Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing P and H solid-state NMR spectroscopy |
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Authors: | Shadi Abu-Baker Justin Newstadt |
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Affiliation: | a Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA b Division and Program of Human Genetics, Cincinnati Children's Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA |
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Abstract: | Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using 2H and 31P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 °C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (SCD) were calculated from the dePaked 2H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The SCD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. SCD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the SCD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The 31P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1]. |
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Keywords: | Sap C, Saposin C SCD, Molecular Order Parameters CSA, Chemical Shift Anisotropy GCase, Glucosylceramidase CL, Cardiolipin DOPG, Dioleoylphosphatidylglycerol DOPS, Dioleoylphosphatidylserine PC, Phosphoatidyecholines PG, Phosphatidylglycerol PS, Phosphatidylserine DSPG-d70, Distearoyl-d70-phosphatidylglycerol DMPC, Dimyristylphosphatidylcholine HCL, Hydrochloric Acid TFE, 2,2,2 Triflouroethanol PLL, Poly( smallcaps" >l-lysine) NMR, Nuclear Magnetic Resonance CP-MAS, Cross-Polarization Magic-Angle Spinning HPLC, High Performance Liquid Chromatography MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry MLVs, Multilamellar vesicles |
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