Naslov
Endosomal pathway of full and empty Ld molecules on nonpolarized P815 cell line

Autori
Mahmutefendić, Hana ; Kučić, Natalia ; Blagojević, Gordana ; Lučin, Pero

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Abstract book, (oral presentation 6, P28) / Hrvatsko imunološko društvo - Rijeka : Hrvatsko imunološko društvo, 2003

Skup
Annual meeting, Croatian Immunological Society

Mjesto i datum
Brijuni, Hrvatska, 17.10.2003. - 19.10.2003

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Endocytosis; MHC class I molecules; vesicular transport; alternative peptide presentation

Sažetak
Objectives: Although majority of MHC class I molecules arise in classical pathway, some of them follow the alternative ones. The aim of our study was to investigate the endosomal traffic of spontaneously internalized full and empty Ld molecules on P815 cell line as well as the the role of acid compartments in their transformation. Materials and methods: In order to achieve the conditions of spontaneous endocytosis on mentioned cell lines, we used cycloheximide (CHX) as an inhibitor of protein synthesis, and different chemical inhibitors of vesicular transport. The cell surface expression of MHC class I molecules was followed by flow cytometry by using monoclonal antibodies 30-5-7 (full Ld molecules) and 64-3-7 (empty Ld molecules). In order to investigate the fate of the internalized molecules, cell surface glycoproteins were biotinylated and determined by immunoprecipitation and chemiluminiscence. Furthermore, the conditions of endosomal pH were simulated outside the cell and the relationship between full and empty Ld molecules, as well as the switch in their conformation in those conditions was studied. Results: The Ld molecules internalized from the cell surface with the half-life of about 6-10 hrs. Inhibitors of endosomal acidification (monensin, concanamycin A and bafilomycin A1) maintained primary surface expression of the empty Ld molecules, but had no effect on expression of the full ones. Furthermore, we found that following biotinilation and immunoprecipitation monensin and NH4Cl decreased the total expression of full Ld molecules but saved empty molecules from deterioration even after 20 hrs. Using the acidification of culture medium to a pH (5.5 – 6.0), we concluded that lower pH increases the expression of empty, and proportionally and reversibly decreases the expression of full Ld molecules at the cell surface. Conclusion: Full and empty Ld molecules are able to reversibly change their conformations in a way that is dependent of pH and the conditions of the environment (beta2-microglobulin, presence of exogenous peptide, chaperons etc.). We propose that internalized MHC molecules change their conformation in acid endosomal compartments where they achieve transitional state that can be transformed into stabile trimolecular complexes if exogenous peptide and beta2-microglobuline are present.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA