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of June 14, 2017.
A CD46-like Molecule Functional in Teleost
Fish Represents an Ancestral Form of
Membrane-Bound Regulators of
Complement Activation
Masakazu Tsujikura, Takahiro Nagasawa, Satoko Ichiki,
Ryota Nakamura, Tomonori Somamoto and Miki Nakao
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Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2015; 194:262-272; Prepublished online 1
December 2014;
doi: 10.4049/jimmunol.1303179
http://www.jimmunol.org/content/194/1/262
The Journal of Immunology
A CD46-like Molecule Functional in Teleost Fish
Represents an Ancestral Form of Membrane-Bound
Regulators of Complement Activation
Masakazu Tsujikura, Takahiro Nagasawa, Satoko Ichiki, Ryota Nakamura,
Tomonori Somamoto, and Miki Nakao
C
omplement system is one of humoral innate immune
factors that play a main role in tagging and killing of
microbe invaders, as well as modulation of inflammatory
and adaptive immune responses (1, 2). Three distinct activation
cascades with different mechanisms in pathogen sensing trigger
the complement activation, as follows: the classical, lectin, and
alternative pathways. These proteolytic cascades form two types
of enzyme complexes, or C3 convertases (C3bBb and C4b2a), to
cleave C3 into biologically active fragments, C3a and C3b. Although C3a induces inflammatory anaphylatoxic responses, C3b
can covalently bind target microbes, playing a role as a tag for
opsonization and as an anchor for C5 convertase, which triggers
Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology,
Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
Received for publication November 25, 2013. Accepted for publication November 2,
2014.
This work was supported in part by Japan Society for the Promotion of Science
KAKENHI Grant 22380111 (to M.N.).
The sequences presented in this article have been submitted to the DNA Data Base in
Japan/European Molecular Biology Laboratory/GenBank (http://www.ncbi.nlm.nih.
gov/genbank) under the following accession numbers: zTecrem, AB723859 and cTecrem,
AB723858.
Address correspondence and reprint requests to Dr. Miki Nakao, Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University,
Hakozaki, Fukuoka 812-8581, Japan. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: C4bp, C4-binding protein; CHO, Chinese hamster
ovary; cTecrem, carp Tecrem; DAF, decay-accelerating factor; GGVB, glucose gelatin veronal buffer; GVB, gelatin veronal buffer; MCP, membrane-cofactor protein;
NTR, netrin domain; RCA, regulator of complement activation; rcTecrem, recombinant carp Tecrem; SCR, short consensus repeat; STP, Ser/Thr/Pro-rich; Tecrem,
teleost complement-regulatory membrane protein; zTecrem, zebrafish Tecrem.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303179
formation of the cytotoxic membrane-attack complex. These potent physiological activities are crucial for effective pathogen
elimination, but may potentially cause damage of host cells in
case of excessive or misdirected activation, resulting in allergic or
autoimmune pathology (1, 3–6).
The activation of C3 is therefore tightly controlled by a redundant set of regulatory factors, such as members of the regulator of
complement activation (RCA) protein family. RCA proteins are
characterized by their modular architecture consisting of the
multiple copies of short consensus repeat (SCR) modules, each of
which is composed of ∼60-aa residues containing typical distribution of four Cys residues and some other consensus residues (1,
7, 8). In humans, secreted RCA proteins such as factor H and C4binding protein (C4bp) and membrane-bound proteins including
membrane-cofactor protein (MCP or CD46), decay-accelerating
factor (DAF or CD55), and complement receptors type 1 and 2
(CR1 or CD35, and CR2 or CD21) have been identified (8). Their
modes of action are categorized into two, as follows: decay acceleration of C3 convertase complex and degradation of C3b/C4b
fragments as cofactor of the degradation protease, or factor I (8).
CR1/CD35 acts as a regulator in both modes and also as a receptor
to mediate phagocytosis (9) and immune adherence (10–12),
whereas CR2/CD21 functions as a C3d receptor with no regulatory role in the complement activation (8). In addition to the roles
in innate immunity, some membrane-bound RCA, MCP/CD46
and CR2/CD21, have been recognized as mediators of adaptive
immune responses, affecting Th1/Th2 balance, Ag presentation,
and B cell activation in mammals (13–18).
Phylogenetically, genes encoding the RCA proteins are classified
into two groups, as follows: group 1 composed of factor H and its
related factors, and group 2 containing the others in the human
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In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement
activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned
from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors.
Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little
is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized
for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus
repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus,
as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory
role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher
tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than
control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on
autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2
RCA in bony fish, implying the host–cell protection is an evolutionarily conserved mechanism in regulation of the complement
system. The Journal of Immunology, 2015, 194: 262–272.
The Journal of Immunology
Identification of Tecrem protein of zebrafish
TBLASTN searches of Zebrafish genome database (http://www.ncbi.nlm.
nih.gov/projects/genome/guide/zebrafish/) were conducted using amino
acid sequence of the human CR1/CD35, CR2/CD21, factor H, DAF/CD55,
MCP/CD46, and C4bp (a-chain and b-chain). Intron–exon organization
was predicted using Softberry program (http://www.softberry.com/berry.
phtml).
Preparation of cDNA
Various organs (intestine, gill, kidney, spleen, hepatopancreas, brain, skin,
muscle, and heart) were isolated from the common carp and zebrafish. Total
RNA was extracted from the organs using ISOGEN reagent (Nippon Gene,
Tokyo, Japan), according to the manufacturer’s instructions, and 1 mg RNA
was reverse transcribed using Moloney murine leukemia virus reverse
transcriptase (Invitrogen, Life Technologies, Carlsbad, CA) and oligo-dT
primer. The first-strand cDNA was used as template for expression analysis.
Cloning and sequencing of Tecrem from zebrafish and carp
Gene-specific primers (Table I) for 59- and 39-RACE, designed according
to the coding sequence of zebrafish Tecrem (zTecrem) predicted from the
database, were used for RACE PCR, using SMART RACE cDNA Amplification Kit (Clontech, Tokyo, Japan) and first-strand cDNA synthesized
from total RNA mixture from the intestine, gill, kidney, gonad, spleen,
hepatopancreas, brain, skin, muscle, and heart. Nested PCR amplifications
were performed using Phusion DNA polymerase (New England Biolab,
Tokyo, Japan) under the following conditions: for first 39-RACE, 30 cycles
of 98˚C for 10 s, 65˚C for 30 s, and 72˚C for 1.5 min; for nested 39-RACE,
30 cycles of 98˚C for 10 s, 60˚C for 20 s, and 72˚C for 1.5 min; for first 59RACE, 30 cycles of 98˚C for 10 s, 55˚C for 30 s, and 72˚C for 1.5 min; and
nested 59-RACE, 30 cycles of 98˚C for 10 s, 60˚C for 20 s, and 72˚C for
1.5 min. The final amplified products were subcloned into pGEM-T vector
(Promega, Madison, WI). More than five clones were independently sequenced on both strands to correct possible PCR errors, using CEQ8800
DNA Analysis system and Dye terminator cycle sequencing kit (Beckman
Coulter, Tokyo, Japan).
RACE PCR of carp Tecrem (cTecrem) was performed similarly but
using primers based on a carp expressed sequence tag (accession number EH649440) homologous to zTecrem, and sequenced essentially as
above.
Materials and Methods
Southern hybridization
Materials
Genomic DNA (10 mg), isolated from carp erythrocytes according to
a standard procedure, was digested to completion at 37˚C for 16 h with 100
U PstI (New England Biolab, Tokyo, Japan) and electrophoresed on a 1%
agarose gel. The DNA fragments were transferred to a Hybond N+
membrane and hybridized with digoxigenin-labeled cDNA probe specific
for cTecrem, detected using chemiluminescent substrate of alkaline
phosphatase, closely, as described previously (38).
