Purification, Identification, and Cloning of Lysoplasmalogenase, the Enzyme That Catalyzes Hydrolysis of the Vinyl Ether Bond of Lysoplasmalogen*

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent Km values of ∼50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent Vm values of 24.5 and 17.5 μmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (Ki ∼20 μm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells

Systemic TAK-242 prevents intrathecal LPS evoked hyperalgesia in male, but not female mice and prevents delayed allodynia following intraplantar formalin in both male and female mice: The role of TLR4 in the evolution of a persistent pain state

OBJECTIVE: Pain resulting from local tissue injury or inflammation typically resolves with time. Frequently, however, this pain may unexpectedly persist, becoming a pathological chronic state. Increasingly, the innate and adaptive immune systems are being implicated in the initiation and maintenance of these persistent conditions. In particular, Toll-like receptor 4 (TLR4) signaling has been shown to mediate the transition to a persistent pain state in a sex-dependent manner. In the present work, we explored this contribution using the TLR4 antagonist, TAK-242. METHODS: Male and female C57Bl/6 mice were given intravenous (IV), intrathecal (IT), or intraperitoneal (IP) TAK-242 prior to IT delivery of lipopolysaccharide (LPS), and tactile reactivity was assessed at regular intervals over 72-hours. Additional groups of mice were treated with IP TAK-242 prior to intraplantar formalin, and flinching was monitored for 1-hour. Tactile reactivity was assessed at 7-days after formalin delivery. RESULTS: LPS evoked TNF release from male and female macrophages and RAW267.4 cells, which was blocked in a concentration dependent fashion by TAK-242. In vivo, IT LPS evoked tactile allodynia to a greater degree in male than female mice. TAK-242, given by all routes, prevented development of IT LPS-induced tactile allodynia in male animals, but did not reverse their established allodynia. TLR4 deficiency and TAK-242 treatment attenuated IT LPS-induced allodynia in male, but not female mice. In the formalin model, pre-treatment with TAK-242 did not affect Phase 1 or Phase 2 flinching, but prevented the delayed tactile allodynia in both male and unexpectedly in female mice (Phase 3). CONCLUSIONS: Together, these results suggest that TAK-242 is a TLR4 antagonist that has efficacy after systemic and intrathecal delivery and confirms the role of endogenous TLR4 signaling in triggering the development of a delayed allodynia in both male and female mice