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*
Department of Biological Sciences, George Washington University, Washington, D.C. 20052; and
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. There
are cysteines in conserved positions for interchain disulfide bonding,
and there is a conserved thioester site in the -chain with an
associated histidine. There are five consensus N-linked
glycosylation sites, and putative cleavage sites for factor I and C3
convertase. Partially purified SpC3 on protein gels shows a nonreduced
size of 210 kDa and, under reducing conditions, reveals an -chain of
130 kDa and a ß-chain of 80 kDa. These sizes are larger than the
deduced sizes, suggesting that the protein has carbohydrates added to
most of the consensus N-linked glycosylation sites.
Phylogenetic analysis of SpC3 compared with other members of the
thioester protein family, which includes C3, C4, C5, and
2-macroglobulin, shows that SpC3 is the first divergent
complement protein, falling at the base of the complement protein
clade. Transcripts from the SpC3 gene (Sp064) are 9 kb, and the gene is
expressed specifically in coelomocytes, which are the immunocytes in
the sea urchin. Genome blots suggest that SpC3 is encoded by a single
copy gene per haploid genome. This is the first identification of a
complement component in an invertebrate, and suggests homology of the
innate immune system within the deuterostome lineage of animals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complement component C3, the central component in all cascades, is the most versatile and multifunctional molecule of the complement proteins (8). Not only does it function in the complement cascades, but it interacts with cell surface proteins on self and foreign cells to initiate and augment immune responsiveness, and it interacts with an array of proteins that regulate its activity (9, 10, 11). C3 also has opsonin functions and is important in immune surveillance and host protection against microbial infection by augmenting phagocytic removal and destruction of invading pathogens by phagocytic cells bearing receptors for C3b. Furthermore, the breakdown product of complement component C3, C3d, when bound to the Ag, is remarkably effective in augmenting the immunogenicity of foreign Ags to activate a specific immune response (12). Clearly, C3 functions effectively in both the adaptive and innate immune responses and acts to link them together. Yet because of the complex and essential activities of C3 in the higher vertebrate, this creates problems in studying each C3 function independent of the others. One means to understand the complexities and evolution of the higher vertebrate complement system and the immune system in general has been to investigate animals that are phylogenetically related, yet have simpler immune responses. The deuterostome lineage of animals is composed of two major phyla, the chordata that includes mammals, and the echinodermata that includes sea urchins, sea stars, sea cucumbers, and other groups. Because of this relationship, the echinoderms are an appropriate choice to understand innate immune responses without the added complexities of interactions between the innate and adaptive systems.
Coelomocytes are cells found in the coelomic cavity of the adult sea urchin that function as mediators of the immune system (for review, see Ref. 13 and references cited therein). Previously, we found that small doses of LPS would significantly activate the coelomocytes from the purple sea urchin, Strongylocentrotus purpuratus (14). This study was then followed by an investigation of genes expressed in these LPS-activated coelomocytes (15). Of the 307 expressed sequence tags (ESTs)3 that were reported, one (EST064) encoded an amino acid sequence that showed significant similarities to the thioester family of complement proteins. We report in this work the completed sequence of the EST064 cDNA, hereafter called Sp064, which shows that it encodes a homologue of complement component C3, called SpC3. The Sp064 gene appears to be single copy per haploid genome, and transcripts are present only in coelomocytes. The encoded protein, SpC3, is found in the coelomic fluid and is composed of two chains. This is the first identification of a complement component homologue expressed in an invertebrate. Because sea urchins are phylogenetically related to vertebrates, both groups being deuterostomes, these data indicate that a simple complement system was present in the deuterostome ancestor rather than the vertebrate ancestor and that it is a far more ancient system than had been previously assumed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of total RNA from coelomocytes and other adult tissues was done according to Smith et al. (16). Briefly, coelomic fluid (40 ml) was poured through sterile cheese cloth and mixed into 10 ml of cold Ca2+- and Mg2+-free sea water (17) containing 30 mM EDTA, pH 7.4 (CMFSW-E). Coelomocyte pellets and minced solid tissues were vortexed and homogenized using a dounce homogenizer in guanidinium thiocyanate extraction buffer (5 M guanidinium thiocyanate, 50 mM sodium acetate, 50 mM EDTA, 50 mM Tris, pH 7.4, and 5% ß-mercaptoethanol), to which was added N-lauroyl sarcosine to a final concentration of 2%. Total RNA was pelleted through a cushion of 5.7 M CsCl containing 50 mM sodium acetate and 50 mM EDTA at 105 x g in either a Ti60 fixed angle rotor (Beckman Instruments, Fullerton, CA) or a swinging bucket rotor (Sorvall, Newtown, CT) at 20°C for 20 h. Pellets were washed in 70% ethanol, resuspended in RNase-free water, extracted in phenol/sevag (1:1) (sevag is 24 parts chloroform, 1 part isoamyl alcohol), precipitated, and resuspended in RNase-free water. Poly(A)+ RNA was isolated using oligo(dT) magnetic beads (Dynal, Great Neck, NY).
