Abstract
Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes, EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study, we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans, whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity, in combination with the IdeS protease, and developed a LC separation method to quantify high mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs and that this can be used for rapid quantification of high mannose content.
Highlights
Bacterial interaction with host glycosylation is widespread, and a vast number of bacteria use enzymes for modulation of the immune system or nutrient acquisition (Garbe and Collin 2012; Sjögren and Collin2014)
The antibodies were incubated with EndoS, EndoS2 or phosphate-buffered saline (PBS) and analyzed on nonreducing sodium dodecyel sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after proteolytic cleavage with the IgG-specific protease immunoglobulin G degrading enzyme of Streptococcus pyogenes (IdeS), which generated F(ab′)2 and IdeS-cleaved fragment crystallizable (Fc) fragments (Figure 1)
It was clear that EndoS and EndoS2 had activity on all the selected antibodies, the degree of hydrolysis differed between the enzymes
Summary
Bacterial interaction with host glycosylation is widespread, and a vast number of bacteria use enzymes for modulation of the immune system or nutrient acquisition (Garbe and Collin 2012; Sjögren and Collin2014). Two enzymes that recently have attracted attention for glycoengineering of therapeutic antibodies are EndoS and EndoS2 from the human pathogen Streptococcus pyogenes (Collin and Olsén 2001; Sjögren et al 2013). Immune evasion factors that abolish the effector functions of immunoglobulin G (IgG) by hydrolyzing N-linked glycans on the antibody (Collin et al 2002; Sjögren et al 2011). IgG carries one complex N-linked oligosaccharide on each CH2 domain, and this glycan is crucial for the structure of the Fc region and the interaction with the Fc receptors (Krapp et al 2003; Woof and Burton 2004). The enzymes are only 37% identical, both EndoS and EndoS2 catalyze the hydrolysis of the β-1,4 linkage between the two N-acetylglucosamines (GlcNAcs) in the core of the N-linked glycan of human IgG. EndoS2 was found to cleave biantennary sialylated glycans of the acute-phase protein α1-acid glycoprotein (Sjögren et al 2013)
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