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ABSTRACT: A proteomics survey of human placental syncytiotrophoblast (ST) apical plasma membranes revealed peptides corresponding to flotillin-1 (FLOT1) and flotillin-2 (FLOT2). The flotillins belong to a class of lipid microdomain-associated integral membrane proteins that have been implicated in clathrin- and caveolar-independent endocytosis. In the present study, we characterized the expression of the flotillin proteins within the human placenta. FLOT1 and FLOT2 were coexpressed in placental lysates and BeWo human trophoblast cells. Immunofluorescence microscopy of first-trimester and term placentas revealed that both proteins were more prominent in villous endothelial cells and cytotrophoblasts (CTs) than the ST. Correspondingly, forskolin-induced fusion in BeWo cells resulted in a decrease in FLOT1 and FLOT2, suggesting that flotillin protein expression is reduced following trophoblast syncytialization. The flotillin proteins co-localized with a marker of fluid-phase pinocytosis, and knockdown of FLOT1 and/or FLOT2 expression resulted in decreased endocytosis of cholera toxin B subunit. We conclude that FLOT1 and FLOT2 are abundantly coexpressed in term villous placental CTs and endothelial cells, and in comparison, expression of these proteins in the ST is reduced. These findings suggest that flotillin-dependent endocytosis is unlikely to be a major pathway in the ST, but may be important in the CT and endothelium.
Histochemie 10/2012; · 2.59 Impact Factor
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ABSTRACT: It has long been known that the ITIM-bearing IgG Fc receptor (FcγRIIb, RIIb) is expressed on liver sinusoidal endothelial cells (LSEC) and that the liver is the major site of small immune complex (SIC) clearance. Thus, we proposed that RIIb of LSEC eliminates blood-borne SIC, thereby controlling immune complex-mediated autoimmune disease. Testing this hypothesis, we found most RIIb of the mouse, fully three-quarters, to be expressed in liver. Moreover, most (90%) liver RIIb was expressed in LSEC, the remainder in Kupffer cells. An absent FcRγ in LSEC implied that RIIb is the sole FcγR expressed. Testing the capacity of liver RIIb to clear blood-borne SIC, we infused mice intravenously with radio-iodinated SIC made of OVA and rabbit IgG anti-OVA. Tracking decay of SIC from the blood, we found the RIIb knockout strain to be severely deficient in eliminating SIC compared with the wild-type strain, terminal half-lives being 6 and 1.5 h, respectively. RIIb on LSEC, a major scavenger, keeps SIC blood concentrations low and minimizes pathologic deposition of inflammatory immune complex.
The Journal of Immunology 10/2012; · 5.79 Impact Factor
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ABSTRACT: Correlative microscopy has meant different things over the years; currently, this term refers to imaging the same exact structures with two or more imaging modalities. This commonly involves combining fluorescence and electron microscopy. Much of the recent work related to correlative microscopy has been done using cell culture models. However, many biological questions cannot be addressed in these models, but require instead the 3-dimensional organization of cells found in tissues. Herein, we discuss some of the issues related to correlative microscopy of tissues including the major reporter systems presently available for correlative microscopy. We present data from our own work in which we have focused on the use of ultrathin cryosections of tissues as the substrate for immunolabeling to combine immunofluorescence and electron microscopy of the same sub-cellular structures.
Methods in cell biology 01/2012; 111:37-57. · 2.05 Impact Factor
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ABSTRACT: The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.
PLoS Pathogens 09/2011; 7(9):e1002281. · 9.13 Impact Factor
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ABSTRACT: Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are functional RNA molecules that have recently emerged as important regulators of gene expression at the posttranscriptional or translational level. The RNA interference effects of siRNA on gene expression make it a valuable research tool for knocking down the expression of genes in mammalian cells in vitro and in vivo enabling the elucidation of molecular mechanisms underlying human diseases. Endogenous miRNAs are involved in a variety of physiological and pathological processes in humans. In this mini-review we first address the synthesis, mechanisms of action, and functions of siRNAs. Then, we focus on recent advances and technologies in miRNA and protein research of the human placenta. Next, we discuss the clinical applications of miRNA in lung cancer. We also touch on "long" noncoding RNAs from intergenic regions of the human genome. This review article is based on a presentation given at a symposium entitled Basic and Clinical Studies on Functional RNA Molecules for Advanced Medical Technologies held at Nippon Medical School in Tokyo, Japan, on November 7, 2009.
Journal of Nippon Medical School 04/2010; 77(2):71-9.
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ABSTRACT: While generally accepted that FcRn of the human syncytiotrophoblast and the mouse yolk sac endoderm is the major IgG transporter, the finding of a different Fc receptor FcgammaRIIb (RIIb) in the human placental endothelium has suggested the existence of an additional IgG transporter. Testing our hypothesis in mouse, we found that while RIIb is expressed in the yolk sac vasculature, IgG concentrations in fetuses of wild-type mice (RIIb(+/+)) and mice with a null mutation in the gene encoding RIIb (RIIb(-/-) mice) are not different, and we thus reject our hypothesis that yolk sac RIIb transports IgG in utero in the mouse. However, the capillary bed in the mouse yolk sac is structurally more complex than in human placenta, consisting of three types of cells: an RIIb-negative endothelium, a unique RIIb-bearing cell that also expresses 2 out of 4 macrophage markers but not endothelial cell or pericyte markers, and pericytes. As in the human placenta the b2 isoform of RIIb predominates in the mouse yolk sac. Remarkably only a single capillary channel rather than 2 channels with a loop is found in each yolk sac villus, which, along with intracapillary erythrocytes, suggests that blood flow is peristaltic, mediated by pericytes. It is not clear whether RIIb in the human placental villus might mediate an IgG transport function in light of the mouse yolk sac equivalent failing to do so.
Journal of Reproductive Immunology 12/2009; 84(2):133-44. · 2.97 Impact Factor
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ABSTRACT: In adults, the nonclassical MHC class I molecule, FcRn, binds both IgG and albumin and rescues both from a degradative fate, endowing both proteins with high plasma concentrations. FcRn also transports IgG from mother to young during gestation. Anticipating that a detailed understanding of gestational IgG transport in the mouse may give us a useful model to understand FcRn function in the human placenta, we have studied FcRn in the mouse yolk sac placenta in detail. Analyzing day 19-20 fetuses of the three FcRn genotypes resulting from matings of FcRn(+/-) parents, we found that FcRn(-/-) fetuses showed negligible IgG concentrations (1.5 microg/ml), whereas IgG concentrations in FcRn(+/-) fetuses were about a half (176 microg/ml) that of FcRn(+/+) fetuses (336 microg/ml), indicating that FcRn is responsible for virtually all IgG transport from mother to fetus. Immunofluorescence and immunoblotting studies indicated that FcRn is expressed in the endoderm of the yolk sac placenta but not in other cells of the yolk sac placenta or in the chorioallantoic placenta. IgG was found in the endoderm of both FcRn(+/+) and FcRn(-/-) yolk sac placentas and in the mesenchyme of FcRn(+/+) but was missing from the mesenchyme of FcRn(-/-) yolk sac placentas, indicating that IgG enters the endoderm constitutively but is moved out of the endoderm by FcRn. The similarities of these results to human placental FcRn expression and function are striking.
The Journal of Immunology 04/2009; 182(5):2583-9. · 5.79 Impact Factor
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ABSTRACT: The syncytiotrophoblast is a specialized epithelium derived from mononuclear cytotrophoblasts that fuse to form this extensive syncytium. Dysferlin is expressed primarily in the apical plasma membrane of the syncytiotrophoblast in the human placenta. Here, we document the presence of another member of the ferlin family, myoferlin, in the placenta and show that it too is expressed primarily in the syncytiotrophoblast. Additionally, we examined the trophoblastic cell lines BeWo, JAR, and JEG-3 for the expression of dysferlin and myoferlin and determined the extent to which their expression was modulated by cell-cell fusion. In trophoblastic cells, there was a positive correlation between cell fusion and increased dysferlin expression but not myoferlin expression. Regarding expression, these trophoblastic cell lines recapitulate the distribution of dysferlin in mononuclear cytotrophoblasts and the syncytiotrophoblast in vivo.
