Genes and Immunity (2001) 2, 119–127 2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene The human gene for mannan-binding lectin-associated serine protease-2 (MASP-2), the effector component of the lectin route of complement activation, is part of a tightly linked gene cluster on chromosome 1p36.2–3 C Stover1, Y Endo2, M Takahashi2, NJ Lynch1, C Constantinescu1, T Vorup-Jensen3, S Thiel3, H Friedl4, T Hankeln4, R Hall5, S Gregory5, T Fujita2 and W Schwaeble1 1Department of Microbiology and Immunology, University of Leicester, Leicester, UK; 2Department of Biochemistry, Fukushima Medical University, School of Medicine, Fukushima, Japan; 3Department of Medical Microbiology and Immunology, The Bartholin Building, University of Århus, Århus, Denmark; 4Genterprise, Institut fu¨r Molekulargenetik und Gentechnische Sicherheit, Johannes Gutenberg University Mainz, Mainz, Germany; 5The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK The proteases of the lectin pathway of complement activation, MASP-1 and MASP-2, are encoded by two separate genes. The MASP1 gene is located on chromosome 3q27, the MASP2 gene on chromosome 1p36.23–31. The genes for the classical complement activation pathway proteases, C1r and C1s, are linked on chromosome 12p13. We have shown that the MASP2 gene encodes two gene products, the 76 kDa MASP-2 serine protease and a plasma protein of 19 kDa, termed MAp19 or sMAP. Both gene products are components of the lectin pathway activation complex. We present the complete primary structure of the human MASP2 gene and the tight cluster that this locus forms with non-complement genes. A comparison of the MASP2 gene with the previously characterised C1s gene revealed identical positions of introns separating orthologous coding sequences, underlining the hypothesis that the C1s and MASP2 genes arose by exon shuffling from one ancestral gene. Genes and Immunity (2001) 2, 119–127. Keywords: complement; lectin pathway; serine proteases; gene cluster; MASP2 gene Introduction vertase, C4b2b, and the C5 convertase, C4b2b(C3b)n. The alternative pathway forms an amplification loop of comp- The complement system is an integral part of the innate lement activation and is initiated by binding of the comp- antimicrobial immune defence and mediates humoral lement activation product C3b to Factor B. The sub- and cellular interactions within the immune response, sequent cleavage of Factor B by Factor D forms an including chemotaxis, phagocytosis, cell adhesion, and B enzymatically active complex, C3bBb, which converts 1 cell differentiation. Complement may be activated via native C3 to C3b. Accumulation of multiple C3b mol- three different routes, the classical pathway, the alterna- ecules in proximity of the alternative pathway convertase tive pathway, and the recently described lectin pathway. C3bBb allows the complex to cleave and activate the fifth The classical activation pathway is initiated by binding of component of complement, C5. the hexaoligomeric recognition molecule C1q to immune The lectin pathway can be activated in the absence of complexes via its globular heads. This effects a confor- immune complexes and is initiated by the recognition of mational change of its collagenous stalks which in turn carbohydrate structures present on microbial organisms leads to the activation of the C1q-associated serine pro- via macromolecular complexes present in body fluids. tease complexes C1r2 and C1s2. Of these, C1r cleaves and These complexes are composed of a multivalent pattern activates the zymogen C1s. Activated C1s subsequently recognition subunit and associated serine proteases cleaves C4 and C4b bound C2 to generate the C3 con- which initiate complement activation upon binding of the recognition subunits to microbial oligosaccharide struc- tures. To date, two pattern recognition components of the Correspondence: Dr WJ Schwaeble, Department of Microbiology and Immunology, University of Leicester, University Road, Leicester LE1 lectin activation pathway have been described, the well 9HN, UK. E-mail: ws5Ȱle.ac.uk characterised plasma glycoprotein mannan-binding lectin This work was supported by the Wellcome Trust, the German (MBL)2 and ficolin p35,3 both members of the collectin Research Foundation (Deutsche Forschungsgemeinschaft), the family,4,5 yet differing in their binding specificities to Japanese Society for the Promotion of Science, and the EMDO microbial surface oligosaccharides. As these vary con- Foundation, Switzerland. siderably amongst members of the collectin family, the Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers presence of more than one recognition subunit may result AL109811, AB033742, AJ297949, AJ299718, and AJ300188. in a wider spectrum of microbial surfaces recognised by Received 4 December 2000; accepted 1 February 2001 the lectin pathway activation