Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer.
Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC(4) and its metabolites LTD(4) and LTE(4) stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC(4) is formed by addition of glutathione to LTA(4), catalyzed by the integral membrane protein, LTC(4) 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.
Svartz
Jesper
J
Division of Cell Biology, Department of Biomedicine and Surgery, Linköping University, SE-581 85, Linköping, Sweden. jeppa@ibk.liu.se
Blomgran
Robert
R
Hammarström
Sven
S
Söderström
Mats
M
D002460:Cell Line; D004795:Enzyme Stability; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005982:Glutathione Transferase / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase