Composition of the Lectin Pathway of Complement in Gallus gallus: Absence of Mannan-Binding Lectin-Associated Serine Protease-1 in Birds This information is current as of September 28, 2021. Nicholas J. Lynch, Saeed-ul-Hassan Khan, Cordula M. Stover, Sara M. Sandrini, Denise Marston, Julia S. Presanis and Wilhelm J. Schwaeble J Immunol 2005; 174:4998-5006; ; doi: 10.4049/jimmunol.174.8.4998 Downloaded from http://www.jimmunol.org/content/174/8/4998

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Composition of the Lectin Pathway of Complement in Gallus gallus: Absence of Mannan-Binding Lectin-Associated Serine Protease-1 in Birds1

Nicholas J. Lynch,2* Saeed-ul-Hassan Khan,2* Cordula M. Stover,* Sara M. Sandrini,* Denise Marston,† Julia S. Presanis,‡ and Wilhelm J. Schwaeble3*

The lectin pathway of complement is activated by multimolecular complexes that recognize and bind to microbial polysaccharides. These complexes comprise a multimeric carbohydrate recognition subunit (either mannan-binding lectin (MBL) or a ficolin), three MBL-associated serine proteases (MASP-1, -2, and -3), and MAp19 (a truncated product of the MASP-2 ). In this study we report the cloning of chicken MASP-2, MASP-3, and MAp19 and the organization of their and those for chicken MBL and a novel ficolin. Mammals usually possess two MBL genes and two or three ficolin genes, but chickens have only one of each, both Downloaded from of which represent the undiversified ancestors of the mammalian genes. The primary structure of chicken MASP-2 is 54% identical with those of the human and mouse MASP-2, and the organization of its gene is the same as in mammals. MASP-3 is even more conserved; chicken MASP-3 shares ϳ75% of its residues with human and Xenopus MASP-3. It is more widely expressed than other lectin pathway components, suggesting a possible function of MASP-3 different from those of the other components. In mammals, MASP-1 and MASP-3 are alternatively spliced products of a single structural gene. We demonstrate the absence of

MASP-1 in birds, possibly caused by the loss of MASP-1-specific exons during phylogeny. Despite the lack of MASP-1-like http://www.jimmunol.org/ enzymatic activity in sera of chicken and other birds, avian lectin pathway complexes efficiently activate C4. The Journal of Immunology, 2005, 174: 4998–5006.

he lectin pathway provides an Ab-independent route of very similar, being homotrimers of a single polypeptide chain, complement activation. It is an ancient system that ante- with an N-terminal -like domain and a C-terminal carbo- T dates the evolution of adaptive immunity and the classical hydrate recognition domain (CRD). In MBL, the CRD is a C-type complement pathway, and it is thought to be particularly important lectin domain, whereas the CRD in ficolins is a fibrinogen-like in lower animals such as the urochordates and cephalochordates, domain. In plasma, the basic homotrimers form higher order oli- which lack an adaptive immune system. In vertebrates, the lectin gomers, which, in turn, form complexes with the MBL-associated by guest on September 28, 2021 pathway provides a first line of defense during the lag phase that serine proteases, MASP-1, -2, and -3 (13–16). Only MASP-2 precedes the onset of an adaptive response (1–3). Deficiencies of cleaves C4 and C4b-bound C2, generating C4b2a, the same C3 the lectin pathway are associated with susceptibility to infectious convertase that results from activation of the classical pathway (17, disease, particularly in infants, young children, and those with an- 18). The roles of MASP-1 and -3 in these complexes are still other acquired immunodeficiency, such as cancer patients under- unclear. going chemotherapy (4–8). All three MASPs share a conserved domain structure. Five N- Activation of the lectin pathway occurs when a multimolecular terminal domains (the C1r/C1s/Uegf/bone morphogenetic I fluid phase activation complex, comprising a recognition subcom- (CUBI), epidermal growth factor (EGF)-like, CUBII, complement ponent and associated serine proteases, binds to carbohydrate control protein I (CCPI), and CCPII domains) precede a C-termi- structures present on microbial surfaces. Two types of fluid phase nal serine protease domain. When activated, the enzymes split into recognition molecule have been described, the mannan-binding two disulfide-linked chains, the A chain, which contains the five lectin(s) (MBL),4 and the ficolins (9–12). Structurally they are N-terminal domains, and the B chain, which contains the serine protease domain (2, 19). The mRNAs encoding MASP-1 and -3 are alternatively spliced products of one single gene. The A chain *Department of Infection, Immunity, and Inflammation, University of Leicester, Le- is common to both , the B chain of MASP-1 is encoded by icester, United Kingdom; †Institute for Animal Health, Compton, United Kingdom; and ‡Medical Research Council Immunochemistry Unit, Department of Biochemistry, five or six exons (depending upon the species), and the B chain of University of Oxford, Oxford, United Kingdom MASP-3 is encoded by a single exon, located between the exons Received for publication November 24, 2004. Accepted for publication January encoding the shared A chain and those coding for the B chain of 31, 2005. MASP-1 (16, 19, 20). This genomic arrangement is found in am- The costs of publication of this article were defrayed in part by the payment of page phioxus (a cephalochordate) and in all vertebrates investigated to charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. date, but not in the ascidians, indicating that it arose after the divergence of the urochordates, but before the divergence of the 1 This work was supported by Wellcome Trust Grants 060574 and 062696. cephalochordates (21). The lectin pathway activation complexes 2 N.J.L. and S.-u.H.K. contributed equally to this work. also contain MAp19, a protein composed of the two N-terminal 3 Address correspondence and reprint requests to Dr. Wilhelm J. Schwaeble, Depart- ment of Infection, Immunity, and Inflammation, University of Leicester, University Road, Leicester, U.K. LE1 9HN. E-mail address: [email protected] domain; CUB, C1r/C1s/Uegf/bone morphogenetic protein; EGF, epidermal growth 4 Abbreviations used in this paper: MBL, mannan-binding lectin; AMC, 7-amino-4- factor; MASP, MBL-associated serine protease; qRT-PCR, quantitative RT-PCR; UT, methylcoumarin; CCP, complement control protein; CRD, carbohydrate recognition untranslated.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 4999 domains of MASP-2 encoded by a truncated mRNA transcript that Northern blotting arises through alternative splicing of the MASP2 gene (19, 22–24). For Northern blot analysis, 10 ␮g of total RNA or 100 ng of poly(Aϩ) MAp19 has no enzymatic activity, and its function within the lec- mRNA was separated on formaldehyde-containing 1.2% agarose gels, tin pathway activation complexes remains unclear. transferred to nylon membrane, and hybridized with [␣-32P]dCTP-labeled Commercial poultry are vulnerable to bacterial, viral, and cDNA probes using standard methods (35). The cDNA probes used were: parasitic infections that cause considerable mortality and eco- for the A chain of MASP-2, a 376-bp RT-PCR product amplified from chicken liver cDNA with primers M2A-ch-F and M2A-ch-R (Table I); for nomic loss. They are also reservoirs for transmissible human the B chain of MASP-2, a 430-bp RT-PCR product generated with primers pathogens, including Salmonella, Campylobacter, and poten- M2dg-F2 and M2dg-R1; for the A chain of MASP-3, a 740-bp RT-PCR tially dangerous stains of influenza, such as the H5N1 (Guang- product amplified with primers M1&3dg-F and M1&3dg-R; and for the B dong) virus that caused the 1997 bird flu outbreak in Hong chain of MASP-3, a 1200-bp NcoI fragment of clone CM3/5, encompass- ing the last 500 bp of the coding sequence and the first 700 bp of the Kong (25, 26). Although the adaptive immune system of the 3Ј-untranslated (3ЈUT) region. For controls, rat liver RNA was hybridized chicken has been extensively studied (27), the complement sys- with