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* Division of Cell Biology and Immunology, The Wellcome Trust Biocentre, University of Dundee, Dundee, United Kingdom; and
Centre d’Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique-Institut National de la Sante et de la Recherche Médicale-Université de la Méditerranée, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ITIMs have been found in the cytoplasmic tail of many inhibitory receptors, such as CD158/killer cell Ig-like receptor (KIR), Ly49, and CD85/Ig-like transport/leukocyte inhibitory receptor molecules expressed on various hemopoietic cells including monocytes and NK cells (reviewed in Refs. 7 and 8). ITIMs were functionally defined as specific amino acid sequences that, once tyrosine-phosphorylated, provide a docking site for the Srchomology 2 (SH2)-domain bearing cytoplasmic phosphatases (8). ITIM-related motifs have also been described in many other inhibitory receptors (9). Their amino acid sequences differ from the consensus ITIM sequence by the presence of a threonine in position –2 or an isoleucine in position + 2. For some of the CD33-related Siglecs (CD33, Siglecs-5, -6, and -9), the membrane-distal motif (TEYSEI/VK/R) is similar to the immunoreceptortyrosine-based switch motif (ITSM) TxYxxI/V (10, 11) found in CD150/signaling lymphocyte activation molecule (SLAM), CD66/carcinoembryonic Ag cell adhesion molecule, leukocyte-associated Ig-like receptor-1 (LAIR-1), and CD31/PECAM-1 molecules (10, 12). The discovery of CD33-related Siglecs and the presence of these two tyrosine motifs raise the possibility that these proteins are involved in regulating cellular activation within the immune system.
Siglecs-7 and -9 share more than 80% sequence identitythroughout the extracellular, transmembrane, and intracellular regions (13) but differ in their expression pattern (1). Siglec-7 is highly expressed on NK cells and weakly on monocytes and a minor subpopulation of CD8 T lymphocytes (14, 15). In contrast, Siglec-9 is broadly expressed with high expression on monocytes and low-level expression on neutrophils and subpopulations of NK, B, and T cells (13). They also differ in sialic acid binding specificity: Siglec-7 possesses a unique preference for 
(2,8)-linked disialic acids and branched (2,6)-linked sialic acids, whereas Siglec-9 does not bind to (2,8)-linked disialic acids but prefers terminal (2,3)- and (2,6)-linkages (16, 17). Regardless of the role of sialic acid recognition on their function, Siglec-7 acts as an inhibitory receptor in human NK cells after engagement by Abs (14) or binding with sialic acid-containing ligands (18) and once phosphorylated can recruit the SH2 domain-bearing protein tyrosine phosphatase (PTP) SHP-1 (14). Anti-Siglec-7 Abs also inhibit the proliferation of myeloid cells (19). Concerning the function of Siglec-9, there have been no reports so far, although its high similarity with Siglec-7 strongly suggests Siglec-9 has a similar inhibitory function.
In this study, we demonstrate for the first time that Siglec-9 acts as an inhibitory receptor using the rat basophilic leukemia (RBL) model, which has been widely used to analyze KIR functions in particular. We show that Siglec-9, as well as Siglec-7, can inhibit FcRI-mediated serotonin release of RBL cells and recruit the tyrosine phosphatases SHP-1 and SHP-2. We then show that the membrane-proximal ITIM is essential for the inhibitory activities of Siglecs-7 and -9, for their tyrosine phosphorylation, and for the recruitment of the PTPs SHP-1 and SHP-2. Furthermore, mutation of the membrane-proximal motif leads to an increase in their RBC binding ability, indicating that signaling involving SHP-1 and SHP-2 may modulate sialic acid recognition mediated by Siglecs-7 and -9.
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Materials and Methods |
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Unless otherwise specified, all reagents and chemicals were purchased from Sigma-Aldrich (Poole, U.K.). [3H]Serotonin (hydroxytryptamine creatine sulfate, 5-[1,2-[3H](N)]) was obtained from NEN (Hounslow, U.K.). Vibrio cholerae sialidase was obtained from Calbiochem (Nottingham, U.K.).
The cDNAs encoding human Siglecs-7 and -9 were described in Refs. 13 and 15). Site-directed mutagenesis of Siglecs-7 and -9 cDNAs was performed with sets of mutagenic primers (Table I) using the QuickChange kit (Stratagene, Amsterdam, The Netherlands) according to the manufacturer’s instructions. The tail deletion form of Siglecs-7 (7
) and -9 (9) were obtained by PCR using sets of primers described in Table I and cloned into the pcDNA3 vector (Invitrogen, Paisley, U.K.). The cDNA-encoding human tyrosine phosphatases SHP-1 and SHP-2 were a kind gift from Benjamin Neel (Harvard Medical School, Boston, MA) and were cloned into the pEGFP-N1 vector (Clontech, Basingstoke, U.K.). Catalytically inactive forms of SHP-1 (C453S, mutSHP-1) and SHP-2 (C459S, mutSHP-2) were generated as above using sets of mutagenic primers described in Table I. All the PCRs were performed using PFU Turbo DNA polymerase (Stratagene), and the presence of introduced mutations was confirmed by DNA sequencing.
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Cells
The RBL-2H3 cells expressing KIR2DL3 (20) were cultured in DMEM supplemented with 10% FCS, penicillin (100 IU/ml), and streptomycin (100 µg/ml). Stable cell lines expressing wild-type (WT) and mutant forms of Siglecs-7 and -9 were generated by electroporation and selection with geneticin at 1 mg/ml (G418) (Roche, East Sussex, U.K.).
Flow cytometry
All incubations were conducted on ice. Cells were incubated with primary mAbs (10 µg/ml) for 30 min, washed, and incubated with the FITC-conjugated rabbit anti-mouse Ig F(ab')2 for 30 min. Cells were analyzed using a FACSCalibur (Becton Dickinson, Oxford, U.K.).
Serotonin release assay
The serotonin release assay was performed as described in Ref.20 . Briefly, [3H]serotonin-labeled RBL cells were incubated with mouse IgE (at 0.1 µg/ml) in the absence or presence of various amount of anti-MHC class I molecules (OX-18), anti-Siglec-7 (S7.5a), anti-Siglec-9 (K8), or anti-KIR2DL3 (GL183) mAbs. After washing, cells were incubated for 30 min at 37°C with goat anti-mouse IgG F(ab')2 (at 50 µg/ml). Supernatants were collected, and the released radioactivity was analyzed using the WinSpectral liquid scintillation counter (Wallac, Milton Keynes, U.K.). Each assay was set up in triplicate, and the results were expressed as a percentage of specific serotonin release: (cpm test – cpm spont)/(cpm max – cpm spont) x 100, where cpm spont is the spontaneous release of [3H]serotonin obtained with cells incubated in absence of Abs and cpm max is the maximum release of [3H]serotonin obtained with cells lysed using Triton X-100. For comparative analysis, results were expressed as a mean of the percentage of inhibition of serotonin release: 100 – (% test x 100/% IgE) obtained in experiments performed with n different clones, where % IgE is the percentage of specific serotonin release obtained with cells incubated with mouse IgE alone.
Immunoprecipitation and immunoblotting
Immunoprecipitation and immunoblotting were performed as previously described (21). Briefly, cells were treated or not with pervanadate (0.5 mM) for 10 min at 37°C. Pre-cleared cell lysates were immunoprecipitated with S7.5a (anti-Siglec-7) or K8 (anti-Siglec-9) mAbs and then analyzed by immunoblotting with sheep anti-Siglecs-7 or -9 polyclonal Abs (PAbs), with 4G10 mAb anti-phosphotyrosine or with rabbit PAbs C-19 anti-SHP-1 or C-18 anti-SHP-2.