Mouse IgG1 mAb (AD1.1.10) conjugated with FITC against 63His tag was
purchased from Abcam. Zebrafish were provided by Y. Yoshiura (National
Research Institute of Aquaculture, Fisheries Research Agency, Mie, Japan)
and kept at 27˚C fed with commercial diets. Common carp (Cyprinus
carpio) weighing 0.2–1 kg was purchased from a local fish farm. Carp
serum was isolated, as described previously (36). Ginbuna crucian carp
(Carassius auratus langsdorfii, S3N strain), a close relative species of the
common carp, weighing ∼50 g, has been maintained in our laboratory, as
described elsewhere (37). CHO cells were a gift of H. Tachibana (Department of Bioscience and Biotechnology, Kyushu University) and were
maintained in Ham’s F12 medium supplemented with 10% FBS. KF-1
cells (Koi carp fin cell line) were maintained in MEM supplemented
with 2 mM glutamine, 14 mM HEPES, and 10% FBS at 20˚C in 5% CO2.
Preparation of anti-CHO carp IgM
CHO cells (1.0 3 107) were collected in 10 mM EDTA in PBS, washed with
PBS three times, and suspended in 1 ml PBS. Then the CHO suspension was
emulsified in 1 ml CFA and i.p. injected into carp six times at weekly
intervals. Next, the carp was bled to separate antiserum. Anti-CHO carp IgM
was purified from the antiserum by gel filtration on a Superdex 200 pg
column (1.6 3 60 cm) equilibrated with 2.5 mM veronal-buffered isotonic
saline (pH 7.4) containing 2.5% glucose, and stored at 280˚C until use.
Preparation of anti-carp erythrocyte ginbuna crucian carp
serum
Carp erythrocytes were separated from heparinized blood, suspended in
PBS at 1.0 3 107 cells/ml, and i.p. injected into ginbuna crucian carp four
times at weekly intervals at a dose of 0.5 ml suspension/100 g body weight.
The fish were bled 7 d after the last injection, and the sera separated were
heat inactivated at 50˚C for 20 min. Sera from three fish, showing agglutination titer of .128 against carp erythrocytes, were pooled and stored
at 280˚C until use.
Tissue expression analysis
Expression sites of Tecrem were analyzed by RT-PCR with primers GSP2
and GSP3 for zebrafish and GSP9 and GSP10 for carp (Table I). Zebrafish
GAPDH and carp 40S ribosomal protein S11 subunit cDNA served as
a positive control for respective species (Table I). After reverse transcription from the total RNA with Moloney murine leukemia virus reverse
transcriptase and oligo-dT primer, PCRs were performed under the following conditions: initial denaturation for 2 min at 94˚C and 30 cycles of
94˚C for 30 s, 55˚C for 30 s, and 72˚C for 1 min, followed by a final
extension at 72˚C for 2 min. The amplified products were run on a 2%
agarose gel and stained with 0.5 mg/ml ethidium bromide.
Cloning and sequencing of zTecrem splicing variants
cDNA fragments encoding from SCR3 to 39-UT of zTecrem were amplified
from various tissues as above and subcloned into pGEM-T vector. Resultant clones containing inserts with four different lengths, ranging from
∼770 to ∼900 bp, were sequenced, and the nucleotide sequences were
compared with zebrafish genomic DNA sequences to identify exons, using
blastn search at Ensembl genome server (http://asia.ensembl.org/Multi/
Tools/Blast).
Flow cytometry
Recombinant carp Tecrem (rcTecrem) transformant and mock transformant
of CHO cells, carp leukocytes, and KF-1 cells were treated with appropriate
first Ab and FITC-labeled anti-mouse IgG, and analyzed with Beckman
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RCA members (19). Loci of each group form a compact cluster,
and, in the human genome, the group 1 cluster and the group 2 loci
are also located in proximity, forming a RCA gene cluster (20–
22). Genomic arrangement of the RCA genes and their clustering
are diverse among species analyzed to date, such as mice (23, 24),
chicken (25, 26), frog (27), and teleost, which is the most common
group of the ray-finned bony fish (28–34).
In a functional point of view, the secreted RCAs are considered
to control excessive complement activation on target surface,
whereas the membrane-bound RCAs should protect host cells from
misdirected complement attack. Both are in combination essential
to prevent autoimmune damages of bystander host tissues from the
anaphylatoxic and cytotoxic attack by the complement (1).
Phylogenetic and genomic lines of evidence have indicated
that RCA had emerged in cyclostome, the most ancient class of
vertebrate (35), and that bony fish are equipped with both group
1 and group 2 RCAs, because factor H–like secreted RCAs and
membrane-bound RCAs have been found in zebrafish genome (33,
34). As for function of RCA in the lower vertebrates, regulatory
roles in deposition of C3 and degradation of C3b/C4b have been
described only for secreted RCAs in lamprey (35) and barred sand
bass (28–30). No functional data, in contrast, have been reported
for membrane-bound RCA protein in any lower vertebrate species.
The present study has aimed at obtaining functional data for
complement-regulatory roles of membrane-bound RCA molecule
at both mRNA and protein levels. Molecular cloning of membranebound RCA, designated teleost complement-regulatory membrane
protein (Tecrem), from zebrafish and carp, tissue distribution of
Tecrem mRNA, recombinant Tecrem expression on Chinese hamster
ovary (CHO) cells, and functional assay using the Tecrem-bearing
CHO have gained a line of evidence that Tecrem is expressed
ubiquitously in fish tissues playing a protective role from possible
host damage by complement.
263
264
TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS
Table I. Primers used for this study
Primer Name
Sequence
Information Regarding Primers
GSP1
GSP2
GSP3
GSP4
GSP5
GSP6
GSP7
GSP8
Universal primer
Nested universal primer
GAPDH forward
GAPDH reverse
40S rRNA forward
40S rRNA reverse
GSP9
GSP10
GSP11
GSP12
59-AAAGAGTGTGCCGCGATGGA-39
59-GCGGCACCACCATCCATAGAA-39
59-TTTGCGGCGTGAAATCAGTC-39
59-GGTGCGGAAGACGATGATGA-39
59-CCCTGATCTCGGCATGTTCTTGT-39
59-GCTTTTGGGTCAACTGGCTTGTG-39
59-GCCCTCAATCTGAATCCAGCACA-39
59-CAAGATGGGCTGTAAGGTGC-39
59-CTAATACGACTCACTATAGGGC-39
59-AAGCAGTGGTATCAACGCAGAGT-39
59-AGGCCTCTCACAAACGAGGA-39
59-GCATGACCATCAATGACCAG-39
59-AGACGGGACTACTTGCATTA-39
59-ATTGAACCTCACTGTCTTGC-39
59-CCCAAGCCTCCTAAAAGGAC-39
59-CAGTTGCCACCTTGTCAGAA-39
59-GTGAAATGTTCAGCACCTCCAG-39
59-TGACAATGCATTGTGGTGGTTC-39
Cloning
Cloning/expression analysis (zebrafish)
Cloning/expression analysis (zebrafish)
Cloning
Cloning
Cloning
Cloning
Cloning
Cloning
Cloning
Expression analysis
Expression analysis
Expression analysis
Expression analysis
Expression analysis (carp)
Expression analysis (carp)
DIG-probe synthesis and genomic PCR
DIG-probe synthesis and genomic PCR
DIG, digoxigenin.
