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N E Simister

Publications and source records attributed to N E Simister.

At least 19 recordsLinked to original sources
PubMed2001

Effects of mutations in potential phosphorylation sites on transcytosis of FcRn.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across intestinal epithelial cells of suckling rats and mice from the lumenal surface to the serosal surface. In cell culture models FcRn transports IgG bidirectionally, but there are differences in the mechanisms of transport in the two directions. We investigated the effects of mutations in the cytoplasmic domain of FcRn on apical to basolateral and basolateral to apical transport of Fc across rat inner medullary collecting duct (IMCD) cells. Basolateral to apical transport did not depend upon determinants in the cytoplasmic domain. In contrast, an essentially tailless FcRn was markedly impaired in apical to basolateral transport. Using truncation and substitution mutants, we identified serine-313 and serine-319 as phosphorylation sites in the cytoplasmic domain of FcRn expressed in Rat1 fibroblasts. Mutations at Ser-319 did not affect transcytosis across IMCD cells. FcRn-S313A was impaired in apical to basolateral transcytosis to the same extent as tailless FcRn, whereas FcRn-S313D transported at wild-type levels. FcRn-S313A recycled more Fc to the apical medium than the wild-type receptor, suggesting that Ser-313 is required to allow FcRn to be diverted from an apical recycling pathway to a transcytotic pathway.

Animals↗
PubMed2000

Tryptophan- and dileucine-based endocytosis signals in the neonatal Fc receptor.

The neonatal Fc receptor, FcRn, transports immunoglobulin G across intestinal cells in suckling rats. FcRn enters these cells by endocytosis and is present on the apical and basolateral surfaces. We investigated the roles of aromatic amino acids and a dileucine motif in the cytoplasmic domain of rat FcRn. We expressed mutant FcRn in which alanine replaced Trp-311, Leu-322, and Leu-323, or Phe-340 in the inner medullary collecting duct cell line IMCD. Individual replacement of the aromatic amino acids or the dileucine motif only partially blocked endocytosis of (125)I-Fc, whereas uptake by FcRn containing alanine residues in place of both Trp-311 and the dileucine motif was reduced to the level obtained with the tailless receptor. Leu-314 was required for the function of the tryptophan-based endocytosis signal, and Asp-317 and Asp-318 were required for the dileucine-based signal. Nonvectorial delivery of newly synthesized FcRn to the two cell surfaces was unaffected by loss of the endocytosis signals. However, the steady-state distribution of endocytosis mutants was predominantly apical, unlike wild-type FcRn, which was predominantly basolateral. This shift appeared to arise because the loss of endocytosis signals inhibited apical to basolateral transcytosis of FcRn more than basolateral to apical transcytosis.

Amino Acid Motifs↗
PubMed2000

Analysis of the promoter region of the human FcRn gene.

The 5'-flanking region of the human FcRn alpha-chain gene was analyzed for its ability to directly express the chloramphenicol acetyltransferase (CAT) reporter gene in NIH3T3 and Lu106 cells. Transient transfection of the CAT constructs revealed that there was promoter activity in the region -660 to +300 of the 5'-flanking sequence. Electrophoretic mobility-shift assays showed that there are functional binding sites for Sp1 or Sp1-like factors, AP1 or a related factor, and additional unidentified proteins in the promoter region.

Gene Expression Regulation↗
PubMed2000

Cloning and characterization of the bovine MHC class I-like Fc receptor.