Biology of Reproduction 03/2009; 81(1):33-9. · 4.01 Impact Factor
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ABSTRACT: The human placenta is a complex organ whose proper function is crucial for the development of the fetus. The placenta contains within its structure elements of the maternal and fetal circulatory systems. The interface with maternal blood is the lining of the placenta, that is a unique compartment known as the syncytiotrophoblast. This large syncytial structure is a single cell layer in thickness, and the apical plasma membrane of the syncytiotrophoblast interacts directly with maternal blood. Relatively little is known about the proteins that reside in this unique plasma membrane or how they may change in various placental diseases. Our goal was to develop methods for isolating highly enriched preparations of this apical plasma membrane compatible with high-quality proteomics analysis and herein describe the properties of these isolated membranes.
Analytical Biochemistry 02/2009; 387(1):87-94. · 3.00 Impact Factor
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ABSTRACT: FcRn, a nonclassical MHC-I protein bound to beta 2-microglobulin (beta 2m), diverts IgG and albumin from an intracellular degradative fate, prolonging the half-lives of both. While knockout mouse strains lacking either FcRn-alpha-chain (AK) or beta 2m (BK) show much shorter half-lives of IgG and albumin than normal mice, the plasma IgG half-life in the BK and AK strains is different, being shorter in the BK strain. Since beta 2m does not affect the IgG production rate, we tested whether an additional beta 2m-associated mechanism protects IgG from catabolism. First, we compared the fractional disappearance rate in plasma of an intravenous dose of radioiodinated IgG in a mouse strain deficient in both FcRn-alpha-chain and beta 2m (ABK), in the two parental knockout strains (AK and BK), and in the background wild-type (WT) strain. We found that IgG survived longer in the beta 2m-expressing AK strain than in the beta 2m-lacking ABK and BK strains, whereas the IgG half-lives between the ABK and BK strains were identical. Then we compared endogenous concentrations of four typical plasma proteins among the four strains and found that steady-state plasma concentrations of both IgG and albumin were higher in the AK strain than in either the BK or the ABK strain. These results suggest that a beta 2m-associated effect other than FcRn prolongs the survival of both IgG and albumin, although leaky gene transcription in the AK strain cannot be ruled out.
Experimental Biology and Medicine 06/2008; 233(5):603-9. · 2.64 Impact Factor
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ABSTRACT: The human amnion is a major intrauterine source of prostaglandin (PG) E(2), a potent mediator of uterine contractions and cervical ripening. During parturition, inflammatory cytokines promote PGE(2) production through increased prostaglandin-endoperoxide synthase-2 (PTGS2, also known as cyclooxygenase-2) expression. This is mediated, in part, through activation of the transcription factor nuclear factor kappa B (NFkappaB). Prostaglandin E synthase (PTGES, also known as microsomal PGE synthase-1) acts downstream of PTGS2 and is inducibly expressed in most systems. We hypothesized that NFkappaB might regulate cytokine-induced PTGES expression in amnion cells. With amnion mesenchymal cells, we found that proinflammatory cytokines coordinately upregulated PTGS2 and PTGES mRNA expression. In parallel, increased expression of the PTGS2 and PTGES proteins was observed. In comparison, the expression of two other PGE synthases (PTGES2 and PTGES3) was unmodified. PTGES induction was blocked both in the presence of pharmacological NFkappaB inhibitors and following adenovirus-mediated overexpression of a dominant-negative NFkappaB pathway protein. In cells transiently transfected with a luciferase reporter bearing a portion (-597/+33) of the human PTGES gene promoter, interleukin-1beta (IL1B) produced a moderate increase in luciferase activity; this effect was abrogated in the presence of an indirect NFkappaB inhibitor (MG-132). Finally, a kappaB-like regulatory element was identified that, when mutated, markedly attenuated IL1B-responsive PTGES promoter activity. In conclusion, our results support a role for NFkappaB in cytokine-induced PTGES expression in amnion mesenchymal cells in vitro. By coordinately regulating PTGS2 and PTGES, NFkappaB may contribute to an inducible PGE(2) biosynthesis pathway during human parturition.
