Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: novel sialoside probe for murine CD22

Brian E. Collins2, Ola Blixt2, Nicolai V. Bovin3, Claus-Peter Danzer4, Daniel Chui5, Jamey D. Marth5, Lars Nitschke4 and James C. Paulson1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The interaction of CD22 with glycoprotein ligands bearing the Sia{alpha}2,6Gal-R sequence is believed to modulate its function as a regulator of B cell signaling. Although a commercial sialoside-polyacrylamide (PAA) probe, NeuAc- {alpha}2,6Gal-PAA, has facilitated studies on ligand binding by human CD22, murine CD22 binds instead with high affinity to NeuGc{alpha}2,6Gal-R. A multivalent probe with this sequence was constructed to facilitate investigations of ligand binding in CD22 function using genetically defined murine models. The probe is based on the sialoside-PAA platform, which is then biotinylated for easy detection. A series of sialoside probes were constructed with two different length linker arms between the sialoside and the backbone and three different sialoside to PAA molar ratios. The NeuGc{alpha}2,6Gal-PAA probe is specific for CD22: it binds to sialidase-treated B cells of wild-type mice but not B cells of CD22-null mice. Additionally, because the probe only binds to sialidase-treated wild-type cells, it confirms that CD22 is constitutively "masked" on most B cells from wild-type mice by binding to ligands in cis. In contrast, the probe bound equally well to native or sialidase-treated B cells from the immunocompromised ligand-deficient ST6Gal I knockout mice, demonstrating that CD22 is constitutively "unmasked" in these cells.

Key words: CD22/NeuGc/probe/Siglec/ST6Gal I


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The Siglecs are a recently defined family of carbohydrate-binding proteins with conserved structural and sequence motifs. All 11 of the members reported to date mediate binding to sialic acid–containing oligosaccharides, and 9 are found on cells of the immune system (reviewed in Crocker and Varki, 2001Go; Angata et al., 2002Go). Though the expression profile of each Siglec is highly restricted, some cell types contain more than one Siglec on their cell surface (Crocker and Varki, 2001Go). With few exceptions, little is known about the biological roles of the Siglecs because most are newly discovered. All but two have cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs) suggesting their potential involvement in regulation of cell signaling events (reviewed in Crocker and Varki, 2001Go).

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., 1999Go; Campbell and Klinman, 1995Go; Cornall et al., 1999Go; Cyster and Goodnow, 1997Go; Doody et al., 1995Go; Law et al., 1996Go; Smith et al., 1998Go). In support of this, mice deficient in CD22 are hyperresponsive to BCR stimulation, as indicated by increased Ca2+ influx, proliferation (Nitschke et al., 1997Go; O'Keefe et al., 1996Go; Otipoby et al., 1996Go; Sato et al., 1996Go), and the presence of anti-DNA auto-antibodies at older age (O'Keefe et al., 1999Go).

As a sialic acid lectin, CD22 has high specificity toward sialic acids in {alpha}2,6 linkages to galactose (Kelm et al., 1994aGo,b; Powell et al., 1995Go; Powell and Varki, 1994Go; Stamenkovic et al., 1992Go). Recently, using a polyvalent probe containing this oligosaccharide, Razi and Varki (1998)Go 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, 1998Go). 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, 1994Go; Lo and Lau, 1999Go; Wang et al., 1993Go; Wuensch et al., 2000Go). Hennet et al. (1998)Go 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 I–deficient 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., 1998Go). 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{alpha}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, 2000aGo; Cornish et al., 1998Go; Patel et al., 1999Go; Zhang et al., 2000Go). Alternatively, the deficient immune response of ST6Gal I-/- mice may be Siglec-independent.

