A Novel Chitin-binding Protein from the Vestimentiferan Riftia pachyptila Interacts Specifically with beta -Chitin

CLONING, EXPRESSION, AND CHARACTERIZATION*

Luc ChamoyDagger §, Maryse NicolaïDagger §, Juliette Ravaux, Brigitte QuennedeyDagger , Françoise Gaill, and Jean DelachambreDagger ||

From Dagger  Unité Mixte de Recherche CNRS 5548, Développement-Communication Chimique, Université de Bourgogne, 6 boulevard Gabriel, 21000 Dijon, France and  Unité Mixte de Recherche CNRS 7622, Biologie Moléculaire et Cellulaire du Développement, Université Pierre et Marie Curie, 9 Quai Saint Bernard, 75005 Paris, France

Received for publication, October 10, 2000, and in revised form, November 30, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A cDNA from Riftia pachyptila was cloned. It encodes a novel 21.3-kDa protein from the worm protective tube, named RCBP (for Riftia chitin-binding protein). On the basis of partial tube-peptide sequences previously obtained, experiments using reverse transcriptase-mediated polymerase chain reaction and rapid amplification of cDNA ends led to the complete cDNA sequence. Analysis of its deduced amino acid sequence shows the presence of two chitin-binding domains. These domains are closely related to type 2 chitin-binding domains that are restricted to the animal kingdom. We showed by affinity assay and immunogold labeling that RCBP is the first protein so far known that binds specifically beta -chitin and that is unable to bind the most common alpha -form found in chitin secreting animals. The RCBP mRNA was found to be present in specific epidermal cells from the worm body wall, but never in the chitin-secreting gland cells. This unexpected result clearly indicates that this tube protein is synthesized in specialized areas of the outer epithelium and that at least two different tissues are involved in this exoskeleton synthesis.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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, beta -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 beta  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 beta -chitin associated with proteins (7-9). Previous studies on R. pachyptila have shown that large beta -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 alpha -chitin (12), when others recognize both alpha  and beta -chitin forms (19), but until the present work, no beta -chitin-specific protein have been reported.

Because R. pachyptila is a producer of large quantities of beta -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 alpha  and beta -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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-- beta -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 beta -chitin extracted from R. pachyptila was controlled by electron microscopy (data not shown) prior to use for both type of binding assays.

Crab shell alpha -chitin and microgranular cellulose used for affinity experiments were purchased from Sigma (C3641 and C6413 references, respectively).

For microscopy grid preparation, alpha -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 alpha - and beta -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 beta -chitin from R. pachyptila or alpha -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 alpha - or beta -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 beta -chitin grid was treated without first antibody and another one treated without RBCP lysate, both steps replaced by a corresponding PBS incubation. A third beta -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 alpha -chitin and treated with RCBP lysate to test chitin form specificity.

Depletion experiments were also performed with RCBP lysate on beta -chitin grids. The crude RCBP lysate was pretreated by overnight shaking at 4 °C with an excess of either alpha -chitin or beta -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 [alpha -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 [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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 (beta -sheets indicated by arrows and alpha -helical turn by a helix). Solid shading indicates amino acid identity, and stippled shading indicates amino acid similarity.

Chitin Affinity Assays-- To check if CBDs found in RCBP sequence are functional, in vitro affinity assays were performed with R. pachyptila beta -chitin. Experiments have been also carried on alpha -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 beta -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 alpha -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 beta -chitin but neither to alpha -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 beta -chitin, alpha -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, beta -chitin blot. B, alpha -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.

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 beta -chitin crystallites (Fig. 5A).



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Fig. 5.   Electron microscope immunochemistry. Electron micrographs of chitin (alpha  or beta ) incubated with recombinant RCBP revealed by immunogold labeling. The scale bar is 96.5 nm for A-D. A, beta -chitin-coated grid incubated with RCBP lysate. B, alpha -chitin-coated grid incubated with RCBP lysate. C, alpha -chitin depletion: beta -chitin-coated grid incubated with alpha -chitin-pretreated RCBP lysate (preincubation with an excess of alpha -chitin). D, beta -chitin depletion: beta -chitin-coated grid incubated with beta -chitin-pretreated RCBP lysate (preincubation with an excess of beta -chitin).

Control experiments showed no label with alpha -chitin (Fig. 5B) and on beta -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 alpha -chitin did not affect RCBP binding (Fig. 5C) and a normal labeling was observed with alpha -chitin pre-treated lysate on beta -chitin as in an unpretreated lysate experiments (Fig. 5A). On the contrary, a beta -chitin pre-incubation eliminates RCBP from the lysate and no label was then observed on beta -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 alpha -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.

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 alpha -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.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -Chitin-specific Binding Protein-- Almost all reported binding studies on CBD proteins used the most common alpha -chitin. However, in some cases (12, 19, 41, 42), alpha -chitin and beta -chitin were used, and the binding specificity observed was either for alpha -chitin (12, 41) or for both forms (19, 42). RCBP is thus the first protein with a strong beta -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 beta -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 beta -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 beta -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 alpha -chitin fibers and more numerous proteins (up to 80 in some insect species (Ref. 44)).


    ACKNOWLEDGEMENTS

We thank the chief scientists Jim Childress, Horst Felbeck, Lauren Mullineaux, and François Lallier for help during cruises and tubeworms sampling. We also thank Bruce Shillito, who helped in tissue collection.


    FOOTNOTES

* This work was supported by C. N. R. S., I. N. S. U. and Dorsales fundings.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) AF266752.

§ These authors contributed equally to this paper

|| To whom correspondence should be addressed. E-mail: jdelach@ u-bourgogne.fr.

Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009244200


    ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; RCBP, R. pachyptila chitin-binding polypeptide; RCBP, transcript or cDNA of R. pachyptila chitin-binding polypeptide; RT, reverse transcription; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; bp, base pair(s); ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; TBS, Tris-buffered saline; TBS-T, Tris-buffered saline plus Triton X-100; PBS, phosphate-buffered saline; CBD1, type 1 chitin-binding domain; CBD2, type 2 chitin-binding domain; PBS, phosphate-buffered saline.


    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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