Použití α-N-Acetylgalactosaminidasy jako nástroje vhodného pro syntézu komplexních oligosacharidových stimulátorů imunitního systému

α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators Introduction: Alpha-N-acetylgalactosaminidases are exoglycosidases specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chain. They occur widely in microorganisms, plants and animals, and have considerable potential in various industrial application. Stability and activity at high temperatures are important properties of α- N-acetylgalactosaminidases. A large screening study for extracellular α-N- acetylgalactosaminidase activity in a library of filamentous fungi (42 strains), led to the identification of the best constitutive producer, Aspergillus niger CCIM K2. The enzyme from Aspergillus niger has certain unique properties, and it was chosen for further investigations. Methods: Alpha-N-acetylgalactosaminidase from Aspergillus niger CCIM K2 was partially sequenced by Edman degradation and MALDI MS. Degenerated PCR primers were designed based on the obtained N-terminal and internal sequences. A gene fragment encoding a putative part of the α-N-acetylgalactosaminidase was amplified using cDNA prepared from Aspergillus niger CCIM K2. The full-length coding sequence of α-N- acetylgalactosaminidase was cloned into pYES-2CT vector and the recombinant protein was expressed in...Použití α-N-acetylgalaktosaminidasy jako nástroje vhodného pro syntézu komplexních oligosacharidových stimulátorů imunitního systému Úvod: Alfa-N-acetylgalaktosaminidasy jsou exoglykosidasy specificky štěpící terminálně vázaný α-N-acetylgalaktosamin v různých sacharidových řetězcích neboz aminokyselin serin nebo threonin. Tyto enzymy jsou široce rozšířené, vyskytují se téměř v každém organismu. Stabilita a aktivita v extremních podmínkách je důležitá vlastnost, které se hojně využívá při mnoha průmyslových aplikací. Studium velkého množství extracelulárních α-N-acetylgalaktosaminidas izolováných z vlaknitých hub byl nalezen nejlepší producent Asperillus niger CCIM K2. Enzym izolovaný z vlaknité houby Aspergillus niger vykazoval jedinečné vlastnosti, které byly dále zkoumány. Metody: Alfa-N-acetylgalaktosaminidasa izolována z A.niger byla částečně sekvenována pomocí Edmanova odbourávání a MALDI MS. Na základě zjištěných sekvencí byly navrženy degenerované oligonukleotidy. Gen kódujíci α-N-acetylgalaktosaminidasu byl amplifikovan pomocí PCR. Jako templát byla použita cDNA izolovaná z A.niger. Získaný gen byl ligován do expresního vektoru pYES-2CT a rekombinantní protein byl exprimován v kvasinkovém expresním systému Saccharomyces cerevisiae. Byly optimalizovány podmínky exprese tohoto proteinu. Protein byl...Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Použití α-N-Acetylgalactosaminidasy jako nástroje vhodného pro syntézu komplexních oligosacharidových stimulátorů imunitního systému

1 Charles University in Prague Faculty of Science Department of Biochemistry α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators Summary of Ph. D. Thesis Mgr. Hynek Mrázek Supervisor: Prof. RNDr. Karel Bezouška, Dsc. Prague 2011 2 Introduction Glycoproteins Glycoproteins consist of proteins to which carbohydrate are covalently linked. The distinction between proteoglycans and glycoproteins residues is in the level and types of carbohydrate modification. Carbohydrates are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The N-glycosidic linkage is through the amide group of asparagine. The O- glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. The site of carbohydrate attachment to N- linked glycoproteins is found within a consensus sequence of amino acids, N-X- S(T), where X is any amino acid except proline. While the N-glycosylation is governed by the above rules, no exact rules were found for glycosylation of O- type. This process is the step-wise addition of the sugar residues directly onto the polypeptide chain. Biosynthesis of glycoproteins occurs via protein glycosylation (the addition of...1 Univerzita Karlova Přírodovědecká Fakulta Katedra Biochemie Použití α-N-acetylgalaktosaminidasy jako nástroje vhodného pro syntézu komplexních oligosacharidových stimulátorů imunitního systému Autoreferát dizertační práce Mgr. Hynek Mrázek Školitel: Prof. RNDr. Karel Bezouška, Dsc. Praha 2011 2 Úvod Glykoproteiny jsou proteiny obsahující kovalentně vázané sacharidové struktury. Sacharidy můžou být připojeny k proteinovému řetězci dvojím způsobem: O-glykosidickou nebo N-glykosidickou vazbou. N-glykosidicky spojené sacharidy jsou vázany k aminokyselině asparaginu, zatím co O-glykosidicky vázané sacharidy jsou spojeny k aminokyselinám serinu nebo threoninu. V glykoproteinech vyšších obratlovců se převážně vyskytují N-glykosidicky vázané sacharidové struktury. V případě N-glykosylace je sacharidový řetězec přenesen na aminokyselinu asparagin, který se nachází v aminokyselinové sekvenci N-X-ST, kde X může být jakákoliv aminokyselina kromě prolinu. Zatímco syntéza N-glykoproteinů se řídí určitými pravidly, u syntézy O-glykoproteinů nebyla žádná taková pravidla pro zatím nalezena. Proteinové glykosylace, která je nejčastější posttranslační modifikací, se účastní velké množství enzymů a enzymových komplexů. Celý proces glykosylace probíhá v endoplazmatickém retikulu a Golgiho aparátu. Glykosidasy hydrolasy Podle...Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Investigation of binding specificities of isoforms of the rat NKR-P1 receptor

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators

1 Charles University in Prague Faculty of Science Department of Biochemistry α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators Summary of Ph. D. Thesis Mgr. Hynek Mrázek Supervisor: Prof. RNDr. Karel Bezouška, Dsc. Prague 2011 2 Introduction Glycoproteins Glycoproteins consist of proteins to which carbohydrate are covalently linked. The distinction between proteoglycans and glycoproteins residues is in the level and types of carbohydrate modification. Carbohydrates are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The N-glycosidic linkage is through the amide group of asparagine. The O- glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. The site of carbohydrate attachment to N- linked glycoproteins is found within a consensus sequence of amino acids, N-X- S(T), where X is any amino acid except proline. While the N-glycosylation is governed by the above rules, no exact rules were found for glycosylation of O- type. This process is the step-wise addition of the sugar residues directly onto the polypeptide chain. Biosynthesis of glycoproteins occurs via protein glycosylation (the addition of..

Investigation of binding specificities of isoforms of the rat NKR-P1 receptor

Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

Effective Removal of Nonionic Detergents in Protein Mass Spectrometry, Hydrogen/Deuterium Exchange, and Proteomics

International audienceDetergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex

The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques