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

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.