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INTRODUCTION |
Most invertebrates possess an extracellular matrix
(ECM)1 surrounding their
epidermis. In such ECM, chitin microfibrils are embedded in a
proteinaceous matrix.
Chitin is a polymer of N-acetyl-D-glucosamine,
but naturally occurring forms vary in the length and arrangement of
their chains. The major form of chitin in living kingdom is
-chitin,
as encountered in mushrooms and arthropods (1). This common form, the
more stable one, is characterized by an anti-parallel joining of
polysaccharidic chains. In contrast,
-chitin is only present in few
species like in squid pen, diatoms, some protists (2) and in tubes of
pogonophorans as Riftia pachyptila (3-5). This rare
form of chitin corresponds to a parallel joining of chains. The giant
vestimentiferan worm R. pachyptila (up to 2 meters in
length), found around the deep sea hydrothermal vents (6), inhabits a
tube which is made of
-chitin associated with proteins (7-9).
Previous studies on R. pachyptila have shown that large
-chitin secreting glands (present in all the four main regions of
the animal except obturaculum) were involved in the tube wall
production (10, 11).
Proteins, the other component of ECMs, were mostly studied in
arthropods and some of them are supposed to interact with chitin by
means of specific domains. Chitin-binding proteins form a highly diversified group. They are encountered in bacteria (12), (13), plants
(14, 15), invertebrates (16, 17), and vertebrates (18). They can have a
single (14, 18) or multiple chitin-binding domains (16) linked or not
to a catalytic subunit. Functions of these proteins can thus range from
a simple binding, like lectins, to chitinase activity, or even
antimicrobial and antifungal properties in case of tachycitin (17).
Substrate specificity of several chitin-binding proteins has been
studied, and results showed that some proteins bind only
-chitin
(12), when others recognize both
and
-chitin forms (19), but
until the present work, no
-chitin-specific protein have been reported.
Because R. pachyptila is a producer of large quantities of
-chitin, we have undertaken the molecular cloning of a cDNA
encoding a protein whose deduced amino acid sequence presents
chitin-binding domains and is thus called RCBP (Riftia
chitin-binding polypeptide). Furthermore, to check that the
chitin-binding domains predicted by search in data bases are
functional, RCBP was expressed in Escherichia coli and
tested for in vitro chitin binding using
and
-chitin
as ligands. To specify the origin of this putative tube component,
Northern blot and in situ hybridization analysis were
performed using tissues from the different regions of this deep sea worm.
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EXPERIMENTAL PROCEDURES |
Materials--
R. pachyptila samples were collected
on the East Pacific Rise depths by the Nautile or Alvin submersibles
(HERO 94, LARVAE 95, HOT 96, and HOPE 99 cruises, 2600-m depth). The
body of this worm presents four successive main anatomical regions from
the upper to the most basal part (for review, see Ref. 6). The obturacular region, with its branchial plume, is the primary exchange surface of the animal. The vestimentum serves to hold the plume out of
the tube opening in the external seawater. The trunk houses endosymbiotic bacteria, and the opisthosome acts as a holdfast, against
which the worm may move into the tube. Once dissected immediately after
arrival on board, the tissues from the four main regions of the worm
(obturaculum, vestimentum, trunk, and opisthosome) were frozen in
liquid nitrogen and stored at
80 °C.
RNA Isolation--
Total RNA from tissues of the four main
regions of the worm were extracted by the phenol/chloroform/isoamyl
alcohol method as described previously (20). Poly(A)-rich RNA were
purified by affinity with oligo(dT)-cellulose (21). The amount and the purity of each RNA sample were estimated using spectrophotometric measurements at 260 and 280 nm.
RT-PCR--
Poly(A)-rich RNA from the four different main
regions were used as templates for reverse transcription using
Superscript II reverse transcriptase (Life Technologies, Inc.) and an
oligo(dT) anchor primer (22). This anchor has the peculiarity to
hybridize specifically at the beginning of the polyadenylated region of the mRNAs. The resulting cDNAs were used as templates for
different PCRs with degenerated primers A and B (primer A, 5'-TGR TCN
GCN GGN GCY TC-3'; primer B, 5'-GGN AAY WSN GCN TAY GT-3'). These two
primers, designed from the amino acid sequences of previously microsequenced peptides E1 and C (23), corresponded to the uncoding strand of the peptide E1 cDNA and to the coding strand of
the peptide C cDNA.
The products were amplified by 30 cycles of PCR. Each cycle comprises 1 min at 94 °C, 1 min at different annealing temperatures, and 2 min
at 72 °C, and then 10 min at 72 °C for the last cycle extension
step. After testing the annealing temperatures from 42 °C to
56 °C, the optimal conditions to obtain a single specific band were
30 cycles at 46 °C of annealing temperature. Reaction was performed
in 20 µl containing 50 pmol for each primer, 0.3 mM
deoxynucleotides, and 0.1 unit of Taq polymerase (Life
Technologies, Inc.) in the appropriate enzyme buffer. Separation of PCR
products was performed on 1% agarose gel, and DNA purification was
carried out on an anion exchange column after solubilization of the
gel-containing band (Sigmapur®). Fragments were subcloned
in a T/A cloning plasmid (pGEM-T Easy, Promega) and sequenced on both
strands by the dideoxy chain termination method (24).
Rapid Amplification of the 3' and 5' cDNA Ends (RACE)
PCR--
Based on the sequence of the fragment obtained by RT-PCR, two
sets of specific primers (C and D and E and F) were synthesized and
used for RACE.
For 3' RACE, poly (A)-rich RNA (2 µg) was reverse transcribed using
an oligo(dT) anchor primer (22). A 30-cycle nested PCR was carried out
using the anchor primer in combination with a specific primer (primer
C, 5'-CGT GTG TTC TGC GAG GGT GGA TTC G-3'), then with a second
specific primer (primer D, 5'-CGC ACG TTT GCT GCC GCA CAT ACA C-3').
Each of the 30 cycles comprised 1 min at 94 °C, 1 min at annealing
temperature (50 °C for the 5 first cycles and 60 °C for the 25 lasting cycles), and 3 min at 72 °C. The amplified product was
purified, subcloned, and sequenced on both strands as described above
for RT-PCR.
For 5' RACE, the first strand cDNA was 3'-tailed using terminal
deoxynucleotidyltransferase and dCTP. This matrix was used for the
second strand cDNA synthesis, using 1 µM poly(G)
adapter-primer, 50 µM dNTPs in one PCR cycle with 0.1 unit of Taq polymerase (Life Technologies, Inc.). A 30-cycle
nested PCR was carried out using the poly(G) adapter-anchor primer in
combination with a specific primer (primer E, 5'-AGT AGG CGA CAC AGG
TGG AC-3'), then with a second specific primer (primer F, 5'-GTG TAT
GTG CGG CAG CAA ACG TGC G-3'). The PCR conditions and product analysis
were the same as for 3' RACE.
Sequence Analysis--
The signal peptide was located using the
SignalP program (25). Data base searches were performed using Gapped
BLAST and PSI-BLAST (26). Multiple sequence alignments of
chitin-binding domains were carried out using the CLUSTALW
(Windows®) program (27).
Chitin Extraction--
-Chitin was extracted from R. pachyptila tube according to a protocol derived from Ref. 28.
Dissected frozen R. pachyptila tube fragments were stirred
overnight in a 5% (w/v) aqueous KOH solution at room temperature. The
specimens were then rinsed with distilled water and treated for 2 h with a chlorite solution at 70 °C. The chlorite solution was
prepared by mixing 3 volumes of distilled water with 2 volumes of the
following solutions mixed (v/v) (solution A, 17 g·liter
1 NaClO2 solution in
water; solution B, aqueous 27 g·liter
1 NaOH
solution in 7.5% (v/v) acetic acid). The chlorite treatment was
followed by thorough washing with distilled water and immersion again
in 5% aqueous KOH overnight. Three new chlorite treatments were then
applied, each treatment being followed by washing and overnight
immersion in aqueous KOH. After washing, the preparation was boiled
2 h in 2.5 N HCl, sonicated 15 min, and submitted to a
final step of 4 h in boiling 2.5 N HCl. The samples
were then neutralized with 1 N NaOH, washed thoroughly with
distilled water, and either dried (for affinity experiments) or stored
in methanol (to prepare grids for electron microscopy). The quality of
-chitin extracted from R. pachyptila was controlled by
electron microscopy (data not shown) prior to use for both type of
binding assays.
Crab shell
-chitin and microgranular cellulose used for affinity
experiments were purchased from Sigma (C3641 and C6413 references, respectively).
For microscopy grid preparation,
-chitin was resuspended by boiling
crab shell chitin for 4 h in 1 N HCl (2 h two times
separated by 24 h at room temperature). The samples were then
neutralized with 1 N NaOH, washed thoroughly with distilled water, and
stored in methanol.
Expression of RCBP--
Primers were designed from the
full-length cDNA sequence to amplify a 550-bp fragment of
RCBP from the beginning of the open reading frame (ORF)
minus signal peptide (base position 113), to base position 662 after
Stop codon. Amplification was achieved with a 30-cycle PCR. Each cycle
comprised 1 min at 94 °C, 2 min at 62 °C, and 2 min at 72 °C,
repeated 30 times.
Restriction enzyme sites, i.e. BamHI and
PstI, were added to specific sequences of primers (primer G,
5'-TAA GGA TCC TGC GGA GGC CCG TGT GAG GC-3'; primer H, 5'-TAA CTG CAG
CTC TCT AGC CTC TGC CTA CG-3') to subclone PCR products directionally
in an expression cloning plasmid that has the peculiarity to add a
6-histidine tag to the N terminus of the recombinant protein (pQE-30,
QIAexpressionist®, Qiagen). Reading frame and integrity of
the cloned cDNA were checked by double strand sequencing.
Transformed E. coli produced recombinant RCBP following kit
recommendations except that 2% (w/v) glucose and phenylmethylsulfonyl
fluoride to 1 mM were added to Luria-Bertani medium to
avoid recombinant protein degradation.
All lysis steps were done at 4 °C, including centrifugation. Induced
cultures were centrifuged and harvested bacteria were lysed under
native condition as follows; cells were resuspended in lysis buffer (10 mM phosphate buffer, pH 6, containing a protease inhibitor
mixture (40 µg·ml
1 bacitracin, 40 µg·ml
1 bestatin, 1 µg·ml
1 pepstatin, 1 mM
phenylmethylsulfonyl fluoride)) and sonicated for 12 s at room
temperature before a centrifugation (10,000 × g for 20 min at 4 °C), to remove cellular lysis fragments. The supernatant, a
complete bacterial lysate, was used in binding experiments so that
recombinant RCBP was in competition with bacterial proteins for chitin binding.
Affinity Assay--
RCBP predicted chitin binding activity was
tested against the two
- and
-chitin natural forms, to reveal a
possible form specificity. Cellulose was used as a negative
polysaccharide control.
The affinity assay binding step was done overnight at 4 °C by
shaking RCBP lysate with one polymer (20 mg of
-chitin from R. pachyptila or
-chitin from crab shell or cellulose, as obtained previously, resuspended in an equal minimal volume of phosphate buffer
10 mM, pH 6). Flow-through of RCBP lysate was collected by
centrifugation. After a wash with 10 mM phosphate buffer,
pH 6, added with 1 M NaCl, three successive elution steps
(by mixing followed of a 30-min 4000 × g 4 °C
centrifugation) were undergone with aqueous acetic acid solutions of
increasing concentration: 10 mM, 500 mM, and 2 M. The five fractions collected were dried in
SpeedVac® concentrator and used for further analysis. The
polysaccharide matrices were then stripped of possible remaining
proteins by boiling with SDS-PAGE 5-fold concentrated sample
buffer. Protein concentrations were determined using bovine
serum albumin (BSA) as a standard (29).
Eluate Electrophoresis and Western Blotting--
The protein
pellets from different fractions were resuspended in SDS-PAGE sample
buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 0.02% bromphenol
blue, 10% glycerol, and 4% 2-mercaptoethanol), then heated for 3 min
at 90 °C and separated on SDS-slab gels according to Laemmli (30),
using a 12.5% polyacrylamide running gel. Polypeptides were blotted
onto nitrocellulose sheets (Schleicher & Schull, 0.2-µm pore size)
(31) using a semidry electroblotter (Bio-Rad). Pre-stained protein
molecular mass standards from 14 to 94 kDa (Bio-Rad) were used to
estimate the apparent molecular mass of the separated polypeptides.
The pQE-30 plasmid used has the peculiarity to add a six-histidine tag
at the N-terminal end of the expressed recombinant protein, thus
allowing recombinant RCBP detection with an anti-histidine tag
monoclonal antibody.
Unspecific sites on Western blots were blocked for 1 h at room
temperature under shaking in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2) containing 0.4% Triton X-100 and 3%
bovine serum albumin (TBS-T-BSA). Blots were incubated 1 h at room
temperature under shaking with the anti-histidine tag mouse antibody
(Amersham Pharmacia Biotech) diluted 1/3000 in TBS-T-BSA.
Nitrocellulose sheets were washed five times for 2 min with TBS-T and
then incubated for 1 h at room temperature under shaking with
alkaline phosphatase-conjugated rabbit anti-mouse secondary antiserum
(Sigma), diluted 1/10,000 in TBS-T-BSA. The blot was washed four times
for 2 min with TBS-T and once with TBS. Positive bands were detected
using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
as substrates (Promega).
Electron Microscope Immunochemistry--
Methanol suspensions of
either
- or
-extracted chitins (0.1 g·liter
1 suspensions, prepared as described
above) were applied on 400-mesh carbon-coated nickel grids and
air-dried. Grids were incubated overnight at 4 °C under soft shaking
with RCBP lysate (binding step). Two washes were done at 4 °C with
fresh lysis buffer added with 1 M NaCl.
The grids were treated mainly according to Whitehouse et al.
(32) for immunogold labeling. They were deposited onto drops of
phosphate-buffered saline (PBS) containing 10% of BSA (PBS-BSA) for 30 min, rinsed twice with PBS, and transferred onto drops of
anti-histidine tag mouse antibody (Amersham Pharmacia Biotech) diluted
1/3000 in PBS-BSA for 1 h 30 min. After being washed two times in
PBS, grids were deposited for 45 min on drops of anti-mouse goat
IgG-colloidal gold (10 nm in diameter, British BioCell
Tebu®) diluted 1/10 in PBS. They were then rinsed twice in
PBS, twice in distilled water, allowed to dry on filter paper without
further staining, and observed using a Hitachi H-600 electron
microscope at 75 kV.
For control grids, all steps described above were achieved except one.
A
-chitin grid was treated without first antibody and another one
treated without RBCP lysate, both steps replaced by a corresponding PBS
incubation. A third
-chitin grid was treated by a lysate of another
histidine-tagged recombinant protein without chitin binding activity
(33), replacing the RCBP lysate. In addition, several grids were
prepared with
-chitin and treated with RCBP lysate to test chitin
form specificity.
Depletion experiments were also performed with RCBP lysate on
-chitin grids. The crude RCBP lysate was pretreated by overnight shaking at 4 °C with an excess of either
-chitin or
-chitin (as obtained previously). Depleted lysates were recovered by
centrifugation and then used for overnight binding step as a normal
RCBP lysate.
Northern Blot Analysis--
Total RNA was separated on a
formaldehyde-agarose (0.8%) gel and transferred onto a
Hybond-N+ nylon membrane (Amersham Pharmacia Biotech). The
equal RNA loading (10 µg) on gel lanes was UV-checked after ethidium
bromide staining of ribosomal RNA. Prehybridization and hybridization
were carried out at 65 °C in 5× NaCl/sodium citrate (with
1× NaCl/sodium citrate defined as 0.15 M NaCl/0.015
M sodium citrate), 5× Denhardt's solution, 10% dextran
sulfate, 0.5% SDS, 250 µg·ml
1 salmon
sperm DNA, and an [
-32P]dCTP random primed DNA probe
of 550 bp (as defined above). After hybridization, membranes were
successively washed twice (20 min each) in 2× NaCl/sodium citrate,
0.1% SDS at room temperature and in 0.2× NaCl/ sodium citrate, 0.1%
SDS at 65 °C. Autoradiography was performed using Kodak Biomax MR film.
In Situ Hybridization--
The 550-bp
[
-32P]dCTP random primed DNA was used as a probe for
in situ hybridization to mRNA on R. pachyptila cryosections of the four main regions deposited on
poly-L-lysine-coated slides. After three washes, sections
were incubated in 0.2 N HCl for 10 min and acetylated with
anhydrous acetic acid, then dehydrated in an ethanol series (70%,
95%, and 100%). Dehydrated sections were incubated for 1 h at
25 °C in prehybridization buffer (4× NaCl/sodium citrate, 1×
Denhardt's solution), and the probe, dissolved in hybridization buffer
(50% formamide (v/v), 10% dextran sulfate, 1× Denhardt's solution,
4× NaCl/sodium citrate, 500 µg·ml
1
salmon sperm DNA), was deposited on each section for 16 h at 42 °C. After six washes (20 min each) in 4× NaCl/sodium citrate, slides were dehydrated, coated with Ilford K5® emulsion
and exposed for 4 days, then stained with light green dye (34) before examination.
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RESULTS |
Isolation and Sequence Analysis of RCBP Full-length
cDNA--
In preliminary studies on R. pachyptila
exoskeleton, in situ trypsination of an electrophoretically
purified tube protein led to five microsequenced peptides (23). To test
by PCR experiments all combinations of arrangements, degenerated
primers were designed in the both strands of cDNAs deduced from
each of this five amino acid sequences. Among these arrangements, some
allowed us to obtain the RP43 cDNA (23) and another one (degenerate
primers A and B defined under "Experimental Procedures") resulted
in the amplification of a single 265-bp fragment in different R. pachyptila tube-secreting tissues (opisthosome, trunk, and
vestimentum). Searches in sequences and motif data bases (BLAST,
BLOCKS, PROSITE, Swissprot, ProDom, PRINTS) showed that the deduced
peptidic sequence of this cDNA was unrelated to RP43, and contained
a partial chitin-binding domain and was thus named RCPB
(Riftia chitin-binding polypeptide). This interesting
peculiarity incited us to perform 5' and 3' RACE-PCR with specific
primers deduced from the 265-bp cDNA fragment to obtain the
full-length cDNA. RACE-PCR experiments lead to overlapping fragments of 325 bp for 3' RACE-PCR and 576 bp for the 5' one. The
complete 876-bp cDNA obtained (GenBankTM/EBI Data Bank accession no. AF266752) revealed an ORF of 573 bp, starting with an ATG codon at
position 56 and ending with a TAG codon at position 629 (Fig.
1). This ORF encodes a protein with a
putative N-terminal 19 amino acid signal peptide (sequence analysis by
SignalP program). The predicted protein, named RCBP, is composed of 191 amino acids, with a calculated molecular mass of 21.3 kDa and a pI of
8.26. After removal of the signal sequence, these protein
characteristics become, respectively, 172 amino acids for a molecular
mass of 19.4 kDa and a pI of 8.21.

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Fig. 1.
Nucleic acid and deduced amino acid sequences
of cDNA encoding RCBP. The predicted signal peptide sequence
is double-underlined, and the putative cleavage site is
indicated by an upward arrowhead. The putative
polyadenylation signal is in bold letters, and
the termination codon is marked by an asterisk. Positions of
the two CBDs are enclosed in shaded boxes.
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The 3' end sequences of the two degenerated primers A and B were found
in the full-length cDNA (data not shown), indicating that the stop
codon observed in the first 265-bp fragment was in fact the actual end
of RCBP. BLAST searches in public data bases using the complete
cDNA sequence revealed no significant similarity with any known DNA
sequence. Similar searches with deduced amino acid sequence of full
RCBP, tested against Swissprot data base, showed a significant
similarity of the central part of RCBP (residues 79-123, Fig. 1) with
the chitin-binding domain (residues 420-463) of human chitotriosidase
precursor (GenBankTM/EBI Data Bank accession no. U29615) (44% of
identities and 66% of positivity, (23)) (18) but no significant
similarity with its catalytic region.
Domain search in Pfam (Sanger) data base upon peptidic RCBP full
sequence localized two occurrences of chitin-binding domains close to
Pfam type 2 chitin-binding domain (CBD2), specific to the animal
kingdom, at positions 29-75 and 79-130 (Fig. 1) but not any type 1 chitin-binding domain (CBD1), only found in plants. A multiple sequence
alignment (Fig. 2) was performed with the two RCBP chitin-binding domains (RCBPA and RCBPB, respectively, 46 and
51 amino acids long) and three known CBD2s. Alignment of the two RCBP
CBDs with the consensus amino acids described for invertebrate CBD2s by
Shen and Jacobs-Lorena in 1999 (35) (Fig. 2, CBD2
line) showed that, in both RCBP CDBs, 50% of consensus amino acids are present. When only the two RCBP CBDs were considered in
the alignment, one can observe 36.8% pairwise amino acid identity and
49.1% positivity between them (Fig. 2). These results can be compared
with the 44.1% pairwise identity and 59.3% positivity obtained
between the two domains of peritrophin 1 (APER1A and -B in Fig. 2), a multi-CBD2 protein of Anopheles
gambiae.

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Fig. 2.
Multiple alignment of the two RCBP CBDs with
CBD2 proteins. The two CBD of RCBP (RCBPA/Rifpa and RCBPB/Rifpa)
were aligned with CHIT/Human (Q13231; Homo sapiens (human)
chitotriosidase) and APER 1A/Anoga and APER1B/Anoga (O76217;
A. gambiae (African malaria mosquito) adult type
I peritrophic matrix protein chitin-binding domains A and B). The
Swissprot/TrEMBL accession numbers are given in brackets.
CBD2 conserved amino acids observed by Shen and Jacobs-Lorena (35) in
type 2 chitin-binding domains are at the last
line. Solid shading indicates amino acid
identity, and stippled shading indicates amino acid
similarity. Identical amino acids in the two CBDs of RCBP are indicated
by an X and similar ones by a circle.
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Recently, Suetake et al. (36) noticed that tachycitin and
hevein CBDs share, a common secondary (Fig.
3) and three-dimensional structure, and
three characteristic amino acids (marked by an asterisk in
Fig. 3) involved in interactions with chitin. Alignment of the
corresponding sequences of the two RCBP CBDs with CBDs of these two
proteins (Fig. 3) shows that RCBPA possesses all the three conserved
residues of tachycitin, but little sequence similarity with either
tachycitin or hevein. In case of RCBPB, only the two first positions
are positives (with Asn-69 of tachycitin and Trp-38 of hevein) but
similarities with tachycitin and hevein are higher.

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Fig. 3.
Alignment of the two RCBP CBDs with two CBDs
of known three-dimensional structures. The two CBD of RCBP
(RCBPA/Rifpa and RCBPB/Rifpa) were aligned with: TACHYC/Tactr (P91818;
Tachypleus tridentatus (Japanese horseshoe crab) tachycitin)
and HEVE/Hevbr (P02877; Hevea brasiliensis (Para rubber
tree) hevein). The Swissprot/TrEMBL accession numbers are given in
parentheses. The three conserved amino acids involved in chitin binding
observed by Suetake et al. (36) in three-dimensional
structures of tachycitin and hevein CBDs are indicated by an
asterisks at the bottom. Secondary structure
observed for tachycitin and hevein (36) is noted in the last
line ( -sheets indicated by arrows and
-helical turn by a helix). Solid shading
indicates amino acid identity, and stippled shading
indicates amino acid similarity.
