From the Institut de Biologie et Chimie des Protéines, CNRS, Unité Mixte de Recherche 5086, Université Claude Bernard, 7 passage du Vercors, 69367 Lyon cedex 07, France
Received for publication, October 20, 2000, and in revised form, March 9, 2001
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ABSTRACT |
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The Sea URchin Fibrillar (SURF) domain is a
four-cysteine module present in the amino-propeptide of the sea urchin
2 Collagens are a large family of extracellular matrix proteins
present in all animal phyla. Among the 19 collagen types hitherto identified, five of them, types I-III, V, and XI, constitute the fibrillar collagens (1). Each procollagen molecule is made of three In this study, we sought to obtain new information concerning SURF
modules in sea urchin. We characterized a new gene coding for a
multidomain protein of the extracellular matrix consisting of a series
of EGF-like and SURF modules. Its general structure is
reminiscent of sea urchin fibropellins. This new protein is present in
several soft tissues of the mineralized part of the adult. Its
co-localization with the 2 Embryo Culture and Nucleic Acid Purification and
Analysis--
Paracentrotus lividuswere purchased from the
Arago laboratory (Banyuls-sur-mer, France). Gamete collection,
fertilization, and embryo culture were done as previously described.
Total RNA from embryonic or adult tissues was purified according to a
published protocol (13). For adult RNA, a supplementary purification
step was performed prior to RACE experiments consisting of pelleting the RNA by ultracentrifugation through a 5.7 M cesium
chloride cushion (14). Poly(A)+ RNA was purified by two
passages through an oligo(dT)-cellulose column (Roche Molecular
Biochemicals). Northern blot, Southern blot, and screening procedures
were done according to conventional techniques (15). The genomic DNA
library was kindly provided by Dr. Christian Gache, marine station,
Villefranche-sur-Mer, France. Hybridization and washing of filters with
moderate stringencies were performed as described (16).
cDNA Synthesis and PCR--
For all RT-PCR experiments, 200 ng of plutei poly(A)+ RNA were reverse transcribed using
random primers and the reverse Expand kit (Roche Molecular
Biochemicals) according to the manufacturer's recommendations. For
PCR, several sets of primers were used and 35 cycles of amplification
of the target single strand cDNA were done using the Taq
Expand polymerase kit (Roche Molecular Biochemicals). The conditions
were: 94 °C for 3', then 10 cycles consisting of 94 °C for
15 s, 55-65 °C (depending on the primers) for 30 s, and
68 °C for 1-2 min. For the last 25 cycles, 15 s were added for
each cycle during the elongation step. After PCR, fragments were
purified from the gel and cloned using the TA-Topo 2-1 cloning kit from
Invitrogen (Groningen, The Netherlands) according to the
manufacturer's instructions. For RACE experiments, we used the 5' and
3' RACE kits from Life Technologies, Inc., and total RNA from the test
was used instead of poly(A)+ RNA extracted from plutei. All
the oligonucleotides used are listed in Fig. 1 and were synthesized by
Isoprim (Toulouse, France). Both DNA strands were sequenced using the
dideoxynucleotide chain termination procedure (Sequenase kit, Amersham
Pharmacia Biotech), and universal primer or synthetic oligonucleotides.
Computer Analysis--
DNA sequences were analyzed by the DNAid
computer program (17). Blast (18) and Prosite (19) searches were
performed using the IBCP site server accessible via the World Wide
Web.2
Antibody Production--
To prepare anti-fibrosurfin monoclonal
antibodies, the DNA insert encoding the SURF module R8 was
generated by PCR using the RT-PCR fragment RT3 (see Fig. 1) as template
with Goldstar DNA polymerase (Eurogentec, Seraing, Belgium). The
5' primer (5'-TATGGATCCGCCGTTGAGGTCACAAGCAC-3') and the 3' primer
(5'-TATCTGCAGACCTGTGCACGTGACAGCTTC-3') included a BamHI and
a PstI site, respectively. We used a derivative of pT7/7
(U S Biochemical Corp.) as an overproducing plasmid in which six His
codons had been included between the PstI and
HindIII sites with a stop codon following the last His codon
(20). Production and purification were done as previously described
(7). Mouse monoclonal antibody production, titration by enzyme-linked
immunosorbent assay, and characterization by immunoblotting was
performed using established protocols (21).
