1. Glycosylation of recombinant rabbit immunoglobulins influences protease susceptibility as shown by comprehensive mass spectrometric glycan analysis.
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Friligou, Irene, Gassner, Jana, Knoblauch, Dominic, Kagerer, Gabriele, Popp, Franziska, Voit, Susanne, Engel, Alfred M, Leinenbach, Andreas, Steffen, Wojtek, Haberger, Markus, and Tabarés, Glòria
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RECOMBINANT antibodies , *GLYCANS , *GLYCOSYLATION , *STERIC hindrance , *GENETIC engineering , *THREONINE , *IMMUNOGLOBULIN G - Abstract
Recombinant immunoglobulins (rIgGs) have become increasingly important as therapeutic agents and diagnostic tools in recent years. Genetic engineering allows the introduction of non-natural features such as the Sortase motif for site-directed labeling. In this study, the enzyme Sortase A (SrtA) was used for the proteolytic cleavage of rIgGs to produce their biotinylated Fab fragments by locating the cleavage site close to the hinge region. However, SrtA cleavage of engineered rabbit IgGs (rRb-IgGs) derived from human embryonic kidney (HEK) 293 cells showed significantly lower yields compared with their mouse counterparts. Nonrecombinant Rb-IgGs have N- and O-glycans, and the presence of O-glycans close to the hinge region of the rRb-IgGs might affect the susceptibility of these antibodies to SrtA cleavage. In addition, the glycosylation pattern of rIgGs differs depending on the host cell used for expression. Therefore, we analyzed the N- and O-glycans of various rRb-IgGs expressed in HEK293 cells, detecting and quantifying 13 different N-glycan and 3 different O-glycan structures. The distribution of the different detected glycoforms in our rRb-IgG N-glycan analysis is in agreement with previous studies on recombinant human IgG N-glycans, confirming the hypothesis that the host cell defines the glycosylation of the recombinant produced IgGs. O-glycosylation could be mapped onto the threonine residue within the hinge region sequence XPTCPPPX, as already described previously for nonrecombinant Rb-IgGs. Substitution of this threonine allowed an almost complete Fab fragment cleavage. Therefore, we could confirm the hypothesis that the O-glycans affect the SrtA activity, probably due to steric hindrance. [ABSTRACT FROM AUTHOR] more...
- Published
- 2021
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