Glycobiology Advance Access originally published online on June 30, 2004
Glycobiology 2004 14(11):959-968; doi:10.1093/glycob/cwh120
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Glycobiology vol. 14 no. 11 © Oxford University Press 2004; all rights reserved.
Evolution of sialic acid–binding proteins: molecular cloning and expression of fish siglec-4
Centre for Biomolecular Interactions Bremen, Department of Biology and Chemistry, University Bremen, 28334 Bremen, Germany
Received on March 16, 2004; revised on June 15, 2004; accepted on June 28, 2004
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Abstract |
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Siglecs are the largest family of sialic acid–recognizing lectins identified so far with 11 members in the human genome. Most of these siglecs are exclusively expressed by cells of the immune system. Comparison of different mammalian species has revealed differential and complex evolutionary paths for this protein family, even within the primate lineage. To understand the evolution of siglecs, in particular the origin of this family, we investigated the occurrence of corresponding genes in bony fish. Interestingly, only unambiguous orthologs of mammalian siglec-4, a cell adhesion molecule expressed exclusively in the nervous system, could be identified in the genomes of fugu and zebrafish, whereas no obvious orthologs of the other mammalian siglecs were found. As in mammals, fish siglec-4 expression is restricted to nervous tissues as demonstrated by northern blot. Expressed as recombinant protein, fish siglec-4 binds to sialic acids with a specificity similar to the mammalian orthologs. Relatively low sequence similarities in the cytoplasmic tail as well as an additional splice variant found in fish siglec-4 suggest alternative signaling pathways compared to mammalian species. Our observations suggest that this siglec occurs at least in the nervous system of all vertebrates.
Key words: Danio rerio / evolution / myelin-associated glycoprotein / siglecs / Takifugu rubripes
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Introduction |
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Sialic acids (Sias) are a diverse family of acidic ketoses with nine-carbon backbones being present in all deuterostomes as well as on some protostomes, protozoa, bacteria, and viruses. Based on the role of Sia as recognition determinants for various pathogenic agents, such as viruses, bacteria, protozoa, and toxins, the unusual structural complexity of Sia has been interpreted as a result of evolutionary arms race between the hosts and pathogens (Angata and Varki, 2002
; Karlsson, 1998; Varki, 1997).
More recent studies have revealed another prominent role of Sias, namely, their functions as ligand determinants for endogeneous lectins (Kelm and Schauer, 1997). A group of structurally related proteins recognizing Sia has been identified as playing an important role in cellular communication of mammalian cells. These siglecs (sialic acid–binding immunoglobulin-like lectins) are the largest family of Sia-recognizing lectins discovered so far. Structurally, all siglecs are type I membrane proteins, consisting of typical Ig-like domains characterized by two opposing ß-sheets held together by a disulfide bridge (Barclay and Brown, 1997), one N-terminal V-set Ig domain, a variable number of C2-set Ig domains, a single-pass transmembrane domain, and a cytoplasmic tail. The N-terminal domains of all siglecs share several similarities in their amino acid sequences. In contrast to the regular pattern of cysteins found in typical members of the Ig superfamily (IgSF), which form an intersheet disulfide bridge between the B and F strands, siglecs contain an intrasheet bridge between the B and E strands. This allows a somewhat larger distance between the two ß-sheets, which appears to be important for the binding of Sia residues. One additional cystein residue occurs at the C-terminal end of the B strand and builds an interdomain bridge to the following domain 2. Whereas most of the other conserved amino acids are important for the overall structure of the Ig-fold, three amino acid residues in domain 1 are critical for Sia recognition (May et al., 1998). Many of the siglecs have potential tyrosine phosphorylation sites, in most cases in the context of an immunoreceptor tyrosine-based inhibitory motif (ITIM), in their cytoplasmic tail, suggesting their involvement in intracellular signaling pathways (Crocker, 2002).
