2 Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MEM-L71, La Jolla, CA 92075, USA; 3 Shemyakin Institute of Bioorganic Chemistry, ul Miklukho-Maklaya 16/10, 117997 GSP-7, V-437 Moscow, Russian Federation; 4 Institute of Virology and Immunobiology, University of Würzburg, Versbacherstr. 7, 97078 Würzburg, Germany; and 5 HHMI Department of Cellular and Molecular Medicine, University of California San Diego, CMM-W Bldg., Room 333, 9500 Gilman Dr., 0625, La Jolla, CA 92073, USA
Received on March 15, 2002; revised on May 13, 2002; accepted on May 15, 2002
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Abstract |
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Key words: CD22/NeuGc/probe/Siglec/ST6Gal I
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Introduction |
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Best understood in this regard is CD22, Siglec 2, a B-cell surface Siglec, which is known as an accessory protein and negative regulator of the B cell receptor (BCR). It is believed that the negative regulation by CD22 occurs through its recruitment of Src homology 2 domain-containing-protein tyrosine phosphatase-1 (SHP-1) to the BCR complex, leading to dephosphorylation and dampening of the signaling cascade (Blasioli et al., 1999; Campbell and Klinman, 1995
; Cornall et al., 1999
; Cyster and Goodnow, 1997
; Doody et al., 1995
; Law et al., 1996
; Smith et al., 1998
). In support of this, mice deficient in CD22 are hyperresponsive to BCR stimulation, as indicated by increased Ca2+ influx, proliferation (Nitschke et al., 1997
; O'Keefe et al., 1996
; Otipoby et al., 1996
; Sato et al., 1996
), and the presence of anti-DNA auto-antibodies at older age (O'Keefe et al., 1999
).
As a sialic acid lectin, CD22 has high specificity toward sialic acids in 2,6 linkages to galactose (Kelm et al., 1994a
,b; Powell et al., 1995
; Powell and Varki, 1994
; Stamenkovic et al., 1992
). Recently, using a polyvalent probe containing this oligosaccharide, Razi and Varki (1998)
showed that the lectin activity of human CD22 is "masked" by cis cell surface sialic acids on resting B cells and can be "unmasked" by enzymatically removing cell surface sialic acids with a sialidase. It was further shown that some resting B cells could become unmasked following stimulation with anti-IgM/CD40 (Razi and Varki, 1998
). These results suggested a role for sialic acids in the regulation of lectin binding of the Siglec CD22 and other members of the Siglec family.
The terminal sialoside structure recognized by CD22 is synthesized in vivo by the enzyme ST6Gal I. Significantly, expression of this enzyme is tightly controlled with high levels seen in B cells (Kitagawa and Paulson, 1994; Lo and Lau, 1999
; Wang et al., 1993
; Wuensch et al., 2000
). Hennet et al. (1998)
observed that a mouse deficient in the enzyme ST6Gal I was immunosuppressed in response to immunization with T-dependent or T-independent antigens. Moreover, ST6Gal Ideficient mice have low serum IgM levels, and their B cells show decreased proliferation and calcium influx following surface IgM (sIgM) cross-linking (Hennet et al., 1998
). These observations have suggested the possibility that CD22 may be exerting stronger negative regulation of B cell signaling in the absence of its primary ligand, Sia
2,6Gal. As many as four Siglecs, however, have been detected on human B lymphocytes and could also be influenced by the absence of this sialoside ligand (Angata and Varki, 2000a
; Cornish et al., 1998
; Patel et al., 1999
; Zhang et al., 2000
). Alternatively, the deficient immune response of ST6Gal I-/- mice may be Siglec-independent.
Investigations of the sialic acidbinding activity of murine CD22, unlike the human ortholog, have been hampered by the lack of an appropriate sialoside probe. Both murine and human CD22 have strong preference for the sialoside sequence Sia2,6Galß1,4GlcNAc, but they were found to be very different in their preference for the N-acetylneuraminic acid and N-glycolylneuraminic acid forms of sialic acid. Human CD22 binds both the N-acetyl and N-glycolyl forms, whereas murine CD22 strongly prefers the N-glycolyl form (Brinkman-Van der Linden et al., 2000
; Kelm et al., 1994b
). Therefore, to investigate the functional status of CD22 in genetically defined mice, a polyvalent probe containing the NeuGc derivative of sialic acid was needed.
