Jinghua Lu

Structural recognition and functional activation of FcγR by innate pentraxins

Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway through C1q (refs 3 and 4). More recently, members of the pentraxin family were found to interact with cell-surface Fcγ receptors (FcγR) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin’s binding to FcγR and its functional activation of FcγR-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and FcγRIIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and FcγRIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for FcγR isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for FcγR binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the FcγR pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.

Structure of FcγRI in complex with Fc reveals the importance of glycan recognition for high-affinity IgG binding

Significance Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. It is the sole high-affinity receptor for IgG and plays a significant role in immunotherapy. To date, there is no structural information available on how the receptor recognizes its antibody ligands, however. Consequently, the mechanism of its high-affinity IgG binding remains unclear. We report the first structure of the high-affinity Fc receptor in complex with IgG-Fc. The structural work reveals a direct receptor recognition of Fc glycan as a major factor in receptor affinity. This is the first example of Fc receptor making direct glycan contact through protein residues. The results have implications for the use of glycan engineering in immunotherapy. Fc gamma receptor I (FcγRI) contributes to protective immunity against bacterial infections, but exacerbates certain autoimmune diseases. The sole high-affinity IgG receptor, FcγRI plays a significant role in immunotherapy. To elucidate the molecular mechanism of its high-affinity IgG binding, we determined the crystal structure of the extracellular domains of human FcγRI in complex with the Fc domain of human IgG1. FcγRI binds to the Fc in a similar mode as the low-affinity FcγRII and FcγRIII receptors. In addition to many conserved contacts, FcγRI forms additional hydrogen bonds and salt bridges with the lower hinge region of Fc. Unique to the high-affinity receptor-Fc complex, however, is the conformation of the receptor D2 domain FG loop, which enables a charged KHR motif to interact with proximal carbohydrate units of the Fc glycans. Both the length and the charge of the FcγRI FG loop are well conserved among mammalian species. Ala and Glu mutations of the FG loop KHR residues showed significant contributions of His-174 and Arg-175 to antibody binding, and the loss of the FG loop–glycan interaction resulted in an ∼20- to 30-fold decrease in FcγRI affinity to all three subclasses of IgGs. Furthermore, deglycosylation of IgG1 resulted in a 40-fold loss in FcγRI binding, demonstrating involvement of the receptor FG loop in glycan recognition. These results highlight a unique glycan recognition in FcγRI function and open potential therapeutic avenues based on antibody glycan engineering or small molecular glycan mimics to target FcγRI for certain autoimmune diseases.

Crystal Structure of Fcγ Receptor I and Its Implication in High Affinity γ-Immunoglobulin Binding

Background: FcγRI plays important roles in antibody functions. Results: We report the first crystal structure of the extracellular human FcγRI. Conclusion: The receptor D3 domain is positioned away from the IgG binding site, and its shorter D2 domain FG-loop is important for its high affinity. Significance: This work provides insights to the mechanism of FcγRI function and helps to design therapeutic reagents. Fcγ receptors (FcγRs) play critical roles in humoral and cellular immune responses through interactions with the Fc region of immunoglobulin G (IgG). Among them, FcγRI is the only high affinity receptor for IgG and thus is a potential target for immunotherapy. Here we report the first crystal structure of an FcγRI with all three extracellular Ig-like domains (designated as D1, D2, and D3). The structure shows that, first, FcγRI has an acute D1-D2 hinge angle similar to that of FcϵRI but much smaller than those observed in the low affinity Fcγ receptors. Second, the D3 domain of FcγRI is positioned away from the putative IgG binding site on the receptor and is thus unlikely to make direct contacts with Fc. Third, the replacement of FcγRIII FG-loop (171LVGSKNV177) with that of FcγRI (171MGKHRY176) resulted in a 15-fold increase in IgG1 binding affinity, whereas a valine insertion in the FcγRI FG-loop (171MVGKHRY177) abolished the affinity enhancement. Thus, the FcγRI FG-loop with its conserved one-residue deletion is critical to the high affinity IgG binding. The structural results support FcγRI binding to IgG in a similar mode as its low affinity counterparts. Taken together, our study suggests a molecular mechanism for the high affinity IgG recognition by FcγRI and provides a structural basis for understanding its physiological function and its therapeutic implication in treating autoimmune diseases.

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis

Significance Serum amyloid A (SAA) is a major serum acute-phase protein and a cause of secondary amyloidosis, which impacts ∼1% of patients with chronic inflammation such as rheumatoid arthritis and neoplastic diseases. The lack of structural information has hampered our understanding of SAA-mediated amyloidosis and the development of effective therapies. Here we report a crystal structure of human SAA1.1 as a prototypic member of the family. SAA1.1 exists as a hexamer with subunits displaying a unique four-helix bundle fold. We further defined binding sites for heparin and high-density lipoprotein, identified major amyloidogenic epitopes, and visualized SAA-mediated protofibril formation using electron microscopy. These studies provide mechanistic insights into amyloidogenic conformational transition of SAA. Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation.

