Glycobiology Advance Access originally published online on April 18, 2007
Glycobiology 2007 17(7):774-783; doi:10.1093/glycob/cwm044
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Molecular and biochemical characterization of galectin from amphioxus: primitive galectin of chordates participated in the infection processes
State Key Laboratory of Biocontrol, The Open Laboratory for Marine Functional Genomics of State High-Tech Development Program, Guangdong Province Key Laboratory for Pharmaceutical Functional Genes, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xingangxi Road, Guangzhou 510275, People's Republic of China
1 To whom correspondence should be addressed; Tel: +86 20 84113655; Fax: +86 20 84038377; e-mail: lssxal{at}mail.sysu.edu.cn
Received on February 5, 2007; revised on March 20, 2007; accepted on April 12, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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A novel F4-carbohydrate recognition domain (CRD)–linker–F3-CRD-type bi-CRD Branchiostoma belcheri tsingtauense galectin (BbtGal)-L together with its alternatively spliced mono-CRD isoform BbtGal-S from amphioxus intestine was encoded by a 9488-bp unique gene with eight exons and seven introns. The recombinant proteins of BbtGal were found to have ß-galactoside-binding activity, indicating that BbtGal was a member of the galectin family. Phylogenetic analysis of this gene along with its splicing form and genome structure suggested that the BbtGal gene was the primitive form of the chordate galectin family. Real-time polymerase chain reaction analyses (PCR) indicated that BbtGal mRNA was expressed during all stages of embryonic development. In terms of tissue distribution, BbtGal-L mRNA was mainly expressed in the immunity-related organs, such as hepatic diverticulum, intestine, and gill, but BbtGal-S was ubiquitously expressed in all tissues. The expression of BbtGal-L mRNA was elevated after acute challenge with various microorganisms, but BbtGal-L only bound to specific bacteria. The immune function of BbtGal was consistent with its localization both outside and inside the cell. Our study on amphioxus galectin may help further understanding of the evolution of chordate galectin in terms of host–pathogen interaction in the immune system.
Key words: amphioxus / evolution / galectin / infection
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Galectins are evolutionarily conserved carbohydrate-binding proteins with highly conserved amino acid sequences in their carbohydrate recognition domain (CRD), and are found to be widely distributed in nature as they have been identified in vertebrates, invertebrates, and protists (Shoji et al. 2003
; Houzelstein et al. 2004).
All known galectins are classified into three types in terms of molecular architecture, i.e. mono-CRD type, chimera type, and bi-CRD type (Hirabayashi and Kasai 1993). In mammals, 15 galectins have been sequenced and characterized (Liu and Rabinovich 2005). Galectin-1, -2, -5, -7, -10, -11, -13, -14, and -15, which belong to the mono-CRD type and are found in many cells and tissues, have molecular weights of 14–18 kDa with a single galactoside-binding domain. Galectin-3 with an apparent molecular weight between 26 and 30 kDa is the only chimera-type galectin found to date, has a collagenous domain, and is abundant in activated macrophages and epithelial cells. Galectin-4, -6, -8, -9, and -12, which belong to the bi-CRD type, have molecular weights of 33–39 kDa with two CRDs linked by a hinge peptide and are present in various cells and tissues. Recent studies have shown that the mRNA of the galectin-8 gene encodes six different isoforms, and three of them belong the mono-CRD group (Bidon et al. 2001). The vertebrate galectin CRDs are always encoded by three exons with two subtypes and are defined by the exon–intron structure (F4-CRD and F3-CRD). The F4-CRD-linker-F3-CRD gene structure is shared among all vertebrate bi-CRD galectins, one Ciona intestinalis galectin (Houzelstein et al. 2004), and the Stronglocentrotus purpuratus galectin (RL-30). The chordate galectins share a common ancestor, and bi-CRD galectins are derived from an ancestral tandem-duplication of mono-CRD galectin either before or in early chordate evolution (Houzelstein et al. 2004).
