N-Linked Glycan Structures of the Human Fcγ Receptors Produced in NS0 Cells

Immune recognition of nonself is coordinated through complex mechanisms involving both innate and adaptive responses. Circulating antibodies communicate with effector cells of the innate immune system through surface receptors known as Fc gamma receptors (Fc gamma Rs). The Fc gamma Rs are single-pass transmembrane glycoproteins responsible for regulating innate effector responses toward antigenic material. Although immunoglobulin G (IgG) antibodies bind to a range of receptors, including complement receptors and C-type lectins, we have focused on the Fc gamma receptors. A total of five functional Fc gamma Rs are broadly classified into three families (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) and together aid in controlling both inflammatory and anti-inflammatory responses of the innate immune system. Due to the continued success of monoclonal antibodies in treating cancer and autoimmune disorders, research is typically directed toward improving the interaction of antibodies with the Fc gamma Rs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies using recombinant forms of the Fc gamma Rs are often used to quantitate changes in binding affinity, a key indicator of a likely biological outcome. However, analysis of the Fc gamma Rs themselves is imperative as recombinant Fc gamma Rs differ greatly from those observed in humans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG-Fc gamma R interaction. Here, we present data for the N-linked glycans present on Fc gamma Rs produced in NS0 cells, namely, Fc gamma RIa, Fc gamma RIIa, Fc gamma RIIB, Fc gamma RIIIa, and Fc gamma RIIIb. Importantly, these Fc gamma Rs demonstrate typical murine glycosylation in the form of alpha-galactose epitopes, N-glycolylneuraminic acid, and other key glycan properties that are generally expressed in murine cell lines and therefore are not typically observed in humans.