Establishment of stable CHO transformants expressing
rcTecrem
A stable CHO cell line expressing rcTecrem (CHO-rcTecrem) was established as follows: cDNA encoding the cTecrem cDNA lacking putative
signal peptides was amplified from the plasmid containing full-length
cTecrem cDNA, using a sense primer with a 63His tag-coding adapter
and an antisense primer corresponding to the stop codon. A sequence
encoding the cTecrem signal peptide was also PCR amplified. These
cDNA segments were cloned into the sites BamHI and EcoRI of pcDNA
3.1 Myc/His A vector (Invitrogen). The recombinant plasmid and corresponding empty vector were separately introduced to CHO cells using
Lipofectamin and Plus reagent (Invitrogen), and neomycin-resistant
transformed cells were selected by culture in the presence of 0.4 mg/ml
G418 (Invitrogen).
Calcein release assay for complement-mediated cytotoxicity
Calcein release cytotoxicity test was performed, as described elsewhere
(35). Briefly, transfected CHO cells (1.0 3 104 cells) were seeded in each
well of 96-well plates and cultured overnight at 37˚C in 5% CO2. Cells
were then washed with 0.1 ml PBS three times and labeled with 10 mM
calcein-AM (Molecular Probes, Eugene, OR) by incubating at 37˚C for
30 min. The labeled cells were washed three times with 0.1 ml isotonic
veronal buffered saline (pH 7.4) containing 2.5% glucose, 0.1% gelatin,
0.15 mM CaCl2, and 1 mM MgCl2 (glucose gelatin veronal buffer
[GGVB]2+) and incubated with 25 ml 70 mg/ml anti-CHO carp IgM at
25˚C for 30 min. Then 25 ml normal carp serum diluted with GGVB2+ was
added, and the plate was incubated at 25˚C for 60 min. The cytolytic reaction was halted by adding 100 ml GGVB containing 10 mM EDTA, and
the plate was centrifuged at 1200 rpm for 5 min. Supernatant (100 ml) was
transferred to a new plate to measure fluorescent intensity with excitation
at 495 nm and emission at 535 nm. CHO cells incubated without normal
carp serum were used for measuring spontaneous calcein release (cont),
and cells lysed with 1% SDS were used for measuring maximum calcein
release (max). The experiment was performed three times in quadruplicate.
Cytotoxicity was calculated as follows:
Cytotoxicityð%Þ ¼ 100 3 Fsample Fcont ðFmax Fcont Þ
where F stands for fluorescent intensity.
Preparation of recombinant carp C4 isotypes and their specific
Abs
The netrin domain (NTR)-encoding cDNA segments were amplified from
respective full-length cDNA clones of carp C4-1 and C4-2 isotypes (39),
using primers with adaptor sequences containing BamHI and SacI sites.
The amplified product was subcloned into pCold-I vector and, after se-
quence confirmation, introduced to Origami B strain. Recombinant C4-1NTR and C4-2-NTR of carp were expressed for 24 h at 15˚C in the
presence of 0.1 mM isopropyl b-D-thiogalactoside and purified from the
culture supernatant by affinity chromatography on Ni-NTR–agarose columns, essentially following the manufacturer’s instructions (Supplemental
Fig. 1A).
The purified rC4-1-NTR and rC4-2-NTR were emulsified in CFA and s.c.
injected into rabbits, four times at weekly intervals. The rabbits were
boosted and 1 wk later bled for separation of antiserum and purification of
IgG on a HiTrap protein A column (GE Health Science, Tokyo, Japan).
Anti-carp C4-1 and anti-carp C4-2 are monospecific and showed no crossreactivity (Supplemental Fig. 1B, 1C).
C3-deposition assay on CHO cells
CHO and CHO-rcTecrem (1.0 3 107 cells) were harvested in PBS containing 10 mM EDTA (pH 7.4), resuspended in GGVB2+ or EDTA-GVB
at 2.0 3 106 cells/ml, and incubated at 25˚C for 30 min with 200 ml
normal carp serum diluted 1/10–1/80 with GGVB2+ or EDTA-GVB. Each
cell suspension was divided into two aliquots, which were incubated at
room temperature for 30 min with 100 ml anti-carp C3 isotype mAb (5C7,
1 mg/ml; 3H11, 10 mg/ml) (40). The cells were then washed with PBS
(pH 7.4) in an Ultrafree-MC centrifugal filter tube (Millipore), incubated
with 120 ml 1/60-diluted anti-mouse IgG (whole molecule) F(ab9)2 fragment–FITC conjugate (Sigma-Aldrich), and analyzed by flow cytometry,
as above.
Preparation of mAb to cTecrem
BALB/c mice were immunized four times at weekly intervals by i.p.
injections of CHO-rcTecrem (1.0 3 107 cells) suspended in PBS, and, 2 d
after a booster injection with the same dose, spleen cells harvested from
the mice were fused with Sp2/O-Ag14 myeloma cells using PEG4000,
following a standard method. Hybridomas grown in GIT medium containing hypoxanthine/aminopterin/thymidine supplements were screened
by ELISA of the culture supernatants using microtiter plates coated with
CHO-rcTecrem, CHO, and KF-1 cell lines. Hybridomas positive to both
CHO-rcTecrem and KF-1, but negative to CHO, were chosen and cloned
by the limit-dilution method.
mAb was purified from the culture supernatant using HiTrap protein G
(GE Health Science, Tokyo, Japan), according to the manufacturer’s
instructions.
Immunoprecipitation
CHO-rcTecrem cells (2.0 3 107 cells) were lysed by sonication in 3 ml
PBS containing 3.3% Triton X-100 and 1 mM PMSF on ice, and the lysate
was cleared by centrifugation. The supernatant was, after absorption with
40 ml protein G-Sepharose, incubated with 16 mg anti-cTecrem mAb 1F12
at 4˚C for 1 h. The immune complexes formed were trapped in 40 ml
protein G-Sepharose, which were washed with 1% Nonidet P-40 in 10 mM
Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, and 10 mM NaF (pH 7.4), and
boiled in 50 mM Tris-HCl, 2% SDS, 5% 2-ME, and 10% glycerol (pH 6.8)
for SDS-PAGE analysis.
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Coulter Epics XL Flow Cytometer equipped with System II software
(Beckman Coulter). Obtained histogram data files were analyzed with
EXPO 32 MultiCOMP software (Beckman Coulter).
The Journal of Immunology
265
Flow cytometric deposition assays of carp C1q, C3-S, C4-1,
and C4-2 on carp erythrocytes
Phylogenetic relationship of Tecrem and other RCA family
members
Carp erythrocyte suspension (2 3 108 cells/ml) was incubated with 1/10diluted anti-carp erythrocyte ginbuna crucian carp antiserum in RPMI
1640 at 25˚C for 30 min and washed with RPMI 1640 three times. The
sensitized carp erythrocyte suspension was incubated at 25˚C for 30 min
with 0.5 mg/ml anti-cTecrem mAb 1F12 in RPMI 1640 or with normal
murine IgG1 as a control, washed twice with RPMI 1640, and then with
normal carp serum (1/10 diluted in RPMI 1640) at 25˚C for 60 min to
activate the classical pathway of carp complement. The serum-treated carp
erythrocytes were incubated with anti-carp C1q A-chain (41), anti-carp C3,
anti-carp C4-1, and anti-carp C4-2 rabbit IgG (10 mg/ml) at 4˚C for
30 min, and then with FITC-labeled anti-rabbit IgG (1/200 dilution), and
analyzed by flow cytometry, as above.
