Biochemistry zyxwvu 1992,31, zyxwvu 9045-9055 9045 Microstructural Polymorphism in Bovine Brain Galactocerebroside and Its Two Major Subfractionst Douglas D. Archibald* Department of Chemistry, University of Washington, Seattle, Washington 98195 Paul Yager' Molecular Bioengineering Program, Center zyxwvut for Bioengineering, FL-20, University of Washington, Seattle, Washington 98195 Received December 2, 1991; Revised Manuscript Received June 1, 1992 ABSTRACT: Aqueous suspensions of either brain galactocerebrosides or its subfraction consisting of a- hydroxyacyl galactocerebrosides are mainly composed of vesicles or granular lipid with occasional mul- tilamellar sheets. In aqueous media the other subfraction consisting of non-hydroxyacyl galactocerebrosides forms some helical structures, but most of the lipid remains as granules or vesicles. It is demonstrated that thermal cycling of non-hydroxyacyl galactocerebrosides in polar nonaqueous solvents can greatly enhance the degree of conversion to helical ribbons about 100 nm in diameter. These structures appear to be a stable dehydrated crystalline form of this lipid and are morphologically similar to helical microstructures produced by a few synthetic lipids. On the other hand, similar treatment of unfractionated bovine brain cerebroside and its a-hydroxy fatty acyl subfraction quantitatively produces straight needles that appear to be cochleate cylinders. While their dimensions depend on formation conditions, a typical suspensionhas uniform particles with diameters close to 100 nm and lengths variable from one to a few hundred micrometers. This is the first report demonstrating the quantitative formation of crystalline high axial ratio microstructures from complex mixtures of natural lipids. The different microstructures formed by the two components appear related to the various forms of lipid deposits occurring in lipid storage diseases. The similarity of these "synthetic" microstructures to biological structures in which they are found (such as myelin and intestinal brush border microvilli) strengthens the possibility that galactocerebrosides have a role in stabilizing cylindrical biological structures. Recently there has been significant interest in regular high axial ratio microstructures that spontaneously self-organize. The research has focused on synthetic or semisynthetic two- chain amphiphiles that, when dispersed in aqueous media, form bilayer rods, tubules, or helices rather than liposomes (Yager et al., 1992). Technological interest in high axial ratio lipid microstructures was sparked by experiments on several synthetic systems that form hollow bilayer tubules or helices; the lipids include lecithins containing diacetylene moieties in both acyl chains (Georger et al., 1987; Yager zyxwvut & Schoen, 1984; Yager et al., 1985), surfactants in which a glutamic or aspartic acid residue is substituted with two alkyl chainsand an alkylammoniumchain (Nakashima et al., 1984, 1985), N-octylaldonamide single-cbain surfactants (Fuhrhop et al., 1987, 1988), amphiphiles containing a headgroup of oligoglutamic acid (Yamada et al., 1984), and perhaps also mixturesof 1,2-dimyristoyl-sn-glycero-3-phosphocholine and its chemical degradation products (Servuss, 1988). In all these tubular, helical, and rod-like microstructures, the bilayer is believed to be in a crystalline state, perhaps one with extremely long range helical order (Blechner et al., 1990; Rhodes et al., 1987, 1988). While several theoretical approaches toward predicting the structure of tubules and helices have been made (Chappell & Yager, 1991a,b,c; de Gennes 1987; Helfrich, 1986; Helfrich & Prost, 1988), none of these models allows a detailed specific prediction as 7 This work was supported by National Science Foundation Grant * To whom correspondence should be addressed. CTS-88 15027 (P.Y.). Current address: School of Chemistry, University of Bath, Claver- ton Down, Bath BA2 7AY, England. 0006-2960/92/043 1-9045$03.00/0 to the nature of the molecular packing in the tubule or helix bilayer, zyxwv so there is no a priori method for either picking or designing a lipid that might form tubular or helical micro- structures. Promising applications may exist for high axial ratio lipid microstructures (Rudolph et al., 1988; Schnur et al., 1987), and therefore there is interest in finding novel methods of preparing microstructures, as well as microstructures with new chemical properties. This work examines conditions of formation and the properties of microstructures formed by biologically derived cerebrosides. Cerebrosides are involved in a number of high axial ratio biological structures such as myelin, and moreover it forms the anisotropic deposit in the lipid storagediseases globoid-cell leukodystrophy (GLD) (Su- zuki & Suzuki, 1989) and Gaucher's disease (Lee et al., 1973). The process of formation of various kinds of GLD deposits is not completely understood. The cerebroside that forms the GLD deposits is referred to as galactosylceramideor galactocerebroside (Gal-Cer). Cere- broside itself is a complex class of lipids characterized by a sphingosine backbone with an amide-linked long-chain fatty acid and a single glycosidic-linked sugar residue (see Figure 1). Adult human brain cerebrosides are almost exclusively galactose-containing with more than 95% CIS-sphingosine [D(+)-erythro- 1,3-dihydroxy-2-amin0-4-trans-octade- cene] . The remainder is mostly CIE-dihydrosphingosine and a much smaller amount of C16-sphingosine. The amide-linked fatty acids are generally long (c2&26) and not polyunsat- urated, and approximately two-thirds are a-hydroxylated. For bovine brain, the acyl composition of the extract has been reported (Johnston & Chapman, 1988). Compared 0 1992 American Chemical Society