Wayne I. Lencer

Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin.

Cholera toxin is assembled from two subunits in the periplasm of Vibrio cholerae and disassembled in the analogous compartment of target cells, the lumen of the endoplasmic reticulum (ER), before a fragment of it, the A1 chain, is transported into the cytosol. We show that protein disulfide isomerase (PDI) in the ER lumen functions to disassemble and unfold the toxin once its A chain has been cleaved. PDI acts as a redox-driven chaperone; in the reduced state, it binds to the A chain and in the oxidized state it releases it. Our results explain the pathway of cholera toxin, suggest a role for PDI in retrograde protein transport into the cytosol, and indicate that PDI can act as a novel type of chaperone, whose binding and release of substrates is regulated by a redox, rather than an ATPase, cycle.

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.

Gangliosides are receptors for murine polyoma virus and SV40

Polyoma virus (Py) and simian virus 40 (SV40) travel from the plasma membrane to the endoplasmic reticulum (ER) from where they enter the cytosol and then the nucleus to initiate infection. Here we demonstrate that specific gangliosides can serve as plasma membrane receptors for these viruses, GD1a and GT1b for Py and GM1 for SV40. Binding and flotation assays were used to show that addition of these gangliosides to phospholipid vesicles allowed specific binding of the respective viruses. The crystal structure of polyoma VP1 with a sialic acid‐containing oligosaccharide was used to derive a model of how the two terminal sugars (sialic acid‐α2,3‐galactose) in one branch of GD1a and GT1b are recognized by the virus. A rat cell line deficient in ganglioside synthesis is poorly infectible by polyoma and SV40, but addition of the appropriate gangliosides greatly facilitates virus uptake, transport to the ER and infection. Lipid binding sites for polyoma are shown to be present in rough ER membranes, suggesting that the virus travel with the ganglioside(s) from the plasma membranes to the ER.

Receptor-mediated immunoglobulin G transport across mucosal barriers in adult life : functional expression of FcRn in the mammalian lung

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain the immunoglobulins (Ig)G and secretory IgA (sIgA) that function together in host defense. Exactly how IgG crosses epithelial barriers to function in mucosal immunity remains unknown. Here, we test the idea that the MHC class I–related Fc-receptor, FcRn, transports IgG across the mucosal surface of the human and mouse lung from lumen to serosa. We find that bronchial epithelial cells of the human, nonhuman primate, and mouse, express FcRn in adult-life, and demonstrate FcRn-dependent absorption of a bioactive Fc-fusion protein across the respiratory epithelium of the mouse in vivo. Thus, IgG, like dimeric IgA, can cross epithelial barriers by receptor-mediated transcytosis in adult animals. These data show that mucosal surfaces that express FcRn reabsorb IgG and explain a mechanism by which IgG may act in immune surveillance to retrieve lumenal antigens for processing in the lamina propria or systemically.

Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure

Epithelial tight junctions form a barrier against passive paracellular flux. This barrier is regulated by complex physiologic and pathophysiologic signals that acutely fine-tune tight junction permeability. Although actomyosin contraction and myosin light chain phosphorylation are clearly involved in some forms of tight junction regulation, the contributions of other signaling events and the role of myosin light chain phosphorylation in this response are poorly understood. Here we ask if activation of myosin light chain kinase alone is sufficient to induce downstream tight junction regulation. We use a confluent polarized intestinal epithelial cell model system in which constitutively active myosin light chain kinase, tMLCK, is expressed using an inducible promoter. tMLCK expression increases myosin light chain phosphorylation, reorganizes perijunctional F-actin, and increases tight junction permeability. TJ proteins ZO-1 and occludin are markedly redistributed, morphologically and biochemically, but effects on claudin-1 and claudin-2 are limited. tMLCK inhibition prevents changes in barrier function and tight junction organization induced by tMLCK expression, suggesting that these events both require myosin light chain phosphorylation. We conclude that myosin light chain phosphorylation alone is sufficient to induce tight junction regulation and provide new insights into the molecular mechanisms that mediate this regulation.

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells.

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the β2-microglobulin (β2m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical FcγRs: FcγRI, FcγRII, and FcγRIII.

MEMBRANE TRAFFIC AND THE CELLULAR UPTAKE OF CHOLERA TOXIN

In nature, cholera toxin (CT) and the structurally related E. coli heat labile toxin type I (LTI) must breech the epithelial barrier of the intestine to cause the massive diarrhea seen in cholera. This requires endocytosis of toxin-receptor complexes into the apical endosome, retrograde transport into Golgi cisternae or endoplasmic reticulum (ER), and finally transport of toxin across the cell to its site of action on the basolateral membrane. Targeting into this pathway depends on toxin binding ganglioside GM1 and association with caveolae-like membrane domains. Thus to cause disease, both CT and LTI co-opt the molecular machinery used by the host cell to sort, move, and organize their cellular membranes and substituent components.

Dependence of antibody-mediated presentation of antigen on FcRn

The neonatal Fc receptor for IgG (FcRn) is a distant member of the MHC class I protein family. It binds IgG and albumin in a pH-dependent manner and protects these from catabolism by diverting them from a degradative fate in lysosomes. In addition, FcRn-mediated IgG transport across epithelial barriers is responsible for the transmission of IgG from mother to infant and can also enhance IgG-mediated antigen uptake across mucosal epithelia. We now show a previously undescribed role for FcRn in mediating the presentation of antigens by dendritic cells when antigens are present as a complex with antibody by uniquely directing multimeric immune complexes, but not monomeric IgG, to lysosomes.

Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria

The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. Selective expression of FcRn in the epithelium is shown here to be associated with secretion of IgG into the lumen that allows for defense against an epithelium-associated pathogen (Citrobacter rodentium). This pathway of host resistance to a bacterial pathogen as mediated by FcRn involves retrieval of bacterial antigens from the lumen and initiation of adaptive immune responses in regional lymphoid structures. Epithelial-associated FcRn, through its ability to secrete and absorb IgG, may thus integrate luminal antigen encounters with systemic immune compartments and as such provide essential host defense and immunoregulatory functions at the mucosal surfaces.

Neonatal Fc receptor: from immunity to therapeutics.

The neonatal Fc receptor (FcRn), also known as the Brambell receptor and encoded by Fcgrt, is a MHC class I like molecule that functions to protect IgG and albumin from catabolism, mediates transport of IgG across epithelial cells, and is involved in antigen presentation by professional antigen presenting cells. Its function is evident in early life in the transport of IgG from mother to fetus and neonate for passive immunity and later in the development of adaptive immunity and other functions throughout life. The unique ability of this receptor to prolong the half-life of IgG and albumin has guided engineering of novel therapeutics. Here, we aim to summarize the basic understanding of FcRn biology, its functions in various organs, and the therapeutic design of antibody- and albumin-based therapeutics in light of their interactions with FcRn.