Biology of Reproduction 02/2008; 78(1):68-76. · 4.01 Impact Factor
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ABSTRACT: A proteomics screen of human placental microvillous syncytiotrophoblasts (STBs) revealed the expression of dysferlin (DYSF), a plasma membrane repair protein associated with certain muscular dystrophies. This was unexpected given that previous studies of DYSF have been restricted to skeletal muscle. Within the placenta, DYSF localized to the STB and, with the exception of variable labeling in the fetal placental endothelium, none of the other cell types expressed detectable levels of DYSF. Such restricted expression was recapitulated using primary trophoblast cell cultures, because the syncytia expressed DYSF, but not the prefusion mononuclear cells. The apical plasma membrane of the STB contained approximately 4-fold more DYSF than the basal membrane, suggesting polarized trafficking. Unlike skeletal muscle, DYSF in the STB is localized to the plasma membrane in the absence of caveolin. DYSF expression in the STB was developmentally regulated, because first-trimester placentas expressed approximately 3-fold more DYSF than term placentas. As the current literature indicates that few cell types express DYSF, it is of interest that the two major syncytial structures in the human body, skeletal muscle and the STB, express this protein.
Biology of Reproduction 10/2007; 77(3):533-42. · 4.01 Impact Factor
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ABSTRACT: Histochemical techniques have contributed significantly to advances in placental biology and cell biology. In this mini-review, we describe recent advances in histochemical technologies and show how these technologies can profoundly improve our understanding of placenta morphological function related to health and disease. Fundamental theories and applications of five separate methods discussed here are 1) tissue-based polymerase chain reaction by laser microdissection, 2) a novel antigen retrieval method using citraconic anhydride plus heating, 3) immunohistochemical detection of Lewis-related antigen expression and galectin-1 binding in the human placenta, 4) confocal microscopy analysis of IgG transport in placental trophoblasts, and 5) high-resolution immunofluorescence and correlative microscopy using ultrathin cryosections in placental research. This review article is based on a presentation given in a workshop entitled Histochemistry: Theory and Application at the 12th International Federation of Placenta Associations Meeting held in Kobe, Japan, on September 9, 2006.
Journal of Nippon Medical School 09/2007; 74(4):268-73.
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ABSTRACT: The nonclassical MHC class-I molecule, FcRn, salvages both IgG and albumin from degradation. Here we introduce a mechanism-based kinetic model for human to quantify FcRn-mediated recycling of both ligands based on saturable kinetics and data from the literature using easily measurable plasma concentrations rather than unmeasurable endosomal concentrations. The FcRn-mediated fractional recycling rates of IgG and albumin were 142% and 44% of their fractional catabolic rates, respectively. Clearly, FcRn-mediated recycling is a major contributor to the high endogenous concentrations of these two important plasma proteins. While familial hypercatabolic hypoproteinemia is caused by complete FcRn deficiency, the hypercatabolic IgG deficiency of myotonic dystrophy could be explained, based on the kinetic analyses, by a normal number of FcRn with lowered affinity for IgG but normal affinity for albumin. A simulation study demonstrates that the plasma concentrations of IgG and albumin could be dynamically controlled by both FcRn-related and -unrelated parameters.
Clinical Immunology 03/2007; 122(2):146-55. · 4.05 Impact Factor
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ABSTRACT: Chorionic villi in the human placenta serve as essential structures in fetomaternal exchanges. According to the embryology and placentology literature, during the first trimester, the cytotrophoblast (CTB) layer that is subjacent to the syncytiotrophoblast (STB) and supported by a basal lamina is nearly complete, but later, it becomes discontinuous. In the present study, we investigated the structural integrity of the CTB layer in the normal villous tree by advanced microscopy techniques using an antibody to hepatocyte growth factor (HGF) activator inhibitor type 1 (SPINT1), a potent inhibitor of HGF activators expressed exclusively on villous CTB. In full-term placenta, the cell surface of the CTB layer was spread over the basal lamina but was not interrupted. Morphometric analysis showed that throughout the villous tree, 80% of the continuity of the CTB layer of full-term placenta and 90% of that of first-trimester placenta were preserved. Gestation was accompanied by unique structural change in the basal domain of the trophoblast layer. The initially cuboidal-shaped CTB cells were transformed to flat cells with many cellular processes that, together with those of the adjacent STB, eventually covered the trophoblast basal lamina in a complex network of interdigitations. In addition, the expression levels of SPINT1, ST14, HGF, and MET mRNAs in the villous tree increased over the course of gestation. These results suggest that the structural integrity of the SPINT1-positive CTB layer may play an important role in villous differentiation and in maintenance of the villous tree via the HGF signaling system during gestation.
Biology of Reproduction 02/2007; 76(1):164-72. · 4.01 Impact Factor
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ABSTRACT: HFE, a major histocompatibility complex class I-related protein, is implicated in the iron overload disease, hereditary hemochromatosis. Whereas patients with hereditary hemochromatosis have low serum transferrin levels, little is known about transferrin turnover in HFE deficiency states. We injected mice intravenously with radioiodinated transferrin and compared plasma transferrin decay and steady-state endogenous transferrin concentration in the plasma between HFE-deficient and wild-type C57BL/6 mouse strains. HFE-deficient mice degraded transferrin faster than normal (P < 0.001) and had lower plasma transferrin concentrations (P < 0.001). Both HFE-deficient and wild-type mice were then fed diets with 3 different iron concentrations that we designated deficient (2-5 mg/kg of iron), control (0.2 g/kg), and overload (20 g/kg) for 6 wk immediately after weaning to create a range of serum iron concentrations and resultant transferrin saturations ranging from 16 to 78%. We found an inverse correlation between transferrin saturation and transferrin half-life (P < 0.0001, r = -0.839) for both HFE-deficient and wild-type mice, which suggests that HFE does not have a direct effect on transferrin catabolism; rather, HFE may influence transferrin half-life indirectly through its effect on transferrin saturation, which in turn enhances transferrin decay in HFE-deficient mice.
Journal of Nutrition 01/2007; 136(12):2993-8. · 3.92 Impact Factor
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ABSTRACT: The MHC-related Fc receptor for IgG (FcRn) protects albumin and IgG from degradation by binding both proteins with high affinity at low pH in the acid endosome and diverting both from a lysosomal pathway, returning them to the extracellular compartment. Immunoblotting and surface plasmon resonance studies show that both IgG and albumin bind noncooperatively to distinct sites on FcRn, that the affinity of FcRn for albumin decreases approximately 200-fold from acidic to neutral pH, and that the FcRn-albumin interaction shows rapid association and dissociation kinetics. Isothermal titration calorimetry shows that albumin binds FcRn with a 1:1 stoichiometry and the interaction has hydrophobic features as evidenced by a large positive change in entropy upon binding. Our results suggest that the FcRn-albumin interaction has unique features distinct from FcRn-IgG binding despite the overall similarity in the pH-dependent binding mechanism by which both ligands are protected from degradation.