With the exception of siglec-4, all siglecs are expressed on the cells of the immune system, mainly on those involved in innate immunity, such as monocytes, macrophages, natural killer cells, and granulocytes (Crocker and Varki, 2001a, 2001b). By virtue of their differential tissue expression and specificities for Sia, and regarding the fact that they are the only proteins specifically recognizing sialylated glycans found in deuterostomes so far, siglecs are thought to play important roles in a wide array of recognition and signaling events (Crocker, 2002; Crocker and Varki, 2001a, 2001b).
Although Sia occur in all deuterostomia, siglecs have only been documented in mammals and birds so far (Crocker, 2002; Dulac et al., 1992). Interestingly, the comparison of siglec-related genes from human and great apes revealed differential and complex evolutionary paths indicating a high rate of evolution within this gene family (Angata et al., 2001; Brinkman-Van der Linden et al., 2000; Gagneux and Varki, 2001; Varki, 2001a, 2001b).
So far, only siglec-4 has been described from a nonmammalian species, from quail, where it is known as Schwann cell myelin protein due to its restricted expression pattern (Dulac et al., 1992). In mammals, siglec-4 is exclusively expressed by myelinating glia cells in the nervous system and is known for over 20 years as myelin-associated glycoprotein (MAG). Several lines of evidence, including those from gene knockout experiments, suggest important roles of siglec-4/MAG in the maintenance of myelin integrity and the regulation of neuronal growth (Filbin, 2003; Hunt et al., 2002; Spencer et al., 2003).
To understand the evolution of siglecs on a deeper time scale, it is of particular interest to evaluate deuterostomian branches that diverged early in evolution. Here we report the molecular cloning and characterization of siglec orthologs expressed in three fish species. They exhibit all structural features typical for siglecs, including all amino acid residues known to be essential for the interaction with Sia. Apparently, these proteins represent the fish orthologs of siglec-4, because high levels of sequence similarities were only found with this siglec.
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Results |
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Identification of putative siglec sequences in fish genomes
As a part of systematic in silico analysis of the siglec family, the Takifugu rubripes whole genome shotgun assembly and the Ensembl Zebrafish Genome Browser were searched using complete sequences of different mammalian siglecs as well as sequences encoding only the first N-terminal domains as queries. Scaffolds producing expect-values below 10–5 were assembled into contigs, and the resulting genomic sequences were analyzed for putative protein-coding sequences. Searches using sequences of siglec-4/MAG as queries lead to significant alignments with 56% identical amino acids in the N-terminal domain. Eight exons were predicted for the coding region of putative fish siglec-4. Also against the Xenopus tropicalis genome at JGI homology searches with mammalian siglec-4/MAG sequences revealed a fragment showing high similarity values to mammalian siglec-4/MAG (scaffold_35387).
In addition, in the fish databases we identified several sequences with low degrees of sequence similarity to domain 1 of mammalian siglecs (10.6–27.0% identical amino acids). In an attempt to identify further siglecs, the predicted amino acid sequences were aligned with the N-terminal domains of mammalian siglecs and analyzed more closely. Some of the candidates (sequences I, II, VII, and VIII; see Table I) contained the main characteristic features of siglecs, such as the unusual distribution of cysteines and the typical amino acids found in the binding site. In contrast, the other genes (sequences III, IV, V, VI, and IX; see Table I) are very unlikely to encode siglecs, because their pattern of cysteine residues did not reflect the situation found in siglecs, nor could we assign the residues typical for siglecs. The potential additional fish siglecs mentioned above could not be assigned as relatives to any of the known mammalian siglecs due to the low sequence similarity to these proteins. Even the degree of similarity between fugu and zebrafish genes (12–32% identical amino acids) was to low to allow assignments. Therefore, a conclusive phylogenetic analysis of all putative siglec sequences is not possible without additional information on related genes in amphibians, birds, and reptiles.
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Structure prediction analysis of fish siglec-4
An analysis of the amino acid sequences of putative fish siglec-4 predicted a structure with five characteristic Ig-like domains suggesting their affiliation with the IgSF (Gough et al., 2001
). Using the complete cDNA of the putative fish siglec-4 orthologs as well as the sequence from X. tropicalis, a sequence alignment was produced together with the sequences of the known mammalian representatives of siglec-4/MAG (for domains 1 and 2 see Figure 1). A comparison of complete fish siglec-4 sequences with mammalian orthologs revealed 38% identical amino acids, whereas the similarity to other mammalian siglecs was much lower (6.6% to 21.0% identical amino acids). The degree of conservation is higher in the extracellular domains, especially in the first N-terminal domain with a successive decrease of conservation from first to fifth domain (56% to 35% identical amino acids). The fragment of X. tropicalis siglec-4 shows a slightly higher degree of similarity to mammalian counterparts (52% identical amino acids) than to fish representatives of siglec-4 (about 44% identical amino acids). The cytoplasmic tail is less conserved (21% identical amino acids between fish and mammalian siglec-4). Both fish and putative X. tropicalis siglec-4 cDNAs encode a protein with the typical features of all siglecs previously described. Particularly, the three amino acid residues critical for sialic acid recognition are the arginine in ß-strand F (Arg99 in fugu siglec-4) and two aromatic amino acids, one in ß-strand A (Trp2 in fugu siglec-4) and one in ß-strand G (Tyr108 in fugu siglec-4). Also the cysteine residues forming the characteristic intrasheet bridges between the B and F strands of domain 1 and between the C-terminal end of the B-strand in domain 1 and domain 2 are present in the fish sequences. Furthermore, they show siglec-4-characteristic features like the absence of a salt bridge found in domain 1 of all other siglecs and one additional disulfide bridge in domain 5.
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Molecular cloning of fugu siglec-4 cDNA
From the in silico analysis described, most parts of a mRNA coding for fugu siglec-4 could be predicted. However, the 5'- and 3'-ends remained completely unclear. To confirm the predicted exons and to obtain the 5'- and 3'-exons of fugu siglec-4 mRNA, rapid amplification of cDNA ends (RACE) experiments were performed using fugu brain RNA as template and sequenced. The cDNA finally cloned contained all exons predicted from the in silico analysis plus one additional exon encoding the signal peptide leading to an open reading frame of 1902 nucleotides (Figure 2). In addition, evidence for alternative splicing at the 3'-end leading to three different C-termini was obtained as will be described shortly.
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KxE with Genomic organization of fish siglec-4
The genomic organization of siglec-4 from fugu and zebrafish and the different mammalian siglec-4/MAG representatives was obtained by comparing the cloned cDNA with the genomic sequences of these genes (Figure 3). All exon/intron boundaries were defined precisely. The signal peptide is encoded by a separate exon, followed by five exons encoding the Ig-like domains. The transmembrane domain is encoded by a separate exon and is followed by a cytoplasmatic domain, which is encoded by two or more exons as will be described. Only minor differences were found between the genomic organization of fish siglec-4 orthologs and the mammalian representatives (Figure 3). Notably, the exons encoding domains one to four have identical length in fish and mammalian siglec-4. Only small differences were found in the lengths of exons coding for domain 5 and the membrane anchor (Figure 3). However, substantial size differences were observed for the corresponding introns of zebrafish, fugu, and mammalian siglec-4 genes. In accordance with the overall situation in these genomes, most of the fugu introns were significantly smaller than those from the other species (Figure 3).
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Splice variants of fish siglec-4
Sequencing of 3' RACE–polymerase chain reaction (PCR) products revealed the presence of at least three splice variants at the 3'-end of the coding sequence leading to three different C-termini as shown schematically in Figure 4A. The cytoplasmic region of the variant shown in Figure 2 was termed L-tail (accession numbers AJ628724 for fugu and AJ628726 for zebrafish) because it resembles mammalian L-MAG. The second splice variant contains one additional sequence of 54 nucleotides (exon 9 in Figure 4A) coding for additional 18 amino acids and consequently the corresponding cytoplasmic region has been termed XL-tail (accession numbers AJ628725 for fugu and AJ628727 for zebrafish). The third splice variant of the gene contains all exons present in the XL-tail variant plus the 252 nucleotides between exons 7 and 9 (exon 8 in Figure 4A). Because this splice form introduces a termination codon at the beginning of exon 8, the resulting truncated C-terminus has been called S-tail (accession number AJ628729), corresponding to mammalian S-MAG.
