Dissertation (Metadaten)
Titel:Biochemische Charakterisierung der 5-Hydroxyeikosanoid-Dehydrogenase und biologische Funktion der 5-Hydroxy- und 5-oxo-Eikosatetraensäure
 
Autor:Shing Kit Lee
 
URN:NBN:urn:nbn:de:gbv:8-diss-9997
 
Fakultät:Mathematisch-Naturwissenschaftliche Fakultät
DDC Sachgebiet:570 Biowissenschaften, Biologie
 
Datum der mdl. Prüfung:12.02.2004
 
Referent(in):Prof. Dr. K. Krupinska
Korreferent(en) Korreferentin:Prof. Dr. J.-M. Schröder
 
Beschreibung (original):keine dt.-spr, Zusammenfassung vh.
 
(übersetzt):Epithelia like human skin are always exposed to an abundance of harmful environmental factors like microorganisms and parasites. Thus, there are a lot of host-defense mechanisms against these pathogens. In addition to the barrier function of the stratum corneum, human skin is capable of protecting against potential pathogens by antimicobrial substances and natural bacteria flora. If skin injuries occur, leukocyte infiltrate out of blood in the site of inflammation. Thereby, neutrophilic and eosinophilic granulocytes are the mainly important effector cells of the immune system. 5-(S)-hydroxyeicosatetraenoic acid (5-(S)-HETE) and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) represent chemotactic eicosanoids which are synthesized by eosinophils and neutrophils. The stereospecific 5-hydroxyeicosanoid dehydrogenase (5-HEDH) converts 5-(S)-HETE to the biologically active compound 5-oxo-ETE in the presence of NADP+ as a cofactor. In contrast to eosinophils and neutrophils, dendritic cells appear to be a potential source of 5-oxo-ETE in human skin. 5-(S)-HETE and 5-oxo-ETE activate primarily eosinophils and also neutrophils. The selective chemotactic properties of 5-oxo-ETE on eosinophils confirm the hypothesis that this lipid could take part in parasite defense or in other eosinophil-related diseases like asthma and atopic dermatitis. Since the molecular structure of 5-HEDH is still unknown and there is only little information about regulation of 5-HEDH, the aim of the present work was to isolate and to characterize the 5-HEDH from human neutrophils. For these purposes, 5-HEDH was isolated from neutrophil supernantant and solubilized microsomal fraction. After partial purification step by high pressure liquid chromatography (HPLC), 5-HEDH was detected indirectly by an established chromatographic assay. Herein, the 5-HEDH activity was measured by converting 5-(S)-HETE to 5-oxo-ETE. The structural characterization of 5-HEDH was performed by using SDS-polyacrylamide gel electrophoresis and aminoterminal sequence analysis (Edman analysis) and mass spectroscopy after a proteolytic digestion. After combination of the anion exchanger and with a reversed phase (RP) chromatography, we could detect cytoskeleton proteins (e.g. actin, alpha-actin, alpha-actinin-1) as well as regulatory proteins like protein kinase C-inhibitor protein-1 (KCIP-1) and anti-microbial peptids like calprotectin and lactoferrin in partial purified HPLC-fractions which contained 5-HEDH activity. With the increasing degree of purification, especially by the RP-chromatography, the 5-HEDH activity was lost completely. Therefor, the partial purified 5-HEDH could be determined only by means of the intensity of their protein bands after the SDS gel electrophoresis. As structure proteins like actin und alpha-actin were indentified frequently in a partial purified sample, it is assumed that 5-HEDH may be able to form a complex with these components. Literature and protein data bank research indicate that the indentified peptids calprotectin und lactoferrin possess catalytic regions and putative NAD(H) and NADP(H) binding sites for converting 5-(S)-HETE to 5-oxo-ETE. Moreover, beta-actin as well as an unknown fragment with the amino acid sequence „APARVFPSLS” were detected in purified 5-HEDH preparations by mass spectroscopy. This peptid shows highly strong homology to a bovine NADH-ubiquinone oxidoreductase and to a putative iron alcohol dehydrogenase from the yeast Schizosaccharomyces pombe. Data bank research suggests that this sequence is found in a human mitochondrial oxidoreductase as well as in a human zinc finger protein and in a GTP-binding protein. According to this indentified amino acid sequence, it was supposed that the determined NADH-ubiquinone oxidoreductase could represent the 5-HEDH. Using RACE-PCR-technique (RACE: Rapid Amplification of cDNA Ends) we have tried to indentify the gene coding for the indentified peptid fragment, which could display 5-HEDH. However, it was impossible to amplify the putative 5-HEDH gene by the RACE-PCR. The reason for these v ain efforts may base on the high degree of degradation of the used oligo nucleotides. Furthermore, keratinocytes and fibroblasts were stimulated with 5-(S)-HETE und 5-oxo-ETE to examine the induction of proinflammatory cytokines and a potential synergism between eicosanoids and chemokines. Using reverse transcription and polymerase chain reaction technique (RT-PCR) we could show a dosis- and time-depending interleukin-1beta; (IL-1beta) induction in fibroblasts after stimulation with 5-oxo-ETE and 5-(S)-HETE. Besides, we observed the expression of interleukin-1alpha (IL-1alpha) and growth related oncogene-alpha (Gro-alpha) after stimulation of fibroblasts with 5-oxo-ETE. In contrast to the fibroblasts, in keratinocytes cytokine expression could not be induced after stimulation with 5-oxo-ETE und 5-(S)-HETE. Moreover, the CC-chemokines „Eotaxin“, which activates eosinophils and provides primarily parasite defense, could not be induced in keratinocytes neither by 5-(S)-HETE nor by 5-oxo-ETE. This observation could be an evidence that 5-oxo-ETE regulate the cytokine synthesis in dermal fibroblasts. Besides, through the induction of IL-1 expression, it is possible that the eosinophil migration as well as the persistence of eosinophils in the site of inflammation will be enhanced by the post synthesis of inflammatory and growth factors, e.g. “granulocyte/macrophage colony stimulating factor” (GM-CSF). Since Gro-alpha expression belongs to the group of CXC-Chemokine and activates primarily neutrophils, the induction of Gro-alpha by 5-oxo-ETE stands for a new aspect in the pathophysiology of eosinophil-related diseases. We postulate that 5-oxo-ETE, which is synthesized in Langerhans cell, is first of all responsible for the induction of eosinophil chemotaxis but also for the attraction and activation of neutrophils. The timely shift in the infiltration of eosinophils and neutrophils and the presumable time-limited specific activation of cells after stimulation by 5-oxo-ETE could have a fundamental meaning in eosinophil-related inflammatory processes.
 
Schlagworte:5-oxo-ETE, 5-Hydroxyeikosanoid Dehydrogenase, 5-HETE, Eosinophilen-assoziierte Erkrankung, Eikosanoide, proentzündliche Zytokine, Eosinophiler Granulozyt, humane Fibroblasten, Entzündung, Neutrophiler Granulozyt
 
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