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Developmental and Comparative Immunology 25 (2001) 11±24
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Cloning and structure of three rainbow trout C3 molecules:
a plausible explanation for their functional diversity p
Ioannis K. Zarkadis a, 1, Maria Rosa Sarrias b, 1, Georgia Sfyroera a,
J. Oriol Sunyer b, 2, John D. Lambris b,*
a
Department of Biology, School of Medicine, University of Patras, Patras, Greece
Protein Chemistry Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania,
402 Stellar Chance Building, Philadelphia, PA 19104-6100, USA
b
Received 23 March 2000; received in revised form 25 May 2000; accepted 26 May 2000
Abstract
We have previously identi®ed and characterized three distinct trout C3 proteins (C3-1, C3-3 and C3-4) that di€er
in their electrophoretic mobility, glycosylation patterns, reactivity with monospeci®c C3 antibodies, partial amino
acid sequence and binding to various complement activators. To study the structural elements that determine the
observed functional di€erences, we have cloned and sequenced the three C3 isoforms. Comparison of the deduced
amino acid sequences showed that the sequence identity/similarity of C3-3 to C3-4 is 76/81%, whereas those of C3-3
and C3-4 to C3-1 are 55/67% and 54/67%, respectively. It is interesting that the b-chain of C3-4 contains two
insertions of 65 (residues 504±569) and 23 amino acids (residues 123±146), while the b-chain of C3-1 contains a 14amino acid insertion (residues 143±157). The C3 convertase cleavage site (Arg±Ser) is conserved in the three trout
isoforms; however, the factor I cleavage sites are Arg±Ala (for C3-1 and C3-4) and Arg±Thr (C3-3) instead of Arg±
Ser at position 1281 of human C3, and Arg±Thr (C3-1, C3-3) instead of Arg±Ser for C3-4 at position 1298 of
human C3. Of special interest is the absence of the His1126 and Glu1128 (human C3 numbering) from C3-4 and of
Glu1128 from C3-3. These residues are thought to play an important role in determining the binding speci®city of
the thioester-containing proteins. Accordingly, we postulate that the distinct binding reactions of the trout C3
isoforms with various complement activators could be due at least in part to the observed changes in the His and
Glu residues. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Comparative immunology; Evolution; Complement; C3; Diversity
This work was supported by National Institutes of Health Grant GM 56698, and Cancer and Diabetes Centers' Core Support
Grants CA 16520 and DK 19525. The sequences described in this paper have been deposited in the GenBank database under accession numbers AF271079 (Trout C3-3) and AF271080 (Trout C3-4).
* Corresponding author. Tel.: +1-215-746-5765; fax: +1-215-573-8738.
E-mail address: [email protected] (J.D. Lambris).
1
Contributed equally.
2
Present address: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 307 Rosenthal Building, Philadelphia, PA 19104, USA.
p
0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 5 - 3 0 5 X ( 0 0 ) 0 0 0 3 9 - 2
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1. Introduction
The rainbow trout, a tetraploid ®sh in the process of diploidization, has a complement system
similar to that of mammals [1,2]. However, unlike
the situation in mammals, four di€erent C3 isoforms have been described in this species (C3-1,
C3-2, C3-3 and C3-4) [3,4]. C3-1, C3-3 and C3-4,
which we have puri®ed from trout plasma and
subsequently characterized [3], resemble mammalian C3 in their structure and properties and each
consists of two disul®de-linked polypeptide chains
and contains a thioester site. Of special interest
are the di€erences encountered between C3-1, C33 and C3-4, concerning their ability to bind to
various complement activators. Of all three isoforms, only C3-1 binds to zymosan. Furthermore,
C3-3 and C3-4 bind to sheep and rabbit erythrocyte ghosts to a similar degree, while C3-1 binds
better to rabbit than sheep erythrocyte ghosts [3].
The C3-2 isoform, described by Nonaka et al.,
was found unable to promote the hemolysis of
sensitized sheep erythrocytes, although it contains
a thioester site [4]. Only N-terminal amino acid
sequence of the a- and b-chains is available for
this isoform.
In order to identify the structural basis for the
di€erences in binding eciencies of trout C3-1,
C3-3 and C3-4, we have cloned the cDNAs
encoding the C3-3 and C3-4 isoforms; the complete C3-1 cDNA sequence has been previously
obtained [5]. Here we present the deduced amino
acid sequences of the three functional trout C3
isoforms, and we compare these sequences to
those of C3s from other species, and provide an
explanation for the observed functional di€erences. We found that the His1126 and Glu1128
(human C3 numbering) which have been implicated in thioester speci®city [6,7], while conserved
in C3-1, have been substituted in C3-3 and C3-4.
2. Materials and methods
2.1. RNA extraction and cDNA library
construction
RNA was isolated from a single trout liver by
the guanidine thiocyanate method, and polyadenylated RNA was puri®ed using the polyA Tract
mRNA isolation kit (Promega) according to the
manufacturer's instructions. The construction of
random or oligo-dT-primed trout liver cDNA
libraries has been described. In brief, total RNA
was isolated by the guadinine isothicyanate
method, and centrifuged through a CsCl cushion.
Polyadenylated RNA was puri®ed with the
PolyA Tract mRNA isolation kit (Promega).
After treatment with EcoRI methylase and addition of synthetic EcoRI linkers (using an Amersham cDNA synthesis kit), the cDNA was
digested with EcoRI and size fractionated on a
Sepharose CL4B column. cDNAs larger than 500
bp were ligated into EcoRI-digested, dephosphorylated lgt11 arms and packaged in vitro
using the Amersham cloning system.
2.2. Determination of trout C3-1, C3-3 and C3-4
sequences
Determination of the trout C3-1 cDNA
sequence has been previously described [5].
During the searching for whole length C3-1, a 1kb clone was isolated that showed a sequence
di€erent to that of C3-1 [3]. To complete this
sequence, the cDNA library was screened under
high stringency conditions using the 1-kb fragment as a probe. One of the clones, C336A,
encoded the C3-3 molecule. An insert of 7.5 kb
was cloned into the pIBI31 vector and subcloned
into the pGEM vector, and each strand was then
sequenced at least two times.
A C3-3 cDNA probe was used to screen the
trout liver libraries under low stringency conditions to obtain a trout C3-4 cDNA. Brie¯y, the
probe was obtained by PCR ampli®cation of a
C3-3 clone using oligos J3 (5 '-AAGGGAATCTTCATAGTC-3 ') and J6 (5 '-GAGGCACCATGAAGGCT-3 ') and was labeled with
[a-32P] dATP by using the random primed labeling kit (BMB) according to the manufacturer's
instructions. Membranes (Nylon Duralon UV,
Stratagene) containing 1 105 clones were hybridized under low stringency conditions, following
the manufacturer's directions (hybridization at
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
328C and washing at 588C). Nine clones were isolated. Analysis by PCR indicated that three
clones encoded the C3-3 molecule and one
encoded the C3-4 clone. An insert of 5.4 kb was
cloned into the pIBI31 vector and subcloned into
the pGEM vector for ease of sequencing.
2.3. DNA sequencing
DNA sequences were determined twice for
both strands by the Sanger method [8].
2.4. Computer methods
The deduced amino acid sequences of trout
C3-1, C3-3, C3-4, as well as several C3 sequences
from other species, were aligned using the Clustal
W 1.5 program [9], and the resulting alignments
were manually corrected. The obtained alignment
was used to calculate Poisson-corrected distance
matrixes to construct trees by the neighbor-joining method [10].
3. Results
3.1. Isolation of trout C3-1, C3-3 and C3-4 cDNA
and nucleotide sequencing
To obtain cDNA clones encoding trout C3-1,
C3-3 and C3-4 cDNA clones, a trout lgt11
library was screened by several methods. The C31 cDNA was previously described [5]. Screening
of the trout C3-1 sequence by PCR also resulted
in the isolation of a partial clone encoding the
C3-3 molecule (C33A), whose deduced amino
acid sequence was 50% identical to the corresponding area in C3-1 and which matched the
C3-3 protein sequence [3]. Subsequently, a new
screening of the cDNA library was performed,
and a C3-3 clone was obtained (C336). The trout
C3-4 cDNA sequence was obtained by screening
the trout lgt11 libraries with a C3-3 probe under
low-stringency conditions. This method produced
a 5.4-kb clone. A single open reading frame was
identi®ed for each cDNA.
