Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

Abstract

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca 2+ -ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca 2+ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca 2+ -dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca 2+ -flush is mainly needed for the recruitment of 5LO to the membrane.Our results also showed that the addition of Ca 2+ -ions promoted binding of 5LO on the FLAPnanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LO in vitro.

Helical remodeling augments 5-lipoxygenase activity in the synthesis of proinflammatory mediators

“…This hypothesis is supported by the earliest activation studies of 5-LOX in the 1980s that established that the addition of calcium ( 36 ) and phospholipids ( 37 ) in cell-free assays enhanced 5-HPETE and LT production ( 38 ), respectively. More recently, just the addition of exogenous AA ( 39 ), or more interestingly competitive inhibitors ( 40 ), is sufficient for the translocation of 5-LOX to the membrane and FLAP, with the later hinting at membrane-binding determinants and FLAP-interacting motifs near the active site. We have potentially circumvented the requirement of these molecular activators by strategically removing the side chains required for restricting access to the catalytic machinery.…”