Glycobiology Research and Training Center, Departments of Medicine and Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0687
Received on February 18, 2004; revised on June 15, 2004; accepted on June 15, 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: B lymphocytes / endocytosis / sialic acids / Siglecs / toxin conjugates
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CD22/Siglec-2 is also an I-type lectin belonging to the Siglec (sialic acid binding Ig-like lectin) family (Crocker and Varki, 2001). It binds to glycans containing sialic acid (Sia) in a highly linkage specific manner. Human and mouse CD22 bind selectively to
2-6-linked Sias (Engel et al., 1993
; Kelm et al., 1994
; Powell et al., 1993
, 1995
; Powell and Varki, 1994
; Sgroi et al., 1993
). The amino-terminal Ig-like V-set domain is critical in this binding (Engel et al., 1995
), requiring a conserved arginine residue that likely forms a salt bridge with the carboxylate groups of the Sia ligand (Van der Merwe et al., 1996
). Like most of the other Siglecs, CD22 is natively bound to sialylated cis ligands on the same cell surface (Razi and Varki, 1998
, 1999
). This "masking" effect is abolished by sialidase treatment, and a small amount of "unmasking" has been found on activated human B cells (Razi and Varki, 1998
). Using different approaches, two groups showed that this cis binding of Sia by CD22 is required for its optimal function as an inhibitory regulator of the BCR (Jin et al., 2002
; Kelm et al., 2002
). These data confirmed the biological importance of Sia recognition in CD22 function. However the mechanism of how cis Sia binding affects CD22 biological functions is not clear. In this article we test two hypotheses regarding to this matter: (1) that cis Sia binding helps keep specific partner proteins close to or away from CD22; and (2) that cis Sia binding restricts CD22 turnover from the cell surface by endocytosis, thus modulating CD22 cell surface levels.
The physiological cis ligands for CD22 are not well defined (Tedder et al., 1997). Many prior studies explored the nature of the sialylated ligands responsible for this cis masking of CD22 Sia binding activity. Several molecules that carry
2-6-linked Sias, for example, cell surface IgM (sIgM) or CD45, as well as circulating glycoproteins such as IgM and haptoglobin have been suggested as candidate ligands (Hanasaki et al., 1995a
; Sgroi et al., 1993
; Stamenkovic et al., 1991
). Currently published models hypothesize that associations of CD22 with sIgM and CD45 are mediated by CD22 recognition of their Sia residues (Collins et al., 2002
; Cyster and Goodnow, 1997
). Indirect support for this hypothesis comes from the finding that B cells from mice deficient in sialylated CD22 ligands show constitutive unmasking of CD22 and altered sIgM signaling responses (Hennet et al., 1998
). However, there is as yet no direct proof for a role of Sias in forming or maintaining specific interactions of other proteins with CD22. In addition, these candidate ligands have been suggested mostly based on the ability of recombinant soluble CD22 to bind them in a Sia-dependent manner in vitro. However, the dimeric CD22-Fc chimeras used for such studies can interact with any molecule with a high density of
2-6-linked Sias, especially when the chimera is immobilized on beads. Indeed, we have previously shown that although a low-density CD22-Fc column selectively interacted with sIgM and haptoglobin from blood plasma, a high-density column could bind most of the
2-6 sialylated glycoproteins in the sample (Hanasaki et al., 1995a
). Also, CD22 expressed on Chinese hamster ovary or COS cells (which are physiologically irrelevant to CD22 biology) can be masked if these cells are also induced to express
2-6-linked Sias by transfection with ST6Gal-I (Braesch-Andersen and Stamenkovic, 1994
; Hanasaki et al., 1995b
).
On the other hand, attempts to study the native interactions in CD22-expressing B cells by coimmunoprecipitation from lysates have been fraught with difficulties. Mild detergent approaches have only detected very limited (12%) interactions of CD22 with sIgM (Law et al., 1994
; Leprince et al., 1993
; Peaker and Neuberger, 1993
). Also, because the single-site binding affinity of CD22 to
2-6-linked Sias is relatively poor (Powell et al., 1995
), it is unlikely to survive the repeated washing steps involved in standard immunoprecipitation protocols. Surface labeling followed by cross-linking thus provides the best hope of quantitatively detecting native interactions (Powell et al., 1995
). However, labeling and cross-linking are typically carried out in a stepwise fashion, and there is a serious risk of perturbing critical interactions during the first labeling step, prior to the cross-linking. We have therefore developed a novel approach for simultaneous biotinylation and cross-linking of cell surface molecules on rapidly chilled cells, which can circumvent many of the problems. We apply this approach to study the nature of CD22-associated cell surface molecules and further examine if these associations are Sia-dependent to test the first hypothesis.