rat MASP-3 A and B chain-encoding cDNA probes, as previously tem and, in particular, the lectin pathway, is less well under- described (20). stood. It is known that Ab-independent activation of the com- plement system plays an important role in the host response to In situ hybridization fowlpox virus; fowlpox virus infection is aggravated in chick- 35 ens treated with cobra venom factor (28). mRNA was localized by in situ hybridization using S-labeled antisense RNA probes generated by in vitro transcription. The templates for in vitro The classical pathway components C1 and C3 through C9 have transcription were RT-PCR products, amplified from liver cDNA and been found in chickens (29). Because a homologue for C2 was not cloned into pGEM-Teasy. For the A chain of chicken MASP-3 and for the Downloaded from found in these early studies, it was assumed that the role of C2 may B chain of chicken MASP-2, the cloned RT-PCR products were those be fulfilled by the chicken factor B-like protease, an evolutionary described above. For the B chain of chicken MASP-3, a 564-bp RT-PCR remnant of a common C2/factor B ancestor described previously product was generated using primers M3SP-F1 and -R1 (Table I); for chicken MAp19, a 170-bp product was obtained using primers MAp19-F1 (29, 30). However, subsequent studies identified and mapped the and -R1; for chicken MBL, a 330-bp product was generated with primers chicken homologues of both the C2 and factor B genes and showed MBL-F1 and MBL-R1; for chicken ficolin, a 324-bp product was amplified that chickens are not deficient in C2 (K. Skjoedt and J. Kaufman, using primers ChkFCN_F1 and ChkFCN_R1; for the B chain of rat http://www.jimmunol.org/ unpublished observations). MBL is the only lectin pathway com- MASP-1, a 400-bp product was generated using primers RM1sp-F1 and RM1sp-R1; and for the B chain of rat MASP-3, a 407-bp product was ponent that has been described in the chicken to date. Although obtained with primers RM3-BCF1 and RM3-BCR1. Antisense RNA most mammals have two separate genes for MBL (31), only one probes were transcribed from the cloned templates using the method de- gene was found in the chicken, and phylogenetic analysis of its scribed by Melton et al. (36). For controls, the templates were transcribed amino acid sequence indicated that it may represent an undiversi- in the opposite direction, generating sense RNA probes. Twenty-micron- thick tissue sections were cut using a cryostat, mounted on poly-lysine- fied ancestor of the mammalian MBLs (32). The concentration of 35 ␮ coated microscope slides (Merck), and hybridized with the S-labeled MBL in chicken serum ranges from 0.4 to 37.8 g/ml (mean, 5.8 probes according to the method of Scha¨fer et al. (37). After hybridization ␮g/ml), with no strain-to-strain variation and no deficiencies de- and washing, the signals were detected by exposing the sections to Kodak tected (33). MBL levels increase ϳ2-fold in chickens infected with BioMax MR x-ray film (Sigma-Aldrich). by guest on September 28, 2021 infectious bronchitis virus, infectious laryngotracheitis virus, and infectious bursal disease virus, indicating that MBL is a mild acute phase protein (34). This report describes the molecular cloning of chicken MASP-2, Table I. Oligonucleotides used in this study MASP-3, and MAp19 and the organization of their genes and those for chicken MBL and a novel ficolin. We demonstrate the Primer Sequence (5Ј-3Ј) apparent absence of MASP-1 and MASP-1-like enzymatic activity ATTGACAACACCCTCACGGTC in chickens and other birds. M2A-ch-F M2A-ch-R CCACATTAAAGGTCTCCAC M2dg-F2 TGACAATGAYATWGCWYTGAT M2dg-R1 ATTTYGTGTAKACHCCATAYTG Materials and Methods M1&3dg-F SARRGARASCACHGAYACHGARC RNA extraction and cDNA synthesis M1&3dg-R CRTTATCCTTYAGCACTYKGTAS AGGACGTGCACAGCGTGA Total RNA was prepared using TRIzol reagent (Invitrogen Life Technol- MAp19-F1 ϩ GCTGGAGGAAGGAGGTCTGA ogies), according to the manufacturer’s instructions. Poly(A) mRNA was MAp19-R1 TGACCTGCACCGACAAATAACTGAT purified from total RNA using the Oligotex mRNA Midi kit, from Qiagen. MBL-F1 GTTGCTGTAAGTTAAAGGCCCACCA For cDNA synthesis, ϳ8 ␮g of total RNA was digested with RNase-free MBL-R1 ACTGTCTTCTGCGATATGGACA DNase I (Promega), extracted once with phenol/chloroform/isoamyl alco- ChkFCN-F1 GTACGATAAGGAGTCTCCAGCA hol (25/24/1), precipitated, washed with ethanol, then redissolved in water. ChkFCN-R1 CATAGTGGTGGAAGACACATCA The concentrations of the RNA samples were determined by measuring the RM3-BCF1 CTGTCACATTAGGGTTGGAGA absorbance at 260 nm. Three micrograms of each RNA sample was primed RM3-BCR1 TGACGGCTGCTCACTGTCTC with oligo(dT) , and single-stranded cDNAs were synthesized using RM1sp-F1 12–18 GTGTAGGCCTCTTGGCAGGTA the SuperScript First-Strand Kit (Invitrogen Life Technologies) according RM1sp-R1 GACTCCTATCCCAGCGACTC to the supplier’s instructions. M3Ach-F1 M3Ach-R1 GTTGGAGAAGTCGGACCTGA M3SP-F1 GATCATCAAGCGCATCATCG cDNA cloning M3SP-R1 CTTGCACTCTGCATGCAGC CCAGCTTTGATCTTGATGTA A ␭ZAP chicken liver cDNA library (Stratagene) was screened by filter M3race-R1 CAGGCAGTCCGAGCTCTTGGGGTAGG hybridization using [␣-32P]dCTP-labeled cDNA probes prepared with the M3race-R2 CGGCCGTGTAATGTGCGTCGAATCC Random Primed Labeling kit (Roche). Isolated ␭ZAP clones were con- M3race-R3 Ϫ GTAGCTGCAGGTGCTG verted into pBluescript SK plasmid clones by in vivo excision, following M2race-R1 GGCTGGCAATTTCTCCAGTTC the supplier’s protocol, and sequenced (Genterprise). The 5Ј and 3Ј ends of M2race-R3 GTTCTGACACTGGGCTGTGCA some cDNA sequences were extended by RACE, using chicken liver RNA M2race-R4 TTCCTGCCCCCAGGGCTC and kits (Invitrogen Life Technologies). RACE products were cloned into MAp19-F2 CAACAAGTAATGGCCTCTTTATTG pGEM-Teasy (Promega) and sequenced. MAp19-R2 5000 LECTIN PATHWAY IN G. gallus

