Features of N-Glycosylation of Immunoglobulins from Knockout Pig Models

For the first time, the N-glycosylation patterns of immunoglobulin G (IgGs) isolated from the serum of two varieties of knockout pigs (lacking N-glycolylneuraminic acid (Neu5Gc) and/or α 1,3 galactose) were examined for the presence of potential glycan xenoantigens and compared to N-glycosylation patterns obtained for wild-type (WT) pig IgGs.

Glycopeptide analysis was chosen over glycan release, as protein-A eluates from pig serum may contain IgA and IgM as shown previously. The experiments focused on the analysis of tryptic glycopeptides EEQFNSTYR and AEQFNSTYR from IgGs, and excluded IgA and IgM, in which N-glycosylated peptides have different sequences and masses. WT pig IgG glycopeptides showed the presence of N-glycolylneuraminic acid (Neu5Gc) and absence of N-acetylneuraminic acid (Neu5Ac). Released glycans from the protein-A eluate, however, showed the presence of both types of sialic acids, allowing Neu5Ac to be attributed to IgA and/or IgM.

The WT IgG samples also showed the presence of glycans that could by composition have been α-galactosylated, but treatments with α- and β-galactosidases produced inconclusive results as to the linkage nature of the terminal Gal residues. Single knockout (α-Gal transferase) pig IgG was shown to contain Neu5Gc residues, and there was a definite absence of α-Gal.