Using chemoenzymatic synthesis techniques, the sialoside sequence NeuGc2,6Galß1,4GlcNAc-R (NeuGc
2,6Gal-R) was synthesized and covalently attached to a polyacrylamide (PAA) polymer. This report demonstrates that the resulting sialoside probe is able to specifically detect CD22 on sialidase-treated murine B cells. Furthermore, it is used to show that CD22 is predominately "masked" on B cells of wild-type mice and constitutively "unmasked" on B cells of ST6Gal I knockout mice. Implications of these results for regulation of B cell signaling are discussed.
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Results |
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Characterization of NeuGc2,6Gal-PAA binding to selected Siglecs
The relative binding of several sialoside-PAA probes to Siglec-Fc chimeras of hCD22, mCD22 and sialoadhesin (Siglec-1) in enzyme-linked immunosorbent assay (ELISA)-type assays are shown in Figure 1. In this assay, the Siglec-Fc chimeras are immobilized by binding to protein A adsorbed to the surface of the wells in a 96-well ELISA plate. The sialoside-PAA probes are then added to the wells; following an incubation period, the wells are washed extensively. After washing, the bound probe, which is labeled with biotin, is detected using streptavidinalkaline phosphatase.
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Although these results were qualitatively predicted by previous reports, hCD22 is reported to recognize NeuAc2,6Gal and NeuGc
2,6Gal with equal affinity and mCD22 is reported to bind to the NeuGc
2,6Gal sequence with 1050-fold higher affinity (Brinkman-Van der Linden et al., 2000
; Kelm et al., 1994b
). The quantitative differences in the specificity of these Siglecs is probably due to the different assays used and the manner of presentation of the sialoside sequence (e.g., polyvalent, monomer, cell surface glycoprotein, etc.).
Optimizing the binding of NeuGc2,6Gal-PAA to murine B cells
To optimize probe binding to cell-expressed CD22, a series of derivatives of the NeuGc2,6Galß1,4GlcNAc sialoside on the PAA backbone were synthesized and tested. Sialosides were synthesized using one of two different linker arms; a shorter two-carbon linker of NH2CH2CH2-R, or a longer four-carbon linker arm of NH2CH2CH2OCH2CH2-R. These sialosides were then attached to the PAA backbone to a final ratio of 10, 20, or 40 mol% sialoside.
Freshly isolated murine B cells were treated with sialidase to remove endogenous sialic acids that have been shown previously to block CD22-mediated binding on human B cells by acting as cis ligands (Razi and Varki, 1998). Binding of the biotinylated probe was detected by flow cytometry using PE-conjugated streptavidin. Although all probes bound to the sialidase-treated cells, those carrying the shorter linker arm bound slightly better or equal to those with the longer linker arm (Figure 2). Additional binding was observed when the molar ratio was increased from 10 to 20 mol% sialoside, but no increase was observed at 40 mol%. From these data, probes containing the shorter linker arm with 20 mol% were used in the rest of the studies.
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A comparison of the binding of several sialoside-PAA probes to native B cells of ST6Gal Inull mice is shown in Figure 6. Although there was clear binding of the NeuGc2,6Gal probe, no significant binding of NeuAc
2,3 or NeuAc
2,6Gal probes relative to background controls was detected. Either the binding of these probes to other Siglecs is masked or binding is too low to be detected over background.
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Discussion |
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CD22 was observed to be largely masked on the surface of murine B cells, as evidenced by the dramatically enhanced binding of the NeuGc2,6Gal specific probes to sialidase-treated B cells relative to wild-type B cells. This observation confirms and extends the similar finding of Razi and Varki (1998)
for human CD22 on peripheral blood B cells. In contrast to the wild-type cells, CD22 was found to be constitutively unmasked on the B cells of ST6Gal Inull mice, which are lacking the enzyme required to elaborate the Sia
2,6Gal linkage. These B cells are devoid of Sia
2,6Gal linkage because they fail to bind the plant lectin SNA that recognizes this sequence (see Figure 5). In contrast, the Maackia amurensis lectin, which recognizes Sia
2,3Gal linkages, was previously shown by Hennet et al. (1998)
to bind equally well to B cells of both wild-type and ST6Gal Inull mice. The aforementioned data demonstrate that the deficiency of the ST6Gal I gene does not significantly affect the expression of other sialoside linkages on B cells. Because only the Sia
2,6Gal linkage is missing from these cells, and CD22 is constitutively unmasked as a result, we conclude that the Sia
2,6Gal sequence is the primary in vivo carbohydrate determinant responsible for masking of CD22 on B cells of wild-type mice.
