2005
Mutagenesis of PA-IIL lectin from human pathogen Pseudomonas aeruginosa and its effect on the saccharide specificity
POKORNÁ, Martina, Edward P. MITCHELL, Jan ADAM, Martin PROKOP, Zdeněk KŘÍŽ et. al.Základní údaje
Originální název
Mutagenesis of PA-IIL lectin from human pathogen Pseudomonas aeruginosa and its effect on the saccharide specificity
Název česky
Mutagenese PA-IIL lektinu z lidského patogenu Pseudomonas aeruginosa a jeji vliv na specifitu
Autoři
POKORNÁ, Martina (203 Česká republika, garant), Edward P. MITCHELL (250 Francie), Jan ADAM (203 Česká republika), Martin PROKOP (203 Česká republika), Zdeněk KŘÍŽ (203 Česká republika), Jaroslav KOČA (203 Česká republika), Anne IMBERTY (250 Francie) a Michaela WIMMEROVÁ (203 Česká republika)
Vydání
Rydzyna, European Congress of Young Chemists, s. 45-45, 2005
Nakladatel
Chemical Scientific Society Flogiston
Další údaje
Jazyk
angličtina
Typ výsledku
Stať ve sborníku
Obor
10600 1.6 Biological sciences
Stát vydavatele
Polsko
Utajení
není předmětem státního či obchodního tajemství
Kód RIV
RIV/00216224:14310/05:00014102
Organizační jednotka
Přírodovědecká fakulta
Klíčová slova anglicky
lectin; mutagenesis; saccharide; Pseudomonas aeruginosa
Štítky
Změněno: 4. 11. 2005 10:05, Mgr. Martina Pokorná, Ph.D., MBA
V originále
Pseudomonas aeruginosa is an opportunistic human pathogen that can infect almost every human tissue if immunity barriers are lowered. Pseudomonas aeruginosa produces two lectins associated with its virulence, D galactose and L-fucose binding lectins, PA-IL and PA-IIL respectively. In contrast with most lectins that display only weak affinity for monosaccharide ligands, the equilibrium dissociation constant of PA-IIL/L-fucose interaction is in the micromolar range [1]. Database searching in newly sequenced bacterial genomes revealed the presence of PA-IIL like proteins within limited number of other opportunistic pathogens. One of them, the close sequence homologue from Ralstonia solanacearum (called RS-IIL), has been recently characterised. Crystal structure of RS-IIL lectin has been solved and showed very high structure similarity with PA-IIL. Both are tetramers with two calcium-mediated sugar binding. The only difference is in a three amino acid motif of the ligand binding loop that is responsible for lectin reverse order preferences toward different monosaccharides. PA-IIL prefers L-fucose and L-galactose whilst RS-IIL prefers D-mannose and D-fructose [2]. In order to characterise the contribution of particular amino acids for such a difference, mutants of PA-IIL concerning Ser22, Ser23 and Gly24 that are responsible for the high preference of PA-IIL to fucose have been designed. Mutants have been prepared by site-directed mutagenesis and purified by affinity chromatography on Mannose-agarose. Interaction of mutants with different monosaccharides has been evaluated using isothermal titration microcalorimetry and some complexes have been crystallised. Experiments showed the importance of amino acid residue on position 22 and opened the feasibility for prediction of specificity for the other PA-IIL-like lectins. Simultaneously, the program TRITON [3], a graphical tool for modelling protein mutants, which has been under development in our laboratory, was used for designing protein mutants in silico. Further docking studies allowed prediction of changes in binding abilities of designed proteins. [1] Mitchell E.P., Sabin C., Šnajdrová L., Pokorná M., Perret S., Gautier C., Hofr C., Gilboa-Garber N., Koča J., Wimmerová M., Imberty A.: Proteins: Structure, Function, and Bioinformatics, 58, 735 - 746 (2005) 2 Sudakevitz D., Kostlánová N., Blatman-Jan G., Mitchell E., Lerrer B., Wimmerová M., Katcoff D.J., Imberty A., Gilboa-Garber N.: Mol. Microbiol. 52, 691-700 (2004) [3] Prokop M., Damborský J., Koča J.: Bioinformatics. 16, 845-846 (2000).
Česky
Pseudomonas aeruginosa is an opportunistic human pathogen that can infect almost every human tissue if immunity barriers are lowered. Pseudomonas aeruginosa produces two lectins associated with its virulence, D galactose and L-fucose binding lectins, PA-IL and PA-IIL respectively. In contrast with most lectins that display only weak affinity for monosaccharide ligands, the equilibrium dissociation constant of PA-IIL/L-fucose interaction is in the micromolar range [1]. Database searching in newly sequenced bacterial genomes revealed the presence of PA-IIL like proteins within limited number of other opportunistic pathogens. One of them, the close sequence homologue from Ralstonia solanacearum (called RS-IIL), has been recently characterised. Crystal structure of RS-IIL lectin has been solved and showed very high structure similarity with PA-IIL. Both are tetramers with two calcium-mediated sugar binding. The only difference is in a three amino acid motif of the ligand binding loop that is responsible for lectin reverse order preferences toward different monosaccharides. PA-IIL prefers L-fucose and L-galactose whilst RS-IIL prefers D-mannose and D-fructose [2]. In order to characterise the contribution of particular amino acids for such a difference, mutants of PA-IIL concerning Ser22, Ser23 and Gly24 that are responsible for the high preference of PA-IIL to fucose have been designed. Mutants have been prepared by site-directed mutagenesis and purified by affinity chromatography on Mannose-agarose. Interaction of mutants with different monosaccharides has been evaluated using isothermal titration microcalorimetry and some complexes have been crystallised. Experiments showed the importance of amino acid residue on position 22 and opened the feasibility for prediction of specificity for the other PA-IIL-like lectins. Simultaneously, the program TRITON [3], a graphical tool for modelling protein mutants, which has been under development in our laboratory, was used for designing protein mutants in silico. Further docking studies allowed prediction of changes in binding abilities of designed proteins. [1] Mitchell E.P., Sabin C., Šnajdrová L., Pokorná M., Perret S., Gautier C., Hofr C., Gilboa-Garber N., Koča J., Wimmerová M., Imberty A.: Proteins: Structure, Function, and Bioinformatics, 58, 735 - 746 (2005) 2 Sudakevitz D., Kostlánová N., Blatman-Jan G., Mitchell E., Lerrer B., Wimmerová M., Katcoff D.J., Imberty A., Gilboa-Garber N.: Mol. Microbiol. 52, 691-700 (2004) [3] Prokop M., Damborský J., Koča J.: Bioinformatics. 16, 845-846 (2000).
Návaznosti
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