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Chitin Affinity Assays--
To check if CBDs found in RCBP
sequence are functional, in vitro affinity assays were
performed with R. pachyptila
-chitin. Experiments have
been also carried on
-chitin, as a potential ligand and cellulose,
assumed to be a negative control. RCBP was produced in the expression
vector pQE30 and used as unpurified lysate for binding experiments.
In the case of an excess of
-chitin, results showed that all RCBP
molecules are bound, as no detection was possible in flow through,
neither in wash nor in intermediate acetic acid concentrations (Fig.
4A, lanes F,
W, E1, and E2). RCBP needed a high
acetic acid concentration (2 M) (Fig. 4A,
lane E3) to be completely eluted (no more RCBP was observed
in Fig. 4 (lane SB)). In contrast, when RCBP was
incubated with
-chitin and cellulose, it was all recovered in
flow-through or in the first buffered-wash (Fig. 4, B
(lane F) and C (lanes
F and W)). These results indicate that RCBP
strongly binds to
-chitin but neither to
-chitin nor to cellulose.

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Fig. 4.
Affinity Assay of RCBP on
polysaccharides. Recombinant RCBP was expressed in E. coli and tested for in vitro binding using -chitin,
-chitin, and cellulose as ligands. Total soluble proteins were
incubated with polysaccharides. Flow-through were collected, and
matrices were then washed and eluted. All this solutions were separated
by SDS-PAGE and transferred onto nitrocellulose sheets. Western blots
were probed with an anti-histidine tag antibody. A,
-chitin blot. B, -chitin blot. C, cellulose
blot. RCBP is indicated by an arrowhead. Lane
M, the migration of protein size markers (given in kDa on
left of upper panel); lane
F, flow-through (nonbinding proteins); lane
W, first wash (10 mM phosphate buffer, 1 M NaCl), lane E1, first elution step
with 10 mM aqueous acetic acid solution; E2,
second elution step with 500 mM aqueous acetic acid
solution; E3, third level elution step with 2 M
aqueous acetic acid solution; lane SB, proteins
recovered from the matrices by boiling with SDS-PAGE 5-fold
concentrated sample buffer.
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Electron Microscope Immunochemistry--
To confirm the binding
specificity of RCBP, transmission electron microscope immunochemistry
was undertaken (Fig. 5). RCBP immunogold
label appeared clearly related to
-chitin crystallites (Fig.
5A).

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Fig. 5.
Electron microscope immunochemistry.
Electron micrographs of chitin ( or ) incubated with recombinant
RCBP revealed by immunogold labeling. The scale
bar is 96.5 nm for A-D. A,
-chitin-coated grid incubated with RCBP lysate. B,
-chitin-coated grid incubated with RCBP lysate. C,
-chitin depletion: -chitin-coated grid incubated with
-chitin-pretreated RCBP lysate (preincubation with an excess of
-chitin). D, -chitin depletion: -chitin-coated grid
incubated with -chitin-pretreated RCBP lysate (preincubation with an
excess of -chitin).
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Control experiments showed no label with
-chitin (Fig.
5B) and on
-chitin grids without first antibody, neither
with RCBP lysate-lacking experiments nor with another unrelated
recombinant protein lysate. In depletion experiments, a pre-incubation
of RCBP lysate with an excess of
-chitin did not affect RCBP binding (Fig. 5C) and a normal labeling was observed with
-chitin
pre-treated lysate on
-chitin as in an unpretreated lysate
experiments (Fig. 5A). On the contrary, a
-chitin
pre-incubation eliminates RCBP from the lysate and no label was then
observed on
-chitin fibers (Fig. 5D).
Distribution of the RCBP Transcript--
The expression pattern of
RCBP was examined in the different anatomical regions of the worm.
Northern blot hybridizations were performed on total RNA isolated from
obturacular, vestimental, trunk body wall, and opisthosomal regions
(Fig. 6). Using the full-length cDNA,
digested from pGEM-T Easy® clone as a probe, a single
transcript of 1.3 kilobase pairs was observed in vestimentum, trunk
body wall, and opisthosome, but no signal was detected in the
obturaculum (Fig. 6A).

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Fig. 6.
Northern hybridization analysis of R. pachyptila RNA with RCBP cDNA.
Total RNA (10 µg on each lane) of tissues from the four main regions
of the worm (obturacular region (Ob), vestimentum
(V), trunk body wall (T), and opisthosome
(Op)) were loaded. A, blot hybridized with an
-32P-labeled RCBP cDNA probe
(upper panel). The approximate length of the
detected transcript, estimated with RNA molecular mass markers, is
indicated at right. B, UV detection of ethidium
bromide-stained total RNA in the formaldehyde-agarose gel before
transfer, showing equal loading.
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To determine the spatial distribution of the RCBP
transcript, in situ hybridization analysis was performed on
R. pachyptila sections using the full-length cDNA probe.
RCBP mRNA is present in particular areas of vestimentum,
trunk and opisthosome (Fig. 7,
B-F) but was absent from the obturaculum (Fig.
7A). In the vestimentum, label was restricted to all outer
epithelium cells of the lateral regions except under apical cuticular
plaques and in the ventral ciliated region (Fig. 7, B and
C), both regions known for lacking chitin-secreting glands.
In contrast, the RCBP transcript was poorly represented in
trunk body wall and in opisthosome (Fig. 7, D-F), where
labeled epithelial cells were grouped in limited portions of the outer
epidermis. Like in vestimentum, the chitin-secreting glands of these
regions were not labeled.

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Fig. 7.
In situ hybridization analysis
with RCBP cDNA. Light micrographs of
transversal cryosections of R. pachyptila: hybridized with
an -32P-labeled RCBP cDNA probe.
A, obturaculum; B, semi-vestimentum;
C, enlargement of epidermis in vestimentum; D,
trunk; E, enlargement of epidermis in trunk; F,
enlargement of epidermis in opisthosome. Arrowheads
highlight labeling. The scale bar is 280 µm
(for A, B, and D) or 70 µm (for
C, E, and F). bl, branchial
lamellae; cp, apical cuticular plaque; ec,
epidermal cells; lm, longitudinal muscle; obr,
obturaculum region; pg, pyriform gland, also called chitin
secreting gland; rm, ring muscle; vcr, ventral
ciliated region.
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DISCUSSION |
RCBP, a Novel Protein of the R. pachyptila Tube--
In this work,
we characterized a chitin-binding protein that has no significant whole
length similarity with a complete DNA or protein in data bases and can
be considered as a novel polypeptide whose deduced amino acid sequence
presents a signal peptide confirming that synthesizing cells excrete
this ECM-binding protein.
The apparent size of the RCBP transcript detected by
Northern blot analysis seems larger than that of the cloned cDNA,
but all clones obtained and length estimation of cDNA ends by
primer extension analysis (data not shown) confirm the 876-bp size of the cDNA. This difference may be due to inter-individual variations between animals used for successive experiments. Such an individual variability in the protein pattern from different tubes has been observed (37). Moreover, considerable phenotypic variations (6) and
relatively high percentage of polymorphic loci (52.4%) was noted in
allozymes studies (38).
RCBP, a Chitin-binding Domain-containing
Protein--
Chitin-binding proteins from plants have been studied
extensively, particularly chitinases and lectins (15). All known plant chitin-binding proteins contain a common structural motif of 30-43 amino acids with eight cysteines, three aromatic residues, and several
conserved glycines (15). This highly conserved motif called type 1 chitin-binding domain or CBD1 (according to Pfam denomination) is only
encountered in plants. In contrast, invertebrates chitin-binding
domains have been characterized only recently in protein 44 from the
peritrophic matrix of Lucilia cuprina (39). Data bases like
Pfam (entry by H. Hutter and A. Bateman) and screening studies (35)
defined a type 2 chitin-binding domain or CBD2 (34 proteins for 84 known domains) restricted to the animal kingdom. CBD2 is less conserved
than plant CBD1, but three charged positions, six cysteines, and four
aromatic residues are encountered in almost all members of the family
(35). The two RCPB CBDs possess both one of the charged position,
respectively six and five cysteines, two and three of the four aromatic
residues. In addition, all pairwise identity and positivity percentages
deduced for the two RCBP CBDs (Fig. 2) are similar but a little lower
than those between CBD of known multi-CBD2 proteins, like A. gambiae peritrophin 1. RCBP CBDs can be thus considered as closely
related to CBD2s but not as real members of the CBD2 family.
Functionality of RCBP Chitin-binding Domain--
Only the first
RCBP CBD (RCBPA) possesses the six consensus cysteines that form
disulfide bonds in the classical CBD2 (17, 35). In the second CBD
(RCBPB), only five cysteines are aligned. Two additional cysteines
(positions 120 and 125) are present in RCBPB but not aligned at the
conserved position 21 in CBD2 line (Fig. 2). A recent study using
cysteine mutational analysis on human chitotriosidase (a protein with
only four cysteines aligned with conserved CBD2 residues in Fig. 2)
demonstrated that the six cysteines of this CBD are essential to chitin
binding activity (40). Both RCBPA and RCBPB are thus potentially
active. Similar mutational studies on RCBP could determine which
cysteines in one or both CBDs are indispensable for chitin binding activity.