Phylogenetic relationship of Tecrems with other RCA protein family
members was assessed by drawing a neighbor-joining tree, as shown
in Fig. 3. In fair agreement with a previous report, cTecrem and
zTecrem form a tight cluster branched from a common ancestor of
the group 2 genes of vertebrate RCA, suggesting that Tecrem may
represent an ancestral form of group 2 RCA molecules, such as
MCP/CD46, CR1/CD35, CR2/CD21, DAF/CD55, and C4bps.
Results
Molecular cloning of membrane-type RCA from zebrafish and
carp
Splicing variants of zTecrem
On the result of RT-PCR, we found doublet bands in several tissues,
suggesting that they are generated by alternative splicing of
mRNA. To test the possibility, a cDNA region from SCR3 domain
to 39-UTR of zTecrem was amplified by RT-PCR with the primer
set GSP2 and CSP3 and subcloned into pGEM-T vector, and
randomly selected 24 clones were sequenced. As a result, cDNA
inserts of four different lengths (823, 773, 916, and 866 bp) were
obtained and designated splicing variants 1, 2, 3, and 4, respectively. Comparison of these nucleotide sequences with that of
a zebrafish genomic DNA contig, NW_001878409, revealed an
additional 93-bp exon encoding 31 aa rich in Ser/Thr/Pro, and the
four types of alternative splicing variants differing in lengths of
the STP region and cytoplasmic region were identified, as summarized in Fig. 2; zTecrem and ZRC1 (34) correspond to types 1
and 2, respectively.
We have examined a possibility of having multiple Tecrem genes
in carp genome, because carp is known as a pseudotetraploid species (42, 43), probably generated by allotetraploidization. Thus, copy
number of Tecrem gene was analyzed by Southern hybridization and
PCR analysis of carp genome DNA. Genomic Southern hybridization
with a 352-bp digoxigenin-labeled probe covering entire SCR3 and
SCR4 gave three bands with approximate size of 16, 12, and 9 kbp
(Supplemental Fig. 2A).
In contrast, a genomic DNA segment corresponding to the third
and fourth SCR was PCR amplified using primers 11 and 12
(Table I) and subcloned into pGEM-T vector. Sequencing of 12
clones, randomly selected, resulted in identification of three distinct sequences, designated types A, B, and C (Supplemental Fig.
2B). The appearance of three hybridization bands and three different Tecrem genomic sequences strongly suggests the presence
of at least two copies of Tecrem genes in carp genome.
Establishment of anti-cTecrem mAb
A hybridoma clone, 1F12, producing IgG1 reactive to CHO-rcTecrem
and KF-1, but not to CHO-mock, was established (Fig. 4A). Specificity of 1F12 was assessed by ELISA and immunoprecipitation
of the Ag from CHO-rcTecrem. As shown in Fig. 4B, 1F12
trapped a 66-kDa polypeptide that was also recognized by anti63His tag. Difference in the observed molecular mass and theoretical value (37.6 kDa) of rcTecrem (including 63His tag, but
excluding the signal peptide) suggests that the mature rcTecrem
protein expressed on CHO cell is heavily O-glycosylated at the
STP-like region, as reported for mammalian CD46 (44).
Expression of Tecrem protein on carp blood cells was examined
by flow cytometry using 1F12 mAb. As shown in Fig. 4C and 4D,
all the erythrocytes and leukocytes from peripheral blood and
leukocytes from the kidney carry Tecrem on their surfaces with no
significant population of Tecrem-negative cells.
Expression sites of Tecrem genes
Tecrem expression in various tissues of zebrafish and carp was
investigated by RT-PCR. As a result, Tecrem mRNA was detected
from all the tissues examined, suggesting that Tecrem is expressed
ubiquitously in both species (Fig. 5). Doublet bands of zTecrem
were detected in several tissues, probably representing the alternatively spliced variants.
Inhibition of complement activation by Tecrem on CHO cells
Predicted complement-regulatory function of Tecrem was examined
using a cytolytic reaction by carp complement. Calcein-labeled
CHO cells were sensitized with carp Abs and attacked by normal
carp serum mainly via the Ab-dependent classical pathway. Flow
cytometry of rcTecrem trasformants using anti-His tag Ab showed
that .70% of cells were rcTecrem positive, whereas there is
a substantial variation in the expression level (Fig. 6A).
Inhibitory effect of Tecrem on complement-mediated cytolysis
was assessed by calcein-release assay using CHO-rcTecrem and
CHO mock sensitized with anti-CHO carp Ab. As shown in
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Our database search resulted in identification of two RCAencoding loci essentially identical to those reported in the preceding paper. One of them, corresponding to a membrane protein
previously named ZRC1, was subjected to cDNA cloning from
zebrafish mixed organ mRNA. A full-length cDNA sequence
(2281 bp) containing an open reading frame encoding 385 aa was
cloned by using RACE PCR. The predicted primary structure
specified a type I membrane protein composed of, from the N
terminus, a signal peptide, five SCR modules, a short Ser/Pro-rich
region, a transmembrane region, and a cytoplasmic domain
(Fig. 1A), thus designated teleost complement-regulatory membrane protein (Tecrem). The translated product of zTecrem is
different from that of previously reported ZRC1 in that zTecrem
possesses a cytoplasmic domain sequence containing a few
phosphorylation sites, whereas ZRC1 lacks the cytoplasmic domain (34).
The zTecrem amino acid sequence was used to search its carp
homolog by TBLASTN program, gaining two expressed sequence
tag fragments (EG649440 and CA964873) that share ∼50% amino
acid sequence identity with zTecrem. On the basis of these nucleotide sequences, primers for 59-, 39-RACE PCR were designed,
and a complete cDNA sequence (2341 bp) encoding 362 aa of
cTecrem was isolated (Fig. 1B). The primary structure of cTecrem
specifies a domain structure homologous to that of zTecrem, except that cTecrem has four SCR modules, followed by a region
rich in Thr and Pro residues in the extracellular domains. Based on
the overall domain architecture, zTecrem and cTecrem molecules
are most similar to MCP/CD46, a type I membrane protein containing four SCR modules and a Ser/Thr/Pro-rich (STP) region,
when compared with the human RCA homologs.
Duplication of Tecrem gene in carp
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TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS
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FIGURE 1. Nucleotide and deduced
amino acid sequences of Tecrem from
zebrafish (A) and carp (B). Asterisks
indicate stop codons. The region
encoding SCR modules, transmembrane
(TM), and cytoplasmic region (CYR) is
indicated by arrows. The putative phosphorylation sites for protein kinase C
(PKC), casein kinase 2 (CK2), mitogenactivated kinase kinase (MAP2K), and
G-coupled receptor kinase (GRK) are
shaded. The polyadenylation signals are
shown by italic and bold.
Fig. 6B, CHO-rcTecrem showed significantly lower level of cytolysis by carp complement than that of mock-control cells, suggesting that Tecrem negatively regulates complement activation.
In the C3-deposition assay, incubation of normal carp serum led
to deposition C3-S isotype on the target cells, and this binding was
substantially lower on Tecrem transformant than on mock control,
indicating that Tecrem downregulates deposition of C3-S isotype
(Fig. 6C). This assay system did not work for C3-H1, another
major isotype of carp C3, because C3-H1 did not show any significant binding to CHO under the conditions that allowed complement activation (data not shown).
Tecrem inhibits deposition of autologous C3 and C4 after the
classical pathway activation
Regulation of C3 and C4 deposition by Tecrem on autologous cells
was assayed using carp erythrocytes sensitized with antiserum
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267
raised in ginbuna crucian carp, a close relative of the common carp.