Protein Detection--
Tissues were dissected from adult
P. lividus. Test, Aristotle's lantern, digestive tract,
spines, base of spines, and peristomial membrane were collected.
Sequential 24 h extractions at 4 °C in 2 M urea and
then in 8 M urea with protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 10 mM
N-ethylmaleimide and 0.5 mM dithiothreitol) were performed on embryos or crushed tissues with ~5 ml of extraction buffer/g of wet material. Supernatants were analyzed by Western blotting. Crude extracts were separated on 6% SDS-polyacrylamide gel
electrophoresis (PAGE) followed by electrotransfer to polyvinylidene difluoride membranes (Immobilon-P, Millipore, St. Quentin en Yvelines, France) overnight at 4 °C in 10 mM CAPS pH 11, 5%
methanol Blots were exposed to 23-2D4- (anti-SURF module R8,
fibrosurfin) and 11-4E11- (anti-SURF module R2, 2
Protein extracts (2 M urea) from test were dialyzed against
20 mM Tris, pH 8, and chromatographed on a DE52 anionic
exchanger. Proteins were eluted with a linear gradient of 0-1
M NaCl.
Immunological Methods--
Test and spine bases were dissected
from individual P. lividus. Samples were rinsed with
artificial sea water (ASW, 480 mM NaCl, 10 mM
KCl, 26 mM MgCl2, 29 mM
MgSO4, 10 mM CaCl2, 2.4 mM NaHCO3, pH 8) and fixed for 4 h at
4 °C in 2.5% paraformaldehyde in ASW. After rinsing with ASW,
calcified tissues were demineralized with 0.5 M EDTA at
4 °C. Finally, all samples were rinsed with phosphate-buffered
saline and frozen in liquid nitrogen. Thin sections (5-10 µm) of
frozen tissue were cut on a Cryostat (Leitz), picked up on slides, or
maintained floating in solution and handled with Pasteur pipettes for
electron microscopy. Sections were immunolabeled with 23-2D4 and
11-4E11 (undiluted hybridoma supernatants) as primary antibodies.
Negative controls were performed by omitting the primary antibody.
Sections were then incubated with secondary antibodies:
fluorescein-conjugated goat anti-mouse IgG (diluted 1/400; Jackson
ImmunoResearch, West Grove, PA) or goat anti-mouse IgG-conjugated to 5 nm gold particles (diluted 1/20, British Biocell International, Cardiff, UK) for electron microscopy. Immunofluorescence observations were performed on a Zeiss Universal microscope. For electron microscopy, immunolabeled sections were fixed for 1 h at
room temperature in 2% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4). Samples were rinsed in the same buffer and
post-fixed for 1 h at room temperature in 1% osmium tetroxide in
1,4-piperazinediethanesulfonic acid buffer (0.1 M, pH 7.4).
After rapid washing in water, sections were dehydrated in a graded
ethanol series and embedded in Epon. Ultrathin sections were cut on a
Reichert-Jung Ultracut ultramicrotome and contrasted with methanolic
uranyl acetate and lead citrate. Samples were observed with a CM120
Philips electron microscope at the "Center de Microscopie
Electronique Appliquée à la Biologie et à la
Géologie" (CMEABG, Université Claude Bernard, Lyon I).
The P. lividus Genome Can Potentially Encode Several Proteins
Possessing SURF Modules--
From previous work we have shown that
SURF modules are present in the N-propeptide of the sea urchin 2 RX1 Encodes a Modular Protein with the General Structure of
Sea Urchin Fibropellins--
To obtain the complete coding sequence,
5' and 3' RACE were performed using total test RNA (Fig. 1). Two new
overlapping RT-PCR fragments (RT7 and RT8) covering the complete
reading frame were prepared and analyzed to confirm the primary
structure. Analysis of RACE and RT-PCR cDNA clones revealed that
the composite sequence presented an open reading frame, which could
encode a protein of 2656 amino acids (Fig.
3). From the amino to the carboxyl
termini, the conceptual open reading frame contained a putative signal peptide of 16 or 24 amino acids, one EGF repeat, a 122-amino acid domain with two cysteine residues, two EGF motifs, 13 SURF modules, 14 EGF repeats, and a short 29-amino acid region with two cysteines. Two
possible translation start sites were present, of which the sequence
flanking the Met codon, numbered 1 in Fig. 3, better matched the
consensus motif for the translation initiation (22). Blast searches
indicated that the 17 EGF motifs gave the best scores with the
comparable domains of fibropellin (23, 24) and Notch (25) proteins.
Like these proteins, 11 of the 17 EGF repeats presented the consensus
signature of calcium-binding EGF modules (cbEGF), i.e.,
(DEQN)X(DEQN)2CXnCXnCX(DN)X4 (FY)XC
(PROSITE, PDOC00913). The 122-amino acid domain gave the best scores
with the CUB domain of fibropellins (23, 24). In comparison,
highest percentage identities were at least 75% for the EGF modules
and 23% for the CUB domains. Blast searches were also performed using the carboxyl-terminal domain, but no significant scores were obtained with any data bank analyzed. A schematic representation of the new
protein resembles the general structure of sea urchin fibropellins (24)
with the exception of 13 SURF modules between EGF repeats 3 and 4 and
the replacement of the avidin-like domain of fibropellins with a
29-amino acid domain (Fig. 1). From the common modular organization
with fibropellin and the presence of SURF modules, we called this
protein fibrosurfin. From its primary structure, fibrosurfin is an
acidic protein with an estimated isoelectric point of 4.12 and a
calculated molecular mass of 276 kDa. The net charge is Fibrosurfin Is Detected in the Unmineralized Part of the Adult
Test--
As indicated above, monoclonal antibodies against a
recombinant protein sharing SURF module 8 of fibrosurfin were prepared. Unlike EGF repeats, most of the SURF modules present a low level of
identity between them. Hence, the SURF module 8 of fibrosurfin shows
the highest identity with SURF modules 5 of fibrosurfin (39%) and 12 of the 2 Immunolocalization of Fibrosurfin and the 2 In this report, we clearly demonstrate that several genes in sea
urchin could encode SURF modules. In addition to the previously described 2 From Fig. 1, a common origin for genes encoding fibropellins and
fibrosurfin is strongly suggested. Firstly, highest identity scores
were obtained between these two proteins for two types of modules, the
CUB and EGF domains. Secondly, their general structures are closely
related with the exception of the carboxyl-terminal domain and the
insertion of a series of SURF modules between two EGF motifs (24). Both
these features greatly support the notion of exon shuffling (28), which
accounts for considerable variety among multimodular proteins. Even
though the general structures of these proteins are similar, it is
difficult to obtain any co-linearity between their EGF modules as has
been observed between sea urchin fibropellins. This suggests that these
genes had diverged early during evolution or that they have evolved
rapidly. Although the 2 In the course of this study, we have compared the results obtained
using anti-2 In fibrosurfin, 11 of the 17 EGF domains could potentially bind
calcium. Proteins that contain EGF domains are often developmentally important (31, 32). Hence, roles in protein-protein or protein-cell interactions have been demonstrated or inferred for these proteins. Stretches of cbEGF are observed in fibrosurfin, and it has been demonstrated that tandemly repeated cbEGF modules display higher affinities than isolated cbEGF for calcium (33). In the same way,
CUB-cbEGF pairs of two complement components, C1s and C1r, show high
affinity for calcium (34). These two proteins form a tetrameric
sub-unit C1s-C1r-C1r-C1s, and their assembly is
calcium-dependent. Thus, the presence of a CUB-cbEGF region
in fibrosurfin reinforces the idea that these domains might promote a
homotypic association, whereas stretches of cbEGF might be involved in
homotypic and heterotypic protein-protein interactions. EGF modules are
located at the two extremities of fibrosurfin and correspond to the
most anionic part of this protein. The interfibrillar matrix of these collagenous ligaments contains several polyanionic glycosaminoglycans (8). Moreover, several acidic glycoproteins that have a strong negative
charge seem to be important in the aggregation properties of the
collagen fibrils (35, 36). Both fibrosurfin and the 2 From this potential capacity to bind calcium and its localization in
collagenous ligaments as an interfibrillar component, fibrosurfin could
be one of the factors responsible for the unusual properties of these
collagenous tissues. In fact, echinoderm ligaments are quite unique and
have been called mutable collagenous tissues or catch connective
tissues (8, 37). These animals possess a mechanism to alter the
transfer properties of the interfibrillar matrix of their ligaments
(35, 37), which permits modulation of both the shape and stiffness of
collagenous tissues. A recent report indicates than one or more
secreted molecules induce the aggregation of fibrils in the presence of
calcium. For the sea cucumber dermis, stiparin is one of these
stiffening factors (35). Modulation of these properties by
anti-stiparin molecules has also been described (36). A more recent
study indicates that stiffening and plasticizing factors seem to be
located inside the cells of the holothurian dermis rather than in
compartments of the extracellular matrix (38). One of their hypotheses
is that the effect of these reagents could be amplified by matrix macromolecules like stiparin. From its extracellular matrix
location and its biochemical characteristics, fibrosurfin might play a similar function.
From the uniqueness of mutable collagenous tissues in echinoderms, an
evolutionary origin for these functions has been proposed (39). SURF
modules have been characterized only in sea urchin despite the numerous
international genome and expressed sequence tag programs. In
Caenorhabditis elegans, more than 20 modules seem unique to
this phylum (40). Because the three proteins harboring SURF modules
appear to be specific to the mutable collagenous tissues, it is
tempting to speculate that this module is one of the evolutionary
elements responsible for this echinoderm feature. A search of SURF
modules in other echinoderms and further analysis of SURF-containing
proteins will permit us, in the future, to define more precisely the
relationship between SURF modules and the so-called mutable collagenous tissues.
fibrillar collagen chain. Despite numerous international genome
and expressed sequence tag projects, computer searches have so far
failed to identify similar domains in other species. Here, we have
characterized a new sea urchin protein of 2656 amino acids made up of a
series of epidermal growth factor-like and SURF modules. From
its striking similarity to the modular organization of fibropellins, we
called this new protein fibrosurfin. This protein is acidic with a
calculated pI of 4.12. Eleven of the 17 epidermal growth factor-like
domains correspond to the consensus sequence of calcium-binding type. By Western blot and immunofluorescence analyses, this protein is not
detectable during embryogenesis. In adult tissues, fibrosurfin is
co-localized with the amino-propeptide of the 2
fibrillar collagen
chain in several collagenous ligaments, i.e., test sutures, spine ligaments, peristomial membrane, and to a lesser extent, tube feet. Finally, immunogold labeling indicates that fibrosurfin is
an interfibrillar component of collagenous tissues. Taken together, the
data suggest that proteins possessing SURF modules are localized in the
vicinity of mineralized tissues and could be responsible for the unique
properties of sea urchin mutable collagenous tissues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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chains, each of which can be identical or not. Each
chain contains
a triple helical region of 1014 amino acids constructed of an
uninterrupted series of GXY triplets. Two non-collagenous regions, the amino- and the carboxyl-propeptide flank this domain. During extracellular maturation of procollagen into collagen molecules, the N- and the C-propeptides are generally removed by the action of
specific proteases. The resulting collagen molecule consists of a
central triple helix flanked by two short non-collagenous segments, the
N- and the C-telopeptides (1, 2). Although the size of the central
