MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Expression of the cell surface mucin gene family in adenocarcinomas

Cell surface mucins are complex glycoproteins expressed on the apical membrane surface of mucosal epithelial cells. In malignant epithelial cells they are thought to influence cell adhesion, and are clinical targets for tumor immunotherapy and serum tumor marker assays. We have compared expression of MUC1, MUC3, MUC4, MUC11, MUC12 and MUC13 mRNA in epithelial cancers and/or cell lines with non-malignant tissues. In non-malignant tissues, MUC3, 4, 11, 12 and 13 were expressed at highest levels in gastrointestinal tissues, whereas MUC1 was more widely distributed. Significant down-regulation of the MUC4, MUC12 and MUC13 genes was observed in colonic cancers compared with normal tissue, whereas MUC1 was upregulated. In rectal cancers, levels of all six mucin genes were not significantly different to those in normal rectal tissues. Both MUC1 and MUC4 were down-regulated in gastric cancers, whereas cancer and normal tissue levels were similar for MUC3, 11, 12 and 13. In esophageal cancers there was a general trend toward higher levels than in normal tissue for MUC1, 3, 12 and 13. In ovarian cancers MUC1 levels were very high, whereas only low levels of all other mucins were observed. We also report expression in renal cell carcinomas, bladder carcinomas and breast cancer cell lines. The reported expression profiles of the cell surface mucin gene family will help direct biological and clinical studies of these molecules in mucosal biology, and in malignant and inflammatory diseases of epithelial tissues.

The LFA-1-associated molecule PTA-1 (CD226) on T cells forms a dynamic molecular complex with protein 4.1G and human discs large

Clustering of the T cell integrin, LFA-1, at specialized regions of intercellular contact initiates integrin-mediated adhesion and downstream signaling, events that are necessary for a successful immunological response. But how clustering is achieved and sustained is not known. Here we establish that an LFA-1-associated molecule, PTA-1, is localized to membrane rafts and binds the carboxyl-terminal domain of isoforms of the actin-binding protein 4.1G. Protein 4.1 is known to associate with the membrane-associated guanylate kinase homologue, human discs large. We show that the carboxyl-terminal peptide of PTA-1 also can bind human discs large and that the presence or absence of this peptide greatly influences binding between PTA-1 and different isoforms of 4.1G. T cell stimulation with phorbol ester or PTA-1 cross-linking induces PTA-1 and 4.1G to associate tightly with the cytoskeleton, and the PTA-1 from such activated cells now can bind to the amino-terminal region of 4.1G. We propose that these dynamic associations provide the structural basis for a regulated molecular adhesive complex that serves to cluster and transport LFA-1 and associated molecules.

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA ( ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multi-vesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

Cytokine-related genes identified from the RIKEN full-length mouse cDNA data set

To identify novel cytokine-related genes, we searched the set of 60,770 annotated RIKEN mouse cDNA clones (FANTOM2 clones), using keywords such as cytokine itself or cytokine names (such as interferon, interleukin, epidermal growth factor, fibroblast growth factor, and transforming growth factor). This search produced 108 known cytokines and cytokine-related products such as cytokine receptors, cytokine-associated genes, or their products (enhancers, accessory proteins, cytokine-induced genes). We found 15 clusters of FANTOM2 clones that are candidates for novel cytokine-related genes. These encoded products with strong sequence similarity to guanylate-binding protein (GBP-5), interleukin-1 receptor-associated kinase 2 (IRAK-2), interleukin 20 receptor alpha isoform 3, a member of the interferon-inducible proteins of the Ifi 200 cluster, four members of the membrane-associated family 1-8 of interferon-inducible proteins, one p27-like protein, and a hypothetical protein containing a Toll/Interleukin receptor domain. All four clones representing novel candidates of gene products from the family contain a novel highly conserved cross-species domain. Clones similar to growth factor-related products included transforming growth factor beta-inducible early growth response protein 2 (TIEG-2), TGFbeta-induced factor 2, integrin beta-like 1, latent TGF-binding protein 4S, and FGF receptor 4B. We performed a detailed sequence analysis of the candidate novel genes to elucidate their likely functional properties.

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 ( neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.

Dissecting the EphA3/ephrin-A5 interactions using a novel functional mutagenesis screen

The EphA3 receptor tyrosine kinase preferentially binds ephrin-A5, a member of the corresponding subfamily of membrane-associated ligands. Their interaction regulates critical cell communication functions in normal development and may play a role in neoplasia. Here we describe a random mutagenesis approach, which we employed to study the molecular determinants of the EphA3/ephrin-A5 recognition. Selection and functional characterization of EphA3 point mutants with impaired ephrin-A5 binding from a yeast expression library defined three EphA3 surface areas that are essential for the EphA3/ephrin-A5 interaction. Two of these map to regions identified previously in the crystal structure of the homologous EphB2-ephrin-B2 complex as potential ligand/receptor interfaces. In addition, we identify a third EphA3/ephrin-A5 interface that falls outside the structurally characterized interaction domains. Functional analysis of EphA3 mutants reveals that all three Eph/ephrin contact areas are essential for the assembly of signaling-competent, oligomeric receptor-ligand complexes.