Fluorescence microscopy
Siglecs-7 and -9 RBL clones were transiently transfected with SHP-1-GFP, SHP-2-GFP, mutSHP-1-GFP, or mutSHP-2-GFP cDNA by electroporation and cultured overnight on coverships (BDH, Lutterworth, U.K.). Cells were then stained with anti-Siglec-7 or anti-Siglec-9 sheep PAbs (10 µg/ml) for 30 min at 4°C and incubated for 30 min at 37°C with tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-sheep Ig F(ab')2 to generate patched Siglecs. Cells were then treated or not with pervanadate (0.5 mM) for 10 min at 37°C, fixed with 4% paraformaldehyde, and analyzed by fluorescence microscopy. For the FcRI-mediated activation of RBL cells, GFP-transfected cells were stained with mIgE (0.1 µg/ml) and S7.5a or K8 mAbs (10 µg/ml) for 30 min at 4°C, incubated with goat anti-mouse Ig F(ab')2 (50 µg/ml) for 30 min at 37°C and then labeled with anti-Siglec-7 or anti-Siglec-9 sheep PAbs (10 µg/ml) and TRITC-conjugated rabbit anti-sheep Ig F(ab')2 (10 µg/ml) for 15 min at 37°C to create clusters. Images were acquired using the AxioVision imaging system (Imaging Associates, Bicester, U.K.) and a Zeiss immunofluorescence microscope (Jena, Germany).
RBC binding assays
RBC binding assays were performed as described in (13). Briefly, RBL cells were treated or not with sialidase for 1 h at 37°C, washed, and incubated with human RBC for 1 h at 4°C. Unbound RBC were gently washed, and cells were fixed with 0.25% glutaraldehyde and rosetting assessed by microscopy. To quantify binding, the percentage of RBL cells forming rosettes (defined as RBL cells binding >5 RBC) was scored from counting at least 200 RBL cells per field in five different fields per experiment. Results are expressed as a mean percentage, obtained from three independent experiments.
Statistics
Values represent the mean ± SD of n different experiments. Student’s t test was applied using a two-tailed distribution of two samples of equal or unequal variances.
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Results |
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RI-mediated serotonin release in RBL cells
It has been shown previously that Siglec-7 acts as a NK cell inhibitory receptor in cytotoxicity assays (14, 18). In contrast, there have been no reports on the inhibitory function of Siglec-9. To investigate the potential inhibitory functions of Siglecs-7 and -9 in parallel, we used the well-defined RBL model in which co-engagement of inhibitory receptors and the activatory receptor, FRI, inhibits serotonin secretion by RBL cells incubated with mIgE. We generated RBL cell lines stably transfected with Siglec-7 or -9 cDNA (designated RBL-7WT and RBL-9WT, respectively) using parental RBL-2H3 cells that had already been transfected with the well-characterized inhibitory receptor KIR2DL3. The expression of KIR2DL3 provided a useful internal reference control in the inhibition assays. As shown in Fig. 1A on a representative clone, expression of Siglec-7 or -9, KIR2DL3, MHC class I (used as a negative control in the inhibition assays), and FcRI molecules assessed by binding of IgE was demonstrated by flow cytometry analysis (Fig. 1A). RBL-7WT and -9WT clones were then incubated with a range of dilutions of mAbs anti-MHC class I molecules (irrelevant mAb), anti-KIR2DL3 (positive control), anti-Siglec-7 or anti-Siglec-9 in the presence of mIgE and used in a serotonin release assay. As described previously (20), cross-linking of KIR2DL3 with FcRI dramatically reduced the level of serotonin release of RBL-7WT and -9WT cells in a dose-dependent manner, whereas no significant effect was observed in the presence of an irrelevant mAb that bound MHC class I molecules (Fig. 1B). In contrast, cross-linking of Siglecs-7 and -9 substantially reduced the serotonin release of RBL-7WT and -9WT cells (Fig. 1B). These results shows that Siglec-9 is as potent as Siglec-7 in delivering inhibitory signals to RBL cells.
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To address the question of whether Siglecs-7 and -9 can recruit the phosphatases SHP-1 and SHP-2 under conditions of activation in RBL cells, we transfected RBL-7WT and -9WT cells with SHP-1-GFP or SHP-2-GFP cDNA and performed co-localization experiments using fluorescence microscopy. Transfected cells were stained with anti-Siglec-7 or -9 sheep PAbs and then incubated with TRITC-conjugated rabbit anti-sheep Ig F(ab')2 for 1 h at 37°C to create patches of Siglec-7 and -9. The cells were then treated or not with pervanadate to inhibit tyrosine phosphatases and increase phosphorylation, fixed, and analyzed. As shown in Fig. 2, no co-localization of Siglec-7 or -9 with SHP-1 or SHP-2 was observed with untreated cells, whereas SHP-1 and SHP-2 co-localized with Siglecs-7 and -9 when the cells were treated with pervanadate. To investigate if the co-localization occurs in a more physiological situation, mIgE and anti-Siglec-7 or -9 mAbs were cross-linked using GFP-transfected cells in a similar way as in the inhibition assay, and then clusters were created by adding sheep PAbs anti-Siglec-7 or -9 followed by TRITC-conjugated rabbit anti-sheep Ig F(ab')2. As shown in Fig. 2, SHP-1 and SHP-2 co-localized with Siglecs-7 and -9 when the cells were activated via engagement of the FcRI receptor.
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To investigate SHP-1 and SHP-2 association biochemically, parental RBL, RBL-7WT, and -9WT cells were treated or not with pervanadate and Siglec-7 or -9 immunoprecipitates from RBL lysates were then analyzed by immunoblotting with anti-Siglec-7 or -9 PAbs, anti-phosphotyrosine mAb, anti-SHP-1, or anti-SHP-2 PAbs (Fig. 3). As expected, specific bands of 
77 and 78 kDa corresponding to Siglecs-7 and -9, respectively were observed with untreated and pervanadate-treated RBL-7WT and -9WT lysates, whereas no signal was detectable with parental RBL lysates (Fig. 3). Tyrosine-phosphorylated Siglecs-7 and -9 and co-immunoprecipitated SHP-1 and SHP-2 were readily detectable in pervanadate-treated RBL-7WT and -9WT lysates, but no signal was seen with untreated RBL-7WT, -9WT, or the parental RBL lysates (Fig. 3).
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The membrane-proximal tyrosine motif is necessary for the inhibitory function, tyrosine phosphorylation, and recruitment of the PTPs SHP-1 and SHP-2 by Siglecs-7 and -9
To examine the roles of individual tyrosine motifs within the cytoplasmic tail of Siglecs-7 and -9 on the inhibitory function of these molecules, we generated tyrosine to phenylalanine mutants in both the membrane-proximal and membrane-distal tyrosine motifs either individually or in combination (Table II). Truncated forms of Siglecs-7 and -9 in which the cytoplasmic tails were deleted were also used (Table II). RBL clones expressing similar levels of Siglec-7 or -9 were selected for comparative analysis in inhibition assays (Fig. 4A). Comparable levels of expression of MHC class I molecules, KIR2DL3, and FcRI were also confirmed by flow cytometry (Fig. 4A). Using RBL-7Y2 and -9-Y2 cells in which the proximal tyrosine motif only was retained, strong inhibition was still observed following co-cross-linking of Siglecs-7 and -9 with FcRI (Fig. 4, B and C). In contrast, mutation of the membrane-proximal motif in RBL-7Y1, -7Y1Y2, -9Y1, or -9Y1Y2 or truncation of the cytoplasmic tail resulted in a complete loss of inhibition (Fig. 4, B and C). These results demonstrate that the membrane-proximal tyrosine motif is both necessary and sufficient to mediate the inhibitory function of Siglecs-7 and -9.
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, RBL-9Y1Y2, and RBL-9 lysates (Fig. 3). In addition, mutation of the membrane-proximal tyrosine motif completely abrogated the tyrosine phosphorylation, whereas substantial phosphorylation was retained with the mutation of the membrane-distal motif (Fig. 3). Concerning the recruitment of the PTPs, all mutations completely abrogated the recruitment of SHP-1, whereas only the membrane-distal mutation could still recruit significant levels of SHP-2 (Fig. 3). To confirm our findings in co-localization experiments, Siglecs-7 and -9 RBL mutants were transfected with mutSHP-1-GFP and mutSHP-2-GFP and patches of Siglec-7 or -9 were created. Cells were then fixed and analyzed by fluorescence microscopy. No co-localization of Siglec-7 or -9 with mutSHP-1 was observed with any of RBL mutants (data not shown). However, in approximately one-third of RBL-7Y2 or RBL-9Y2 cells, Siglecs-7 and -9 clearly co-localized with mutSHP-2 (Fig. 5), whereas no association was observed with the other Siglecs mutants (Fig. 5). These results demonstrate that the membrane-proximal tyrosine motif is essential for the tyrosine phosphorylation of Siglecs-7 and -9 and the recruitment of the PTPs SHP-1 and SHP-2, whereas tyrosine phosphorylation and association with SHP-2 can still occur after mutation of the distal-motif of Siglecs-7 and -9.
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As members of the Siglec family, Siglecs-7 and -9 have the ability to recognize sialic acid residues, either in cis on the Siglec-expressing cell, or in trans on other cells (reviewed in Ref.1). In a recent study, we have shown that the Siglec-7 binding site is masked on NK cells, but can be unmasked following sialidase treatment to destroy the cis-interacting sialic acids (18). Human RBC are a very convenient indicator of sialic acid-dependent binding of most Siglecs, including Siglecs-7 and -9 (13, 15). To investigate if mutations in the tyrosine motifs affect recognition of sialylated glycans by Siglecs-7 and -9, RBL-7WT, -9WT, and respective mutants were treated or not with sialidase and tested in RBC binding assays. As expected, no rosettes occurred with untreated RBL cells (Fig. 6), suggesting that cis-interactions between Siglec-7 or -9 and sialic acids occurs on the membrane of RBL cells and abrogate any trans-interaction. In contrast, sialidase-treated RBL-7WT and -9WT mediated robust RBC binding activity. A striking increase in binding was observed with Y1 and Y1Y2 mutants, but no effect was observed with Y2 mutants (Fig. 6). These results indicate that the membrane-proximal tyrosine motif modulates the ligand binding activity of Siglecs-7 and -9. One explanation could be that the Y1 and Y1Y2 mutations directly affect the distribution of Siglecs-7 and -9 on the membrane of the cells. To investigate this possibility, RBL-7WT, -7Y1Y2, -9WT, and -9Y1Y2 cells were treated or not with sialidase, fixed, stained with anti-Siglec-7 or -9 mAbs, and analyzed by confocal microscopy. No obvious difference was observed between RBL-7WT or -9WT cells and RBL-7Y1Y2 or -9Y1Y2 cells, respectively (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-mediated activation of RBL cells, is tyrosine-phosphorylated after pervanadate-treatment, and recruits the PTPs SHP-1 and SHP-2 after pervanadate-treatment and FcRI-mediated activation, it fulfills the criteria for inclusion in the inhibitory receptor superfamily (5, 7, 8).
Previous reports on CD33 (22, 23, 24) and mouse Siglec-E (25, 26), demonstrated the importance of the membrane-proximal tyrosine motif in recruitment of SHP-1 and SHP-2 by co-immunoprecipitation, but this study on Siglecs-7 and -9 provides the first analysis of the role of tyrosine-based motifs in the inhibitory function for CD33-related Siglecs. Mutation of the membrane-proximal tyrosine motif that conforms to the consensus ITIM led to a complete abrogation of tyrosine-phoshorylation of the receptors, recruitment of the PTPs SHP-1 and SHP-2, and the inhibition of FcRI-mediated activation of RBL cells. Similar results have been obtained with CD158/KIR molecules that contain two ITIMs, demonstrating that the proximal ITIM is critical for the inhibition of T cell activation by CD158a/KIR2DL1 (27), the FcRI-mediated activation of RBL cells by CD158b/KIR2DL3 (28) or NK cytotoxicity function by CD158b/KIR2DL3 (29), CD158e1/KIR3DL1 (30), and CD158/KIR2DL5 (31). Some of these studies also showed that tyrosine phosphorylation of these receptors and recruitment of the PTPs were dependent on the membrane-proximal motif (28, 29, 30). In addition, mutation of the membrane-proximal ITIM of LAIR-1 molecule, which also has two tyrosine-based motifs, resulted in abrogation of its inhibitory function in NK cytotoxicity using a NK cell line (32), whereas both motifs contributed equally to the inhibition function using RBL cells. Both motifs contributed to the inhibition mediated by NKG2A, which has also two ITIMs, using the RBL model (33), with a dominant role of the membrane-distal motif probably due to the opposite orientation of the ITIMs in type II vs type I transmembrane proteins (33). Interestingly, the fact that tyrosine phosphorylation of Siglecs-7 and -9 did not occur after mutation of the membrane-proximal motif strongly suggests that this motif is the main binding site of tyrosine kinases. However, we cannot exclude the possibility that the anti-phosphotyrosine mAb used is unable to detect the tyrosyl-phosphorylated membrane-distal motif present in the Y1 mutants. Although the identity of the kinase(s) involved remains unknown, it is worth noting that Siglec-3/CD33 can be phosphorylated in vitro and in vivo by Src family kinase c-Src (22) and Lck (24), the membrane-proximal motif playing a key role (24).