In mammalian siglec-4, the cytoplasmic regions of L- and S-MAG contain different phosphorylation sites. Because these are likely to be relevant for signal transduction, the three cytoplasmic tails of fish siglec-4 were analyzed for consensus sequences with the potential for signal transduction. Whereas no such sites were found in the S-tail of fish siglec-4, the L-tail and the XL-tail sequences contain three putative tyrosine-based phosphorylation sites with two of them found in the context of an ITIM (LYY545SAV and LNY581ASL in the L-form of fugu, SDY566QSV and LNY582AAL in the L-form of zebrafish) (Figure 4B). Interestingly, these phosphorylation sites are significantly more similar to such sites in several other siglecs than to the phosphorylation sites in mammalian L-MAG, whereas outside of these sites the sequences of the cytoplasmic regions have only very low similarities (Figure 4B).
Surprisingly, exons 7 and 9 encode for two potential sumoylation sites (S I and S II; Figure 4C). Whereas S I occurs in all splice variants, S II occurs only in the XL-tail of siglec-4. In none of the known mammalian or avian siglecs a corresponding sequence has been identified.
Expression pattern of fish siglec-4
For northern blot analysis we used RNA from carp tissues because of the size of this fish, the possibility to obtain sufficient amounts of RNA from different tissues, and its close relationship to zebrafish. For this purpose an authentic probe from carp siglec-4 was synthesized and sequenced (accession number AJ628728). It covered exons 2–4 showing 87% and 73% sequence identity to zebrafish and fugu, respectively. As shown in Figure 5 the expression of siglec-4 appears to be restricted to nervous tissues with higher levels in lobus opticus, cerebellum, and spinal cord and lower levels in bulbus olfactorius, lobus inferior, and rhombencephalon. The major mRNA species in carp is estimated to be about 6.0 kb, which is twice as large as in avian or mammalian species (Dulac et al., 1992; Fujita et al., 1989).
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Fish siglec-4 as Sia-dependent cell adhesion molecules
To investigate whether the predicted fish siglec-4 orthologs represent true siglecs, it was important to demonstrate their ability to bind Sia-containing glycoconjugates. Therefore, cell adhesion assays were performed with COS-7 fibroblasts transfected with full-length fugu siglec-4 cDNA and human erythrocytes as target cells. These experiments clearly demonstrated that fugu siglec-4 mediates Sia-dependent adhesion of erythrocytes (Figure 6), because binding was completely abolished by pretreating erythrocytes with sialidase. Notably, sialidase pretreatment of COS-7 cells was not required for erythrocyte binding, indicating that at least in this cell system fugu siglec-4 is not occupied by Sia-containing glycoconjugates on the same cell surface (cis-ligands).
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Fc-chimeras of siglecs are commonly used to characterize their binding specificity (Kelm, 2001
). Therefore recombinant Fc-chimeras containing the extracellular three or five domains of fugu and zebrafish siglec-4 were prepared. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Fc-chimeras of fish siglec-4 exhibited a higher apparent molecular weight than the corresponding murine protein despite identical sequence length (data not shown). This could be explained by a different glycosylation of these proteins, as fish siglec-4 contains an additional potential N-glycosylation site in domain three (see Figure 2).
Complexed with anti-human IgG, Fc-chimeras of fugu and zebrafish siglec-4 showed robust binding to the GlycoWell plates derivatized with Neu5Ac at levels similar to the corresponding murine protein. All siglecs investigated so far require an intact extracyclic glycerol side chain of Neu5Ac for binding. Also, the binding of fish siglec-4 to periodate oxidized Neu5Ac on GlycoWell plates is reduced to background levels (Figure 7). Mammalian siglec-4/MAG binds preferentially to 
2,3-linked Sia (Strenge et al., 1998). For a further investigation on the glycan specificity, hapten inhibition assays were performed with increasing concentrations of 2,3- or 2,6-sialyllactose. Similar to murine siglec-4/MAG, fugu siglec-4 binding is reduced by 2,3-sialyllactose in a concentration-dependent manner, whereas no inhibition by 2,6-sialyllactose was observed (Figure 7). These data provide evidence that also fish siglec-4 binds with high preference to 2,3-linked Sia.
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In all the binding experiments, no differences were found between murine and fugu siglec-4. However, whereas murine siglec-4/MAG Fc-chimeras bind well to immobilized fetuin or polyacrylamide derivatives containing
2,3-linked Sia (unpublished data), no specific binding of fish siglec-4 to these glycoconjugates was observed (data not shown). |
Discussion |
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In mammals, siglecs appear to play critical roles in mediating cell–cell interactions and signaling functions in the hematopoietic, immune, and nervous systems (Crocker and Varki, 2001b
; Crocker, 2002). This study has been initiated to explore the occurrence of siglecs in fish because these represent the most distant vertebrates sharing with mammals basically all blood cells, a nervous system with myelinated axons, and a complex immune system. We were able to identify genomic sequences of a putative siglec in two fish species (T. rubripes and Danio rerio). The encoded proteins were clearly identified as fish orthologs of mammalian siglec-4/MAG based on the degree of sequence identity and exhibit all structural features described for siglecs in general as well as those specific for siglec-4.
To identify these putative orthologs as authentic siglecs, binding studies have been performed providing direct evidence that these proteins are true fish orthologs of mammalian siglec-4/MAG by showing specificity for 2,3-linked Sia. Although in this respect the binding specificity appears identical in fish and mammalian siglec-4, the observation that fish siglec-4 failed to bind to immobilized fetuin suggests that fish and mammalian orthologs have different selectivities for specific glycan structures. Further studies will be necessary to investigate these aspects of fine specificity, in particular toward the modifications of sialic acids, because besides Neu5Ac other Sia such as Neu5Gc and KDN are present in high amounts in fish glycoconjugates (Inoue and Inoue, 1997).
In mammals siglec-4/MAG is expressed exclusively by myelinating glia cells in the central and peripheral nervous system. It has been proposed to regulate interactions between glia cells and neurons involved in events like myelination, axonal growth, and signal transduction (Domeniconi et al., 2002; Schachner and Bartsch, 2000; Vinson et al., 2003; Wong et al., 2002). However it has been well established that in contrast to mammalian myelin, fish myelin does not inhibit neuronal growth, and this difference has been addressed to myelin composition (Klinger et al., 2004; Schweitzer et al., 2003). As demonstrated by northern blot analysis, in fish the expression of siglec-4 also appears to be restricted to the nervous system. This is in good agreement to the tissue distribution described for mammalian siglec-4/MAG. However, to clarify with certainty whether in fish the expression is also restricted to myelinating cells, additional histochemical experiments will be necessary.
Interestingly, in contrast to the two splice variants found for mammalian siglec-4 (Fujita et al., 1989; Heape et al., 1999; Ishiguro et al., 1991) in fish alternative splicing leads to three isoforms with different cytoplasmic tails with two of them containing potential phosphorylation and sumoylation sites. Notably, two of the three potential tyrosine-based phosphorylation sites are found in the context of ITIMs, a characteristic feature of the siglecs found in the immune system but absent in the mammalian and avian representatives of siglec-4. The existence and high conservation of those motifs potentially involved in signal transduction in fish suggest important biological functions different from those described for mammalian siglec-4. Furthermore, the existence of ITIMs in fish siglec-4 indicates that ITIMs may be an ancestral feature of siglecs, which persisted in most siglecs (Figure 4B) but was lost later during the evolution of siglec-4 at least in mammals and birds.
This brings up the question of whether siglec-4 represents the common ancestor also of immune system–related siglecs. In this respect it is interesting that we could identify several additional potential siglec genes in both the fugu and the zebrafish genomes representing additional candidates for such a common ancestor. However, the low degree of sequence similarities does not allow a reasonable phylogenetic analysis. Additional insight in this topic could be expected to come from the analysis of other deuterostomian genomes, for example, from amphibians, reptiles, birds, or nonvertebrate deuterostomes evolutively older than fish. If compared with the much higher degree of conservation found for the enzymes of the Sia metabolism, the results of this study suggest that the siglec representatives known to date are relatively new inventions in Sia evolution. In this context, our observation that siglec-4 is the only siglec known to date that shows a high conservation from fish to mammals is of particular interest. This indicates a high evolutive pressure for this protein suggesting an indispensable role for siglec-4 in the maintenance of a nervous system with myelinated axons. In contrast, other putative siglecs apparently maintained a high degree of structural and possibly functional flexibility during evolution. It may be speculated that this reflects the adaptive potential of the immune system and its flexibility during evolution.
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Materials and methods |
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Fugu brain RNA was obtained from MRC Gene Service (Cambridge, UK). Tissue culture media were obtained from Invitrogen. Unless otherwise specified, all other reagents were purchased from Sigma (Munich, Germany).
Homology searches for siglecs in fish and frog genomes
The sequences of human and murine siglecs were used as templates in homology searches of T. rupripes whole genome shotgun assembly version 2.0 at the DOE Joint Genome Institute (JGI) Web site (Aparicio et al., 2002), the Ensembl Zebrafish Genome Browser at the Sanger Institute, and JGI X. tropicalis Web site using the tblastn program. Scaffolds showing e-values below 10–5 were assembled using VectorNTI and analyzed for putative protein-coding sequences using the GENSCAN Web Server at the MIT Department of Biology (Burge and Karlin, 1997). The multiple protein sequence alignment was constructed by VectorNTI using ClustalW algorithm (Thompson et al., 1994) and adjusted manually if necessary to accommodate structural restrictions, such as the positions of conserved cystein residues and predictable ß-strands. Protein sequences of putative siglec-4 orthologs were analyzed using the ExPASy Molecular Biology Server, the superfamily1.63 HMM library and genome assignments server, and the prediction servers available at the CBS home page. Genomic structures were deduced from comparison of cDNAs with genomic DNA sequences.
Molecular cloning of a full-length cDNA encoding fugu siglec-4
To obtain 5'-ends of the cDNA, RACE-PCR was performed using the GeneRacer Kit (Invitrogen, Karlsruhe, Germany) and gene-specific primer 5'-TAGCCATTGTCTCCGACGCAAGTATAGA-3'. To obtain the entire coding region, 5'-end and 3'-ends were first amplified separately by PCR using the primer sets 5'-GCTCTAGAGTGGAAACCATGTGGTGTTT-3', complementary to nucleotides 1–20 (Figure 2) and 5'-TAGCCATTGTCTCCGACGCA AGTATAGA-3', complementary to nucleotides 914–941 for the 5'-end; 5'-TCAAGTATGCTCCTCGCTC TGTG-3', complementary to nucleotides 716–738 and 5'-CCGGATCCGGGCCCTCATTTGGCTTTGATTTCCCTATAGTTG-3', complementary to nucleotides 1875–1902, for the 3'-end.
Fugu brain cDNA generated using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) was used as template. The PCR was performed as follows: 94°C for 2 min, 25 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 90 s, and 72°C for 2 min. The resulting 5'-fragment was digested with XbaI and XhoII, the resulting 3'-fragment with XhoII and BamHI and dephosphorylated with shrimp alkaline phosphatase (Fermentas). Both fragments were ligated into XbaI/BamHI cut pcDNA 3.1 vector (Invitrogen) and sequenced.
Cell adhesion assays
Fugu siglec-4/pcDNA 3.1 constructs were transfected using ExGen 500 in vitro transfection reagent (Fermentas) into COS-7 cells. Erythrocyte adhesion assays were performed 48 h after transfection, with or without Vibrio cholerae sialidase (Dade Behring, Liederbach, Germany) pretreatment of COS-7 cells or erythrocytes as described previously (Kelm et al., 1994).
Production of recombinant fish siglec-4-Fc
A cDNA fragment encoding the three N-terminal Ig-like domains of fugu siglec-4 was amplified by PCR using 5'-GCTCTAGAGTGGAAACCATGTGGTGTTT-3' (nucleotides 1–20) and 5'-CCGGATCCACTTACCTGTCTTGACCGCCAGATACATGGAGGT-3' (complementary to nucleotides 955–978) as primers and fugu siglec-4/pcDNA 3.1 as template. The corresponding fragment coding for the three N-terminal domains of zebrafish siglec-4 was cloned using primers 5'-GCTCTAGAATGAAGGGCTTAGAGCTGCT-3' and 5'-CCGGATCCACTTACCTGTATTTACTGCCAGATACAT-3' and zebrafish brain cDNA as template. The fish siglec-4d1–3 fragments were digested with XbaI and BamHI and ligated to XbaI/BamHI cut pIgBOS vector (van der Merwe et al., 1995). Sequences of both inserts were verified by sequencing. The constructs leading to soluble proteins were transfected as described, culture supernatants were collected, and the chimeras were purified on protein A–Sepharose (Amersham Biosciences, Freiburg, Germany) as described previously (Crocker and Kelm, 1996).
Northern blot analysis
Total RNA was prepared from different carp (Cyprinus carpio) tissues using TriFast reagent (Peqlab, Erlangen, Germany) following the manufacturer's recommendations. Northern blots were prepared as described (Gieselmann et al., 1989) using 7 µg RNA per lane. Probes for carp siglec-4 domains 1 and 2 were prepared by nested PCR using carp brain cDNA, generated as described, as template and the following primers: 5'-CAGTGGAATGTGTGGATGCCN-3' (sense primer 1), 5'-ATTTCAGCCATGACAAACTCCTN-3' (sense primer 2), 5'-GGGGCAGG ATTACTGTCTACATCACN-3' (antisense primer 1) and 5'-GTGTTTGGGAAGTTGACCCGGCAACCCN-3' (antisense primer 2) giving a product of the expected size (587 base pairs) that was subcloned into pCR 2.1-TOPO (Invitrogen) and sequenced. Hybridization was performed using ULTRAhyb reagent (Ambion, Lund, Sweden) following the manufacturer's recommendations.
Binding specificity of fish siglec-4
GlycoWell plates with covalently linked Neu5Ac were used for siglec-4 binding studies (SW-01–004; Lundonia Biotech, Lund, Sweden). As negative control wells were treated with 150 µl 10 mM sodium periodate in 0.1 M sodium acetate pH 5.5 for 1 h at 4°C, rinsed with H2O, incubated for 30 min at 4°C with sodium borohydride (5 mg/ml), and thoroughly washed with H2O. Purified siglec-4 Fc-chimeras (0.5 µg/ml final concentration) were complexed with affinity-purified AP-conjugated anti-human IgG (0.3 µg/ml final concentrations; Dianova, Hamburg, Germany) and incubated in the presence of 0–5 mM 2,3- or 2,6-sialyllactose in the GlycoWell plates (20 µl/well). After an incubation for 4 h at 4°C, the wells were washed with HBS/0.05% Tween-20 (5 x 200 µl). Bound AP was quantified kinetically with 15 µM fluorescein diphosphate (50 µl/well, MoBiTec, Gottingen, Germany) in 50 mM Tris–HCl pH 8.5/10 mM MgCl2 as substrate (excitation at 485 nm, emission at 520 nm; Fluoroskan Ascent SL, Thermo Labsystems, Dreieich, Germany). Assays were performed in duplicate and repeated at least three times. Inhibition curves were calculated using the ligand module of SigmaPlot (SPSS, Chicago, IL) assuming a single binding site for the inhibiting oligosaccharides.
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Acknowledgements |
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We thank Nicolai Bovin for the kind donation of sialyllactose-PAA-derivatives and Nazila Isakovic for technical assistance. The financial support by Zentrale Forschungsförderung der Universität Bremen and Volkswagen Foundation is acknowledged.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: flehmann{at}uni-bremen.de
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Abbreviations |
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Ig, immunoglobulin; IgSF, immunoglobulin superfamily; ITIM, immunoreceptor tyrosine-based inhibitory motif; MAG, myelin-associated glycoprotein; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; Sia, sialic acid; siglecs, sialic acid–binding immunoglobulin-like lectins; Sn, sialoadhesin
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References |
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