Open Access

Stavroula Markoutsa

• 5-lipoxygenase (5-LO) is an enzyme with a substantial role in inflammatory processes. In vitro kinase assays using [32P]-ATP in combination with mutagenesis have revealed that serine residues 271, 523 and 663 can be phosphorylated by MK2, PKA and ERK2 kinases, respectively. A few available reports regarding 5-LO protein sequence have covered up to 30% of the sequence after amino acid sequencing including Ser663. In LCMS/MS analyses of 5-LO tryptic digests from different cellular sources different peptides have been detected; however, none of the three phosphorylations has been detected and only Ser663 was included in the covered sequence. As there was no comprehensive mass spectrometric analysis of 5-LO, the purpose of this study was to optimize the experimental conditions under which detection of the aforementioned phosphorylation events, as well as other possible post-translational modifications (PTMs), would be feasible. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) was used for peptide analysis of 5-LO cleaved either by chemical reagents or by proteases. Sequence coverage of 5-LO could be enhanced to be close to completion by combination of results from digestions by trypsin, AspN and chymotrypsin. In-gel trypsin digestion followed by in-solution AspN digestion proved to be a useful sample treatment for reproducible detection of the Ser271-containing peptide. Nevertheless, in none of the examined cleavage protocols the sequence around Ser523 was detected reproducibly or with acceptable signal intensity for subsequent peptide fragmentation. Propionic anhydride and sulfo-NHS-SS-biotin cross-linker (EZ-linkTM), were used for derivatization of lysine side chains and hindrance of lysine residue recognition by trypsin. Phosphopeptide enrichment became possible after tryptic digestion of these samples, not only due to formation of an individual Ser523-containing peptide, but also because TiO2-mediated enrichment, which is performed in acidic pH, was not impaired by positively charged free lysine side chains. Additionally, biotinylation of lysine residues was exploited for an intermediate enrichment step of the lysine containing peptides, prior to TiO2 phosphopeptide enrichment. MALDI-MS analysis after in-vitro phosphorylation of 5-LO by the three kinases showed that Ser271 was phosphorylated in the MK2 and PKA kinase assays, while Ser523 was phosphorylated only in the PKA kinase assay. Surpisingly, no phosphopeptides were detected in the in-vitro kinase assays with ERK2, even though the unmodified counterpart of the Ser663-containing peptide was easily detected. The detection limit for each of the three phosphorylation sites was determined by the use of custom made phosphopeptides and an amount of 0.06 pmol of phosphopeptide in 1 μg 5-LO (representing 0.5% phosphorylation rate) was sufficient in all cases for successful enrichment and detection by MS. In-vitro kinase assays with [32P]-ATP were performed for some kinases that were expected to phosphorylate 5-LO according to in-silico data. Three members of the Src tyrosine kinase family (Fgr, Hck and Yes) and the Ser/Thr specific kinase DNA-PK used 5-LO as their substrate and mainly residues at the N-terminal part of 5-LO were detected phosphorylated by MS (e.g. Y42, Y53). Additional in-vitro assays for recombinant 5-LO modification included incubation with glutathione or compound U73122, previously described as inhibitor of 5-LO. Since in-vitro assays might have generated artifacts, a method for 5-LO purification from human cells was sought, in order to examine the modification state of the protein in the cellular context. ATP-agarose affinity purification and anti-5-LO immunoprecipitation proved inappropriate for sample purification for MALDI-MS analysis. Consequently, two human cell lines that are able to express 5-LO (Rec-1 Blymphocytes and MM6 monocytes) were transduced with a DNA cassette that contained recombinant human 5-LO sequence with an attached N-terminal FLAG-tag. Anti-FLAG immunoprecipitation was then performed effectively in cell lysates and the precipitated FLAG-5-LO was separated by SDS-PAGE before MALDI-MS analysis. The examined cell stimuli were expected to result to phosphorylation of 5-LO at Ser523 by PKA in Rec-1 cells and to phosphorylation of Ser271 and/or Ser663 in MM6 cells by activated MK2 and ERK2, respectively. Additionally, under the conditions of MM6 cell stimulation, Fgr, Hck and Yes kinases, which phosphorylated 5-LO in vitro, were expected to be activated and the possibility of 5-LO phosphorylation on tyrosine was investigated. Although immunoblotting results indicated that all the aforementioned phosphorylation events existed in the examined samples, MALDI-MS analysis verified only phosphorylation on Ser271 in differentiated MM6 cells, interestingly regardless of cell stimulation. Finally, the primary amine derivatization procedure by EZ-linkTM was utilized for MS analysis of lysine rich proteins. In the past, chemical propionylation of histones had been employed prior to trypsin digestion; however it was easily confused in MS with combinations of other PTMs (e.g. acetylation, methylation). Moreover, propionylation is a PTM for histone H3 and this information was lost. Consequently, the EZ-link reagent was more useful for analysis of histones, as unambiguous assignment of PTMs and detection of native propionylation on bovine H3 became possible.
• Das Enzym 5-Lipoxygenase (5-LO) nimmt eine Schlüsselrolle im Entzündungsgeschehen ein, wie es beispielsweise bei Erkrankungen wie Asthma, Rheumatoide Arthritis oder Atherosklerose vorkommt. Es ist der Ausgangspunkt des Leukotrien-Stoffwechselwegs, indem es die Oxidation der Arachidonsäure zu proinflammatorischen Mediatoren katalysiert. Gleichzeitig ist die 5-LO, im Zusammenspiel mit den Enzymen 12- und 15-LO, an der Bildung antiinflammatorischer Lipoxine beteiligt. Neuere Ergebnisse zeigen darüber hinaus eine Beteiligung der 5-LO an der Bildung zellulärer Proteinprodukte, die zur Aggregation neigen und die mit der Alzheimerschen Erkrankung in Zusammenhang gebracht werden. Weitere Arbeiten beschreiben eine Korrelation zwischen der Expression von 5-LO und der Entwicklung von Tumorerkrankungen, während andere Ergebnisse auf eine Beteiligung am Transkriptionsgeschehen schließen lassen. Daher wurde die 5-LO zu einem weltweit intensiv beforschten Enzym, und es wurde eine große Zahl von Arbeiten durchgeführt, um das Enzym in seinen biochemischen Eigenschaften zu charakterisieren, seinen zellulären Mechanismus sowie mögliche Interaktionen mit anderen Proteinen aufzuklären sowie chemische Spezies zu entwickeln, die das Enzym inhibieren können. Die Leukotrienbiosynthese durch die 5-LO wird durch mehrere Faktoren, darunter posttranslationale Modifikationen (PTM), strikt reguliert. Zu den bisher bekannten PTMs der 5-LO gehören Phosphorylierungen durch Serin-Threonin-Kinasen. Im Einzelnen handelt sich um die Serinreste an den Positionen 271, 523 und 663, die als Zielstrukturen der Kinasen MK2, PKA und Erk2 bestimmt werden konnten. Diese drei Phosphorylierungen werden als bedeutsam für die Wirksamkeit von Inhibitoren betrachtet und damit als wichtig für Protokolle zur Testung neuer Arzneistoffe. Allerdings basieren die bisherigen Untersuchungen zur Phosphorylierung der 5-LO nicht auf massenspektrometrischen Techniken, so dass ein Beweis auf molekularer Ebene dafür noch zu erbringen ist....