We have also shown that mutation of the membrane-distal motif did not affect the inhibitory function of Siglecs-7 and -9. Interestingly, with this mutation, we were still able to observe tyrosine phosphorylation and SHP-2 recruitment using both immunoprecipitation- and fluorescence microscopy-based approaches, whereas SHP-1 was not recruited. Although an involvement of other signaling partners cannot be excluded, it is tempting to speculate that the inhibition observed after mutation of the membrane-distal motif is mediated by SHP-2. Such a mechanism involving SHP-2 rather than SHP-1 in inhibitory signaling has been suggested in a previous study with KIR2DL3 (28) and has been demonstrated recently with KIR2DL4 in RBL cells (34) and KIR3DL1 and KIR2DL5 in NK cells (30, 31). Based on these studies and the work on LAIR-1 (32), it has been proposed that the conserved sequence, VTYAQL, is sufficient for the recruitment of SHP-2 and the inhibitory capacity of the receptor (32). The involvement of SHP-2 rather than SHP-1 in the inhibitory function could also apply to Siglecs-7 and -9, although they have different ITIM sequences (IQYAPL and LQYASL, respectively) compared with the proposed consensus. Studies using peptides from KIR molecules indicated that the position –2 from the tyrosine was critical for SHP-1 and SHP-2 binding (5, 35, 36). Furthermore, a study using peptides from mast cell function-associated Ag, which has a single ITIM, clearly demonstrates that the nature of the amino acid residue present at position –2 is important for differential binding of SHP-1 or SHP-2 (37). Indeed binding of SHP-1 was only observed with the native phospho-peptide SIpYSTL and the mutation serine to valine, whereas binding of SHP-2 was maintained after mutation of the serine to several amino acid residues and in particular to isoleucine and leucine (37), residues present in Siglecs-7 and -9 ITIMs. Taken together, our results support the concept that SHP-1 requires two functional tyrosine-based motifs, whereas SHP-2 tolerates only one ITIM (28, 29, 32).
Another feature of some CD33-related Siglecs is that the membrane-distal motif is similar to the ITSM TxYxxI/V found in CD150/SLAM and SLAM-related molecules (10, 11, 38). It has been shown that the ITSM of CD150 is involved in recruitment of SLAM-associated protein (SAP) or EAT-2 in T and B cells (10, 39, 40, 41). This prevented an interaction with the phosphatase SHP-2 (10, 39, 41, 42) and facilitated the recruitment of the phosphatase SHIP (10, 43) or the Src family kinases FynT (43). Although this issue has not been addressed directly in this study, it is unlikely that this ITSM serves as a docking site for SAP-family adaptor molecules. Indeed Siglec-10, which contains two membrane-proximal ITIMs and one membrane-distal ITSM-like motif, does not bind to the SAP molecule in a three-hybrid system (44).
