scholarly journals Lectin PLL3, a Novel Monomeric Member of the Seven-Bladed β-Propeller Lectin Family

Molecules ◽  
2019 ◽  
Vol 24 (24) ◽  
pp. 4540 ◽  
Author(s):  
Lukáš Faltinek ◽  
Eva Fujdiarová ◽  
Filip Melicher ◽  
Josef Houser ◽  
Martina Kašáková ◽  
...  
Keyword(s):  
The Other ◽  
Titration Calorimetry ◽  
Gram Negative Bacteria ◽  
Hemagglutination Assay ◽  
Binding Properties ◽  
X Ray Crystallography ◽  
The Family ◽  
Mutualistic Relationship ◽  
Lectin Family

The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed β-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.

scholarly journals Characterization of a porin channel in the endosymbiont of the trypanosomatid protozoan Crithidia deanei

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2818-2830 ◽  
Author(s):  
Iamara da Silva Andrade ◽  
João Lídio Vianez-Júnior ◽  
Carolina Lage Goulart ◽  
Fabrice Homblé ◽  
Jean-Marie Ruysschaert ◽  
...  
Keyword(s):  
3D Structure ◽  
Symbiotic Bacterium ◽  
Gram Negative Bacteria ◽  
Genome Database ◽  
Gram Negative ◽  
Secondary Structure Analysis ◽  
Electrophysiological Measurements ◽  
Mutualistic Relationship ◽  
Obligate Intracellular Bacteria

Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a β-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain β-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.

scholarly journals SapL1: A New Target Lectin for the Development of Antiadhesive Therapy Against Scedosporium apiospermum

2020 ◽  
Author(s):  
Dania Martínez-Alarcón ◽  
Jean-Philippe Bouchara ◽  
Roland J. Pieters ◽  
Annabelle Varrot
Keyword(s):  
Biochemical Characterization ◽  
Present Report ◽  
Binding Mode ◽  
Host Cells ◽  
Titration Calorimetry ◽  
Carbohydrate Binding ◽  
Scedosporium Apiospermum ◽  
X Ray Crystallography ◽  
Opportunistic Fungal Pathogen

AbstractScedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised patients. This fungus exhibits limited susceptibility to all current antifungals and, due its emerging character, its pathogeny and virulence factors remain largely unknown. Carbohydrate binding proteins such as lectins are involved in host-pathogen interactions and may constitute valuable therapeutic targets to inhibit microbial adhesion to the host cells by using carbohydrate mimics. However, such lectins are still unidentified in S. apiospermum. Here, we present the first report of the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 is homologous to the conidial surface lectin FleA from Aspergillus fumigatus known to be involved in the adhesion to host glycoconjugates present in human lung epithelium. The present report includes a detailed strategy to achieve SapL1 soluble expression in bacteria, its biochemical characterization, an analysis of its specificity and affinity by glycan arrays and isothermal titration calorimetry (ITC), as well as the structural characterization of its binding mode by X–ray crystallography. The information gathered here contribute to the understanding of glycosylated surface recognition by Scedosporium species and is essential for the design and development of antiadhesive glycodrugs targeting SapL1.

scholarly journals Mass spectrometry for fragment screening

2017 ◽  
Vol 61 (5) ◽  
pp. 465-473 ◽  
Author(s):  
Daniel Shiu-Hin Chan ◽  
Andrew J. Whitehouse ◽  
Anthony G. Coyne ◽  
Chris Abell
Keyword(s):  
Drug Discovery ◽  
High Sensitivity ◽  
Titration Calorimetry ◽  
Label Free ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Differential Scanning Fluorimetry ◽  
Biophysical Techniques

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening.

scholarly journals Characterization of Six Bacteriophages ofSerratia liquefaciens CP6 Isolated from the Sugar Beet Phytosphere

1999 ◽  
Vol 65 (5) ◽  
pp. 1959-1965 ◽  
Author(s):  
Kevin E. Ashelford ◽  
John C. Fry ◽  
Mark J. Bailey ◽  
Aaron R. Jeffries ◽  
Martin J. Day
Keyword(s):  
Sugar Beet ◽  
Burst Size ◽  
Single Step ◽  
The Other ◽  
Cross Hybridization ◽  
The Family ◽  
Step Growth ◽  
Natural Ecology ◽  
Changes Over Time

ABSTRACT Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the family Siphoviridae, while ΦCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10−9 to 3.9 × 10−7, whereas ΦCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.

scholarly journals Genetic and Transcriptional Analyses of theVibrio cholerae Mannose-Sensitive Hemagglutinin Type 4 Pilus Gene Locus

1999 ◽  
Vol 181 (4) ◽  
pp. 1110-1117 ◽  
Author(s):  
Jane W. Marsh ◽  
Ronald K. Taylor
Keyword(s):  
Gene Locus ◽  
Mobile Genetic Element ◽  
Direct Repeat ◽  
The Other ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
The Family ◽  
Colonization Factors ◽  
El Tor

ABSTRACT The mannose-sensitive hemagglutinin (MSHA) of the Vibrio cholerae O1 El Tor biotype is a member of the family of type 4 pili. Type 4 pili are found on the surface of a variety of gram-negative bacteria and have demonstrated importance as host colonization factors, bacteriophage receptors, and mediators of DNA transfer. The gene locus required for the assembly and secretion of the MSHA pilus has been localized to a 16.7-kb region of the V. cholerae chromosome. Sixteen genes required for hemagglutination, including five that encode prepilin or prepilin-like proteins, have been identified. Examination of MSHA-specific cDNAs has localized two promoters that drive expression of these genes. This evidence indicates that the MSHA gene locus is transcriptionally organized into two operons, one encoding the secretory components and the other encoding the structural subunits, an arrangement unique among previously characterized type 4 pilus loci. The genes flanking the MSHA locus encode proteins that show homology to YhdA and MreB ofEscherichia coli. In E. coli, theyhdA and mreB genes are adjacent to each other on the chromosome. The finding that the MSHA locus lies between these two E. coli homologs and that it is flanked by a 7-bp direct repeat suggests that the MSHA locus may have been acquired as a mobile genetic element.