Prior studies also have suggested that cell surface CD22 undergoes constitutive endocytosis and degradation, with a t1/2 of 8 h and that certain residues in the cytosolic tail are involved in mediating this turnover (Chan et al., 1998
; Shan and Press, 1995
). Because CD22 expression level on B cells is significantly reduced in the ST6Gal-I knockout mouse, where CD22 ligand formation is abolished (Hennet et al., 1998
), we considered the possibility that cis binding Sia ligands might help maintain optimal CD22 levels on the cell surface by restricting its rate of endocytosis. To test this hypothesis, we examined not only the constitutive but also antibody-mediated endocytosis of CD22, an issue that is of importance to the anti-CD22-based immunotherapy of B cell leukemias and lymphomas (Herrera et al., 2000
, 2003
; Mansfield et al., 1997
; Messmann et al., 2000
; Pagel et al., 2002
; Shen et al., 1988
; Tuscano et al., 2003
) and how Sia-binding as well as sIgM ligation affect these processes.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The two primary amine-reactive reagents EZ-link Sulfo-NHS-Biotin and 3,3'-dithiobis (sulfosuccinimidylpropionate) (DTSSP) (for biotinylation and cross-linking, respectively) were mixed in various ratios and applied to cells that had just been rapidly chilled to prevent any lateral movement of molecules in the membrane. The reactive group of these two reagents is identical (a primary amine-reactive NHS ester), and they therefore compete for the same sites on cell surface proteins. We found that a ratio of 1:1 of the reagents allowed both biotinylation and cross-linking of cell surface molecules to proceed efficiently on ice in a short time. The labeled membrane proteins could then be solubilized and immunoprecipitated with anti-CD22 antibody, allowing recovery of biotinylated CD22 along with other associated biotinylated proteins, which could then be detected by streptavidin-based reagents. Because the DTSSP cross-linker is cleavable by reducing reagents, a nonreducing gel can indicate if CD22 forms any complexes, and a reducing gel can show what the individual components are. Surface biotinylation and streptavidin staining also ensures detection of only cell surface molecules that interact in the native situation, either through Sia binding or via other mechanisms. To our knowledge, this is the first attempt to define noncovalent cell surface associations using this approach.
We first examined Daudi cells, a human B cell line that constitutively expresses CD22. A nonreducing gel showed that a CD22-antibody-specific immunoprecipitate comprised a high-molecular-weight smear so large that it hardly entered into the 7.5% separating gel (Figure 1A). A reducing gel indicated that in keeping with previous reports (Engel et al., 1995), CD22 in Daudi cells appears as two bands (which cannot be explained by differential sialylation or N-glycosylation, data not shown, and neither of which is CD22
, based on antibody specificity). The reducing gel also showed that several other proteins are involved in forming this high-molecular weight complex with CD22 (Figure 1A). Some of these could be identified by their typical molecular weights and by parallel immunoprecipitation of the B cell surface proteins CD45 and sIgM (Figure 1B). Sequential immunoprecipitation and control antibody immunoprecipitation were done to confirm the identities of these proteins (data not shown). Comparisons with a small amount of total lysate (without immunoprecipitation) loaded on the same gel indicated that these findings are not simply explained by the fact that sIgM and CD45 are major components of the cell surface (Figure 1B). Because several other major bands in the total lysate were not represented in the immunoprecipitated complexes, the observed associations with CD22 must be specific. Other proteins also coimmunoprecipitated with CD22, and potential candidates are other components of BCR, such as Ig
and Igß (Leprince et al., 1993
). However, in this article we focus only on the higher-molecular-weight proteins sIgM and CD45. A quantitative analysis of the results for these three proteins is presented in Table I.
|
|
|
|
Interactions of CD22 with CD45 and sIgM are not affected with BCR activation
Because the importance of Sia binding in CD22 function has been clearly demonstrated by others with BCR-activated cells, we also examined the associations between CD22, CD45, and sIgM on Daudi cells following BCR activation by a F(ab')2 goat anti-human IgM Fc5u specific antibody (cellular activation was confirmed by enhanced calcium influx, data not shown). The BCR-stimulated Daudi cells were simultaneously biotinylated and cross-linked, and immunoprecipitation was done with anti-CD22, anti-IgM, and anti-CD45. As shown in Figure 3C, no major change in the interactions between the three molecules was detected after BCR activation. Similar results were found with CD22-transfected J2-44 cells following BCR stimulation (with or without arginine mutation of CD22, data not shown). Varying the concentrations of anti-IgM for activation or allowing activation for different periods of time (130 min) also did not show any changes in CD22 interactions with sIgM or CD45.
Constitutive endocytosis and turnover of CD22 is not affected by cell surface Sia
Taken together, the data indicate that the well-documented importance of Sia recognition in CD22 function (Jin et al., 2002; Kelm et al., 2002
) cannot be explained by a primary role in forming associations with sIgM and CD45. We next tested another hypothesis, that cis binding Sia ligands might help maintain optimal CD22 levels on the cell surface by restricting its rate of endocytosis. This hypothesis was suggested by a previous finding that in the ST6Gal-I knockout mouse, where CD22 ligand formation is eliminated, B cell CD22 expression level is significantly reduced (Hennet et al., 1998
). The hypothesis was tested by comparing CD22 endocytosis rates in the presence or absence of Sia-based interactions. In the first approach, sialidase treatment was used to induce a sudden unmasking. Daudi cells were subjected to cell surface biotinylation in the cold, and the constitutive turnover of CD22 at 37°C with the presence or absence of sialidase was then studied by immunoprecipitation at various time points in a pulse-chase format. However, no difference in the rate of turnover was observed (data not shown). Of course, sialidase treatment could also cause multiple functional changes on the cell surface related to a general loss of net negative charge. Thus we also studied the rate of turnover of human CD22 and its arginine-mutated variant expressed by transfection into CD22-negative mouse B cells. Again, no difference in the rate of turnover was seen (data not shown).
In contrast to our surface biotinylation/chase approach, some previous studies describing CD22 endocytosis actually followed the internalization of labeled anti-CD22 antibodies (Chan et al., 1998; Press et al., 1989
). In comparing these reports with those involving other means, such as surface labeling (Shan and Press, 1995
), we noted that the reported rate of internalization for antibody-induced CD22 turnover was much faster than the constitutive one. Thus it appears that antibody binding to CD22 enhances endocytosis via the same or different mechanisms that are involved in the constitutive process. Even if the mechanisms are different, the antibody-mediated process is of practical significance, because antibodies and antibody-based immunotoxins are being investigated for the treatment of CD22-expressing lymphomas and leukemias (Herrera et al., 2000
, 2003
; Mansfield et al., 1997
; Messmann et al., 2000
; Pagel et al., 2002
; Tuscano et al., 2003
). We therefore asked whether CD22 interactions with cell surface sialylated ligands have an impact on antibody-mediated endocytosis.
As shown in Figure 4A, sialidase-treated cells showed a higher rate of antibody-induced CD22 endocytosis than untreated cells. The differences, though small, are reproducible (the differences at 30, 60, and 90 min are statistically significant). A result consistent with this was also seen in an immunotoxin killing assay. RFB4-dgRTA is an immunotoxin (kindly provided by Dr. Ellen S. Vitetta) consisting of a CD22 antibody coupled to the ricin A chain (Shen et al., 1988). Because this toxin is delivered via CD22 endocytosis after binding via the antibody portion (Chan et al., 1998
), we tested the toxin sensitivity of cells in which CD22 was either masked or unmasked. Sialidase treatment requires serum-free media, and we found that the cells were intrinsically more sensitive to the immunotoxin killing in serum-free media than in serum-containing media (data not shown). Regardless, the overall killing efficiency was higher in sialidase-treated cells in comparison with buffer-incubated controls (Figure 4B, the differences seen at 15 pM concentrations are statistically significant, p < 0.05). Taken together, these findings negate the hypothesis that binding of sialylated cis ligands is a major factor regulating the cell-surface half-life of CD22 under native conditions. However, the antibody-induced CD22 endocytosis, and therefore CD22 immunotoxin killing can be altered by the presence of Sia.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Overall, our conclusion is that the steady-state associations of CD22 with sIgM and CD45 occur at much higher levels than previously reported. This is presumably because our approach of simultaneous labeling and cross-linking rapidly chilled cells is more likely to detect relatively labile noncovalent complexes. We also excluded other factors that could have biased the result. First the content of potentially biotinylated lysine residues in the extracellular domain of CD22, CD45, and sIgM (µ) C region are 5.8%, 5.8%, and 4.8%, respectively. Second, the overall efficiency of first pass immunoprecipitation was similar for CD22, CD45, sIgM (µ) (30%, 25%, and 36%, respectively, the numbers probably reflect the fact that not all molecules are accessible for immunoprecipitation after such major cross-linking). Therefore the disparity with previous reports is unlikely to be a result of major differences in biotinylation or immunoprecipitability of these molecules in our experiment. Rather it is likely due to the sensitivity of the protein complexes to detergent lysis and/or surface labeling steps that are typically done as a separate step, prior to cross-linking. Our new approach should be of value in many other biological situations where there are multiple loosely associated components in such cell surface complexes. Potentially, it could even be used to study intracellular protein complexes by simultaneously adding the biotinylation and cross- linking reagents along with a detergent or cell-permeabilizing reagent.
