title: Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing 31P and 2H solid-state NMR spectroscopy.
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 (2)H and (31)P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 degrees 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 (S(CD)) were calculated from the dePaked (2)H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S(CD) profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S(CD) profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S(CD) 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 (31)P 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].
authors.affiliation: Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
authors.forename: Shadi
authors.initials: S
authors.lastname: Abu-Baker
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Qi
authors.forename: Justin
authors.initials: J
authors.lastname: Newstadt
authors.forename: Gary A
authors.initials: GA
authors.lastname: Lorigan
mesh_terms: D000143:Acids; D000595:Amino Acid Sequence; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010715:Phosphatidylglycerols / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry*; D010759:Phosphorus Isotopes; D011994:Recombinant Proteins / Q000737:chemistry; D049231:Saposins / Q000737:chemistry*
chemical_list: D000143:Acids; D008051:Lipid Bilayers; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D010759:Phosphorus Isotopes; D011994:Recombinant Proteins; D049231:Saposins; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C042511:1,2-dioleoylphosphatidylserine; D003903:Deuterium