Recently, galectins have attracted the attention of immunologists as novel regulators of host–pathogen interaction (Almkvist and Karlsson 2004; Rubinstein et al. 2004; Rabinovich and Gruppi 2005). Galectin-1 and galectin-3 are upregulated in gastric epithelial cells infected with Helicobacter pylori, which suggests that galectins might contribute to bacterial invasion (Lim et al. 2003). In addition, galectin-3 has the ability to bind to Gram-negative (G–) bacteria through the recognition of different bacterial lipopolysaccharides (LPSs) (Mey et al. 1996) and induce the death of Candida species expressing specific ß-1,2-linked mannans (Kohatsu et al. 2006). The elevation of galectin-9 is involved in the inflammatory response of periodontal ligament cells exposed to Porphylomonas gingivalis LPS in vitro and in vivo (Kasamatsu et al. 2005) and can recognize the Leishmania major-specific polygalactostosyl epitope (Pelletier et al. 2003).
However, the questions of whether and how the galectins negatively or positively affect microbial invasion, such as bacteria, viruses, fungi, and parasites, and whether they are involved in the defense of different infections remain elusive. Functional complexity of galectins in higher vertebrate hampers further analyses of the biological significance of these genes. To understand the biological significance of galectins in innate immune systems, comparative analyses of their primitive form in a lower chordate may be useful to gain insight into the evolution of vertebrate galectin in the innate immune response. To this end, amphioxus, a cephalochordate that has been regarded as the closest relative to the ancestor of ancient vertebrates (Holland et al. 2004), is used in our study.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Identification of galectins from Chinese amphioxus
An expressed sequence tag with sequence similarity to galectin was identified from the amphioxus intestine cDNA library, and 5'-RACE (rapid amplification of cDNA ends) was performed to amplify the 5'-end of the cDNA of amphioxus intestine. When the PCR products were cloned and sequenced, two different sequences overlapping with known galectin sequence were identified. Analysis by Seqtools8.0 demonstrated that they were alternatively spliced forms of a single galectin gene and possessed the same 131 bp 5'-untranslated region (5'-UTR) and the 251 bp 3'-UTR, including the poly(A) tail (Figure 1). The two full-length cDNA sequences of amphioxus galectins were 1318 and 847 bp in length, including the poly(A) tail, and contained a 936 and 465 bp open-reading frame (ORF), respectively.
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The longer cDNA, named Branchiostoma belcheri tsingtauense galectin-L, encoded a 311 amino acid protein of 34 kDa with homologous N-terminal (136 amino acids) and C-terminal (138 amino acids) CRDs, connected by a 20 amino acid linker peptide. CRDs of BbtGal were formed by two antiparallel ß-sheets, and each ß-sheet was composed of five ß-strands (labeled F1–F5 and S2–S6). The S1 strand, which was found to be present in other galectins of vertebrates, was missing. This subtle structural difference may result in certain functional differences between BbtGal and other galectins in vertebrates. Both domains contained S-type lectin motifs (e.g. H-NRP at position 66/224, WG-EE at position 86/243; Figure 1), which were conserved in all known galectins. N-CRD and C-CRD within the same vertebrate bi-CRD galectins had only 37–40% homology, but the N-CRD domain of BbtGal-L shared 63% homology with C-CRD, and a similar observation was made in the S. purpuratus galectin (RL-30), with 65% homology between the two CRDs (Table I).
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The shorter cDNA, named BbtGal-S and encoding 154 amino acid protein of 18 kDa, was spliced from the repeat site (LQCGAST) of BbtGal-L. The anterior part of the S3 sheet of N-CRD and the posterior part of the S3 sheet of C-CRD of BbtGal-L comprised the alternatively spliced form, BbtGal-S (Figure 1), containing a single CRD domain.
Genomic structure of BbtGal
According to the conserved genomic structure of the galectin family, three pairs of primers (–18F/167R, 81F/721R, and 656F/948R) were designed (Figure 2B). These primers were used to amplify 3-, 7-, and 0.6-kb fragments, respectively. A 9488-bp genomic sequence of amphioxus galectin was obtained by sequencing and editing these fragments. By analysis with GeneScan (http://genes.mit.edu/GENSCAN.html) and comparing the cDNA with the genomic DNA sequence, the structure of exon and intron organization was clearly revealed to contain eight exons and seven introns, as shown in Figure 2A.
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Both CRDs of BbtGal-L were encoded by three exons (Figure 1). The first exon encoded the F2 ß-strand. The second exon, which was referred to as the W exon because it contained a highly conserved tryptophan (W) residue, encoded three adjacent ß-strands (S4, S5, and S6) that form a pocket containing the residues directly involved in carbohydrate binding. The W exon of the N-CRD, encoded by the S3 to F4 sequence, was classified into the F4 subtype, and the W exon of the C-CRD, encoded by the S3 to F3 sequence, belonged to the F3 subtype. The F4-CRD-linker-F3-CRD gene structure of BbtGal-L exhibited the same characteristic organization as all vertebrate bi-CRD galectins which was also found in RL-30 and in one of the bi-CRD galectins, (ciona-a) from C. intestinalis. The BbtGal-S was an F3 subtype CRD galectin as was BbtGal-L-C-CRD.
Sequence alignment and phylogenetic analyses of BbtGal
The human and mouse galectin-8/9 were selected from the GenBank database, and a multiple sequence alignment was made with S3, S4, S5, and S6 ß-sheets, which were involved in the galectin–carbohydrate interactions (Figure 3). From the alignment, it was found that many amino acid residues were conserved in all galectins, such as galectin sequence motifs WG-EE and H-NPR. However, in the BbtGal-L-N-CRD, there was a Phe-to-Met substitution at amino acid position 65. This amino acid was also substituted with Cys in Xenopus galectin IIIa-N-CRD, but the galectin retained the ability to bind ß-galactoside sugars (Shoji et al. 2003). In BbtGal-L-C-CRD, there was a His-to-Gln substitution at amino acid position 223. For most galectin family members, this Gln stabilized galectin–carbohydrate interactions (Lobsanov et al. 1993). These results suggested that each domain of BbtGals differed with respect to the ability to bind sugar ligands.
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On the basis of the alignment, a phylogenetic tree was constructed with CRDs of each galectin (Figure 4). The vertebrate N-CRDs were all clustered into the F4-CRD subtype, whereas C-CRDs were all clustered into the F3-CRD subtype. The phylogenetic analyses showed that the galectins of amphioxus and sea urchin were the primitive forms of the chordate galectins, and the divergence event of vertebrate galectins occurred after echinoderms and cephalochordates.
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Temporal and spatial expression patterns of BbtGal-L and -S mRNAs
BbtGal-L and -S mRNAs were expressed througout the entire embryonic development (Figure 5A). The abundant BbtGal-L mRNA expression in the midblastula stage was consistent with the zygotic gene expression initiating at the midblastula stage. After activation in the midblastula stage, the BbtGal-L mRNA expression decreased to a very low level in the early neurula stage. From the early neurula stage to the adult, the gradual increase in the mRNA expression suggested that the amphioxus galectin might play roles in the organization of various tissues because the formation of various organs was actively underway at these stages.
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We also analyzed the distribution of the galectin mRNA in adult tissues (Figure 5B and C). BbtGal-L had a specific tissue distribution, especially in hepatic diverticulum, intestine, and gill, suggesting that the function of BbtGal-L might be related to the function of these highly expressed tissues. In contrast, BbtGal-S mRNA was detected in almost all tissues. These results demonstrated that BbtGal-L was abundantly expressed in immune organs and might preferentially participate in the immune system.
Upregulation of BbtGal-L expression by pathogenic challenges
The high expression of BbtGal-L in immune organs led us to study its expression pattern after pathogenic challenge. In order to determine the effects of different bacteria strains on amphioxus, the mean lethal dose (LD50) of each microorganism was tested. The LD50 of Vibrio vulnificus (G–) to the amphioxus was 5.24 x 1011 colony-forming units (CFU)/fish, whereas that of Vibrio parahaemolyticus (G–) and Staphylococcus aureus (G+) was 1.02 x 109 and 1.26 x 109 CFU/fish, respectively. The real-time PCR results indicated that BbtGal-L mRNA expression was upregulated after acute immune challenge with these pathogenic stimulations (Figure 6). BbtGal-L could be quickly induced after challenge with V. vulnificus 4 h later, continuously increased for the next 8 h, then kept at a steady level (8.8 times that in the naive animal). In contrast, the mRNA expression was induced later and expressed at a lower level after challenge with V. parahaemolyticus and S. aureus.
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), V. parahaemolyticus (
), and S. aureus (
) at 4, 8, 12, 24, and 48 h compared with unchallenged naive animals. Data were expressed as an increasing ratio to the amphioxus galectin mRNA expression in naive animals. The endogenous control for quantification was cytoplasmic actin. Each experiment of real-time PCR was repeated 3 times and the results were expressed as mean ±SD.The binding activity of BbtGal proteins to ß-galactoside sugars
To study further the biochemical characterization of BbtGal-L and -S, we expressed them as fusion proteins with glutathione S-transferase (GST) using the GST fusion system. In the presence of 5 mM ß-mercapitoethanol (ß-ME), the proteins at and above 25 nM could induce a strong agglutination of three kinds of animal erythrocytes (Table II). Only lactose, galactose, and other ß-galactoside saccharides had an inhibitory effect on the hemagglutination (HA) capabilities of BbtGals (Table III). These data indicated that BbtGals could interact only with the ß-galactoside saccharides, similar to their vertebrate homologs. We also demonstrated this by incubating the entire bacterial supernatant of recombinant proteins with lactosyl-Sepharose 4B beads. A single band, corresponding to the predicted molecular weight of BbtGals, was found to be present in the supernatant eluted off with lactose and in the beads eluted off with sucrose (Figure 7), demonstrating specific binding of BbtGals to lactose and confirming their identity as galectin family members.
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Subcellular localization and secretion of BbtGals
Because galectins were known to be secreted by non-classical (non-ER-Golgi) pathways although they had features of cytosolic proteins (Rubinstein et al. 2004
; Rabinovich and Gruppi 2005), we investigated whether BbtGals were secreted from cells. The pEGFP-N1-BbtGal-L or -S constructs were transiently transfected into HEK293T cells, and proteins were obtained from either cell extracts or conditioned medium at 48 h post-transfection. The cell extract and medium fractions were resolved on a polyacrylamide gel and analyzed by Western blot using an anti-GFP antibody. The results demonstrated that BbtGals were present in both the cell extract and media (Figure 8), indicating that BbtGals could be secreted from the cytosol. We also detected the subcellular localization of BbtGals by laser confocal imaging and found that they were well distributed in the cytoplasm (Supplementary data).
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Bacteria-binding activity of BbtGal-L in vitro
Many studies demonstrated that some members of the galectin family could directly bind with the microorganisms, whereas in our study we found that BbtGal-L could be released into the extracellular medium and upregulated after challenge with the microorganisms, which led us to study its bacterial-binding activity in vitro. We incubated V. vulnificus, V. parahaemolyticus, and S. aureus with the targeted protein, and the bacterial pellets were assessed by Western blot using anti-GST antibody. As shown in Figure 9, GST-BbtGal-L was found to bind to V. vulnificus, but not to V. parahaemolyticus and S. aureus.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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In the present study, a novel F4-CRD-linker-F3-CRD galectin BbtGal-L along with its alternatively spliced mono-CRD isoform BbtGal-S was identified from amphioxus. Recombinant BbtGal bound to the ß-galactoside sugars specifically, confirming their identity as galectin family members. BbtGal-S was encoded by a unique sequence from the spliced exons I, II, VI, and VIII of BbtGal-L. This form of alternative splicing form was also found in human galectin-8 (Bidon et al. 2001
). The similarity between BbtGal and galectin-8 indicated that the alternatively spliced form was conserved from the amphioxus to human galectin genes. This conservation was also found in the F4-CRD-linker-F3-CRD gene structure of BbtGal that exhibited the same characteristic organization as all vertebrate bi-CRD galectins, RL-30, and C. intestinalis galectin-a, but never found in other invertebrates.
It was reported that the chordate mono- and bi-CRD galectins evolved by duplication and divergence from an ancestral mono-galectin on the basis of the phylogenetic analysis of galectin chromosomal localization, exon–intron organization, and CRD sequences. This ancestral mono-CRD galectin gave rise to the first bi-CRD galectin (Houzelstein et al. 2004). BbtGal-L-N-CRD and -C-CRD were very similar to each other with 63% homology, indicating that the two CRDs of BbtGal-L were formed by gene duplication. This phenomenon was also supported by S. purpuratus RL-30, of which the N-CRD also had a high similarity to its C-CRD. The shared exon–intron organization, alternatively spliced form, and high homology between N-CRDs with C-CRDs in amphioxus and sea urchin strongly supported that all vertebrate CRDs originated from a common ancestral CRD of echinoderm or from more primitive species by a mechanism of gene duplication and divergence.
The vertebrate galectins had multiple functions participating in development as well as innate and adaptive immunity. The amino acid sequence similarity between BbtGals and vertebrate galectins suggested that amphioxus galectins might share a similar function to vertebrate homologs, which was indirectly confirmed by the ß-galactoside-binding activity of the recombinant proteins. The potential roles of galectin in development stages, such as notochord development, somitogenesis, and the development of central nervous system in vertebrates, prompted us to study the function of the amphioxus galectin in embryogenesis (Poirier et al. 1992; Fowlis et al. 1995; Shoji et al. 2003). The temporal expression patterns of BbtGals showed that they were expressed during embryogenesis, suggesting that BbtGal participated in embryogenesis. However, the level of BbtGals expression in the adult was higher than that at each stage of embryogenesis, indicating that BbtGal-L had a prominent function in adult animals.
The high expression level of BbtGal-L had a specific tissues distribution, especially in hepatic diverticulum, intestine, and gill, which could synthesize antimicrobial peptides and phagocytose microbes, suggesting that BbtGal-L played an important role in the immune system, consistent with the function of these tissues. In contrast to BbtGal-L, BbtGal-S was ubiquitously expressed in all tissues. The different expression patterns of the two isoforms indicated their different roles in amphioxus.
Like some mammalian galectins, BbtGals were present both inside and outside cells and functioned both intracellularly and extracellularly, which was consistent with the immune function in host–pathogen interactions. BbtGal-L was upregulated after challenge with the microorganisms, indicating that pathogen-mediated regulation of galectin gene expression and galectin-mediated signal transduction pathways also existed in the amphioxus innate immune system. BbtGal might be released from immune cells of amphioxus following an immune challenge, similar to the release of galectin-10 from mammalian eosinophils following stimulation (Leonidas et al. 1995) or the release of galectin-3 from dendritic cell exosomes during antigen presentation (Thery et al. 2001). BbtGals functioned like their vertebrate homologs, being involved in the inflammatory response by cross-linking ß-galactoside glycojugates or glycoprotein receptors on cell surfaces to mediate cell–cell or cell–matrix interactions, responding to the invading pathogens and triggering signal transduction pathways.
Many mammalian galectins functioned as potential pattern-recognition receptors (or danger signals) by transmitting the information of microbial invasion to immune cells (Rubinstein et al. 2004; Rabinovich and Gruppi 2005). In the extracellular medium, BbtGal-L also acted as host receptor for the specific invading microorganisms such as V. vulnificus, which could induce quick and high expression of BbtGal-L compared with other tested microorganisms. It could directly bind to V. vulnificus to reduce the danger to the amphioxus, which might be consistent with the unexpected fact that the increase of BbtGal-L mRNA expression was higher for a weak pathogen.