As shown in Fig. 7, the ginbuna crucian carp IgM was capable of
triggering the classical pathway of carp complement on carp
erythrocytes, as evidenced by binding of C1q A-chain, which was
not affected by treatment with anti-cTecrem mAb. Deposition of
C3 and the two C4 isotypes, C4-1 and C4-2, was also detected on
the sensitized carp erythrocytes, and they were increased by
treatment with anti-cTecrem, suggesting that Tecrem plays a role
in restriction of C3 and C4 deposition on autologous cells.
Discussion
Our attempts to discover membrane-bound types of RCA protein
have been conducted on zebrafish independently from the group
that published recently two RCA molecules (ZRC1 and ZRC2), both
of which belong to group 2 of RCA family (34). We have tentatively
named the membrane-bound RCA as Tecrem and found that
zTecrem and ZRC1 are transcripts from the same gene. We have
also cloned its carp homolog for further phylogenetic and functional studies and revealed that cTecrem is closely similar to
zTecrem, but differs in the domain structure such as number of
SCR modules (4 in carp, 5 in zebrafish). The diversity in the
number of SCR modules is common in the member of RCA family
(45, 46), and a minimum functional unit as a binding site usually
spans only a single or two SCR modules (47–55). Thus, it seems
unlikely that the difference in the number of SCR modules would
significantly affect functions of Tecrem between the two species.
We found four types of splicing variants of zTecrem that differ in
the serine/threonine-rich and the cytoplasmic regions. cTecrem,
which contained a 49-aa residues-long threonine/proline-rich region and cytoplasmic region, resembled splicing variant 3 of
zTecrem in molecular architecture (Fig. 2B). It is intriguing to note
that human MCP/CD46 gene also produces very similar alternative splicing variants differing in the STP-rich region and the
cytoplasmic region (56), implying tight evolutionary conservation
of this type of splicing variant. In the phylogenetic tree analysis,
Tecrem branches from the root of group 2 RCA molecules, which
diverge to mammalian MCP/CD46, DAF/CD55, CR1/CD35, CR2/
CD21, and C4bp subunits. In addition, whereas tetrapod vertebrates such as frogs, birds, and mammals possess both MCP/
CD46-like type I membrane protein RCAs and GPI-anchored
RCAs, there is no indication for the existence of a GPI-anchored
RCA in teleost. Taken together, it is conceivable that Tecrem rep-
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FIGURE 2. Generation of alternative splicing products of zTecrem. (A) Exon–intron organization of zTecrem gene. Closed and open rectangles show
untranslated region and coding region, respectively. Exon and intron size are indicated by number on exon and between exons. Sequences of additional
exons are shown below exon–intron organization. (B) Domain structures of zTecrem splicing variants (Sp var. 1–4) and cTecrem. CYR, cytoplasmic region;
SP, signal peptide; TM, transmembrane region.
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TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS
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FIGURE 3. Phylogenetic tree of RCA proteins. Phylogenetic tree was constructed by neighbor-joining method using amino acid sequence of identified
two kinds of Tecrem, factor H–like molecule, and RCA proteins (C4bp a-chain, C4bp b-chain, factor H, DAF/CD55, MCP/CD46, CR1/CD35, CR2/CD21,
Crry, and Cremp) of higher vertebrates. Database accession numbers for sequences employed are as follows: human factor H, NP_000177; human factor H–
related protein 1, NP_002104; human factor H–related protein 2, NP_005657; human factor H–related protein 3, NP_066303; human factor H–related
protein 4, NP_006675; human factor H–related protein 5, NP_110414; rat factor H, NP_569093; mouse factor H, NP_034018; mouse factor H–related
protein, AAA37416; cow factor H, NP_001029108; wild boar factor H, NP_999446; chicken factor H, XP_426613; Xenopus tropicalis factor H,
NP_989218; barred sandbass sand bass cofactor protein, AAA92556; barred sandbass sand bass cofactor-related protein, CAA67355; rainbow trout factor
H like, CAF25505; human coagulation factor XIII b subunit, NP_001985; rat coagulation factor XIII b subunit, NP_001099426; mouse coagulation factor
XIII b subunit, BAE28851; cow coagulation factor XIII b subunit, NP_001033618; human C4-binding protein a chain, NP_000706; rat C4-binding protein
a chain, NP_036648; mouse C4-binding protein, NP_031602; cow C4-binding protein a chain, NP_776677; guinea pig C4-binding protein a chain,
BAB39739; chicken C4-binding protein a chain, NP_989995; human C4-binding protein b chain, NP_000707; rat C4-binding protein b chain, AAH88152;
cow C4-binding protein b chain, NP_776678; human DAF, NP_000565; rat DAF, BAA88991; mouse DAF, NP_034146; cow DAF, NP_001025474; wild
boar DAF, AAG14413; guinea pig DAF, BAA08398; human MCP, NP_002380; rat MCP, NP_062063; mouse MCP, NP_034908; cow MCP, NP_898903;
wild boar MCP, NP_999053; rat crry, NP_062174; human complement receptor 1, NP_000564; human complement receptor 1 like, NP_783641; chicken
complement-regulatory membrane protein, NP_001028815; human complement receptor 2, NP_001006659; rat, complement receptor 2,NP_001099459;
mouse complement receptor 2, NP_031784: lamprey lamprey complement-regulatory protein, BAC77070.
resents an ancestral form of membrane-bound group 2 RCA molecules. Identification of membrane-bound RCA in more primitive
animals such as agnathans and invertebrates would draw more
convincing conclusion.
More interestingly from a functional point of view, number of
potential O-glycosylation sites seen on serine/threonine-rich
regions on zTecrem splicing variants and their potency were different among splicing variants (Supplemental Fig. 3). The dif-
The Journal of Immunology
269
FIGURE 4. Specificity of anti-cTecrem mAb (1F12) and detection of
cTecrem protein on blood cells. (A) Schematic representation of rcTecrem
protein with a 63His tag. (B) ELISA using anti-cTecrem mAb (1F12).
CHO, CHO-rcTecrem, and KF-1 cells were cultured in 96-well plate. After
culture, each cell was fixed and used for ELISA using anti-cTecrem mAb
(1F12). (C) Immunoprecipitation using anti-cTecrem mAb 1F12. CHOrcTecrem cells were lysed in PBS containing 3.3% of Triton X-100 and 1
mM PMSF. Supernatant of lysate was incubated with anti-cTecrem mAb
1F12. Proteins trapped by protein G-Sepharose were electrophoresed on
10% polyacrylamide gel. After electrophoresis, gel was stained by Coomassie brilliant blue R-250 (left panel) or used for Western blotting using
anti-63His tag mAb (right panel). Molecular masses of detected proteins
are shown between left and right panels. Ig H and Ig L denote H and L
chains, respectively, of mouse Ig. (D) Flow cytometric detection of cTecrem protein on the surface of blood cells. Leukocytes and erythrocytes
from carp peripheral blood were treated with anti-cTecrem mAb (1F12),
followed by FITC-labeled second Ab treatment, and subjected to flow
cytometry. Gray peaks represent negative control samples without anticTecrem mAb.
ference seen on the threonine/proline-rich region may have a
functional effect on the Tecrem molecule, because in mammalian
MCP/CD46, O-glycosylation in the STP region controls its protein
expression and complement-regulatory function (44, 55). In this
regard, it should be noted that these splicing variants contained
different length and potential O-glycosylation sites of threonine/
proline-rich regions and may cause functional divergence of
Tecrem.
In mammals, membrane-bound RCA proteins can be categorized
into two groups: regulators of complement activation by cofactor
(MCP/CD46) or decay acceleration (DAF/CD55) and complement
receptors to recognize C3-tagged Ag (CR1/CD35 and CR2/CD21)
(1, 2). The wide tissue and cellular distribution of Tecrem shown
in the current study suggest its role as complement regulator rather
than complement receptor, of which expression would be limited
to phagocytes and other immune cells.
