Glycobiology Research and Training Center, Department of Medicine and Cancer Center, University of California San Diego, La Jolla, CA 92093, USA
Received on October 6, 1999; revised on November 5, 1999; accepted on November 8, 1999.
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Abstract |
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Key words: receptors/Siglecs/sialic acids/lectins/Ig superfamily
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Introduction |
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Assignment of specific functions to the Sia binding phenotype of the Siglecs is complicated, because these binding sites are often masked by endogenous ligands (Braesch-Andersen and Stamenkovic, 1994; Freeman et al., 1995
; Hanasaki et al., 1995b
; Sgroi et al., 1995
; Sgroi et al., 1996
; Collins et al., 1997b
; Tropak and Roder, 1997
), and can be unmasked by sialidase treatment or cellular activation (Razi and Varki, 1998
). Recently, we reported that human blood leukocytes have Sia binding sites that can be unmasked by sialidase treatment (Razi and Varki, 1999
). Such unmasking occurred not only on B cells, monocytes and neutrophils (known to carry Siglecs) but also on natural killer cells and on a minority of mature T cells. The masking of such sites on unactivated cells may explain why many of these lectins have not been previously discovered. Here we report the discovery and characterization of a seventh member of the Siglec family. While this work was being prepared for submission, an apparent splice-variant of the same gene (p75/AIRM1) was independently reported by another group studying natural killer cells and also shown to bind erythrocytes in Sia-dependent manner (Falco et al., 1999
). We are also aware that yet another group has independently cloned the same gene product (P.Crocker, personal communication).
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Results |
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Cloning of a full-length cDNA encoding the putative Siglec-7
Since the clone lacked a coding sequence for the first half of an Ig-like domain, 5'-RACE and RT-PCR were performed, using human PBMC total mRNA as a template (PBMC were chosen based on preliminary Northern analysis using the partial clone). The full-length clone contained an open reading frame encoding 374 amino acids, starting with a typical signal sequence and two Ig-like domains, followed by a probable transmembrane domain and cytosolic tail (Figure 1). The cytosolic tail of the full-length protein contains two tyrosine residues, of which the first is contained in an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM; S/I/L/VxYxxL/V). The second is in a motif [NEYSEI] similar, but not identical to the binding site on SLAM (Signaling Lymphocyte Activating Molecule) for SLAM-associated protein (SAP) (Sayos et al., 1998). Nucleotide identity with other human Siglecs (first 750 nucleotides) are: Siglec-1, 45.9%; Siglec-2, 48.0%; Siglec-3, 63.6%; Siglec-4, 45.4%; Siglec-5, 64.0%; Siglec-6, 63.0%, and many of the residues typical of Siglecs are conserved (Figure 2a). Siglec-7 formed a closely related cluster with Siglecs 3, 5, and 6 on an unrooted phylogram (Figure 2b).
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Discussion |
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The Sia-binding properties of Siglec-7 are interesting. Among the potential targets studied, the preferred recognition motif is Neu5Ac26Galß14Glc, which is very similar to the naturally occurring Sia
26Galß14GlcNAc sequences of N-glycans in vertebrate systems. This motif is also recognized by Siglec-5 (Cornish et al., 1998
), and is the preferred ligand for Siglec-2 (Powell et al., 1993
; Powell and Varki, 1994
). This may also explain why it was not necessary to treat Siglec-7 transfected COS cells with sialidase to unmask the binding siteCOS cells have low endogenous levels of this structure. As with most other Siglecs, the actual binding affinity is likely to be low, and detection of specificity is achieved by the presentation of the cognate structures in multivalent arrays. While erythrocytes give robust Sia-dependent binding, this also represents an artificial situation. In the in vivo situation, erythrocytes are constantly bathed in plasma proteins that carry at least ~1 mM concentration of glycosidically linked Sia
26Galß14GlcNAc sequences (Hanasaki et al., 1995a
). Thus, the location and nature of the natural sialylated ligands of Siglec-7 remain to be determined.
Site-directed mutagenesis studies of Siglec-1 and Siglec-2 identified many residues in the amino-terminal V-set domain that were suggested to be involved in recognition of sialylated ligands (Van der Merwe et al., 1996; Vinson et al., 1996
). However, the subsequently obtained crystal structure of Siglec-1 in complex with sialyllactose indicated that most of these effects had been due to a general disruption of protein folding (May et al., 1998
). However, one Arg residue (R97) that was originally mutated and led to loss of binding (Crocker et al., 1999
) was in fact found to form a critical salt bridge with the carboxylate of the bound Sia (May et al., 1998
). Mutation of the corresponding Arg residue in Siglec-2 (Van der Merwe et al., 1996
), Siglec-3 (Taylor et al., 1999
), and Siglec-4a (Tang et al., 1997
) also resulted in loss of binding. This Arg residue is conserved among all the remaining Siglecs reported to date (see Figure 2a). We therefore conservatively mutated this residue in Siglec-7 to a Lys residue, giving a complete loss of binding to erythrocytes, and to semi-synthetic ligands. Thus, one can predict that this Arg residue is critical to the binding of Sias by all Siglecs. This also allows us to suggest an in vivo approach to explore the function of the sialic binding properties of Siglecs. Partial or wholesale deletion of a Siglec molecule in the intact mouse is informative, but the results can be difficult to interpret, because more than just the sialic-acid binding property is eliminated. We suggest instead a "knock-in" strategy, where the only change made would be the mutation of the critical Arg residue of a given Siglec into a Lys. This would leave the Siglec with all of its domains intact, but lacking only its Sia binding site. Such an experiment should be more informative with regard to the function of Sia recognition.
