Post-HDX Deglycosylation of Fc Gamma Receptor IIIa Glycoprotein Enables HDX Characterization of Its Binding Interface with IgG

J Am Soc Mass Spectrom. 2021 Jul 7;32(7):1638-1643. doi: 10.1021/jasms.1c00003. Epub 2021 Feb 24.

Abstract

Protein glycosylation is a common and highly heterogeneous post-translational modification that challenges biophysical characterization technologies. The heterogeneity of glycoproteins makes their structural analysis difficult; in particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) often suffers from poor sequence coverage near the glycosylation site. A pertinent example is the Fc gamma receptor RIIIa (FcγRIIIa, CD16a), a glycoprotein expressed on the surface of natural killer cells (NK) that binds the Fc domain of IgG antibodies as a trigger for antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we describe an adaptation of a previously reported method using PNGase A for post-HDX deglycosylation to characterize the binding between the highly glycosylated CD16a and IgG1. Upon optimization of the method to improve sequence coverage while minimizing back-exchange, we achieved coverage of four of the five glycosylation sites of CD16a. Despite some back-exchange, trends in HDX are consistent with previously reported CD16a/IgG-Fc complex structures; furthermore, binding of peptides covering the glycosylated asparagine-164 can be interrogated when using this protocol, previously not seen using standard HDX-MS.

MeSH terms

  • Binding Sites, Antibody
  • Glycoproteins / chemistry
  • Glycoproteins / metabolism
  • Glycosylation
  • Humans
  • Hydrogen Deuterium Exchange-Mass Spectrometry / methods*
  • Immunoglobulin G* / chemistry
  • Immunoglobulin G* / metabolism
  • Models, Molecular
  • Receptors, IgG* / chemistry
  • Receptors, IgG* / metabolism

Substances

  • Glycoproteins
  • Immunoglobulin G
  • Receptors, IgG