cDNA library construction
The LPS-activated coelomocyte cDNA library was constructed using
the Time Saver cDNA kit (Pharmacia, Piscataway, NJ) according to kit
instructions. The first strand of cDNA was made from
Poly(A)+ RNA with a random primer containing a
NotI site. An EcoRI adapter was ligated to the 5'
end, and the cDNA was directionally cloned into 
ExCell phage arms
(Pharmacia), as previously reported (15). An arrayed cDNA library was
directionally constructed from nonactivated coelomocyte
poly(A)+ RNA using the same NotI/random primer,
but with a SalI adapter at the 5' end. The cDNA was ligated
into the pSPORT vector (Life Technologies, Grand Island, NY) and
electroporated into DH10b bacteria. Individual colonies were inoculated
into 384-well microtiter plates according to the methods of Maier et
al. (18). The arrayed library consisted of approximately 92,160 clones.
PCR products of clone inserts (18,432) were spotted in duplicate onto
five Hybond N+ nylon filters, 22 cm x 22 cm
(Amersham, Arlington Heights, IL) for screening.
Screening cDNA libraries
The ExCell library was screened using
32P-labeled RNA probes that were generated according
to technical information from Promega Corp. (Madison, WI) and as
previously reported (16). Filters were prehybridized for 2 h in
hybridization solution (50% formamide, 250 mM phosphate buffer, pH
7.4, 1 mM EDTA, 0.1% BSA, and 7% SDS), and then incubated with the
probe at 42°C overnight in a rotating oven (Robbins Scientific,
Sunnyvale, CA). Final washes were conducted at 68°C in 1x SSC (0.15
M NaCl and 15 mM sodium citrate, pH 7) with 1% SDS. Filters were
exposed overnight to X-OMAT XAR-5 x-ray film (Eastman Kodak, Rochester,
NY), and positive plaques were rescreened once to purify the phage
clones. Phagemids were released according to manufacturers’
instructions (Pharmacia), and in some cases, inserts were subcloned
into Bluescript (Stratagene, La Jolla, CA).
The arrayed library was screened with a random primed
32P-labeled probe generated from a deletion clone that was
made for sequencing, and consisted of approximately 500 bp of the 5'
end of pExCell14 (Fig. 1
). The deletion clone was first used as a PCR
template with the M13-40 primer and a primer specific for Sp064
(TCTAAGCAGGTAGACAGC; see Fig. 2, nucleotides 1988 to 2001). After
amplification of the insert, the 5' polylinker was removed by
EcoRI digestion, producing a 160-bp PCR fragment, which was
isolated by gel electrophoresis and eletroelution. The fragment was
then labeled with 32P by random priming.
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cDNA sequencing
Sequencing was conducted on plasmid DNA according to the
dideoxynucleotide termination protocol (19) using the TaqTrack
sequencing kit (Promega Corp.) and incorporating
[-35S]dATP (DuPont NEN, Boston, MA). Sequencing
reactions were electrophoresed on a 6% acrylamide gel with 0.6x TBE
(10x TBE is 0.9 M Tris, 0.9 M boric acid, and 20 mM EDTA, pH 8.3)
running buffer, after which the gel was dried and exposed overnight to
BioMax MR-1 x-ray film (Eastman Kodak). For clones longer than several
hundred nucleotides, such as the EcoRI to NotI
fragment of pExcell139 and all of pExCell14 (see Fig. 1
), insert
fragments were subcloned into Bluescript (Stratagene), and the
Erase-a-Base kit (Promega Corp.) was used to create a nested set of
deleted clones. Sequences were analyzed using the MacVector sequence
analysis program (Eastman Kodak) and the AssemblyLIGN sequence assembly
program (Eastman Kodak) on a Power Macintosh (Apple Computer,
Cupertino, CA).
Protein alignment and phylogenetic analysis
The cDNA sequence was used to run a basic blast search of
GenBank (20), and several of the matched sequences were used to
construct protein alignments with the Clustal W program (21). Without
altering the default parameters, a number of variations were used to
obtain the best alignment. We defined "best" alignments to be those
in which the thioester site and junctions between chains corresponded
best between the various proteins. It was noticed that the order in
which the proteins were listed slightly affected the alignment results
using Clustal W. Therefore, the order was changed in several runs to
obtain the best alignment. These variations included 1) listing SpC3
first, followed by the other proteins arranged from lower to higher
phylogenetic order, 2) keeping multiple proteins from the same species
together, and 3) listing the 2-macroglobulin
(2 M) protein family last. The best alignment on Clustal
W was verified by using a second alignment program, DNASIS version 2.1
(Hitachi, San Bruno, CA). To optimize the alignment on DNASIS, various
gap penalties were tried (5, 10, 25, 50) and then kept at 5. The fixed
gap penalty (10, 25, 50, 100) and floating gap penalty (10, 25, 50,
100) were adjusted separately and in combinations. We found that
alignments with DNASIS could not be improved over that generated by
Clustal W, and results from the two programs agreed best when the gap
penalties on DNASIS were maintained at the default settings.
The PAUP program (version 3.1.1) (22) was used with standard set parameters (character types were set as unordered, and character weights were set as 1) to compare and assemble multiple sequences from the Clustal W alignments into a phylogenetic tree. Slightly different alignments resulting from different ordering of the proteins in Clustal W were used in the PAUP program to identify the shortest tree. The heuristic search method was used with various search options to obtain the shortest tree. The general search options were set to keep minimal trees only and to collapse zero-length branches. The stepwise addition searches were either simple or random with seed numbers of 1, 50, and 100. The degree of support for internal branches was assessed using the bootstrapping method with 1000 bootstrap replications.
Preparation of anti-SpC3 antiserum
A rabbit antiserum was produced against the peptide,
DNAKVQEEVDVSPSIGR (see Fig. 2), which was chosen from the amino
acid sequence deduced from Sp064 and was based on predictions from the
human C3 structure that it would be located on an exposed region of the
-chain and would therefore produce a useful anti-peptide
antiserum. The peptide was synthesized using a 430A peptide synthesizer
(Applied Biosystems, Foster City, CA), conjugated to keyhole limpet
hemocyanin using glutaraldehyde (23), mixed with complete (first
injection) or in CFA (second and third boosts). The rabbit received
three injections given weekly, after which it was bled weekly for 3 wk.
The serum was collected and stored at -70°C.
Specific anti-peptide Abs were obtained by affinity chromatography. The synthetic peptide was coupled to activated CH-Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. The peptide column was equilibrated with PBS containing 10 mM EDTA, and 2 ml of the rabbit antiserum containing 10 mM EDTA was passed over the column twice. Unbound protein was washed with PBS, and the specific Ab was eluted with 0.1 M glycine/HCl, pH 2.5. The pH of the eluted fractions was immediately neutralized by the addition of 1 M Tris-HCl, pH 8. Affinity-purified Ab was stored at -70°C.
Purification of SpC3 protein from coelomic fluid
Sea urchin C3 was partially purified from coelomic fluid using modifications to published methods (24). Coelomic fluid was pooled from several sea urchins to which had been added EDTA (3 to 15 mM, final concentration), PMSF (2 mM, final concentration; Sigma Chemical Co.), and pepstatin A (1 to 100 µM, final concentration; Sigma Chemical Co.). Coelomocytes were pelleted, and the cell-free fluid was stored at -70°C until further purification could be conducted. Forty milliliters of sea urchin coelomic fluid were concentrated with an Amicon filter (10-kDa cut-off) (Amicon, Bedford, MA) to 2 ml, and the sample was passed through a PD-10 gel filtration column (Pharmacia) to exchange the buffer to 10 mM phosphate, pH 7.5. The concentrated coelomic fluid was precipitated with 4% polyethylene glycol, by stirring for 30 min at 4°C, followed by centrifugation at 15,000 x g for 20 min. The supernatant was brought to 16% polyethylene glycol by stirring at 4°C for 30 min and centrifuging as before. The pellet was then resuspended in 10 mM phosphate buffer, pH 7.5, and applied to a Mono Q HR 5/5 anion exchange chromatography column (Pharmacia) that had been equilibrated with the same buffer. Bound proteins were eluted with a linear salt gradient (0–500 mM NaCl), and fractions containing SpC3 were identified by gel electrophoresis and Western blotting using the affinity-purified anti-SpC3 peptide Ab.
Determination of NH2-terminal amino acid sequence
Purified protein was subjected to SDS-PAGE under reducing conditions and electroblotted onto ProBlott membranes (Applied Biosystems), and the NH2-terminal sequences of the protein chains were obtained by using a modification of the method of Matsudaira (25), as previously described (26). The individual SpC3 chains were separated by SDS-PAGE, electroblotted, cut out of the filter, and subjected to Edman degradation, using an 473 protein sequencer (Applied Biosystems).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A cDNA, identified as encoding a putative sea urchin complement
component, was one of 307 clones that were partially characterized from
the LPS-activated coelomocyte ExCell library, and was reported as
expressed sequence tag 064 (EST064) (15). In preliminary BLAST (20)
searches of GenBank, EST064 matched to cobra C3 (27), human and mouse
C3 (28, 29), mouse sex-limited protein (30), in addition to a few
others. This result suggested that EST064 encoded a new member of the
thioester gene family. The 5' end of pExCell064 (Fig. 1) was used to rescreen the same library
to obtain additional clones. The initial screen yielded 144 clones, 43
of which rescreened positive, and 12 of which were chosen for
sequencing based on the size differences of their 5' ends. Sizes were
determined by PCR using Sp6, a primer that hybridizes to the 5'
polylinker of the phagemid, and an insert-specific primer that
hybridized to the 5' end of pExCell064. Because this set of clones had
approximately 100- to 200-bp increment differences in size, this
created a natural deletion set for sequencing. Three
(pExCell139, 063, and 054) of the 12 clones from this set are
shown on Figure 1. The sequence obtained from the 5' ends of these 12
clones was published as the deduced protein, and was shown aligned with
other complement protein family members (see Ref. 15, Fig. 1). However,
at the time, it was not clear whether SpC3 was homologous to C3, C4, or
C5, although it did not appear to be 2 M
(15).
Because the total sequence length of these overlapping clones (shown in
Fig. 1 as pExCell054, pExCell063, pExCell064, and pExCell139) covered
only about one-half of the estimated open reading frame and only about
one-half of the transcript, the ExCell library was
screened a second time. The first 450 bp of pExCell054, the 5'-most
clone of the set (Fig. 1), was used to make another riboprobe that
yielded 84 clones, of which 18 rescreened positive. The clone with the
longest 5' end, pExCell14 (Fig. 1), was sequenced in its entirety using
nested set deletion clones. However, the beginning of the open reading
frame was not obtained. The third library screen used the arrayed
coelomocyte library constructed by Jonathan Rast and Eric Davidson at
the California Institute of Technology (Pasadena, CA). The 32 positive
clones obtained from this screen were analyzed by PCR using the Sp6 and
T7 primer sites in the vector to determine insert sizes, and the Sp6
primer in combination with an internal primer specific for the 5' end
of pExCell14 to determine the sizes of the 5' ends. These PCR-amplified
fragments were also checked by Southern blots using the same probe as
that used to screen the library. Analysis of the 5' end of
pSPORTA22/137, pSPORTG11/211, and pSPORTJ17/96 showed an additional 2
kb 5' of pExCell14. Sequence analysis of two clones,
pSPORTA22/137 and J17/96 (Fig. 1), indicated that the 5' end of the
open reading frame and a short stretch of the 5' untranslated (UT)
region were included.
The complete sequence of Sp064 and the deduced protein are shown
in Figure 2. There
are 7611 nucleotides (nt) in the overlapping cDNAs, which are missing
much of the 5' UT region and part of the 3' UT region of the transcript
since, by RNA gel blot, the transcript is 9 kb (see below). Because a
random primer was used to construct the cDNA library, the
poly(A)+ tail is not expected to be identified in any
clone. However, a consensus polyadenylation signal, GATAAA, is located
93 nt from the 3' end (Fig. 2), which suggests that most of the 3' UT
region may be present. The sequence shows 129 nt in the 5' UT region
(based on the most probable start site), 5097 nt in the open reading
frame, and 2385 nt in the 3' UT region. There are three possible start
sites near the beginning of the sequence shown in Figure 2. The first
ATG is followed by two in-frame stop codons, while the second and third
are in-frame and are not followed by stops. Because the third ATG,
located at nt 130, is surrounded by a Kozak sequence,
ACCATGG, this suggests that this is the most probable
start site for translation. Following the start site, there is a
hydrophobic region of 12 amino acids plus a serine, which is followed
by a hydrophilic region that includes serine, proline, and glycine
(31). This combination of a short hydrophobic and hydrophilic regions
is typical of a leader or signal sequence of 13 amino acids and is
based on the "[-3,-1]-rule" (32). A leader region is expected to
be present since SpC3 is produced in the coelomocytes and appears to be
secreted into the coelomic fluid (see Fig. 5; Gross and Smith,
unpulished). There are six ATTTA repeats in the 3' UT region (Fig. 2).