The fact that the primary ligand of CD22 is missing on the B cells of the ST6Gal I -/- mice is relevant to understanding how the binding of CD22 to its carbohydrate ligand can modulate its function as a regulator of B cell signaling. B cells of these mice exhibit reduced proliferation and calcium mobilization in response to antibody cross-linking of sIgM or CD40, and the mice have attenuated antibody production in response to immunization with T-dependent and T-independent antigens (Hennet et al., 1998). Though these traits have not been unequivocally demonstrated to be mediated by CD22, the finding that CD22 is masked in wild-type B cells and unmasked in the ST6Gal Inull B cells raises the possibility that the immunosuppressed phenotype results from increased inhibition of B cell signaling by CD22 due to the absence of functional ligands.
Using a NeuAc2,6Gal-PAA probe, Razi and Varki (1998)
showed that a small population of human B cells (~4.5%) became unmasked within 2 h following stimulation with anti-IgM plus anti-CD40. These results have suggested that there is a dynamic unmasking of CD22 during B cell activation. We looked extensively for changes in binding of the PAA and SAAP probes to murine B cells following activation under a variety of conditions, including those used in this previous study. However, we have observed no increase in the binding of the two probes to activated murine B cells (not shown). Because both the probes and species of B cells being evaluated differ in these two studies, inability to see unmasking following activation in our hands sheds little light on the importance of this observation to the function of CD22.
It is widely believed that CD22 must physically associate with the BCR to participate in the regulation of signaling (see reviews: Cyster and Goodnow, 1997; DeFranco et al., 1998
; Fearon, 1997
; Justement, 2000
; Tedder et al., 1997
; Tooze et al., 1997
). Following ligation of the BCR, kinases (Lyn/Blk/Fyn) are activated that can then phosphorylate tyrosine residues in ITIMs in the cytoplasmic domain of CD22. In turn, CD22 can then recruit phosphatases, such as SHP-1, which bind to the phosphorylated ITIMs, and attenuate signaling through dephosphorylation of BCR complex proteins. Several models have been suggested for how the interaction of CD22 with its carbohydrate ligand might influence the association of CD22 with the BCR complex and thereby mediate its ability to down-regulate BCR signaling (Cyster and Goodnow, 1997
; Doody et al., 1996
; Fearon, 1997
). As illustrated in Figure 7, they are: (A) CD22 binds directly to carbohydrates on sIgM or other BCR glycoproteins, (B) CD22 binds in cis to B cell glycoproteins (or even CD22 glycans) that sequester CD22 away from the BCR, and (C) CD22 binds in trans to glycans on T cells (or other cells) that simultaneously sequester CD22 and/or recruit T cell help.
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Both of the other models envision that the binding of CD22 to glycoprotein ligands act to sequester it away from BCR. Conversely, in the absence of ligands, increased association with the BCR and down-regulation of signaling would be expected. Both of these models are consistent with the observed phenotype of ST6Gal I -/- and CD22 -/- mice. However, purified B cells from ST6Gal Inull mice exhibit reduced proliferation and increased calcium flux relative to wild type cells (Hennet et al., 1998). Because these phenotypes are independent of T cells, they are most consistent with model (B), involving the cis-interaction of CD22 with glycoprotein ligands on the same cell that sequester it from the BCR. Candidate glycoproteins could be any glycoprotein carrying the NeuGc
2,6Gal linkage, or specific glycoproteins such as CD45 (Sgroi et al., 1995
). In this regard, it is of interest that earlier studies demonstrated that treatment of B cells with anti-CD22 antibodies resulted in enhanced calcium mobilization and increased proliferation (Doody et al., 1995
; Pezzutto et al., 1987
, 1988; Tuscano et al., 1996
). These enhanced B cell responses have been interpreted to be a result of the sequestration of CD22 away from the BCR by the anti-CD22 antibodies.
In view of the role of the Sia2,6Gal linkage in mediating the masked and unmasked states of CD22 and the known activity of CD22 as a negative regulator of BCR signaling, the involvement of CD22 in the B cell phenotype of the ST6Gal Inull mice seems likely. However, it is also possible that the immunosuppressed phenotype may be due in part or entirely to factors unrelated to CD22. For example, human B cells express other Siglecs to a lesser extent, including Siglecs 5, 6, 9, and 10, raising the prospect that other Siglecs could contribute to the phenotype (Angata and Varki, 2000a
,b; Crocker et al., 1998
; Foussias et al., 2000
; Li et al., 2001
; Munday et al., 2001
; Patel et al., 1999
; Zhang et al., 2000
). Significantly, although six murine Siglecs have been described (Angata et al., 2001
; Crocker et al., 1991
; Fujita et al., 1989
; Tchilian et al., 1994
; Torres et al., 1992
; Yu et al., 2001
), none have been reported to date on mature murine B cells. Interestingly, recent data have suggested that activation of B cells with aIgM in the presence of a potent small-molecule sialic acid analog leads to higher signaling and impaired CD22 activation and SHP-1 recruitment (Kelm et al., 2002
). Because the anticipated effect of soluble inhibitors of ligand binding would be to free CD22 from binding to endogenous ligands, these results would be most consistent with release of CD22 from the BCR complex (Figure 7A), which is not readily reconcilable with the phenotype of the ST6Gal Inull mice. Clearly, further investigation into the modulation of CD22 function by its ligands is required to distinguish between these alternative models.
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Materials and methods |
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Synthesis of NeuGc2,6Galß1,4GlcNAcß-R
Sialosides with two different azido spacers were prepared with chemical and enzymatic strategies as described (Blixt et al., 2001a,b). Briefly, glycosides of N-acetylglucosamine were prepared by reacting 2-azidoethanol or 5-azido-3-oxapentanol with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-
-D-glucopyrano)-[2,1-d]-2-oxazoline and sulfuric acid in dichloromethane, followed by deblocking and purification as described elsewhere (Lemieux and Driguez, 1975
) to yield GlcNAcß-R, where R is either CH2CH2N3 or CH2CH2OCH2CH2N3. These glycosides were then used as acceptor substrates in an enzymatic galactosylation reaction using ß4GalT-/UDPGlc-4'-epimerase fusion protein and UDP-glucose (Blixt et al., 2001a
,b), forming the corresponding Galß1,4GlcNAc-R disaccharides in good yield (7080%). These products were then sialylated using CMP-NeuGc as a donor substrate.
CMP-NeuGc was prepared from NeuGc (Kuboki et al., 1997) and CTP using the ST3-CMP NeuAc-synthetase fusion protein (Gilbert et al., 1998
) as demonstrated (Blixt et al., 2001a
,b). Briefly, NeuGc (180 mg, 1 eqv) and CTP (290 mg, 1 eqv) were mixed in Tris-HCl buffer (10 mM, pH 8.5) containing manganese chloride (20 mM), magnesium chloride (20 mM), and ST3-CMP NeuAc-synthetase fusion protein (10 U). After 16 h at room temperature the fusion protein was removed by ultrafiltration (10 K), and the filtrate containing CMP-NeuGc was used without further purification.
For sialylation, the disaccharides (100 mg) were dissolved in 20 ml cacodylate buffer (50 mM, pH 6.5) containing 1.5 eqv CMP-NeuGc and hST6Gal (1 U), and the reaction was allowed to proceed for 12 h. The resulting trisaccharides were purified by ion-exchange and size-exclusion chromatography as reported elsewhere (Blixt et al., 2001a,b) with overall yields of 8993% based on the amount of disaccharide used. Prior to use in constructing multivalent probes, the azido group on the linkers was reduced to an amino group with hydrogenation on 10% palladium on carbon in aqueous methanol.