BbtGal might participate in the innate immune system of the amphioxus by facilitating microbial recognition. Alternatively, BbtGal might be released following an immune challenge and involved in the inflammatory response via galectin-mediated signal transduction pathways. Such a manner of immune regulation was also found for the vertebrate galectins (Sato and Nieminen, 2004). Galectin-3 was upregulated in gastric epithelial cells infected with Helicobacter pylori (Lim et al. 2003) and required for the specific recognition of Candida albicans by macrophages to discriminate Saccharomyces cerevisiae and the association with TLR2 for signaling (Jouault et al. 2006). Moreover, galectin-3 possessed the ability to induce the death of Candida species expressing specific ß-1,2-linked mannans (Kohatsu et al. 2006). The manner of galectin's involvement in inflammatory response was diversified and conserved in chordate evolution. Our study on amphioxus galectin might help further understanding of the interaction between host and pathogens in vertebrates, particularly understanding of the evolution of vertebrate galectin in terms of host–pathogen interaction in the immune system.
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Materials and methods |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Preparation of tissue and embryo samples of amphioxus
Mature adults of Chinese amphioxus, Branchiostoma belcheri tsingtauense (genus Branchiostoma, family Branchiostomidae), were obtained from Kioachow Bay near Qingdao, Shangdong Province, China, and cultured in the tank (at 25 °C) filled with air-pumped circulating sterilize seawater. Spawning females swimming up from the sand to lay eggs were caught with a net and immediately put into a large Petri dish containing naturally inseminated seawater. The embryos of each period were collected and quickly frozen in liquid nitrogen. Adult amphioxus was not fed for 10 days before the experiment. Tissues separated under an optical microscope were fast frozen by liquid nitrogen.
LD50 value of various bacteria to amphioxus and immune challenge of amphioxus
Amphioxus, approximately 2.5–3.5 cm long and 0.7–1.0 g, were injected with different concentrations of live V. vulnificus (G–), V. parahaemolyticus (G–), and S. aureus (G+) phosphate-buffered saline (PBS) suspension from 104 to 1012 CFU. Twenty amphioxus were injected with bacteria for each treatment. The experiment was repeated 3 times. The number of the lethal amphioxus was statistically analyzed 96 h after injection. The LD50 was calculated as following: log LD50 = Xk – d(
Pi – 0.5). Xk was the largest logarithm, d the margin of two boundary doses, Pi the lethal rate, and i the number of treatments. For immune challenge, live bacteria (15 µL/animal, 109 CFU/mL) were injected into the celom of amphioxus. Twenty random samples of amphioxus were collected after injection and frozen by liquid nitrogen similarly to experimental samples.
Full-length cDNA and genomic DNA cloning and sequencing
Gene-specific primers 806R and 758R were designed for amplification of the 5'-end sequence of amphioxus galectin cDNA using the GeneRACETM Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Total RNA of the amphioxus intestine was extracted for cDNA synthesis. The primers, –18F/167R, 81F/721R, and 656F/948R (Figure 2B), were designed according to the genome structure of mammalian and C. elegan galectins to amplify the genomic DNA of the amphioxus galectin using LA Enzyme (Takara, Dalian, China). The amplified fragments were cloned into the pGEX-T easy vector (Promega, Madison, WI) and sequenced by the Perkin Elmer ABI Prism 3730 DNA sequencer.
Phylogenetic and structural analyses
Sequences related to galectin were extracted from GenBank and aligned with the sequence identified in this report using CLUSTAL X (http://bips.u-strasbg.fr/fr/Documentation/ClustalX/, and phylogenetic analysis was performed using MEGA (version 3.1; http://www.megasoftware.net/) with the CRD domain of galectin. The secondary structure of amphioxus galectin was predicated using 3D-PSSM Protein Fold Recognition (Threading) Server prediction methods.
PCR analyses of the galectin expression patterns
Reverse transcriptase (RT)–PCR and real-time PCR were performed as described (Yuan et al. 2007). RT–PCR analyses were performed on cDNAs from various sources by utilizing the primers 99F/723R (Figure 3). Real-time PCR analyses of the BbtGal-L expression were performed with the primers 478F/600R. The cytoplasmic actin gene of Chinese amphioxus was used as an endogenous control.
Section in situ hybridization
The fragment amplified with the primers 99R/600F was cloned into the pGEX-T easy vector and then digoxigenin (DIG)-labeled sense and antisense probes were synthesized. Sexually mature amphioxus were cut into three to four pieces and fixed in freshly prepared 4% paraformaldehyde in 100 mM PBS (pH 7.4) at room temperature for 2 h or at 4 °C overnight. They were dehydrated, embedded in paraffin, and sectioned at 8 µm. The sections were mounted on poly-L-lysine-coated slides, dried at 42 °C overnight, and de-waxed in xylene for 20 min (two changes for 10 min each) followed by immersion in absolute ethanol for 10 min (two changes for 5 min each). They were then rehydrated and brought to double-distilled water containing 0.1% diethypyrocarbonate (DEPC). The hybridization procedure was essentially adapted from the published protocols (Kanazir et al. 1997; Kerner et al. 1998), and care was taken to make sure that all solutions and equipment used for hybridization histochemistry were RNase-free. Briefly, sections were first digested with 10 µg/mL proteinase K (Merck, Darmstadt, Germany) in 100 mM Tris–HCl buffer (pH 8.0) with 50 mM EDTA at 37 °C for 30 min, post-fixed in 4% paraformaldehyde in 10 mM PBS (pH 7.4) at room temperature for 20 min, and then acetylated in freshly prepared 100 mM Tris–HCl (pH 8.0) with 0.25% acetic anhydrite at room temperature for 10 min, de-hydrated with graded ethanol, prehybridized in a hybridization buffer containing 50% deionized formamide (v/v), 100 µg/mL heparin, 5 x SSC, 0.1% Tween-20, 5 mM EDTA, 1 x Denhardt solution, and 1 mg/mL total yeast RNA at 45 °C for 2 h, and hybridized in the same hybridization buffer with 1 µg/mL DIG-labeled sense and antisense probes at 45 °C for 16 h in a moist chamber). The sections were subjected to RNase A (Promega) digestion (20 µg/mL in 2 x SSC) at 37 °C for 30 min, washed 3 times in 100 mM Tris–HCl (pH 7.5) with 150 mM NaCl (15 min each), preincubated in 1% blocking reagent (Roche, Basel, Switzerland) in 100 mM Tris–HCl (pH 7.5) with 150 mM NaCl for 1 h at room temperature, and then incubated with anti-DIG alkaline phosphatase-conjugated antibody (Roche) diluted 1:2000 in 1% blocking reagent in 100 mM Tris–HCl with 150 mM NaCl (pH 7.5) for 2 h at room temperature. After washing three times in 100 mM Tris–HCl (pH 9.5) containing 100 mM NaCl and 50 mM MgCl2 (5 min each), the sections were incubated with a coloring solution consisting of 3.4 µg/mL NBT and 3.5 µg/mL BCIP in 100 mM Tris–HCl (pH 9.5) with 100 mM NaCl and 50 mM MgCl2 for 4–12 h in the dark. The coloring reaction was stopped in Tris–HCl (pH 8.0) with 1 mM EDTA for 10 min, and the sections were then rinsed in distilled water, dehydrated, mounted in Canada balsam, and photographed under a Nikon microscope.
Construction of expression vectors and preparation of recombinant proteins in Escherichia coli
Recombinant amphioxus galectin proteins were expressed as fusion proteins with GST using the GST fusion system (GE Healthcare, Chalfont St. Giles, UK). The primers 5'-CGGGATCCGATGGCGTACCCAGGATT-3' (forward) and 5'-ATAAGAATGCGGCCGCTTATGTGAAGCGGATCTGCT-3' (reverse) were used to create full-length proteins of BbtGal-L and -S. The amplified fragments were inserted into pGEX-4T between the BamHI and NotI sites (in bold), and the expression plasmids were introduced into E. coli BL21 (DE3). The recombinant proteins were purified with a glutathione–Sepharose column according to the manufacturer's instructions.
