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Biomedical subjects

Wayne I Lencer

Publications and source records attributed to Wayne I Lencer.

At least 19 recordsLinked to original sources
PubMed2006

Rafting with cholera toxin: endocytosis and trafficking from plasma membrane to ER.

Cholera toxin (CT), and members of the AB(5) family of toxins enter host cells and hijack the cell's endogenous pathways to induce toxicity. CT binds to a lipid receptor on the plasma membrane (PM), ganglioside GM1, which has the ability to associate with lipid rafts. The toxin can then enter the cell by various modes of receptor-mediated endocytosis and traffic in a retrograde manner from the PM to the Golgi and the endoplasmic reticulum (ER). Once in the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol so as to induce disease. GM1 is the vehicle that carries CT from PM to ER. Thus, the toxin pathway from PM to ER is a lipid-based sorting pathway, which is potentially meditated by the determinants of the GM1 ganglioside structure itself.

Animals↗
PubMed2006

IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity.

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. 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. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4(+) T cells in regional organized lymphoid structures. FcRn, through its ability to secrete and absorb IgG, thus integrates luminal antigen encounters with systemic immune compartments and, as such, provides essential host defense and immunoregulatory functions at the mucosal surfaces.

Antigens↗
PubMed2006

Protein disulfide isomerase-like proteins play opposing roles during retrotranslocation.

Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.

Cell Culture Techniques↗
PubMed2006

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.

Actins↗
PubMed2006

The Cdc42 inhibitor secramine B prevents cAMP-induced K+ conductance in intestinal epithelial cells.

Cyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl- transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba2+-sensitive K+ channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca2+-mediated chloride secretion pathway, which requires a separate K+ channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K+ conductance in intestinal epithelial cells.

Benzazepines↗
PubMed2006

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.

Animals↗
PubMed2006

A biochemical method for tracking cholera toxin transport from plasma membrane to Golgi and endoplasmic reticulum.

Asiatic cholera is a rapidly progressing disease resulting in extreme diarrhea and even death. The causative agent, cholera toxin, is an AB5-subunit enterotoxin produced by the bacterium Vibrio cholera. The toxin must enter the intestinal cell to cause disease. Entry is achieved by the B-subunit binding to a membrane lipid that carries the toxin all the way from the plasma membrane through the trans-Golgi to the endoplasmic reticulum (ER). Once in the ER, a portion of the A-subunit, the A1 chain, unfolds and separates from the B-subunit to retro-translocate to the cytosol. The A1 chain then activates adenylyl cyclase to cause disease. To study this pathway in intact cells, we used a mutant toxin with C-terminal extension of the B-subunit that contains N-glycosylation and tyrosine-sulfation motifs (CT-GS). This provides a biochemical readout for toxin entry into the trans Golgi (by 35S-sulfation) and ER (by N-glycosylation). In this chapter, we describe the methods we developed to study this trafficking pathway.

Animals↗
PubMed2005

Raft trafficking of AB5 subunit bacterial toxins.

Cholera and the related AB(5)-subunit toxins co-opt plasma membrane (PM) glycolipids to move retrograde into the endoplasmic reticulum (ER) of the host cell where a portion of the toxin is retro-translocated to the cytosol to induce disease. Only glycolipids that associate strongly with detergent insoluble membrane microdomains can sort the toxins backwards from PM to ER. The way certain lipids and proteins are clustered in the plane of the membrane to form lipid rafts likely explains how the glycolipids can function as sorting motifs for the toxins.

Animals↗
PubMed2005

Calnexin and ERp57 facilitate the assembly of the neonatal Fc receptor for IgG with beta 2-microglobulin in the endoplasmic reticulum.

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in noncovalent association with beta(2)-microglobulin (beta(2)m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the beta(2)m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and beta(2)m, CNX bound to the FcRn H chain before the FcRn H chain association with beta(2)m. However, calreticulin only bound to the FcRn H chain-beta(2)m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for beta(2)m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.

Amino Acid Sequence↗
PubMed2005

Role of p97 AAA-ATPase in the retrotranslocation of the cholera toxin A1 chain, a non-ubiquitinated substrate.

The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.

Adenosine Triphosphatases↗
PubMed2005

A passionate kiss, then run: exocytosis and recycling of IgG by FcRn.

The MHC-class-I-like Fcgamma receptor FcRn recycles immunoglobulin (Ig)G from most cells and transports it bi-directionally across epithelial barriers to affect systemic and mucosal immunity. Recent studies have shown that FcRn rescues IgG from intracellular lysosomal degradation by recycling it from the sorting endosome to the cell surface. Most recycling vesicles fuse completely with the plasma membrane in a classical pattern of exocytosis. Similar to the process seen for neurotransmitter release at synaptic junctions, other vesicles fuse only partially, releasing FcRn-IgG complexes to mix into the plasma membrane in cycles of 3-4s over prolonged periods of time.

