Electrospray Ionization Mass Spectrometry and Exogenous Heavy Isotope-labeled Lipid Species Provide Detailed Information on Aminophospholipid Acyl Chain Remodeling

Abstract

Mammalian cells maintain the phospholipid compositions of their different membranes remarkably constant. Beside de novo synthesis, degradation, and intracellular trafficking, acyl chain remodeling plays an important role in phospholipid homeostasis. However, many key details of this process remain unresolved, largely because of limitations of existing methodologies. Here we describe a novel approach that allows one to study metabolism of individual phospholipid species in unprecedented detail. Forty different phosphatidylethanolamine (PE) or -serine (PS) species with a deuterium-labeled head group were synthesized and introduced to BHK21 or HeLa cells using cyclodextrin-mediated transfer. Their metabolism was then monitored in detail by electrospray ionization mass spectrometry. Atypical PE and PS species were rapidly remodeled at both sn1 and sn2 position, yielding a molecular species profile similar to that the endogenous PE and PS. In contrast, remodeling of exogenous species identical or similar to major endogenous ones was more limited and much slower. Major differences in remodeling pathways and kinetics were observed between species within a class, as well as between corresponding PE and PS species. These data along with those obtained with pharmacological inhibitors strongly suggest that multiple lipid class-specific A-type phospholipases and acyl transferases are involved in aminophospholipid remodeling. In conclusion, the approach described here provides highly detailed information on remodeling of exogenously added (amino)glycerophospholipids and should thus be very helpful when elucidating the proteins and processes maintaining molecular species homeostasis.

Substrate Efflux Propensity Plays a Key Role in the Specificity of Secretory A-type Phospholipases

“…It is tempting to speculate that substrate efflux propensity could be a critical factor regulating homeostatic phospholipases as well. Supporting this suggestion, exogenous phospholipids with short/less hydrophobic chains were rapidly hydrolyzed in cultured cells (20). The high throughput approach used here provides a powerful tool to study the factors underlying the specificity of homeostatic PLA 2 s, because it allows one to determine the rates of hydrolysis of multiple molecular species in very complex mixtures as found in biological membranes.…”

“…However, it is not clear how those PLAs are regulated (e.g. how they can effectively hydrolyze particular phospholipid species while leaving similar ones intact) (20). It is tempting to speculate that substrate efflux propensity could be a critical factor regulating homeostatic phospholipases as well.…”

“…In conclusion, the relative contributions of the PLA active site accommodation and substrate efflux propensity to phospholipid hydrolysis are poorly understood at present. Such information is necessary to fully understand the mode of action of PLAs as well as to establish why homeostatic PLAs preferentially hydrolyze certain phospholipid species (20), thus helping to maintain the characteristic phospholipid composition of cellular membranes.…”

Introduction of phospholipids to cultured cells with cyclodextrin

“…Previous studies have shown that certain cyclodextrins can be used to transfer hydrophobic lipids to cells ( 5,10,11 ), but the parameters affecting transfer effi ciency and the possible deleterious effects on cells have not been systematically studied. In the present study, we studied these issues in detail and the key result was that with most phospholipids the amount transferred to cells increased systematically with increasing i ) donor vesicle concentration, ii ) time, and iii ) me ␤ -CD concentration.…”

“…Baby hamster kidney (BHK21) and HeLa cells were grown as previously ( 5,18 ). Human skin fi broblasts were grown in RPMI medium containing 15% FBS, 200 U/ml penicillin, 200 µg/ml streptomycin, 2 mM L-glutamine, and 0.1 mM unessential amino acids.…”