To verify that the isolated clones were indeed
C3 cDNAs, the deduced amino acid sequences of
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the clones were aligned with the N-terminal and
internal amino acid sequences of the puri®ed C3
isoforms from trout serum [3] (the b-chain of C31, and the a-chain and several tryptic peptides of
C3-1, C3-3 and C3-4) (Fig. 3).
Several attempts to obtain the sequences of the
N-terminal amino acids and of the signal peptides for the three isoforms were unsuccessful.
Thus, the compiled amino acid sequences that we
obtained for trout C3-1, C3-3 and C3-4 are 1640,
1614 and 1684 amino acids in length, respectively.
DNA sequence analysis indicated that the
sequence identity/similarity of C3-3 to C3-4 is 76/
81%, while those of C3-3, C3-4 to C3-1 are 55/
67% and 54/67%, respectively. We also identi®ed
a potential post-translational processing site,
RRRR, in C3-1, C3-3 and C3-4 in analogous
positions at the junction of the a- and b-chains
of human C3 [11,12]. This ®nding con®rms the
observation at the protein level that the three isoforms are composed of an a-chain and a b-chain.
The number and location of the potential N-glycosylation sites apparently di€er among the three
isoforms: C3-1 contains one potential N-glycosylation site in each chain, whereas C3-3 and C3-4,
four sites in the a-chain and one and ®ve, respectively, in the b-chain. One sequence insertion
was found in the b-chain of C3-1 (14 amino
acids, beginning at residue 143) and two in the bchain of C3-4 (65 amino acids from residue 504,
and 23 amino acids from residue 123). (Fig. 1).
Analysis of the sequence insertions in a protein
database did not yield any signi®cant similarity
to any other protein.
3.2. Alignment of trout C3s with C3s from other
species
The deduced amino acid sequences of trout
C3-1, C3-3 and C3-4 were aligned with those of
C3 molecules from other species using the Clustal
software. When a phylogenetic tree was generated from all available C3 sequences it showed
that all the bony ®sh C3 formed a cluster. To
our surprise, trout C3-3 and C3-4 clustered
together, whereas trout C3-1 clustered with the
medaka ®sh C3s, which was supported by a poor
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bootstrap percentage (58%) (data not shown).
This is due to the slightly higher sequence identity/similarity of trout C3-1 with the medaka C3s
(around 56/71%) than with trout C3-3 and C3-4
(around 55/67%). However, when the phylogenentic tree was generated using only the a-chains
of the aligned C3s, trout C3-1 clustered with
trout C3-3 and C3-4 (Fig. 2). Similar discordances in the drawing of the C3 phylogenetic tree
have been described before [13]. Hughes
suggested that the b-chain may have a higher
pattern of nonsynonymous evolution than the a-
chain, due to a reduced constraint at the amino
acid level on the b-chain region [14]. The alignment, displayed in Fig. 3, showed that the C3
domain which contains the thioester site
(GCGEQ) is totally conserved in all species,
including the trout. Furthermore, the thioester
sites in C3-1, C3-3 and C3-4 are surrounded by
hydrophobic amino acids, as they are in other C3
and C4 proteins. In vitro mutagenesis experiments involving human C3 have suggested that
Pro1007 and Pro1020 are necessary for stable thioester formation [15] and that His1126 determines
Fig. 1. Schematic diagram of three trout C3 isoforms, showing their main structural features. (.) potential N-glycosylation sites.
Numbering of C3-3 and C3-4 is according to C3-1.
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
the thioester binding speci®city [16]. Our analysis
demonstrated that Pro1007 is conserved, whereas
Pro1020 is replaced by Leu in the trout C3-1, C33 and C3-4. However, His1126 is conserved in C31 and C3-3, but it is replaced by Thr in C3-4. In
addition, Glu1128 in human C3 is also thought to
play an important role in determining the binding speci®city of the human C3 thioester [6]. This
residue is conserved in C3-1, whereas the analogous residues in C3-3 and C3-4 are Ser and Thr,
respectively.
