Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity

Abstract

In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.

Altered triglyceride and phospholipid metabolism predates the diagnosis of gestational diabetes in obese pregnancy

“…Alterations in these membrane properties is consistent with general evidence that membrane composition affects protein activity. [53][54][55] There are recognised relationships between membrane composition and protein activity in hyperglycaemia. 56 In addition certain fatty acids, DG and ceramide have been shown to activate certain serine kinases that weaken insulin signalling pathways and therefore cause insulin resistance.…”

Perspectives on the membrane fatty acid unsaturation/pacemaker hypotheses of metabolism and aging

“…Increases in the unsaturation of membranes change the phase behavior of membranes, increasing the propensity of membranes to form hexagonal (H II ) phases (Epand et al, 1991;Furse et al, 2015;Yang et al, 2005). Increases in H II phase propensity increase the activity of multiple classes of signaling proteins due to increasing recruitment of these signaling proteins into membranes (Escribá et al, 1995;Martinez et al, 2005;Vögler et al, 2004;Yang et al, 2005).…”

Relationship between the lipid composition of maternal plasma and infant plasma through breast milk

“…Approximately 12.2% of the total (80% of the DG fraction) are m/z values associated with DGs that have lost one equivalent of water. Although this is well known to occur to ordinary DGs in the ionisation/gas phase used in mass spectrometry (Furse et al 2015b), this fragment can also been ascribed to the TGs exposed to the same conditions. It is therefore not clear what proportion of the DG fraction can be ascribed to DGs of the sample, those produced by lipolytic activity on TGs during handling, or to the TGs of the milk as produced.…”