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UF CHM 6304 - BBA 2005 Lorigan SapC 31P and 2H NMR

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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 spectroscopyIntroductionMaterials and methodsMaterialsSap C preparation and purificationNMR sample preparationNMR spectroscopyNMR data analysisResults and discussion2H NMR study of sap C interacting with a mixed DOPG: DOPS bilayer31P NMR study of sap C interacting with DOPG and DOPS binary mixed bilayersAcknowledgementSupplementary dataReferencesStructural changes in a binary mixed phospholipid bilayer of DOPG andDOPS upon saposin C interaction at acidic pH utilizing31P and2H solid-stateNMR spectroscopyShadi Abu-Bakera, Xiaoyang Qib, Justin Newstadta, Gary A. Lorigana,*aDepartment of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USAbDivision and Program of Human Genetics, Cincinnati Children’s Hospital Medical Center and University of Cincinnati College of Medicine, Cincinnati,OH 45229, USAReceived 15 July 2005; received in revised form 7 September 2005; accepted 12 September 2005Available online 10 October 2005AbstractSaposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates thehydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order tobetter understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine(DOPS) (5:3 ratio) and Sap C were investigated using2H and31P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 -CatpH4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. Themolecular order parameters (SCD) were calculated from the dePaked2H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The SCDprofiles indicate that the addition of Sap C to the negatively chargedphospholipids is concentration dependent. SCDprofiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order.However, the SCDprofiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half ofthe 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 andDOPS mixed bilayers. The31P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and thespectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups ofboth acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree withthe 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].D 2005 Elsevier B.V. All rights reserved.Keywords: Saposin C; Dioleoylphosphatidylglycerol; Dioleoylphosphatidylserine; Solid-state NMR spectroscopy; Molecular order parameter; Mixed bilayer1. IntroductionSaposins are a family of a small 80 amino acid heat stableglycoproteins that are essential for the in vivo hydrolyticactivity of several lysosomal enzymes in the catalytic pathwayof glycosphingolipids [2,3]. Four members of the saposinsfamily, A, B, C and D are proteolytically derived from a singleprecursor protein, named prosaposin [4,5]. Saposin C (Sap C)is known to stimulate the catalytic activity of the lysosomalenzyme Glucosylceramidase (GCase) and thereby to facilitatethe hydrolysis of glucosylceramide to ceramide and glucose[6,7]. In addition, the presence of both Sap C and acidicphospholipids such as phosphatidylserine (PS) is required for0005-2736/$ - see front matter D 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamem.2005.09.014Abbreviations: 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, Dimyristylphospha-tidylcholine; HCL, Hydrochloric Acid; TFE, 2,2,2 Triflouroethanol; PLL,Poly(l-lysine); NMR, Nuclear Magnetic Resonance; CP-MAS, Cross-Polari-zation Magic-Angle Spinning; HPLC, High Performance Liquid Chromatog-raphy; MALDI-TOF, Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry; MLVs, Multilamellar vesicles* Corresponding author. Tel.: +1 513 529 3338; fax: +1 513 529 5715.E-mail address: [email protected] (G.A. Lorigan).Biochimica et Biophysica Acta 1717 (2005) 58 – 66http://www.elsevier.com/locate/bbafull activity of GCase [8–10]. Deficiency in either Sap C orGCase leads to different variant form s of Gaucher’s disease[9–12]. Gaucher’s disease is the most common genetic diseaseaffecting Jewish people of Eastern European ancestry and is themost common lipid-storage disorder [13]. In addition, thedisease course is quite variable, ranging from no outwardsymptoms to severe disability and death [13].Under acidic pH conditions, Sap C is thought todestabilize the phospholipid membrane and facilitate theassociation of GCase with acidic phospholipid [12,14]. Thebinding of Sap C to phospholipid vesicles is a pH-controlledreversible process [14]. Since Sap C is a lysosomal proteinand pH gradients occur in lysosomes in vivo, the degradationof lipids in the lysosome is proposed to be switched on andoff by Sap C [14]. The three-dimensional structure of Sap Chas been determined via solution NMR spectroscopy and SapC was found to have five a-helices [14]. The negativelycharged electrostatic surface of Sap C, resulting from itseleven Glu residues (See Fig. 1), needs to be partiallyneutralized to promote membrane binding [14]. In addition,the solution NMR structure of human Sap C in a detergentenvironment has been determined [15]. Using fluorescenceemission spectroscopy and quenching analysis, a hypotheticalmodel of membrane interactions with Sap C was proposed byXiaoyang Qi and coworkers [1]. In this model, theamphipathic amino terminus of helix 1 and the carboxylterminus of


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