As mentioned before, there are two proposed models regarding CD22 cis ligand binding (Collins et al., 2002) indicating that Sia binding is the major force either keeping CD22 bound to sIgM or away from it (by binding to other sialylated proteins, e.g., CD45). Our result does not support either model, because no major difference was found in the protein components within the CD22-related complex between wild-type and arginine-mutated molecules or when treating cells with sialidase. Our results instead show that Sia binding is not required for mediating the primary interactions of CD22 with CD45 and sIgM (and some other unidentified proteins). Thus we propose that these associations are primarily mediated via proteinprotein interactions. Of course because CD45 and sIgM can themselves carry N-glycans with
2-6 Sias, initial associations with CD22 via proteinprotein interactions may then give them an advantage over other cell surface molecules in developing secondary interactions via Sias. On the other hand, we cannot rule out the possibility that some other minor cell surface molecules associate with CD22 exclusively in a Sia-dependent matter. CD22 may also be masked via low-level interactions with many or all of the
2-6 sialylated molecules on the cell surface, in a dynamic equilibrium, without requiring a favored interaction with a specific ligand. Results consistent with this conclusion come from a very recent publication, in which the authors showed that the degree of CD22 masking on cell surface is affected by local concentration of the ligands and that CD45 is not requisite for CD22 masking (Collins et al., 2004
).
As with all coimmunoprecipitation and cross-linking approaches, ours is based on the vicinity of the proteins to one another. If a Sia residue that binds to CD22 is located on a protein that is physically further away from CD22 than the length of the cross-linker, then that association could be missed using our method. It is also possible that an interacting protein could carry glycan groups that are bulky enough to prevent the access of our cross-linker. In this article, we focused mainly on the two proteins that have been proposed as ligands for CD22 and found their associations with CD22 are not solely based on Sia binding. Defining all of the possible ligands for CD22 remains to be done, but this is outside the scope of the present study.
BCR stimulation is a process during which CD22Sia binding is known to have effects on calcium response, SHP-1 recruitment, CD22 phosphorylation, and so on. Thus it is possible that CD22Sia binding may have different functions following activation. We have done some preliminary experiments using anti-IgM stimulation without seeing any major effects on the associations between CD22 and sIgM or CD45 (Figure 3C). We cannot rule out some very subtle changes that are not detectable with our method, of course. However, our results do not support an extensive change in major partners as a mechanism for how Sia binding affects CD22 function during activation. Fully exploring this issue will require a lot of additional work.
Another point of interest is that the proteinprotein interactions between CD22, sIgM, and CD45 occur even when human CD22 is transfected into mouse B cells. Thus the domains and amino acid residues involved in these interactions have remained conserved since the common ancestor of humans and mice more than 50 million years ago. Given the 5570% conservation of the amino acid sequences of the extracellular domains of CD22 between humans and mice (Torres et al., 1992), it should be possible eventually to define the specific domains and amino acid residues involved in these interactions.
Also of note is the fact that CD22 itself is the main component in the high-molecular-weight immunoprecipitated complexes both in Daudi cells and in transfected J2-44 cells (60% as calculated by fluorescence intensity). This fits with previous findings suggesting that CD22 may form multimers (Powell et al., 1995
). If such multimers do exist, the fact that the high-molecular-weight complexes were seen even with the arginine-mutated CD22 indicates again that proteinprotein interactions rather than Sia recognition are primarily involved in generating them. Of course, considering the relatively low single-site affinity of CD22 for Sias, a multimeric complex mediated by proteinprotein interactions would facilitate any secondary binding avidity via Sias and/or facilitate possible self-masking due to the
2-6-linked Sias carried by N-glycans on CD22 itself. Because of the very high molecular weight of all the cross-linked complexes, we could not accurately analyze whether such CD22 clustering is further assisted by Sia-dependent interactions.
In the second part of the article we tested an alternative hypothesis regarding how cis binding might affect CD22 function. The question we ask is whether the Sia-based interactions of CD22 with other cell surface molecules might restrict its ability to be cleared from the cell surface by endocytosis. We found that although Sia did not affect constitutive CD22 endocytosis and turnover, it did have some effect on antibody-induced CD22 endocytosis. Removing cell surface Sia enhances the rate of antibody-induced CD22 endocytosis. Although the overall effect is modest, it is reproducible and could be of practical relevance in the use of immunotoxins for treatment of lymphomas, where the therapeutic margin (efficacy over toxicity) is likely to be narrow. The transfected J2-44 cells discussed could not be used for a similar study, because toxin sensitivity is critically dependent on cell surface target density, and it was difficult to precisely match the cell surface expression of the wild-type and arginine-mutated CD22.
