Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

Glucose-Induced Regulation of NHEs Activity and SGLTs Expression Involves the PKA Signaling Pathway

The effect of glucose on the intracellular pH (pH(i)) recovery rate (dpH(i)/dt) and Na(+)-glucose transporter (SGLT) localization was investigated in HEK-293 cells, a cell line that expresses endogenous NHE1, NHE3, SGLT1, and SGLT2 proteins. The activity of the Na(+)/H(+) exchangers (NHEs) was evaluated by using fluorescence microscopy. The total and membrane protein expression levels were analyzed by immunoblotting. In cells cultivated in 5 mM glucose, the pH(i) recovery rate was 0.169 +/- A 0.020 (n = 6). This value did not change in response to the acute presence of glucose at 2 or 10 mM, but decreased with 25 mM glucose, an effect that was not observed with 25 mM mannitol. Conversely, the chronic effect of high glucose (25 mM) increased the pH(i) recovery rate (similar to 40%, P < 0.05), without changes in the total levels of NHE1, NHE3, or SGLT1 expression, but increasing the total cellular (similar to 50%, P < 0.05) and the plasma membrane (similar to 100%, P < 0.01) content of SGLT2. Treatment with H-89 (10(-6) M) prevented the stimulatory effect of chronic glucose treatment on the pH(i) recovery rate and SGLT2 expression in the plasma membrane. Our results indicate that the effect of chronic treatment with a high glucose concentration is associated with increased NHEs activity and plasma membrane expression of SGLT2 in a protein kinase A-dependent way. The present results reveal mechanisms of glucotoxicity and may contribute to understanding the diabetes-induced damage of this renal epithelial cell.

Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode

Introduction: Tim-3 is a Th1 lymphocytes membrane protein with inhibitory function. Its ligand, galectin-9, was recently identified and it is expressed in some lymphocyte subpopulation. In addition, endothelial cells and fibroblasts can also express galectin-9 according to the local cytokine milieu. Both molecules can act as important regulatory tools in the immune system. Aim: Evaluate the expression of these immunoregulatory molecules inside kidney allografts during acute rejection episodes. Methods: By using a quantitative polymerase chain reaction assay, we measured the levels of messenger RNA (mRNA) for galectin-9 and Tim-3 in 21 samples obtained at allograft nephrectomy. Five samples received the histological diagnosis of acute non-vascular rejection (ANVR), twelve of acute vascular rejection (AVR), and five of loss of non-immune cause (LNIC; as control). As cytolytic response markers we measured mRNA levels of granzyme B, interferon-gamma and perforin. The statistic analysis was performed using one way analysis of variance (ANOVA) and Pearson correlation. Results: The mean levels of Tim-3 mRNA expression were 13.99 +/- 6.99 for LNIC, 48.13 +/- 54.47 for RACNV and 238.63 +/- 333.14 for RAV (p = 0.004). For galectin-9, the mean values were 0.57 +/- 0.49 for LNIC, 0.66 +/- 0.36 for RACNV and 2.34 +/- 1.62 for RAV (p = 0.006). Furthermore, there was a positive correlation between both molecules (r = 0.526, p = 0.016). Also. granzyme B, perforin and interferon-gamma mRNA expression were different among the three groups. Conclusion: Messenger RNA level expressions of all the studied molecules were higher inside allografts with more severe rejection. Moreover, there was a positive correlation between galectin-9 and Tim-3 mRNA levels. The simultaneous expression of galectin-9 and Tim-3 may indicate an immunoregulatory function, during the ongoing cytotoxic response. (C) 2008 Elsevier B.V. All rights reserved.

Vitellin-binding proteins in the nematode Oscheius tipulae (Nematoda, Rhabditida)

We describe the first application of a non-radioactive ligand-blotting technique to the characterization of proteins interacting with nematode vitellins. Chromatographically purified vitellins from the free-living nematode Oscheius tipulae were labeled with fluorescein in vitro. Ligand-blotting assays with horseradish peroxidase-conjugated anti-fluorescein antibodies showed that labeled vitellins reacted specifically with a polypeptide of approximately 100 kDa, which we named P100. This polypeptide is a specific worm`s vitellin-binding protein that is present only in adult worms. Blots containing purified O. tipulae vitellin preparations showed no detectable signal in the 100 kDa region, ruling out any possibility of yolk polypeptides self-assembling under the conditions used in our assay. Experiments done in the presence of alpha-methyl mannoside ruled out the possibility of vitellins binding to P100 through mannose residues. Triton X-114 fractionation of whole worm extracts showed that P100 is either a membrane protein or has highly hydrophobic regions. (C) 2008 Elsevier Inc. All rights reserved.

Structure and Calcium-Binding Activity of LipL32, the Major Surface Antigen of Pathogenic Leptospira sp.

Leptospixosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as an important emerging infectious disease. LipL32 is the major exposed outer membrane protein found exclusively in pathogenic leptospires, where it accounts for up to 75% of the total outer membrane proteins. It is highly immunogenic, and recent studies have implicated LipL32 as an extracellular matrix binding protein, interacting with collagens, fibronectin, and laminin. In order to better understand the biological role and the structural requirements for the function of this important lipoprotein, we have determined the 2.25-angstrom-resolution structure of recombinant LipL32 protein corresponding to residues 21-272 of the wild-type protein (LipL32(21-272)). The LipL32(21-272) monomer is made of a jelly-roll fold core from which several peripheral secondary structures protrude. LipL32(21-272) is structurally similar to several other jelly-roll proteins, some of which bind calcium ions and extracellular matrix proteins. Indeed, spectroscopic data (circular dichroism, intrinsic tryptophan fluorescence, and extrinsic 1-amino-2-naphthol-4-sulfonic acid fluorescence) confirmed the calcium-binding properties of LipL32(21-272). Ca(2+) binding resulted in a significant increase in the thermal stability of the protein, and binding was specific for Ca(2+) as no structural or stability perturbations were observed for Mg(2+), Zn(2+), or Cu(2+). Careful examination of the crystal lographic structure suggests the locations of putative regions that could mediate Ca(2+) binding as well as binding to other interacting host proteins, such as collagens, fibronectin, and lamixidn. (C) 2009 Elsevier Ltd. All rights reserved.

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (similar to 50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (similar to 30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, We demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64 kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22 kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. (C) 2007 Elsevier Ltd. All rights reserved.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated. (C) 2011 Elsevier Inc. All rights reserved.

Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Mu hiding resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetra-acetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. (HEPATOLOGY 2010;52:327-337)

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 mu L) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced similar to 20% and 10 min after an acute it? vivo stimulus with insulin, the plasma membrane GLUT4 content was similar to 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (similar to 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright (C) 2010 John Wiley & Sons, Ltd.

Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) similar to 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene

Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand. (C) 2009 Elsevier B.V. All rights reserved.