International Immunology

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International Immunology, Vol. 11, No. 5, 859-863, May 1999
© 1999 Japanese Society for Immunology

A truncated form of mannose-binding lectin-associated serine protease (MASP)-2 expressed by alternative polyadenylation is a component of the lectin complement pathway

Minoru Takahashi, Yuichi Endo, Teizo Fujita and Misao Matsushita

Department of Biochemistry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan

Correspondence to: M. Matsushita

	Abstract

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The lectin complement pathway is initiated by binding of mannose-binding lectin (MBL) and MBL-associated serine protease (MASP) to carbohydrates. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4, C2 and C3. Here we report that the human MBL–MASP complex contains a new 22 kDa protein [small MBL-associated protein (sMAP)] bound to MASP-1. Analysis of the nucleotide sequence of sMAP cDNA revealed that it is a truncated form of MASP-2, consisting of the first two domains (i.e. the first internal repeat and the epidermal growth factor-like domain) with four different C-terminal amino acids. sMAP mRNAs are expressed in liver by alternative polyadenylation of the MASP-2 gene, in which a sMAP-specific exon containing an in-frame stop codon and a polyadenylation signal is used. The involvement of sMAP in the MBL–MASP complex suggests that the activation mechanism of the lectin pathway is more complicated than that of the classical pathway.

Keywords: alternative polyadenylation, lectin complement pathway, MASP-2

	Introduction

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Serum mannose-binding lectin (MBL; also called MBP) is a C-type lectin which recognizes certain carbohydrates such as mannose or N-acetylglucosamine on the surfaces of pathogens (1) and plays a crucial role in innate immunity (2). MBL acts as an opsonin (3,4) and also activates the complement system (5,6). Early reports on the mechanism of complement activation by MBL demonstrated in vitro that it forms a complex with C1 subcomponents, C1r and C1s (7,8). Subsequent studies, however, revealed that MBL circulates in serum complexed with a novel serine protease termed MBL-associated serine protease (MASP) but not with C1r and C1s (9). To date two types of MASP (MASP-1 and MASP-2) with structural similarity to C1r and C1s have been identified in human MBL preparations (9,10). MASP, C1r and C1s are all composed of six domains [a first internal repeat, an epidermal growth factor (EGF)-like domain, a second internal repeat, two complement control protein domains (CCP) and a serine protease domain] (11,12). Therefore, they constitute a novel branch of the serine protease family (13). Complement activation via the MBL–MASP complex has been designated the lectin pathway, a third pathway of complement activation (14). In the human lectin pathway, upon binding of the MBL–MASP complex to carbohydrate ligands, the MASP proteins in the complex convert from proenzymes to activated forms, resulting in cleavage of C4, C2 and C3 (9,10,15). Although it was demonstrated that C4 cleavage is ascribed to MASP-2 activity (10), the precise roles of two MASPs in the MBL–MASP complex remain to be elucidated.

The MBL–MASP complex in the lectin pathway and the C1 complex in the classical pathway are very similar in that both are composed of recognition molecules and structurally related serine proteases. This raises the possibility that both pathways have a similar activation mechanism. In this study, we report that unlike the C1 complex human MBL–MASP complex contains a truncated form of the MASP-2 gene by alternative polyadenylation.

	Methods

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Preparation of human MASP-1
Human MASP-1 in a proenzyme form was prepared according to the procedure described previously (16). In brief, human serum was chromatographed on yeast mannan–Sepharose using an imidazole buffer (pH 6.0) containing 0.2 M NaCl, 50 mM CaCl₂, 0.2 mM p-nitrophenyl-p-guanidinobenzoate, 20 µM (amidinophenyl) methanesulfonylfluoride and 2% mannitol. MASP-1 and MASP-2 complexed with MBL was eluted with the above buffer containing 0.3 M mannose. In order to separate MASP-1 and MASP-2 from MBL, fractions containing the complex were added to anti-MBL (3E7)–Sepharose and eluted with imidazole buffer containing 20 mM EDTA. Finally, MASP-1 was separated from MASP-2 using anti-MASP-1 (1E2)–Sepharose in the presence of EDTA.

SDS–PAGE and Western blotting
SDS–PAGE was performed according to the Laemmli method. After transferring proteins from the gels to PVDF membranes, blots were probed with anti-MASP-2 (antiserum raised against a synthetic peptide representing the 19 N-terminal amino acids of MASP-2, which was kindly provided by Dr Jens C. Jensenius, Aarhus University).

Amino acid sequence analysis
A MASP-1 preparation containing a 22 kDa protein (sMAP) was subjected to SDS–PAGE under reducing conditions and transferred to an Immobilon PVDF membrane (Millipore, Bedford, MA). After staining with Coomassie brilliant blue, the band of sMAP was excised and analyzed with a gas-phase protein sequencer (Applied Biosystems, Foster City, CA).

Cloning of sMAP cDNA
We searched the database of expressed sequence tags (dbEST) for proteins homologous to MASP-2. Two clones were found with similarity to MASP-2. The full length of this MASP-2-like cDNA was obtained using normal human liver poly(A)⁺ RNA and 5' RACE with a Marathon kit (Clontech, Palo Alto, CA). The N-terminal amino acid sequence of the protein from the MASP-2-like cDNA was identical to that of sMAP protein.

Northern blotting analysis
Human fetal multiple tissue Northern (MTN) blot II (Clontech) was used as a blotting nylon membrane. Northern hybridization was performed according to the manufacturer's protocol (Clontech) with a sMAP cDNA fragment (nucleotides 16–604) as a probe.

Analysis of genomic DNA and mRNA by PCR
To analyze the genomic structure of sMAP and its relationship to that of MASP-2, a genomic fragment was amplified by PCR using genomic DNA from normal human peripheral blood cells as a template. The primers used were as follows: CU; 5'-CACCTGCGACCACCACTGCCACAAC and P22L; 5'-GGCCGGAGCTCCAGGGGAGGCTAG and M2L; 5'-ACAAAGTCCAGAATGACACTGAACC. The amplified fragment was subcloned into pGEM-T vector (Promega, Madison, WI) and sequenced.

A similar PCR was performed to amplify the corresponding sequences in the cDNA, which was prepared from human liver poly(A)⁺ RNA using a Superscript preamiplification system (Life Technologies, Gibco/BRL, Rockville, MD).

	Results

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Presence of a 22 kDa protein in MASP-1 preparations
Proenzyme MASP-1 preparations which were obtained using mannan–Sepharose, anti-MBL–Sepharose and anti-MASP-1–Sepharose, contained a 22 kDa protein as assessed by SDS–PAGE (Fig. 1). This protein, designated sMAP (small MBL-associated protein), also co-purified with activated MASP-1 (data not shown). After transferring sMAP to a PVDF membrane, the sequence of the seven N-terminal amino acids was determined to be Thr-Pro-Leu-Gly-Pro-Lys-Trp, which completely matches the N-terminal sequence of MASP-2. To determine whether sMAP is generated from MASP-2 during purification or a MASP-2-related protein, we tested human serum for the presence of sMAP by Western blotting using antibody against a peptide consisting of the 19 N-terminal amino acids of MASP-2. As shown in Fig. 2, when human serum was analyzed, a protein with the same mobility as sMAP under both reducing and non-reducing conditions was detected. Similar results were obtained with human plasma containing EDTA, indicating that sMAP is not generated from MASP-2 by proteolysis during purification but is present as such in human blood.

View larger version (38K): [in this window] [in a new window]   Fig. 1. SDS–PAGE of MASP-1 preparations containing sMAP. A MASP-1 preparation containing sMAP from an anti-MASP-1–Sepharose column was subjected to SDS–PAGE (12% gel) under reducing (A) or non-reducing (B) conditions. Proteins were stained with Coomassie brilliant blue R250. Upper and lower arrowheads indicate MASP-1 and sMAP respectively.

 

View larger version (24K): [in this window] [in a new window]   Fig. 2. Western blotting analysis of sMAP in human blood. MASP-1 and MASP-2 preparations containing sMAP from an anti-MBL–Sepharose column (a) or human serum (b) or human plasma containing EDTA (c) were subjected to SDS–PAGE (12% gel) under reducing (A) or non-reducing (B) conditions followed by Western blotting using anti-MASP-2 serum as a probe. (C) Control experiment in which Western blotting was carried out without anti-MASP-2 under non-reducing conditions. Upper and lower arrowheads in (A) and (B) indicate MASP-2 and sMAP respectively.