In the cow, maternal immunity is exclusively mediated by colostral Igs, but the receptor responsible for the IgG transport has not yet been identified. The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. We now report the cloning and characterization of the bovine FcRn (bFcRn). The cDNA and deduced amino acid sequences show high similarity to the FcRn in other species, and it consists of three extracellular domains, a hydrophobic transmembrane region, and a cytoplasmic tail. Despite the high similarity of the extracellular domains with other species, the bovine cytoplasmic tail is the shortest thus far analyzed. Aligning the known FcRn sequences, we noted that the bovine protein shows a 3-aa deletion compared to the rat and mouse sequences in the alpha1 loop. Furthermore, we found a shorter transcript of the bFcRn reflecting an exon 6-deleted mRNA, which results from an inadequate splice acceptor site in intron 5 and produces a transmembrane-deficient molecule, as was previously demonstrated in the related MHC class I gene family in mouse and humans. The presence of bFcRn transcripts in multiple tissues, including the mammary gland, suggests their involvement both in IgG catabolism and transcytosis.

Amino Acid Sequence↗
PubMed2000

Cloning and analysis of the gene encoding the human neonatal Fc receptor.

The neonatal Fc receptor, FcRn, is expressed in human placental syncytiotrophoblast, capillary endothelium, intestinal epithelium, and other tissues. By analogy with its role in the mouse, human FcRn is expected to transport maternal IgG to the foetus, and protect circulating IgG from catabolism. The larger subunit of FcRn is homologous to the alpha chains of the major histocompatibility complex (MHC) class I proteins, but is encoded outside the MHC on chromosome 19. We report the isolation of clones encoding the alpha chain of human FcRn from chromosome 19-specific libraries. The sequence revealed a similar organization to classical and non-classical MHC, and MHC-related genes. Compared with classical MHC class I genes, the human FcRn alpha chain gene has expanded by acquiring many repetitive sequences in its introns, including multiple Alu elements in the fourth intron. Primer extension analysis showed that there are two transcription initiation sites in the upstream flanking sequence.

Amino Acid Sequence↗
PubMed2000

Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia.

The neonatal Fc receptor, FcRn, transports immunoglobulin G (IgG) across cellular barriers between mother and offspring. FcRn also protects circulating IgG from catabolism, probably during transport across the capillary endothelium. Only one cell culture model of transcytosis has been used extensively, the transport of IgA from the basolateral to the apical surface of Madin-Darby canine kidney cells by the polymeric immunoglobulin receptor (pIgR). We report that rat inner medullary collecting duct (IMCD) cells transfected with DNA encoding the (alpha) subunit of rat FcRn specifically and saturably transport Fc when grown as polarized monolayers. Using this system, we have found that transcytosis by FcRn, like transcytosis by the pIgR, depends upon an intact microtubule system. FcRn differs most strikingly from the pIgR in its ability to transport its ligand in both the apical to basolateral and basolateral to apical directions. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibited basolateral to apical transport by FcRn more than apical to basolateral transport, suggesting that there are differences in the mechanisms of transport in the two directions. Lastly, we found that transcytosis by FcRn depends upon vesicular acidification. We anticipate that the IMCD cell culture model will allow further elucidation of the mechanism of IgG transport by FcRn.

Amino Acid Sequence↗
PubMed1999

Bidirectional FcRn-dependent IgG transport in a polarized human intestinal epithelial cell line.

The MHC class I-related Fc receptor, FcRn, mediates the intestinal absorption of maternal IgG in neonatal rodents and the transplacental transport of maternal IgG in humans by receptor-mediated transcytosis. In mice and rats, expression of FcRn in intestinal epithelial cells is limited to the suckling period. We have recently observed, however, clear expression of FcRn in the adult human intestine, suggesting a function for FcRn in intestinal IgG transport beyond neonatal life in humans. We tested this hypothesis using the polarized human intestinal T84 cell line as a model epithelium. Immunocytochemical data show that FcRn is present in T84 cells in a punctate apical pattern similar to that found in human small intestinal enterocytes. Solute flux studies show that FcRn transports IgG across T84 monolayers by receptor-mediated transcytosis. Transport is bidirectional, specific for FcRn, and dependent upon endosomal acidification. These data define a novel bidirectional mechanism of IgG transport across epithelial barriers that predicts an important effect of FcRn on IgG function in immune surveillance and host defense at mucosal surfaces.