Preparation of NeuGc2,6Galß1,4GlcNAcß-PAA
The sialosides with amino terminated linkers were coupled to biotinylated PAA as described previously (Bovin et al., 1993). A solution of 11.6 mg (25 µmol) biotin-NH(CH2)6NH2 in 1 ml dimethyl sulfoxide (DMSO) was added to 96.6 mg (0.5 mg equivalents) of poly[4-nitrophenylacrylate] in 4 ml DMSO. One-tenth of the solution obtained was aliquoted and a solution made of the oligosaccharide with spacer (5, 10, or 20 µmol) in 100 ml DMSO followed by triethylamine (20 µl). The mixture was kept at 4°C for 24 h, and the resulting conjugate was then modified by amidation with 60 µl 2-ethanolamine for 24 h at 20°C. Gel filtration on a Sephadex LH-20 column (1 x 30 cm), elution with acetonitrile/water (1:1 by volume) followed by concentration and freeze-drying gave rise to PAA conjugates, yields 9095%. Carbohydrate content of the conjugates was confirmed by monosaccharide analysis with high-performance liquid chromatography after acid hydrolysis. Biotinylated NeuAc
2,6Galß1,4Glc-PAA and NeuAc
2,3Galß1,4Glc-PAA with 20% mol saccharide were obtained from Glycotech.
Binding of sialoside-PAA probes to immobilized Siglecs
High binding microtiter plates (Corning Costar) were coated with 50 µl of 15 µg/ml protein A (Sigma) in 50 mM Na bicarbonate, pH 9.0, overnight at 4°C. Wells were then washed with buffer (EB = 10 mg/ml bovine serum albumin, 50 mM HEPES, 150 mM NaCl, 1 mM ethylenediamine tetra-acetic acid [EDTA], pH 7.5) and 200 µl Siglec-Fc-containing culture supernatant added with 10 mU V. cholerae sialidase (Roche Molecular Biochemicals) before incubating for 4 h at 37°C. After washing off unbound Siglec-Fc, 100 µl of the sialoside-PAA probe in EB was added at the desired concentration and allowed to bind for 2 h at 37°C. After washing, the bound probe, biotinylated in the PAA carrier, was detected with alkaline phosphataseconjugated streptavidin (Sigma, 1:5000 in EB, 1 h at 37°C) and measured with p-nitrophenyl phosphate (Sigma) read at 405 nm.
Periodate treatment of sialoside probes
Periodate treatment of the PAA-based probe was performed by diluting 10 µg of the probe into a final volume of 100 µl of 2 mM Na metaperiodate (Sigma) in phosphate buffered saline (PBS) and incubating on ice for 90 min in the dark. The resulting aldehydes were reduced by adding 100 µl of a 20 mM Na borohydride (Sigma) in PBS solution and incubating for 1 h at 37°C.
Lymphocyte preparations
Single cell suspensions from spleen, brachial lymph node, and bone marrow were obtained from mice aged 610 weeks. To obtain single cell suspensions, tissues were ground between two frosted glass slides and passed through a 200 µm nylon mesh (Polysciences). After washing, erythrocytes were lysed with a 5-min incubation at room temperature in a solution of 150 mM ammonium chloride, 10 mM potassium carbonate, and 0.1 mM EDTA, pH 7.2. Lysed erythrocytes were removed by passing the suspension over a cotton-plugged Pasteur pipette, the cells washed twice and resuspended in Roswell Park Memorial Institute Media/5% fetal bovine serum/50 µM ß-mercaptoethanol.
Flow cytometry
Single cell suspensions were resuspended in 10 mg/ml BSA in PBS at 1 x 106 cells/ml. Cells (100,000) were incubated with 1 µg of the PAA probe for 2 h on ice, washed twice, and then incubated for 1 h with 1 µg streptavidin-PE (Molecular Probes) and other antibodies at the following concentrations: anti-B220-CyChrome at 2 µg/ml (Pharmingen) and anti-CD22- fluoroscein isothycyanate (FITC) 1 µg/ml (Southern Biotechnology Associates). For sialidase pretreatment, cells were resuspended at 5 x 106cells/ml in PBS containing 2 mg/ml BSA and 400 mU/ml A. ureafaciens sialidase (Kyoto Research Laboratories, Marukin Chuyu). After 2 h at 37°C with end-over-end rotation, the cells were washed three times and then stained. Cells were stained with FITC-labeled SNA (Vector Labs) by incubating 5 x 105 cells in 10 mg/ml BSA in PBS with 2 µg/ml SNA for 30 min on ice. Flow cytometry data was acquired on a FACSCaliber (BD Biosciences) flow cytometer and analyzed using the Cell Quest software system.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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