This work describes the newly developed sialoside BPC-Neu5Ac as a specific inhibitor for the ligand binding domain of hCD22. To unequivocally interpret our experiments on B cell signaling in presence of BPC-Neu5Ac, it was crucial to address the issue of specificity of this compound. The high specificity of BPC-Neu5Ac for hCD22 is supported by the following evidence: first, BPC-Neu5Ac inhibited binding of hCD22-Fc to Sia containing target cells very well, but not the binding of the closely related Siglec-Fc proteins, mCD22-Fc, Sn-Fc, and MAG-Fc (data not shown); second, BPC-Neu5Ac inhibited staining of sialidase-treated human B cells with the synthetic CD22 ligand NeuGc2,6-PAA, while it had no effect on staining of murine B cells. The specificity of the synthetic CD22 ligand for CD22 in this staining protocol was demonstrated with murine wild-type and CD22−/− B cells. A high specificity of the probe also for hCD22 is therefore very likely, although other probe-binding receptors on human B cells cannot be totally excluded; and third, a new crystal structure of BPC-Neu5Ac bound to Sn confirmed the predicted binding site to this Siglec (unpublished data). The higher affinity of BPC-Neu5Ac for hCD22 than for mSn can be explained by molecular modeling of the CD22 binding site. The Val-109 and Leu-107 of Sn which make contact to the biphenyl group of the sialoside are substituted by Arg-111 and Met-109 in hCD22. The biphenyl substituent could be sandwiched between these two side chains in hCD22 contributing a substantial binding affinity.
Together these data clearly suggest that the higher IgM triggered Ca2+
signal of BPC-Neu5Ac treated B cells is due to a specific inhibition of the ligand-binding domain of CD22. This interference with ligand-binding leads to an incomplete activation of the intracellular inhibitory domain of CD22. From the data presented it is obvious that the availability of 2,6Sia ligands on glycoproteins on the cellular surface is important for the function of this Siglec. B cells usually display high levels of 2,6Sia on the surface (13
). Upon in vitro activation, a subset of human peripheral B cells seems to downregulate surface expression of 2,6Sia (13
). This could be due to downregulation of the α2,6 sialyltransferase ST6GalI which is highly regulated in several cell types (22
) or activation of a sialidase (13
). Thus, the inhibitory activity of CD22 could be regulated by the differential expression of 2,6Sia on the B cell surface. The inhibitor BPC-Neu5Ac most likely also affects the cellular distribution of CD22 on the B cell membrane.
All available structural data show that the ligand-binding domains of Siglecs are specific for the sialylated carbohydrate moieties with no involvement of the core protein in binding (16
). Also, recent surface plasmon resonance experiments have shown that the affinity of CD22 for 2,6Sia, coupled to different carriers, is very similar, irrespective of whether the sugar is attached to different protein backbones or even polyacrylamide (Bakker, T., and A. van der Merwe, personal communication). Thus, any glycoprotein on the B cell surface containing 2,6Sia as terminal sugars could be a potential ligand for CD22. Our Ca2+
data argue against the model that ligand binding of CD22 by other surface glycoproteins sequesters CD22 away from the BCR and thereby releases the BCR from CD22 inhibition (18
). In this case, interference with the ligand binding by sialosides would release CD22 from this sequestering and lead to its availability for BCR inhibition, resulting in a lower Ca2+
In contrast, our results support the model that the lectin domain mediates CD22 interaction to specific transmembrane glycoprotein ligands which are positively involved in BCR signaling. Such a specificity would be in contradiction to the only carbohydrate-based binding specificity mentioned above. But Sia-binding of CD22 could support interaction to certain transmembrane proteins to which a specific interaction is accomplished by other mechanisms. Alternatively, CD22 binds to many glycoproteins, but the ones which are abundant, structurally well accessible and carry the highest level of appropriately linked Sia win. There are two likely candidates as ligands for CD22: one is membrane Ig. It has been shown that a low stochiometric amount of CD22 can be coprecipitated with IgM from the B cell surface (23
). CD22 lectin binding to sialylated membraneIgM may contribute to this constitutive association ( A). Proximity to the BCR is likely to be crucial for CD22 because the recruited SHP-1 needs to be near its tyrosine phosphorylated substrates of the antigen receptor signaling complex. A second likely candidate as CD22 ligand is CD45, since CD45 is an abundant protein on the B cell surface, is highly glycosylated and has been detected as a prominent ligand by immunoprecipitation with CD22-Fc (25
). Also, CD45 is known to activate lyn, the src-like kinase responsible for CD22 phosphorylation (26
) ( B). After initial binding to CD45, CD22 could subsequently interact with the BCR or even recruit CD45 to the BCR. These possibilities will be directly tested in future experiments. Additionally to the discussed cis-binding, trans interactions of CD22 to ligands on adjacent cells cannot be excluded. Such interactions which may relieve the B cell from CD22 suppression, could be relevant when B cells are closely packed together such as in primary follicles (5
). Recently, it was shown that trans interactions of CD22 with ligands on target cells can also influence Ag-specific B cell activation (27
Figure 5. Model of possible ligands for CD22 on the B cell surface. (A) Interaction to surface Ig (BCR). (B) Interaction to CD45. Both CD22 interactions would be 2,6 Sia-dependent and could thus be inhibited by BPC-Neu5Ac, leading to impairment of CD22 activation, (more ...)
In summary, we have described a novel role for the CD22-lectin domain. Binding to ligands on the cell surface apparently supports the inhibitory function of CD22 on BCR-triggered Ca2+
flux. Our data are very well compatible with the study of Jin et al. (28
) who have come to similar conclusions with a completely different experimental approach. In addition to addressing the biological role of the adhesion domain of CD22, the newly described sialoside could be potentially very important also therapeutically. Application of this structurally simple compound leads to an enhanced B cell response, which could be valuable for immunocompromised patients. Oligomerization of the sialoside can easily be used in the future to enhance affinity and avidity and improve the biological efficacy even further.