These AU-rich repeats are typical of transcripts that encode
inducible genes and may function to stabilize the transcript after
induction, which results in increased translation (33).
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). The ß-chain has
672 amino acids (without the leader), and the -chain has 1010 amino
acids after removing the RRKR at the ß junction. The deduced m.w.
for the preprocessed SpC3 is 186 kDa, and after processing the deduced
-chain is predicted to be 110 kDa and the ß-chain 73.5 kDa. These
predicted m.w. do not take into consideration the possibility of
N-linked glycosylation, which is known for human C3. There
are five consensus recognition sequences for N-linked
glycosylation in SpC3, four of which are located in the -chain (Fig. 2); however, none are conserved in position compared with glycosylation
sites in human C3 (34). The deduced isoelectric point for SpC3, as
calculated by DNASIS, is 4.76. This is substantially lower than the
range of isoelectric points 5.87 to 7.99, which was calculated for
other C3, C4, and C5 proteins that are listed in Table I.
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Regions of the deduced SpC3 protein that are similar to other
complement proteins can best be estimated through amino acid
alignments. The BLAST search provided a list of proteins to which SpC3
matched best, and we chose a subset of these to construct an alignment
that included some of the complement proteins (Fig. 3). Inspection of
Figure 3 reveals a number of regions in SpC3 that are conserved when
compared with the other complement sequences and other regions that are
not conserved. All proteins in Figure 3 show a ß junction,
including SpC3, whereas only the C4 proteins and the cyclostome C3
components show an 
junction. A conserved thioester site (GCGEQ)
is located in the SpC3 -chain, identical to that seen in vertebrate
C3 and C4 proteins, but that is not present in C5. The histidine
involved in thioester binding to hydroxyl groups (35), located about
100 amino acids (H1090 in Fig. 2) toward the C terminus, is conserved
in SpC3 (Fig. 3). The hydrophobic region surrounding the thioester site
is also conserved in SpC3, the function of which is thought to shield
the thioester from the aqueous environment and nucleophilic attack
(36). In vitro mutagenesis experiments have shown that the two prolines
surrounding the thioester in human C3 are necessary for stable
formation of the activated thioester (37), and these positions are
conserved in SpC3 (Fig. 3). This analysis of SpC3 compared with
vertebrate complement components indicates that it is a two-chain
structure with a conserved thioester site similar to other C3
proteins.
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) showed that five of the SpC3 cysteines are
located in the ß-chain, three of which align with the three conserved
cysteines in the ß-chain of all of the other components. In the
-chain, 18 of the 27 cysteines align with conserved cysteines in
other components. The two cysteines involved in interchain disulfide
bonding in the human sequence (38) are aligned between the sea urchin
sequence and all of the other proteins (Fig. 3). To understand more
fully whether the nonaligned cysteines in SpC3 were due to differences
between the protein sequences or due to errors made by the Clustal W
algorithm, additional alignments were performed to inspect the
positioning of the cysteines in SpC3. Both Clustal W and DNASIS were
used to align the entire thioester protein family and a subset that
included just the complement proteins. The Clustal W alignment of the
complement protein subset is shown in Figure 3, and the Clustal W
alignment for the entire thioester family can be obtained by E-mail
(see legend to Fig. 4; DNASIS alignments
are not shown). Results of these four alignments revealed similar, but
not identical, positioning of the cysteines in SpC3 relative to the
other proteins. Most (22 to 24) of the SpC3 cysteines align with or
near cysteines that are generally conserved in the complement protein
family. There are four to five positions in which cysteines are missing
in SpC3, where the other proteins showed a conserved cysteine, and SpC3
has seven to nine extra cysteines that do not align with other
proteins. In no case did any of the extra cysteines in SpC3 align with
cysteines in the 2 M group rather than the
complement group. Interpretation of these data may suggest that SpC3
has a similar, but perhaps not identical folding pattern compared with
the other complement proteins.
