inwhich the presence of an abundant, small MASP-2 gene product isconserved implies a physiologic requirement for MAp19 and

MAp19 homologues. Likewise, the conservation of the genomicmechanism that generates the small MASP-2 gene product is sup-ported by results of Southern blot analyses of rat genomic DNAindicating that the MASP-2 gene in the rat is similarly organizedas the human gene described in this work (C. Stover, S. Thiel, N.Lynch, and W. Schwaeble, manuscript in preparation), thus favor-ing the view of evolutionary stability (25).

Splice junction sequences in the human MASP-2 gene suggestthat the generation of two mRNA transcripts encoding two differ-ent proteins by the same single structural gene is due to an alter-native splicing/polyadenylation mechanism. Yet, as neither the 59nor the 39 splice sites match the consensus sequences (26), thesesites may be suboptimal for recognition by the nuclear spliceo-some (27). Thus, it is possible that the intron separating exon afrom exon b is retained and that this unspliced transcript is trans-ported to the cytoplasm. Indeed, isolation of cDNA clone phl-8showed the presence of such an intermediate transcript. However,splice sites may be strengthened by certain nucleotides surround-ing the splice junctions: these criteria of primary structure arefound in the human MASP-2 pre-mRNA and comprise cytosines atpositions29, 114, 115 of the first 59 splice site, a branchpointsequence at position272 of the first 39 acceptor site, and poly-pyrimidine 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, downstreamof the respective 39splice junctions. Similarly, the high content ofcytidine nucleotides in exon b may qualify certain stretches asoligo (C) tracts, which were shown by others to support splicing(28). Taken together, it appears possible that additive strengthenersof splice sites concentrate around the first 59and 39splice junctionsthat might make these, together with the polyadenylation signal(AATAAA) at position 1133 of the first 39 acceptor site, moreefficient in the splicing process of exons a and b and overrulefurther downstream splicing signals. By comparison, the polyad-enylation signal used in the generation of the poly(A) tail of the2.6-kb MASP-2 mRNA species, CATAAA, is rather weak (29). Inin vitro systems, this signal sequence has been shown to signifi-cantly decrease polyadenylation and cleavage efficiencies (30),which may engender low levels of mRNA expression due to im-pairments in stability (31). Thus, these structural features withinthe MASP-2 gene may be the molecular basis for the observationthat the 1-kb MASP-2-related mRNA species is abundantly ex-pressed, approximately eightfold more than the 2.6-kb MASP-2mRNA species. When analyzing MBL/MASP preparations by pro-tein staining of SDS-PAGE gels and by Western blotting, it seemsthat MAp19 is present at a higher concentration than MASP-2 (notshown), indicating that the relative amounts of the two mRNAspecies are also reflected by the relative abundance of the respec-tive proteins in plasma.

Table IV. Exon/intron boundaries of the split human MASP-2 genea

Splice Donor Splice AcceptorCodonPhase

Exon a: TCA Ggtgagg Exon b: tctctcttccagAG CA IExon a: TCA Ggtgagg Exon c: cccttccctccttccagCC CT IExon c: C AAGgtctgg Exon d: cttctttctcag ATT C 0

a The nucleotides surrounding the splice donor and acceptor sites for each of thethree introns are indicated. The sequences conforming to the gt/ag rule (21) are in boldletters. Exons are shown in uppercase and introns are shown in lowercase letters.Codon phase refers to the location of the intron within the codon. The pyrimidine-richtracts preceding the 39 splice sites are underlined. While exons c and d are constitu-tively spliced, exon a, b and a,c, respectively, are alternatively spliced (same codonphase) and mutually exclusive (Fig 5D).

