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PHOTOACTIVABLE LACTOSYLCERAMIDE DERIVATIVES: PREPARATION AND USE IN THE COMPREHENSION OF THE GENERATION OF OXIDATIVE SPECIES IN NEUTROPHILS.
Full text linkPhotoactivable 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 devoted to prepare the proper aminoacid derivatives for conjugation to lactosylsphingosine and subsequent derivatization with the photoactivable probe. Both the C-18 ω-aminoacid and the C-18 α,ω-diaminoacid were prepared by chemical synthesis, since they are not commercially available. In addition, the synthesis of the long chain α,ω-diamino acid can be very interesting for the possibility of simultaneous capture of the proteins belonging to the two leaflets.
The C-18 ω-aminoacid was derived from commercially available octadecandioic acid through a eight steps synthetic pathway. On the contrary, the α,ω-diamino homologue was constructed in seven steps by subsequent condensations of shorter building blocks, starting from suitable protected aspartic acid, through a synthetic route that showed several tricky points.
Both aminoacids, activated as pentafluorophenolates, have been coupled to lactosylsphingosine, which has been previously tritiated in order to follow the sphingolipid biological pathway. The obtained proper LacCers were finally derivatized with nitrophenilazide to give the target probes 1 and 2.
To assess the capability of an α,ω-diamino fatty acid functionalized probe, i.e. 2, to be internalized by the cells, we have designed probe 3 in which the hydrophilic moiety is ganglioside GM1.
Previous studies have in fact demonstrated the capability of GM1, with one photoactivable group at the end of the fatty chain, to be inserted in the membrane lipid core in a way that closely resembles that of endogenous gangliosides.9
Photolabelling experiments are now in progress both in Milan (probe 3, GM1) and in Japan (probe 1 and 2, experiments on human neutrophils in collaboration with Professor Iwabuchi)
Photoactivable probes for the study of glycosphingolipid-protein interactions in lipid-rich membrane domains
No full textLipid-rafts are membrane microdomains particularly enriched in glycosphingolipids (GSLs) involved in the information transduction process across the membrane.
The great interest for such domains is due to the observation that some membrane associated proteins are highly concentrated in lipid rafts, even if the overall protein content of these membrane areas is very low. It has been assumed that the trapping of certain proteins in lipid rafts might be somehow functional to their biological role, i.e. their involvement in signal transduction processes.
GSL-protein interactions can be investigated by cell photolabelling experiments using radioactive photoactivable GSLs, which yield, when illuminated, a very reactive intermediate that covalently binds to the molecules in the environment, i.e. proteins.
In this context, we have designed a fatty acid probe with two nitrophenylazide photoactivable groups, one at position 2 and the other at the end of the acyl chain. The conjugation of the fatty acid to a radioactive glycosphingolipid generates a species to be used for photolabelling experiments allowing the simultaneous identification of the proteins belonging to the cytoplasmatic and the extracellular leaflets of the membrane. Herein we describe a general synthetic strategy to obtain not commercially available α,ω-diamino acids, the synthetic precursors of the labelled fatty acids, which we have applied to the synthesis of a C-18 derivative. Furthermore, it will be described the preparation of a photolabelled radioactive GSL as a case study
Synthesis of photoactivable probes for the study of glycosphingolipid-protein interactions
No full textSYNTHESIS OF PHOTOACTIVABLE PROBES FOR THE STUDY OF GLYCOSPHINGOLIPID-PROTEIN INTERACTIONS
Giuseppina Brasile,1 Federica Compostella,1 Laura Mauri,2Sandro Sonnino2 and Fiamma Ronchetti1
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano
1Via Saldini 50, 20133-Milano, Italy; 2Via Fratelli Cervi 93, 20190- Segrate (Milano), Italy
E-mail:[email protected]
It is widely accepted that glycosphingolipids (GSLs) at the level of the plasma membrane can affect the biological functions of protein molecules, such as cell surface receptors or transporters. The interactions between GSLs and proteins belonging to specific membrane microdomains, called lipid rafts, could be responsible for the modulation of the functional properties of membrane proteins participating in signal transduction. GSL-protein interactions can be investigated by cell photolabelling experiments using radioactive photoactivable GSLs, which yield, when illuminated, a very reactive intermediate that covalently binds to the molecules in the environment, i.e. proteins.1,2
In this context, we have designed a fatty acid probe with two nitrophenylazide photoactivable groups, one at position 2 and the other at the end of the acyl chain. The conjugation of the fatty acid to a radioactive sphingoglycolipid generates a species to be used for photolabelling experiments. In this way, the simultaneous identification of the proteins belonging to both the leaflets of the plasma membrane, the cytoplasmatic and the extracellular one, will be realized.
Herein we describe a general synthetic strategy to obtain not commercially available α,ω-diamino acids, the synthetic precursors of the labelled fatty acids, which we have applied to the synthesis of a C-18 derivative. Furthermore, it will be described the preparation of a photolabelled radioactive GSL as a case study.
1. Aureli M., Prioni S., Mauri L. Loberto N., Casellato R., Ciampa M.G., Chigorno V., Prinetti, A.; Sonnino S. J. Lipid Res. 2010, 51, 798-808
2. Mauri L., Prioni S., Loberto N., Chigorno V., Prinetti A., Sonnino S. Glycoconj. J. 2004, 20, 11-23
A practical route to long-chain non-natural α,ω-diamino acids
No full textAn efficient method for the synthesis of long-chain α,ω-diamino acids, starting from natural α-amino acids, has been developed. The long-chain skeleton has been generated through condensation between a protected aldehyde, derived from l-aspartic acid, and an ylide obtained from an ω-hydroxy-alkyl phosphonium salt. After conversion of the ω-hydroxy group into an amine, catalytic hydrogenation produced the N, N′-protected α,ω-diamino acid. The present route to α,ω-diamino acids allows the modulation of the chain length depending on the length of the ylide used for the Wittig olefination reaction
Synthesis of a photoactivable lactosylceramide for the study of transduction process through the membrane in neutrophils
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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
An approach to understand the glycosphingolipid-protein interactions
No full textINTRODUCTION: Lipid-rafts are membrane microdomains particularly enriched in glycosphingolipids (GSLs) and proteins involved in trasduction processes across the membrane.
GSL-protein interactions can be investigated by cell photolabelling experiments using radioactive photoactivable GSLs, which yield, when illuminated, a very reactive intermediate that covalently binds to the molecules in the environment, i.e. proteins (1,2). The preparation of a GSL probe with two photoactivable groups, one at position 2 and the other at the end of the acyl chain of ceramide, could be a tool for the simultaneous identifications of the proteins involved in the biological recognition and belonging to both the extracellular and the cytoplasmatic leaflets of the membrane.
RESULTS: The preparation of an α,ω-diaminoacid as a bifunctional fatty acid useful for the conjugation of GLSs to photoactivable groups, i.e. nitrophenylazide, through the amino functionalities has been performed. This lipid chain has been conjugated to a radioactive lyso-ganglioside GM1, tritium labeled at position 3 of sphingosine, obtained by an enzymatic reaction with a SCDase. The purpose is to verify the capability of this new kind of probes to enter the cells and participate to metabolic processes. So, the photolabelled 3H-GM1 will be administer to cells and its metabolic fate will be studied
Photoactivable lactosylceramide for the study of membrane signal transduction in neutrophils
No full textIdentification of the proteins associated to long chain fatty acid containing lactosylceramide in the transduction of information through the membrane in neutrophils
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