IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic ...surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.
Activation of membrane receptors through clustering is a common mechanism found in various biological systems. Spatial proximity of receptors may be transduced across the membrane to initiate ...signaling pathways or alternatively be recognized by peripheral proteins or immune cells to trigger external effector functions. Here we show how specific immunoglobulin G (IgG) binding induces clustering of monomeric target molecules in lipid membranes through Fc–Fc interactions. We visualize and characterize the dynamic IgG oligomerization process and the molecular interactions involved using high-speed atomic force microscopy, single-molecule force spectroscopy, and quartz crystal microbalance experiments. We found that the Fc–Fc interaction strength is precisely tuned to be weak enough to prevent IgG oligomerization in solution at physiological titers, but enabling IgG oligomerization when Fabs additionally bind to their cognate surface epitopes, a mechanism that ultimately targets IgG-mediated effector functions such as classical complement activation to antigenic membranes.
Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement ...activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of
, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)
:C1q complexes and prevents downstream complement activation on the surface of
Finally, we demonstrate that IgG3 antibodies against
can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.
Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement ...component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol DNP-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.
Vitamin A-derived retinoic acid (RA) signals are critical for the development of several organs, including the pancreas. However, the tissue-specific control of RA synthesis in organ and cell lineage ...development has only poorly been addressed in vivo. Here, we show that retinol dehydrogenase-10 (Rdh10), a key enzyme in embryonic RA production, has important functions in pancreas organogenesis and endocrine cell differentiation. Rdh10 was expressed in the developing pancreas epithelium and surrounding mesenchyme. Rdh10 null mutant mouse embryos exhibited dorsal pancreas agenesis and a hypoplastic ventral pancreas with retarded tubulogenesis and branching. Conditional disruption of Rdh10 from the endoderm caused increased mortality, reduced body weight, and lowered blood glucose levels after birth. Endodermal Rdh10 deficiency led to a smaller dorsal pancreas with a reduced density of early glucagon+ and insulin+ cells. During the secondary transition, the reduction of Neurogenin3+ endocrine progenitors in the mutant dorsal pancreas accounted for fewer α- and β-cells. Changes in the expression of α- and β-cell-specific transcription factors indicated that Rdh10 might also participate in the terminal differentiation of endocrine cells. Together, our results highlight the importance of both mesenchymal and epithelial Rdh10 for pancreogenesis and the first wave of endocrine cell differentiation. We further propose a model in which the Rdh10-expressing exocrine tissue acts as an essential source of RA signals in the second wave of endocrine cell differentiation.
Significance
Antibody-dependent complement activation plays a major role in various pathophysiological processes in our body, including infection, inflammation, autoimmunity, and transplant ...rejection. In order to activate complement, antibodies should bind to target cells and recruit complement component C1. C1 is a large, multimolecular complex that consists of the antibody recognition protein C1q and a heterotetramer of proteases (C1r
2
s
2
). Although it is believed that interactions between C1 and IgGs are solely mediated by C1q, we here show that C1r
2
s
2
proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules. Furthermore, we demonstrate that C1q–IgG stability is influenced by IgG oligomerization and that promoting IgG oligomerization improves phagocytosis of the pathogenic bacterium
Staphylococcus aureus
.
Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr
2
s
2
) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r
2
s
2
). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r
2
s
2
proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol DNP-coated surfaces and pathogenic
Staphylococcus aureus
). The extent to which C1r
2
s
2
contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r
2
s
2
. In addition, hexamer-enhanced IgGs targeting
S. aureus
mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.
X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked ...lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap−/− mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.
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•XIAP protects against TLR- and TNF-driven inflammasome formation and cell death•XIAP contributes to the regulation of RIP1 ubiquitylation in response to TNF•Exaggerated IL-1β secretion and cell death in Xiap−/− dendritic cells are RIP3 dependent•Upon infection, Xiap−/− mice show signs of hyperinflammation similar to XLP-2 patients
Loss of X-linked inhibitor of apoptosis protein (XIAP) function causes the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Yabal et al. now show that in the absence of XIAP, activation of immune cells by TNF leads to the induction of RIP3-dependent cell death and, independently, to aberrant inflammasome activation. In this process, XIAP controls the correct ubiquitylation of RIP1. These findings might hold promise for new therapeutic opportunities for patients with XLP-2.
Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic ...stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts, occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice, tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically.
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