It is possible that antibody-induced endocytosis is representative of the situation when CD22 is being cross-linked, for example, by natural trans ligands after exposure during activation. More work needs to be done to explore this potential connection. Beyond this possibility, the critical role of Sia recognition by CD22 in controlling B cell activation state remains unexplained. Finally, we found that anti-IgM cross-linking, a process involved not only in BCR activation but also in CD22 unmasking, results in a slower rate of antibody-induced CD22 turnover and a less efficient CD22 immunotoxin killing. Whether these effects are due to cell activation, stoichiometric change in associated protein partners, or other mechanisms is not clear. Further studies on this topic are needed to better understand the relationship between BCR activation state and CD22 turnover, and the results could be relevant to clinical usage of CD22 immunotoxin.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Simultaneous cross-linking and biotinylation and CD22 immunoprecipitation
DTSSP and EZ-Link Sulfo-NHS-Biotin were purchased from Pierce (Rockford, IL). Daudi cells (10 x 106) or transfected J2-44 cells were harvested, washed three times in ice-cold phosphate-buffered saline (PBS), pH 8.0, and resuspended to 25 x 106 cells/ml in ice-cold PBS, pH 8.0, containing 1 mM each freshly prepared DTSSP and Sulfo-NHS-Biotin. Cross-linking and biotinylation were allowed to proceed at 4°C for 30 min with gentle shaking. Cells were then pelleted and washed three times in ice-cold PBS, pH 8.0. The pellet was lysed in lysis buffer (PBS, 1% NP-40, 1% Triton X-100, 1 mM ethylenediamine tetra-acetic acid, 2 µg/ml each of leupeptin, aprotinin, and pepstatin A) for 30 min at 4°C with inverting. Supernatants were collected after centrifuging for 30 min at 20,000 x g at 4°C. Aliquots of the lysate were precleared with protein ASepharose (Amersham Biosciences, Little Chalfont, UK) for 1 h at 4°C on a rotating wheel, and protein quantity was determined using the BCA protein assay kit (Pierce). The precleared lysate was subjected to immunoprecipitation with protein ASepharose conjugated with antibodies to CD22 (clone To15, Dako, Denmark), IgM (clone G20-127, BD Biosciences Pharmingen, San Diego, CA), CD45 (clone HI30, BD Biosciences Pharmingen) or isotype-matched mouse IgG (BD Biosciences Pharmingen) as control. Immunoprecipitation was done at 4°C for 2 h on a rotating wheel, and the beads were washed three times with lysis buffer and one time with PBS. Immunoprecipitates were boiled in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) sample buffer with or without 5% ß-mercaptoethanol (2-ME) for 5 min, run on 7.5% SDSPAGE gels, transferred, blocked, and stained by 1:300 Cy5-conjugated streptavidin (Jackson Immuno Research, West Grove, PA). Visualization and quantification were performed using Storm 860 scanner (Amersham Pharmacia Biotech) and ImageQuant software.
Sialidase treatment and BCR stimulation
Arthrobacter ureafaciens sialidase (Sigma, St. Louis, MO) 10 mU was used to unmask CD22 on 1 x 106 CD22- expressing cells. For 2-6 Sia probe binding experiments, sialidase pretreatment was performed in sialidase treatment buffer (20 mM HEPES, 140 mM NaCl) at room temperature for 15 min with shaking. Cells were washed four times in FACS staining buffer (PBS containing 1% bovine serum albumin, 0.02% sodium azide) before staining with the probe. For simultaneous cross-linking and biotinylation of Daudi cells following sialidase treatment, sialidase was used in sialidase treatment buffer for 1 h at 4°C with gentle shaking. For the other experiments, sialidase was added into serum-free culture media at the same concentration. For BCR stimulation, F(ab')2 fragments of goat anti-human IgM, Fc5u specific antibody (Jackson ImmunoResearch) were added to the cell culture media at 1 µg/ml.
Antibody-induced CD22 turnover assay
The whole experiment was done at 4°C unless otherwise indicated. Cells were pelleted, washed with serum-free AIM-V media, and resuspended at 10 x 106/ml in AIM-V media containing 1:100 fluorescein isothiocyanate (FITC)-conjugated anti-human CD22 (clone 4KB128, from Dako). The incubation was done in the dark for 30 min with gentle shaking. Cells were then washed three times to remove unbound antibody and resuspended in culture media with or without serum, depending on the experiment. After being incubated at 37°C for 090 min, 10 volumes of ice-cold media were added to the cells, which were then washed twice, followed by incubation with 0.2 M glycine, pH 2.5, for 5 min to remove any remaining surface-bound antibody. Cells were then washed, and subjected to flow cytometric analysis. FACSCalibur (BD Biosciences) and Flowjo software were used to collect and analyze the data.