Confirming the above assumptions, inhibitory activity of Tecrem
on complement activation was observed at the protein level through
the experiments using two different assay models, as follows: one
employing rcTecrem and another utilizing autologous complement
activation system. In the latter system, anti-carp erythrocyte Ab
raised in a closely related fish species successfully triggered the
classical activation cascade of carp complement on the carp cells,
and it was demonstrated that anti-cTecrem mAb allowed more
deposition of carp C3 and two C4 isotypes on the autologous cells
by inhibiting the regulatory function of Tecrem. It is unclear which
C3 isotypes were bound to carp erythrocytes, because the anti-carp
C3 used in this work was polyclonal Ab reactive to all the C3
isotypes (57). Taken together, the present results suggest Tecrem
can restrict C3 deposition on the host cell, thereby preventing host
cell damage by complement-mediated cytotoxicity. It remains to
be analyzed whether the mode of inhibitory action of Tecrem is
decay acceleration of C3 convertase or C3b degradation as a cofactor for factor I. We have cloned two isotypes of complement
factor I from carp (58), and are now making their recombinant
proteins, which will be useful for functional analysis to examine
detailed inhibitory mechanism of Tecrem.
Teleost fish including carp, zebrafish, and trout possess divergent
isotypes of C3 differing in binding spectrum to various targets (40,
57, 59). It is particularly interesting to examine how the activation
of different C3 isotypes is regulated by RCA. Preceding studies on
comprehensive search of zebrafish RCA genes have revealed only
ZRC1 as a single gene that encodes membrane-bound RCA protein, suggesting that a Tecrem has a versatile regulatory ability to
different C3 isoforms in a single teleost species. In the case of
carp, a pseudotetraploid species, our data suggest the presence of
duplicated genes of Tecrem, possibly encoding two distinct
Tecrem proteins with different inhibition spectra against the diversified C3 isotypes. In these contexts, it is interesting to point
out that pig DAF fails to act on human C3b, which shares 76%
amino acid identity with pig C3b, resulting in complementmediated hyperacute damage of porcine organ transplant in human serum (60, 61). In a single teleost species, such as trout and
carp, C3 isoforms are as divergent as seen between human and
pig, sharing 75–82% identity. If such diverged C3 isoforms can be
regulated by a single kind of RCA protein, the inhibitory mechanism would be intriguing to analyze to better understand evolutionary and functional significance of C3 diversity.
Molecular anatomy to identify functional domains essential for
the complement regulation is also an issue to be addressed. In
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FIGURE 5. Expression of Tecrem transcripts in zebrafish and carp tissues. (A) zTecrem expression analyzed by RT-PCR. GAPDH was used as
positive control. Lane 1, kidney; lane 2, spleen; lane 3, hepatopancreas;
lane 4, heart; lane 5, brain; lane 6, intestine; lane 7, gill; lane 8, muscle;
lane 9, skin; lane 10, no template control. (B) cTecrem expression detected
by RT-PCR. The 40S rRNA S11 was used as a positive control. Lane 1,
head kidney; lane 2, body kidney; lane 3, spleen; lane 4, hepatopancreas;
lane 5, heart; lane 6, brain; lane 7, intestine; lane 8, gill; lane 9, skin; lane
10, PBLs; lane 11, no template control. Five zebrafish and three carp
individuals were independently analyzed, and representative data are
shown.
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TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS
FIGURE 6. Complement-inhibitory activities of cTecrem assayed using
CHO cells expressing rcTecrem. (A) Surface expression of rcTecrem on the
recombinant CHO cells revealed by flow cytometry. CHO-rcTecrem and its
mock transfectant (CHO-mock) were treated with anti-63His FITC-labeled
Ab and analyzed by flow cytometry. Gray and black dotted lines show negative controls without anti-63His of CHO-rcTecrem and CHO-mock, respectively. Gray-filled peak indicates CHO-mock with anti-63His, showing a
nonspecific signal of anti-63His. Solid black line shows CHO-rcTecrem
stained with anti-63His. (B) Inhibition of cytotoxicity by carp complement
against CHO cells by rcTecrem. CHO-rcTecrem and CHO-mock sensitized
with carp anti-CHO were incubated with 1/8-diluted normal carp serum in
GGVB2+ at 25˚C for 1 h to allow the classical pathway activation, and their
cytotoxicity was measured by the calcein-release assay. The experiment was
performed in quadruplicate, and the results are represented as mean 6 SD (p ,
0.05, Student t test). (C) Suppression of C3 deposition by rcTecrem on CHO
cells. CHO-rcTecrem and CHO-mock were attacked by carp complement, as
above, but at noncytolytic concentration (1/80 dilution) at 25˚C for 30 min. The
cells were treated with 1 mg/ml anti-carp C3-S mAb (5C7) and with FITClabeled second Ab, and then analyzed by flow cytometry to measure degree
of C3 deposition. Gray-filled peak is a negative control without anti–C3-S.
Gray and black lines show CHO-rcTecrem and CHO-mock, respectively, indicating a significant inhibition of C3 deposition onto CHO-rcTecrem.
barred sand bass, a factor H–like soluble RCA has been reported to
show a cofactor activity in factor I–mediated degradation of both
human C3b and C4b (30). It would be interesting to analyze
whether Tecrem can regulate both C3b and C4b, and, if yes, which
modules are responsible for the regulation, to gain evolutionary
implication of this protein family through vertebrate lineage.
Mammalian MCP/CD46 has also been reported to have immunomodulatory functions in adaptive immune response. Interaction of
C3b with MCP/CD46 expressed on T cells and macrophages has
been shown to modify adaptive immune response by controlling
T cell activation and inactivation (16–18, 62, 63). The MCP/CD46like molecular architecture of Tecrem and the presence of motifs for
cell signaling, such as putative phosphorylation sites by protein
kinase C, MAPK kinase, and casein kinase 2, in its cytoplasmic
domain, lead us to hypothesize that Tecrem may also play an important role in linking innate immunity and adaptive immune response in teleost, especially in balancing Th1/Th2 responses. We
have cloned orthologs of Tecrem from ginbuna crucian carp, a close
species of carp, in which purified B cells and CD4- and CD8positive T cells can be used for analysis of adaptive immune
responses (64). Evolutionary conservation of the modulation of
adaptive immune response by CD46-like molecules will be explored using the ginbuna crucian carp model.
Acknowledgments
We thank Prof. Hirofumi Tachibana (Department of Bioscience and Biotechnology, Kyushu University) for providing CHO cells and Dr. Yasutoshi
Yoshiura (National Institute of Aquaculture) for supplying zebrafish.
Disclosures
The authors have no financial conflicts of interest.
References
1. Walport, M. J. 2001. Complement: first of two parts. N. Engl. J. Med. 344: 1058–
1066.
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FIGURE 7. Effect of anti-cTecrem mAb on the deposition of carp C1q,
C3, C4-1, and C4-2 to autologous erythrocytes. Carp erythrocytes sensitized
with antiserum raised in ginbuna crucian carp were treated with anticTecrem mAb (1F12) or with control IgG and then incubated with normal
carp serum to allow activation of the classical complement pathway. Deposition of C1q, C3, C4-1, and C4-2 on the sensitized and complement-activated
carp erythrocytes was analyzed by flow cytometry. Gray-filled peak is a negative control without the first Ab. Black and gray lines show carp erythrocytes
treated with anti-cTecrem mAb and with control IgG, respectively.
The Journal of Immunology
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
regulatory activities of both human C4b binding protein and factor H. J. Biol.
Chem. 273: 19398–19404.
Katagiri, T., I. Hirono, and T. Aoki. 1998. Molecular analysis of complement
regulatory cDNA composed of 12 tandem SCRs from the Japanese flounder. Fish
Pathol. 33: 351–355.
Katagiri, T., I. Hirono, and T. Aoki. 1998. Molecular analysis of complement
regulatory protein-like cDNA from the Japanese flounder Paralichthys olivaceus.
Fish. Sci. 64: 140–143.
Sun, G., H. Li, Y. Wang, B. Zhang, and S. Zhang. 2010. Zebrafish complement
factor H and its related genes: identification, evolution, and expression. Funct.
Integr. Genomics 10: 577–587.
Wu, J., H. Li, and S. Zhang. 2012. Regulator of complement activation (RCA)
group 2 gene cluster in zebrafish: identification, expression, and evolution.
Funct. Integr. Genomics 12: 367–377.
Kimura, Y., N. Inoue, A. Fukui, H. Oshiumi, M. Matsumoto, M. Nonaka,
S. Kuratani, T. Fujita, M. Nonaka, and T. Seya. 2004. A short consensus repeatcontaining complement regulatory protein of lamprey that participates in
cleavage of lamprey complement 3. J. Immunol. 173: 1118–1128.
Yano, T., and M. Nakao. 1994. Isolation of a carp complement protein homologous to mammalian factor D. Mol. Immunol. 31: 337–342.
Somamoto, T., T. Nakanishi, and M. Nakao. 2013. Identification of anti-viral
cytotoxic effector cells in the ginbuna crucian carp, Carassius auratus langsdorfii. Dev. Comp. Immunol. 39: 370–377.
Nakao, M., Y. Fushitani, K. Fujiki, M. Nonaka, and T. Yano. 1998. Two diverged
complement factor B/C2-like cDNA sequences from a teleost, the common carp
(Cyprinus carpio). J. Immunol. 161: 4811–4818.
Mutsuro, J., N. Tanaka, Y. Kato, A. W. Dodds, T. Yano, and M. Nakao. 2005.
Two divergent isotypes of the fourth complement component from a bony fish,
the common carp (Cyprinus carpio). J. Immunol. 175: 4508–4517.
Ichiki, S., Y. Kato-Unoki, T. Somamoto, and M. Nakao. 2012. The binding
spectra of carp C3 isotypes against natural targets independent of the binding
specificity of their thioester. Dev. Comp. Immunol. 38: 10–16.
Oladiran, A., and M. Belosevic. 2010. Trypanosoma carassii calreticulin binds
host complement component C1q and inhibits classical complement pathwaymediated lysis. Dev. Comp. Immunol. 34: 396–405.
Ohno, S., J. Muramoto, and L. Christian. 1967. Diploid-tetraploid relationship
among old-world members of the fish family Cyprinidae. Chromosoma 23: 1–9.
Ohno, S., U. Wolf, and N. B. Atkin. 1968. Evolution from fish to mammals by
gene duplication. Hereditas 59: 169–187.
Liszewski, M. K., M. K. Leung, and J. P. Atkinson. 1998. Membrane cofactor
protein: importance of N- and O-glycosylation for complement regulatory
function. J. Immunol. 161: 3711–3718.
Fingeroth, J. D. 1990. Comparative structure and evolution of murine CR2: the
homolog of the human C3d/EBV receptor (CD21). J. Immunol. 144: 3458–3467.
Fingeroth, J. D., M. A. Benedict, D. N. Levy, and J. L. Strominger. 1989.
Identification of murine complement receptor type 2. Proc. Natl. Acad. Sci. USA
86: 242–246.
Gordon, D. L., R. M. Kaufman, T. K. Blackmore, J. Kwong, and D. M. Lublin.
1995. Identification of complement regulatory domains in human factor H. J.
Immunol. 155: 348–356.
Schmidt, C. Q., A. P. Herbert, D. Kavanagh, C. Gandy, C. J. Fenton, B. S. Blaum,
M. Lyon, D. Uhrı́n, and P. N. Barlow. 2008. A new map of glycosaminoglycan
and C3b binding sites on factor H. J. Immunol. 181: 2610–2619.
Blom, A. M., L. Kask, and B. Dahlbäck. 2001. Structural requirements for
the complement regulatory activities of C4BP. J. Biol. Chem. 276: 27136–
27144.
Adams, E. M., M. C. Brown, M. Nunge, M. Krych, and J. P. Atkinson. 1991.
Contribution of the repeating domains of membrane cofactor protein (CD46) of
the complement system to ligand binding and cofactor activity. J. Immunol. 147:
3005–3011.
Iwata, K., T. Seya, Y. Yanagi, J. M. Pesando, P. M. Johnson, M. Okabe, S. Ueda,
H. Ariga, and S. Nagasawa. 1995. Diversity of sites for measles virus binding
and for inactivation of complement C3b and C4b on membrane cofactor protein
CD46. J. Biol. Chem. 270: 15148–15152.
Brodbeck, W. G., D. Liu, J. Sperry, C. Mold, and M. E. Medof. 1996. Localization of classical and alternative pathway regulatory activity within the decayaccelerating factor. J. Immunol. 156: 2528–2533.
Mossakowska, D., I. Dodd, W. Pindar, and R. A. Smith. 1999. Structure-activity
relationships within the N-terminal short consensus repeats (SCR) of human
CR1 (C3b/C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the
classical and alternative pathways of complement activation. Eur. J. Immunol.
29: 1955–1965.
Krych-Goldberg, M., R. E. Hauhart, V. B. Subramanian, B. M. Yurcisin, II,
D. L. Crimmins, D. E. Hourcade, and J. P. Atkinson. 1999. Decay accelerating
activity of complement receptor type 1 (CD35): two active sites are required for
dissociating C5 convertases. J. Biol. Chem. 274: 31160–31168.
Russell, S. M., R. L. Sparrow, I. F. McKenzie, and D. F. Purcell. 1992. Tissuespecific and allelic expression of the complement regulator CD46 is controlled
by alternative splicing. Eur. J. Immunol. 22: 1513–1518.
Purcell, D. F., S. M. Russell, N. J. Deacon, M. A. Brown, D. J. Hooker, and
I. F. McKenzie. 1991. Alternatively spliced RNAs encode several isoforms of
CD46 (MCP), a regulator of complement activation. Immunogenetics 33: 335–
344.
Nakao, M., J. Mutsuro, R. Obo, K. Fujiki, M. Nonaka, and T. Yano. 2000.
Molecular cloning and protein analysis of divergent forms of the complement
component C3 from a bony fish, the common carp (Cyprinus carpio): presence
of variants lacking the catalytic histidine. Eur. J. Immunol. 30: 858–866.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
2. Kinoshita, T. 1991. Biology of complement: the overture. Immunol. Today 12:
291–295.
3. Nicholson-Weller, A., J. P. March, S. I. Rosenfeld, and K. F. Austen. 1983.
Affected erythrocytes of patients with paroxysmal nocturnal hemoglobinuria are
deficient in the complement regulatory protein, decay accelerating factor. Proc.
Natl. Acad. Sci. USA 80: 5066–5070.
4. Noris, M., and G. Remuzzi. 2005. Hemolytic uremic syndrome. J. Am. Soc.
Nephrol. 16: 1035–1050.
5. Noris, M., S. Brioschi, J. Caprioli, M. Todeschini, E. Bresin, F. Porrati,
S. Gamba, and G. Remuzzi, International Registry of Recurrent and Familial
HUS/TTP. 2003. Familial haemolytic uraemic syndrome and an MCP mutation.
