Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function

The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding

“…This section contains a description of multiple methods available to obtain homogeneity that manipulate N-glycans during protein expression, post purification, or a combination of both. These methods have advanced glycoprotein studies, however, it is not always clear what types of glycans are found on glycoproteins in the human body, and proteins expressed by HEK293F cells do not necessarily contain the correct types of N-glycans, thus, caution is suggested (Patel, Roberts, Subedi, & Barb, 2018). Another aspect that is not addressed by any of these techniques is the accessibility of the N-glycosylation site to the oligosaccharyltransferase.…”

“…This technique requires a only small amount of glycoprotein (1-5 μg). In lieu of permethylation, N-glycans are derivatized at the reducing end by reacting with procainamide to form a Schiff base that is reduced with sodium cyanoborohydride (Patel et al, 2018). The reaction mixture is next directly injected onto a HILIC column (Waters) in 75% acetonitrile, 25% H 2 O, 0.1% formic acid and 0.01% trifluoroacetic acid, and eluted with an increasing linear water gradient.…”

Antibody receptors steal the sweet spotlight

“…It has been known for many years that the surface glycome of any given cell is driven by many factors, including the underlying metabolism, nutrient availability, expression pattern of the glycosyltransferases, glycosidases, and nucleotide-sugar transporters, rate of protein synthesis, and others. This study (6) reveals that Fc receptor ligand affinity and possibly function may be tuned in a cell-specific fashion. This makes intuitive sense because it imbues the cell with increased flexibility to regulate its response to specific signals in a context-dependent fashion, while enabling a single receptor to play cell-and tissue-specific roles in responding to a stimulus.…”

“…It is therefore important to remember that nearly every secreted and integral membrane protein is glycosylated, and, as it has been said before (10), ignore glycans at your own peril. (6) have discovered that the glycans on Fc␥RIIIA from primary and uncultured NK cells from human donors carry a much higher proportion of high mannose and hybrid N-glycans than recombinant Fc␥RIIIA expressed by HEK293 cells in culture. HEK293 cells preferentially placed complex-type N-glycans on the receptor, which cause a different conformational change from the NK-mediated glycans and a 12-fold reduction in binding affinity (K d ) for IgG.…”

“…To gain insights into Fc␥RIIIA N-glycans, Patel et al (6) first had to optimize a method to purify the receptor from primary human cells at sufficient quantities for analysis. They used a two-step process, first enriching blood samples for NK cells using leukocyte reduction filters and then employing immunoprecipitation to isolate mature Fc␥RIIIA.…”