Suetake et al. (36) demonstrated recently a striking
similarity of secondary (Fig. 3, last line) and
three-dimensional structures between tachycitin and hevein CBDs. They
also observed that three amino acids (marked by an asterisk
in Fig. 3), known to be involved in interaction with chitin in hevein,
are found, as identical or similar amino acids, at exactly the same
spatial position in tachycitin. Sequence comparison of RCBPA and RCBPB
with tachycitin and hevein (Fig. 3) shows that, in RCBPA, the three
conserved positions are found, but sequence similarities with
tachycitin and hevein are low and numerous gaps have been added to the
alignment. Thus, it is unlikely that the three conserved residues could
be at appropriate spatial position in case of RCBPA because of overly short intermediary sequences. For RCBPB, only two of the three conserved positions are observed but sequence similarities with tachycitin and hevein are higher and very few gaps have been added. Moreover, if sequence similarities with human chitotriosidase (Fig. 2)
are considered, RCBPB is more likely functional, even if lacking some
characteristic amino acids. Change of the third conserved position in
RCBPB may explain
-chitin binding specificity (Figs. 4 and 5).
RCBP presents two CBDs that could, if they are both functional, form
bridges between chitin monomers chains in a single crystallite or
between two crystallites to form and maintain a three-dimensional network within R. pachyptila tube sheets, as it was
postulated for A. gambiae peritrophin 1, that also contains
only two CBD (type 2) (16). RCBP may also interact with glycoprotein
bridging proteins, like RP43, another R. pachyptila tube
protein, that contains three CUB domains supposed to be involved in ECM
stabilization (23). Further studies, like recombinant expression of
separate (full or truncated) RCBP CBDs, are thus needed to know if both RCBP domains are functional.
RCBP, a
-Chitin-specific Binding Protein--
Almost all
reported binding studies on CBD proteins used the most common
-chitin. However, in some cases (12, 19, 41, 42),
-chitin and
-chitin were used, and the binding specificity observed was either
for
-chitin (12, 41) or for both forms (19, 42). RCBP is thus the
first protein with a strong
-chitin binding specificity.
It is, however, noticeable that most in vitro chitin binding
assays published had low strength washing and elution: phosphate buffer
near pH 7 added with 1 M NaCl (12, 19, 41) or low acetate/acetic acid concentration (20 mM) (13). However, in the case of a rye seed chitinase-a study (14) and for RCBP, a high
concentration of acetic acid (5 M for Ref. 14 and 2 M for RCBP) was necessary to strip chitin binding domains
from bound chitin. This signals a strong stability of RCPB binding to
-chitin, a logical feature for a tube protein of a marine animal
living in a chemically aggressive environment like deep sea
hydrothermal vents (8).
Regarding RCBP CBD alignments and chitin binding experiments,
characterization of other CBD proteins from R. pachyptila
and other vestimentiferan tubes should be useful to see if a new CBD type arises in these worms.
Distribution of the RCBP Transcript--
From Northern blot
analysis and PCR products obtained, the RCBP mRNA was
localized in vestimentum, trunk body wall, and opisthosome, i.e. the three regions involved in tube wall synthesis (6). In contrast, the obturacular region, considered to be a nonsecreting exoskeleton tissue (6), was also found to be lacking in RCBP transcript.
The in situ hybridization revealed that the RCBP
mRNA is absent from the obturaculum and restricted to outer
epidermis epithelial cells of the secreting tissues, but is never found
in chitin-secreting glands. All vestimentum areas are heavily labeled,
whereas RCPB transcript is less expressed in trunk body wall
and opisthosome. If the transcript level is correlated to protein
production, this could contribute to explain tube protein profile
variations previously observed in R. pachyptila between tube
portions from the four main regions of the animal (37).
Such a restricted distribution of the RCBP mRNA is
somewhat surprising, since it is generally admitted that the
chitin-secreting glands are the site of tube components secretion (4,
6, 10, 43). Putative protein material linked to chitin microfibrils has
been observed at the ultrastructural level in these tissues, i.e. before their secretion outside the organism (9, 10). Nevertheless, no defined data have been published on protein synthesis by these glands. Present results and similar ones obtained with RP43,
another R. pachyptila tube protein characterized (23), clearly indicate that major tube proteins are synthesized in at least
two specialized areas of the outer epithelium. The chitin secreting
glands seem to be mainly devoted to the chitin production, whereas the
outer epidermis would be specialized in the synthesis of major protein
components. In these conditions, the assembly of these components to
form the tube would occur in the surrounding seawater, thus without a
direct control by the secreting cells, contrary to arthropods.
The present study opens the way to further experiments in two distinct
domains. On one hand, mechanisms of
-chitin binding specificity
could be investigated by functional dissection of RCBP CBDs using
recombinant proteins with targeted deletions or mutations. On the other
hand, protein-protein binding experiments using antibodies against RCBP
may validate the putative interactions described above. Moreover, these
antibodies would be used to define in vivo the exact
location of RCBP in the tube.
Such approaches could lead to address the poorly understood
protein-protein and protein-chitin interactions in ECMs. In view of the
large size of its
-chitin and the relative simplicity of its protein
matrix (37), R. pachyptila could be a suitable model when
compared with arthropod chitinous ECMs, which are built with thin
-chitin fibers and more numerous proteins (up to 80 in some insect
species (Ref. 44)).