Table II. Cross-species comparison of MASP-3, MASP-2, and MAp19 software supplied with the machine (38). Melting curve analysis was used sequences (percentage of identical residues). to confirm the specificity of the products (39). The primers used to amplify the A chain of MASP-3 were M3Ach-F1 and -R1 (generating a 261-bp product); for the B chain of MASP-3, M3SP-F1 and M3SP-R1 (564 bp); Chicken for MAp19, MAp19-F1 and MAp19-R1 (170 bp); for MASP-2, M2A-ch-F MASP-3 MASP-2 and M2A-ch-R (376 bp); for MBL, MBL-F1 and MBL-R1 (330 bp); and for chicken ficolin, ChkFCN-F1 and ChkFCN-R1. Standard curves were Total B chain Total B chain Map19 produced for each analysis using serial dilutions of the corresponding cD- (%) (%) (%) (%) (%) NAs cloned in pBluescript SKϪ (for MASP-2 and -3) or pGEMTeasy (for MAp19 and MBL). Human 75 79 54 50 59 Mouse 76 80 54 50 63 Enzyme assays Xenopus 75a 83a 47 45 Mannan-coated microtiter plates were used to capture MBL-MASP com- a Xenopus MASP3a. plexes from pooled normal human serum, chicken serum (from a pool of five animals), and turkey serum (also pooled from five animals). C4 cleav- age activity of the captured complexes was assayed using the method de- Analysis of by quantitative RT-PCR (qRT-PCR) veloped by Petersen et al. (40), with minor modifications (41). MASP-1- like activity was measured using the fluorescent substrate, butyloxy- cDNA was analyzed by quantitative PCR using a LightCycler (Roche) to carbonyl-Val-Pro-Arg-7-amino-4-methylcoumarin (AMC), as previously follow the incorporation of SYBR Green I into the PCR products in real- described (42). time. Each 15 ␮l of PCR contained 1/100th of the original cDNA synthesis reaction (corresponding to 30 ng of total RNA), 0.5 ␮M of each primer, Sequence analysis and 7.5 ␮l of QuantiTect SYBR Green Master Mix (Qiagen). Forty-five Downloaded from cycles of amplification were performed; the annealing temperature was The chicken genome database is available at ͗www.ensembl.org/Gallus_ reduced from 70 to 58°C during the first 15 cycles and was kept constant gallus/͘. Similarity searches with nucleotide and polypeptide query se- at 58°C thereafter. The fluorescence signal was detected at the end of each quences were run on this server using BLASTN and TBLASTN, respec- cycle, and results were analyzed using the Fit Points option in the LDCA tively. The MutliContigView option was used to create chicken/human http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Alignment of the cDNA-derived peptide sequences of chicken, human, and mouse MASP-3 and X. laevis MASP-3a. Predicted leader are italicized, poten- tial N-glycosylation sites are under- lined, conserved residues are marked with an asterisk, conservative substi- tutions are marked with a colon, and conserved cysteines are shown in bold. The arginine/isoleucine cleav- age site that separates the A and B chains is indicated by an arrow, and the three residues that form the cata- lytic triad are boxed. The Journal of Immunology 5001 synteny maps. Signal peptides and N-linked glycosylation sites were pre- The largest clone obtained using the MASP-2 B chain probe was dicted using SignalP v3.0 and NetNGlyc v1.0, which can be found at 2082 bp long, which did not comprise the full-length coding se- ͗ ͘ www.cbs.dtu.dk/services/ . quence. The 5Ј end of this clone extended to the cDNA encoding Ј Results the CUBII domain. 5 RACE using primers M2race-R1, -R3, and -R4 gave a 601-bp product, which extended the known sequence cDNA cloning by 336 bp and included the initiation codon. The complete Degenerate oligonucleotides were designed to match parts of the MASP-2 cDNA (GenBank accession no. AY567828) is 2449 bp MASP-1/3 A chain and the B chain of MASP-2 that are conserved long and includes a 52-bp 5ЈUT region, a 2058-bp open reading in the human, rat, mouse, and Xenopus laevis sequences. The frame, the stop codon, and a 336-bp 3ЈUT region, with a polyad- MASP-1/3 primers (M1&3dg-F and M1&3dg-R; Table I) were enylation signal 19 bp upstream of the poly(A)ϩ tail. All the im- used to amplify a 740-bp fragment of cDNA from chicken liver portant features found in the primary structure of human, mouse, RNA by RT-PCR, and the MASP-2 specific primers (M2dg-F2 and Xenopus MASP-2 are conserved in chicken MASP-2 (Fig. 2), and M2dg-R1) were used to amplify a 430-bp fragment from the although the degree of identity among the MASP-2 amino acid same source. Both products were cloned into pGEM-Teasy and sequences is less than that among the MASP-3 sequences then sequenced to confirm that they encode the anticipated regions (Table II). of the chicken MASP cDNAs. The RT-PCR products were sub- Chicken MAp19 cDNA was cloned by RT-PCR. First, the 3Ј sequently labeled with [␣-32P]dCTP and used to screen a propri- sequence was obtained by 3ЈRACE using primers located within etary chicken liver cDNA library. the DNA encoding the CUBI domain of MASP-2 (MAp-race-F1

