5 Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia; 6 Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071 Barcelona, Spain
Received on December 10, 2001; revised on March 26, 2002; accepted on March 26, 2002
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Abstract |
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Key words: CTLD/multidomain free lectin/planarian gland cells/planarian
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Introduction |
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The CTLD-containing proteins are divided into several families according to primary protein structure and architecture. The CTLD sequence similarity within members of one family is usually higher than that between families. The classification of CTLD-containing proteins into families is further supported by analysis of the genomic organization of CTLDs. The lectin domains of proteins within a single family are accompanied by similar types of nonlectin domains (Drickamer, 1993a,b; 1999; Gabius, 1997
; Dodd and Drickamer, 2001
).
Within the superfamily of C-type lectinlike proteins, a heterogeneous group of proteins has been described, consisting of a single CTDL found in the absence of other protein domains. Proteins containing isolated CTDLs, known as free lectins, have been reported in both vertebrates and invertebrates (Lasserre et al., 1992; Mann and Siedler, 1999
; Drickamer and Dodd, 1999
; Weiss et al., 2000
; Dodd and Drickamer, 2001
). Additionally, putative genes that encode proteins holding two CTDLs and no other protein domains have been described in invertebrates, like Caenorhabditis elegans (group D1) and Drosophila (group B) (Drickamer and Dodd, 1999
; Dodd and Drickamer, 2001
).
Freshwater planaria are well-known traditional objects for studying the phenomenon of regeneration. This animal is able to restore itself even from a tiny fragment of its body (Brondsted, 1969; Baguna et al., 1994
). Planarian species are represented by different strains employing various modes of reproduction, including exclusively sexual, exclusively asexual, or both sexual and asexual (Benazzi and Benazzi-Lentati, 1976
; Ribas et al., 1989
; Bessho et al., 1997
). Animals from the asexual planarian strains propagate only by fission and differ from sexual planaria in their size and details of coloration. Unlike asexual animals, mature planaria of the sexual strains possess well-distinguished reproductive organs and lay fertile cocoons. Strains that reproduce exclusively asexually are genetically different from strains of the same species able to reproduce both sexually and asexually. In particular, worms belonging to different asexual strains of Girardia (formerly Dugesia) tigrina and Dugesia japonica are triploid or mixoploid, whereas sexual strains are diploid (Ribas et al., 1989
; Bessho et al., 1997
).
We have recently identified a scarf gene encoding a CTLD-containing protein from freshwater planarian Girardia tigrina (Bogdanova et al., 1998). We used a subtractive hybridization to search for difference in gene expression between anteriorly and posteriorly regenerating tissues of the same region of the planarian body. Expression of scarf gene was found to be dramatically decreased in anteriorly regenerating tissues. Although scarf does not participate in regeneration control, analysis of scarf expression allowed us to reveal inductive interaction regulating body patterning in planaria (Bogdanova et al., 1998
).
Analysis of Scarf amino acid sequence showed that this protein contains a signal peptide and a pair of similar CTLDs spaced with a single amino acid residue. Northern blotting of planarian tissues revealed mRNA species of various sizes with close homology to scarf, possibly encoding very similar proteins (Bogdanova et al., 1998).
In this article, we characterize several novel genes homologous to scarf that encode proteins with varying amounts of CTLDs with no other functional domains. Based on sequence analysis, exonintron organization, and the overall architecture of predicted proteins, we conclude that these genes belong to a new family of C-type lectinlike genes. We propose the term multidomain free lectins (MDFLs) for this novel family.
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Results |
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Another transcript, referred to as s-scarf2a (additional s stands for "sexual"), was isolated by PCR (using the same primers already described) from sexual G. tigrina. The s-scarf2a gene, encoding a two CTLD-containing protein, showed 92% sequence identity with scarf2 at the amino acid level.
