Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

Membrane-cytoskeletal crosstalk mediated by myosin-I regulates adhesion turnover during phagocytosis.

Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.

Origin of the apical transcytic membrane system in jejunal absorptive cells of neonates.

We investigated the origin of the apical transcytic membrane system in jejunal absorptive cells of neonatal rats using light, electron, and immunofluorescence microscopy. In rats just after birth, intraluminally injected horseradish peroxidase (HRP), used as a macromolecular tracer, was observed only in the apical endocytic membrane system including the lysosomes, of jejunal absorptive cells in vivo. No tracer, however, was found in the intercellular space between the jejunal absorptive cells and the submucosa. Immunoreactive neonatal Fc receptor (FcRn) was localized in the perinuclear region of these absorptive cells whereas immunoglobulin G (IgG) was not found in these absorptive cells. In contrast, in rats 2 h after breast-feeding, intraluminally injected HRP was observed in the apical endocytic membrane system and in the apical transcytic membrane system of the absorptive cells. Moreover, HRP was found in the intercellular space between the jejunal absorptive cells and the submucosa. Furthermore, FcRn and IgG were widely distributed throughout the absorptive cells, and IgG was detected in both the intercellular space and the submucosa. These data suggest that initiation of breast-feeding induces the transportation of membrane-incorporated FcRn from its perinuclear localization to the apical plasma membrane domain. This transportation is achieved through the membrane system, which mediates apical receptor-mediated transcytosis via the trans-Golgi network. Subsequently, the apical plasma membrane containing the FcRn binds to maternal IgG, is endocytosed into the absorptive cells, and is transported to the basolateral membrane domain.

Structural model of human endoglin, a transmembrane receptor responsible for hereditary hemorrhagic telangiectasia.

Endoglin is a type I membrane protein expressed as a disulphide-linked homodimer on human vascular endothelial cells whose haploinsufficiency is responsible for the dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia (HHT). Structurally, endoglin belongs to the zona pellucida (ZP) family of proteins that share a ZP domain of approximately 260 amino acid residues at their extracellular region. Endoglin is a component of the TGF-beta receptor complex, interacts with the TGF-beta signalling receptors types I and II, and modulates cellular responses to TGF-beta. Here, we have determined for the first time the three-dimensional structure of the approximately 140 kDa extracellular domain of endoglin at 25 A resolution, using single-particle electron microscopy (EM). This reconstruction provides the general architecture of endoglin, which arranges as a dome made of antiparallel oriented monomers enclosing a cavity at one end. A high-resolution structure of endoglin has also been modelled de novo and found to be consistent with the experimental reconstruction. Each subunit comprises three well-defined domains, two of them corresponding to ZP regions, organised into an open U-shaped monomer. This domain arrangement was found to closely resemble the overall structure derived experimentally and the three modelled de novo domains were tentatively assigned to the domains observed in the EM reconstruction. This molecular model was further tested by tagging endoglin's C terminus with an IgG Fc fragment visible after 3D reconstruction of the labelled protein. Combined, these data provide the structural framework to interpret endoglin's functional domains and mutations found in HHT patients.

Expression of functionally active FcRn and the differentiated bidirectional transport of IgG in human placental endothelial cells.

The mechanism of selective transport of the immunoglobulins G from the placental stroma to the lumen of the fetal blood vessels has not been elucidated yet. It was postulated that the specific transport as well as the regulation of IgG level in the blood, involves the MHC class I related receptor FcRn for the Fc domain of IgG. We questioned whether human placental endothelial cells (HPEC) express FcRn and, if present, whether it is in a functionally active form. The experiments were performed on cultured HPEC and as positive control, human trophoblastic (JEG3) and mouse endothelial cells (SVEC) were used. Expression of FcRn, was demonstrated by indirect immunofluorescence and RT-PCR. The role of FcRn was assessed by quantifying the transcellular transport of [(125)I]-hIgG or [(125)I]-rF(ab')(2) fragments from the apical to basolateral surface, and in the reverse direction of HPEC grown on filters in a double chamber system. The intracellular pathway of FcRn or IgG was examined by electron microscopy using the proteins adsorbed to 5 nm and 20 nm colloidal gold particles, respectively. The results showed that: (a) FcRn is expressed by human placental endothelial cells, in a functionally active form; (b) transcytosis of IgG in HPEC is a time-dependent process that takes place preferentially from the basolateral to the apical compartment; and (c) both IgG and FcRn colocalize in an intracellular endocytic compartment, chloroquine sensitive. Together these data suggest that the regulation of IgG level by endothelial cells may result from interplay between salvaging, exocytosis, and transcytosis of the molecules. One can assume that IgG that does not bind to FcRn may be destined for destruction, and this would explain the mechanism by which IgG homeostasis is maintained.

Receptores Fc gamma en salud y enfermedad / Fc gamma receptors in health and disease

Los receptores para la porción Fc de las inmunoglobulinas G (Fc gamma R) forman parte de la superfamilia de las inmunoglobulinas que se expresan en diferentes tipos celulares y que por su peso molecula, afinidad y especificidad por ligandos, se clasifican en tres grupos (Fc gamma RI, Fc gamma RII y Fc gamma RIII). Además, ciertos polimorfismos genéticos son responsables de la expresión de isoformas que aumentan la heterogeneidad de cada grupo. Los Fc gamma R son tema de intensidad investigación: se conoce la organización de sus genes, propiedades bioquímicas y estructurales. Por otro lado, se sabe que funcionalmente lo Fc gamma R juegan un papel importante en la regulación de la respuesta biológicas generadas durante episodios inflamatorios (infección, por ejemplo), ya que actúan como conectores entre las respuestas inmune celular y humoral. En este artículo presentamos ejemplos donde destaca la participación de los Fc gamma R en funciones de regulación de la respuesta inmune, así como los mecanismos intracelulares que se activan cuando los Fc gamma R son entrecruzados por complejos antígeno-antícuerpo. También recibimos el efecto de citocinas y factores de crecimiento en la regulación de la expresión de los Fc gamma R, remarcando la importancia del posible empleo de éstos en situaciones clínicas en donde las alteraciones de sus niveless de expresión se asocian con ciertos padecimientos y enfermedades. Finalmente, se analiza la participación de los Fc gamma R como vía de entrada para agentes infecciosos tales como el VIH

Receptores Fc. Distribución, estructura y función

Muchas de las funciones esenciales del sistema inmune son mediadas por glicoproteínas de superficie conocidas como receptores Fc (FcR), que interaccionan en forma específica con el dominio constante o región Fc de inmunoglobulinas homólogas. Se encuentran ampliamente distribuidos entre las células del sistema inmune y tienen especificidad por diferentes isotipos de inmunoglobulinas. Las cadenas proteicas que componen los FcR contienen una región extracelular, una región transmembranal y una región intracelular. La región extracelular es altamente conservada mientras que la citoplasmática es muy heterogénea, lo que parece explicar las diferencias funcionales existentes entre los FcR expresados por las distintas células. El análisis molecular de genes y proteínas que constituyen los FcR ha mostrado una diversidad de estructuras, subunidades proteicas y vías para la transducción de señales que son compartidas con otros receptores del sistema inmune. Conocer las funciones específicas para cada uno de los FcR y el mecanismo a través del cual ocurre la transducción de señales para la activación celular permitirá un mejor entendimiento del papel inmunorregulador de los FcR y su utilización en el diseño de alternativas terapéuticas para varios desórdenes inmunológicos en los cuales participan.

[The nature of macrophage functional heterogeneity exemplified by Fc receptors]. / Priroda funktsional'noi geterogennosti makrofagov na primere Fc-retseptorov.

It is known that functional activity of macrophages (Mph), adhesion and phagocytosis, depends on the local concentration of Fc receptors (Fc gamma R) on the cell surface. Fc gamma R may have a random distribution on cell membrane or form clusters of different sizes. This process depends on the local activity of microfilaments that, in its turn, reflects peculiarities of macrophage microenvironments. One may conceive that the number of opsonized erythrocytes (OE) subject to adhesion (engulfing) to separate Mph depends on the amount of active centres on the cell surface and their activity. The structures, such as Fc gamma R clusters, phagosomes and others, in whose formation numerous components of Mph (cell skeleton, microvesicles, signal receptive system and others) are involved, are arbitrarily called the structural functional units (SFU). Thus, we used two parameters: the average number of SFU per Mph in population, and the average activity of SFU instead of a generally accepted number of OE per Mph. The number of SFU per Mph can be 0, 1, 2 etc., and one SFU may bind (engulf) 0, 1, 2 etc. EA. For the assessment of the parameters, the approximation of experimental histograms by type-A Neyman's distribution was used. To verify the adequacy of the model, observations of OE adhesion and phagocytosis in different conditions were made. The results show that the parameters of the model fit well the biological processes in this system.

Transmembrane helical interactions: zeta chain dimerization and functional association with the T cell antigen receptor.

Members of the zeta family of receptor subunits (zeta, eta and gamma) are structurally related proteins found as components of the T cell antigen receptor (TCR) and certain Fc receptors. These proteins share the ability to form disulfide-linked dimers with themselves and with other members of the family. Comparison of the amino acid sequences of zeta and gamma reveals a significant degree of homology, which is highest within their membrane-spanning domains. Analysis of their transmembrane sequences on a helical wheel projection suggests that all of the identical amino acids are clustered on one face of a potential alpha-helix. This face contains the only cysteine residue within zeta, suggesting that this conserved region may function to mediate dimerization. Indeed, replacing the transmembrane domain of the Tac antigen (alpha chain of the interleukin-2 receptor) by that of the zeta chain resulted in the formation of disulfide-linked dimers of Tac. The conserved aspartic acid residue found in the zeta and gamma transmembrane sequences was found to play a role in disulfide linkage. Replacing the aspartic acid with a lysine but not with an alanine or valine residue allowed formation of disulfide-linked dimers. The ability of the aspartic acid residue to support dimerization was dependent upon its position within the helix. Thus, these observations indicate that residues within the zeta transmembrane domain play a critical role in the formation of disulfide-linked dimers. Expression of zeta mutants in zeta-deficient T cells revealed that the zeta transmembrane domain is also responsible for reconstituting transport of functional TCR complexes to the cell surface and differentiated the requirements for disulfide-linked dimerization per se from assembly of the TCR complex.

[Fc gamma receptors: structure, function, and clinical significance]. / Fc gamma-Rezeptoren: Struktur, Funktion und klinische Bedeutung.

Fc gamma receptors are a group of three different receptors with several subtypes. They are widely distributed on many cells of the immune system and contribute to the pathogenesis of immune complex- and autoantibody-mediated diseases such as vasculitis, rheumatoid arthritis, idiopathic thrombocytopenic purpura or autoimmune neutropenia. This review focuses on the structure, distribution and function in Fc gamma receptors and their subtypes.

[Cytokines and allergy]. / Cytokines et allergie.

The immune system is a homeostatic system adaptating the internal self to the variations of the external non-self and allowing us to get on well together with the environment. Intercellular communications are necessary for the system well operating and are mediated by either direct contacts or by soluble factors called cytokines. Allergy is the upshot of a disregulation of the immune response that does not discriminate between a potentially harmful antigen (tetanus toxoid) and a rather harmless antigen (pigeon droppings). This could be the result of an imbalance between two helper T lymphocytes subsets called TH1 and TH2: their main produced cytokines, respectively IFN-gamma and IL-4, have opposite effects on IgE synthesis whereas other cytokines (IL-3, IL-5) are more directly involved in the differentiation of eosinophil and mast cells lineages.

Structural requirements of the cytoplasmic domains of the human macrophage Fc gamma receptor IIa and B cell Fc gamma receptor IIb2 for the endocytosis of immune complexes.

