i(3)so3-galactosylceramide and globotriaosylceramide

We have analyzed the interaction of adamantyl Gb(3) (adaGb(3)), a semi-synthetic soluble analog of Gb(3), with HIV-1 surface envelope glycoprotein gp120. In this analog, which was orginally designed to inhibit verotoxin binding to its glycolipid receptor, Gb(3), the fatty acid chain is replaced with a rigid globular hydrocarbon frame (adamantane). Despite its solubility, adaGb(3) forms monolayers at an air-water interface. Compression isotherms of such monolayers demonstrated that the adamantane substitution resulted in a larger minimum molecular area and a more rigid, less compressible film than Gb(3). Insertion of gp120 into adaGb(3) monolayers was exponential whereas the gp120/Gb(3) interaction curve was sigmoidal with a lag phase of 40 min. Adding cholesterol into authentic Gb(3) monolayers abrogated the lag phase and increased the initial rate of interaction with gp120. This effect of cholesterol was not observed with phosphatidylcholine or sphingomyelin. In addition, verotoxin-bound adaGb(3) or Gb(3) plus cholesterol was recovered in fractions of comparable low density after ultracentrifugation through sucrose-density gradients in the presence of Triton X-100. The unique biological and physico-chemical properties of adaGb(3) suggest that this analog may be a potent soluble mimic of Gb(3), providing a novel concept for developing GSL-derived viral fusion inhibitors.

Topics: Adamantane; Animals; Cattle; Centrifugation, Density Gradient; Cholesterol; Erythrocytes; HIV Envelope Protein gp120; Humans; Lipids; Models, Structural; Pressure; Protein Binding; Protein Structure, Tertiary; Serum Albumin, Bovine; Solubility; Substrate Specificity; Sulfoglycosphingolipids; Surface Tension; Trihexosylceramides

Topics: alpha-Galactosidase; beta-Galactosidase; Ceramides; Cerebroside-Sulfatase; Enzyme Activators; G(M1) Ganglioside; G(M2) Activator Protein; G(M2) Ganglioside; Galactosylgalactosylglucosylceramidase; Glucosylceramidase; Glucosylceramides; Glycoproteins; Glycosphingolipids; Hydrolases; Lysosomes; Protein Precursors; Proteins; Saposins; Sphingolipid Activator Proteins; Sulfoglycosphingolipids; Trihexosylceramides

The activator protein for the enzymatic hydrolysis of sulfatide, ganglioside GM1, and globotriaosylceramide was purified from human kidney, brain, and urine. As far as they could be assayed, these three activities cochromatographed during all steps, indicating that they are due to the same protein. This result was corroborated by immunochemical comparison of individually purified activator preparations. In contrast, the activator for ganglioside GM2 hydrolysis could clearly be separated from the other activities. Kinetic data were determined for the interaction of the sulfatide activator with the different glycolipids and hydrolases.

Topics: Brain Chemistry; Chromatography, DEAE-Cellulose; Chromatography, High Pressure Liquid; Enzyme Activation; G(M1) Ganglioside; Globosides; Glycosphingolipids; Humans; Hydrolysis; Immunoelectrophoresis, Two-Dimensional; Isoelectric Focusing; Kidney; Sulfoglycosphingolipids; Trihexosylceramides

The conformations of the neutral glycosphingolipid, globotriaosylceramide, and of the methyl ester of GM1-ganglioside have been predicted by energy-minimization techniques and compared with those previously obtained for GM1- and GM2-ganglioside. A triple-binding-domain model is put forward to explain known specificities of binding between these glycosphingolipids and activator proteins. This model suggests that hydrophobic interactions, electrostatic interactions and hydrogen-bonding between sugar residues are important. The model is discussed in relation to previous studies on the effect of chemical modification of glycosphingolipids on their ligand properties.

Topics: G(M1) Ganglioside; Globosides; Glycoproteins; Glycosphingolipids; Models, Molecular; Protein Conformation; Proteins; Saposins; Sphingolipid Activator Proteins; Sulfoglycosphingolipids; Trihexosylceramides