Sundberg, E. | Search bioRxiv

bioRxiv2023

Mechanism of glycoform specificity and protection against antibody dependent enhancement by an anti-afucosylated IgG nanobody

Immunoglobulin G (IgG) antibodies contain a single, complex N-glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its C{gamma}2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fc{gamma} 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 C{gamma}2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fc{gamma} 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-Fc{gamma}RIIIa interactions and rescue mice in a model of dengue virus infection.

immunology↗

bioRxiv2022

MS1/MMD1 homologs in the moss P. patens are required for male and female gametogenesis and likely for sporogenesis

O_LIThe Arabidopsis Plant HomeoDomain (PHD) proteins AtMS1 and AtMMD1 provide chromatin-mediated transcriptional regulation essential for tapetum-dependent pollen formation. Such pollen-based male gametogenesis is a derived trait of seed plants. Male gametogenesis in the common ancestors of land plants is instead likely to have been reminiscent of that in extant bryophytes where flagellated sperms are produced by an elaborate gametophyte generation. Still, also bryophytes possess MS1/MMD1-related PHD proteins. C_LIO_LIWe addressed the function of two MS1/MMD1-homologs in the bryophyte model moss Physcomitrium patens by the generation and analysis of reporter and loss-of-function lines. C_LIO_LIThe two genes are together essential for both male and female fertility by providing cell autonomous functions in the gamete-producing inner cells of antheridia and archegonia. They are furthermore expressed in the diploid sporophyte generation suggesting a function during sporogenesis, a process proposed related by descent to pollen formation in angiosperms. C_LIO_LIWe propose that the moss MS1/MMD1-related regulatory network required for completion of male and female gametogenesis and possibly for sporogenesis, represent a heritage from ancestral land plants. C_LI

plant biology↗

bioRxiv2021

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain site are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains, as well as the overall flexibility of EndoS2, to the proteins catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulation and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and long-time MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models. Author SummaryThe pathogen Streptococcus pyogenes uses the endoglycosidases S and S2 to cleave the glycans on the Fc portion of IgG antibodies, leading to a decreased cytotoxicity of the antibodies, thereby evading the host immune response. To identify potential structures of the complex of EndoS2 with IgG that could lead to the catalytic hydrolysis of the IgG glycan, molecular modeling and molecular dynamics simulations were applied. The resulting structural models predict that EndoS2 initially interacts through its carbohydrate binding module (CBM) with the IgG with subsequent interactions with the catalytic glycoside hydrolase (GH) domain yielding stable complexes. In the modeled complexes the CBM and the GH interact either simultaneously with the same Fc CH2/CH3 domain or with the two individual Fc CH2/CH3 domains separately to yield potentially catalytically competent species. In addition, apo EndoS2 is shown to assume both open and closed conformations allowing it to directly form either type of complex from which deglycosylation of either mono- or diglycosylated IgG species may occur. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/445403v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@15ffa95org.highwire.dtl.DTLVardef@3c8032org.highwire.dtl.DTLVardef@452bdorg.highwire.dtl.DTLVardef@1d3f85a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