N‑Linked Glycan Structures of the Human Fcγ Receptors Produced in NS0 Cells Eoin F. J. Cosgrave, †,‡ Weston B. Struwe, †,§ Jerrard M. Hayes, † David J. Harvey, ∥ Mark R. Wormald, ∥ and Pauline M. Rudd* ,† † NIBRT Glycobiology Group, National Institute for Bioprocessing Research and Training, Foster’s Avenue, Mount Merrion, Blackrock, County Dublin, Ireland ‡ Pharmaceutical Life Sciences Group, Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States § Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom ∥ Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom ABSTRACT: 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 Fcγ receptors (FcγRs). The FcγRs 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 Fcγ receptors. A total of five functional FcγRs are broadly classified into three families (FcγRI, FcγRII, and FcγRIII) 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 FcγRs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies using recombinant forms of the FcγRs are often used to quantitate changes in binding affinity, a key indicator of a likely biological outcome. However, analysis of the FcγRs themselves is imperative as recombinant FcγRs differ greatly from those observed in humans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG−FcγR interaction. Here, we present data for the N-linked glycans present on FcγRs produced in NS0 cells, namely, FcγRIa, FcγRIIa, FcγRIIB, FcγRIIIa, and FcγRIIIb. Importantly, these FcγRs demonstrate typical murine glycosylation in the form of α-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. KEYWORDS: glycosylation, antibody, IgG, Fcγ receptors, N-linked glycan, α-galactosylation, N-glycolylneuraminic acid, HPLC, mass spectrometry, N-acetyllactosamine, CD16, CD32, CD64 ■ INTRODUCTION Clearance of foreign material from the host system is achieved in part by the activity of immunoglobulins (Igs), an important class of glycoproteins responsible for the recognition of nonself epitopes and the subsequent broadcasting of this identification to the innate and adaptive arms of the immune system. However, recognition of potentially harmful foreign antigens followed by opsonization is insufficient for their removal, and immunoglobulins rely on activation of innate effector cells that carry out the elimination process. Coupling of soluble immunoglobulins to innate host cell membranes is facilitated through binding to membrane-bound receptors (Fc receptors) specific for the Fc region of Igs, which then trigger immune activation. This leads to clustering of receptors, immune complex formation, and triggering of an intracellular signaling cascade involving phosphorylation/dephosphorylation of ITAMs (immuno-tyrosine-like activatory motifs) and ITIMs (immuno-tyrosine-like inhibitory motifs). Key to the ability of immunoglobulin G (IgG) in coordinating immune responses is glycosylation, a non- template-driven post-translational modification that when absent adversely affects the structural integrity of the antibody molecule, therein eliminating or significantly reducing binding to its Fc receptors C1q and MBL. 1−5 It is now widely appreciated that this binding and activation event is mediated by glycosylation. Carbohydrates attached to IgG, the predominant class of Ig found in human serum, play a critical role in its function of linking antigen recognition to immune effector functions. 6 Antibody-mediated immune responses Received: April 14, 2013 Published: June 18, 2013 Article pubs.acs.org/jpr © 2013 American Chemical Society 3721 dx.doi.org/10.1021/pr400344h | J. Proteome Res. 2013, 12, 3721−3737