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UC Irvine UC Irvine Previously Published Works UC Irvine UC Irvine Previously Published Works Title Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence. Permalink https://escholarship.org/uc/item/2601m9d4 Journal Biophysical journal, 75(1) ISSN 0006-3495 Authors Bagatolli, LA Gratton, E Fidelio, GD Publication Date 1998-07-01 DOI 10.1016/s0006-3495(98)77517-4 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Biophysical Journal Volume 75 July 1998 331–341 331 Water Dynamics in Glycosphingolipid Aggregates Studied by LAURDAN Fluorescence L. A. Bagatolli,*# E. Gratton,# and G. D. Fidelio* *Departamento de Quı´mica Biolo´ gica-CIQUIBIC, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´ rdoba, Argentina and #Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, Illinois USA ABSTRACT We have characterized the fluorescence properties of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN) in pure interfaces formed by sphingomyelin and 10 chemically related glycosphingolipids (GSLs).1 The GSLs contain neutral and anionic carbohydrate residues in their oligosaccharide chain. These systems were studied at temperatures below, at, or above the main phase transition temperature of the pure lipid aggregates. The extent of solvent dipolar relaxation around the excited fluorescence probe in the GSLs series increases with the magnitude of the glycosphingolipid polar headgroup below the transition temperature. This conclusion is based on LAURDAN’s excitation generalized polarization (GPex) and fluores- cence lifetime values found in the different interfaces. A linear dependence between the LAURDAN GPex and the intermo- lecular spacing among the lipid molecules was found for both neutral and anionic lipids in the GSLs series. This relationship was also followed by phospholipids. We conclude that LAURDAN in these lipid aggregates resides in sites containing different amounts of water. The dimension of these sites increases with the size of the GSLs polar headgroup. The GP function reports on the concentration and dynamics of water molecules in these sites. Upon addition of cholesterol to Gg4Cer, the fluorescence behavior of LAURDAN was similar to that of pure cerebrosides and sphingomyelin vesicles. This observation was attributed to a change in the interfacial hydration as well as changes in the shape and size of the Gg4Cer aggregates in the presence of cholesterol. After the addition of cholesterol to gangliosides, the changes in the LAURDAN’s spectral parameters decrease progressively as the polar headgroup of these lipids becomes more complex. This finding suggests that the dehydration effect of cholesterol depends strongly on the curvature radius and the extent of hydration of these lipid aggregates. In the gel phase of phrenosine, GalCer, Gg3Cer, sulfatide, and sphingomyelin, the excitation red band (410 nm) of LAURDAN was reduced with respect to that of LAURDAN in the gel phase of pure phospholipids. This observation indicates a local environment that interacts differently with the ground state of LAURDAN in GSLs when compared with LAURDAN in phospholipids. INTRODUCTION Glycosphingolipids (GSLs) are ubiquitous components of (Pascher, 1976; Boggs, 1987; Curatolo, 1987). Neutral animal cell membranes that are particularly abundant in the GSLs and gangliosides are implicated in cell recognition nervous system (Tettamanti, 1988). GSLs are involved in a and signal transduction processes (Tettamanti, 1988; Mag- large variety of physiologically important phenomena (Tet- gio, 1994). These lipids occur in relatively large amounts in tamanti, 1988; Maggio, 1994). For example, the simple specialized membranes such as myelin (Norton and Cam- polar headgroup GSLs play a role in stabilizing membranes mer, 1984; Ledeen, 1989; Yu and Saito, 1989; Maggio et (Pascher, 1976), perhaps through interlipid hydrogen bonds al., 1986). In particular, gangliosides are enriched in nerve cells where they represent between 2% and 10% of the total lipids. On the neuronal surface .30% of the sialic acid Received for publication 22 December 1997 and in final form 7 April 1998. content is supplied by these lipids (Maggio, 1994; Curatolo, Address reprint requests to Dr. Luis A. Bagatolli, Laboratory for Fluores- 1987). However, the effects of these lipids on the structure cence Dynamics, 184 Loomis Lab., 1110 West Green, Urbana, IL 61801. Tel.: (217) 244-5620; Fax: (217) 244-7187; E-mail: [email protected]. and function of cell membranes remain obscure. uiuc.edu. Previous work from our laboratory has elucidated several This work is dedicated to the loving memory of Prof. Gregorio Weber. biophysical properties of a series of chemically related Abbreviations used: GSLs, glycosphingolipids; Cer, ceraminde: (N-acyl- GSLs ranging from simple cerebrosides to complex gan- sphingosine); DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DSC, gliosides (Maggio, 1994; Maggio et al., 1981, 1986). Gan- b 3 9 differential scanning calorimetry; GalCer, Gal 1 1 Cer; Gg3Cer (asialo gliosides contain one or more sialic acids attached to a b 3 b 3 b 3 9 GM2), Gal-NAc 1 4Gal 1 4Glc 1 1 Cer; Gg4Cer (asialo GM1), b 3 b 3 b 3 b 3 9 a3 neutral oligosaccharide chain resulting in a series of polar Gal 1 3Gal-NAc 1 4Gal 1 4Glc 1 1 Cer; GM3, NeuAc2 b b 3 9 b 3 4 a b 3 headgroups of different complexity. The hydrophilic por- 3Gal 1 4Glc 1 1 Cer; GM2, Gal-NAc 1 4Gal(3 2 NeuAc) 1 b 3 9 b 3 b 3 4 a b 3 4Glc 1 1 Cer; GM1, Gal 1 3Gal-NAc 1 4Gal(3 2 NeuAc) 1 tion of these gangliosides reaches a length similar to that of b 3 9 a3 b 3 b 3 4Glc 1 1 Cer; GD1a, NeuAc2 3Gal 1 3Gal-NAc 1 4Gal the hydrocarbon portion and, in turn, dramatically influ- (342aNeuAc)b134Glcb1319Cer; G , NeuAc2a33Galb13 T1b ences the surface, thermotropic, and topological properties 3GalNAcb1 34Gal(342aNeuAc842aNeuAc)b134Glcb1319Cer; LAURDAN, 6-dodecanoyl-2-dimethylamine-naphthalene; Phre, of ganglioside micelles (Maggio, 1994; Maggio et al., 1981, phrenosine: Galb1319(N-[a-OH]acyl-sphingosine); Sphin, egg yolk 1985, 1986, 1988; Fidelio et al., 1986). The number and 3 b 3 9 sphingomyelin; Sulf, HSO3 3Gal 1 1 Cer. type of carbohydrates in the polar headgroup of these GSLs © 1998 by the Biophysical Society also have a direct influence on the extent of the interfacial 0006-3495/98/07/331/11 $2.00 hydration or micropolarity (Montich et al., 1985 and 1988) 332 Biophysical Journal Volume 75 July 1998 and on the polarity gradient taken from the polar headgroup related to the number and motional freedom of water mol- to the hydrophobic core of the lipid aggregate (Bagatolli et ecules around the fluorescent group (Parasassi et al., 1991). al., 1995). (The term aggregate was used in the text as a In a recent study by two-photon fluorescence microscopy, general term. In Table 3 the shape of the aggregates is heterogeneous GP distributions in pure DLPC and DOPC specified for each lipid.) vesicles in the liquid crystalline state were reported (Paras- To study the polarity of the lipid interfaces, we used sasi et al., 1997). This heterogeneity was found in individual LAURDAN, which is an amphiphilic fluorescence probe vesicles and was attributed to a distribution of different synthesized by Prof. G. Weber to study the dipolar relax- LAURDAN sites, characterized by a different number of ation processes (Weber and Farris, 1979). In solvents of adjacent water molecules. high polarity, LAURDAN shows a considerable shift of its In a previous report we described the fluorescence prop- emission spectrum to longer wavelengths due to dipolar erties of LAURDAN in pure GM1 and Gg4Cer aggregates relaxation processes (Parasassi et al., 1991). When the local and in mixed aggregates of DPPC/Gg4Cer, and we com- environment of LAURDAN is a phospholipid phase, the pared these results with the calorimetric data measured for emission depends strongly on the packing of the lipid these interfaces (Bagatolli et al., 1997). For pure GM1 and chains. At temperatures below the phase transition (gel Gg4Cer dispersions, we demonstrated that LAURDAN ex- state) the emission maximum is near 440 nm. At tempera- hibits a progressive red shift of its emission below the tures above the phase transition (liquid crystalline state) the gel-to-liquid crystalline transition temperature depending on emission maximum is red-shifted to ;490 nm (Parasassi et the size of the polar headgroup of these GSLs. This effect al., 1991; Parasassi and Gratton, 1995). The steady-state was attributed to an increase in the water content of these fluorescence parameter known as the generalized polariza- interfaces compared to phospholipids (Bagatolli et al., tion (GP) quantitatively relates these spectral changes by 1997). LAURDAN shows a high partition coefficient into taking into account the relative fluorescence intensities of lipid aggregates and is virtually insoluble in water. The the blue and red edge regions of the emission and excitation fluorescent moiety of LAURDAN has been reported to be spectra, respectively (Parasassi et al., 1991; Parasassi and located at the hydrophilic-hydrophobic interface of the phos- Gratton, 1995). For phospholipids, a characteristic GP value pholipid bilayer (Parasassi et al., 1991). In GSLs the loca- was found for the gel and liquid crystalline phases. How- tion of the LAURDAN fluorescent group is in the hydrophilic- ever, the observation of a GP value intermediate between hydrophobic interface near the upper part of the ceramide the gel
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