Seven clones were obtained using the MASP-1/3 A chain probe. and MAp-race-F2). A 440-bp RACE product was obtained, cloned Downloaded from Sequencing showed that all seven were incomplete MASP-3 cD- into pGEM-Teasy, and sequenced. Next, primers representing the NAs, the longest of which (clone CM3/5) was 3343-bp long and 5Ј end of MASP-2 (MAp19-F2) and the 3Ј end of the MAp19 was complete at its 3Ј end, but began with DNA encoding the RACE product (MAp19-R2) were used to amplify full-length middle of the CUBI domain, ϳ300 bp downstream of the expected MAp19 cDNA from chicken liver cDNA. The first 588 bp of transcription start. Where they overlapped, the sequences of the chicken MAp19 cDNA are identical with those of the MASP-2 Ј other six clones were identical with that of CM3/5. The 5 end of cDNA and encode the CUBI and EGF-like domains (Fig. 2). The http://www.jimmunol.org/ the MASP-3 sequence was completed by RACE, using chicken remainder of the cDNA is unique to MAp19 and includes the third liver RNA and the primers M3race-R1, -R2, and -R3. A 565-bp nucleotide of the codon for the last residue, the stop codon, and a RACE product was obtained, extending the known sequence by 3ЈUT region of 177 bp (GenBank accession no. AY567830). 293 bp. Mammalian MAp19 proteins have a four-residue C-terminal ex- The complete chicken MASP-3 cDNA (GenBank accession no. tension (EQSL) that is absent in the corresponding MASP-2 se- AY567829) is 3636 bp long and comprises a 159-bp 5ЈUT region, quences (23, 24). Chicken MAp19 lacks this feature; its entire followed by a 2190-bp open reading frame, a stop codon TGA, and amino acid sequence is contained within the MASP-2 sequence. a3ЈUT region of 1284 bp. A putative polyadenylation signal is ϩ located 13 bp upstream of the poly(A) tail. Analysis of the Organization of the lectin pathway genes by guest on September 28, 2021 cDNA-derived amino acid sequence revealed extensive similarity The genes for chicken MASP-2 and MASP-3 were characterized between chicken MASP-3 and MASP-3 from other species. The by comparing the cDNA sequences with the recently completed chicken sequence shares 75% of its residues with the human se- chicken genome sequence. quence, 76% with the mouse sequence, and 75% with the Xenopus The MASP-2 gene occupies ϳ14 kb of 21, and its laevis MASP-3a sequence (Table II). The isoleucine/arginine intron/exon structure is identical with that reported for the human cleavage site that separates the A and B chains is conserved, as are and mouse MASP-2 genes (23, 24). Exon 1 encodes the 5ЈUT three residues that are important for catalytic activity (His, Asp, region and the initiation codon, exons 2 and 3 encode the signal and Ser, the so-called catalytic triad). All the N-glycosylation sites peptide and the CUBI domain, and exon 4 encodes the EGF-like are also conserved, as are all the cysteine residues, including the domain. Splicing between exons 4 and 5 generates the MAp19 two that form the methionine loop, a cystine bond around a me- mRNA. In mammals, the fifth exon encodes the last four residues thionine residue in the serine protease domain (Fig. 1). of MAp19 (EQSL), the stop codon, and the 3ЈUT region. In the No MASP-1-like clones were obtained, either directly from the chicken, exon 5 only contains the last nucleotide of the coding cDNA library or by 3ЈRACE using primers located in the A chain sequence, followed by the stop codon and the 3ЈUT region. Exons of MASP-3 (which is shared with MASP-1 in other species). 6 and 7 encode the CUBII domain of MASP-2, exons 8–11 encode

