Modulation of Nanotube Formation by Structural Modifications of Sphingolipids

Abstract

Galactosylceramides (GalCers) containing nervonoyl (24:1Δ15(cis)) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (Kulkarni et al., 1995. Biophys. J. 69:1976–1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1(X-9). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1Δ15(cis) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25–30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1Δ15GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1Δ15Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1Δ15GalCer. 22:1Δ13GalCer dispersions consisted of long, smooth tubules (35–40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1Δ11GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30–35nm, with helical pitches of 80–90nm. 18:1Δ9GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation.