Transmembrane Insertion of the Toxoplasma gondii GRA5 Protein Occurs after Soluble Secretion into the Host Cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.

Fibroblast Growth Factor (FGF) Soluble Receptor 1 Acts as a Natural Inhibitor of FGF2 Neurotrophic Activity during Retinal Degeneration

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

Defining Extracellular Integrin α-Chain Sites That Affect Cell Adhesion and Adhesion Strengthening without Altering Soluble Ligand Binding

It was previously shown that mutations of integrin α4 chain sites, within putative EF-hand-type divalent cation-binding domains, each caused a marked reduction in α4β1-dependent cell adhesion. Some reports have suggested that α-chain “EF-hand” sites may interact directly with ligands. However, we show here that mutations of three different α4 “EF-hand” sites each had no effect on binding of soluble monovalent or bivalent vascular cell adhesion molecule 1 whether measured indirectly or directly. Furthermore, these mutations had minimal effect on α4β1-dependent cell tethering to vascular cell adhesion molecule 1 under shear. However, EF-hand mutants did show severe impairments in cellular resistance to detachment under shear flow. Thus, mutation of integrin α4 “EF-hand-like” sites may impair 1) static cell adhesion and 2) adhesion strengthening under shear flow by a mechanism that does not involve alterations of initial ligand binding.

Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions

Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins. The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic. Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin.

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8+ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+ T-cell supernatants; and (iii) the CD8+ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+ T-cell-derived HIV-suppressor factors.

Recombinant scrapie-like prion protein of 106 amino acids is soluble

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.

Use of soluble peptide–DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis

Considerable evidence indicates that CD4+ T cells are important in the pathogenesis of rheumatoid arthritis (RA), but the antigens recognized by these T cells in the joints of patients remain unclear. Previous studies have suggested that type II collagen (CII) and human cartilage gp39 (HCgp39) are among the most likely synovial antigens to be involved in T cell stimulation in RA. Furthermore, experiments have defined dominant peptide determinants of these antigens when presented by HLA-DR4, the most important RA-associated HLA type. We used fluorescent, soluble peptide–DR4 complexes (tetramers) to detect synovial CD4+ T cells reactive with CII and HCgp39 in DR4+ patients. The CII-DR4 complex bound in a specific manner to CII peptide-reactive T cell hybridomas, but did not stain a detectable fraction of synovial CD4+ cells. A background percentage of positive cells (<0.2%) was not greater in DR4 (DRB1*0401) patients compared with those without this disease-associated allele. Similar results were obtained with the gp39-DR4 complex for nearly all RA patients. In a small subset of DR4+ patients, however, the percentage of synovial CD4+ cells binding this complex was above background and could not be attributed to nonspecific binding. These studies demonstrate the potential for peptide–MHC class II tetramers to be used to track antigen-specific T cells in human autoimmune diseases. Together, the results also suggest that the major oligoclonal CD4+ T cell expansions present in RA joints are not specific for the dominant CII and HCgp39 determinants.

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose

The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA. Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C. thermocellum endoglucanase CelD (1,4-β-d endoglucanase, EC3.2.1.4) toward various cellulosic substrates. The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBDCBH1 and CBDCBH2) (1,4-β-d glucan-cellobiohydrolase, EC3.2.1.91), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBDCex) (β-1,4-d glucanase, EC3.2.1.8), and from C. thermocellum CipA (CBDCipA). The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes. Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBDCBH1 < CBDCBH2 ≈ CBDCex < CBDCipA. In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD. Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs. Furthermore, burst of activity also was observed upon adding new batches of CelD–CBD complexes that contained a CBD differing from the first one. This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping. The only exception was CBDCipA, whose sites appeared to overlap all of the other sites.

Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

The α1- and β1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Genomic organization of α1 and β1 subunits of the mammalian soluble guanylyl cyclase genes

The structures of the genes encoding the α1 and β1 subunits of murine soluble guanylyl cyclase (sGC) were determined. Full-length cDNAs isolated from mouse lungs encoding the α1 (2.5 kb) and β1 (3.3 kb) subunits are presented in this report. The α1 sGC gene is approximately 26.4 kb and contains nine exons, whereas the β1 sGC gene spans 22 kb and consists of 14 exons. The positions of exon/intron boundaries and the sizes of introns for both genes are described. Comparison of mouse genomic organization with the Human Genome Database predicted the exon/intron boundaries of the human genes and revealed that human and mouse α1 and β1 sGC genes have similar structures. Both mouse genes are localized on the third chromosome, band 3E3-F1, and are separated by a fragment that is 2% of the chromosomal length. The 5′ untranscribed regions of α1 and β1 subunit genes were subcloned into luciferase reporter constructs, and the functional analysis of promoter activity was performed in murine neuroblastoma N1E-115 cells. Our results indicate that the 5′ untranscribed regions for both genes possess independent promoter activities and, together with the data on chromosomal localization, suggest independent regulation of both genes.

Generation of mucosal cytotoxic T cells against soluble protein by tissue-specific environmental and costimulatory signals

We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover, distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand, B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator, B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent. The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because of distinct costimulatory and inflammatory signals in the mucosa.

Mechanism for fetal globin gene expression: Role of the soluble guanylate cyclase–cGMP-dependent protein kinase pathway

Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the β-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the γ-globin gene. sGC, an obligate heterodimer of α- and β-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different β-like globin genes showed that, whereas the β-subunit is expressed at similar levels, high-level expression of the α-subunit is preferentially observed in erythroid cells expressing γ-globin but not those expressing β-globin. Also, the levels of expression of the γ-globin gene correlate to those of the α-subunit. sGC activators or cGMP analogs increased expression of the γ-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with β-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the γ-globin gene. Furthermore, increased expression of the γ-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC–PKG pathway constitutes a mechanism that regulates expression of the γ-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the β-globin disorders.

Sephadex-binding RNA ligands: rapid affinity purification of RNA from complex RNA mixtures

Sephadex-binding RNA ligands (aptamers) were obtained through in vitro selection. They could be classified into two groups based on their consensus sequences and the aptamers from both groups showed strong binding to Sephadex G-100. One of the highest affinity aptamers, D8, was chosen for further characterization. Aptamer D8 bound to dextran B512, the soluble base material of Sephadex, but not to isomaltose, isomaltotriose and isomaltotetraose, suggesting that its optimal binding site might consist of more than four glucose residues linked via α-1,6 linkages. The aptamer was very specific to the Sephadex matrix and did not bind appreciably to other supporting matrices, such as Sepharose, Sephacryl, cellulose or pustulan. Using Sephadex G-100, the aptamer could be purified from a complex mixture of cellular RNA, giving an enrichment of at least 60 000-fold, compared with a non-specific control RNA. These RNA aptamers can be used as affinity tags for RNAs or RNA subunits of ribonucleoproteins to allow rapid purification from complex mixtures of RNA using only Sephadex.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

A method for soluble overexpression of the α7 nicotinic acetylcholine receptor extracellular domain

We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the α7 subunit of the nicotinic acetylcholine receptor. The approach was to fuse the α7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli. An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the α7 ECD. This protein is soluble upon expression in bacteria and is purified by affinity chromatography. It binds the competitive nicotinic antagonist α-bungarotoxin with 2.5 μM affinity and displays a CD spectrum corresponding to a folded protein. The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments.