TG2, a novel extracellular protein with multiple functions

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca(2+)-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for ß integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCa. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.

Glycogen, the preferred energy source for extracellular protein formation and growth of Aeromonas salmonicida

A study was made of the effect of supplementing a rich 3% (w/v) tryptone soya broth (TSB) medium and a poorer 1.7% (w/v) tryptone-based medium with glucose, maltose and glycogen, as carbon sources, on growth and exoprotein formation by Aeromonas salmonicida. In TSB, glucose inhibited growth and repressed exoprotein formation whilst maltose and glycogen had little effect, up to 20 h, when compared with an unsupplemented control. By contrast, in the poorer medium, over a 24-h incubation period, growth was stimulated three-fold by glycogen, and whilst exoprotein formation was low in comparison with that observed in TSB, the greatest production was observed in the presence of glycogen. Extracellular alpha-amylase was measured in the tryptone medium in the presence of the three carbon sources and the highest level, produced in the presence of glycogen, was 1.6 times that with added maltose whilst none was detectable with glucose present. This pattern was repeated in the case of the maltose-inducible porin, LamB, of the outer membrane.

A coiled-coil region of an insect immune suppressor protein is involved in binding and uptake by hemocytes

Polydnaviruses are associated with certain parasitoid wasps and are introduced into the body cavity of the host caterpillar during oviposition. Some of the viral genes are expressed in host tissues and corresponding proteins are secreted into the hemocoel causing suppression of the host immune system. The Cotesia rubecula polydnavirus gene product, CrV1, effectively inactivates hemocytes by mediating cytoskeleton break-down. A precondition for the CrV1 function is the incorporation of the extracellular protein by hemocytes. Here, we show that a coiled-coil domain containing a putative leucine zipper is required for CrV1 function, since removal of this domain abolishes binding and uptake of the CrV1 protein by hemocytes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Spatial pattern of prion protein deposits in patients with sporadic Creutzfeldt–Jakob disease

The spatial pattern of the prion protein (PrP) deposits was studied in the cerebral cortex and cerebellum in 10 patients with sporadic Creutzfeldt–Jakob disease (CJD). In all patients the PrP deposits were aggregated into clusters and, in 90% of cortical areas and in 50% of cerebellar sections, the clusters exhibited a regular periodicity parallel to the tissue boundary; a spatial pattern also exhibited by ß-amyloid (Aß) deposits in Alzheimer's disease (AD). In the cerebral cortex, the incidence of regular clustering of the PrP deposits was similar in the upper and lower cortical laminae. The sizes of the PrP clusters in the upper and lower cortex were uncorrelated. No significant differences in mean cluster size of the PrP deposits were observed between brain regions. The size, location and distribution of the PrP deposit clusters suggest that PrP deposition occurs in relation to specific anatomical pathways and supports the hypothesis that prion pathology spreads through the brain via such pathways. In addition, the data suggest that there are similarities in the pathogenesis of extracellular protein deposits in prion disease and in AD.

The spatial patterns of prion protein deposits in Creutzfeldt-Jakob disease:Comparison with β-amyloid deposits in Alzheimer's disease

Similar pathological processes may be involved in the deposition of extracellular proteins in the brains of patients with Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease (AD). Hence, this study compared the spatial patterns of prion protein (PrP) deposits in the cerebral cortex and hippocampus in cases of sporadic CJD with those of β-amyloid (Aβ) deposits in sporadic AD. PrP and Aβ deposits were aggregated into clusters and, in 90% of brain areas in CJD and 57% in AD, the clusters were regularly distributed parallel to the tissue boundary. In a significant proportion of cortical analyses, the mean diameter of the clusters of PrP and Aβ deposits were similar to those of the cells of origin of the cortico-cortical pathways. Aβ deposits in AD were distributed more frequently in larger-sized clusters than PrP deposits in CJD. In addition, in the hippocampus and dentate gyrus, clustering of Aβ deposits was observed in AD but PrP deposits were rare in these regions in CJD. The size, location and distribution of the extracellular protein deposits within the cortex of both disorders was consistent with the degeneration of the cortico-cortical pathways. Furthermore, spread of the pathology along these pathways may be a pathogenic feature common to CJD and AD. © 2001 Elsevier Science Ireland Ltd.

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

Hepatocyte [H-3]-palmitate uptake: effect of albumin surface charge modification

The role of plasma proteins on the cellular uptake of lipophilic substrates has perplexed investigators for many years. We tested the hypothesis that an ionic interaction between the protein-ligand complex and hepatocyte surface may be responsible for supplying more ligand to the cell for uptake. The surface-charged groups on albumin were modified to yield proteins having a range of isoelectric points (ALB, ALBs, ALBm, ALBe had values of 4.8-5.0, 4.5-4.7, 3.0-3.5, 8.4-8.6, respectively). [H-3]-Palmitate uptake studies were performed with adult rat hepatocyte suspensions using similar unbound ligand fractions in the presence of the different binding proteins. Mass spectrometry, isoelectric focusing (pI), and heptane : water partitioning were used to determine protein molecular weight, pI, and protein-palmitate equilibrium binding constant, respectively. Hepatocyte [H-3]-palmitate clearance in the presence of ALBs and ALBm were significantly lower (p < 0.05) than ALB, whereas [H-3]-palmitate clearance in the presence of ALBe was significantly higher (p < 0.05) than ALB. The data were consistent with the notion that ionic interactions between extracellular protein-ligand complexes and the hepatocyte surface facilitate the uptake of long-chain fatty acids.

Overexpression of a Slit homologue impairs convergent extension of the mesoderm and causes cyclopia in embryonic zebrafish

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation. (C) 2001 Academic Press.

Conserved modularity and potential for alternate splicing in mouse and human Slit genes

The vertebrate Slit gene family currently consists of three members;Slit1,Slit2 and Slit3. Each gene encodes a protein containing multiple epidermal growth factor and leucine rich repeat motifs, which are likely to have importance in cell-cell interactions. In this study, we sought to fully define and characterise the vertebrate Slit gene family. Using long distance PCR coupled with in silico mapping, we determined the genomic structure of all three Slit genes in mouse and man. Analysis of EST and genomic databases revealed no evidence of further Slit family members in either organism. All three Slit genes were encoded by 36 (Slit3) or 37 (Slit1 and Slit2) exons covering at least 143 kb or 183 kb of mouse or human genomic DNA respectively. Two additional potential leucine-rich repeat encoding exons were identified within intron 12 of Slit2. These could be inserted in frame, suggesting that alternate splicing may occur in Slit2 A search for STS sequences within human Slit3 anchored this gene to D5S2075 at the 5' end (exon 4) and SGC32449 within the 3' UTR, suggesting that Slit3 may cover greater than 693 kb. The genomic structure of all Slit genes demonstrated considerable modularity in the placement of exon-intron boundaries such that individual leucine-rich repeat motifs were encoded by individual 72 by exons. This further implies the potential generation of multiple Slit protein isoforms varying in their number of repeat units. cDNA library screening and EST database searching verified that such alternate splicing does occur.

Regional and cellular patterns of reelin mRNA expression in the forebrain of the developing and adult mouse

The reelin gene encodes an extracellular protein that is crucial for neuronal migration in laminated brain regions. To gain insights into the functions of Reelin, we performed high-resolution in situ hybridization analyses to determine the pattern of reelin expression in the developing forebrain of the mouse. We also performed double-labeling studies with several markers, including calcium-binding proteins, GAD65/67, and neuropeptides, to characterize the neuronal subsets that express reelin transcripts. reelinexpression was detected at embryonic day 10 and later in the forebrain, with a distribution that is consistent with the prosomeric model of forebrain regionalization. In the diencephalon, expression was restricted to transverse and longitudinal domains that delineated boundaries between neuromeres. During embryogenesis,reelin was detected in the cerebral cortex in Cajal-Retzius cells but not in the GABAergic neurons of layer I. At prenatal stages, reelin was also expressed in the olfactory bulb, and striatum and in restricted nuclei in the ventral telencephalon, hypothalamus, thalamus, and pretectum. At postnatal stages, reelin transcripts gradually disappeared from Cajal-Retzius cells, at the same time as they appeared in subsets of GABAergic neurons distributed throughout neocortical and hippocampal layers. In other telencephalic and diencephalic regions,reelin expression decreased steadily during the postnatal period. In the adult, there was prominent expression in the olfactory bulb and cerebral cortex, where it was restricted to subsets of GABAergic interneurons that co-expressed calbindin, calretinin, neuropeptide Y, and somatostatin. This complex pattern of cellular and regional expression is consistent with Reelin having multiple roles in brain development and adult brain function.

Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections

Here we examine the role of Reelin, an extracellular protein involved in neuronal migration, in the formation of hippocampal connections. Both at prenatal and postnatal stages, the general laminar and topographic distribution of entorhinal projections is preserved in the hippocampus of reeler mutant mice, in the absence of Reelin. However, developing and adult entorhinal afferents show severe alterations, including increased numbers of misrouted fibers and the formation of abnormal patches of termination from the medial and lateral entorhinal cortices. At perinatal stages, single entorhinal axons in reeler mice are grouped into thick bundles, and they have decreased axonal branching and decreased extension of axon collaterals. We also show that the number of entorhino-hippocampal synapses is lower in reeler mice than in control animals during development. Studies performed in mixed entorhino-hippocampal co-cultures combining slices from reeler and wild-type mice indicate that these abnormalities are caused by the lack of Reelin in the target hippocampus. These findings imply that Reelin fulfills a modulatory role during the formation of layer-specific and topographic connections in the hippocampus. They also suggest that Reelin promotes maturation of single fibers and synaptogenesis by entorhinal afferents.