Finally, we have shown here that mutation of the membrane-proximal ITIM of Siglecs-7 and -9 leads to an increase in the sialic acid-dependent binding of RBC, whereas mutation of the membrane-distal motif has no effect. Similar results have been reported previously with Siglec-3/CD33 using transiently transfected COS cells (22) and may be a general feature of human CD33-related Siglecs. The fact that SHP-2 was only recruited with Y2 mutants, which displayed low levels of RBC binding similar to the WT forms of Siglecs-7 and -9, raised the possibility that interactions of this phosphatase could negatively regulate the ligand binding activity of Siglecs-7 and -9. Binding of sialic acid residues by monomeric Siglecs is generally of very low affinity and therefore clustering of receptors within the plasma membrane is essential for high-avidity interactions with ligands on other cells. Although we failed to see gross changes in distribution of Siglecs-7 or -9 comparing WT and mutant forms of the proteins expressed on transfected RBL cells by confocal light microscopy, there could be subtle changes that would require higher resolution imaging techniques for their visualization.
In conclusion, we demonstrate that Siglec-9 is a new member the inhibitory receptor superfamily and that the membrane-proximal ITIM is essential for the inhibitory function of both Siglecs-7 and -9 molecules.
Note added in proof.
A recent publication demonstrated the inhibitory activity of Siglec-7 and Siglec-9 in transfected Jurkat cells (Ikehara, Y., S. K. Ikehara, and J. C. Paulson. 2004. Negative regulation of T cell receptor signaling by Siglec-7 (p70/AIRM) and Siglec-9. J. Biol. Chem. 279:43117.).
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Acknowledgments |
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Footnotes |
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1 Research in the PRC laboratory is supported by the Wellcome Trust. 
2 Address correspondence and reprint requests to Dr. Paul R. Crocker, The Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. E-mail address: p.r.crocker{at}dundee.ac.uk
3 Abbreviations used in this paper: Siglec, sialic acid-binding Ig-like lectin; ITIM, immunoreceptor tyrosine-based inhibition motif; ITSM, immunoreceptor tyrosine-based switch motif; KIR, killer cell Ig-like receptor; LAIR-1, leukocyte-associated Ig-like receptor-1; MAG, myelin-associated glycoprotein; PAb, polyclonal antibody; PTP, protein tyrosine phosphatase; RBL, rat basophilic leukemia; SAP, SLAM-associated protein; SH2, Src homology 2; SHP, SH2 domain-containing phosphatase; SLAM, signaling lymphocyte activation molecule; TRITC, tetramethylrhodamine isothiocyanate, WT, wild type.
Received for publication July 21, 2004. Accepted for publication September 21, 2004.
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References |
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2,8-disialyl and branched 2,6-sialyl residues. A comparison with Siglec-9. J. Biol. Chem. 277:6324.This article has been cited by other articles:
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  D. J. Song, J. Y. Cho, S. Y. Lee, M. Miller, P. Rosenthal, P. Soroosh, M. Croft, M. Zhang, A. Varki, and D. H. Broide Anti-Siglec-F Antibody Reduces Allergen-Induced Eosinophilic Inflammation and Airway Remodeling J. Immunol., October 15, 2009; 183(8): 5333 - 5341. [Abstract] [Full Text] [PDF]
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 A. F. Carlin, S. Uchiyama, Y.-C. Chang, A. L. Lewis, V. Nizet, and A. Varki Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response Blood, April 2, 2009; 113(14): 3333 - 3336. [Abstract] [Full Text] [PDF]
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 P. Gunnarsson, L. Levander, P. Pahlsson, and M. Grenegard The acute-phase protein {alpha}1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (Siglecs) FASEB J, December 1, 2007; 21(14): 4059 - 4069. [Abstract] [Full Text] [PDF]
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 H. Tateno, H. Li, M. J. Schur, N. Bovin, P. R. Crocker, W. W. Wakarchuk, and J. C. Paulson Distinct Endocytic Mechanisms of CD22 (Siglec-2) and Siglec-F Reflect Roles in Cell Signaling and Innate Immunity Mol. Cell. Biol., August 15, 2007; 27(16): 5699 - 5710. [Abstract] [Full Text] [PDF]
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M. Zhang, T. Angata, J. Y. Cho, M. Miller, D. H. Broide, and A. Varki Defining the in vivo function of Siglec-F, a CD33-related Siglec expressed on mouse eosinophils Blood, May 15, 2007; 109(10): 4280 - 4287. [Abstract] [Full Text] [PDF]
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 A. F. Carlin, A. L. Lewis, A. Varki, and V. Nizet Group B Streptococcal Capsular Sialic Acids Interact with Siglecs (Immunoglobulin-Like Lectins) on Human Leukocytes J. Bacteriol., February 15, 2007; 189(4): 1231 - 1237. [Abstract] [Full Text] [PDF]
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 S. J. Orr, N. M. Morgan, R. J. Buick, C. R. Boyd, J. Elliott, J. F. Burrows, C. A. Jefferies, P. R. Crocker, and J. A. Johnston SOCS3 Targets Siglec 7 for Proteasomal Degradation and Blocks Siglec 7-mediated Responses J. Biol. Chem., February 9, 2007; 282(6): 3418 - 3422. [Abstract] [Full Text] [PDF]
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H. Attrill, A. Imamura, R. S. Sharma, M. Kiso, P. R. Crocker, and D. M. F. van Aalten Siglec-7 Undergoes a Major Conformational Change When Complexed with the {alpha}(2,8)-Disialylganglioside GT1b J. Biol. Chem., October 27, 2006; 281(43): 32774 - 32783. [Abstract] [Full Text] [PDF]
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 T. Avril, S. J. North, S. M. Haslam, H. J. Willison, and P. R. Crocker Probing the cis interactions of the inhibitory receptor Siglec-7 with {alpha}2,8-disialylated ligands on natural killer cells and other leukocytes using glycan-specific antibodies and by analysis of {alpha}2,8-sialyltransferase gene expression J. Leukoc. Biol., October 1, 2006; 80(4): 787 - 796. [Abstract] [Full Text] [PDF]
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 T. Avril, E. R. Wagner, H. J. Willison, and P. R. Crocker Sialic Acid-Binding Immunoglobulin-Like Lectin 7 Mediates Selective Recognition of Sialylated Glycans Expressed on Campylobacter jejuni Lipooligosaccharides Infect. Immun., July 1, 2006; 74(7): 4133 - 4141. [Abstract] [Full Text] [PDF]
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 D. H. Nguyen, N. Hurtado-Ziola, P. Gagneux, and A. Varki Loss of Siglec expression on T lymphocytes during human evolution PNAS, May 16, 2006; 103(20): 7765 - 7770. [Abstract] [Full Text] [PDF]
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J. Zhang, A. Raper, N. Sugita, R. Hingorani, M. Salio, M. J. Palmowski, V. Cerundolo, and P. R. Crocker Characterization of Siglec-H as a novel endocytic receptor expressed on murine plasmacytoid dendritic cell precursors Blood, May 1, 2006; 107(9): 3600 - 3608. [Abstract] [Full Text] [PDF]
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 A. Varki and T. Angata Siglecs--the major subfamily of I-type lectins Glycobiology, January 1, 2006; 16(1): 1R - 27R. [Abstract] [Full Text] [PDF]
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T. Yamaji, M. Mitsuki, T. Teranishi, and Y. Hashimoto Characterization of inhibitory signaling motifs of the natural killer cell receptor Siglec-7: attenuated recruitment of phosphatases by the receptor is attributed to two amino acids in the motifs Glycobiology, July 1, 2005; 15(7): 667 - 676. [Abstract] [Full Text] [PDF]
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T. Avril, S. D. Freeman, H. Attrill, R. G. Clarke, and P. R. Crocker Siglec-5 (CD170) Can Mediate Inhibitory Signaling in the Absence of Immunoreceptor Tyrosine-based Inhibitory Motif Phosphorylation J. Biol. Chem., May 20, 2005; 280(20): 19843 - 19851. [Abstract] [Full Text] [PDF]
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) or with serial dilutions with an irrelevant mAb OX-18 (
), S7.5a (
), K8 (
), or GL183 (*) mAbs and then challenged with goat anti-mouse Ig F(ab')2. The serotonin released in supernatants was measured. Results shown are expressed as the mean percentage of specific serotonin release of triplicate wells from one experiment representative of at least three independent experiments.