Table III. Polymorphic nucleotide sequences in exons b and d of the human MASP-2 gene(pgM-2 A/pgM-2 B)a

pgM-2 A, pos. 1034: CGGCCTG pgM-2 B: CGGCTG (deletion)pgM-2 A, pos. 1047: CAGTCA pgM-2 B: CAGGTCA (insertion)pgM-2 A, pos. 1068: TGCCTGCT pgM-2 B: TGCTGGCCT (deletion/insertions)pgM-2 A, pos. 1097: TGGCTGTGCCCA pgM-2 B: TGCTGTGCCCCA (deletion/insertion)pgM-2 A, pos. 4360: CCACCC pgM-2 B: CCGCCC (transition)

a The differences are confined to the exons containing the 39 UT sequences of the 1.0 kb and 2.6 kb mRNA transcripts(Fig. 6).

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Our data suggest that the major translational product of theMASP-2 gene is a plasma protein, which is devoid of the serineprotease domain and consists solely of the N-terminal CUB do-main and the EGF-like module with four unique amino acids at theC terminus. Its presence in the MBL-MASP complex implies anarchitecture of the MBL/MASP complex that is different from theinitiation complex of the classical complement activation pathway(C1qC1r2C1s2).

The function of MAp19 remains to be elucidated. A trypticderivative of C1s consisting mainly of N-terminal regulatorydomains was shown to bind to C1q, compete with the bindingof native C1s, and thus inhibit hemolysis of Ab-coated eryth-rocytes (32). A CUB domain motif present in calreticulin andexpressed as a recombinant protein was shown in an in vitroassay to bind to C1q and to MBL (33). Thus, MAp19 may havea modulating role in the activation of complement via the MBLpathway.

The primary structure of the human gene of MASP-2 presentedin this study indicates that both MASP-2 and MAp19 are encodedby two differentially processed mRNA species encoded by onesingle structural gene. In its genomic organization, the MASP-2gene is quite distinct from those of MASP-1 and the classical path-way serine proteases. The human MASP-1 gene is located on chro-mosome 3q27-q28 (34). Its size was estimated at approximately 50kb. Sequence analysis showed that there are 16 exons, 6 of whichcode for the serine protease domain (35–37). Each of the regionscoding for the CUB-I, CUB-II, CCP-I, and CCP-II structural do-mains of MASP-1 is encoded by two exons each, while the regioncoding for the EGF-like domain is encoded by one exon only (37).C1r and C1s are closely linked on chromosome 12p13 and thoughtto have arisen by gene duplication from a common ancestor (38).The gene for C1s spans about 13 kb, comprises 12 exons, andcompares with the MASP-1 gene in exon/intron structure (37),except for the region coding for the serine protease domain: it isencoded by one exon only, as is also the case for C1r and hapto-globin (37, 39). By comparative analysis of cDNA and genomicDNA amplification products for the coding sequence of theserine protease domain of human MASP-2, it was suggestedthat it is encoded by one exon (37). This work assigns the re-gions coding for the CUB-I domain and the EGF-like domain toone exon (exon a), the sequence coding for the C terminus ofMAp19 to exon b, that for the N-terminal portion of CUB-IIdomain to exon c, and assigns the sequence coding for the C-terminal part of the CUB-II domain, the two CCP domains, andthe serine protease domain to one exon (exon d). Interestingly,the position of the intron within the region coding for theCUB-II domain is conserved between the genes for C1s,MASP-1 (37), and MASP-2 (Fig. 6). The generation of twofunctionally distinct gene products, the structural features of thetranslational product of the 2.6-kb mRNA species (12), theexon-intron structure of the gene (see above), and the localiza-tion on human chromosome 1p36.2-3 (40) may imply a distinctbranch for MASP-2 on the evolutionary tree of complementserine proteases.

AcknowledgmentsWe thank Dr. Keith Whaley for his continuous support, and Drs. KennethB. M. Reid and Robert B. Sim for helpful discussions. The human liverspecimen used for preparation of RNA was gratefully received from TheInternational Institute for the Advancement of Medicine (Director: Mr. R.Anderson), University of Leicester (Leicester, U.K.).

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