The protein sequence alignment further indicated that the properdin binding and C3a receptor (C3aR) sites are highly conserved, whereas
the region of human C3 to which CR1 binds
shows only low similarity. The C3 convertase
cleavage site (Arg±Ser) is conserved in the three
trout C3s, as it is in the C3s from virtually all
species, with the exception of lamprey C3 convertase cleavage site (Arg±Asn). Likewise, the residues Cys559 and Cys816 that link the a- and bchains through a disul®de bond, are conserved in
the C3s from all species. In contrast, the factor I
cleavage sites are Arg±Ala (C3-1, C3-4) and
15
Arg±Thr (C3-3), instead of Arg±Ser at residues
1281-2 in human C3, and Arg±Thr (C3-1, C3-3)
instead of Arg±Ser at residues 1298-9 in human
C3; the Arg±Ser sequence is conserved in C3-4.
4. Discussion
In the present study we have completed the
cDNA sequences of two trout C3 isoforms, C3-3
and C3-4, in order to compare their deduced
amino acid sequences, and thereby, identify
structural variations capable of explaining their
dissimilar biochemical and functional properties.
We are con®dent that the cDNA clones we have
obtained represent the sequences of C3-3 and C34, because they match all the available protein
sequences for these proteins.
Careful analysis of the trout C3 sequences has
yielded many interesting observations; structurally, trout C3-1, C3-3 and C3-4 are similar to
mammalian C3 in having two chains (a and b),
with a thioester site in the a-chain. The potential
b±a processing signal RRRR is present in all
Fig. 2. Phylogenetic tree of C3 protein sequences. The tree was generated by the neighbor-joining method, based on the a-chain
sequences and the alignment of Fig. 3. Numbers on the branches show the percent recovery in 1000 bootstrap replications.
Fig. 3. Alignment of trout C3 proteins with C3 proteins from other species, performed with the Clustal program. Several important functional domains and amino
acids are indicated. Underlined are the C3-3 and C3-4 amino acid residues that match the protein sequences obtained by Edman degradation, and in bold and italic
are the potential N-glycosylation sites. Human C3 numbering is shown, which includes amino acids of the signal peptide. Discrepancies in the numbering of some residues are due to the signal peptide, but they were not changed accordingly for clearer referral to the original reference.
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I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
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Fig. 3 (continued).
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
Fig. 3 (continued).
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19
Fig. 3 (continued).
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
Fig. 3 (continued).
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I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
Fig. 3 (continued).
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I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
three isoforms. However, the location and number of the potential N-glycosylation sites in C3-1
di€er from those in C3-3 and C3-4, which are
highly similar to each other. The putative glycosylation sites in both the a- and b-chains of C3-3
and C3-4 molecules are consistent with the presence of Concavalin A (Con A)-binding carbohydrate moieties in these isoforms. In contrast,
C3-1 apparently lacks Con A-binding carbohydrates in its a-chain, where an N-glycosylation
site is predicted. Since Con A recognizes only
high-mannose carbohydrates, the presence of
carbohydrates that do not bind Con A cannot be
excluded. Because the a-chain carbohydrate moiety in human C3 is involved in the binding to
conglutinin, it would be of interest to see whether
the trout C3 isoforms have any related activity
[17].
The occurrence of sequence insertions in C3-1
and C3-4 raises the possibility that these insertions confer particular functions upon the proteins, although no similarity to any other protein
was found when these sequences were submitted
to protein databases.
Amino acid sequence comparisons of the three
isoforms revealed that C3-3 and C3-4 are more
similar to each other than to C3-1. Since these
amino acid di€erences between C3-1, C3-3 and
C3-4 were scattered throughout the proteins, it is
very unlikely that they are the result of di€erential mRNA splicing. Therefore, although the relevant genes have not been mapped yet, we
suggest that these trout C3 isoforms are the product of at least three di€erent genes.
The thioester site and surrounding hydrophobic amino acids are highly conserved in trout
C3-1, C3-3 and C3-4 isoforms, which emphasizes
the importance of the thioester site, which is
necessary for the attachment of C3 and C4 molecules to surfaces, and its surroundings to protect
it from the aqueous environment [18,19]. In vitro
mutagenesis studies have shown that the presence
of H1126 (human C3 numbering) determines the
speci®city of the thioester for hydroxyl, rather
than amino groups, as a result of the formation
of an intramolecular acyl-imidazole bond [16].
This residue is conserved in all C3 and C4 molecules, except for cobra venom factor, human
C4A, and trout C3-4; in these proteins His is
substituted by Ser, Asp and Thr, respectively.