Investigations of the sialic acid–binding 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 Sia{alpha}2,6Galß1,4GlcNAc, 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., 2000Go; Kelm et al., 1994bGo). 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 NeuGc{alpha}2,6Galß1,4GlcNAc-R (NeuGc{alpha}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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Design of a multivalent sialoside probe specific for CD22
The sialoside NeuGc{alpha}2,6Galß1,4GlcNAcß-R, anticipated to be a ligand for mCD22, was synthesized using a chemoenzymatic strategy (see Materials and methods). To facilitate the construction and evaluation of multivalent probes, the sialoside was prepared as a glycoside with two different amino terminated linkers (R = OCH2CH2NH2, or R = OCH2CH2OCH2CH2NH2). Sialosides coupled to a PAA backbone have previously been documented to bind to cell surface Siglecs (Angata et al., 2001Go; Angata and Varki, 2000aGo,b; Cornish et al., 1998Go; Floyd et al., 2000aGo,b; Galanina et al., 2001Go; Munday et al., 2001Go; Nicoll et al., 1999Go; Yu et al., 2001Go; Zhang et al., 2000Go). Accordingly, the NeuGc{alpha}2,6Galß1,4GlcNAcß-R sialoside with both linker arms were coupled to a PAA backbone at 10, 20, and 40 mol% to yield the corresponding PAA probe (NeuGc{alpha}2,6Gal-PAA). The PAA backbone was also labeled with biotin to aid in detection of the probe using phycoerythrin (PE)-labeled or alkaline phosphatase–labeled streptavidin.

Characterization of NeuGc{alpha}2,6Gal-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 streptavidin–alkaline phosphatase.



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Fig. 1. Differential binding of the NeuGc{alpha}2,6Galß1,4GlcNAc-PAA probe to Siglec-Fc chimeras. Siglec-Fc chimeras of human CD22, murine CD22 and sialoadhesin (Siglec 1) were immobilized on protein A–coated wells of a 96-well plate and then incubated with the indicated sialoside-PAA construct for 1 h at 37°C. After washing, the bound biotinylated probes were detected using alkaline phosphatase–conjugated streptavidin as described in Materials and methods. Data are the average ± SD of triplicate determinants.

 
As shown in the middle panel of Figure 1, of the three sialoside-PAA probes examined, mCD22 bound the NeuGc{alpha}2,6Gal-PAA with highest affinity. Conversely, hCD22 preferentially recognized NeuAc{alpha}2,6Gal-PAA (Figure 1). Though neither mCD22 nor hCD22 recognized the NeuAc{alpha}2,3Gal-PAA, sialoadhesin (Siglec-1) preferentially bound this probe.

Although these results were qualitatively predicted by previous reports, hCD22 is reported to recognize NeuAc{alpha}2,6Gal and NeuGc{alpha}2,6Gal with equal affinity and mCD22 is reported to bind to the NeuGc{alpha}2,6Gal sequence with 10–50-fold higher affinity (Brinkman-Van der Linden et al., 2000Go; Kelm et al., 1994bGo). 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 NeuGc{alpha}2,6Gal-PAA to murine B cells
To optimize probe binding to cell-expressed CD22, a series of derivatives of the NeuGc{alpha}2,6Galß1,4GlcNAc 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, 1998Go). 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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Fig. 2. Effect of spacer length and sialoside content on binding of NeuGc{alpha}2,6Gal-PAA probe to sialidase-treated murine B cells. Freshly isolated splenocytes were mock (dotted line) or A. ureafaciens sialidase-treated (solid line) for 2 h to unmask Siglecs. After washing, cells were incubated with 1 µg of a NeuGc{alpha}2,6Galß1,4GlcNAcß-R-PAA conjugate for 2 h on ice. Six conjugates were compared for their binding to murine B cells with the sialoside attached to PAA through either a short linker arm, R = OCH2CH2NH2 (panels A, B, and C) or a long linker arm, R = OCH2CH2OCH2CH2NH2 (panels D, E, and F), and coupled at 10 mol% (panels A and D), 20 mol% (panels B and E), or 40 mol% (panels C and F). Bound probe was detected by flow cytometry with PE-conjugated streptavidin as described in Materials and methods. Data are of B220+ cells only, and mean channel fluorescence of the sialidase-treated B cells is indicated in the figure.