Recognition and functional activation of the human IgA receptor (FcαRI) by C-reactive protein

C-reactive protein (CRP) is an important biomarker for inflammatory diseases. However, its role in inflammation beyond complement-mediated pathogen clearance remains poorly defined. We identified the major IgA receptor, FcαRI, as a ligand for pentraxins. CRP recognized FcαRI both in solution and on cells, and the pentraxin binding site on the receptor appears distinct from that recognized by IgA. Further competitive binding and mutational analysis showed that FcαRI bound to the effector face of CRP in a region overlapping with complement C1q and Fcγ receptor (FcγR) binding sites. CRP cross-linking of FcαRI resulted in extracellular signal-regulated kinase (ERK) phosphorylation, cytokine production, and degranulation in FcαRI-transfected RBL cells. In neutrophils, CRP induced FcαRI surface expression, phagocytosis, and TNF-α secretion. The ability of CRP to activate FcαRI defines a function for pentraxins in inflammatory responses involving neutrophils and macrophages. It also highlights the innate aspect of otherwise humoral immunity-associated antibody receptors.

The structure of the TLR5-flagellin complex: a new mode of pathogen detection, conserved receptor dimerization for signaling.

TLR5 exhibits a distinct ligand-binding mechanism compared with those used by other TLRs. Knowledge about how Toll-like receptors (TLRs) recognize pathogenic ligands is critical to understanding how these receptors are activated and to designing therapeutic compounds that target this family of receptors for inflammatory diseases. The crystal structure of TLR5 in complex with its bacterial ligand flagellin revealed that the ligand-binding mode for TLR5 is distinct from that of previously characterized TLRs. Nevertheless, like other TLRs, TLR5 forms a dimer in response to ligand binding. This work contributes to our current knowledge of TLR function and further demonstrates the ability of TLRs to couple versatile ligand recognition to a conserved receptor signaling mechanism.

T cell receptor repertoires of mice and humans are clustered in similarity networks around conserved public CDR3 sequences

Diversity of T cell receptor (TCR) repertoires, generated by somatic DNA rearrangements, is central to immune system function. However, the level of sequence similarity of TCR repertoires within and between species has not been characterized. Using network analysis of high-throughput TCR sequencing data, we found that abundant CDR3-TCRβ sequences were clustered within networks generated by sequence similarity. We discovered a substantial number of public CDR3-TCRβ segments that were identical in mice and humans. These conserved public sequences were central within TCR sequence-similarity networks. Annotated TCR sequences, previously associated with self-specificities such as autoimmunity and cancer, were linked to network clusters. Mechanistically, CDR3 networks were promoted by MHC-mediated selection, and were reduced following immunization, immune checkpoint blockade or aging. Our findings provide a new view of T cell repertoire organization and physiology, and suggest that the immune system distributes its TCR sequences unevenly, attending to specific foci of reactivity. DOI: http://dx.doi.org/10.7554/eLife.22057.001

Plasmodium falciparum merozoite surface protein 1 blocks the proinflammatory protein S100P

The malaria parasite, Plasmodium falciparum, and the human immune system have coevolved to ensure that the parasite is not eliminated and reinfection is not resisted. This relationship is likely mediated through a myriad of host–parasite interactions, although surprisingly few such interactions have been identified. Here we show that the 33-kDa fragment of P. falciparum merozoite surface protein 1 (MSP133), an abundant protein that is shed during red blood cell invasion, binds to the proinflammatory protein, S100P. MSP133 blocks S100P-induced NFκB activation in monocytes and chemotaxis in neutrophils. Remarkably, S100P binds to both dimorphic alleles of MSP1, estimated to have diverged >27 Mya, suggesting an ancient, conserved relationship between these parasite and host proteins that may serve to attenuate potentially damaging inflammatory responses.

Structural mechanism of high affinity FcγRI recognition of immunoglobulin G.

Antibody‐based immunotherapies are becoming powerful means of modern medicine for treating cancers and autoimmune diseases. The increasing popularity of antibody‐based treatment demands a better understanding of antibody functions and in particular, their interaction with Fc receptors as effectiveness of antibodies often depends on their ability to activate or avoid effector cell functions through Fc receptors. Until recently, our understanding of antibody recognition by Fc receptors is based on the structures of low affinity Fc receptor in complex with Fc. These structural studies provided significant insights to our understanding of how an IgG antibody generally docks on Fcγ receptor and the requirement of immune complex formation for effector cell activations. They are less informative, however, to the molecular forces underlying the vast different affinities between antibodies and their Fcγ receptors. Recently, the structure of the high affinity FcγRI in complex with IgG‐Fc has been determined. This review will focus on the knowledge learned from the high affinity complex structural work and a potential receptor–glycan interaction as an important contribution to the receptor affinity.

Toll-like receptor 9 antagonizes antibody affinity maturation

Key events in T cell–dependent antibody responses, including affinity maturation, are dependent on the B cell’s presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells’ capture and presentation of antigen.The presentation of antigen by germinal-center B cells to follicular T cells engenders the process of antibody affinity maturation and humoral memory. Pierce and colleagues show that TLR9 signaling in B cells antagonizes B cell–mediated antigen presentation, which leads to the enhanced generation of short-lived plasma cells and the production of lower-affinity antibodies.