Crystal Structure of CBD2 from the Drosophila Na/Ca Exchanger: Diversity of Ca Regulation and Its Alternative Splicing Modification

Na⁺/Ca²⁺ exchangers (NCXs) promote the extrusion of intracellular Ca²⁺ to terminate numerous Ca²⁺-mediated signaling processes. Ca²⁺ interaction at two Ca²⁺ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca²⁺ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca²⁺ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca²⁺ binding. The carboxylate residues that coordinate two Ca²⁺ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca²⁺ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca²⁺ binding site to change Ca²⁺ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca²⁺ regulatory behavior.     

Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer

The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipi-dated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9-17 in the extra-cellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Fran-cisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.     

Recombination in Tula Hantavirus Evolution: Analysis of Genetic Lineages from Slovakia

To examine the evolution of Tula hantavirus (TUL), carried by the European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from Slovakia, the country where the virus is endemic. Phylogenetic analysis (PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or complete N-protein-encoding sequences divided Slovakian TUL variants into two main lineages: (i) strains from eastern Slovakia, which clustered with Russian strains, and (ii) strains from western Slovakia situated closer to those from the Czech Republic. We found genetic diversity of 19% between the two groups and 4% within the western Slovakian TUL strains. Phylogenetic analysis of the 3′ noncoding region (3′-NCR), however, placed the eastern Slovakian strains closer to those from western Slovakia and the Czech Republic, with a greater distance to the Russian strains, suggesting a recombinant nature of the S segment in the eastern Slovakian TUL lineage. A bootscan search of the S-segment sequences of TUL strains revealed at least two recombination points in the S sequences of eastern Slovakian TUL strains (nt 400 to 415 and around 1200) which agreed well with the pattern of amino acid substitutions in the N protein and deletions/insertions in the 3′-NCR of the S segment. These data suggest that homologous recombination events occurred in the evolution of hantaviruses.     

Crystal structure of a beta-catenin/Tcf complex

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear beta-catenin/Tcf complex, activating Wnt target genes. A crystal structure of beta-catenin bound to the beta-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to beta-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in beta-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.     

Enzymatic activity, protein expression, and gene sequence of cruzipain in virulent and attenuated Trypanosoma cruzi strains.

Protein expression, characterized in Western blots and gelatinolytic activity, of cruzipain (Cr), the major Trypanosoma cruzi cysteine proteinase, was compared among 3 attenuated T. cruzi strains (TUL 0, TCC, and Y null) and their virulent counterparts (TUL 2, Tulahuen, and Y). All attenuated strains displayed a weaker gelatinolytic activity as compared with their virulent counterparts. The electrophoretic mobility and immunological reactivity revealed quantitative and qualitative differences, with the attenuated parasites showing bands of less density in all strains and lower mobility in 2 of them, as compared with the virulent strains. Sequence analysis of 1 Cr gene in the Tulahuen and TCC strains indicated 37/1404 base pair substitutions, corresponding to 20 amino acid changes in the attenuated strain. A similar comparative analysis of 1 Cr gene between Y and Y null strains showed 13/1404 base pair substitutions, corresponding to 8 amino acid changes in the attenuated strain. Although enough variability exists in the Cr gene to allow for less- or nonfunctional isoforms of the protein, further clones should be analyzed to establish whether attenuation is regularly associated with specific sequence changes of this enzyme.     

Recombinant Antigens E6 and E7 of Human Papilloma Virus Type 16: Preparation,Purification, and Assessment of the Immunogenicity and Antitumor Effects In Vivo

Applied Biochemistry and Microbiology - The recombinant proteins E6-CBD and E7-CBD, which are antigens E6 and E7 of human papillomavirus type 16 (HPV16), were connected by a glycine-serine spacer...     

Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.     

Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines

Background

There is a continuous demand for new immunosuppressive agents for organ transplantation. Galectin-9, a member of the galactoside-binding animal lectin family, has been shown to suppress pathogenic T-cell responses in autoimmune disease models and experimental allograft transplantation. In this study, an attempt has been made to develop new collagen matrices, which can cause local, contact-dependent immune suppression, using galectin-9 and collagen-binding galectin-9 fusion proteins as active ingredients.

Methods

Galectin-9 and galectin-9 fusion proteins having collagen-binding domains (CBDs) derived from bacterial collagenases and a collagen-binding peptide (CBP) were tested for their ability to bind to collagen matrices, and to induce Jurkat cell death in solution and in the collagen-bound state.

Results

Galectin-9-CBD fusion proteins exhibited collagen-binding activity comparable to or lower than that of the respective CBDs, while their cytocidal activity toward Jurkat cells in solution was 80 ~ 10% that of galectin-9. Galectin-9 itself exhibited oligosaccharide-dependent collagen-binding activity. The growth of Jurkat cells cultured on collagen membranes treated with galectin-9 was inhibited by ~ 90%. The effect was dependent on direct cell-to-membrane contact. Galectin-9-CBD/CBP fusion proteins bound to collagen membranes via CBD/CBP moieties showed a low or negligible effect on Jurkat cell growth.

Conclusions

Among the proteins tested, galectin-9 exhibited the highest cytocidal effect on Jurkat cells in the collagen-bound state. The effect was not due to galectin-9 released into the culture medium but was dependent on direct cell-to-membrane contact.

General significance

The study demonstrates the possible use of galectin-9-modified collagen matrices for local, contact-dependent immune suppression in transplantation.     

Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation.

The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated. The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined. At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively. The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M. These results are discussed with regard to the properties of the various substrates. The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain [CelA2 and CelA3, respectively]) in cellulose degradation was also studied. Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested. This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area. In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation.     

Domain coupling in a multimodular cellobiohydrolase CbhA from Clostridium thermocellum

Cellobiohydrolase A (CbhA) from Clostridium thermocellum is composed of an N-terminal carbohydrate-binding domain 4 (CBD4), an immunoglobulin-like domain (Ig), a glycoside hydrolase 9 (GH9), X1(1) and X1(2) domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X1(2), X1(1)X1(2), CBD3, X1(1)X1(2)-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X1(1)X1(2), and Ig-GH9-X1(1)X1(2)-CBD3. Interactions between domains in (1) buffer, (2) with Ca2+, or (3) ethylenediaminetetraacetic acid (EDTA) were studied by differential scanning calorimetry. Thermal unfoldings of all constructs were irreversible. Calcium increased T(d) and cooperativity of unfolding. Multi-domain constructs exhibited more cooperative unfolding in buffer and in the presence of EDTA than did individual domains. They denatured by mechanism simpler than expected from their modular architecture. The results indicate that domain coupling in thermophilic proteins constitutes a significant stabilizing factor.     

Domain engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg ^–1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.     

Tuning the properties of elastin mimetic hybrid copolymers via a modular polymerization method

We have synthesized elastin mimetic hybrid polymers (EMHPs) via the step-growth polymerization of azide-functionalized poly(ethylene glycol) (PEG) and alkyne-terminated peptide (AKAAAKA)(2) (AK2) that is abundant in the cross-linking domains of the natural elastin. The modular nature of our synthesis allows facile adjustment of the peptide sequence to modulate the structural and biological properties of EMHPs. Therefore, EMHPs containing cell-binding domains (CBDs) were constructed from α,ω-azido-PEG and two types of alkyne-terminated AK2 peptides with sequences of DGRGX(AKAAAKA)(2)X (AK2-CBD1) and X(AKAAAKA)(2)XGGRGDSPG (AK2-CBD2, X = propargylglycine) via a step-growth, click coupling reaction. The resultant hybrid copolymers contain an estimated five to seven repeats of PEG and AK2 peptides. The secondary structure of EMHPs is sensitive to the specific sequence of the peptidic building blocks, with CBD-containing EMHPs exhibiting a significant enhancement in the α-helical content as compared with the peptide alone. Elastomeric hydrogels formed by covalent cross-linking of the EMHPs had a compressive modulus of 1.06 ± 0.1 MPa. Neonatal human dermal fibroblasts (NHDFs) were able to adhere to the hydrogels within 1 h and to spread and develop F-actin filaments 24 h postseeding. NHDF proliferation was only observed on hydrogels containing RGDSP domains, demonstrating the importance of integrin engagement for cell growth and the potential use of these EMHPs as tissue engineering scaffolds. These cell-instructive, hybrid polymers are promising candidates as elastomeric scaffolds for tissue engineering.     

Increased antifungal and chitinase specific activities of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> CECT 2413 by addition of a cellulose binding domain

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.     

Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBD(Cel5). Firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.     

Relationship between long-term resistance to Trypanosoma cruzi and latent infection,examined by antibody production and polymerase chain reaction in mice

Trypanosoma cruzi infections persist for the lifetime of humans and laboratory animals as either latent or pathogenic parasitism. Mice inoculated with a nonpathogenic, attenuated strain (TCC) display resistance against virulent challenge, with a strong control of parasitemia and protection against tissue lesions for more than 12 mo. Three main approaches were used to test whether protection by TCC inocula is based on a latent infection or on a "sterile" immunological memory: curative Benznidazole (Bzl) treatment, serological reactions, and detection of infection by polymerase chain reaction (PCR). If resistance is maintained in the absence of infection, it should not be reduced by Bzl treatment and TCC-inoculated animals should not maintain long-term serological or PCR reactivity. The Bzl treatment after TCC inoculations did not reduce, after periods of up to 420 days, TCC-induced resistance to challenge. But TCC inocula given during Bzl treatment conferred short-term, but not long-term. protection. Maintenance of high antibody levels and protection were better in the virulent Tulahuen (TUL) strain than in the attenuated TCC strain infections, and trypomastigote inocula of either strain were better inducers of antibodies and resistance than epimastigotes. PCR detection of T. cruzi DNA was positive in almost all TUL strain-inoculated animals and negative in immunocompetent animals inoculated with TCC epimastigotes, although high numbers of TCC trypomastigotes produced persistent PCR signals of infection in newborn BALB mice. Thus, 2 polar models were developed, where latent infection by TCC was either demonstrated or excluded. In both, resistance to virulent challenge was maintained during long periods. But late declination of antibody titers (>200 days) and resistance to challenge (>350 days) was observed in animals displaying clearance of all signals of infection.     

Structural and dynamic aspects of Ca2+ and Mg2+ binding of the regulatory domains of the Na+/Ca2+ exchanger

Intracellular Ca2+ regulates the activity of the NCX (Na+/Ca2+ exchanger) through binding to the cytosolic CBD (Ca2+-binding domain) 1 and CBD2. In vitro studies of the structure and dynamics of CBD1 and CBD2, as well as studies of their kinetics and thermodynamics of Ca2+ binding, greatly enhanced our understanding of NCX regulation. We describe the fold of the CBDs in relation to other known structures and review Ca2+ binding of the different CBD variants from a structural perspective. We also report on new findings concerning Mg2+ binding to the CBDs and finally we discuss recent results on CBD1-CBD2 interdomain interactions.     

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Changes in the Levels of IAA,ABA, and Cytokinins in Wheat Seedlings Infected with Tilletia caries

The seedling growth and the content of endogenous phytohormones in wheat seedlings were estimated 3, 6, and 9 days after infection with the bunt pathogen (Tilletia caries) (DC.)TUL. The infection of a pathogen-susceptible species Triticum aestivum L. and a resistant species T. timopheevii Zhuk. resulted, respectively, in an increase and a decrease in the seedling growth and the IAA content as compared to the control. The cytokinin content increased in both species, and the increase in T. timopheevii was more rapid. The pathogen-induced increase in auxin content is suggested to enhance fungal invasion of plants. In the susceptible species, a high ABA concentration was retained for a longer period of time and could act as a factor of virulence. At the same time, in the resistant species, an increase in ABA content was transient and seems to trigger plant defense mechanisms.