Two isotypes of the monocyte/macrophage as well as B cell Fc gamma receptor type II (FcRIIa and FcRIIb2, respectively) mainly differ in the length (76 vs. 44 amino acids) and amino acid sequence of their cytoplasmic domains. Only the eight amino acids just behind the putative transmembrane region are identical. Despite this marked difference, both FcRII mediate endocytosis of immune complexes. To determine the functional significance of the cytoplasmic domains, we expressed truncated FcRIIa and FcRIIb2 in FcR- BHK-21 cells. Mutants of both receptors containing only one amino acid (tail-minus) of the cytoplasmic domain failed to mediate immune complex uptake. The significance of the cytoplasmic domain of the receptors could be further demonstrated using a chimeric FcRIII-FcRIIa construct. Therefore we expressed an FcRIII lacking the hydrophobic carboxyl terminus (containing the putative phosphatidyl - inositol - glycan anchor site) fused inframe to the transmembrane and cytoplasmic domain of the FcRIIa in BHK-21 cells. In contrast to the wild type FcRIII, this chimeric receptor mediated immune complex uptake indistinguishable from that mediated by the FcRIIa. Receptor mutants with relatively short cytoplasmic domains (FcRIIb2: 13, and FcRIIa: 16 amino acids) revealed, that these short amino acid stretches are sufficient to allow reduced receptor-mediated endocytosis of bound ligand. Furthermore, using FcRIIa deletion mutants with a cytoplasmic domain consisting of 62, 46, and 28 amino acids, respectively, we found that the capability of these mutants to mediate immune complex uptake decreased gradually with the truncation of the cytoplasmic tails. Thus, only short amino acid sequences of the cytoplasmic domain are sufficient to enable an, albeit reduced, receptor-mediated endocytosis.

Conformations of IgE bound to its receptor Fc epsilon RI and in solution.

Previous resonance energy transfer studies suggested that murine immunoglobulin E (IgE) is bent near the junction of its Fc and Fab segments when bound to its high-affinity receptor (Fc epsilon RI) on RBL cells. To examine further the conformations of IgE, both bound to this receptor and in solution, a mutant recombinant IgE (epsilon/C gamma 3*) was prepared that has a cysteine replacing a serine near the C-terminal ends of the heavy chain. The introduced cysteine residues provide a means for specific modification of IgE, and the sulfhydryl groups were selectively labeled with fluorescein-5-maleimide (FM-epsilon/C gamma 3*). This IgE also binds a 5-(dimethylamino)naphthalene-1-sulfonyl (DNS) group in the antigen-binding sites. Resonance energy transfer experiments carried out on receptor-bound FM-epsilon/C gamma 3* yielded a distance of 53 A between fluorescein near the C-terminal end of the Fc segment and amphipathic acceptor probes at the membrane surface. The average distance between this C-terminal fluorescein and acceptor eosin-DNS in the antigen-binding sites at the N-terminal ends of the Fab segments was found to be 69 A. These results combine with those from previous structural studies to provide an unprecedented detailed description of the bent geometry of IgE bound to its receptor on the membrane. Energy transfer measured for FM-epsilon/C gamma 3* in solution between fluorescein near the C-terminal end of the Fc segment and eosin-DNS at the N-terminal ends of the Fab segments indicates that the average distance between these probes is about 71 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopy of Fc receptors on the surface of clinical isolates of streptococci.

The distribution of Fc receptors on the surface of clinical isolates of streptococci was examined by immunoelectron microscopy. A unique two-layered ferritin tagging was observed on the surface of isolate MS-4 (group A). The spacing between the layers (10-40 nm) was narrower than that of the layered protein A (30-70 nm) on Staphylococcus aureus. Labeling on the other isolates, IP-28 (group A) and ES-21L (group C), showed similar rough layers, but the labeling were clearly different in thickness and density. The labeling on isolate IP-28 was also similar to that on strain AR1 (group A), reported previously, while the labeling on ES-21L was similar to that on protein G producing strain G148 (group G) in thickness. However, it was obviously thinner than that on IP-28 and AR1. These results strongly suggest that there are at least three distribution patterns of Fc receptors on streptococci, and these are distinct from that of protein A.

Fc receptors and membrane immunoglobulin.

