B-cell-independent sialylation of IgG

Abstract

IgG carrying terminal α2,6-linked sialic acids added to conserved N-glycans within the Fc domain by the sialyltransferase ST6Gal1 accounts for the anti-inflammatory effects of large-dose i.v. Ig (IVIg) in autoimmunity. Here, B-cell-specific ablation of ST6Gal1 in mice revealed that IgG sialylation can occur in the extracellular environment of the bloodstream independently of the B-cell secretory pathway. We also discovered that secreted ST6Gal1 is produced by cells lining central veins in the liver and that IgG sialylation is powered by serum-localized nucleotide sugar donor CMP-sialic acid that is at least partially derived from degranulating platelets. Thus, antibody-secreting cells do not exclusively control the sialylation-dependent anti-inflammatory function of IgG. Rather, IgG sialylation can be regulated by the liver and platelets through the corresponding release of enzyme and sugar donor into the cardiovascular circulation.

Keywords: B cell; IVIg; IgG; sialylation; sialyltransferase.

Fig. 1.

B-cell–specific, ST6Gal1-deficient cKO mice have normal IgG sialylation. (A) Cell-surface glycosylation of circulating lymphocytes was validated using α2,6 sialic-acid–specific (SNA) and terminal galactose-specific (ECL) lectins by flow cytometry with gated CD19⁺ cell histograms shown in B and the percentage of CD19⁺SNA^hi cells in wild-type and cKO mice shown in C (n = 10). (D) Total splenic CD19⁺ cells were quantified to confirm the lack of a B-cell developmental defect, and (E) CD138⁺B220^lo long-lived plasma cell flow cytometry from the bone marrow confirmed that antibody-producing cells were similar to circulating CD19⁺ B cells in glycosylation. Serum IgG from untreated animals was separated by SNA chromatography and quantified by concentration (F) and percentage (G) of sialylated IgG from wild type (n = 12) compared with cKO (n = 23). (H) LC-MS analysis of serum IgG glycans from wild-type and cKO animals (Top) with the percentage of each glycan calculated and compared for 8- and 52-wk-old animals (Bottom). All observed sialic acids in MS structures were α2,6-linked. Mean ± SEM and P values are from a two-sided t test: *P < 0.05, ØP > 0.05.

Fig. 2.

OVA-specific IgG sialylation in mice with wild-type and ST6Gal1 cKO B cells is indistinguishable. Wild-type and cKO mice were injected i.p. with 50 µg of OVA/Alum and boosted at days 14 and 28 to measure IgG sialylation in response to immunization. Total IgG sialylation by concentration (A) or percentage (B) from serum of preimmunization (total: wild type, n = 6; cKO, n = 15) and postimmunization (hatched bars; wild type, n = 12; cKO, n = 18) was quantified on day 42. The percentage of IgG that was OVA-specific was also quantified (C), as was OVA-specific IgG sialylation, shown by concentration (D) and percentage of total (E). Mean ± SEM and P values are two-sided t test: *P < 0.05, ØP > 0.05.

Fig. 3.

Adoptive transfer of cKO B cells into B-cell–deficient recipients yields normal IgG sialylation. FACS sorted CD19⁺SNA^hi wild-type or CD19⁺SNA^lo cKO cells from OVA-immunized animals were transferred i.v. into CD19-CRE^+/+ B-cell–deficient mice. After 24 h, recipients were boosted with OVA/Alum. Total IgG sialylation (wild type, n = 6; cKO, n = 15) by concentration (A) and percentage (B) from recipient serum is shown, as is OVA-specific IgG (wild type, n = 12; cKO, n = 18) by concentration (C) and percentage (D). (E) Flow analysis of B cells (CD19) and sialylation status (SNA) from immunized wild-type and cKO mice before flow sorting and transfer into B-cell–deficient recipients. (F) Flow analysis of IgD⁺ memory B-cell sialylation by SNA staining in recipients following immunization. Mean ± SEM and P values are two-sided t test: *P < 0.05, ØP > 0.05.

Fig. 4.

Secreted ST6Gal1 is predominantly expressed by the liver. (A) Total organ lysates were Western-blotted with anti-ST6Gal1 (red; Left) and SNA (green; Middle) and merged on Right. (B) Confocal microscopy (10×) of liver sections from untreated wild-type mice stained with anti-ST6Gal1 (red; Left) and SNA (green; Middle panels) and merged on Right. (C) A 30± image of a central (CV) and portal (PV) vein. Further magnified images of central (D) and portal (E) veins demonstrating ST6Gal1 fine localization, including luminal vein wall visualization by 3D reconstruction of z-axis stacks (Bottom Left) and rotated 45° (Bottom Right). Images are representative of at least five mice.

Fig. 5.

Secreted ST6Gal1 is concentrated near hepatic central veins. Laser capture microdissected liver tissue from central (CV) and portal (PV) veins was probed for ST6Gal1 mRNA levels by qRT-PCR (n = 3) in comparison with GAPDH and normalized to the portal vein (A). (B) Extracts were also Western-blotted and quantified by fluorescence for ST6Gal1 protein and molecular mass. Mean ± SEM are reported.

Fig. 6.

ST6Gal1 is active in the circulation. (A) After removal of sialic acids by mild acid hydrolysis, serum proteins were blotted and incubated with recombinant ST6Gal1 (rST6) or raw serum with and without 100 µM CMP-SA and stained with SNA (green) or MAL-I (red) to quantify α2,6- and α2,3-linked sialic acid products, respectively. (B) rST6-mediated sialylation of immobilized and neuraminidase (NA)-treated fetuin and its inhibition by CMP, all quantified by SNA staining (n = 3). (C) Untreated and heat-inactivated (Inact.) serum-mediated sialylation of immobilized and NA-treated fetuin with and without competitive inhibition by CMP (n = 3). (D) Untreated (Unt) and NA-treated (NA) glycan array chips from the Consortium for Functional Glycomics were stained with fluorescent SNA as a baseline. NA-treated arrays were incubated with rST6 and CMP-SA (rST6), mouse serum, or serum with CMP as an inhibitor and then quantified with SNA. The top nine glycan targets after serum incubation are shown with the corresponding glycan structures (n = 2 arrays with three internal replicates per array). (E) Similarity of serum and rST6 specificity were analyzed by Pearson correlation of SNA signals from all array glycans. Mean ± SEM and P values are two-sided t test: *P < 0.05, ØP > 0.05.

Fig. 7.

CMP-SA from platelet granules fuels ST6Gal1 reactions. (A) Serum from two independent resting mice was inactivated and then used with rST6 to quantify the concentration CMP-SA within each serum sample by quantifying the liberated CMP following incubation with rST6 as described in Materials and Methods (n = 5 for each serum; ***P < 0.0008; **P < 0.008). (B) rST6 sialylation of immobilized NA-treated fetuin using platelet releaseates as a source of CMP-SA was quantified by adding either resting or thrombin-activated platelet supernatant (PS) at physiological blood volumes with and without CMP inhibition (n = 3). (C) Percentage of SNA⁻ and SNA⁺ IgG from resting platelet degranulation-deficient Tomosyn^−/− (Mut) compared with wild-type controls (n = 4). Mean ± SEM and P values are two-sided t test: *P < 0.05, ØP > 0.05.