Fig. 3. Acetylated sialic acid localizes to Fab N-glycans.
(a) Percentage IgG2 conserved Fc N-glycan sites with each indicated glycoform. (b) Relative mass peak area for Acetyl-Neu5Gc determined by tandem mass spectroscopy oxonium ion filtering on three unique Fab glycopeptides from Lm-specific IgG purified from virgin or pregnant mice. (c) Fold-reduction in Acetyl-Neu5Gc for each Fab glycopeptide in pregnant compared with virgin Lm-specific IgG described in panel b. (d) Fold increase in Acetyl-Neu5Gc after pepsin treatment for Fab glycopeptides relative to untreated full-length IgG. (e, f) Bacterial burdens (e) and anti-Lm F(ab’)2 titers (f) after virulent Lm infection in neonatal mice transferred pepsin-treated IgG from: naive mice [nF(ab’)2], pregnant/postpartum Lm-primed mice [pF(ab’)2], or virgin Lm-primed mice [vF(ab’)2] treated with SIAE to remove acetylation from sialic acid residues. Pups were infected with virulent Lm 4 days after birth, 24 hours after F(ab’)2 transfer, with enumeration of bacterial burden 72 hours post-infection. Mass spectroscopy experiments were performed 3-4 times using purified IgG pooled from virgin or pregnant mice and data from each replicate experiment is shown (a-d). For infection and anti-Lm titers, each symbol represents an individual mouse, with graphs showing data combined from at least 2 independent experiments each with 3-5 mice per group per experiment (e, f). Bar, mean ± standard error. P values between key groups are shown as determined by one-way ANOVA adjusting for multiple comparisons (e, f) or unpaired t-test (c, d).