FIGURE 2. The cDNA-derived amino acid se- quences of chicken MASP-2 and MAp19. Predicted leader peptides are italicized. Features that are con- served in other species include the two N-glycosylation sites (underlined), all the cysteine residues shown in bold, the three residues that form the catalytic triad in MASP-2 (boxed), and the arginine/isoleucine cleavage site that separates the A and B chains of MASP-2 (in- dicated by an arrow). 5002 LECTIN PATHWAY IN G. gallus

Table III. Structure of the chicken MASP2 gene GALG00000007474 and TFR1_chick are found 3Ј of the chicken MASP-3 gene, but their human homologues (PCYTIA and TFRC) Size Codon Next Intron are located ϳ9 Mb upstream of the human MASP1/3 gene. These Exon Encodes (bp) Phase (bp) findings suggest that an early translocation event separated the 15Ј UT and ATG 57 II 270 MASP-1-specfic exons from the 3Ј end of the original MASP-1/3 2 Signal peptide and CUBI 220 0 1369 gene, and that the MASP-1-specific exons together with the ho- 3 CUBI 178 I 193 mologues of TM7L_HUMAN and RPL39L were either lost during 4 EGF-like 132 I 1437 5 Stop codon and 3Ј UT of MAp19 183 NAa 4611 translocation or disappeared at a later stage. 6 CUBII 197 0 559 We also identified genes encoding the two lectin pathway rec- 7 CUBII 145 I 554 ognition molecules in the chicken, MBL and a ficolin. Most mam- 8 CCPI 119 0 997 mals have two functional MBL genes, located on separate chro- 9 CCPI 79 I 311 10 CCPII 141 I 401 mosomes, although in humans, MBL1 is a pseudogene, and only 11 CCPII 72 I 782 MBL2 produces a protein (31). The chicken has just one MBL 12 Serine protease 1091 NAa gene, which is located on chromosome 6 in a conserved cluster that a NA, not applicable. includes the gene for pulmonary surfactant protein D. It is homol- ogous to the human pseudogene (MBL1) and the mouse and rat genes (Mbl1 and MABA_RAT). the two CCP domains, and exon 12 encodes the serine protease Humans have two ficolin genes, FCN1 and FCN2, arranged Downloaded from domain (Table III). The codon phases are conserved between the back-to-back on chromosome 9q34, and a third, FCN3, on chro- chicken and human genes (43). The region of chromosome 21 that mosome 1p35. FCN1 encodes M-ficolin, which is expressed on the contains the chicken MASP2 gene is syntenic with the MASP2 loci surface of immature macrophages, whereas FCN2 and FCN3 en- on human chromosome 1p36, mouse chromosome 4E1, and rat code L-ficolin and H-ficolin, which are both plasma proteins. The chromosome 5q36. All four loci contain the genes TARDBP, mouse homologues of FCN1 and FCN2 (Fcna and Fcnb, respec- MASP2, SRM, PMSCL2, and FRAP1 (or their homologues), in that tively) are arranged back-to-back on chromosome 2. Chickens http://www.jimmunol.org/ order (43, 44). have a single ficolin gene located on chromosome 17, which ap- The gene for chicken MASP-3 is located on . It pears to represent an undiversified ancestor of FCN1 and FCN2. Its comprises 11 exons, the organization of which is identical with putative protein sequence is 60% identical with that of L-ficolin that of the first 11 exons in the human and mouse MASP1/3 genes. (the product of FCN2) and 57% identical with that of M-ficolin Ј The first 10 exons encode the 5 UT region and the A chain of (the product of FCN1). MASP-3, and the 11th exon encodes the serine protease domain, stop codon, and 3ЈUT region. In the mammalian genes, exon 11 is followed by six additional exons, which encode the (TCN-type) Expression of the lectin pathway components serine protease domain of MASP-1. We looked for the equivalent In situ hybridization was used to analyze mRNA expression in by guest on September 28, 2021 exons in the chicken using the peptide sequences from the serine protease domains of human, mouse, and X. laevis MASP-1 as embryonic tissue (Fig. 4); Northern blotting and qRT-PCR were query sequences for TBLASTN searches of the chicken genome used to analyze adult tissue (Figs. 5–7). database. There is no evidence for MASP-1-specific exons in the In the chicken, as in other species (23, 24), MASP-2 and MAp19 chicken genome, either 3Ј of the MASP-3 gene or elsewhere in the mRNA expression is restricted to liver tissue (Fig. 4, A and B). Ј genome. (The closest hits were actually MASP-2 and anticoagu- Northern blotting using a cDNA probe comprising the 5 -coding lant protein C.) sequence for the A chain of MASP-2 revealed two mRNA species ϳ ϳ Examination of the sequence surrounding the MASP-3 gene re- in adult liver, the 2.5-kb MASP-2 message and the 0.8-kb vealed a possible explanation for the absence of MASP-1-specific MAp19 mRNA (Fig. 5A). Using a probe comprising the coding exons in the chicken genome. The region 5Ј of the chicken sequence for the B chain, only the 2.5-kb MASP-2 message was MASP-3 gene, which includes the chicken homologues of hsBCL6 seen (Fig. 5B). The qRT-PCR analysis showed that MAp19 and hsSST, is syntenic with the human MASP1/3 locus on chro- mRNA is ϳ3.5 times more abundant than the MASP-2 message in mosome 3q27, but the synteny ends immediately 3Ј of the adult liver (data not shown). MASP-3 gene (Fig. 3). TM7L_HUMAN is 20 kb downstream of Chicken MBL mRNA is restricted to the liver in both embryos the human MASP1/3 gene, followed by RPL39L and SIAT1.Inthe (Fig. 4I) and adult tissue. This is in contrast to murine and human chicken, the homologues of TM7L_HUMAN and RPL39L are MBL, which is expressed in hepatic and extrahepatic tissues (45). missing altogether, and the homologue of SIAT1 (SAI1_chick)is The putative chicken ficolin gene is expressed in embryonic and found 5.8 Mb 5Ј of the chicken MASP-3 gene. Likewise, ENS- adult liver tissue only (see Fig. 4J).

