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Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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2000 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 480, no 2-3, 239-243 p.Article in journal (Refereed) Published
Abstract [en]

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein–protein interaction may contribute to the regulation of LTC4 production.

Place, publisher, year, edition, pages
2000. Vol. 480, no 2-3, 239-243 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-47589DOI: 10.1016/S0014-5793(00)01885-8ISI: 000089209900035OAI: oai:DiVA.org:liu-47589DiVA: diva2:268485
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Leukotriene C4 synthase: studies on oligomerization and subcellular localization
Open this publication in new window or tab >>Leukotriene C4 synthase: studies on oligomerization and subcellular localization
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Leukotrienes (LTs) are polyunsaturated fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 can be hydrolyzed to LTB4, or be conjugated with glutathione forming LTC4. LTC4 together with its metabolites LTD4 and LTE4, formed by amino acid removal from the glutathione moiety, constitute the cysteinyl LTs that are the active compounds of "slow reacting substance of anaphylaxis" (SRS-A). LTC4 and LTD4 are involved in several inflammatory conditions, e.g. asthma and allergic rhinitis. The conversion of LTA4 to LTC4 is catalyzed by an integral membrane protein, LTC4 synthase (LTC4S), localized on the endoplasmic reticulum (ER) and nuclear envelope. This 150 amino acid protein has four transmembrane helices and two hydrophilic loops oriented to the lumen side of the ER membrane. LTC4S belongs to a family of proteins called membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG).

We have shown that LTC4S and another MAPEG member, microsomal glutathione S-transferase (MGST)-1, interact and colocalize in transiently transfected cells. Coexpression decreased their catalytic activities indicating functional significance of the interaction between LTC4S and MGST1. LTC4S was demonstrated to form homo-oligomers in cell free assays using GST pulldown assays, as well as in living cells using bioluminescence resonance energy transfer (BRET) technique. When testing various truncated variants of LTC4S in BRET assays two hydrophobic regions were mapped as interaction domains: amino acids 6-27 and 114-135. GFP-fusion proteins containing the latter sequence also showed distinct ER/nuclear envelope localization and a minimal ER/nuclear envelope localization sequence was mapped to amino acids 117-132. In cell free assays we also demonstrated interactions between 5-LO, fivelipoxygenase activating protein (FLAP) and LTC4S. The second hydrophilic loop of LTC4S was found to be important for interaction with 5-LO, whereas the N-terminal part of LTC4S gave the strongest interaction with FLAP. LTC4 diminished the interaction between 5-LO and FLAP suggesting a feed-back regulatory mechanism. Our results concerning LTC4S oligomer formation and mapping of interaction domains may provide novel means to rational design of LTC4S inhibitors.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2005. 73 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 913
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-31123 (URN)16857 (Local ID)91-85299-23-5 (ISBN)16857 (Archive number)16857 (OAI)
Public defence
2005-10-14, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
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Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2014-06-13Bibliographically approved

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Svartz, JesperMagnusson, Karl-EricHammarström, SvenSöderström, Mats

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