Exocytosis↗
PubMed2004

Trafficking of cholera toxin-ganglioside GM1 complex into Golgi and induction of toxicity depend on actin cytoskeleton.

Intestinal epithelial lipid rafts contain ganglioside GM1 that is the receptor for cholera toxin (CT). The ganglioside binds CT at the plasma membrane (PM) and carries the toxin through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER). In the ER, a portion of the toxin unfolds and translocates to the cytosol to activate adenylyl cyclase. Activation of the cyclase leads to an increase in intracellular cAMP, which results in apical chloride secretion. Here, we find that an intact actin cytoskeleton is necessary for the efficient transport of CT to the Golgi and for subsequent activation of adenylyl cyclase. CT bound to GM1 on the cell membrane fractionates with a heterogeneous population of lipid rafts, a portion of which is enriched in actin and other cytoskeletal proteins. In this actin-rich fraction of lipid rafts, CT and actin colocalize on the same membrane microdomains, suggesting a possible functional association. Depolymerization or stabilization of actin filaments interferes with transport of CT from the PM to the Golgi and reduces the levels of cAMP generated in the cytosol. Depletion of membrane cholesterol, which also inhibits CT trafficking to the TGN, causes displacement of actin from the lipid rafts while CT remains stably raft associated. On the basis of these observations, we propose that the CT-GM1 complex is associated with the actin cytoskeleton via the lipid rafts and that the actin cytoskeleton plays a role in trafficking of CT from the PM to the Golgi/ER and the subsequent activation of adenylyl cyclase.

Actins↗
PubMed2004

Pulmonary delivery of an erythropoietin Fc fusion protein in non-human primates through an immunoglobulin transport pathway.

Administration of therapeutic proteins by methods other than injection is limited, in part, by inefficient penetration of epithelial barriers. Therefore, unique approaches to breaching these barriers are needed. The neonatal constant region fragment (Fc) receptor (FcRn), which is responsible for IgG transport across the intestinal epithelium in newborn rodents, is expressed in epithelial cells in adult humans and non-human primates. Here we show that FcRn-mediated transport is functional in the lung of non-human primates and that this transport system can be used to deliver erythropoietin (Epo) when it is conjugated to the Fc domain of IgG1. FcRn-dependent absorption was more efficient when the EpoFc fusion protein was deposited predominantly in the upper and central airways of the lung, where epithelial expression of FcRn was most prominently detected. To optimize fusion protein absorption in the lung, we created a recombinant "monomeric-Epo" Fc fusion protein comprised of a single molecule of Epo conjugated to a dimeric Fc. This fusion protein exhibited enhanced pharmacokinetic and pharmacodynamic properties. The bioavailability of the EpoFc monomer when delivered through the lung was approximately equal to that reported for unconjugated Epo delivered s.c. in humans. These studies show that FcRn can be harnessed to noninvasively deliver bioactive proteins into the systemic circulation in therapeutic quantities.

Absorption↗
PubMed2004

Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans-Golgi network but not the Golgi apparatus in Exo2-treated cells.

Cholera toxin (CT) follows a glycolipid-dependent entry pathway from the plasma membrane through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER) where it is retro-translocated into the cytosol to induce toxicity. Whether access to the Golgi apparatus is necessary for transport to the ER is not known. Exo2 is a small chemical that rapidly blocks anterograde traffic from the ER to the Golgi and selectively disrupts the Golgi apparatus but not the TGN. Here we use Exo2 to determine the role of the Golgi apparatus in CT trafficking. We find that under the condition of complete Golgi ablation by Exo2, CT reaches the TGN and moves efficiently into the ER without loss in toxicity. We propose that even in the absence of Exo2 the glycolipid pathway that carries the toxin from plasma membrane into the ER bypasses the Golgi apparatus entirely.

Animals↗
PubMed2004

Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways.

Cholera toxin (CT) and related AB(5) toxins bind to glycolipids at the plasma membrane and are then transported in a retrograde manner, first to the Golgi and then to the endoplasmic reticulum (ER). In the ER, the catalytic subunit of CT is translocated into the cytosol, resulting in toxicity. Using fluorescence microscopy, we found that CT is internalized by multiple endocytic pathways. Inhibition of the clathrin-, caveolin-, or Arf6-dependent pathways by overexpression of appropriate dominant mutants had no effect on retrograde traffic of CT to the Golgi and ER, and it did not affect CT toxicity. Unexpectedly, when we blocked all three endocytic pathways at once, although fluorescent CT in the Golgi and ER became undetectable, CT-induced toxicity was largely unaffected. These results are consistent with the existence of an additional retrograde pathway used by CT to reach the ER.

ADP-Ribosylation Factor 6↗