FIGURE 3. Comparison of the chicken MASP-3 and human MASP1/3 loci. Homologous genes are joined by dashed lines, and arrowheads indicate the di- rection of transcription. This figure is not to scale. The Journal of Immunology 5003

FIGURE 5. Expression of MASP-2 and MAp19 mRNA in chicken liver. Northern blots, prepared from chicken liver RNA, were hybridized with cDNA probes corresponding to the A chain (A) and the B chain (B) of MASP-2. Downloaded from (Fig. 4, F and G) (N. J. Lynch, unpublished observations). In day 12 chicken embryos, MASP-3 mRNA was detected by in situ hy- bridization in the liver, spleen, thymus, gizzard, mesonephros, and neuronal areas of the brain, but not in the lung. The same distri- bution and intensity of expression were observed using separate

cRNA probes for the A and B chains of MASP-3 (Fig. 4, C and D). http://www.jimmunol.org/ This result underlines the findings of the Northern blot analysis, showing that no evidence for a MASP-1-like transcript can be found. This is also confirmed by the results obtained by qRT-PCR anal- ysis of adult chicken tissues: MASP-3 expression was most abun- dant in the liver, followed by spleen, kidney, tonsil, duodenum, and brain. Again, using a combination of oligonucleotides that amplifies mRNA sequences specific for either the A chain or the B chain shows that identical abundances for MASP-3 A chain- and by guest on September 28, 2021 MASP-3 B chain-encoding mRNAs were detected and provided no evidence for the presence of a MASP-1-like transcript. More- over, the amount of A and B chain message was identical in all tissues tested (Fig. 6). Northern blot analysis using separate probes for the A and B chains of MASP-3 revealed a single 3.7-kb message in chicken liver, reinforcing the evidence that the MASP-1 transcript is miss- ing (Fig. 7, first lane). This is in contrast to what was seen in mammals, where both MASP-3 and MASP-1 mRNA transcripts were detected (Fig. 7, upper last lane) (20). Analyzing liver

FIGURE 4. Localization of lectin pathway mRNAs by in situ hybrid- ization. Cryostat sections (20 ␮m) were hybridized with 35S-labeled cRNA probes and exposed to Kodak BioMax MR film for 48 h. A–D, Day 12 chicken embryos hybridized with antisense probes for: MASP-2 B chain, MAp19, MBL, MASP-3 A chain, and MASP-3 B chain. E, Control section hybridized with a sense probe for MBL. F and G, Day 19 rat embryos hybridized with antisense probes for the B chains of MASP-1 and MASP-3. H, Control section hybridized with a sense probe for MASP-1 B chain. I and J, In situ hybridization results for chicken embryo sections obtained with antisense probes specific for the chicken lectin pathway rec- ognition molecules MBL (I) and ficolin (J). FIGURE 6. The qRT-PCR analysis of MASP-3 expression in adult chicken tissues. cDNA was prepared from the tissues indicated and was analyzed by real-time PCR using a LightCycler instrument. Results shown In humans, rats, and mice, MASP-1 expression is liver specific, are copies per microgram of RNA and are the means of four separate whereas MASP-3 is expressed in the liver and in nonhepatic tis- experiments (two cDNA syntheses per sample, two analyses per cDNA). sues, including the spleen, lung, small intestine, thymus, and brain Error bars represent the SD. 5004 LECTIN PATHWAY IN G. gallus

FIGURE 8. Enzymatic activity of MBL-MASP complexes from human and avian sera. Mannan-coated microtiter plates were used to capture MBL-MASP complexes from serum. The immobilized complexes were assayed for MASP-1-like activity using the fluorescent substrate Val-Pro- Arg-7-AMC. To assay MASP-2, human C4 was added to the plates, and the deposition of C4b was measured. Results shown are percentages of the activity measured in human serum and are the means of duplicate determinations. Downloaded from

matic form of MASP-2). Only one of these molecules has previously been reported in the chicken, namely MBL. In this study we present the most complete description of the lectin path- way in the chicken to date.

Laursen et al. (31) cloned the cDNA for chicken MBL and http://www.jimmunol.org/ FIGURE 7. Expression of MASP-3 mRNA in the livers of different (based on a phylogenetic comparison of its deduced amino acid birds. Poly(A)ϩ mRNA was prepared from the species indicated and an- sequence with those of its mammalian homologues) concluded that alyzed by Northern blotting. Blots were hybridized with ␣-32P-labeled it represents an undiversified ancestor of the two different forms of cDNA probes corresponding to the A chain (A) and the B chain (B; serine MBL found in mammals. Our findings support their conclusion; protease domain) of chicken MASP-3. As a control, rat liver RNA was the chicken has a single MBL gene located on chromosome 6. hybridized with the corresponding rat-specific probes encoding either the A Genomic analysis showed that it is more closely related to MBL1, or B chain of rat MASP-3 (right lanes). Distinct signals for MASP-1 (5 kb) Mbl1, and MABA-RAT than to MBL2, Mbl2, and MABC_RAT, sug- and MASP-3 (3.5 kb) were seen using the A chain probe. gesting that the first group represents the common ancestor. Like- wise, the chicken has a single ficolin gene that appears to represent by guest on September 28, 2021 an undiversified ancestor of FCN1 and FCN2. It is not particularly mRNA from other birds (including turkey, duck, goose, ostrich, surprising that the chicken has so few lectin pathway recognition and pigeon) on Northern blots suggested that the absence of molecules; the gene duplications that lead to the diversification of MASP-1 may be a general phenomenon in birds (Fig. 7). MBL and the ficolins were relatively recent evolutionary events (1), and such chromosomal rearrangements are thought to occur Enzymatic activity of MBL-MASP complexes in avian sera much more slowly in birds (and reptiles) than in mammals or am- We tested the enzymatic activity of MBL-MASP complexes cap- phibians (46, 47). Interestingly, chicken C1q (the recognition mol- tured from chicken and turkey sera. Serum samples were diluted in ecule of the classical pathway) appears to be a homotrimer, en- a high salt buffer, which dissociates C1, thus avoiding interference coded by a single gene on chromosome 21. In this respect, it is from the classical pathway serine proteases, C1r and C1s (40). The more closely related to the ancestral form found in the lamprey diluted sera were added to ELISA plates coated with mannan, a than to mammalian C1q, which is a heterotrimer of three different polysaccharide that binds MBL-MASP complexes, then the bound polypeptides encoded by three separate genes (48). complexes were assayed for MASP-1 and MASP-2. MASP-1-like The serine proteases found in the classical and lectin pathway activity was measured using the fluorescent substrate, Val-Pro- activation complexes can be divided into two evolutionary groups: Arg-7-AMC, which is cleaved by MASP-1, but not by MASP-2 the ancestral TCN type (which includes MASP-1 and the ascidian (42). The activity of MASP-2 was measured by following the MASPs) has a TCN codon for the serine in its catalytic triad and cleavage of human C4. split exons for the protease domain, whereas the AGY type (which MASP-1-like activity could be detected in MBL-MASP com- includes MASP-2, MASP-3, and C1s) uses AGY to encode its plexes from human serum, which was included as a control, but active serine and has a single exon for the protease domain (49). not in the avian sera, whereas MASP-2-like activity was present in The AGY type evolved after, and appears to be derived from, the all sera tested (Fig. 8). Subsequent experiments showed that MBL- gene rearrangement that gave rise to the alternatively spliced MASP complexes from goose and ostrich sera also lack MASP- MASP-1/3 gene (1, 21). 1-like enzymatic activity (data not shown). As expected, chicken MASP-2 is an AGY-type serine protease. Its primary structure shows 54% identity with those of human and Discussion mouse MASP-2. The organization of its gene is highly conserved; Most vertebrates have three or four different lectin pathway rec- the intron-exon structure and codon phases are identical with those ognition molecules, with differing, but overlapping, carbohydrate of the human and mouse genes (Tables II and III). As in other specificities that allow them to recognize a broad spectrum of mi- species, chicken MAp19 is an alternatively spliced product of the crobial polysaccharides. The recognition molecules form com- MASP-2 gene. It is slightly unusual in that it lacks the four-residue plexes with three MASPs and MAp19 (the truncated, nonenzy- C-terminal extension found in mammalian MAp19 (22–24). In The Journal of Immunology 5005 mammals these four residues (EQSL) are encoded by exon 5, Jim Kaufman (Institute for Animal Health, Compton, U.K.) for kind at- which is unique to MAp19; in the chicken, exon 5 encodes only the tention and most valuable comments on the manuscript. third nucleotide of the last residue (which is identical with that of MASP-2), the stop codon, and the 3ЈUT region. Disclosures MASP-3 is the most highly conserved protein in the activation The authors have no financial conflict of interest. complexes; the primary structure of the chicken MASP-3 is ϳ75% identical with those of the human, mouse, and Xenopus proteins. References Its expression is more widespread than that of the other lectin 1. Fujita, T. 2002. Evolution of the lectin-complement pathway and its role in innate pathway components; significant amounts of MASP-3 mRNA are immunity. Nat. Rev. Immunol. 2:346. 2. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. 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