Similarly, the Glu residue located two amino
acids downstream, Glu1128 in human C3, is
highly conserved, but is replaced by Thr in C3-3
and Ser in C3-4. Since this residue forms a
hydrogen bond with H1126, it is thought to render
H1126 a stronger nucleophile, thus increasing the
rate of acyl-imidazole formation in C3 and promoting its speci®city for hydroxyl nucleophiles,
as compared with C4B (H1126, Ser1128) [6]; C4B
shows a considerable reactivity with amino as
well as hydroxyl nucleophiles. The three C3 isoforms contain the residues His, Glu (C3-1); His,
Thr (C3-3); and Thr, Ser (C3-4) at these positions
which are important for the reactivity of the thioester site. We are at the present investigating
whether these di€erences in the isoforms indeed
correlate with di€erences in thioester reactivity
among the trout C3 molecules. Di€erences in the
binding properties of the thioester among the
di€erent isoforms would enable the complement
system in trout to react with a wider repertoire
of substrates and would thus enhance its mechanism of defense against pathogens. In fact, we
have recently hypothesized that the C3 diversity
present in trout and other teleosts may provide a
mechanism for generating immune diversity that
would allow these animals to expand their innate
immune recognition capabilities [20].
The coexistence of several functional isoforms
of C3 in the same animal and the distinct nature
of their binding speci®cities raises the question,
what mechanisms regulate complement activation
in the trout. Unfortunately, these studies are not
possible at the present time, as only C5 [21], C9
[2], and factors B and D [22] have thus far been
characterized in these ®sh.
The comparison of the C3 sequences unveils
other interesting features concerning the factor
I and convertase cleavage sites. The high
degree of conservation of Arg726±Ser727, the C3
convertase cleavage site in the a-chain, suggests
that the C3 convertases from many species have
similar binding speci®cities to that of human
complement. It would be of interest to determine
whether the formation of the convertase in
species that possess multiple C3 isoforms is C3-
I.K. Zarkadis et al. / Developmental and Comparative Immunology 25 (2001) 11±24
type-speci®c, i.e., does a C3-1-containing convertase bind C3-3 or C3-4 in trout? Based on protein alignment, the factor I cleavage sites in the
trout isoforms are Arg±Ala (C3-1, C3-4) and
Arg±Thr (C3-3) instead of Arg±Ser at position
1281 of human C3; and Arg±Thr (C3-1, C3-3)
instead of Arg±Ser at position 1298 of human
C3, where C3-4 maintains the Arg±Ser residues.
By sequencing zymosan-eluted trout C3-1 fragments we have previously found that C3-1 is
cleaved by trout factor I at the Arg±Thr bond, a
®nding that indicates that factor I in the presence
of adequate cofactor can cleave either Arg±Thr
or Arg±Ser bonds [23].
The mechanism which led to the generation
of multiple C3 isoforms remains unknown.
Trout are tetraploid ®sh in the process of
diploidization
[24].
We
have
previously
suggested that during the tetraploidization
event, some duplicated loci (including those
encoding complement proteins) did not
undergo diploidization, and therefore gave rise
to several protein isoforms [3]. The appearance
of multiple C3 genes is not exclusive of the
trout, in fact, it seems to be a general feature
of teleost ®sh. In the carp (Cyprinus carpio ), a
tetraploid teleost ®sh, eight di€erent PCR
clones highly homologous to C3 have been
ampli®ed from a cDNA library derived from a
single ®sh [25,26]. Furthermore, multiple C3
forms are not exclusive of tetraploid teleost
®sh, since ®ve di€erent isoforms of C3 have
been puri®ed and characterized in the gilthead
sea bream (Sparus aurata ) [27,28], and medaka
®sh (Orzias latipes ) have been shown to possess three C3 genes [29]. Both medaka and the
gilthead sea bream ®sh are diploid teleost ®sh.
We have previously suggested that because of
their functional relevance, the emerging genes
apparently remained active, and thus generated a
C3 protein family that appears to have expanded
the immunorecognition capabilities of ®sh [20].
Acknowledgements
We thank Drs. A. Sahu and W. T. Moore for
23
helpful suggestions and Dr. D. McClellan for editorial assistance.
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