Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids.

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A2, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (Vmax∼15.5 µmol/min/mg; Km∼83 µM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall-deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.

The YhhN protein of Legionella pneumophila is a Lysoplasmalogenase.

Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein (LpYhhN), and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 µmol/min/mg protein and a Km of 45 µM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.

Linkages between mitochondrial lipids and life history in temperate and tropical birds.

Temperate birds tend to have a fast pace of life and short life spans with high reproductive output, whereas tropical birds tend to have a slower pace of life, invest fewer resources in reproduction, and have higher adult survival rates. How these differences in life history at the organismal level are rooted in differences at the cellular level is a major focus of current research. Here, we cultured fibroblasts from phylogenetically paired tropical and temperate species, isolated mitochondria from each, and compared their mitochondrial membrane lipids. We also correlated the amounts of these lipids with an important life history parameter, clutch size. We found that tropical birds tended to have less mitochondrial lipid per cell, especially less cardiolipin per cell, suggesting that cells from tropical birds have fewer mitochondria or less inner mitochondrial membrane per cell. We also found that the mitochondria of tropical birds and the species with the smallest clutch sizes had higher amounts of plasmalogens, a lipid that could serve as an antioxidant. Overall, our findings are consistent with the idea that there are underlying molecular and cellular physiological traits that could account for the differences in whole-animal physiology between animals with different life histories.

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 K(m) values of ∼50 µm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) 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 (K(i) ∼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.

beta-Glucocerebrosidase activity in the stratum corneum of house sparrows following acclimation to high and low humidity.

Skin is an important avenue of water loss in terrestrial birds, so environmental conditions that necessitate water conservation should favor physiological mechanisms that reduce cutaneous water loss (CWL). Skin resistance to CWL is conferred by a barrier of lipid molecules located in the stratum corneum (SC), the outer layer of the epidermis. In mammals, SC barrier function depends on the conversion of cerebrosides to ceramides by the enzyme beta -glucocerebrosidase ( beta -GlcCer'ase). Avian SC contains both cerebrosides and ceramides, suggesting that observed plasticity in CWL may be mediated by changes in beta -GlcCer'ase activity and resultant SC lipid composition. We tested the hypothesis that changes in ambient humidity would alter beta -GlcCer'ase activity by acclimating house sparrows (Passer domesticus) to either dry (6.5 g H(2)O m(-3) absolute humidity) or humid (31 g H(2)O m(-3)) conditions for 5 and 21 d at 30 degrees C and then measuring beta -GlcCer'ase activity from SC homogenates. Our results provide the first characterization of beta -GlcCer'ase activity in any nonmammalian vertebrate. Relative to nonacclimated controls, both dry- and humid-acclimated sparrows had significantly elevated beta -GlcCer'ase activity at 21 d postacclimation. Across individuals, we observed negative correlations between beta -GlcCer'ase activity and both CWL and SC ceramide content. Although dry- and humid-acclimated sparrows did not differ in beta -GlcCer'ase activity, these results are consistent with our findings that both humidity treatments caused a reduction in CWL and similar changes in SC lipid composition. Our results demonstrate physiological plasticity in CWL and provide tentative support for a role of beta -GlcCer'ase in mediating this response.