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PubMed2006

Localization of StarD5 cholesterol binding protein.

Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.

Adaptor Proteins, Vesicular Transport↗
PubMed1997

Microtubule-dependent transport of secretory vesicles visualized in real time with a GFP-tagged secretory protein.

Biosynthetic transport from the trans-Golgi network (TGN) to the plasma membrane (PM) is mediated by secretory vesicles. We analyzed secretory vesicle transport in real time using a GFP-tagged secretory protein, hCgB-GFP, consisting of human chromogranin B (hCgB) and green fluorescent protein (GFP). The fusion protein was expressed transiently in Vero cells or in a stable clone after induction with butyrate. After arrest of the biosynthetic protein transport at 20 degrees C, fluorescent hCgB-GFP colocalized with TGN38, a marker of the TGN. Subsequent release of the secretion block at 37 degrees C led to the formation of green fluorescent vesicles. Confocal analysis revealed that these vesicles were devoid of TGN38 and of Texas Red-coupled transferrin and cathepsin D, markers of the endosomal/lysosomal pathway. As determined by fluorometry and metabolic labelling hCgB-GFP was secreted from the TGN to the PM with a t(1/2) of 20-30 minutes. Video-microscope analysis of green fluorescent vesicles showed brief periods of rapid directed movement with maximal velocities of 1 microm/second. Vesicle movement occurred in all directions, centrifugal, centripetal and circumferential, and 50% of the vesicles analyzed reversed their direction of movement at least once within an observation period of 45 seconds. In the presence of nocodazole the movement of fluorescent vesicles ceased. Concomitantly, secretion of hCgB-GFP was slowed but not completely blocked. We suggest that microtubules (MT) facilitate the delivery of secretory vesicles to the PM by a stochastic transport, thereby increasing the probability for a vesicle/target membrane encounter.

Animals↗
PubMed1999

Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes.

PA2.26 antigen was identified as a cell-surface protein induced in epidermal carcinogenesis and skin remodeling processes. PA2.26 is expressed in carcinoma cell lines and cultured fibroblasts but absent in nontumorigenic keratinocytes. In tissues, PA2.26 is present in epithelial cells of the choroid plexus, ependyma, glomerulus and alveolus, in mesothelial cells, and in endothelia of lymphatic vessels. Biochemical characterization of PA2.26 protein and sequence analysis of the isolated cDNA demonstrate that PA2.26 antigen is a mucin-like transmembrane glycoprotein. Confocal and immunoelectron microscopy analysis in cultured cells reveal that PA2. 26 is concentrated in actin-rich microvilli and plasma membrane projections, such as filopodia, lamellipodia and ruffles, where it colocalizes with members of the ERM (ezrin, radixin, moesin) family protein. Ezrin and moesin, but not radixin, can be coimmunoprecipitated together with PA2.26 from cell lysates. Ectopic expression of PA2.26 in immortalized, nontumorigenic, keratinocytes induces an epithelial-fibroblastoid morphological conversion with increased plasma membrane extensions, concomitantly to a major reorganization of the actin cytoskeleton, redistribution of ezrin to cell-surface projections, and enhanced motility. These findings suggest an involvement of PA2.26 in cell migration.

Actins↗
PubMed2001

Decreased leptin uptake in hypothalamic nuclei with ageing in Wistar rats.

Leptin interacts with specific receptors in hypothalamic nuclei and modulates energy balance. Growing evidence has shown the association of obesity and hyperleptinaemia with non-insulin-dependent diabetes mellitus and insulin resistance. The aged Wistar rat shows peripheral insulin resistance in the absence of obesity and alterations of glucose homeostasis. However, it is not known whether, in these animals, the leptin action is altered. Here we studied the effect of ageing on plasma leptin concentration and the ability of hypothalamic nuclei to capture i.c.v.-injected digoxigenin-labelled leptin. Our data indicate that 24-month-old animals are hyperleptinaemic. However, daily food intake was greater in old animals, suggesting that they are leptin resistant. Leptin uptake in the hypothalamus was reduced in old rats. This uptake was a receptor-mediated process as demonstrated by displacement. Leptin accumulation in hypothalamic nuclei was partially colocalized with neuropeptide Y fibres. Immunohistochemical and western blot analyses showed a lower amount of the long form of leptin receptors in the hypothalamus of aged rats. Analysis by RT-PCR also demonstrated a decreased expression of leptin receptor mRNA in old animals. We conclude that the lower leptin uptake may be explained, at least in part, by a decreased amount of receptors in hypothalamic neurones of the aged rats.

Aging↗
PubMed2000

A genome scan using a novel genetic cross identifies new susceptibility loci and traits in a mouse model of rheumatoid arthritis.

Proteoglycan-induced arthritis (PGIA) is a murine model for rheumatoid arthritis (RA) both in terms of its pathology and its genetics. PGIA can only be induced in susceptible mouse strains and their F(2) progeny. Using the F(2) hybrids resulting from an F(1) intercross of a newly identified susceptible (C3H/HeJCr) and an established resistant (C57BL/6) strain of mouse, our goals were to: 1) identify the strain-specific loci that confer PGIA susceptibility, 2) determine whether any pathophysiological parameters could be used as markers that distinguish between nonarthritic and arthritic mice, and 3) analyze the effect of the MHC haplotype on quantitative trait loci (QTL) detection. To identify QTLs, we performed a genome scan on the F(2) hybrids. For pathophysiological analyses, we measured pro- and antiinflammatory cytokines such as IL-1, IL-6, IFN-gamma, IL-4, IL-10, IL-12, Ag-specific T cell proliferation and IL-2 production, serum IgG1 and IgG2 levels of both auto- and heteroantibodies, and soluble CD44. We have identified four new PGIA-linked QTLs (Pgia13 through Pgia16) and confirmed two (Pgia5, Pgia10) from our previous study. All new MHC-independent QTLs were associated with either disease onset or severity. Comprehensive statistical analysis demonstrated that while soluble CD44, IL-6, and IgG1 vs. IgG2 heteroantibody levels differed significantly between the arthritic and nonarthritic groups, only Ab-related parameters colocalized with the QTLs. Importantly, the mixed haplotype (H-2(b) and H-2(k)) of the C3H x C57BL/6 F(2) intercross reduced the detection of several previously identified QTLs to suggestive levels, indicating a masking effect of unmatched MHCs.

Animals↗
PubMed2002

IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.

Targeted disruption of either c-Src or TNFR-associated factor 6 (TRAF6) in mice causes osteoclast dysfunction and an osteopetrotic phenotype, suggesting that both molecules play important roles in osteoclastic bone resorption. We previously demonstrated that IL-1 induces actin ring formation and osteoclast activation. In this study, we examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in the activation of osteoclast-like cells (prefusion cells (pOCs); multinucleated cells) formed in the murine coculture system. In normal pOCs, IL-1 induces actin ring formation and tyrosine phosphorylation of p130(Cas), a known substrate of c-Src. However, in Src-deficient pOCs, p130(Cas) was not tyrosine phosphorylated following IL-1 treatment. In normal pOCs treated with IL-1, anti-TRAF6 Abs coprecipitate p130(Cas), protein tyrosine kinase 2, and c-Src. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is required for formation of the TRAF6, p130(Cas), and protein tyrosine kinase 2 complex. Moreover, an immunocytochemical analysis revealed that in osteoclast-like multinucleated cells, IL-1 induced redistribution of TRAF6 to actin ring structures formed at the cell periphery, where TRAF6 also colocalized with c-Src. Taken together, these data suggest that IL-1 signals feed into the tyrosine kinase pathways through a TRAF6-Src molecular complex, which regulates the cytoskeletal reorganization essential for osteoclast activation.

Actins↗
PubMed1995

Expression, secretion, and age-related downregulation of pigment epithelium-derived factor, a serpin with neurotrophic activity.