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in Lambris et al. (36),
and are indicated in Figure 3 for comparisons between SpC3 and human
C3. There are nine matches between SpC3 and human C3 in a span of 43
amino acids (20.9%) within the region in which human C3 interacts with
the C3a receptor, CR1 and CR3, factor H and factor B. This region is
generally not well conserved in vertebrate C3 proteins (8), except for
the C3 convertase cleavage site (Arg, Ser), which is conserved in most
of the C3 proteins including SpC3. The second factor H binding site
located farther down in the -chain, within which is located the CR2
binding site, reveals 18 amino acids in SpC3 that match to human C3 in
a span of 76 (23.7%). Eleven of thirty-two amino acids (34.3%)
match between SpC3 and human C3 within the properdin binding site that
is known as a region of high conservation (8). There are five
factor I cleavage sites labeled in Figure 3 that are positioned
relative to the human C3 sequence (10). Of those sites, none match
perfectly to SpC3; however, there are two Arg/Ser sequences in the SpC3
that align near factor I sites 1 and 5 (Fig. 3). In general, the
alignment with other complement proteins and the comparison with
functional sites in human C3 suggest that the sea urchin protein may
have a different folding pattern and fewer functions, and may not
interact with as many other proteins as is known for human C3. Phylogenetic relationships between SpC3 and other thioester family proteins
Our first approach to understanding the phylogenetic
relationships between SpC3 and other thioester family proteins was to
generate pairwise alignments between SpC3 and 25 sequences that
included several C3, C4, C5, and 2 M proteins from
a number of different species. We then calculated the percentages of
amino acids that showed identical matches and the percentages that were
identical plus similar between the two proteins. The results, shown in
Table I, indicate that SpC3 showed greater similarity to the complement
proteins (23.1–27.9% identical; 41.1–44.9% similar) than to the
2 M family (20.4–21.4% identical; 36.6–39.2%
similar) in general agreement with our previous report (15). However,
these results could have been due to the shorter length of
2 M proteins and not due to fewer amino acid identities
and similarities between SpC3 and the 2 M proteins.
Consequently, we generated a large alignment using all of the sequences
listed in Table I with the Clustal W program (21) (available by E-mail,
see legend to Fig. 4). The alignment was inspected at each amino acid
position, and SpC3 was scored as identical to each group of proteins
based on the number of sequences to which it matched. SpC3 was scored
as similar to C3 when it matched to six of nine of the vertebrate C3
sequences; similar to C4 when it matched to four of five of the C4
sequences; similar to C5 when it matched to both of the C5 sequences;
and similar to 2 M when it matched to six of nine of the
2 M sequences. Results indicated that SpC3 was equally
similar to all of the complement components (19%) and was less similar
to the 2 M proteins (15%) for this alignment set. A
similar result was found when the matches were calculated for a shorter
region that corresponded only to the 2 M protein length
(20% similar to complement components and 17% similar to the
2 M group). These alignment analyses suggest that
SpC3 is a complement protein and is not an 2 M
protein.
Since the paired alignments (Table I) were not informative as to the
relationships between SpC3 and the other complement protein family
members, a phylogenetic analysis was done using the PAUP program (22).
This program is designed to compare and assemble related sequences
into phylogenetic trees that can then be used to infer evolutionary
relationships. We used the same alignment between SpC3 and the 25 other
thioester proteins listed in Table I in a phylogenetic analysis using
PAUP. We were able to repeatedly identify the shortest tree
(length = 13614) when we used a number of variations in generating
the alignment with Clustal W, which were then used in the PAUP program.
Based on the lower percentage identity between SpC3 and the
2 M group (Table I), these proteins were selected as the
outgroup. The phylogenetic tree (Fig. 4) shows that SpC3 is positioned
basal to the complement clade that includes C3, C4, and C5 proteins.
Furthermore, the branch arrangement within the vertebrate complement
clade generally agrees with other published trees (26, 39, 40);
however, the internal details of the positioning of some of the
proteins differed in our trees depending on the protocols used. For
example, the hagfish component clustered with either the C3 or the C4
clade in different analyses. It should be noted that the hagfish C3
position in Figure 4 is not supported by bootstrapping and the position
of the lamprey C3 is poorly supported. The important result revealed in
Figure 4 is that SpC3 appears as the first diverging member of the
complement protein family.
We also investigated the -chains of the thioester proteins because
this chain includes many important functional regions. We chose 490
amino acids that started at the beginning of the -chain, included
the thioester site, and terminated with the end of the 2
M proteins. We used this region in another alignment and phylogenetic
analysis, again employing the 2 M proteins as the
outgroup. The resulting tree had a different appearance from that seen
in Figure 4. The complement clade had six unresolved groups that
included 1) sea urchin C3, 2) hagfish C3, 3) lamprey C3, 4) higher
vertebrate C3, 5) vertebrate C4, and 6) mammalian C5 (results not
shown). This decreased resolution perhaps reflects the similarity of
the -chains that contain significant sequence conservation in all of
the complement proteins.
