Figure 9.

Serum mannosidase activity may account for preferential targeting of glycan core by elicited antibodies. (a) Depiction of glycan residues preferentially targeted by the glycopeptide-elicited response. Antibodies elicited in this study bind preferentially to the cyclohexyl and core mannose residues of the glycopeptide immunogens, but not to the Manα1 → 2Man termini. (b) Hypothesis that serum mannosidase trimming of Manα1 → 2Man in vivo leads to lack of antibodies elicited against Manα1 → 2Man. Similar trimming of Man₉GlcNAc₂ on kif-Rituximab has been measured in vivo.⁶⁹ (c, d) Experiment to assess mannosidase trimming of 10F6 glycopeptide conjugate in serum. An alkyne-tagged version of the 10F6–CRM₁₉₇ glycoconjugate was incubated in serum, and aliquots were “quenched” by addition of mannosidase inhibitors kifunensine and swainsonine. Conjugate was retrieved from samples by “click” biotinylation, affinity capture on NeutrAvidin beads, and elution by dilute hydrazine cleavage of the linker. Trypsin digestion followed by nanoUPLC-MS and MS/MS enabled identification and relative quantification of glycoforms of 10F6 tryptic glycopeptides, showing cleavage of ca. 3 mannose residues per glycan after 2 days. Similar data for a different tryptic glycopeptide can be found in the SI, Figure S18 and Table S10.