triple helical region is conserved, with one glycine residue for every
three amino acids, the sequence of the C-propeptide domain is the most
conserved among the
chains. In contrast, the N-propeptide domain is
the most variable region among procollagen molecules. Three different
N-propeptide configurations have been characterized in vertebrates (3),
and a fourth structure has been defined in sea urchin (4). All of them
contain a short triple helical region at the carboxyl terminus. In sea
urchin, the N- propeptide consists, from the amino to the carboxyl
terminus, of a cysteine-rich region or tsp-2 module, 12 repeats of a
four-cysteine domain, and a short triple helical region connected to
the N-telopeptide. The four-cysteine module or
SURF,1 for Sea URchin
Fibrillar, domain has been described for the first time in the 2
fibrillar collagen chain, but the sea urchin genome possesses at least
one other region that could potentially encode several SURF modules (4,
5). The consensus sequence of this 140-145 amino acid motif is
X(40)GX2LWX11GXGX39CX6CX2(L/F)X(23)CX(4)CX1 (where the numbers in parentheses represent an average number of
residues). In situ hybridization reveals that 2
transcripts are detected in mesenchymal cells at the late gastrula
stage and in spicule- and gut-associated cells in plutei (6).
Immunostaining indicates the presence of this protein around the
skeleton spicules and as a thin meshwork in the extracellular matrix
surrounding mesenchymal cells (7). In adults, collagen fibrils have
been detected in the soft connective tissues of the test, the dermal outer appendages or spines, the Aristotle's lantern or echinoid jaw,
the tube feet, and the peristomial membrane that bridges the gap
between the jaw and the skeleton (8-12).
fibrillar collagen chain, its biochemical
properties, the presence of EGF-like motifs that might bind calcium,
and its interfibrillar localization suggest a function for this protein
in the so-called mutable collagenous ligaments of sea urchin.
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DISCUSSION
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chain; Ref. 7)
purified antibodies at a concentration of 1 µg/ml. Alkaline
phosphatase-conjugated goat anti-mouse IgG (Bio-Rad) were used as
secondary antibody and developed using the substrate kit from Bio-Rad
(Ivry-sur-Seine, France).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
fibrillar collagen chain and that another part of the sea urchin genome
could encode several SURF modules (4, 5). Until now, however, we have had no evidence that this region is part of an active gene or pseudogene. Moreover, a Southern blot of P. lividus genomic
DNA under moderate stringency revealed that several parts of the sea urchin genome could encode SURF modules (data not shown). From these
results, we used the same hybridization conditions to screen a P. lividus genomic DNA library. Among 60,000 clones, 54 positive clones exhibiting variable intensities of labeling were detected. Shotgun sequencing analyses were done for several weakly positive clones, two of which overlap, that possess sequences coding for SURF
modules. Blast search analyses revealed that these SURF modules shared
20-30% identity with comparable domains of the 2
chain and the
putative 5
protein. RT-PCR experiments were done using poly(A)+ RNA extracted from plutei embryos. As presented in
Fig. 1, six overlapping RT-PCR fragments
(RT1-RT6) lead to the characterization of 11 SURF modules and three EGF
repeats. Northern blots performed using poly(A)+ from
plutei with the RT-PCR fragment RT4 as probe failed to give any
detectable signals (Fig. 2A).
Moreover (as described below in more detail) no positive bands were
obtained during embryogenesis when monoclonal antibodies against the
SURF modules R8 were used in Western blotting, though a positive
reaction was obtained with adult test. By Northern blotting using the
RT4 DNA fragment, a 13-kilobase mRNA was detected with total test
RNA (Fig. 2A). As a control, multiple 2
transcripts were
detected either with plutei or test RNA (Fig. 2B).
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Fig. 1.
Schematic representation of fibrosurfin.
A, RT-PCR and RACE cDNA clones are depicted
above the modular organization of sea urchin fibrosurfin.
B, modular structure of sea urchin fibropellins. The common
modular organization between fibrosurfin and fibropellins is evident
with the dotted lines indicating the insertion of 13 SURF
modules between EGF repeats 3 and 4 in fibrosurfin. Note that the
carboxyl-terminal domain of fibrosurfin is replaced by an
avidin-like domain in fibropellins. C, sequences of the
synthetic oligonucleotides used for the RT-PCR and RACE experiments.
Fw, forward primer; Rev, reverse primer.
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Fig. 2.
Northern blot comparisons of fibrosurfin
(A) and 2
(B) mRNAs. Probes for fibrosurfin (RT4
cDNA) and 2
(DNA coding for SURF modules R6-R8) were hybridized
to plutei (P) poly(A)+ RNA (1 µg) or total RNA
(10 µg) from test (T). The positions of 28S and 18S rRNA
markers are indicated on the left.
211 (10.7%
of Asp + Glu), but EGF domains are the most anionic part of
fibrosurfin (13.15-21.05% of Asp + Glu). From its amino acid
composition, fibrosurfin is rich in serine and threonine residues
(20.5%), especially the SURF domains (up to 28.3%). Finally, five
consensus N-linked glycosylation sites are present, two in
the CUB domain, one between EGF-repeats 2 and 3, and the
remainder within SURF modules 5 and 13 (Fig. 3).
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Fig. 3.
Complete amino acid sequence of
fibrosurfin. Asterisks indicate the two putative Met.
The sequence surrounding the first Met codon (GGTTCCATGG) is related
more to the Kozak sequence (GCC(A/G)CCATGG, Ref. 22) than that
surrounding the second Met codon (TTTAAGATGG). In the repeating
subdomains, boundaries and identities of each repeat are indicated by
the horizontal arrows and numbers, respectively.
Vertical arrow indicates the putative signal peptide
cleavage site. Putative N-linked glycosylation sites are
underlined.
chain (33%). Moreover, the monoclonal antibody used in
this study did not cross-react with several previously produced
recombinant proteins harboring SURF modules of the 2
chain (data not
shown and Ref. 7). These antibodies were used to examine the expression
of fibrosurfin in sea urchin tissues. Because the gene coding for this
protein was expressed in test, Western blotting was performed using
different protein extracts from demineralized tests (Fig.
4). After urea treatment, several immunoreactive bands were detected between 80-160 kDa. Positive bands
with a molecular mass higher than 120 kDa disappeared rapidly upon
short term storage at 4 °C or
20 °C (results not shown). Using
the chemical properties of fibrosurfin, urea protein extracts from test
were submitted to anionic exchange chromatography, and eluted fractions
were separated by SDS-PAGE (Fig.
5A). The major bands present
in the 0.36 M NaCl fraction were recognized by the anti-fibrosurfin monoclonal antibody (Fig. 5B), whereas 2
immunoreactive bands were detected in the 0.04 and 0.2 M
NaCl fractions (Fig. 5C). Edman degradation sequencing of
the 0.36 M NaCl bands specific from fibrosurfin was
performed, but their amino termini were blocked. Nevertheless, in some
experiments, a highest molecular mass band (280-300 kDa) was
recognized by the anti-fibrosurfin monoclonal antibody (Fig.
5D, 0.25 and 0.3 M NaCl fractions). As a next
step, Western blots were performed using protein extracts from embryos using the anti-fibrosurfin monoclonal antibody (Fig.
6). In these blots, no immunoreactive
bands were detected except for the positive control consisting of
proteins extracted from test. Finally, several tissues from adult
animals were analyzed by Western blotting using anti-fibrosurfin (Fig.
7A) or anti 2
(Fig.
7B) monoclonal antibodies. From these blots, the 2
N-propeptide and fibrosurfin were present in the same tissues,
i.e. test, spine ligament, and peristomial membrane.
Traces of the proteins were detected in the tube feet, whereas
no detectable signals were obtained in extracts of the digestive tract
or of spine tips. The 2
chain is also detected in the Aristotle's
lantern. As for fibrosurfin, several immunoreactive bands were exposed
using anti-2
N-propeptide monoclonal antibodies. For both proteins,
the patterns of positive bands were slightly different in the different
tissues analyzed, especially for fibrosurfin. High molecular mass
immunoreactive bands were obtained for both proteins in extracts from
the peristomial membrane.
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Fig. 4.
Extraction of fibrosurfin from
demineralized test. Crushed tests were sequentially extracted at
4 °C in 1 M NaCl (1), 50 mM
CAPS (2), 2 M urea (3), 8 M urea (4), and 0.1% SDS (5).
Extracts were separated on 3.5-15% SDS-PAGE, transferred to the
membrane, and reacted with the purified monoclonal antibody 23-2D4
(anti-fibrosurfin).
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Fig. 5.
Anion-exchange chromatographic analysis of
fibrosurfin. Urea protein extracts from test were separated by
anionic exchange chromatography (DE52) under a linear gradient of NaCl,
and fractions were separated by 8% SDS-PAGE followed by Coomassie Blue
staining (A) and analyzed using the monoclonal antibody
(23-2D4) against fibrosurfin (B) or analyzed using the
monoclonal antibody (11-4E11) against the 2 chain (C). In
an other experiment, a stepwise NaCl elution was performed, and Western
blot was carried out using the monoclonal antibody (23-2D4) against
fibrosurfin (D). (*) indicates the three major bands in 0.36 M NaCl fraction from A that are positively
stained in B.
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Fig. 6.
Immunoblot analysis of fibrosurfin during the
early embryogenesis. Urea extracts from eggs to plutei were
analyzed by Western blotting using the monoclonal anti-fibrosurfin
antibody 23-2D4. A urea extract from test (T) was used as a
positive control.
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Fig. 7.
Immunoblot analysis of fibrosurfin
(A) and 2 chain
(B) in different adult tissues. 8 M
urea extracts from different adult tissues were
analyzed by Western blotting using the monoclonal anti-fibrosurfin
antibody 23-2D4 or the monoclonal anti-2
antibody 11-4E11.
T, test; Sb, base of the spine; St,
top of the spine; PM, peristomal membrane;
Tf, tube feet; AL, Aristotle's lanthern;
I, intestine. V corresponds to the volume (X.10
µl) of each urea extract that was analyzed (5 ml of extraction
buffer/g of tissue).
N-propeptide--
Two positive tissues, the catch apparatus and the
test, were analyzed by immunostaining using the same antibodies. In
Fig. 8A, a section of the
catch apparatus consists of three regions: the mineralized tissues of
the spine, the collagenous ligaments, and the external region, which is
not depicted and contains mainly muscle cells and the epidermis.
The sutural ligaments that link the calcite plates are composed of
collagen fibrils (Fig. 8A). We can distinguish the
meridional or zigzag sutures from the circumferential sutures between
the test plates. Using monoclonal antibodies against fibrosurfin or the
2
N-propeptide, immunofluorescence studies indicated that 2
and
fibrosurfin were co-localized in the collagenous ligaments of the catch
apparatus (Fig. 8, B and C) and in the sutural
ligaments (Fig. 8, D and E). Zigzag sutures were
more intensively stained than circumferential sutures. As shown in Fig.
8F, a strong autofluorescence was detected within the
mineralized plates. To better localize fibrosurfin and the 2
N-propeptide in the spine ligament, preembedding immunoelectron
microscopy was performed to preserve antigenicity. For fibrosurfin,
gold particles were observed between or in close proximity to collagen fibrils, indicating that fibrosurfin is an interfibrillar component (Fig. 9, A and C).
For the 2
N-propeptide, gold particles accumulated at the periphery
of the bundles made of collagenous fibrils aligned in parallel. These
gold particles were generally in the vicinity of cells (Fig.
9D) and rarely observed at the surface of collagen fibrils
(Fig. 9E). No signal was observed for the negative control (data not shown).
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Fig. 8.