Four other planarian transcripts encoding CTLD-containing proteins, called gtlec1, gtlec2a, gtlec2b, and gtlec2c (from G. tigrina lectin) were identified independently with the ordered differential display (Matz et al., 1997; Mats et al., 1998
). This method was used to search for planarian genes that are expressed in different body regions. A single planarian was cut transversely into six pieces of approximately equal size, and samples of total RNA from each part were prepared and used for comparison. Several clones that were differentially distributed along anteriorposterior axis of planarian body were selected. Some of these appeared to be homologs of C-type animal lectins.
Full-length cDNAs corresponding to cloned fragments were obtained using the SMART rapid amplification of cDNA ends (RACE) method (Matz et al., 1999). The cDNA fragments comprising full coding sequences of MDFL genes were thus cloned, except for gtlec1. The isolated gtlec1 cDNA contains the 3' end of the coding sequence, which comprises a number of tandem CTLDs separated by linkers. seven identical repeat CTLDs were in the 5' end of the gtlec1 cDNA. Such repetitive sequences hinder the further cloning of gtlec1 using the RACE technique.
MDFLs are C-type lectinlike proteins
CTLDs in overall protein architecture.
Sequences of predicted proteins were examined using SMART (Schultz et al., 2000) and SignalP V2.0 software (Nielsen et al., 1999
). Protein structures are illustrated schematically in Figure 1. All proteins are made up of a signal peptide and different numbers of CTLDs interconnected by linkers. The scarf1 gene encodes a one CTLD-containing protein; scarf2 and s-scarf2a, two CTLD-containing proteins; scarf3a and scarf3b, three CTLD-containing proteins; gtlec2a, b, c, five CTLD-containing proteins, whereas gtlec1 codes for at least 13 CTLD-containing proteins. No other protein domains were found.
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Analysis of potential Ca
2+ sites. To evaluate the potential Ca2+-binding capacity of MDFLs, we analyzed the residues involved in calcium binding in known C-type lectins. Most known CTLD structures contain a conserved site (site 2) for calcium and sugar-coordinated binding. The presence of the five-amino-acid motif of site 2 is an key criterion for predicting whether a particular CTLD-like domain binds carbohydrates in a manner analogous to mannose-binding CTLD (Weis et al., 1991, 1992). An additional site (site 1), originally identified in mannose-binding protein (MBP-A), is less conserved (Weis et al., 1992
; Ng et al., 1996
).
Multiple sequence alignments were employed to identify CTLDs of planarian containing some of the conserved residues that make up the calcium-binding site (Figure 2). The amino acids involved in site 1 or in site 2 organization are enumerated in Figure 2.
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The fourth residue corresponding to Asn205 in MBP-A is replaced by Phe in s-Scarf2a and Trp in gtLec2a-D, gtLec2b-D, and gtLec2c-D CTLDs. The same substitutions were described in tetranectin and a tetranectin family member, stem cell growth factor (Nielsen et al., 1997; Mio et al., 1998
). Tetranectin contains two sites for calcium binding, analogous to MBP-A. The first and second residues of site 2 in tetranectin are Gln and Asp, whereas in MBP-A and MDFL CTLDs contain Glu and Asn, respectively. According to published data, these substitutions do not affect the calcium-binding capacity of the domains (Drickamer, 1992
; Iobst and Drickamer, 1994
). CTLDs of Scarf1, Scarf2 and Scarf3a, b also contain Phe in the fourth position of site 2. However, the third residue of site 2 is Gln instead of Glu, which may influence the calcium-binding capacity of the protein. Two CTLDs (gtLec2c-B and gtLec2a-B) contain positively charged amino acids in site 2 that may hinder calcium binding. Other MDFL CTLDs exhibit significantly dissimilar site 2 sequences, which rules out speculations on the capacity to bind carbohydrates in a calcium-dependent manner.
To create a functional Ca2+-binding site analogous to site 2 of the vertebrate carbohydrate recognition domains, the conserved amino acids of the site should be presented in the proper context. Several key residues (marked with asterisks on Figure 2) establish the geometry of the binding site in mannose-binding proteins. Two residues are conserved in all MDFL CTLDs: the Trp preceding the fourth residue of site 2 (Trp204 in MBP-A) and Pro, located between the first and second residues of site 2 (Pro186 in MBP-A). However, the residue analogous to Trp181 of MBP-A is replaced by Phe in a number of MDFL CTLDs. In MBP-A, this tryptophan residue packs with Trp204 those indole side chain projects into the hydrophobic core of the protein (Weis et al., 1991). We suggest that the Phe side chain makes the same contact within the hydrophobic core.
Thus, analyses of MDFL CTLDs indicate that most of these domains may bind sugar in a Ca2+-dependent manner through site 2, similarly to MBP-A. Some MDFL CTLDs additionally contain a putative site 1 for calcium binding. Predicted CTLDs of all scarf-like genes (except Scarf3a-B CTLD) contain a site 1 identical to that of MBP-C (Ng et al., 1996). The only residue equivalent to Glu193 in MBP-A that is involved in both site 2 and site 1 binding is Gln, although this substitution still permits Ca2+ binding through site 1. The site 1 sequence of gtLec2 CTLDs (except C-terminal CTLD) is identical to that of tetranectin, with experimentally proven capacity to bind calcium (Nielsen et al., 1997
).
Phylogenetic analysis.
Construction of phylogenetic trees with PHYLIP (Phylogeny Inference Package) version 3.5c based on various animal CTLD sequences showed that MDFLs form a separate clade within the C-lectin-like protein superfamily, with >90% bootstrap frequency support (Figure 3A). Construction of maximum likelihood (ML) tree with TREE-PUZZLE software resulted in the tree with same topology.
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A phylogenetic tree constructed on the basis of CTLD sequences of MDFLs revealed that all MDFL CTLDs can be subdivided into three subclades (Figure 3B). The first comprises Scarf-like and gtLec1 CTLDs, the second includes CTLDs from A to D of gtLec2 proteins, and the third involves C-terminal CTLDs (CTLD-E) of gtLec2 proteins.
For gtLec2 proteins, the similarity between CTLDs within one protein is significantly lower than that between equivalent CTLDs at the same positions (numbered from the N-termini) in different gtLec2 proteins. This supports the hypothesis that gtlec2 genes originate from a common five-CTLD-encoding ancestor.
Comparison of scarf2 CTLDs (from asexual planaria) with s-scarf2a CTLDs (from sexual planarian) indicates that scarf CTLDs emerged before the two planarian races diverged.
Exonintron organization of MDFLs.
The exonintron organization of several C-type lectin genes has been described (Bezouska et al., 1991). In each case, the CTLD-coding region is separated from the rest of the gene by an intron. Usually the CTLD coding region is interrupted by two introns in cartilage and fibroblast proteoglycan core proteins (group I of the C-type lectins) and type II asialoglycoprotein receptors (group II), and by three introns in free lectins. The intron positions are conserved within each C-type lectin group but shifted between any two groups. CTLD regions of the several families of C-type lectin-like proteins are encoded on uninterrupted exons.
PCR with gene-specific primers and the genome walking technique were used to isolate the genomic sequences for scarf1, scarf2, s-scarf2a, and tree gtlec2. We also cloned the genomic sequences for three gtlec1 CTLDs. Genomic sequences were compared with cDNA sequences to detect intron positions. The scheme of exonintron organization of the genes is presented in Figure 4. All exonintron junctions obey the GT-AG rule. In all cases, each CTLD is interrupted by two introns. Signal peptide coding sequences and linker stretch coding sequences are also divided by introns.
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Introns interrupting CTLD coding sequences are in the same positions for all MDFL genes. In every case, the first intron divides the coding sequence between codons and the second, between the second and third nucleotides of each codon. Introns of MDFL CTLDs coincide with the intron positions of type II receptor CTLDs (Bezouska et al., 1991). The identical intronexon organization of these lectins points to the evolutionary relatedness between the groups.
MDFL genes are differentially expressed in the planarian body
The spatial pattern of scarf-2 transcription in the whole planarian organism has been described as bilaterally symmetric and comprising two "spots" in the "neck" body region and two posterior longitudinal "branches" (Bogdanova et al., 1998). All novel scarf-like genes of asexual planaria (scarf-1, scarf-3a and b) have identical expression patterns, as revealed by RT-PCR (data not shown). The levels of transcription of both scarf-3a and b are extremely low.
The transcription pattern of s-scarf2a in sexual planaria, detected by whole-mount in situ hybridization, is shown in Figure 5. The pattern is similar but not identical to that of scarf-like genes of asexual planaria. The general shape is similar, with two anterior "spots" and two posterior longitudinal "branches" of s-scarf2a-expressing cell clusters. Unlike scarf genes in asexual planaria, the branches converge at the tail region. Moreover, no changes in the density of s-scarf-expressing cell clusters are observed along the branches, whereas in asexual planaria, density decreases toward the posterior, making the branches look dotted or absent. Asexual planaria normally propagate by severing the tail region, which occurs once a week, and the tail region remains in a state of continuous growth. The daily increase of the tail region or of the proportion of tail size to the rest of the body may affect the scarf branches pattern in this region.
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To examine the expression pattern of gtlec2 genes, we used a common probe for all three genes (gtlec2a, gtlec2b and gtlec2c). Cells stained with the gtlec2 probe form a closed curve stretching neatly between scarf-expressing cells and the edge of the planarian body (body edge). The anterior arch of this curve is actually a stripe (neck stripe) across the body that overlaps the scarf spot location. The curve is much wider at this point than in the vicinity of the body edge and it is reminiscent of a "beak" inserted into the planarian head as it extends toward the anterior. Moreover, discrete islets of gtlec2-expressing cells are scattered all over the planarian body (Figure 5). Using RT-PCR with primers specific for distinct gtlec2 genes, we observed a differential gene expression pattern along the planarian body. The gtlec2a gene is expressed in a neck stripe and beak, gtlec2b in body stretches and discrete islets and gtlec2c in the bottom part of the neck stripe.
MDFLs are excreted by planarian gland cells to the mucus
Polyclonal antibodies against recombinant Scarf-2 and gtLec1 proteins were used to detect these proteins in planarian tissue (Figure 6). Whole-mount immunostaining revealed the presence of both proteins in elongated cell processes extending from respective mRNA locations. The gtLec1-containing cell processes accumulate forward from the site of detection of gtlec1 mRNA and reach the head edge.
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Many flatworms use glandular secretion to adhere to substratum. G. tigrina belongs to a systematic group of turbellaria with adhesive glands or duo-glands of two types (Tyler, 1976, 1988). The first type, known as viscid glands due to their presumed role in forming an adhesive substance, contains ovoid electron-dense granules. The second type, originally called releasing glands due to a reputed role in allowing release of mucous products, has smaller, less dense granules. Some turbellarian glands contain openings in a specialized collar of epidermal cells individual for each gland neck, and the necks of viscid glands comprise a collar of microvilli around their openings. Therefore, MDFL-containing cells are long-neck glands similar to viscid glands. Cell bodies are located in parenchyma and long necks directed to the planarian body edge, where they open outward onto the body surface.
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Discussion |
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MDFL genes are expressed by specialized long-neck glands and transported in granules to the planarian body edge. There is an essential similarity between identified glands from G. tigrina and the viscid glands described in some turbellaria (Tyler, 1976, 1988). MDFL proteins are detected in planarian mucus that generally consist of complex carbohydrates. The colocalization of these carbohydrates and MDFL proteins suggest that they may interact and that MDFL proteins may participate in the formation of planarian mucus. It is presumed that viscid glands ensure planarian adherence to substratum. It is possible that MDFL proteins are used by planaria to form an adhesive substance. However, other putative functions, including defense against infection and self/nonself recognition processes inherent in C-type lectins, cannot be excluded.
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Materials and methods |
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Isolation of MDFL cDNA
Total RNA was purified using homogenization with guanidine isothiocyanate and phenol/chloroform extraction (Chomczynski and Sacchi, 1987). A SMART RACE cDNA amplification kit (Clontech) was used for cDNA synthesis and amplification of 5' ends of mdfl cDNAs, as described previously (Matz et al., 1999
). To isolate scarf-2 homologs, amplified cDNA for SMART 5'-RACE was used as a template for PCR with scarf-2-specific primers (5'-cttgttgggttaattttccaact-3' and 5'-catttaatcacattcgtcctc-3'). Sequences of oligonucleotides used for amplification of 5'-ends were as follows.
gtlec15'-acagtgtttattcccgccattatt-3'; 5'-ccaagttccatctacggcacg-3'; 5'-tcatattcttcattagaatcgtc-3'; 5'-tcattattgtcattatcagtatc-3'; 5'-gtcattttcgtattacccatgt-3'
gtlec2a5'-attcattttcagactgaagaac-3'; 5'-ttctttctcgacctcca
cttg-3'; 5'-tgtattgggatcggggccat-3'
gtlec2b5'-acttcaatatccgtttgaga-3'; 5'-acatccaaagcagtatcatc-3'; 5'-caatcgaaataccaacgcaga-3'
gtlec2c5'-acttcaatatccgtttgaga-3'; 5'-tacatccaatgaactgtcac-3';
5'-atttactcgatataccattag-3'
scarf-like genes5'-catttaatcacattcgtcctc-3' and 5'-gacccaagctcaatattat-3'
All PCR reactions were performed in a 10 µl reaction mixture (1x PC-II buffer: 50 mM Tricine-KOH, pH 8.7; 16 mM ammonium sulfate; 2.5 mM MgCl2;150 µg/ml bovine serum albumin [BSA]; 50 µM dATP, dCTP, dTTP, dGTP each; 0.4 µM each primer; and KlenTaq polymerase [Ab Peptides] at a concentration of 25 U per 100 µl reaction mixture).
Isolation of MDFL genomic sequences
Total DNA was isolated from planaria following a rapid extraction method (Davis et al., 1980). This DNA was used as template in PCR reactions with primers specific for the 5' and 3' ends of the corresponding MDFL cDNA. To isolate 5' genomic regions, the Universal Genome Walker Kit (Clontech) was used, as specified (Siebert et al., 1995
). Sequences of oligonucleotides used for amplification were as follows.
gtlec2a5'-tgacgattgttggtaactgt-3' and 5'- attcattttcagactgaagaac-3'
gtlec2b5'-tggcgattgttggtaactgt-3' and 5'-acttcaatatccgtttgaga-3'
gtlec2c5'-tgacgattgttggtaactgt-3' and 5'-acttcaatatccgtttgaga-3'
scarf-like genes5'-catttaatcacattcgtcctc-3' and 5'-gacccaagctcaatattat-3'
To isolate genomic sequences of the three CTLDs of the gtlec1 the pairs of specific primers were as follows: 5'-tatcacaattaaaatcatcccag-3' and 5'-gacactggaacagcttcagga-3'; 5'-tatcacaattaaaatcatcccag-3' and 5'-ggtaatacgaaaatgacttcca-3'; 5'-tatcacaattaaaatcatcccag-3' and 5'-ggtaatacgaaaatgacttcca-3'.
Cloning and sequencing
All standard DNA procedures were performed in accordance with standard laboratory protocols (Sambrook et al., 1989). DNA sequence was determined using a Beckman SEQ-2000 automated sequencer and FS dye terminator chemistry.
Sequence analysis and comparisons
Sequences were analyzed using BLAST software. Domain organization was verified using Web-based SMART tool (http://smart.embl-heidelberg.de) and SignalP V2.0 software (http://www.cbs.dtu.dk/services/SignalP-2.0). Multiple sequence alignment was performed with the Clustal W version 1.8 software, with following eye optimization. For phylogenetic tree construction, truncated CTLD sequences from the first to last conserved cysteine residues were used for alignment. Phylogenetic analysis was performed using PHYLIP version 3.57c (Retief, 2000) and Tree-PUZZLE, version 5.0 (Strimmer and von Haeseler, 1997
). For analysis by PHYLIP, the neighbor-joining algorithm with 500 bootstrap replications was used. ML trees obtained with Tree-PUZZLE were inferred by using quartet puzzling with 10,000 puzzling steps. We used the discrete G-distribution model (with eight categories) for site heterogeneity (Yang, 1996
), and Whelan and Goldman model of substitution (Whelan and Goldman, 2001
). Neighbor-joining tree was used to estimate the parameters.
Whole-mount in situ hybridization
Whole-mount in situ hybridization for both asexual and sexual planaria was carried out essentially as described elsewhere (Bogdanova et al., 1998). Digoxigenin-labeled RNA probes were prepared from cDNA fragments generated in PCR reactions with gene-specific primers and cloned in pT-Adv cloning vector (Advantage PCR Cloning Kit, Clontech).
RT-PCR analysis
For RT-PCR analysis, various regions of the planarian body were isolated and used for RNA preparation, as described. First-strand cDNA was synthesized using Advantage RT-for-PCR Kit (Clontech) and employed for PCR with gene-specific primers. As a control for RNA quality, the homeobox-containing gene Dth-2, expressed uniformly along the planarian body in peripheral parenchyma cells, was selected (Garcia-Fernandez et al., 1993). Sequences of oligonucleotides used for RT-PCR were as follows.
gtlec15'-gaaaggtgaacctaacaacg-3' and 5'-acagtgtttattcccgccattatt-3'
gtlec2a5'-ggtgggatatagcttgcaca-3' and 5'-attcattttcagactgaagaac-3'
gtlec2b5'-ggtgggatatagcttgcaca-3' and 5'-acttcaatatccgtttgaga-3'
gtlec2c5'-ttgataatttgatgatatcaacc-3' and 5'-gtataatgtacatccaatgaact-3'
scarf-like genes5'-catttaatcacattcgtcctc-3' and 5'-gacccaagctcaatattat-3'
dth25'-ttggttccaaatcggcttcc-3' and 5'-ccagaacgtgagcatttggc-3'
Expression constructs for immunization
Polyclonal antibodies were prepared to His-target Scarf2 and gtLec1 proteins produced in Escherichia coli. cDNA fragments encoding Scarf2 mature protein and two C-terminal CTDLs of gtLec1 were generated in PCR reactions with gene-specific primers and cloned in the His-target expression vector pQE-30 (Qiagen). The resulting plasmids were transformed into the E. coli strain Ad 494 (Novagen). Recombinant proteins, sequestered into inclusion bodies, were further purified by immobilized metal affinity chromatography using Talon Resin (Clontech) under denaturing conditions.
Preparation of polyclonal antibodies against scarf2 and gtLec1
Rabbits were immunized and boosted four times at monthly intervals with recombinant Scarf2 or gtLec1 polypeptides emulsified in complete Freunds adjuvant. Animals were bled 10 or 1 days after each boost. Polyclonal antiserum to Scarf2 and gtLec1 was tested with an enzyme-linked immunosorbent assay (ELISA) kit (Haematologic Technologies) and used for the preparation of Fab fragments. Immunoglobulins were purified by chromatography with protein A sepharose (Pharmacia) as described (Harlow and Lane, 1988) and dialyzed against 100 mM sodium acetate, pH 5.5. Fab fragments of antibodies were generated by pepsin digestion following Harlow and Lane (1988)
, purified by chromatography with a protein A column, and dialyzed against phosphate buffered saline (PBS) (120 mM NaCl; 7 mM Na2HPO4; 3 mM NaH2PO4; 2.7 mM KCl) overnight at 4°C. The Fab fragments obtained were tested by ELISA and western immunoblotting.
Immunostaining with planarian mucus
For protein detection in mucus, planaria were placed on a nitrocellulose membrane (Schleicher and Schuell) in a drop of 10x Locke solution (8% NaCl, 0.2% KCl, 0.2% CaCl2, 0.2% NaHCO3) to increase mucus secretion. Mucus proteins were immobilized on membrane preincubated in PTw buffer (0.1% Tween-20 in PBS) with 3% BSA (Sigma) for 40 min at room temperature. Primary Fab fragments were diluted (1:300) in the same BSA solution and incubated with nitrocellulose membrane for 1 h at room temperature with gentle shaking. Commercially available alkaline phosphataseconjugated goat Fab fragments to rabbit immunoglobulin (Burlingame) were diluted (1:25,000) in BSA solution and incubated for 1 h. After washing, immunoreactivity was visualized with nitrotetrazolium blue (Sigma) and 5-bromo-4-chloro-3-indolylphosphate (Sigma).
Whole-mount immunostaining
Planaria were fixed in 2% formaldehyde diluted in macerating mixture (water, methanol, glycerin, and acetic acid; 14:3:2:1, respectively), as for whole-mount in situ hybridization (Bogdanova et al., 1998). After fixation, animals were washed three times in methanol and subjected to ultrasound for 57 min at Soniprep-150 (amplitude 1022 micron) to break cell walls and increase tissue permeability. Following sonication, planaria were washed in PTw and blocked for 2 h at room temperature in 3% BSA in PTw. Primary Fab fragments were diluted (1:100) in1% BSA solution and incubated with planaria overnight at 4°C with gentle shaking.
After washing in PTw for a few hours, samples were blocked with 5% goat serum in PTw for 2 h at 4°C with gentle shaking. Alkaline phosphataseconjugated secondary Fab fragments to rabbit immunoglobulins (Burlingame) were diluted (1:25,000) in BSA solution and incubated with animals overnight at 4°C with gentle shaking. After washing, immunoreactivity was visualized with nitrotetrazolium blue and 5-bromo-4-chloro-3-indolylphosphate.
Immunoelectron microscopy
Planaria were fixed with 2% paraformaldehyde, 0.5% glutaraldehyde, and 0.1% cacodylate for 40 min, washed three times in PBS and once in 0.5% glycine, and incubated with 1% osmium in PBS for 1 h. They were then dehydrated with a graded series of ethanol and embedded in LR White resin (London Resin, London) according to the manufacturers instructions. Ultra-thin sections were cut with a microtome using a diamond knife and placed on Ni grids. Immunogold labeling of LR White-embedded sections was performed by floating the grid section side-down on the following aqueous solutions: 3% hydrogen peroxide for 3 min, five washes in distilled water, blocking in 3% BSA (in PBS) for 30 min; primary antibody (1:100) in 1% BSA (in PBS) for 2 h; five washes with 1% BSA (in PBS); 20 nm gold-conjugated anti-rabbit-IgG 1:100 in 1% BSA (in PBS) for 1 h; and five washes in distilled water. Sections were poststained with 0.5% aqueous uranylacetate for 30 min, 0.02% aqueous lead citrate for 5 min, and then examined under an electron microscope at 80 kV.
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Acknowledgments |
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The nucleotide sequences reported here have been submitted to the GeneBank/EMBL/Data Bank with accession numbers AY057969 (gtlec1), AY057970 (gtlec2a), AY057971 (gtlec2b), AY057972 (gtlec2c), AY057980 (scarf1), AY057974 (scarf2, exons 14), AY057975 (scarf2, exons 57), AY057976 (scarf3a), AY057977 (scarf3b), AY057978 (s-scarf2a, exons 14), AY057979 (s-scarf2a, exons 57), and AY057973 (s-scarf2b).
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Abbreviations |
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Footnotes |
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2 Present address: Department of Preclinical Veterinary Sciences, The University of Edinburgh, UK
3 Present address: Laboratory of Cellular and Molecular Biology; National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
4 To whom correspondence should be addressed; E-mail: luk@ibch.ru
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