Regarding the biological significance of Sia recognition, the best studied example is Siglec-2. Genetic elimination in the intact mouse gives a phenotype of B cell dysregulation (O'Keefe et al., 1996; Otipoby et al., 1996
; Sato et al., 1996
; Cornall et al., 1998
). Genetic disruption of the ST6Gal I sialyltransferase that generates the CD22 ligand (Sia
26Galß14GlcNAc) also gives suppression of B cell function, which is however more severe than that obtained by disruption of CD22 itself (Hennet et al., 1998
). This may now be explained by the fact that there are other Siglecs (3, 5, and 7) that can recognize the ST6Gal I product. The functions of Siglec-1 remain unclear, although it may be involved in macrophage interactions with developing myeloid cells (Crocker et al., 1990
) and/or cellular trafficking (Shi et al., 1996
). Regarding Siglec-4a, genetic disruption gives alterations in myelin sheath stability (Filbin, 1995
), which correlates with the Wallerian degeneration seen upon genetic elimination of its cognate ganglioside ligands (Sheikh et al., 1999
).
The biological functions of the CD33-related Siglecs (3, 5, 6, and 7) remain to be elucidated. There is a conserved consensus sequence surrounding the intracellular tyrosine residues of all four of these molecules [E(I/L)xYAxL(1218 residues)(T/N)EYSE(I/V)(K/R)] suggesting that they may associate with common intracellular signal transducers in their respective cell types. Indeed, as with Siglec-2 and 4a, the cytoplasmic tail of Siglec-3 is tyrosine-phosphorylated upon engagement (Taylor et al., 1999). Once the tyrosines are phosphorylated, the first one is part of an ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif; consensus: L/I/V/SxYxxL/V), which is found in many members of the Ig superfamily (including Siglec-2/CD22), and forms a potential docking site for the SHP-1 tyrosine phosphatase. Of note, the second tyrosine-containing motif [(T/N)EYSE(I/V)] is similar to the sequence (TxYxxI/V) that has been reported in SLAM (Sayos et al., 1998
), an immunoregulatory molecule of the Ig superfamily. This motif is the docking site in SLAM for an SH2-containing molecule called SAP (SLAM-associated protein), which blocks recruitment of the tyrosine phosphatase SHP-2 to its docking site in the SLAM cytoplasmic region. We have postulated that a similar interplay occurs between the recruitment of phosphatases to the ITIM motif in Siglecs and the presence of SAP or SAP-like inhibitors to the SAP binding motif (Patel et al., 1999
).
Given the combination of an extracellular Sia binding site and an intracellular ITIM motif, it is reasonable to suggest that Siglec-7 is involved in transmembrane regulatory signaling of the types mentioned above. In this regard, a paper has just appeared reporting what appears to be the identical gene (the major cDNA reported has an additional extracellular C2-set Ig domain) (Falco et al., 1999). These authors isolated this cDNA based on a monoclonal antibody directed against human natural killer cells, and showed that the protein negatively regulates the natural killer cell activity upon cross-linking by antibody. They also showed that the protein, when expressed on COS-7 cell, binds erythrocytes in Sia-dependent manner. While the relationship between Sia-binding at extracellular domain and signal transduction via intracellular domain of Siglec-7 is yet the be established, our recent finding that Sia binding sites on peripheral blood natural killer cells are masked by endogenous Sias (Razi and Varki, 1999
) is of note in this context. Thus, if Siglec-7 indeed serves as an inhibitory receptor on natural killer cells, the Sias that regulate the signaling could be either on the same cell surface as the Siglec, or on another cell type. It also remains to be seen if Siglec-7 is expressed and functionally important on other cell types outside the hematopoietic system.
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Materials and methods |
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Cloning and mutation of a full-length cDNA for Siglec-7
RT-PCR was performed using primers based on the DNA sequence of the EST clone and 5' RACE product. First strand cDNA was synthesized by reverse transcription of 1 µg of PBMC total RNA with random DNA hexamers as primer. The reaction product was subjected to two rounds of PCR using Pwo DNA polymerase (Roche), using SLX-5'UTR (5'-CTCGGATCCCTGGCACCTCTAACCC-3') and SLX-3'UTR (5'-GGTCTAGAACCCTCAAACAAGCCC-3') as primers. The PCR products were digested with BamHI and XbaI, ligated to BamHIXbaI sites of pBluescript II KS(-) (Stratagene) and sequenced. A point mutation converting an Arg residue (R124) to Lys was introduced using QuickChange Site-Directed Mutagenesis Kit (Stratagene), following the manufacturers protocol.
Northern blot analysis
A human 12-lane Multiple Tissue Nothern Blot (Clontech) was probed with the NcoIXhoI fragment of the EST clone labeled with the Strip-EZ DNA kit (Ambion) and [-32P]dATP (NEN). Hybridization signals were visualized using a Phosphor Imager.
Phylogenetic analysis of Siglecs
DNA sequences of human Siglecs 17 encoding the first two Ig-like domains (750 nt; the human Siglec-1 sequence was kindly provided by P.Crocker, Dundee, UK) were aligned by Clustal W at the European Bioinformatics Institute web site, and also subjected to phylogenetic analysis using PAUP* 4.0 (Sinauer Associates). The phylogenetic tree was constructed using the neighbor joining method (Saitou and Nei, 1987). The distance matrix was based on Tamura-Nei genetic distances (Tamura and Nei, 1993
).
Expression of full-length Siglec-7 on COS-7 cells and erythrocyte rosetting
The full-length coding sequence of Siglec-7 (wild-type and R124K mutant) was amplified by PCR using the primers SLX 5'Chi (5'-CCTGTCGACGCCACCATGCTGCTGCTGCTGCTGCTGCCC-3') and SLX 3'UTR. The PCR fragment was treated with SalI and subcloned into the XhoIEcoRV sites of pcDNA3.1(-) (Invitrogen), and sequenced. Constructs were transfected using LipofectAMINE reagent (Life Technologies) into COS-7 cells. After 48 h, cells were washed twice with PBS, treated with or without 10 mU Arthrobacter ureafaciens sialidase at 37°C for 1 h, and washed three times with rosetting assay solution (DMEM with 0.25% bovine serum albumin). Human erythrocytes (0.25% v/v; also pretreated with or without sialidase) in rosetting assay solution were layered on the COS-7 cells and incubated at 37°C for 30 min. Unbound erythrocytes were gently washed away, and rosettes observed under a microscope.
Production of recombinant chimeric proteins (Siglec-7-Fc)
A DNA fragment of Siglec-7 (wild-type and R124K) encoding the first two Ig-like domains was amplified by PCR using SLX 5'Chi and SLX 3'Chi (5'-CCTCATTTTGCCTGTGTACTCCTG-3') as primers. The fragment was cloned into the expression vector EK-Fc-pEdDC (prepared in this laboratory by Hui-ling Han), giving rise to a fusion protein of Siglec-7 extracellular domains and a human IgG Fc tail with a FLAG epitope tag (DYKDDDDK) in between. The constructs were transfected using LipofectAMINE into COS-7 cells or CHO-TAg cells, culture supernatants collected, and the chimeric proteins purified on Protein ASepharose (Amersham Pharmacia Biotech).
Binding specificity of Siglec-7
Binding to sialylated oligosaccharides on polyacrylamide arrays (Glycotech) was performed as described (Patel et al., 1999). Briefly, microtiter plate wells (Nunc, catalog #269620) were coated with protein A (0.5 µg/well) in 50 mM sodium carbonate-bicarbonate buffer, pH 9.5, at 4°C overnight, washed three times with ELISA buffer (20 mM HEPES, 1% bovine serum albumin, 125 mM NaCl, 1 mM EDTA, pH 7.45), blocked with ELISA buffer (RT, 1 h), and sequentially incubated at RT with the following (each incubation followed by 3 washes with ELISA buffer): Siglec-7-Fc (0.5 µg/well, human gamma globulin as negative control), 2 h; probes (1 µg/well), 2 h; streptavidin-conjugated alkaline phosphatase (1/1000 diluted from stock solution, Life Technologies), 1 h. After a final wash, p-Nitrophenyl Phosphate Liquid Substrate (Sigma) was added, incubated at RT for varying times (product formation was linear up to 18 h), and absorbance measured at 405 nm.
Mild periodate treatment of probes
The polyacrylamide probe carrying Neu5Ac26Galß14Glc was subjected to mild periodate treatment, to truncate the glycerol-like side chain of Sia (Van Lenten and Ashwell, 1971
). Typically, 10 µg of the probe was incubated in 100 µl of 2 mM NaIO4 in PBS for 30 min on ice in the dark. After the incubation, 100 µl of 20 mM NaBH4 in PBS was added to the mixture and further incubated at RT for 1 h in the dark, to reduce aldehydes generated by periodate treatment. The mixture was diluted with 800 µl ELISA buffer and directly used in the assay.
Chromosomal localization of Siglec-7
From the physical map of human chromosome 19 (Human Genome Center, Laurence Livermore National Laboratory), BAC clones contiguously covering the region of chromosome 19 containing the Siglec-3/CD33 gene were identified and obtained through Research Genetics. The BACs were prepared by a modified alkaline lysis method (BACPAC Resources, Rosewell Park Cancer Institute), and subjected to PCR using SP1 and SP-5' (5'-AAACTCGGGACCGATTCCAC-3') as primers. PCR products were purified and directly sequenced.
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Acknowledgments |
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Abbreviations |
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Note added in proof |
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Footnotes |
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References |
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