Flow cytometric analysis on transfected J2-44 cells
Cells (1 x 106) were collected, washed, and suspended in FACS staining buffer (PBS containing 1% bovine serum albumin, 0.02% sodium azide). 1:100 FITC-conjugated anti-human CD22 (clone 4KB128, from Dako) was used to stain cell surface CD22 for 1 h at 4°C. 2-6-Sialyllactose-PAA-biotin (biotinylated polyacrylamide arrays from GlycoTech [Rockville, MD], carrying multiple copies of
2-6-sialyllactose and biotin) was used to detect Sia binding by CD22.
2-6-Sialyllactose-PAA-biotin (10 µg/ml) was incubated with cells for 1 h at 4°C, washed, and followed by 1:100 R-phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch) staining for 30 min at 4°C. FACSCalibur (BD Biosciences) and Flowjo software were used to collect and analyze the data.
Immunotoxin killing assay
CD22 immunotoxin (RFB4-dgRTA) was a kind gift from Dr. Ellen S. Vitetta (Shen et al., 1988). Cells (5 x 104) were incubated with RFB4-dgRTA 1012 to 109 M and cultured at 37°C for 2448 h in 96-well plate in triplicate. Cells were harvested, washed, and resuspended in PBS containing 1% bovine serum albumin, 0.02% sodium azide. Propidium iodide (Roche, Indianapolis, IN) was added to the suspension immediately before analysis to a final concentration of 2 µg/ml. Samples were subjected to flow cytometric analysis.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chan, C.H., Wang, J., French, R.R., and Glennie, M.J. (1998) Internalization of the lymphocytic surface protein CD22 is controlled by a novel membrane proximal cytoplasmic motif. J. Biol. Chem., 273, 2780927815.
Chan, C.S., Soehnlen, F., and Schechter, G.P. (1990) Differential response of malignant human B-cells to anti-IgM immunoglobulin (anti-mu) and B-cell growth factor: unique direct cytotoxicity of anti-mu on prolymphocytic leukemia cells. Blood, 76, 16011606.[Abstract]
Chaouchi, N., Vazquez, A., Galanaud, P., and Leprince, C. (1995) B cell antigen receptor-mediated apoptosis: Importance of accessory molecules CD19 and CD22, and of surface IgM cross-linking. J. Immunol., 154, 30963104.
Collins, B.E., Blixt, O., Bovin, N.V., Danzer, C.P., Chui, D., Marth, J.D., Nitschke, L., and Paulson, J.C. (2002) Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: novel sialoside probe for murine CD22. Glycobiology, 12, 563571.
Collins, B.E., Blixt, O., DeSieno, A.R., Bovin, N., Marth, J.D., and Paulson, J.C. (2004) Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact. Proc. Natl Acad. Sci. USA, 101, 61046109.
Crocker, P.R. and Varki, A. (2001) Siglecs, sialic acids and innate immunity. Trends Immunol., 22, 337342.[CrossRef][ISI][Medline]
Cyster, J.G. and Goodnow, C.C. (1997) Tuning antigen receptor signaling by CD22: integrating cues from antigens and the microenvironment. Immunity, 6, 509517.[ISI][Medline]
Doody, G.M., Justement, L.B., Delibrias, C.C., Matthews, R.J., Lin, J., Thomas, M.L., and Fearon, D.T. (1995) A role in B cell activation for CD22 and the protein tyrosine phosphatase SHP. Science, 269, 242244.[ISI][Medline]
Engel, P., Nojima, Y., Rothstein, D., Zhou, L.J., Wilson, G.L., Kehrl, J.H., and Tedder, T.F. (1993) The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes. J. Immunol., 150, 47194732.
Engel, P., Wagner, N., Miller, A.S., and Tedder, T.F. (1995) Identification of the ligand-binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid-dependent ligand. J. Exp. Med., 181, 15811586.[Abstract]
Hanasaki, K., Powell, L.D., and Varki, A. (1995a) Binding of human plasma sialoglycoproteins by the B cell-specific lectin CD22. Selective recognition of immunoglobulin M and haptoglobin. J. Biol. Chem., 270, 75437550.
Hanasaki, K., Varki, A., and Powell, L.D. (1995b) CD22-mediated cell adhesion to cytokine-activated human endothelial cells. Positive and negative regulation by alpha26-sialylation of cellular glycoproteins. J. Biol. Chem., 270, 75337542.
Hennet, T., Chui, D., Paulson, J.C., and Marth, J.D. (1998) Immune regulation by the ST6Gal sialyltransferase. Proc. Natl Acad. Sci. USA, 95, 45044509.
Herrera, L., Farah, R.A., Pellegrini, V.A., Aquino, D.B., Sandler, E.S., Buchanan, G.R., and Vitetta, E.S. (2000) Immunotoxins against CD19 and CD22 are effective in killing precursor-B acute lymphoblastic leukemia cells in vitro. Leukemia, 14, 853858.[CrossRef][ISI][Medline]
Herrera, L., Yarbrough, S., Ghetie, V., Aquino, D.B., and Vitetta, E.S. (2003) Treatment of SCID/human B cell precursor ALL with anti-CD19 and anti-CD22 immunotoxins. Leukemia, 17, 334338.[CrossRef][ISI][Medline]
Jin, L., McLean, P.A., Neel, B.G., and Wortis, H.H. (2002) Sialic acid binding domains of CD22 are required for negative regulation of B cell receptor signaling. J. Exp. Med., 195, 11991205.
John, B., Herrin, B.R., Raman, C., Wang, Y.N., Bobbitt, K.R., Brody, B.A., and Justement, L.B. (2003) The B cell coreceptor CD22 associates with AP50, a clathrin-coated pit adapter protein, via tyrosine-dependent interaction. J. Immunol., 170, 35343543.
Kelm, S., Pelz, A., Schauer, R., Filbin, M.T., Tang, S., De, B.M.-E., Schnaar, R.L., Mahoney, J.A., Hartnell, A., Bradfield, P., and Crocker, P.R. (1994) Sialoadhesin, myelin-associated glycoprotein and CD22 define a new family of sialic acid-dependent adhesion molecules of the immunoglobulin superfamily. Curr. Biol., 4, 965972.[ISI][Medline]
Kelm, S., Gerlach, J., Brossmer, R., Danzer, C.P., and Nitschke, L. (2002) The ligand-binding domain of CD22 is needed for inhibition of the B cell receptor signal, as demonstrated by a novel human CD22-specific inhibitor compound. J. Exp. Med., 195, 12071213.
Law, C.-L., Sidorenko, S. P., and Clark, E.A. (1994) Regulation of lymphocyte activation by the cell-surface molecule CD22. Immunol. Today, 15, 442449.[CrossRef][ISI][Medline]
Leprince, C., Draves, K.E., Geahlen, R.L., Ledbetter, J.A., and Clark, E.A. (1993) CD22 associates with the human surface IgM-B-cell antigen receptor complex. Proc. Natl Acad. Sci. USA, 90, 32363240.[Abstract]
Mansfield, E., Amlot, P., Pastan, I., and Fitzgerald, D.J. (1997) Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors. Blood, 90, 20202026.
Messmann, R.A., Vitetta, E.S., Headlee, D., Senderowicz, A.M., Figg, W.D., Schindler, J., Michiel, D.F., Creekmore, S., Steinberg, S.M., Kohler, D., and others. (2000) A phase I study of combination therapy with immunotoxins IgG-HD37-deglycosylated ricin A chain (dgA) and IgG-RFB4-dgA (Combotox) in patients with refractory CD19(+), CD22(+) B cell lymphoma. Clin. Cancer Res., 6, 13021313.
Nitschke, L., Carsetti, R., Ocker, B., Köhler, G., and Lamers, M.C. (1997) CD22 is a negative regulator of B-cell receptor signalling. Curr. Biol., 7, 133143.[ISI][Medline]
O'Keefe, T.L., Williams, G.T., Davies, S.L., and Neuberger, M.S. (1996) Hyperresponsive B cells in CD22-Deficient mice. Science, 274, 798801.
Otipoby, K.L., Andersson, K.B., Draves, K.E., Klaus, S.J., Farr, A.G., Kerner, J.D., Perlmutter, R.M., Law, C.L., and Clark, E.A. (1996) CD22 regulates thymus-independent responses and the lifespan of B cells. Nature, 384, 634637.[CrossRef][ISI][Medline]
Pagel, J.M., Matthews, D.C., Appelbaum, F.R., Bernstein, I.D., and Press, O.W. (2002) The use of radioimmunoconjugates in stem cell transplantation. Bone Marrow Transplant., 29, 807816.[CrossRef][ISI][Medline]
Peaker, C.J. and Neuberger, M.S. (1993) Association of CD22 with the B cell antigen receptor. Eur. J. Immunol., 23, 13581363.[ISI][Medline]
Powell, L.D. and Varki, A. (1994) The oligosaccharide binding specificities of CD22beta, a sialic acid-specific lectin of B cells. J. Biol. Chem., 269, 1062810636.
Powell, L.D., Sgroi, D., Sjoberg, E.R., Stamenkovic, I., and Varki, A. (1993) Natural ligands of the B cell adhesion molecule CD22beta carry N-linked oligosaccharides with alpha-2,6-linked sialic acids that are required for recognition. J. Biol. Chem., 268, 70197027.
Powell, L.D., Jain, R.K., Matta, K.L., Sabesan, S., and Varki, A. (1995) Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding. J. Biol. Chem., 270, 75237532.
Press, O.W., Farr, A.G., Borroz, K.I., Anderson, S.K., and Martin, P.J. (1989) Endocytosis and degradation of monoclonal antibodies targeting human B-cell malignancies. Cancer Res., 49, 49064912.[Abstract]
Razi, N. and Varki, A. (1998) Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes. Proc. Natl Acad. Sci. USA, 95, 74697474.
Razi, N. and Varki, A. (1999) Cryptic sialic acid binding lectins on human blood leukocytes can be unmasked by sialidase treatment or cellular activation. Glycobiology, 9, 12251234.
Sato, S., Miller, A.S., Inaoki, M., Bock, C.B., Jansen, P.J., Tang, M.L.K., and Tedder, T.F. (1996) CD22 is both a positive and negative regulator of B lymphocyte antigen receptor signal transduction: Altered signaling in CD22-deficient mice. Immunity, 5, 551562.[ISI][Medline]
Schulte, R.J., Campbell, M.A., Fischer, W.H., and Sefton, B.M. (1992) Tyrosine phosphorylation of CD22 during B cell activation. Science, 258, 10011004.[ISI][Medline]
Sgroi, D., Varki, A., Braesch-Andersen, S., and Stamenkovic, I. (1993) CD22, a B cell-specific immunoglobulin superfamily member, is a sialic acid-binding lectin. J. Biol. Chem., 268, 70117018.
Shan, D. and Press, O.W. (1995) Constitutive endocytosis and degradation of CD22 by human B cells. J. Immunol., 154, 44664475.
Shen, G.L., Li, J.L., Ghetie, M.A., Ghetie, V., May, R.D., Till, M., Brown, A.N., Relf, M., Knowles, P., Uhr, J.W. and others. (1988) Evaluation of four CD22 antibodies as ricin A chain-containing immunotoxins for the in vivo therapy of human B-cell leukemias and lymphomas. Int. J. Cancer, 42, 792797.[ISI][Medline]
Stamenkovic, I., Sgroi, D., Aruffo, A., Sy, M.S., and Anderson, T. (1991) The B lymphocyte adhesion molecule CD22 interacts with leukocyte common antigen CD45RO on T cells and alpha 26 sialyltransferase, CD75, on B cells. Cell, 66, 11331144.[ISI][Medline]
Sykes, D.B. and Kamps, M.P. (2001) Estrogen-dependent E2a/Pbx1 myeloid cell lines exhibit conditional differentiation that can be arrested by other leukemic oncoproteins. Blood, 98, 23082318.
Tedder, T.F., Tuscano, J., Sato, S., and Kehrl, J.H. (1997) CD22, A B lymphocyte-specific adhesion molecule that regulates antigen receptor signaling. Annu. Rev. Immunol., 15, 481504.[CrossRef][ISI][Medline]
Torres, R.M., Law, C.L., Santos-Argumedo, L., Kirkham, P.A., Grabstein, K., Parkhouse, R.M., and Clark, E.A. (1992) Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule. J. Immunol., 149, 26412649.
Tuscano, J.M., O'Donnell, R.T., Miers, L.A., Kroger, L.A., Kukis, D.L., Lamborn, K.R., Tedder, T.F., and DeNardo, G.L. (2003) Anti-CD22 ligand-blocking antibody HB22.7 has independent lymphomacidal properties and augments the efficacy of 90Y- DOTA-peptide-Lym-1 in lymphoma xenografts. Blood, 101, 36413647.
Van der Merwe, P.A, Crocker, P.R., Vinson, M., Barclay, A.N., Schauer, R., and Kelm, S. (1996) Localization of the putative sialic acid-binding site on the immunoglobulin superfamily cell-surface molecule CD22. J. Biol. Chem., 271, 92739280.
Wilson, G.L., Fox, C.H., Fauci, A.S., and Kehrl, J.H. (1991) cDNA cloning of the B cell membrane protein CD22: a mediator of B-B cell interactions. J. Exp. Med., 173, 137146.[Abstract]