Identification and characterization of a new family of C-type lectin–like genes from planaria Girardia tigrina

Dmitry A. Shagin5, Ekaterina V. Barsova5, Ekaterina A. Bogdanova5, Olga V. Britanova5, Nadia G. Gurskaya5, Konstantin A. Lukyanov5, Mikhail V. Matz1,5, Natalia I. Punkova5, Natalia Y. Usman2,5, Eugene P. Kopantzev3,5, Emili Salo6 and Sergey A. Lukyanov4,5

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


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A novel family of C-type lectin–like genes, denoted multidomain free lectin (MDFL), was identified in the freshwater planaria Girardia (Dugesia) tigrina. We cloned several genes that encode proteins comprising a signal peptide and a number of consecutive C-type lectin–like domains (CTLDs) interconnected by short linker stretches. Analyses of genomic organization, CTLD amino acid sequences, and the overall architecture of these proteins indicate that planarian proteins are a separate family of C-type lectin–like proteins. These genes are expressed in specifically differentiated gland cells of planaria and the corresponding proteins are excreted as components of the planarian body surface mucus.

Key words: CTLD/multidomain free lectin/planarian gland cells/planarian


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Animal C-type lectins are generally multidomain proteins with diverse overall architecture, containing one or more homologous domains (named carbohydrate-recognition domains) that provide Ca2+-dependent sugar recognition activity. Other protein modules determine a broad range of biological functions of C-type lectins (Drickamer, 1993aGo, 1999; Dodd and Drickamer, 2001Go). Carbohydrate-recognition domains of C-type lectins form a subgroup of a larger family of protein domains that are called C-type lectin–like domains (CTLDs) (Dodd and Drickamer, 2001Go). The CTLDs share a common sequence motif that determines their overall structure. Some CTLDs bind sugar and calcium, but many CTLDs serve functions other than sugar recognition.

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, 1993aGo,b; 1999; Gabius, 1997Go; Dodd and Drickamer, 2001Go).

Within the superfamily of C-type lectin–like 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., 1992Go; Mann and Siedler, 1999Go; Drickamer and Dodd, 1999Go; Weiss et al., 2000Go; Dodd and Drickamer, 2001Go). 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, 1999Go; Dodd and Drickamer, 2001Go).

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, 1969Go; Baguna et al., 1994Go). 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, 1976Go; Ribas et al., 1989Go; Bessho et al., 1997Go). 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., 1989Go; Bessho et al., 1997Go).

We have recently identified a scarf gene encoding a CTLD-containing protein from freshwater planarian Girardia tigrina (Bogdanova et al., 1998Go). 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., 1998Go).

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., 1998Go).

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, exon–intron organization, and the overall architecture of predicted proteins, we conclude that these genes belong to a new family of C-type lectin–like genes. We propose the term multidomain free lectins (MDFLs) for this novel family.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of MDFL genes from planaria
Reverse transcription polymerase chain reaction (RT-PCR) with scarf-specific primers (the first, corresponding to part of the signal peptide sequence, and the second, to the 3'-untranslated region sequence) of total RNA of asexual planaria yielded several products of various lengths, in addition to scarf. Cloning and sequencing of these products revealed three additional scarf-like proteins with different numbers of CTLDs. One encodes a polypeptide comprising one CTLD, whereas the two others encode three CTLD-containing polypeptides. We propose the names scarf1, scarf2 (previously called scarf), and scarf3 (a and b) for one, two, and three CTLD-coding genes, respectively.

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., 1997Go; Mats et al., 1998Go). 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 anterior–posterior 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., 1999Go). 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 lectin–like proteins
CTLDs in overall protein architecture.
Sequences of predicted proteins were examined using SMART (Schultz et al., 2000Go) and SignalP V2.0 software (Nielsen et al., 1999Go). 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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Fig. 1. Overall architecture of predicted MDFL proteins. L, signal peptide; S, linkers dividing CTLDs. CTDLs are numbered (from A to G) from the N-termini. Digits at the bottom indicate the number of amino acids in the corresponding protein part. All scarf-like predicted proteins contain very similar CTLDs (more than 80% identity at the amino acid level). Scarf-like CTLDs are separated mostly by a single Ile residue (or Glu in scarf-3a between B and C CTLDs). All scarf-like proteins contain signal peptides and short (nine residues long) identical C-terminal sequences. The gtlec2 gene group (gtlec2a, gtlec2b, and gtlec2c) encodes very similar proteins (more then 75% identity at amino acid level) with identical overall architectures. In the gtLec1 protein, CTDLs from A1 to A7 and interconnecting linkers are identical to each other.

 

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., 1991Go, 1992). An additional site (site 1), originally identified in mannose-binding protein (MBP-A), is less conserved (Weis et al., 1992Go; Ng et al., 1996Go).

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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Fig. 2. Multiple alignment of MDFL CTLDs. CTDLs are numbered (from A to G) beginning at the N-termini. Amino acid numbering is based on rat mannose-binding protein A. Introduced gaps are represented by dots. Residues of the sequence motif characteristic of CTLD are highlighted in black (strictly conserved) or gray. The motif is indicated at the top of each section: H, hydrophobic; F, aromatic; A, aliphatic; C, P, and W follow the one-letter amino acid code. Amino acid compositions of Ca2+-binding sites 1 and 2 are specified by numbers. Residues that function in establishing the geometry of the binding site in mannose-binding protein are specified by asterisks. Abbreviations and references for the vertebrate sequences are as follows: MBP-A, rat mannose-binding protein a (GenBank Accession P19999); MBP-C, rat mannose-binding protein C (P08661); CD23, rat IgE Fc receptor II (S34198); RHL-1, rat hepatic lectin 1 (NP_036635); AGC1, human aggrecan 1 (NP_037359); CSPG3, human neurocan (NP_004377); E-Sel, human selectin E (NP_000441); P-Sel, human selectin P (CAA18142).

 
The site 2 of the first CTLD (from N-termini) of gtLec2a and gtLec2c (gtLec2a-A; gtLec2c-A), the second of gtLec2b (gtLec2b-B), and the third of gtLec2a, gtLec2b, and gtLec2c (gtLec2a-C, gtLec2b-C, and gtLec2c-C) is identical to that of MBP-A. All CTLDs of gtLec1, except the C-terminal domain (gtLec1-G), comprise potential calcium-binding site 2, analogous to CTLDs of C. elegans (Drickamer and Dodd, 1999Go). Although sequence compositions differ from those of known mannose- and galactose-binding proteins, the Ca2+-binding capacity was predicted.

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., 1997Go; Mio et al., 1998Go). 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, 1992Go; Iobst and Drickamer, 1994Go). 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., 1991Go). 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., 1996Go). 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., 1997Go).

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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Fig. 3. Phylogenetic trees of the C-type lectin–like proteins. (A) Unrooted neighbor-joining tree constructed with PHYLIP software based on CTLD sequences of a number of MDFL proteins, vertebrate C-type lectins from Figure 2, and C. elegans lectins: C14A6.1-A and C14A6.1-B first and second CTLD of C14A6.1 protein (CAB03881); B0218.8-A first CTLD of B0218.8 protein (AAB00670). Numbers at branch nodes indicate percentage bootstrap support for the particular node based on 500 replications. (B) Unrooted ML tree constructed with TREE-PUZZLE software based on CTLD sequences of MDFL proteins. The ML tree was inferred by using quartet puzzling with 10,000 puzzling steps. Numbers at nodes indicate support values for each branch.

 

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.

Exon–intron organization of MDFLs.
The exon–intron organization of several C-type lectin genes has been described (Bezouska et al., 1991Go). 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 exon–intron organization of the genes is presented in Figure 4. All exon–intron 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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Fig. 4. Scheme of exon–intron organization of MDFL genes. L, signal peptide; S, linkers dividing CTLDs. CTDLs are numbered from the N-termini. Digits indicate intron size (bp).

 
Besides s-scarf2a, an additional sequence (designated s-scarf2b, the closest homolog of s-scarf2a) was cloned from the genomic DNA of sexual planaria. The s-scarf2b gene is intron-containing but includes a stop codon in the coding region. No cDNA corresponding to s-scarf2b was identified. Within the s-scarf2b sequence, introns are located at the same positions as s-scarf2a, the only difference being that the fourth intron is significantly shorter (227 bp).

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., 1991Go). The identical intron–exon 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., 1998Go). 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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Fig. 5. Analysis of MDFL gene expression by whole-mount in situ hybridization. (A) Whole-mount in situ hybridization with antisense s-scarf2a riboprobe on sexual planaria. (B) Whole-mount in situ hybridization with antisense scarf2 riboprobe on asexual planaria. (C) Whole-mount in situ hybridization with antisense gtlec1 riboprobe. (D) Whole-mount in situ hybridization with antisense gtlec2 riboprobe. Arrows indicate the regions in which different gtlec2 genes are expressed. Ph, pharynx; e, eyes. Whole-mount in situ hybridization with a sense riboprobes for MDFL genes had not revealed any signal (not shown).

 
Whole-mount in situ hybridization was also used to study gtlec1 and gtlec2 gene expression patterns. The gtlec1 gene expression pattern is outlined as a single stripe across the body that overlaps the observed scarf "spots" (Figure 5).

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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Fig. 6. MDFL detection in planarian body by whole-mount immunostaining. (A) Immunostaining using rabbit polyclonal antibodies to Scarf2 recombinant protein. Positive Scarf2 immunostaining is observed in the head region anterior to the area where scarf2 mRNA is detected and the body edge. (B) Immunostaining with rabbit polyclonal antibodies to gtLec1 recombinant protein. Positive gtLec1 immunostaining is noted anterior to the region where gtlec1 mRNA is detected and in the head edge, as illustrated with larger magnification in C. Control immunostaining with rabbit immunoglobulins had not revealed any signal (not shown).

 
Scarf-containing cell processes spread distally from scarf mRNA locations and reach the body edge. Most parts modulate their route to approach the body edge under the right angle. Immunoelectron microscopy revealed that the processes of scarf-expressing cells have a developed microtubular system at the periphery and contain electron-dense granules. Scarf2 protein was detected within these granules (Figure 7). Each process opening at the body edge is surrounded by a characteristic collar of microvilli. Scarf-2 and gtLec1 proteins were also detected in the mucus normally abundantly released by planaria (data not shown). Therefore, we propose that MDFL proteins are produced by specific gland cells and secreted as components of planarian mucus.



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Fig. 7. Immunoelectron microscopy using a gold-conjugated anti-scarf2 antiserum. Scarf-expressing cells have a developed microtubular system at the periphery and contain electron-dense granules. mt, microtubules; black arrow heads indicate Scarf-containing granules, and white arrowheads specify granules that do not contain Scarf.

 
Various types of glands have been described for several flatworm species (Rieger et al., 1991Go). These glands are unicellular, with deeply embedded cell bodies and long processes (denoted gland necks) projecting to the body surface and opening outward through channels within or between epidermal cells. Gland necks can reach 600 µm in an 1100-µm-long Paromalostomum atratum (Rieger, 1971Go).

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, 1976Go, 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.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The MDFL genes identified in the planarian G. tigrina encode proteins that belong to the C-type lectin–like protein superfamily. Predicted proteins contain a number of C-type lectin–like domains with a characteristic conserved residue motif. All predicted MDFLs contain a signal peptide linked to a number of CTLDs with short linker sequences in between. No other functionally relevant sequences are associated with CTLDs. Comparisons of these CTLD amino acid sequences with known animal C-type lectins and analyses of protein architecture and genome organization studies support their sorting into a separate family. Only the Scarf1 protein shows substantial overall architecture similarity to free lectins. However, the exon–intron organization of the scarf1 gene is different to that of free lectins. Moreover, sequence analyses (using coding sequences as well as introns) of scarf-like genes suggest that scarf1 and scarf3 genes originate from unequal homologous recombination in the scarf2 region (Figure 8). Because the initial scarf isoform (scarf2) was retained, we hypothesize that scarf2 duplication occurred before recombination. The presence of at least two scarf-like genomic sequences in sexual planaria supports this hypothesis.



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Fig. 8. Scheme of homologous recombination yielding a number of scarf-like genes. The central areas of scarf-like CTLD sequences exhibit the closest similarity (marked by white), whereas the 3' ends contain C-terminal amino acid motifs; "YNA" in C-terminal CTLDs (Scarf1 CTLD; scarf2 CTLD-B; scarf3a,b CTLD-C) or "EIV" in other CTLDs (marked in red and blue, respectively). The 5' ends comprise two characteristic amino acid sequences: the first (marked by yellow) is present in N terminal domains (Scarf1 CTLD; scarf2 CTLD-A; scarf3a,b CTLD-A), and the second (marked by green) is observed in other domains (scarf2 CTLD-B; scarf3a,b CTLD-B, and CTLD-C). Intron positions are marked by arrowheads. Nearly identical intron sequences are specified by the same arrowhead color.

 
Unlike C. elegans lectins of group D1 that contain two CTLD-containing proteins, of which only one maintains the calcium-binding capacity (Drickamer and Dodd, 1999Go), most MDFL predicted proteins are made up of more than one CTLD that potentially binds calcium.

