PHOTOACTIVABLE LACTOSYLCERAMIDE DERIVATIVES: PREPARATION AND USE IN THE COMPREHENSION OF THE GENERATION OF OXIDATIVE SPECIES IN NEUTROPHILS.
Tesi di Dottorato
Data di Pubblicazione:
2012
Citazione:
PHOTOACTIVABLE LACTOSYLCERAMIDE DERIVATIVES: PREPARATION AND USE IN THE COMPREHENSION OF THE GENERATION OF OXIDATIVE SPECIES IN NEUTROPHILS / G. Brasile ; docenti guida: S. Sonnino, F. Compostella ;
collaboratore: Iwabuchi ; coordinatore : F. Bonomi. Universita' degli Studi di Milano, 2012 Feb 10. 24. ciclo, Anno Accademico 2011. [10.13130/brasile-giuseppina_phd2012-02-10].
Abstract:
Photoactivable lactosylceramide derivatives: preparation and use in the comprehension of the generation of oxidative species in neutrophils.
Glycosphingolipids (GSL) are anphiphilic membrane components consisting in a hydrophobic moiety, ceramide, and a hydrophilic oligosaccharide headgroup. Ceramide is responsible for their insertion into the outer layer of plasma membranes, with the oligosaccharide chain protruding in the extracellular environment. GSLs participate to the signaling processes across the membrane1 determining the lateral organization of cellular membranes and modulating the function of several classes of membrane proteins.2 Their function rely to the ability they have to form clusters with sphingomyelin, ceramide, cholesterol and signal trasduction proteins such as GPI-anchored and acylated proteins (including Src family-kinases), to form specialized membrane domains called “lipid-rafts”.3
In mature neutrophils, which play the first line of defense against invading microorganisms and have an important role in acute inflammatory reaction, more than 70% of GSLs are LacCers, which are aberrantly expressed at high levels on mature neutrophils. It is possible that LacCer activates NADPH oxidase, thereby affecting the functions of superoxide-producing cells. However, the mechanisms by which LacCer activates NADPH oxidase in neutrophils have not yet been well characterized.
It is known that lactosylceramide (LacCer) is specifically coupled with Src family kinase Lyn in plasma membrane microdomains of human neutrophils. Ligand binding to LacCer activates Lyn, resulting in neutrophils functions, such as superoxide generation and migration.4 The β-D-Gal-(1→4)-β-D-Glc structure is necessary but it is not sufficient for LacCer-mediated Lyn activation. For this function, the presence of a LacCer molecular species with a ceramide containing a very long fatty acid is also required.
GSLs containing very long fatty acids might participate in reducing the membrane thickness through interdigitation of the two membrane leaflets. The interdigitation is proposed to be the switch for the transduction of information throughout the membrane perhaps by allowing contact and interaction between proteins belonging to the two layers of the plasma membrane.5 Anyway the molecular mechanism by which the interactions between GSLs and protein influence the cell functions has still to be elucidated.
The aim of this thesis is to give a contribute in the comprehension of the intermediate steps of the signaling process mediated by Lyn proteins. In particular, the purpose is to explore the role of long chain LacCer in this process, with a particular interest in the identification of the proteins associated with LacCer in the immune response to several microorganisms in human neutrophils.
The cross-linkage between gangliosides and proteins can be investigated by cell photolabeling using radioactive photoactivable gangliosides carrying the reactive group at different positions of the molecule. In fact the photoactivable group, when illuminated, yield a very reactive intermediate that covalently binds to the molecules in the environment.6,7
With the final aim to identify LacCer associated proteins, we have developed probes 1 and 2, containing one or two photoactivable groups, located at specific points of the molecule and, in principle, capable to interact with proteins belonging to the cytoplasmic and/or to the extracellular membrane layer.
Probes 1 e 2 display an acyl chain long enough for LacCer-mediated Lyn activation, as preliminary experiments have shown.8 The initial task of the thesis has been the preparation of the acyl derivatives, suitable for the construction of probes 1 and 2. The synthetic efforts were mainly de
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
G. Brasile
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