Analysis of SpC3 protein by SDS-PAGE and N-terminal sequencing
The presence of a single junction in the deduced, unprocessed
protein predicted that SpC3 would have two chains. To test this and to
characterize the size of the protein, SpC3 was partially purified from
coelomic fluid and was separated by SDS-PAGE under reducing and
nonreducing conditions (Fig. 5). The
nonreduced protein is 210 kDa (Fig. 5, lane 1), and
under reducing conditions (lane 2), two chains
are resolved as the -chain (130 kDa) and the ß-chain (80 kDa).
These observed sizes are larger than the sizes deduced from the cDNA
sequence, suggesting that some or all of the consensus
N-linked glycosylation sites are filled during SpC3
processing by the coelomocyte.
A rabbit antiserum was raised against a peptide designed from the
deduced sequence in the -chain (see Fig. 2). On Western blots of
reducing gels, the antiserum bound to the larger -chain (Fig. 5, lane 3). To ensure that the protein isolated from the
coelomic fluid was encoded by Sp064, the N terminus of both chains was
sequenced. The ß-chain was found to be blocked; however, the
-chain gave the peptide, SIDRDQLXLYDP. In sequencing the cDNA
encoding the N terminus of the -chain, we found the following
peptide: SIDRDQLCLYDP (see Fig. 2). This identical match is evidence
that the protein isolated from the coelomic fluid is the same as that
encoded by Sp064.
Sp064 gene expression in sea urchin tissues
To determine whether coelomocytes were the only tissue to
express the Sp064 gene, we probed poly(A)+ RNA
isolated from the major adult tissues (coelomocyte, ovary, testis, gut)
and found that Sp064 is expressed exclusively in the coelomocytes (Fig. 6). The transcript size is approximately
9 kb, which is longer than the total length of the overlapping cDNAs
that we have sequenced (9-kb transcript, 7.6-kb cDNA sequence). Very
weak bands appeared in all lanes after very long exposures (data not
shown), with expression in gonads being higher than that in the gut.
Although the other tissues appear to have low expression of the Sp064
gene, it is likely that coelomocytes were present in or on these organs
at the time of dissection and total RNA isolation. Since it is not
possible to wash or remove all coelomocytes from other sea urchin
tissues during RNA isolation, these cells may account for the weak
bands (Fig. 6). These data indicate that coelomocytes are the major or
perhaps the only source of Sp064 gene expression in the adult sea
urchin.
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In most animals, C3 is a single copy gene; however, gene
duplication events are known to have occurred in some organisms.
Examples include C4A and C4B in humans (41), C4 and sex-limited protein
in mice (30, 42), and multiple gene copies of C3 in trout (24, 43) and
cobra (27, 44). To determine whether Sp064 is a single or multiple copy
gene per haploid genome, we analyzed a genome blot. Three male sea
urchins were treated to 15 V (direct current) and shaken to induce
spawning. Sperm was collected and DNA was isolated according to Lee et
al. (45). Each sample was digested with three endonucleases
(EcoRI, KpnI, BamHI), and the
genome blot was analyzed with a riboprobe that corresponded to the
-chain region of the message (500-bp fragment from the 5' end of
pExCell054). Only one or two bands were seen in each lane, indicating
that Sp064 is a single copy gene (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nonspecific, activatible immune response in the sea urchin is very
effective at maintaining a healthy animal; however, little is known
about the molecular mechanisms of gene expression or protein function
through which this system functions. Our preliminary study of sea
urchin ESTs was the first molecular evidence that a simple complement
system exists in the sea urchin (15), and we report in this work the
molecular characterization of SpC3. This is the first identification of
a C3 homologue that is expressed in a sea urchin, and furthermore, it
is the first complement component to be identified in an invertebrate.
Homology of SpC3 with vertebrate C3 proteins is based on several pieces
of evidence. 1) Sequence analysis reveals a ß junction, the
absence of an junction (which is present in C4), and the
conserved thioester site (which is absent in C5). 2) Protein gel
analysis shows that SpC3 is composed of two chains. The combination of
a two-chain molecule with a conserved thioester site suggests that SpC3
is a C3 complement homologue. Calculations of identities and
similarities of paired alignments shown on Table I plus the analysis of
the alignment and the phylogenetic tree all show that SpC3 is not an
2 M homologue and that it is the first diverging
member of the thioester complement protein family.