 
Sequence analysis of sMAP cDNA
To determine the cDNA sequence of sMAP we searched for homologues of MASP-2 in a database of human expressed sequence tags (dbEST) and found that two EST clones (EST ID: 85164 and 129432) showed homology to MASP-2. Sequence analysis revealed that in addition to a predicted open reading frame, these clones contained a polyadenylation signal (AATAAA) in their 3'-untranslated regions, but no initiation codons were found. The full-length cDNA of the MASP-2-like sequence was obtained by 5' RACE. The N-terminal amino acid sequence deduced from this cDNA was identical to the seven N-terminal residues of sMAP. The cDNA obtained consisted of 725 bp, encoding 185 amino acid residues (Fig. 3). The predicted mol. wt of the mature protein was calculated to be 20,629, which matched that of sMAP. We compared the cDNA sequences of sMAP and MASP-2, and found that the sMAP cDNA sequence of nucleotides 11–570 deduced is identical to nucleotides 21–580 of MASP-2 cDNA (10) and that only four deduced C-terminal amino acids of sMAP are different from the corresponding deduced sequence of MASP-2. The predicted sMAP protein is composed of only the regions encoding the first two domains (first internal repeat and EGF-like domain) in the N-terminal half of MASP-2 and lacks the second internal repeat, the two CCPs and a serine protease domain.

View larger version (58K): [in this window] [in a new window]   Fig. 3. The nucleotide sequence of sMAP cDNA and its deduced amino acid sequence. The predicted amino acid sequence is shown in single-letter codes below the nucleotide sequence. The N-terminal signal sequence is doubly underlined. The arrowhead shows an exon boundary. The nucleotide sequence before the arrowhead from nucleotide 11 to 570 is identical to the 5' terminus of the MASP-2 sequence. Underlined sequence indicates the 155 bp sMAP-specific exon (exon II). A polyadenylation signal sequence is boxed. The nucleotide sequence data of the cDNA will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases (accession no. AB008047).

 
Presence of a sMAP-specific exon in the MASP-2 gene
The cDNA sequence corresponding to nucleotides 571–725 of sMAP is not found in MASP-2 cDNA and the termination codon (TAG) is present in nucleotides 582–584 of sMAP cDNA (Fig. 3). We analyzed the genomic structure of sMAP and its relationship to that of MASP-2. First, primers (CU and P22L) were designed for PCR to amplify the region between the open reading frame and the 3'-untranslated region of sMAP cDNA. By sequencing a 587 bp PCR product thus obtained, it was found that this fragment contained 454 bp intron. A similar PCR to amplify a genomic fragment with primers CU and M2L yielded an ~2.0 kb fragment. Based on sequence analysis of this fragment, a partial genomic map was constructed as shown in Fig. 4(A). A sMAP-specific 155 bp exon (exon II) exists between a common exon (exon I) and a MASP-2-specific exon (exon III). The facts that the sMAP-specific exon contains an in-frame stop codon and a polyadenylation signal suggest that the mRNAs of both sMAP and MASP-2 are derived from the same gene by alternative polyadenylation.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Exon organization of the sMAP and MASP-2 genes and their transcripts. (A) Schematic diagram of a partial genomic structure of sMAP and MASP-2. Exons are shown in three differently designed boxes. Exon I and exon III are incomplete, lacking a 5' and a 3' terminus respectively. Arrowheads show the positions of primers (CU, P22L and M2L) used for RT-PCR. S; SacI, P; PstI. (B) RT-PCR amplification of sMAP and MASP-2 transcripts. PCR was performed by starting with human liver poly(A)+ RNA (lane 1). In a control PCR experiment, a plasmid containing sMAP cDNA (lane 2) and a plasmid containing MASP-2 cDNA (lane 3) were used as template DNAs. X174 DNA–HaeIII digest marker was loaded into lane M (upper band corresponds to 310 bp). Schematic representation of the PCR products are shown on the left.

 
Analysis of transcriptional products of the sMAP and MASP-2 gene
It has been reported that the MASP-1 gene is expressed in liver (11). To investigate the tissue-specific expression of the MASP-2/sMAP gene, we carried out Northern blot analyse. The region of cDNA shared by sMAP and MASP-2 was used as a probe. As shown in Fig. 5, several bands which differed in size were observed in fetal liver but not in brain, lung or kidney. Similar expression of the MASP-2/sMAP gene was observed in adult liver but not in heart, brain, placenta, lung, skeletal muscle, kidney or pancreas (data not shown). The major transcript was estimated to be 0.9 kb long. This presumably corresponds to the sMAP transcript, which was expressed at least 8 times higher than the other products. The 4.8, 3.4 and 2.4 kb MASP-2 transcripts may be produced by the use of different polyadenylation signals in the MASP-2 gene.

