The alpha,alpha-(1-->1) linkage of trehalose is key to anhydrobiotic preservation

Abstract

This study compares the efficacy of six disaccharides and glucose for the preservation of solid supported lipid bilayers (SLBs) upon exposure to air. Disaccharide molecules containing an alpha,alpha-(1-->1) linkage, such as alpha,alpha-trehalose and alpha,alpha-galacto-trehalose, were found to be effective at retaining bilayer structure in the absence of water. These sugars are known to crystallize in a clam shell conformation. Other saccharides, which are found to crystallize in more open structures, did not preserve the SLB structure during the drying process. These included the nonreducing sugar, sucrose, as well as maltose, lactose, and the monosaccharide, glucose. In fact, even close analogs to alpha,alpha-trehalose, such as alpha,beta-trehalose, which connects its glucopyranose rings via a (1-->1) linkage in an axial, equatorial fashion, permitted nearly complete delamination and destruction of supported bilayers upon exposure to air. Lipids with covalently attached sugar molecules such as ganglioside GM1, lactosyl phosphatidylethanolamine, and glucosylcerebroside were also ineffective at preserving bilayer structure. The liquid crystalline-to-gel phase transition temperature of supported phospholipid bilayers was tested in the presence of sugars in a final set of experiments. Only alpha,alpha-trehalose and alpha,alpha-galacto-trehalose depressed the phase transition temperature, whereas the introduction of other sugar molecules into the bulk solution caused the phase transition temperature of the bilayer to increase. These results point to the importance of the axial-axial linkage of disaccharides for preserving SLB structure.

Figure 1

Structure of the mono- and disaccharides employed in this study: (a) α,α-trehalose, (b) α,β-trehalose, (c) maltose, (d) α,α-galacto-trehalose, (e) sucrose, (f) lactose, and (g) glucose.

Figure 2

The dehydration of supported phospholipid membranes. Upper-Right Panel: In the absence of a lipopreservative the thin lipid film spontaneously reorganizes and delaminates from the solid support. Lower-Right Panel: The presence of a lipoprotectant suppresses damage and delamination.

Figure 3

(a) A FRAP curve for a fully hydrated POPC bilayer. Inserts: Fluorescence micrographs of the same bilayer showing the laser bleach spot both before and after recovery (red circles). (b) A fluorescence micrograph of the POPC bilayer after exposure to air.

Figure 4

Images of supported POPC lipid membranes after drying from 20 w/w % solutions of (a) α,α-trehalose, (b) α,β-trehalose, (c) maltose, (d) α,α-galacto-trehalose, (e) sucrose, (f) lactose, and (g) glucose.

Figure 5

Images of rehydrated POPC lipid membranes exposed to 20 w/w % solutions of (a) α,α-trehalose, (b) α,β-trehalose, (c) maltose, (d) α,α-galacto-trehalose, (e) sucrose, (f) lactose, and (g) glucose.

Figure 6

Structures of three glycolipids: (a) GM₁, (b) lactosyl-PE, and (c) glucosylcerebroside.

Figure 7

Fluorescence micrographs of dehydrated POPC lipid membranes containing: (a) 10 mol % GM₁, (b) 10 mol % lactosyl-PE, and (c) 10 mol % glucosylcerebroside.

Figure 8

Plots of the relative diffusion constant of DMPC bilayers vs. temperature in the presence of 20 w/w % sugar solutions: (a) control DMPC membrane in PBS buffer, (b) α,α-trehalose, (c) maltose, (d) α,β-trehalose, (e) α,α-galacto-trehalose, (f) sucrose, (g) lactose, and (h) glucose.