Germinal center marker GL7 probes activation-dependent repression of N-glycolylneuraminic acid, a sialic acid species involved in the negative modulation of B-cell activation

Abstract

Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the alpha2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing alpha2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.

FIG. 1.

Involvement of Sia in the GL7 epitope. (A) GL7 staining in flow cytometry. Mouse B-cell lines (70Z/3, WEHI231, X16c8.5, and A20) and human B-cell lines (KMS-12 BM, KMS-12 PE, Daudi, and Ramos) were stained with FITC-conjugated GL7. Black solid lines indicate staining with GL7, and gray dashed lines indicate nonstaining controls. (B) The effect of sialidase treatment on GL7 staining. Daudi cells were treated with sialidase before staining with FITC-conjugated GL7, mSn-Fc, or hCD22-Fc. Gray dashed lines indicate negative controls (nonstaining for GL7 and R-PE-conjugated anti-human IgG for the others), and black thin lines indicate the results without sialidase treatment. Black bold lines indicate the results with A. ureafaciens sialidase treatment, and gray bold lines indicate results with S. enterica serovar Typhimurium sialidase treatment. Sialidase from A. ureafaciens releases α2-3,6,8-linked Sia, whereas sialidase from S. enterica serovar Typhimurium is specific to the α2-3 linkage. To confirm the effect of sialidase treatment, changes in cell surface expression of α2,3-linked Sia and α2,6-linked Sia were detected with mSn-Fc and hCD22-Fc chimeric probes precomplexed with R-PE-conjugated anti-human IgG, respectively. (C and D) Effect of free sugars on GL7 binding. Daudi cells were stained with FITC-conjugated GL7 in the presence of 50 mM free sugars (C) or the indicated concentrations of Neu5Ac (D). The data are shown as the relative MFI of each staining. Gal, galactose; Glc, glucose; Fuc, fucose; GlcNAc, N-acetylglucosamine; GlcA, glucuronic acid. (E) GL7 blotting of human B-cell lines. Membrane fractions of human B-cell lines (KMS-12 BM, KMS-12 PE, Daudi, and Ramos) were analyzed by GL7 immunoblotting. The addition of 100 mM Neu5Ac during incubation with GL7 reduced most of the staining on blotted membranes.

FIG. 2.

Involvement of α2,6-linked Neu5Ac in the GL7 epitope. (A) Numerical comparison of GL7 staining among human B-cell lines. The results of GL7 staining of human B-cell lines were numerically compared using MFI values in flow cytometry. To normalize the binding in different cells, the endogenous fluorescence of sample cells (gray dashed lines) was adjusted to an MFI of around 5. For comparison with the gene expression profile, GL7-stained MFI values were divided by the background value. The relative values indicated on the top of each staining were used as the GL7 determinant expression profile. (B) Appearance of the GL7 determinant by ST6GAL1 expression. CHO-K1 clones stably transfected with rat St6gal1 or an empty vector (as a control) were stained with FITC-conjugated GL7 or FITC-conjugated SSA. The results from two such clones are shown. (C) Carbohydrate binding assay of GL7. Carbohydrate binding was measured using ELISA. Data are shown as the means of triplicate samples, and the bars represent standard errors of the mean. LSTa, Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc; LSTb, Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc; LSTc, Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc; GD3, Neu5Acα2-8Neu5Acα2-3Galβ1-4Glc; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glc; Neu5Ac α2-3, Neu5Acα2-3Galβ1-4Glc; Neu5Ac α2-6, Neu5Acα2-6Galβ1-4Glc; Neu5Gc α2-3, Neu5Gcα2-3Galβ1-4Glc.

FIG. 3.

Change in Sia species in germinal centers. (A) Structural differences between two major molecular species of Sia. The metabolic precursor Neu5Ac and its modified form Neu5Gc differ only by an oxygen atom at the C-5 position. The conversion of CMP-Neu5Ac to CMP-Neu5Gc is catalyzed by the enzyme Cmah. (B) Biosynthesis of sialylated glycoproteins destined for the cell surface. Cytosolic metabolism of Sia is responsible for the abundance of the molecular species of Sia on the cell surface, as a given ratio of cytosolic CMP-Sia is imported into the Golgi apparatus and then used by the sialyltransferases for the biosynthesis of glycoproteins en route to the plasma membrane. (C) Loss of CD22 ligand in germinal centers. Spleen sections of SRBC-immunized mice (10 days after immunization) were costained with FITC-conjugated GL7 and mCD22-Fc precomplexed with R-PE-conjugated anti-human IgG. The mCD22-Fc is a chimeric probe that binds to the CD22 ligand. Arrows indicate germinal centers. (D) Downregulation of Cmah expression in germinal center B cells. GL7-positive germinal center cells and GL7-negative cells were prepared from a B-cell-enriched fraction derived from the spleen of a mouse 12 days after immunization with SRBC. Ultracentrifugation supernatant fractions (cytosolic fractions) of untreated mouse B cells (nonimmunized; control), GL7-positive B cells (GL7+), and GL7-negative B cells (GL7−) were subjected to immunoblotting with anti-mouse Cmah antibody and antiactin antibody (to demonstrate equal loading of samples). The Neu5Gc/(Neu5Ac+Neu5Gc) ratio of the ultracentrifugation pellets (membrane fractions) of each cell type was measured by HPLC.

FIG. 4.

Downregulation of Cmah mRNA in primary cultured B cell blasts, causing GL7 epitope expression. (A and B) Cmah repression caused by in vitro B-cell activation. Splenic B cells were stimulated with 30 μg/ml LPS for the indicated times. Reverse-transcribed cDNAs prepared from total RNA of these cells were subjected to real-time PCR analysis. The right box shows capillary electrophoresis analysis results indicating the lack of RNA degradation in the RNA used for cDNA synthesis. The expression levels of the mRNA of Gapdh, Actb (beta actin), Cmah, Cd22, and St6gal1 are shown as the relative change compared with the mRNA expression in untreated B cells (A). The same set of cells that was used to prepare total RNA was stained with FITC-conjugated GL7, SSA, and anti-CD22 (B). The MFI of each stain is indicated at the right of each panel. (C) Reduced expression of the GL7 epitope by ectopic Cmah expression. Cmah was ectopically expressed in LPS-stimulated splenic B blasts using retrovirus. Retrovirus-infected cells were sorted and stained with FITC-conjugated GL7.

FIG. 5.

Generation and biochemical analyses of Cmah knockout mice. (A) Allele for targeted Cmah. A targeting vector was created by inserting the PGK-neoR cassette into the NspV site of the second coding exon (exon 5) of the Cmah gene. (B) Genotype of homologous recombination of selected ES cell lines. The genotypes of G418-selected cell lines were determined by Southern blotting analysis of genomic DNA digested with BglI, using both radiolabeled 5′ internal and 3′ external probes. The genetic status of the Cmah allele is indicated as follows: +/+, wild type; +/−, heterozygote; and −/−, null (B to F). (C) Loss of Cmah enzyme demonstrated by immunoblotting analysis of liver cytosolic fractions. Ultracentrifugation supernatant fractions of livers were assessed for the expression of Cmah using anti-mouse Cmah immunoblotting. Staining of a ∼67-kDa band (arrowhead) in wild-type and heterozygote livers represents the signal of Cmah, which is not detectable in Cmah-null liver samples. (D) Loss of Neu5Gc production throughout the body in mutated mice. Acid-hydrolyzed Sia from the indicated tissues was derivatized using DMB, and the ratios of Neu5Ac and Neu5Gc to total Sia were measured by reverse-phase HPLC. Solid columns represent the percentage of Neu5Gc in various tissues, and open columns represent the percentage of Neu5Ac. The detection limit for Neu5Gc in this assay was around 0.1%. (E) Flow cytometry profile of Cmah-null mice splenocytes. The expression of IgM, MHC-II (I-A and I-E), and CD22 on splenocytes from wild-type and Cmah-null mice was detected by flow cytometry. In anti-MHC-II and anti-CD22 staining, splenocytes were costained with anti-B220, a marker for B cells. (F) Strong expression of the GL7 epitope on Cmah-null mice B cells. Splenocytes from wild-type and Cmah-null mice were costained with anti-B220 and GL7 and subjected to flow cytometry.

FIG. 6.

Hyperresponsive phenotypes of Cmah-null mice. (A) T-independent hyperresponse of Cmah-null mice. DNP-Ficoll was used to immunize 8-week-old mice. Serum was collected each week and analyzed for reactivity with DNP-conjugated BSA coated on ELISA plates. The titer of hapten-reacting mouse Igs from each animal was determined by isotype-specific ELISA. The measured optical density at 405 nm was normalized to anti-DNP units by comparison with the value from standard pooled serum against DNP on the same plate. The results are presented as the mean responses of 10 animals for each genotype measured in two sets of experiments. The bars represent standard errors of the means. Open circles indicate the responses of wild-type mice, and filled diamonds indicate the responses of Cmah-null mice for each isotype. Genotypes are indicated as follows: +/+, wild-type; −/−, Cmah-null (A and B). (B) Normal T-dependent immune response of Cmah-null mice. DNP-KLH in complete Freund's adjuvant was used to immunize 8-week-old mice. The titers of hapten-reacting mouse Igs from each animal were determined by isotype-specific ELISA as above. Arrows indicate the time of secondary immunization with DNP-KLH. Open circles indicate the responses of wild-type mice, and filled diamonds indicate the responses of Cmah-null mice for each isotype. (C) In vitro hyperproliferation response of Cmah-null B cells. Splenic B cells from wild-type (open columns) and Cmah-null (filled columns) mice were assessed for proliferation using the F(ab′)₂ fragment of anti-mouse IgM (μ chain) or anti-IgM plus 2 ng/ml IL-4 as stimulating reagents. After stimulation for 24 h, BrdU was added. Following incubation overnight, incorporated BrdU was detected by ELISA. Data are shown as the means of triplicate cultures, and the bars represent standard errors of the means. The results shown here were obtained in one of the experiments using 10% FBS-containing medium.

FIG. 7.

Rescue of augmented proliferation of Cmah-null B cells by Cmah expression. (A) In vitro hyperproliferation response of Cmah-null B cells to LPS. Splenic B cells from wild-type (open columns) and Cmah-null mice (filled columns) were assessed for proliferation using LPS from S. enterica serovar Enteritidis as the stimulating reagent. Proliferation assays were performed as described in the legend of Fig. 6C. Data are shown as the means of triplicate cultures, and the bars represent standard errors of the means. (B) Reduction of B-cell proliferation by retrovirus-mediated Cmah expression. Cmah was ectopically expressed by mouse stem cell virus in Cmah-null splenic LPS B blasts. After being cultured for 2.5 days in the presence of 30 μg/ml LPS, the virus-infected B cells were subjected to a proliferation assay. As a control, cells were infected with an empty vector. Data are shown as the means of triplicate cultures, and the bars represent standard errors of the means.

FIG. 8.

Changes in staining of germinal center markers in normal or SRBC-immunized Cmah-null mice. (A) Histochemical analyses of spleen sections without immunization. Spleen sections from wild-type and Cmah-null mice were costained with FITC-conjugated GL7 and biotin-conjugated PNA visualized by R-PE-conjugated streptavidin. (B) Histochemical analyses of spleen sections after T-dependent immunization. Wild-type and Cmah-null mice were immunized with SRBC, and the spleens were removed 8 days after immunization. The frozen spleen sections were costained with FITC-conjugated GL7 and biotin-conjugated PNA followed by R-PE-conjugated streptavidin. Arrows indicate germinal centers. Genotypes are indicated as follows: +/+, wild type; −/−, Cmah null.

FIG. 9.

Loss of optimal CD22 ligand and normal immediate response upon BCR cross-linking in Cmah-null mice. (A) Loss of optimal ligand for CD22 in Cmah-null mice. The expression of surface ligands for sialoadhesin and CD22 was detected by flow cytometry. Splenocytes from wild-type and Cmah-null mice were costained with FITC-conjugated anti-B220 and mSn/mCD22-Fc precomplexed with R-PE-conjugated anti-human IgG. Wild-type B cells were strongly stained with mCD22-Fc. In contrast, the level of mCD22-Fc staining showed a marked decrease in Cmah-null mice. The weak signal found on Cmah-null splenocytes was detected only with the chimeric probe mCD22-Fc prepared from Lec2 cell culture medium and not with the probe prepared from COS7 cells, possibly because of the autosialylation. (B) Histochemical analyses of CD22 ligand expression in spleen sections. Spleen sections from wild-type and Cmah-null mice 8 days after SRBC immunization were costained with FITC-conjugated GL7 and mCD22-Fc precomplexed with R-PE-conjugated anti-human IgG. Arrows indicate germinal centers. (C) Overall tyrosine phosphorylation upon anti-IgM stimulation. Splenic B cells from wild-type and Cmah-null mice were stimulated with the F(ab′)₂ fragment of anti-mouse IgM (μ chain) for the indicated times. Whole-cell lysates were subjected to immunoblotting with antiphosphotyrosine antibody (PT-66). (D) Phosphorylation of CD22. Splenic B cells were stimulated with the F(ab′)₂ fragment of anti-mouse IgM (μ chain) for the indicated times. The cell lysates were subjected to immunoprecipitation with anti-CD22 antibody (Cy34.1). The precipitated proteins were immunoblotted with antiphosphotyrosine (pTyr) antibody (PT-66) and then reprobed with anti-CD22 polyclonal antibody. (E) In vitro hyperproliferation response of Cmah-null B cells to calcium signaling. Splenic B cells were assessed for proliferation using tetradecanoyl phorbol acetate (10 ng/ml) plus ionomycin (5 μg/ml) as stimulating reagents. The proliferation assay was performed as described in the legend of Fig. 6C. The open column represents the mean proliferation of wild-type B cells, and the filled column represents the mean proliferation of Cmah-null B cells. The bars represent the standard errors of the mean for triplicate cultures. +/+, wild type; −/−, Cmah null; IP, immunoprecipitation.