Cloning and functional characterization of a high-affinity Na+/dicarboxylate cotransporter from mouse brain

2001 ◽  
Vol 280 (5) ◽  
pp. C1215-C1223 ◽  
Author(s):  
Ana M. Pajor ◽  
Rama Gangula ◽  
Xiaozhou Yao
Keyword(s):  
Electrical Properties ◽  
Mouse Brain ◽  
Functional Characterization ◽  
The Other ◽  
Acidic Ph ◽  
High Affinity ◽  
The Family ◽  
Electrode Voltage ◽  
Menten Constant

Neurons contain a high-affinity Na+/dicarboxylate cotransporter for absorption of neurotransmitter precursor substrates, such as α-ketoglutarate and malate, which are subsequently metabolized to replenish pools of neurotransmitters, including glutamate. We have isolated the cDNA coding for a high-affinity Na+/dicarboxylate cotransporter from mouse brain, called mNaDC-3. The mRNA coding for mNaDC-3 is found in brain and choroid plexus as well as in kidney and liver. The mNaDC-3 transporter has a broad substrate specificity for dicarboxylates, including succinate, α-ketoglutarate, fumarate, malate, and dimethylsuccinate. The transport of citrate is relatively insensitive to pH, but the transport of succinate is inhibited by acidic pH. The Michaelis-Menten constant for succinate in mNaDC-3 is 140 μM in transport assays and 16 μM at −50 mV in two-electrode voltage clamp assays. Transport is dependent on sodium, although lithium can partially substitute for sodium. In conclusion, mNaDC-3 likely codes for the high-affinity Na+/dicarboxylate cotransporter in brain, and it has some unusual electrical properties compared with the other members of the family.

scholarly journals Enhanced affinity of racemic phosphorothioate DNA with transcription factor SATB1 arising from diastereomer-specific hydrogen bonds and hydrophobic contacts

2020 ◽  
Vol 48 (8) ◽  
pp. 4551-4561 ◽  
Author(s):  
Kazuhiko Yamasaki ◽  
Yukie Akutsu ◽  
Tomoko Yamasaki ◽  
Makoto Miyagishi ◽  
Tomomi Kubota
Keyword(s):  
Transcription Factor ◽  
Hydrogen Bonds ◽  
Salt Concentration ◽  
The Other ◽  
Titration Calorimetry ◽  
Racemic Mixture ◽  
Dna Binding Domain ◽  
X Ray ◽  
X Ray Crystallography ◽  
Modified Dna

Abstract Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.

scholarly journals Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins

2021 ◽  
Vol 22 (7) ◽  
pp. 3482
Author(s):  
Christopher Mendoza ◽  
Sai Harsha Nagidi ◽  
Dario Mizrachi
Keyword(s):  
Tertiary Structure ◽  
Extracellular Domain ◽  
The Other ◽  
Immunoglobulin Superfamily ◽  
Adhesive Properties ◽  
The Family ◽  
Heterotypic Interactions ◽  
Hydrophobic Nature ◽  
And Function

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.

scholarly journals Cloning and Characterization of tdhA, a Locus Encoding a TonB-Dependent Heme Receptor fromHaemophilus ducreyi

1998 ◽  
Vol 66 (9) ◽  
pp. 4254-4262 ◽  
Author(s):  
Christopher E. Thomas ◽  
Bonnie Olsen ◽  
Christopher Elkins
Keyword(s):  
Agar Medium ◽  
Gram Negative Bacteria ◽  
Outer Membrane Receptor ◽  
Gene Encoding ◽  
Reading Frame ◽  
The Family ◽  
Independent Mechanism ◽  
Low Levels

ABSTRACT Haemophilus ducreyi is unable to synthesize heme and must acquire it from its only known host, humans. We cloned and sequenced a gene encoding an outer membrane receptor for heme. It was designated tdhA (for TonB-dependent heme receptor A) since it was related by sequence homology to the family of TonB-dependent receptors. TdhA was strikingly similar to open reading frame HI0113 from the genome of Haemophilus influenzae Rd and also shared homology with five other heme receptors, including HxuC, HemR, HmuR, ChuA, and ShuA, from gram-negative bacteria. AnEscherichia coli hemA tonB mutant strongly expressingH. ducreyi tdhA grew on low levels of heme as a source of heme only when an intact H. ducreyi Ton system plasmid was present, formally demonstrating functional TonB dependence.tdhA was expressed poorly in vitro by H. ducreyi and only under conditions of heme limitation. A survey ofH. ducreyi revealed that all tested strains but one synthesized small amounts of TdhA in vitro under heme-limiting conditions. Surprisingly, an isogenic mutant of tdhA as well as its parent, 35000, both required the same high levels of heme for growth (50 μg/ml [77 μM] on agar medium). This result, together with previous findings, suggests that in vitro, the uptake of heme by H. ducreyi is mediated by a TonB- and TdhA-independent mechanism, possibly diffusion.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

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