The determination of a suitable buffer environment for a protein of interest is not an easy task. The requirements of advanced techniques, the demands on the biological material and the researcher time needed for buffer optimization, as well as personal inflexibility, lead frequently to the use of sub-optimal buffers. Here, we demonstrate the design of a 48-condition buffer screen that can be used to determine an appropriate environment for downstream studies. By the combination of several techniques (differential scanning fluorimetry, dynamic light scattering, and bio-layer interferometry), we are able to assess the protein stability, homogeneity and binding activity across the screen with less than half a milligram of protein in 1 day. The application of this screen helps to avoid unsuitable conditions, to explain problems observed upon protein analysis and to choose the most suitable buffers for further research. The screen can be routinely used as a primary screen for buffer optimization in labs and facilities.

• Keywords
• Bio-layer interferometry, Buffer, Differential scanning fluorimetry, Dynamic light scattering, Protein stability, Screening,
• MeSH
• Dynamic Light Scattering MeSH
• Fluorometry MeSH
• Proteins MeSH
• Buffers MeSH
• Protein Stability * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteins MeSH
• Buffers MeSH

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.

• Keywords
• O-methylated saccharides, Photorhabdus, l-fucose, lectin,
• MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Fructose metabolism   MeSH
• Calorimetry MeSH
• Crystallography, X-Ray MeSH
• Lectins chemistry   genetics   metabolism   MeSH
• Photorhabdus metabolism   MeSH
• Surface Plasmon Resonance MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Fructose MeSH
• Lectins MeSH
• Recombinant Proteins MeSH

Series of multivalent α-l-fucoside containing glycoclusters and variously decorated l-fucosides were synthesized to find potential inhibitors of fucose-specific lectins and study the structure-binding affinity relationships. Tri- and tetravalent fucoclusters were built using copper-mediated azide-alkyne click chemistry. Series of fucoside monomers and dimers were synthesized using various methods, namely glycosylation, an azide-alkyne click reaction, photoinduced thiol-en addition, and sulfation. The interactions between compounds with six fucolectins of bacterial or fungal origin were tested using a hemagglutination inhibition assay. As a result, a tetravalent, α-l-fucose presenting glycocluster showed to be a ligand that was orders of magnitude better than a simple monosaccharide for tested lectins in most cases, which can nominate it as a universal ligand for studied lectins. This compound was also able to inhibit the adhesion of Pseudomonas aeruginosa cells to human epithelial bronchial cells. A trivalent fucocluster with a protected amine functional group also seems to be a promising candidate for designing glycoconjugates and chimeras.

• Keywords
• cystic fibrosis, glycoclusters, hemagglutination, l-fucosides, lectins, multivalency,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Fucose chemistry   metabolism   MeSH
• Fungal Proteins chemistry   metabolism   MeSH
• Hemagglutination MeSH
• Lectins chemistry   metabolism   MeSH
• Humans MeSH
• Hemagglutination Inhibition Tests MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Fungal Proteins MeSH
• Lectins MeSH

Photorhabdus luminescens is known for its symbiosis with the entomopathogenic nematode Heterorhabditis bacteriophora and its pathogenicity toward insect larvae. A hypothetical protein from P. luminescens was identified, purified from the native source, and characterized as an l-fucose-binding lectin, named P. luminescens lectin (PLL). Glycan array and biochemical characterization data revealed PLL to be specific toward l-fucose and the disaccharide glycan 3,6-O-Me2-Glcβ1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 PLL was discovered to be a homotetramer with an intersubunit disulfide bridge. The crystal structures of native and recombinant PLL revealed a seven-bladed β-propeller fold creating seven putative fucose-binding sites per monomer. The crystal structure of the recombinant PLL·l-fucose complex confirmed that at least three sites were fucose-binding. Moreover, the crystal structures indicated that some of the other sites are masked either by the tetrameric nature of the lectin or by incorporation of the C terminus of the lectin into one of these sites. PLL exhibited an ability to bind to insect hemocytes and the cuticular surface of a nematode, H. bacteriophora.

• Keywords
• Galleria mellonella, Photorhabdus luminescens, bacterial pathogenesis, crystal structure, hemocytes from insect larvae, host/pathogen interaction, lectin, structural biology,
• MeSH
• Bacterial Proteins chemistry   isolation & purification   MeSH
• Fucose chemistry   MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• Lectins chemistry   isolation & purification   MeSH
• Photorhabdus chemistry   MeSH
• Protein Domains MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Fucose MeSH
• Lectins MeSH

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.

• MeSH
• Aspergillus fumigatus immunology   pathogenicity   MeSH
• Adult MeSH
• Fluorescent Antibody Technique MeSH
• Fucose metabolism   MeSH
• Fungal Proteins immunology   metabolism   MeSH
• Lectins immunology   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Macrophages immunology   metabolism   MeSH
• Disease Models, Animal MeSH
• Mucins immunology   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Pulmonary Aspergillosis immunology   metabolism   MeSH
• Flow Cytometry MeSH
• Immunity, Mucosal immunology   MeSH
• Spores, Fungal immunology   MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• fucose-binding lectin MeSH Browser
• Fucose MeSH
• Fungal Proteins MeSH
• Lectins MeSH
• Mucins MeSH