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, 1976Go, 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Animals
Experiments were conducted on both asexual and sexual planarian G. tigrina (Platyhelminthes, Turbellaria, Tricladida). One-week-starved organisms (7–13 mm long) were used in all experiments and the temperature was maintained at 22 ± 1°C.

Isolation of MDFL cDNA
Total RNA was purified using homogenization with guanidine isothiocyanate and phenol/chloroform extraction (Chomczynski and Sacchi, 1987Go). 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., 1999Go). 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.

gtlec1—5'-acagtgtttattcccgccattatt-3'; 5'-ccaagttccatctacggcacg-3'; 5'-tcatattcttcattagaatcgtc-3'; 5'-tcattattgtcattatcagtatc-3'; 5'-gtcattttcgtattacccatgt-3'

• gtlec2a—5'-attcattttcagactgaagaac-3'; 5'-ttctttctcgacctcca

cttg-3'; 5'-tgtattgggatcggggccat-3'

• gtlec2b—5'-acttcaatatccgtttgaga-3'; 5'-acatccaaagcagtatcatc-3'; 5'-caatcgaaataccaacgcaga-3'

gtlec2c—5'-acttcaatatccgtttgaga-3'; 5'-tacatccaatgaactgtcac-3';

5'-atttactcgatataccattag-3'

scarf-like genes—5'-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., 1980Go). 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., 1995Go). Sequences of oligonucleotides used for amplification were as follows.

• gtlec2a—5'-tgacgattgttggtaactgt-3' and 5'- attcattttcagactgaagaac-3'

• gtlec2b—5'-tggcgattgttggtaactgt-3' and 5'-acttcaatatccgtttgaga-3'

gtlec2c—5'-tgacgattgttggtaactgt-3' and 5'-acttcaatatccgtttgaga-3'

• scarf-like genes—5'-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., 1989Go). 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, 2000Go) and Tree-PUZZLE, version 5.0 (Strimmer and von Haeseler, 1997Go). 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, 1996Go), and Whelan and Goldman model of substitution (Whelan and Goldman, 2001Go). 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., 1998Go). 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., 1993Go). Sequences of oligonucleotides used for RT-PCR were as follows.

• gtlec1—5'-gaaaggtgaacctaacaacg-3' and 5'-acagtgtttattcccgccattatt-3'

gtlec2a—5'-ggtgggatatagcttgcaca-3' and 5'-attcattttcagactgaagaac-3'

• gtlec2b—5'-ggtgggatatagcttgcaca-3' and 5'-acttcaatatccgtttgaga-3'

gtlec2c—5'-ttgataatttgatgatatcaacc-3' and 5'-gtataatgtacatccaatgaact-3'

• scarf-like genes—5'-catttaatcacattcgtcctc-3' and 5'-gacccaagctcaatattat-3'

• dth2—5'-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 Freund’s 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, 1988Go) and dialyzed against 100 mM sodium acetate, pH 5.5. Fab fragments of antibodies were generated by pepsin digestion following Harlow and Lane (1988)Go, 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 phosphatase–conjugated 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., 1998Go). After fixation, animals were washed three times in methanol and subjected to ultrasound for 5–7 min at Soniprep-150 (amplitude 10–22 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 phosphatase–conjugated 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 manufacturer’s 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We are grateful to I.M. Sheiman for providing animals and helpful discussions and to N.V. Bovin for consultations. We also thank N.N. Luchinskaya for considerable assistance in electronic microscopic experiments. This work was supported by the Russian Foundation for Fundamental Research (grant no. 97-04-50123-a), the Russian Foundation for Support Domestic Science (grant to S.L.), and by a FEBS short-term fellowship.

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 1–4), AY057975 (scarf2, exons 5–7), AY057976 (scarf3a), AY057977 (scarf3b), AY057978 (s-scarf2a, exons 1–4), AY057979 (s-scarf2a, exons 5–7), and AY057973 (s-scarf2b).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; CTLD, C-type lectin–like domain; ELISA, enzyme-linked immunosorbent assay; MDFL, multidomain free lectin; ML, maximum likelihood; MBP, mannose-binding protein; PBS, phosphate buffered saline; RACE, rapid amplification of cDNA ends; RHL, rat hepatic lectin; RT-PCR, reverse transcription polymerase chain reaction.


    Footnotes
 
1 Present address: Whitney Laboratory, University of Florida, FL, USA Back

2 Present address: Department of Preclinical Veterinary Sciences, The University of Edinburgh, UK Back

3 Present address: Laboratory of Cellular and Molecular Biology; National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Back

4 To whom correspondence should be addressed; E-mail: luk@ibch.ru Back


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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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