This year there have been three major highlights in this field: genetic sequencing of the principal Fc receptors has been completed; new proteins associated with both Fc gamma receptors and B-cell membrane immunoglobulins have been identified; and there has been an initial analysis of structural-functional relationships in these receptors using genetic engineering.

Neutrophil activation through high-affinity Fc gamma receptor using a monomeric antibody with unique properties.

The high-affinity, type I Ig Fc receptor (Fc gamma RI) for human IgG1, human IgG3, murine IgG2a, and murine IgG3 is highly expressed on monocytes, neutrophils (PMN) in certain disease states, and phagocytes treated with interferon-gamma (IFN-gamma). We studied the activation of the human PMN oxidative burst and stimulated fluid pinocytosis induced by three monoclonal antibodies (MoAbs) directed against Fc gamma RI (CD64) to study the role of this receptor in Fc-mediated cellular activation. All three MoAbs were capable of triggering PMN activation from IFN-gamma-treated PMN when cross-linked with goat antimurine Ig reagents. However, MoAb 197 alone demonstrated a concentration-dependent activation of the oxidative burst without the use of a second cross-linking antibody. The oxidative burst and stimulated fluid pinocytosis responses induced by monomeric MoAb 197 could be elicited only after the IFN-gamma induction of approximately 8,000 Fc gamma RI receptor equivalents and did not occur in freshly isolated or control-cultured PMN. Competitive blocking of Fc binding of MoAb 197 by human IgG or purified Fc fragments inhibited cellular activation. We believe the ability of MoAb 197 to activate these oxidative burst and fluid pinocytic responses was because of its murine IgG2a subclass, which allowed it to function as a trivalent anti-Fc gamma RI antibody binding through the combination of its two FAB regions and the Fc domain. This study demonstrates that the cross-linking of CD64 can activate PMN oxidative and endocytic responses and supports a role for Fc gamma RI in the human neutrophil inflammatory response.

[Does induced Heinz body formation lead to the liberation of IgG receptors on the erythrocyte membrane?]. / Führt induzierte Heinz-Körper-Bildung zur Freisetzung von IgG-Rezeptoren an der Erythrozytenmembran?

Immunofluorescence microscopical and biochemical studies led other authors to the conclusion that the formation of membrane-bound Heinz bodies at these parts of the plasmalemma of erythrocytes leads to clustering of band 3-protein and increased binding of IgG. We failed to detect immunocytochemically an increased IgG binding over phenylhydrazine induced membrane-bound Heinz bodies in otherwise intact erythrocytes using transmission electronmicroscopy and we also did not notice any clustering of intramembraneous particles over the numerous Heinz bodies by means of freeze etching. Especially at higher phenylhydrazine concentrations the erythrocytes show formation of vesicles and hemolysis accompanied by increased IgG binding and clustering of the intramembraneous particles. Photographs of such cells obtained by immunofluorescence microscopy are very similar to the pictures known from the literature.

Membrane activity and topography of F-Met-Leu-Phe-Treated polymorphonuclear leukocytes. Acute and sustained responses to chemotactic peptide.

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) causes a dramatic stimulation of membrane ruffling and a fluid pinocytosis in polymorphonuclear leukocytes (PMNs). These responses are maximal by 1 minute and subside within 5-10 minutes. The same immediate responses characterize cells exposed to several peptide hormones and may thus represent an essential component of target cell activation by peptides. The stimulation of the whole membrane following f-Met-Leu-Phe binding is succeeded by the development of a polarized cell shape with a posterior uropod and a broad anterior lamellipodium, both subtended by microfilaments. Membrane components and functions segregate into distinct domains on polarized PMNs. Thus, succinyl concanavalin A-receptor complexes are capped and internalized by receptor-mediated endocytosis at the uropod; the uptake by fluid pinocytosis of fluorescein-dextran is restricted to the uropod; and coated pits and coated vesicles are concentrated at the uropod. The lamellipodium excludes coated pits and lacks pinocytic activity but shows preferential binding of immunoglobulin aggregates, presumably to Fc receptors. The origin and physiologic implications of these asymmetries of membrane molecular and functional topography on polarized cells are discussed.