complexes. MBL forms a Organisation of the human MASP2 gene locus C Stover et al 120 hexoidal macromolecular complex of six structural sub- sequence coding for the signal peptide is split by an units each composed of three identical polypeptide intron of 83 bp (Figure 1a). Exon b codes for the remain- chains. This associates with two distinct serine proteases, der of the signal peptide (R)LLTLLGLLCGSVA and the the MBL-associated serine proteases-1 and -2 (ie, MASP- N-terminal 63 residues of the CUB I domain. The C-ter- 1, MASP-2), and a 19 kDa MASP-2 related plasma pro- minal 59 residues of the CUB I domain are encoded by tein, termed MAp19 or sMAP.6 No detailed information exon c, separated from exon b by 157 bp and from exon is yet available on the stoichiometry of lectin pathway d, the single exon coding for the EGF-like domain, by activation complexes formed with ficolin p35. It is, how- 1016 bp. The alternative splice/polyadenylation exon e ever, clear that ficolin p35 complexes associate with (which contains coding sequence for the four C-terminal MASP-1, MASP-2 and MAp19/sMAP and initiate the lec- amino acids of MAp19 not found in MASP-2, namely glu- tin pathway via the aforementioned serine proteases.3 In tamic acid, glutamine, serine, and leucine, and sequence vitro, purified and recombinant MASP-2 was shown to of the 3Ј UT region specific for MAp19 mRNA) follows cleave the fourth and second component of complement after 454 bp of intronic sequence. The 3Ј boundary of (ie, C4 and C2) in absence of MASP-1.6–8 The sequential exon e is determined by the position of the poly(A) proteolytic cleavage of C4 and C4b bound C2 to form the addition site14 noted in the human MAp19 mRNA tran- C3 convertase, C4b2b, and the C5 convertase, scripts with the accession numbers Y18281–Y18284. 1262 C4b2b(C3b), respectively, is the essential step by which bp further downstream, sequence for the second CUB the lectin pathway activation complex as well as the structural domain is split by an intron of 316 bp (exon f, classical pathway activation complex initiate further 197 bp; exon g, 148 bp, respectively). An intron of 1997 downstream activation of complement.7,8 The potential of bp follows (AJ299718). The two CCP domains of MASP- MASP-2 to activate complement in absence of MASP-1 in 2 are encoded by two exons each over a stretch of 7.6 kb, vitro is supported by the analysis of sera of a recently exon h (119 bp) and exon i (79 bp), exon j (135 bp) and established MASP-1 deficient mouse strain, which exhib- exon k (75 bp), respectively. 2527 bp further downstream, its effective lectin pathway activation under physiological exon l encodes the serine protease domain of MASP-2 9 conditions. Thus, MASP-2 is the effector component of and contains the 3Ј UT region specific for MASP-2 the lectin pathway of complement activation. MASP-1 mRNA. The 3Ј boundary of exon l is determined from and MAp19 are constitutive components of the lectin the position of the poly (A) addition site14 found in cDNA pathway activation complexes, their specific functional clones Y09926, phl-1, phl-2, phl-3.15 Exons b-l are pre- role(s), however, have yet to be defined. ceded by polypyrimidine rich tracts. Sequence compari- We have previously shown that a single structural son of the clones aligned in Figure 1 revealed only two MASP2 gene of approximately 20 kb on chromosome variants (transitions) within the coding sequence of the 1p36.23–31 is transcribed and processed to generate two MASP2 gene: the codon for the amino acid position 362 gene products, a 2.6 kb MASP-2 mRNA, encoding the 76 of the mature MASP-2 protein is GTG (valine) in kDa MASP-2 serine protease, or an abundant 1.0 kb AL109811 and GCG (alanine) in AJ300188, respectively; mRNA transcript, encoding a truncated MASP-2 related the codon for amino acid position 478 is TCC (serine) in plasma protein of 19 kDa devoid of a serine protease AL109811 and TCT (serine) in AJ300188, respectively. domain, termed MAp19 or sMAP.10–13 The codon phases (positions of introns within the The present report amends and completes our pre- codon) are given in Table 1. They are identical for the viously presented organisational map (based solely on human MASP2, MASP1, and C1s genes in the ortholog- genomic PCR)10,11 by a comparative analysis of overlap- ous exonic composition represented in MASP2 exons a, ping sequences obtained from human genomic DNA iso- 16 Ј lated as independent YAC, BAC and ␭FixII clones. These b, c, d, f, g, h, i, j, and k. The codon phase at the 5 end of exon l is also identical for the orthologous position in represent approximately 222 kb of the chromosomal area 17 1p36.2–3. Furthermore, we present putative promoter the human C1r gene. A GC level of 50.37% was determined for sequence elements responsible for the strict hepatic expression pat- − tern of MASP-2 and MAp19 mRNA.