Biochemistry 05/2006; 45(15):4983-90. · 3.42 Impact Factor
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ABSTRACT: We compared the z-axis resolutions achieved by immunofluorescence (IF) microscopic imaging of tissue sections of different thicknesses (ultrathin cryosections, optical sections of cryostat sections and conventional cryostat sections). We used these images to determine the distribution of caveolin-1alpha (CAV-1alpha) and CD31 in endothelial cells of full-term, human placenta. Anti-CAV-1alpha antibody was used to visualize caveolae, which are among the smallest organelles. By using ultrathin cryosections as substrates for IF microscopy, we were able to resolve discrete caveolae that were primarily present immediately beneath the endothelial cell surface. In contrast, neither conventional nor confocal images from cryostat sections were able to resolve individual caveolae, despite dramatic reductions in the confocal image degradation that arises from out-of-focus fluorescence signals. Anti-CD31 antibody labeled the endothelial cell surface exclusively. Quantitative analysis of ultrathin cryosections showed that about 2.5 times more CD31 was expressed on the luminal surface of cells than on the abluminal surface. Our results demonstrate that ultrathin cryosections can serve as excellent substrates for ultrahigh-resolution IF microscopy.
Journal of Electron Microscopy 05/2006; 55(2):107-12. · 1.31 Impact Factor
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ABSTRACT: It is now understood that the nonclassical major histocompatibility complex-I molecule FcRn binds albumin and retrieves it from an intracellular degradative fate. Whether FcRn in the liver modulates albumin turnover through effects on biosynthesis and production is not known. Thus we quantified the appearance of biosynthetically labeled albumin in plasma after an intravenous bolus injection of [(3)H]leucine in FcRn-deficient mice. The production rates for both albumin (FcRn substrate) and transferrin (nonsubstrate) are increased by approximately 20% in FcRn-deficient mice compared with normal mice, likely compensating for the lowered plasma oncotic pressure caused by hypoalbuminemia in FcRn-deficient mice. Determining the magnitude of FcRn-mediated effects on albumin turnover, we then measured the steady-state plasma concentrations of biosynthetically labeled albumin and transferrin during [(3)H]leucine infusion. The concentration of albumin was approximately 40% lower in FcRn-deficient mice compared with normal mice. Furthermore, the approximately 40% lower plasma albumin concentration in FcRn-deficient mice along with the approximately 20% increase in albumin production indicate, by the mass-balance equation, that albumin degradation in FcRn-deficient mice is twice that of normal mice. These studies of biosynthetically labeled, and thus native, albumin support our previous finding that FcRn protects albumin from degradation. Permitting quantification of the magnitude of FcRn-mediated recycling, they further indicate that FcRn has extraordinary capacity: the amount of albumin saved from degradation by FcRn-mediated recycling is the same as that produced by the liver.
AJP Gastrointestinal and Liver Physiology 03/2006; 290(2):G352-60. · 3.43 Impact Factor
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ABSTRACT: Two siblings, products of a consanguineous marriage, were markedly deficient in both albumin and IgG because of rapid degradation of these proteins, suggesting a lack of the neonatal Fc receptor, FcRn. FcRn is a heterodimeric receptor composed of a nonclassical MHC class I alpha-chain and beta(2)-microglobulin (beta(2)m) that binds two ligands, IgG and albumin, and extends the catabolic half-lives of both. Eight relatives of the siblings were moderately IgG-deficient. From sera archived for 35 years, we sequenced the two siblings' genes for the heterodimeric FcRn. We found that, although the alpha-chain gene sequences of the siblings were normal, the beta(2)m genes contained a single nucleotide transversion that would mutate a conserved alanine to proline at the midpoint of the signal sequence. Concentrations of soluble beta(2)m and HLA in the siblings' sera were <1% of normal. Transfection assays of beta(2)m-deficient cultured cells with beta(2)m cDNA indicated that the mutant beta(2)m supported <20% of normal expression of beta(2)m, MHC class I, and FcRn proteins. We concluded that a beta(2)m gene mutation underlies the hypercatabolism and reduced serum levels of albumin and IgG in the two siblings with familial hypercatabolic hypoproteinemia. This experiment of nature affirms our hypothesis that FcRn binds IgG and albumin, salvages both from a degradative fate, and maintains their physiologic concentrations.
Proceedings of the National Academy of Sciences 03/2006; 103(13):5084-9. · 9.68 Impact Factor