Comparisons of physical separation methods of Kunjin virus-induced membranes

The two sets of connected membranes induced in Kunjin virus-infected cells are characterized by the presence of NS3 helicase/protease in both, and by RNA-dependent RNA polymerase (RdRp) activity plus the associated double-stranded RNA (dsRNA) template in vesicle packets (VP), or by the absence of both the VP-specific markers in the convoluted membranes/paracrystalline arrays (CM/PC). Attempts were made to separate flavivirus-induced membranes by sedimentation or flotation analyses in density gradients of sucrose or iodixanol, respectively, after treatment of cell lysates by sonication, osmotic shock, or tryptic digestion. Only osmotic shock treatment provided suggestive evidence of separation. This was explored by flow cytometry analysis (FCA) of RdRp active membrane fractions from a sucrose gradient, using dual fluorescent labelling via antibodies to NS3 and dsRNA. FCA revealed the presence of a dual labelled membrane population indicative of VP, and in a faster sedimenting fraction a membrane population able to be labelled only in NS3, representative of CM/PC and associated (R)ER. It was postulated that osmotic shock ruptured the bounding membrane of the VP, releasing the enclosed small vesicles associated with the Kunjin virus replication complex characterized previously. Notably, the presence of the full spectrum of nonstructural proteins in some membrane fractions was not a reliable marker for RdRp activity. These experiments may provide the opportunity for isolation of relatively pure flavivirus replication complexes in their native membrane-associated state by fluorescence-activated cell sorting. (C) 2004 Elsevier B.V. All rights reserved.

Investigation of the immunocompetent cells that bind early pregnancy factor and preliminary studies of the early pregnancy factor target molecule

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. It has been shown to suppress the delayed-type hypersensitivity response in mice as well as acute and chronic forms of experimental autommume encephalomyelitis in rats and mice, respectively. In previous studies, we have demonstrated that EPF binds to a population of lymphocytes and we hypothesized that it mediates its suppressive effects by binding to CD4(+) T cells. In the present study, we isolated monocytes and subpopulations of lymphocytes and labelled them with fluoresceinated EPF in order to determine which populations bind EPF. We demonstrated that EPF binds specifically to CD4(+), CD8(+), CD14(+) (monocytes) and CD56(+) NK cells but not to CD19(+) B cells. The identity of the molecule(s) on the cell surface that is targeted by EPF is unknown, but as EPF is an extracellular homologue of the intracellular protein chaperonin 10 (Cpn 10), we examined the possibility that the EPF receptor is a membrane-associated form of chaperonin 60 (Cpn60), the functional associate of Cpn 10 within the cell. The EPF target molecule on lymphocytes was visualized by chemical cross-linking of exogenous iodinated Cpn10 to cells and probed with anti-Cpn60. The effect of anti-Cpn60 on activity in the EPF bioassay, the rosette inhibition test, was also examined. In both instances, no specific interaction of this antibody and the putative receptor was observed. It was concluded that the cell surface molecule targeted by EPF is unlikely to be a homologue of Cpn60.

In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

MUC13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells

Transmembrane mucins are glycoproteins involved in barrier function in epithelial tissues. To identify novel transmembrane mucin genes, we performed a tblastn search of the GenBank(TM) EST data bases with a serine/ threonine-rich search string, and a rodent gene expressed in bone marrow was identified. We determined the cDNA sequence of the human orthologue of this gene, MUC13, which localizes to chromosome band 3q13.3 and generates 3.2-kilobase pair transcripts encoding a 512-amino acid protein comprised of an N-terminal mucin repeat domain, three epidermal growth factor-like sequences, a SEA module, a transmembrane domain, and a cytoplasmic tail (GenBank(TM) accession no. AF286113), MUC13 mRNA is expressed most highly in the large intestine and trachea, and at moderate levels in the kidney, small intestine, appendix, and stomach, In situ hybridization in murine tissues revealed expression in intestinal epithelial and lymphoid cells. Immunohistochemistry demonstrated the human MUC13 protein on the apical membrane of both columnar and goblet cells in the gastrointestinal tract, as well as within goblet cell thecae, indicative of secretion in addition to presence on the cell surface. MUC13 is cleaved, and the beta -subunit containing the cytoplasmic tail undergoes homodimerization, Including MUC13, there are at least five cell surface mucins expressed in the gastrointestinal tract.

Syntaxin 7 complexes with mouse Vps10p tail interactor 1b, Syntaxin 6, vesicle-associated membrane protein (VAMP)8, and VAMP7 in B16 melanoma cells

Syntaxin 7 is a mammalian target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane transport between late endosomes and lysosomes. The aim of the present study was to use immunoaffinity techniques to identify proteins that interact with Syntaxin 7. We reasoned that this would be facilitated by the use of cells producing high levels of Syntaxin 7, Screening of a large number of tissues and cell lines revealed that Syntaxin 7 is expressed at very high levels in B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in these cells as they underwent melanogenesis. From a large scale Syntaxin 7 immunoprecipitation, we have identified six polypeptides using a combination of electrospray mass spectrometry and immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin 6, mouse Vps10p tail interactor 1b (mVti1b), alpha -synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and the protein phosphatase 1M regulatory subunit. We also observed partial colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE Syntaxin 13. Based on these and data reported previously, we propose that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes with either VAMP7 or VAMPS to regulate fusion events within the late endosomal pathway and that these events may play a critical role in melanogenesis.