Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer
2003 (English)In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, ISSN 1388-1981, E-ISSN 1388-1918, Vol. 1633, no 2, 90-95 p.Article in journal (Refereed) Published
Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC4 and its metabolites LTD4 and LTE4 stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC4 is formed by addition of glutathione to LTA4, catalyzed by the integral membrane protein, LTC4 synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.
Place, publisher, year, edition, pages
Elsevier, 2003. Vol. 1633, no 2, 90-95 p.
eicosanoid; fusion protein; green fluorescent protein; human embryonic kidney cell; oligomerization
Biochemistry and Molecular Biology Cell Biology
IdentifiersURN: urn:nbn:se:liu:diva-25056DOI: 10.1016/S1388-1981(03)00091-XISI: 000184499300003Local ID: 9484OAI: oai:DiVA.org:liu-25056DiVA: diva2:245382