 
Specific binding of NeuGc{alpha}2,6Gal-PAA probe to CD22 on murine B cells
Evaluation of the sequence specificity on the binding of PAA-based sialoside probes to sialidase-treated murine B cells is shown in Figure 3. NeuGc{alpha}2,6Gal-PAA probe bound well to sialidase-treated unmasked B cells as compared to the cells mock-treated (Figure 3), which showed a similar background level of binding obtained with streptavidin alone (data not shown). These data support the notion that CD22 on murine B cells is predominantly in a masked state, unable to bind to sialoside probes. Though the experiment shown in Figure 3 depicted the weak binding of the NeuAc{alpha}2,3Gal and NeuAc{alpha}2,6Gal probes to wild-type cells, binding was not observed in all experiments. No binding of any of the three probes was observed in a single experiment with CD22 -/- B cells (data not shown). Thus, weak binding of the NeuAc{alpha}2,6Gal-PAA and NeuAc{alpha}2,3Gal-PAA probes to murine B cells may be due either to weak binding to murine CD22 (Floyd et al., 2000bGo), another murine Siglec, or both.



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Fig. 3. Preferential binding of NeuGc{alpha}2,6Gal-PAA to sialidase-treated B lymphocytes. Freshly isolated splenocytes were mock (dotted line) or A. ureafaciens sialidase-treated for 2 h as described in Materials and methods. Following washing, 100,000 cells were incubated with 1 µg of either NeuGc{alpha}2,6Gal-PAA (dotted and thick solid lines), NeuAc{alpha}2,6Gal-PAA (thin solid line), or NeuAc{alpha}2,3Gal-PAA (hashed line) probes for 2 h and then detected by flow cytometry with PE-conjugated streptavidin. B cells were detected with Cy-chrome labeled anti-B220, and the data are of B220+ cells only. Details are provided in Materials and methods.

 
To determine if the binding of NeuGc{alpha}2,6Gal-PAA probe was specific to CD22, the ability of the probe to bind to B cells of wild-type or CD22-null mice was compared. B cells pretreated with or without sialidase were incubated with the NeuGc{alpha}2,6Gal-PAA probe, and bound probe was detected with PE-conjugated streptavidin (Figure 4). Background levels of probe binding were observed with native (untreated) B cells from both wild-type and CD22-deficient mice. Following sialidase pretreatment, although significant binding of probe was observed with the wild-type cells, no additional binding of probe was observed with the sialidase-treated CD22-null cells (Figure 4). Additionally, mild periodate treatment of the probe, which abolishes the sialic acid glycerol side chain required for Siglec binding, reduced probe binding to wild-type cells to background levels (data not shown). The absence of probe binding to CD22-deficient cells and dependence of the glycerol side chain for binding indicate that the probe is specific for the murine CD22 molecule.



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Fig. 4. NeuGc{alpha}2,6Gal-PAA probe binding to murine B cells is CD22 dependent. B cells were isolated from wild-type (WT) and CD22 -/- mice and then mock or A. ureafaciens sialidase-pretreated for 2 h as described in Materials and methods. Cells (100,000) were incubated with 1 µg of the NeuGc{alpha}2,6Gal-PAA probe on ice for 1 h, and bound probe was detected by flow cytometry with PE-streptavidin as described in Materials and methods.

 
Unmasked CD22 in ST6Gal I -/- mice
The sialyltransferase ST6Gal I is the only transferase capable of making the Sia{alpha}2,6Gal linkage (Tsuji et al., 1996Go), the preferred ligand of CD22. Having established that the NeuGc{alpha}2,6Gal-PAA probe is specific for CD22, it was possible to evaluate unambiguously the functional status of CD22 on B cells of the ST6Gal I–deficient mice. Results shown in Figure 5 compare the ability of native and sialidase-treated B cells of wild-type (top) and ST6Gal I–null (bottom) mice to bind the Sambucus nigra agglutinin (SNA) and the NeuGc{alpha}2,6Gal-PAA probe. SNA specifically binds the Sia{alpha}2,6Gal linkage and binds avidly to wild-type native B cells. Following pretreatment with the sialidase from Arthrobacter ureafaciens there is a >10-fold decrease in SNA binding, reflecting a loss of the preferred ligand for CD22. As shown in the top right panel, there is a concomitant increase in binding of the probe, reflecting unmasking of CD22 following treatment with sialidase (see also Figures 3 and 4).



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Fig. 5. CD22 is constitutively unmasked on B cells of ST6Gal I -/- mice. Splenocytes freshly isolated from WT or ST6Gal I-/- mice were mock (dotted line) or A. ureafaciens sialidase-treated (solid line) prior to incubation for on ice with either FITC-labeled SNA or the NeuGc{alpha}2,6Gal -PAA probe. Washed cells were incubated with PE-streptavidin to detect the bound PAA probe and with Cy-chrome labeled anti-B220 to label B cells and subjected to flow cytometry as described in Materials and methods. Shown are results of SNA staining (left panels) and binding of the NeuGc{alpha}2,6Gal-PAA probe (right panels) to B220+ splenocytes from WT (top panels) and ST6Gal I -/- (bottom panels) mice. Data are of B220+ cells only.

 
In contrast to wild-type B cells, native B cells of ST6Gal I–null mice bind >1000-fold less SNA than B cells of wild-type mice, and there is no effect of sialidase treatment on SNA binding (bottom left). Thus the product of ST6Gal I (Sia{alpha}2,6Gal) appears to be responsible for virtually all SNA binding to B lymphocytes. Binding of the NeuGc{alpha}2,6Gal-PAA probe to native B cells of ST6Gal I–null mice was three- to fourfold higher than observed with the B cells from wild-type mice, and no additional probe binding was observed following sialidase treatment. The 2–2.5 overall lower probe binding observed with the sialidase treated ST6Gal I -/- B cells compared to the wild-type B cells can be attributed to the lower levels of CD22 found in these cells, which is 38% relative to wild-type littermate controls (data not shown) (Hennet et al., 1998Go). Considered together, the results show that CD22 on B cells of ST6Gal I–null mice is constitutively unmasked. Furthermore, the data suggest that masking of CD22 on native B cells of wild-type mice is due to the activity of ST6Gal I.

A comparison of the binding of several sialoside-PAA probes to native B cells of ST6Gal I–null mice is shown in Figure 6. Although there was clear binding of the NeuGc{alpha}2,6Gal probe, no significant binding of NeuAc{alpha}2,3 or NeuAc{alpha}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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Fig. 6. Only NeuGc{alpha}2,6Galß1,4GlcNAc-PAA probe binding is demonstrated in ST6Gal I -/- B cells. ST6Gal I -/- B220+ splenocytes were incubated with NeuGc{alpha}2,6Gal-PAA (thick line), NeuAc{alpha}2,6Gal-PAA (thin line) ,or NeuAc{alpha}2,3Gal-PAA (dotted lnie) for 2 h on ice. Bound probe was detected with PE-streptavidin. NeuAc{alpha}2,6Gal-PAA and NeuAc{alpha}2,3Gal-PAA binding were equal to PAA carrier alone binding to ST6Gal I -/- cells or NeuGc{alpha}2,6Gal-PAA binding to masked wild-type cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CD22 is well documented to be an accessory protein of the BCR complex that negatively regulates B cell signaling. However, little is known about how the binding to its sialoside ligand can modulate its function in B cells (Crocker and Varki, 2001Go; Cyster and Goodnow, 1997Go; Doody et al., 1995Go; Nitschke et al., 2001Go; Smith and Fearon, 2000Go). Several groups have developed multivalent sialoside constructs that bind to cell surface Siglecs (Floyd et al., 2000bGo; Galanina et al., 2001Go; Sliedregt et al., 2001Go; van Rossenberg et al., 2001Go), but none have been demonstrated to bind specifically to CD22 on the murine B cell. With an interest in investigating the role of ligand binding in CD22 function in the murine model, we developed a probe containing the NeuGc{alpha}2,6Galß1,4GlcNAc sequence, the preferred carbohydrate ligand of mCD22. The probe was found to bind specifically to CD22 on murine B cells, as shown by the binding to sialidase-treated wild-type B lymphocytes but not to sialidase-treated lymphocytes from CD22-null mice. This is a key finding because five different Siglecs have been reported on human B cells, and the overlapping sialoside specificity of the Siglecs with probes studied to date is well documented (Angata et al., 2001Go; Angata and Varki, 2000aGo; Cornish et al., 1998Go; Floyd et al., 2000aGo,b; Munday et al., 2001Go; Nicoll et al., 1999Go; Patel et al., 1999Go; Yu et al., 2001Go; Zhang et al., 2000Go). Thus, these probes are anticipated to be useful tools in future investigations of the function of the carbohydrate ligand in modulating the role of CD22 in B cell signaling in the murine model.

CD22 was observed to be largely masked on the surface of murine B cells, as evidenced by the dramatically enhanced binding of the NeuGc{alpha}2,6Gal 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)Go 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 I–null mice, which are lacking the enzyme required to elaborate the Sia{alpha}2,6Gal linkage. These B cells are devoid of Sia{alpha}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{alpha}2,3Gal linkages, was previously shown by Hennet et al. (1998)Go to bind equally well to B cells of both wild-type and ST6Gal I–null 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{alpha}2,6Gal linkage is missing from these cells, and CD22 is constitutively unmasked as a result, we conclude that the Sia{alpha}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., 1998Go). 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 I–null 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 NeuAc{alpha}2,6Gal-PAA probe, Razi and Varki (1998)Go 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, 1997Go; DeFranco et al., 1998Go; Fearon, 1997Go; Justement, 2000Go; Tedder et al., 1997Go; Tooze et al., 1997Go). 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, 1997Go; Doody et al., 1996Go; Fearon, 1997Go). 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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Fig. 7. Possible interactions of CD22 with glycoprotein ligands. Shown are three possible modes of CD22 interactions with glycoprotein ligands that could modulate the function of CD22 by influencing its physical proximity to the B cell receptor complex (IgM) and recruitment of phosphatases, such as SHP-1. The three models differ in the presentation of the glycoprotein displaying the preferred Sia{alpha}2,6Gal ligand sequence (green ball), which is displayed (A) on the BCR receptor, (B) on non-BCR glycoproteins, and (C) on T cell glycoproteins.

 
The first model envisions that the carbohydrate ligand is present on BCR complex glycoproteins and is required for CD22 association. It draws support from the observation that CD22 binds selectively and with high affinity to glycans of soluble IgM (Hanasaki et al., 1995Go). However, B cells of the ST6Gal I–null mice are devoid of functional glycan ligands of CD22 and cannot mediate its association with the BCR. This model is therefore inconsistent if CD22 is playing a major role in this immunosuppressed phenotype observed with the ST6Gal I -/- mice.

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 I–null mice exhibit reduced proliferation and increased calcium flux relative to wild type cells (Hennet et al., 1998Go). 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{alpha}2,6Gal linkage, or specific glycoproteins such as CD45 (Sgroi et al., 1995Go). 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., 1995Go; Pezzutto et al., 1987Go, 1988; Tuscano et al., 1996Go). 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 Sia{alpha}2,6Gal 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 I–null 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, 2000aGo,b; Crocker et al., 1998Go; Foussias et al., 2000Go; Li et al., 2001Go; Munday et al., 2001Go; Patel et al., 1999Go; Zhang et al., 2000Go). Significantly, although six murine Siglecs have been described (Angata et al., 2001Go; Crocker et al., 1991Go; Fujita et al., 1989Go; Tchilian et al., 1994Go; Torres et al., 1992Go; Yu et al., 2001Go), 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., 2002Go). 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 I–null mice. Clearly, further investigation into the modulation of CD22 function by its ligands is required to distinguish between these alternative models.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Siglec chimeras and mice strains
All reagents used were of the highest quality commercially available. Antibodies used were obtained from Pharmingen unless otherwise noted. C57Bl/6 mice were obtained from the Scripps Research Institute Custom Breeding Core; ST6Gal I -/- and CD22 -/- mice were generated as described previously (Hennet et al., 1998Go; Nitschke et al., 1997Go). Chinese hamster ovary cells stably expressing mCD22-Fc and sialoadhesin-Fc chimeras (Kelm et al., 1994aGo) were the generous gifts of Dr. Paul Crocker, and the plasmid encoding for hCD22-Fc chimera (Brinkman-Van der Linden et al., 2000Go) was the generous gift of Dr. Ajit Varki.

Synthesis of NeuGc{alpha}2,6Galß1,4GlcNAcß-R
Sialosides with two different azido spacers were prepared with chemical and enzymatic strategies as described (Blixt et al., 2001aGo,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-{alpha}-D-glucopyrano)-[2,1-d]-2-oxazoline and sulfuric acid in dichloromethane, followed by deblocking and purification as described elsewhere (Lemieux and Driguez, 1975Go) 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., 2001aGo,b), forming the corresponding Galß1,4GlcNAc-R disaccharides in good yield (70–80%). These products were then sialylated using CMP-NeuGc as a donor substrate.

CMP-NeuGc was prepared from NeuGc (Kuboki et al., 1997Go) and CTP using the ST3-CMP NeuAc-synthetase fusion protein (Gilbert et al., 1998Go) as demonstrated (Blixt et al., 2001aGo,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., 2001aGo,b) with overall yields of 89–93% 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 NeuGc{alpha}2,6Galß1,4GlcNAcß-PAA
The sialosides with amino terminated linkers were coupled to biotinylated PAA as described previously (Bovin et al., 1993Go). 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 90–95%. Carbohydrate content of the conjugates was confirmed by monosaccharide analysis with high-performance liquid chromatography after acid hydrolysis. Biotinylated NeuAc{alpha}2,6Galß1,4Glc-PAA and NeuAc{alpha}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 phosphatase–conjugated 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 6–10 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We wish to thank Drs. Paul Crocker, Ajit Varki, and Els Brinkman-Van der Linden for the generous gifts of Siglec-Fc chimeras; Dr. Yasuhiro Ohta at Kyoto Research Laboratories, Marukin Chuyu Co. Ltd., for the generous gift of Arthrobacter ureafaciens sialidase; and Ms. Anna Tran-Crie for her help in manuscript preparation. This study was supported by NIH grants GM25042 to B.E.C., GM60938 to J.C.P., and P01-HL57345 to J.D.M. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BCR, B cell receptor; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FITC, fluoroscein isothycyanate; ITIM, immunoreceptor tyrosine-based inhibition motif; NeuGa2,6Gal-Paa, NeuGca2,6Galb1,4GlcNAc-PAA; NeuAca2,6Gal-PAA, NeuAca2,6Galb1,4Glc-PAA; NeuAca2,3Gal-PAA, NeuAca2,3 Galb1,4Glc-PAA; PAA, polyacrylamide; PBS, phosphate buffered saline; PE, phycoerythrin; SNA, Sambucus nigra agglutinin; sIgM, surface IgM; SHP-1, Src homology 2 domain-containing-protein tyrosine phosphatase-1.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail: jpaulson{at}scripps.edu Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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