In mammals, the primary site of C3 biosynthesis and secretion is the
liver, with more than 90% of all C3 being produced by hepatocytes
(10). However, several other cell types appear to produce and secrete
C3 besides the liver, including macrophages, monocytes, fibroblasts, B
lymphocytes, polymorphonuclear leukocytes, type II pneumocytes,
astrocytes, and microglial cells (10, 49, 50, 51, 52, 53). These extrahepatic sites
of complement production are very important in local inflammatory
reactions. Since sea urchins do not have an equivalent of a liver, or a
hepatopancreas that is found in sea stars, the specific expression of
the Sp064 gene in coelomocytes suggests that these cells are involved
in producing SpC3 for both local and systemic function. Although we
cannot rule out the possibility that all major tissues express Sp064
based on the minor bands present in all lanes in Figure 6, one of the
functions of coelomocytes may involve patrolling and invasive
activities within organs when responding to microinjuries or focal
sites of inflammation. This possibility has been suggested previously
and was based on several ESTs encoding putative proteases that may be
capable of degrading the extracellular matrix (15). Consequently, the
presence of coelomocytes in all tissues of the adult sea urchin would
be revealed by low levels of Sp064 expression.
Simple complement systems have been identified in agnathan fishes and consist of a C3-like component (54, 55, 56, 57), factor B (58), and a putative complement receptor on circulating leukocytes (59). Complement in hagfish has been shown to function as an opsonin (60, 61). Although little is known about complement in tunicates, a mannan-binding lectin-associated protease homologue has been characterized from a tunicate, suggesting the presence of a lectin activation pathway (62) and other complement components may be present in these animals. Preliminary sequence data from a PCR fragment have indicated that a thioester protein may be present in the compound tunicate, Botryllus schlosseri (63). Based on what is known about complement function in lower vertebrates, SpC3 may function as an opsonin and be a very important mechanism for host protection against pathogens.
The presence of a C3 protein in sea urchins may suggest that a number of accessory and regulatory proteins may also be present in this organism. If the sea urchin complement acts as an opsonin functioning to identify foreign cells for removal and destruction by phagocytic coelomocytes, this suggests that a C3 receptor should be present on these cells. Preliminary evidence for a receptor putatively associated with the sea urchin complement system has been reported previously, suggesting that it might be involved in augmented phagocytosis (64, 65, 66, 67). In vertebrates, spontaneously activated C3 can be bound to self cells in the form of C3b, which is then inactivated by factor I that functions with a number of cofactors such as membrane cofactor protein, factor H, and CR1 (for review, see Refs. 68, 69). Decay-accelerating factor is an additional cell surface protein that may dissociate the C3b-factor B complex, called C3bBb, thereby deactivating its C3 convertase activity (70). Predictions of regulatory proteins in the sea urchin at present can only be based on the presence of a few conserved cleavage sites that have been identified in deduced SpC3 sequence. These include sites for factor I and C3 convertase, which is formed through an interaction between C3b and factor B. At present, none of these predicted proteins have been cloned and sequenced in the sea urchin, except one. We have characterized recently the complete open reading frame from EST152 (15), and it encodes a factor B-like protein (Smith, unpublished).
Lachmann (71) has proposed that the most primitive complement cascade or "archeo-complement" system would have resembled a simple alternate pathway consisting of a C3-like protein with a thioester site, a factor B-like protein containing short consensus repeats and a serine protease domain, and a complement receptor on phagocytic immune cells. Based on the sequence similarities among various complement components, it has been suggested that several of the complement protein families have been generated by gene duplication from a small number of primordial genes (35, 39, 72). Previous to the current work, the agnathans appeared to fulfill the prediction of an archeo-complement system, inferring not only that the alternate cascade was more ancient than the classical cascade, but that it was present in the common ancestor of the vertebrates. It is now clear, with this first characterization of a C3 homologue from the phylogenetically older deuterostome phylum, the echinodermata, that this protein represents the first diverging complement component. Furthermore, it is interesting to consider the possibility that it may still bear some similarities to the ancestral protein that functioned in the common ancestor of the deuterostomes that gave rise through gene duplication to the complement family of thioester proteins that functions in the higher vertebrates today.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. L. Courtney Smith, Department of Biologic Sciences, George Washington University, 2023 G St. NW, Washington, D.C. 20052. E-mail address:
3 Abbreviations used in this paper: EST, expressed sequence tag; 2 M, 2-macroglobulin; nt, nucleotide; UT, untranslated.
Received for publication September 26, 1997. Accepted for publication November 24, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin family. J. Immunol. 154:2164.[Abstract]
2-macroglobulin family members in the colonial ascidian, Botryllus schlosseri. Dev. Comp. Immunol. 22:147.
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