Immunofluorescence analysis of fibrosurfin
and 2 chain expression in test and spine
ligament. A, schematic representation of sea urchin
test with magnified views of the ligamental sutures, a section of the
catch apparatus, and the peristomial membrane (26, 27).
Immunofluorescence analyses were done in catch apparatus (B
and C) and in test (D-F), using anti-2
chain
antibody 11-4E11 (B and C), anti-fibrosurfin
antibody 23-2D4 (D and E)
and without antibody (F). Bar = 100 µm.
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Fig. 9.
Ultrastructural analysis of the spine ligament
using anti-fibrosurfin (A-C) and anti-2 chain
D-F) antibodies. No signal is observed for the
negative control (E). Bar = 200 nm.
M, muscle; mt, mineralized tissues.
DISCUSSION
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ABSTRACT
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DISCUSSION
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fibrillar collagen chain (4, 5), we have obtained the
primary structure of a new protein, which we call fibrosurfin and
contains a series of 13 SURF modules. Immunolocalization and biochemical studies indicate that fibrosurfin, like the 2
chain, is
one of the components of the collagenous ligaments that link together
the calcite ossicles of the sea urchin skeleton. In addition, preliminary data concerning the previously described
COLP5
gene (5), indicate a similar localization of this
related protein in adult
tissues.3 Taken together,
these results suggest that proteins, including SURF modules, seem to be
located around the mineralized region of the sea urchin and in
so-called adult mutable collagenous tissues.
chain and 5
protein are similar, we could
not detect any similarities between their SURF motifs and those of
fibrosurfin. However, like 2
and 5
, fibrosurfin SURF modules are
acidic. One of the particularities of fibrosurfin SURF modules is their
high serine and threonine residue content. Several clusters of these
amino acids provide potential sites for O-linked
glycosylation (29).
N-propeptide and anti-fibrosurfin antibodies. It is
worth noting that in plutei, we have previously shown the retention of
the N-propeptide of the 2
chain at the surface of thin fibrils (7),
indicating that this domain is not fully processed during
embryogenesis. Here, we could detect several immunoreactive bands by
Western blotting of adult tissue extracts, although immunoelectron
microscopic labeling indicated that the 2
N-propeptide is located
around bundles made of fibrils aligned in parallel with fibrosurfin
located between fibrils. These results suggest that the N-propeptide is
processed in the adult. This is consistent with the observation that
adult fibrils are thicker (124 nm on average) (30). Finally, the
distinct 2
bands could also represent the different 2
N-propeptide isoforms of the 2
chain previously identified (4). For
fibrosurfin, it is apparent that, in most cases, we could not obtain
intact molecules in our extracts. Either this protein is already
cleaved in these tissues or proteolytic events occurred during the
solubilization procedures. It is worth indicating that a similar
complex pattern of bands has been reported for the Notch receptor in
sea urchin (25), a protein containing cbEGF. Moreover, some of the
faster migrating bands probably also represent isoforms of fibrosurfin
despite no alternatively spliced mRNA having been detected during
the RT-PCR procedures and only one hybridizing band revealed by
Northern blotting (pluteus and test RNA). We have yet to investigate
possible alternative splicing events in other adult tissues.
N-propeptide
(pI 4.55) are also acidic and have a strong negative charge.
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FOOTNOTES |
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* This work was supported in part by the European Community Contract Biotechnology BIO4-CT96OG62.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ291489.
Supported by the Fondation Marcel Mérieux and by the
Fondation pour la Recherche Médicale.
§ To whom correspondence should be addressed. Tel.: 33-4-72-72-26- 77; Fax: 33-4-72-72-26-02; E-mail: jy.exposito@ibcp.fr.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M009597200
2 Contact corresponding author for Web address.
3 C. Cluzel, C. Lethias, R. Garrone, and J. Y. Exposito, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SURF, sea urchin fibrillar; EGF, epidermal growth factor; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; RT, reverse transcription; PAGE, polyacrylamide gel electrophoresis; ASW, artificial sea water; cb, calcium-binding.
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