HA inhibition assay
Hemagglutinating activity (HA) of recombinant proteins was tested on trypsin-treated glutaral-dehyde-fixed rabbit, chicken, and carp fish erythrocytes. HA was measured by a series of 2-fold dilutions of purified GST-fusion recombinant proteins of equal protein concentration in 96-well microtiter plates with V-shaped well bottoms. The lowest dilution that caused distinct hemagglutination was determined after 1 h at room temperature. To analyze the inhibitory effect of sugars on HA, the buffer was replaced by different sugar solutions. The reciprocal of the highest dilution of the lectin showing visible hemagglutination was recorded as the titer, and the specificity was determined as the lowest dilution of sugars that inhibited HA of the lectin solution. The final concentration of ß-ME in the assay was 5 mM. PHA as a positive control and GST protein as a negative control were tested in the same way as recombinant proteins.
Lactose-binding assay
One milliliter of recombinant GST-BbtGal-L and -S unpurified proteins in sonicated buffer were incubated with 20 µL of lactosyl-Sepharose 4B at 4 °C for 2 h with constant mixing. After the beads were washed 3 times with 1 mL of binding buffer (50 mM phosphate buffered saline (PBS), 150 mM NaCl, 5 mM EDTA, 10 mM ß-ME), the various proteins were incubated at 4 °C for 1 h with lactosyl-Sepharose 4B in the presence of 50 µL of lactose or sucrose (100 mM). The supernatant eluted by lactose or sucrose was collected. Then the beads were washed 3 times with 1 mL of binding buffer. The supernatant and the beads were subjected to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by immunoblotting. Briefly, proteins separated by SDS–PAGE were transferred to nitrocellulose (NC) membrane and detected with a mouse monoclonal anti-GST antibody and horseradish peroxidase (HRP)-labeled antimouse IgG antibody.
Protein localization assay
The fragments amplified with the primers 5'-GGAATTCATGGCGTACCCAGGATTTG-G-3' (forward) and 5'-CGGATCCTGTGAAGCGGATCTGCTG-3' (reverse) were inserted into pEGFP-N1 (Clontech, Mountain View, CA) between the EcoRI and BamHI sites (in bold). The pEGFP-N1-BbtGal-L or -S constructs were transfected into HEK293T cells, using Lipofectamine2000 (Invitrogen), and expression monitored by immunoblotting analysis. Briefly, the proteins were isolated from cell extracts with lysis buffer (20 mM Tris.–HCl, pH 7.4, 250 mM NaCl, 1 mM EDTA, 0.5% NP-40) or from conditioned medium 48 h post-transfection. The protein from the conditioned medium was concentrated for 1 h with a Centricon-10 concentrator. The proteins (20 µg) from the media and cell extracts were separated by 15% SDS–PAGE, transferred to NC membranes, and probed with rabbit monoclonal anti-GFP antibody (eBioscience, San Diego, CA) and HRP-labeled anti-rabbit IgG antibody. A pEGFP-N1 construct was transfected in parallel as a negative control. The cells were changed to serum-free medium 24 h after transfection.
Bacteria-binding assay
Approximately 5 x 107 bacteria were incubated with 20 µg of recombinant BbtGal-L protein in the binding buffer (50 mM PBS, 10 mM ß-ME) by gentle orbital rotation overnight at 4 °C. In all the cases, bacteria were pelleted and washed stringently with the binding buffer. The washed pellets were then suspended and subjected to 15% SDS–PAGE and analyzed by immunoblotting with anti-GST antibody. The GST protein was treated in parallel as a negative control.
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflict of interest statementAcknowledgmentsReferences |
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This work was supported by Project 2004AA621030 and Project 2003AA626010 of the State High-Tech Development Project (863) of the Ministry of Science and Technology of China, Key Project (0107) of the Ministry of Education, Project 30300264 of the National Natural Science Foundation, and Key Projects of the Commission of Science and Technology of Guangdong Province and Guangzhou City.
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Abbreviations |
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ß-ME, ß-mercapitoethanol; BbtGal, galectin from Branchiostoma belcheri tsingtauense; CFU, colony-forming units; CRD, carbohydrate recognition domain; DEPC, diethypyrocarbonate; DIG, digoxigenin; GST, glutathione S-transferase; HA, hemagglutination activity; LD50, the mean lethal dose; LPS, lipopolysaccharide; NC, nitrocellulose; ORF, open-reading frame; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PHA, phytohemagglutinin; RT–PCR, reverse transcriptase–PCR; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
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