View larger version (63K): [in this window] [in a new window]   Fig. 5. Northern blot of sMAP and MASP-2 transcripts in several human fetal tissues. sMAP cDNA was hybridized to poly(A)+ RNA from human fetal brain (lane 1), lung (lane 2), liver (lane 3) and kidney (lane 4). At least, five signals of 4.8, 3.4, 2.4, 1.7 and 0.9 kb are detected only in fetal liver. The filter was exposed to an autoradiogram imaging screen and the image was then read using Bioimaging Analyzing System (BAS1000; Fujifilm, Tokyo, Japan).

 
To determine whether transcripts of sMAP and MASP-2 are actually produced in normal human liver, we amplified mRNA by RT-PCR using three primers (CU, P22L and M2L). When a cloned plasmid containing sMAP cDNA or MASP-2 cDNA was used as a template, a 129 or 234 bp fragment was detected respectively (Fig. 4B, lanes 2 and 3). In a similar RT-PCR using human liver mRNA both bands were observed, indicating that both sMAP and MASP-2 transcripts are expressed in normal liver.

	Discussion

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We identified a 22 kDa protein (sMAP) in MASP-1 preparations and demonstrated that the N-terminal amino acid sequence of sMAP is identical to that of MASP-2. Thiel and colleagues reported that a 20 kDa protein which co-purified with MASP-1 reacted with the same antibody against a peptide of MASP-2 used in this study (10), suggesting that sMAP and the 20 kDa protein are the same protein. sMAP is not generated from MASP-2 by proteolysis during purification but exists as such in human blood. We also demonstrated the existence of a variant of the MASP-2 gene which is expressed specifically in liver. The calculated mol. wt of the MASP-2 variant is the same as that of sMAP. Therefore, we conclude that sMAP is a truncated form of MASP-2, and that its transcript is generated from exon I and a sMAP-specific exon in the MASP-2 gene by alternative polyadenylation.

It is likely that sMAP binds directly to MASP-1 in a Ca²⁺-independent manner, since sMAP was retained on an anti-MASP-1–Sepharose column in the presence of EDTA and co-eluted with MASP-1. In contrast, MASP-2 was passed through an anti-MASP-1–Sepharose column in the presence of EDTA, resulting in its separation from MASP-1 (data not shown). This suggests that MASP-1 and MASP-2 form a complex in the presence of Ca²⁺ as do C1r and C1s, or they independently form complexes with MBL. In the C1 complex, C1r and C1s form a tetramer, C1s–C1r–C1r–C1s, in which the internal repeat and EGF-like domain of both components are involved in the C1r–C1s interaction in the presence of Ca²⁺. sMAP consists only of these two domains which are shared by MASP-2 except that the four C-terminal amino acids of the sMAP protein are different from the corresponding sequence found in the EGF-like domain of MASP-2. It is conceivable, therefore, that this four amino-acid sequence of sMAP is responsible for the Ca²⁺-independent interaction between sMAP and MASP-1.

The precise composition and stoichiometry of the MBL–MASP-1–MASP-2–sMAP complex and the roles of sMAP remain to be elucidated. In the C1 complex, the C1r₂C1s₂ tetramer binds to C1q. Upon binding of C1 to immune complexes through C1q, C1r autoactivates with concomitant activation of C1s. Activated C1s in turn cleaves C4 and C2. Truncated forms of C1r or C1s such as sMAP have not been found in the C1 complex, suggesting that there are more complicated activation mechanisms of MBL–MASP than those of C1. As discussed above, one possible function of the internal repeats and the EGF-like domains of C1r and C1s is to mediate the C1r–C1s interaction. These domains might also facilitate the binding of C1r₂C1s₂ to C1q. If this is the case, sMAP may be involved in the MBL–MASP-1 interaction. Higher expression of sMAP transcripts than of MASP-2 in human liver suggests a crucial role for sMAP in the MBL–MASP complex. Further investigation of the roles of sMAP in the MBL–MASP complex may clarify the difference in activation mechanisms between C1 and MBL–MASP.

	Abbreviations

 

CCP	complement control protein
EGF	epidermal growth factor
MBL	mannose-binding lectin
MASP	MBL-associated serine protease
sMAP	small MBL-associated protein

	Notes

 
Transmitting editor: K. Sugamura

Received 7 January 1999, accepted 4 February 1999.

	References

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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]

				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]

				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]

				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]

				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]

				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]

				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]

				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]

				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]

				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]

				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]

				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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