RT Journal Article SR Electronic T1 Mechanism of glycoform specificity and protection against antibody dependent enhancement by an anti-afucosylated IgG nanobody JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.01.23.525277 DO 10.1101/2023.01.23.525277 A1 Aaron Gupta A1 Kevin Kao A1 Rachel Yamin A1 Deena A. Oren A1 Yehuda Goldgur A1 Jonathan Du A1 Pete Lollar A1 Eric J. Sundberg A1 Jeffrey V. Ravetch YR 2023 UL http://biorxiv.org/content/early/2023/01/24/2023.01.23.525277.abstract AB Immunoglobulin G (IgG) antibodies contain a single, complex N-glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its Cγ2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper Cγ2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fcγ receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a ‘glycan sensor’, forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection.Competing Interest StatementThe authors have declared no competing interest.