Adult↗
PubMed1997

Human placental Fc receptors and the transmission of antibodies from mother to fetus.

During human pregnancy, maternal IgG is transported across the placenta to the fetus. On the way, some maternal antibodies against fetal antigens are removed as immune complexes. The placenta contains several known Fc receptors and also other proteins that bind immunoglobulins. A consideration of the binding properties and distribution of these proteins suggests that the neonated Fc receptor (FcRn) transports IgG across the syncytiotrophoblast, and possibly the fetal blood vessel endothelium. Fc gamma RI, Fc gamma RII and Fc gamma RIII on Hofbauer cells in the stroma probably clear immune complexes, together with Fc gamma RII on endothelial cells.

Animals↗
PubMed1997

New functions of the MHC class I-related Fc receptor, FcRn.

FcRn was originally identified as the receptor responsible for IgG binding to the intestinal epithelium of neonatal rats and mice. FcRn transports IgG from milk across the intestinal epithelial cells of the suckling animal. Subsequently, FcRn was detected in tissues involved in the transmission of IgG from mother to fetus: rat fetal yolk sac, mouse fetal yolk sac and human placental syncytiotrophoblast. In addition, FcRn mRNA has been detected in many tissues of adult rats, mice and humans, and FcRn is present in several adult tissues and in cell lines. The selective protection from catabolism of IgG compared with other immunoglobulins is lost in mice that lack functional FcRn. One function of FcRn in tissues that do not transport IgG, and beyond the perinatal period, is thus to rescue circulating IgG from degradation. These recent observations reveal a more widespread use of FcRn than had been supposed.

Animals↗
PubMed1997

Expression of the neonatal Fc receptor, FcRn, on human intestinal epithelial cells.

Maternal IgG is transferred to the suckling mouse and rat through a major histocompatibility complex (MHC) class I-related Fc receptor (FcRn) on the brush border of the proximal small intestine. We have previously described a site on the epithelial surface of the human fetal intestine with IgG binding characteristics similar to FcRn. We report here the identification by reverse transcriptase polymerase chain reaction amplification and sequencing of the human orthologue of rat and mouse FcRn in tissue obtained from human fetal and adult intestine. FcRn protein was detected in adult human intestine by western blot. Immunohistochemical studies of sections of human intestine show that the FcRn is localized mostly to the epithelial cells, where it is in the apical region. These data suggest that the binding of IgG previously seen in the fetal intestine is due to the presence of FcRn. Potential roles for this MHC class I-like Fc receptor in the human intestine include the transfer of passive immunity, induction of oral tolerance, and immunosurveillance.

Adult↗
PubMed1996

An IgG-transporting Fc receptor expressed in the syncytiotrophoblast of human placenta.

During normal human pregnancy, maternal IgG crosses the placenta and provides passive immunity for the fetus. In so doing, IgG passes through two cellular barriers: the syncytiotrophoblast and the fetal capillary endothelium. The Fc region of IgG is required for its transport across the placenta, but the Fc receptors responsible have not been identified definitively. We recently reported the isolation from a placental cDNA library of clones encoding the alpha chain of a human homologue of the major histocompatibility complex class I-related Fc receptor, the neonatal Fc receptor (FcRn). In mice, FcRn is essential for the transport of maternal IgG to the fetus and the neonate. We report here the localization of human FcRn mRNA within the placenta by in situ hybridization, and of human FcRn protein by immunohistochemistry. Both methods show that human FcRn is expressed in syncytiotrophoblast, and is, thus, appropriately located to transport maternal IgG across the first barrier. We confirm previous findings that specific binding of IgG to placental membranes is greater at pH 6.0 than pH 7.5. This corresponds with the pH dependence of IgG binding to FcRn and is consistent with the presence of FcRn in syncytiotrophoblast. We propose a transport model in which maternal IgG binds FcRn at low pH in endosomes within the syncytiotrophoblast. FcRn is not expressed in fetal capillary endothelia, and the mechanism of IgG transport across the second barrier remains unknown.

Amino Acid Sequence↗
PubMed1996

Increased clearance of IgG in mice that lack beta 2-microglobulin: possible protective role of FcRn.

The mechanisms that regulate immunoglobulin G (IgG) catabolism are little understood. We have previously found unusually low IgG concentrations in sera of mice homozygous for a targeted disruption of the beta 2-microglobulin gene. We therefore investigated whether this might result, at least in part, from increased clearance of IgG from the systemic circulation in mice lacking beta 2-microglobulin. We compared the half-lives of radiolabelled mouse IgG1 injected intravenously into beta 2-microglobulin-/- mice and wild-type or heterozygous siblings. The clearance of 125I-labelled IgG1 was strikingly more rapid in the mice lacking beta 2-microglobulin. beta 2-microglobulin-/- mice lack functional molecules of the MHC class I-related Fc receptor, FcRn. Some mutations in mouse IgG1 that increase its clearance have recently been shown to prevent binding to FcRn in the gut. To determine whether the slower degradation of immunoglobulin in mice with beta 2-microglobulin correlated with the ability of the antibody to bind FcRn, we measured the clearance of chicken IgY, which does not bind this receptor. The 125I-labelled IgY was catabolized equally rapidly in beta 2-microglobulin-deficient and wild-type mice. We compared the half-lives of the four subclasses of mouse IgG in beta 2-microglobulin-/-, +/-, and +/+ mice to determine whether the difference we had noted for IgG1 was peculiar to this subclass. The 125I-labelled IgG of all subclasses, with the possible exception of IgG2b. was degraded more rapidly in the beta 2-microglobulin-deficient mice than in heterozygous or wild-type siblings. These data suggest that FcRn can protect IgG from degradation, and is therefore important in maintaining IgG levels in the circulation.

Animals↗
PubMed1995

Requirement for a beta 2-microglobulin-associated Fc receptor for acquisition of maternal IgG by fetal and neonatal mice.

There is considerable evidence to suggest that an FcR similar in structure to class I MHC Ags, neonatal Fc receptor (FcRn), transports IgG across the intestinal epithelium of suckling mice. However, this has not previously been shown definitively, nor has it been shown whether FcRn is the only, or even the major, IgG transporter in the neonatal mouse gut. We report here that neonatal mice homozygous for a targeted disruption of the beta 2microglobulin (beta 2m) gene, which encodes one subunit of FcRn, had reduced FcRn alpha-chain at the lumenal plasma membrane of intestinal cells. These mice had strikingly lower serum IgG levels during the first month after birth than littermates that possessed functional FcRn. Furthermore, we found by fostering mice on mothers with a different IgG allotype that all of the IgG in sera of beta 2m-/- mice was endogenous, and that none was obtained from milk. We conclude that FcRn is the only transporter of IgG from mother to young in the mouse. The onset of IgG synthesis in mice that received no milk IgG lagged behind that in siblings with normal IgG transport, suggesting that maternal IgG stimulates Ab production in the neonate. We noted no difference between the IgG concentrations in the milk of beta 2m-/- and beta 2m+/- mice, indicating that FcRn is not involved in the secretion of IgG into milk.

Animals↗
PubMed1995

A major histocompatibility complex class I-related Fc receptor for IgG on rat hepatocytes.

Intestinal epithelial cells of the neonatal rat and mouse have been shown to express a major histocompatibility complex (MHC) class I-like Fc receptor, or FcRn, which transports IgG in an apical to basolateral direction. Previous studies have suggested the possible expression of this receptor beyond the neonatal period within the liver. Since bile contains high levels of IgG, we sought to determine whether the FcRn was functionally expressed by adult rat hepatocytes. Using primers specific for FcRn, which did not cross hybridize with MHC class I transcripts, FcRn DNA was amplified by reverse transcriptase polymerase chain reaction from RNA of adult rat hepatocytes. This RNA contained functional FcRn transcripts as it encoded a beta 2-microglobulin-associated cell surface protein as determined by immunoprecipitation of biotinylated cell surface proteins with a polyclonal anti-FcRn specific antiserum. Western blotting of hepatocyte canalicular (apical) and sinusoidal (basolateral) plasma membranes with an FcRn-specific monoclonal antibody further confirmed the protein expression and suggested that FcRn was enriched on the canalicular surface membranes. FcRn, on the surface of hepatocytes, was biologically functional as it bound Fc fragments of IgG at pH 6.0 but not 8.0, which is the same pH dependence observed for FcRn in rat neonatal enterocytes. Thus, FcRn is functionally expressed outside of the neonatal period on the canalicular cell surface of adult hepatocytes. This suggests that hepatocyte FcRn may bind luminal IgG, providing a potential functional communication between parenchymal immune cells and bile.

Animals↗
PubMed1994

A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus.

The acquisition of maternal antibodies is critical for immunologic defense of the newborn. In humans, maternal IgG is actively transported across the placenta. The receptor responsible for this transport has not been identified definitively. We report the isolation from a placental cDNA library of clones encoding the alpha-chain of an immunoglobulin G (IgG)-Fc receptor (hFcRn) that resembles a class I major histocompatibility complex antigen. The DNA and predicted amino acid sequences are very similar to those of the neonatal rat and mouse intestinal Fc receptors, rFcRn and mFcRn. These receptors mediate transport of maternal IgG from milk to the blood-stream of the suckling rat or mouse. Like rat and mouse FcRn, hFcRn binds IgG preferentially at low pH, which may imply that IgG binds hFcRn in an acidic intracellular compartment during transport across the placenta.

Amino Acid Sequence↗
PubMed1994

Crystal structure at 2.2 A resolution of the MHC-related neonatal Fc receptor.

The three-dimensional structure of the rat neonatal Fc receptor (FcRn) is similar to the structure of molecules of the major histocompatibility complex (MHC). The counterpart of the MHC peptide-binding site is closed in FcRn, making the FcRn groove incapable of binding peptides. A dimer of FcRn heterodimers seen in the crystals may represent a receptor dimer that forms when the Fc portion of a single immunoglobulin binds. An alternative use of the MHC fold for immune recognition is indicated by the FcRn and FcRn/Fc co-crystal structures.

Animals↗
PubMed1993

Mouse MHC class I-like Fc receptor encoded outside the MHC.

In many mammalian species antibodies transmitted from the mother provide humoral immunity to the young. Maternal IgG from milk is transported across the intestinal epithelium of neonatal rats by an Fc receptor (FcRn) that comprises an alpha-chain similar to the class I Ag of the MHC and beta 2-microglobulin. Suckling mice also acquire antibodies by uptake from the gut. We made a neonatal mouse intestinal cDNA library and screened it with a probe encoding rat FcRn alpha-chain. The nucleotide and predicted amino acid sequences of the two positive clones were very similar to those of rat FcRn. Comparison of the FcRn domains to various MHC class I and CD1 molecules suggests a divergence of FcRn from MHC early in the mammalian lineage. We expressed one of these cDNA in mouse 3T3 fibroblasts. Cells that expressed the cDNA product bound the Fc fragment of IgG with the same pH dependence as neonatal rat intestinal epithelium. We detected RNA that hybridize with the mouse cDNA only in neonatal small intestine and fetal yolk sac, two tissues involved in IgG transport. These data show that the mouse cDNA code for FcRn alpha-chain. The mouse FcRn alpha-chain is similar in sequence to the class I MHC Ag, encoded on chromosome 17 in the mouse. However, we find that the mouse FcRn gene lies outside the MHC, on chromosome 7.

Amino Acid Sequence↗