Lancet 362: 1542–1547.
6. Józsi, M., C. Licht, S. Strobel, S. L. H. Zipfel, H. Richter, S. Heinen, P. F. Zipfel,
and C. Skerka. 2008. Factor H autoantibodies in atypical hemolytic uremic
syndrome correlate with CFHR1/CFHR3 deficiency. Blood 111: 1512–1514.
7. Norman, D. G., P. N. Barlow, M. Baron, A. J. Day, R. B. Sim, and
I. D. Campbell. 1991. Three-dimensional structure of a complement control
protein module in solution. J. Mol. Biol. 219: 717–725.
8. Morgan, B. P., and C. L. Harris. 1999. Complement Regulatory Proteins. Academic Press, San Diego, CA.
9. Gasque, P. 2004. Complement: a unique innate immune sensor for danger signals. Mol. Immunol. 41: 1089–1098.
10. Fearon, D. T. 1980. Identification of the membrane glycoprotein that is the C3b
receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte,
and monocyte. J. Exp. Med. 152: 20–30.
11. Nelson, R. A., Jr. 1953. The immune-adherence phenomenon; an immunologically specific reaction between microorganisms and erythrocytes leading to
enhanced phagocytosis. Science 118: 733–737.
12. Li, J., J. P. Wang, I. Ghiran, A. Cerny, A. J. Szalai, D. E. Briles, and
R. W. Finberg. 2010. Complement receptor 1 expression on mouse erythrocytes
mediates clearance of Streptococcus pneumoniae by immune adherence. Infect.
Immun. 78: 3129–3135.
13. Carter, R. H., and D. T. Fearon. 1992. CD19: lowering the threshold for antigen
receptor stimulation of B lymphocytes. Science 256: 105–107.
14. Kopf, M., B. Abel, A. Gallimore, M. Carroll, and M. F. Bachmann. 2002.
Complement component C3 promotes T-cell priming and lung migration to
control acute influenza virus infection. Nat. Med. 8: 373–378.
15. Carroll, M. C. 2004. The complement system in regulation of adaptive immunity.
Nat. Immunol. 5: 981–986.
16. Fuchs, A., J. P. Atkinson, V. Fremeaux-Bacchi, and C. Kemper. 2009. CD46induced human Treg enhance B-cell responses. Eur. J. Immunol. 39: 3097–3109.
17. Cardone, J., G. Le Friec, P. Vantourout, A. Roberts, A. Fuchs, I. Jackson,
T. Suddason, G. Lord, J. P. Atkinson, A. Cope, et al. 2010. Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells. Nat. Immunol. 11: 862–871.
18. Cardone, J., G. Le Friec, and C. Kemper. 2011. CD46 in innate and adaptive
immunity: an update. Clin. Exp. Immunol. 164: 301–311.
19. Krushkal, J., O. Bat, and I. Gigli. 2000. Evolutionary relationships among proteins encoded by the regulator of complement activation gene cluster. Mol. Biol.
Evol. 17: 1718–1730.
20. Cole, J. L., G. A. Housley, Jr., T. R. Dykman, R. P. MacDermott, and
J. P. Atkinson. 1985. Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines. Proc. Natl.
Acad. Sci. USA 82: 859–863.
21. Carroll, M. C., E. M. Alicot, P. J. Katzman, L. B. Klickstein, J. A. Smith, and
D. T. Fearon. 1988. Organization of the genes encoding complement receptors
type 1 and 2, decay-accelerating factor, and C4-binding protein in the RCA locus
on human chromosome 1. J. Exp. Med. 167: 1271–1280.
22. Krushkal, J., C. Kemper, and I. Gigli. 1998. Ancient origin of human complement factor H. J. Mol. Evol. 47: 625–630.
23. Kingsmore, S. F., D. P. Vik, C. B. Kurtz, P. Leroy, B. F. Tack, J. H. Weis, and
M. F. Seldin. 1989. Genetic organization of complement receptor-related genes
in the mouse. J. Exp. Med. 169: 1479–1484.
24. Holers, V. M., T. Kinoshita, and H. Molina. 1992. The evolution of mouse and
human complement C3-binding proteins: divergence of form but conservation of
function. Immunol. Today 13: 231–236.
25. Inoue, N., A. Fukui, M. Nomura, M. Matsumoto, Y. Nishizawa, K. Toyoshima,
and T. Seya. 2001. A novel chicken membrane-associated complement regulatory protein: molecular cloning and functional characterization. J. Immunol. 166:
424–431.
26. Oshiumi, H., K. Shida, R. Goitsuka, Y. Kimura, J. Katoh, S. Ohba, Y. Tamaki,
T. Hattori, N. Yamada, N. Inoue, et al. 2005. Regulator of complement activation
(RCA) locus in chicken: identification of chicken RCA gene cluster and functional RCA proteins. J. Immunol. 175: 1724–1734.
27. Oshiumi, H., Y. Suzuki, M. Matsumoto, and T. Seya. 2009. Regulator of complement activation (RCA) gene cluster in Xenopus tropicalis. Immunogenetics
61: 371–384.
28. Dahmen, A., T. Kaidoh, P. F. Zipfel, and I. Gigli. 1994. Cloning and characterization of a cDNA representing a putative complement-regulatory plasma
protein from barred sand bass (Parablax neblifer). Biochem. J. 301: 391–397.
29. Zipfel, P. F., C. Kemper, A. Dahmen, and I. Gigli. 1996. Cloning and
recombinant expression of a barred sand bass (Paralabrax nebulifer) cDNA: the
encoded protein displays structural homology and immunological crossreactivity
to human complement/cofactor related plasma proteins. Dev. Comp. Immunol.
20: 407–416.
30. Kemper, C., P. F. Zipfel, and I. Gigli. 1998. The complement cofactor protein
(SBP1) from the barred sand bass (Paralabrax nebulifer) mediates overlapping
271
272
TELEOST COMPLEMENT-REGULATORY MEMBRANE PROTEINS
58. Nakao, M., S. Hisamatsu, M. Nakahara, Y. Kato, S. L. Smith, and T. Yano. 2003.
Molecular cloning of the complement regulatory factor I isotypes from the
common carp (Cyprinus carpio). Immunogenetics 54: 801–806.
59. Sunyer, J. O., I. K. Zarkadis, and J. D. Lambris. 1998. Complement diversity: a mechanism for generating immune diversity? Immunol. Today 19:
519–523.
60. Dalmasso, A. P., G. M. Vercellotti, J. L. Platt, and F. H. Bach. 1991. Inhibition of
complement-mediated endothelial cell cytotoxicity by decay-accelerating factor:
potential for prevention of xenograft hyperacute rejection. Transplantation 52:
530–533.
61. Auchincloss, H., Jr., and D. H. Sachs. 1998. Xenogeneic transplantation. Annu.
Rev. Immunol. 16: 433–470.
62. Heeger, P. S., and C. Kemper. 2012. Novel roles of complement in T effector cell
regulation. Immunobiology 217: 216–224.
63. Kemper, C., J. W. Verbsky, J. D. Price, and J. P. Atkinson. 2005. T-cell stimulation and regulation: with complements from CD46. Immunol. Res. 32: 31–43.
64. Nur, I., H. Harada, M. Tsujikura, T. Somamoto, and M. Nakao. 2013. Molecular
characterization and expression analysis of three membrane-bound complement
regulatory protein isoforms in the ginbuna crucian carp Carassius auratus
langsdorfii. Fish Shellfish Immunol. 35: 1333–1337.
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