Retinal pigment epithelial (RPE) cells form a functional complex with photoreceptor neurons of the retina, interacting through the interphotoreceptor matrix (IPM). We now provide evidence that the gene for pigment epithelium-derived factor (PEDF), a protein possessing neurotrophic and neuronal-survival activities, is highly expressed by both fetal and young adult RPE cells. PEDF mRNA is present in RPE cells of the human eye at 17 weeks of gestation, demonstrating its potential for action in vivo during early retinal development. The PEDF protein is secreted in vivo where it constitutes a part of the fetal and adult IPM surrounding photoreceptor outer segments. A polyclonal PEDF antibody recognizes at least four isoforms of secreted human and bovine PEDF by two dimensional gel analysis, and detects a similar 50 kDa protein in the IPM of several other vertebrate species. Within soluble extracts of RPE cells, however, where little, if any, of the 50 kDa species can be detected, an immunoreactive 36 kDa protein is observed by Western blot analysis. By immunofluorescence, PEDF is localized intracellularly in association with the nucleus, presumptive secretory granules, and cytoskeletal elements of cultured RPE cells with PEDF and actin antibodies colocalizing to the same cytoskeletal structures. During initial stages of attachment, PEDF and actin also concentrate at the tips of pseudopods extended by the cultured RPE cells. However, with successive passages, synthesis, and secretion of the PEDF protein as well as transcription of its mRNA decrease and are lost by about 10 passages. In parallel, cultured RPE cells lose their proliferative potential and change from an epithelial-like morphology in early passages to a more fibroblast-like appearance by about the 10th passage. PEDF is thus apparently present intracellularly and extracellularly in both fetal and early adult periods where it could be involved in cellular differentiation and survival and with its loss, in the onset of senescence.

Aging↗
PubMed1998

A novel AP180-related protein in vesicles that concentrate at acetylcholine receptor clusters.

Monoclonal antibodies were generated to vesicular membranes of clathrin coated vesicles enriched for acetylcholinesterase (AChE). One of these, C172, recognizes vesicles which accumulate in muscle cells around nuclei associated with acetylcholine receptor AChR clusters. Immunoblots of muscle extracts and brain purified clathrin coated vesicles show that C172 recognizes a 100 kd band in muscle, but a 180 kd band in brain. Western blots of purified AP180 protein stained with the two antibodies AP180.1 and C172 displayed the same staining pattern. Tryptic digests probed with peptide antibodies (PS26 and PS27) generated to known sequences of AP180 were used to map the epitope for C172 within the brain AP180 sequence. On immunoblots of digested AP180, all AP180 antibodies and C172 recognized a 100 kd tryptic fragment, however only C172 recognized a smaller 60 kd. Our results suggest that the C172 epitope is located within amino acids 305-598 of the AP180 sequence. Confocal fluorescence microscopy of myoblasts and myotubes stained with the C172 antibody gives a punctate immunofluorescence pattern. Myoblasts stained with C172 revealed a polarized distribution of vesicles distinct from that observed when cells are stained with gamma adaptin antibody which is known to localize to trans Golgi network. Myotubes stained with C172 antibody reveal a linear array of vesicular staining. Quantitative analysis of C172 reactive vesicles revealed a significant increase in number of vesicles present around the nuclei associated with the acetylcholine receptor clusters. These vesicles did not colocalize with the Golgi cisternae. These results indicate that a protein with homology to the neuron-specific coated vesicle protein AP180, is present in muscle cells associated with vesicles showing significant concentration around postsynaptic nuclei present in close proximity to AChR clusters.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

Stable complexes involving acetylcholinesterase and amyloid-beta peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils.

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.

Acetylcholinesterase↗
PubMed1993

Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are mitogenic polypeptides that may contribute to cancer cell proliferation. In the present study we examined aFGF and bFGF expression in human pancreatic cancer. Northern blot analysis of total RNA isolated from 12 pancreatic cancers revealed elevated aFGF and bFGF mRNA levels in 12 and 10 samples, respectively, by comparison with the normal human pancreas. Immunostaining demonstrated the presence of aFGF and bFGF in many cancer cells and in the atrophic acini and ducts adjacent to the cancer cells, but to a much lesser extent in the surrounding fibroblasts. By in situ hybridization, both mRNA moieties colocalized with their respective proteins and were abundant in many cancer cells. Immunoblotting confirmed that cancer tissues with increased aFGF and bFGF immunoreactivity contained elevated levels of both proteins. To determine the significance of aFGF and bFGF expression in the pancreatic cancer cells, immunohistochemical analysis of 78 human pancreatic carcinomas was performed. aFGF and bFGF immunoreactivity was present in the cancer cells in 47 (60%) and 44 (56%) of the tumors, respectively. There was a significant correlation between the presence of either aFGF or bFGF in the cancer cells and advanced tumor stage, and the presence of bFGF and shorter patient survival. These data suggest that aFGF and bFGF are overexpressed in a significant proportion of human pancreatic carcinoma cells and that this overexpression may contribute to disease progression.

Adult↗
PubMed2004

Functional analysis of the NPDC-1 gene.

Mouse NPDC-1 (Neural Proliferation Differentiation and Control-1) is specifically expressed in neural cells when they stop dividing and start to differentiate. The NPDC-1 protein has been shown to interact with the E2F1 transcription factor, D-type cyclins and Cdk2. Immunocytochemical studies and subcellular fractionation of rat brains disclosed a partial colocalization of NPDC-1 with synaptic vesicle proteins, suggesting additional functional interactions. Here, we report the characterization of the mouse and human genes that were found to display very similar structures. We mapped the human gene to chromosome 9q34.3. No obvious pathological defect has been previously linked to this region. In order to gain further insights into its function(s), we generated null mice for the NPDC-1 gene. We did not detect any macroscopic phenotypical defect. Analysis of the upstream sequence of the mouse NPDC-1 gene delineated two regions involved in its negative and positive transcriptional regulation. Evidence for the regulation of NPDC-1 by Krox family transcription factors is presented.

Animals↗
PubMed2005

Cellular localization of IL-18 and IL-18 receptor in pig anterior pituitary gland.

Pro-inflammatory cytokine interleukin 18 (IL-18) has been proposed to have a role in modulating immuno-endocrine functions. Our previous study showed that IL-18 and IL-18 receptor (IL-18R) colocalized in somatotrophs of the bovine anterior pituitary gland, and the possibility that IL-18 acts on somatotrophs as an autocrine factor. In the present study, we investigated the localization of IL-18 and IL-18R in the pig anterior pituitary gland. RT-PCR analysis showed the expression of IL-18 and IL-18R mRNAin the pig anterior pituitary gland. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs, mammotrophs, thyrotrophs and gonadotrophs. IL-18R was localized in somatotrophs and thyrotrophs. Furthermore, the somatotrophs immunoreactive for IL-18 did not contain IL-18R. Thus, IL-18R and IL-18 were not colocalized in an identical somatotroph. These findings suggest that the localization of IL-18 in pig somatotrophs is different from that in bovine somatotrophs, although IL-18 closely associates with somatotrophs in the anterior pituitary glands in both species.

Animals↗
PubMed1986

Comparative distribution of 3H-GABA uptake and GAD immunoreactivity in goldfish retinal amacrine cells: a double-label analysis.

The comparative distribution of 3H-GABA uptake and glutamic acid decarboxylase immunoreactivity (GAD-IR) in amacrine cells of goldfish retina was studied simultaneously by using a combined autoradiographic/immunocytochemical technique in order to determine the degree of colocalization of these two markers of GABAergic neurons; 3H-GABA was taken up most intensely by large pyriform Ab amacrine cells (3% of inner nuclear layer (INL) somata), and less intensely by smaller, polyform amacrine cells (12% of INL somata), cell bodies in the ganglion cell layer (one-half as common as Ab cells), and cell bodies in the inner plexiform layer (very rare). GAD-IR was observed in 25% of amacrine cells in the INL, the vast majority of which were polyform in shape, and cell bodies in the ganglion cell layer. Twice as many cells were labeled for GAD-IR as for 3H-GABA uptake. Of the cells that took up 3H-GABA, colocalization of 3H-GABA uptake and GAD-IR was observed in 90% of the polyform amacrine cells, 80% of the cells in the ganglion cell layer, and none of the pyriform Ab amacrine cells or cells in the inner plexiform layer. We suggest that the polyform cells compose the major population of GABAergic amacrine cells in the goldfish retina, rather than the pyriform Ab cells. GABAergic displaced amacrine cells or ganglion cells are also indicated by our data. The implications of these data with regard to the physiology of the goldfish retina are discussed as well.

Animals↗
PubMed1990

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Amino Acid Sequence↗
PubMed2004

Molecular analysis of the interaction between palladin and alpha-actinin.

Palladin is a novel component of stress fiber dense regions. Antisense and transient overexpression studies have indicated an important role for palladin in the regulation of actin cytoskeleton. Palladin colocalizes and coimmunoprecipitates with alpha-actinin, a dense region component, but the molecular details and functional significance of the interaction have not been studied. We show here a direct association between the two proteins and have mapped the binding site within a short sequence of palladin and in the carboxy-terminal calmodulin domain of alpha-actinin. Using transfection-based targeting assays, we show that palladin is involved in targeting of alpha-actinin to specific subcellular foci indicating a functional interplay between the two actin-associated proteins.

Actinin↗
PubMed2003

Association of gephyrin and glycine receptors in the human brainstem and spinal cord: an immunohistochemical analysis.

Gephyrin is a postsynaptic clustering molecule that forms a protein scaffold to anchor inhibitory neurotransmitter receptors at the postsynaptic membrane of neurons. Gephyrin was first identified as a protein component of the glycine receptor complex and is also colocalized with several GABAA receptor subunits in rodent brain. We have studied the distribution of gephyrin and glycine receptor subunits in the human brainstem and spinal cord using immunohistochemistry at light and confocal laser scanning microscopy levels. This study demonstrates the novel localization of gephyrin with glycine receptors in the human brainstem and spinal cord. Colocalization of immunoreactivities for gephyrin and glycine receptor subunits was detected in the dorsal and ventral horns of the spinal cord, the hypoglossal nucleus and the medial vestibular nucleus of the medulla. The results clearly establish that gephyrin is ubiquitously distributed and is colocalized, with a large proportion of glycine receptor subunits in the human brainstem and spinal cord. We therefore suggest that gephyrin functions as a clustering molecule for major subtypes of glycine receptors in the human CNS.

Aged↗
PubMed1998

CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts.

Chemokines as well as the signaling through the adhesion molecules intercellular adhesion molecule (ICAM)-3 and CD43 are able to induce in T lymphocytes their switching from a spherical to a polarized motile morphology, with the formation of a uropod at the rear of the cell. We investigated here the role of CD43 in the regulation of T-cell polarity, CD43-cytoskeletal interactions, and lymphocyte aggregation. Pro-activatory anti-CD43 monoclonal antibody (MoAb) induced polarization of T lymphocytes with redistribution of CD43 to the uropod and the CCR2 chemokine receptor to the leading edge of the cell. Immunofluorescence analysis showed that all three ezrin-radixin-moesin (ERM) actin-binding proteins localized in the uropod of both human T lymphoblasts stimulated with anti-CD43 MoAb and tumor-infiltrating T lymphocytes. Radixin localized at the uropod neck, whereas ezrin and moesin colocalized with CD43 in the uropod. Biochemical analyses showed that ezrin and moesin coimmunoprecipitated with CD43 in T lymphoblasts. Furthermore, in these cells, the CD43-associated moesin increased after stimulation through CD43. The interaction of moesin and ezrin with CD43 was specifically mediated by the cytoplasmic domain of CD43, as shown by precipitation of both ERM proteins with a GST-fusion protein containing the CD43 cytoplasmic tail. Videomicroscopy analysis of homotypic cell aggregation induced through CD43 showed that cellular uropods mediate cell-cell contacts and lymphocyte recruitment. Immunofluorescence microscopy performed in parallel showed that uropods enriched in CD43 and moesin localized at the cell-cell contact areas of cell aggregates. The polarization and homotypic cell aggregation induced through CD43 was prevented by butanedione monoxime, indicating the involvement of myosin cytoskeleton in these phenomena. Altogether, these data indicate that CD43 plays an important regulatory role in remodeling T-cell morphology, likely through its interaction with actin-binding proteins ezrin and moesin. In addition, the redistribution of CD43 to the uropod region of migrating lymphocytes and during the formation of cell aggregates together with the enhancing effect of anti-CD43 antibodies on lymphocyte cell recruitment suggest that CD43 plays a key role in the regulation of cell-cell interactions during lymphocyte traffic.

Antigens, CD↗
PubMed2004

Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin.

Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.

Animals↗