Format

Send to

Choose Destination
J Immunol Methods. 2019 Apr;467:58-62. doi: 10.1016/j.jim.2019.02.005. Epub 2019 Feb 8.

The enzymatic removal of immunoglobulin variable domain glycans by different glycosidases.

Author information

1
Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands; Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands. Electronic address: sannevandebovenkamp@hotmail.com.
2
Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands; Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Abstract

About 15% of immunoglobulin G (IgG) molecules contain glycans linked to the antigen-binding fragments (Fab arms) in addition to the glycans linked to the crystallizable fragment (Fc tail) of all IgGs. Fab glycosylation appears to be an important feature of antibodies, for example by influencing antigen binding and antibody stability. The reliable generation of antibodies that either have or lack Fab glycans would be very helpful to study the role of Fab glycans in more detail. In this study, we set out to remove Fab glycans by treating polyclonal and monoclonal human IgG antibodies with two commonly used glycosidases and an improved version of one of the two (Endo F3, PNGase F, and Rapid™ PNGase F). Fc glycans can be removed using PNGase F and Rapid™ PNGase F, but not with Endo F3. For most antibody clones, Endo F3 partially cleaved off the Fab glycans. In contrast, PNGase F left the Fab glycans of most clones unaffected, but could remove glycans of some clones. Rapid™ PNGase F showed a higher glycosidase efficacy than PNGase F, and more clones could be deglycosylated using this enzyme. In summary, not all Fab glycans can be cleaved off by the tested glycosidases (under non-denaturing conditions), suggesting that Fab glycans are exposed to different degrees.

PMID:
30742813
DOI:
10.1016/j.jim.2019.02.005

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center