Mary Ellen K. Salyan

Synthesis and characterization of lyso-GM3 (II3Neu5Ac lactosyl sphingosine), de-N-acetyl-GM3 (II3NeuNH2 lactosyl Cer), and related compounds

Various GM3 derivatives which are present in A431 cells have different effects on the activity of the EGF receptor kinase. In order to systematically study these effects, the following GM3 derivatives have been synthesized: de-N-acetyl-GM3 (D1), de-N-acetyl-lyso-GM3 (D2), lyso-GM3 (D3), de-N-acetyl-GM3 with N-acetylsphingosine (D4), and GM3 with N-acetylsphingosine (D3). A crucial step for the preparation of D1 is the use of mild alkaline conditions of hydrolysis under which the N-acetyl group of sialic acid is preferentially hydrolyzed. For the preparation of D3, conditions which allowed preferential N-acetylation of the amino group of the neuraminic acid moiety were devised, i.e., D2 was incorporated in a dipalmitoyl-phosphatidylcholine (dpPC) liposome in which the sphingosine moiety was protected and the amino group of neuraminic acid was N-acetylated with acetate and a water-soluble catalyst, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). When an aqueous micellar solution of D2 was treated with acetic anhydride and sodium hydrogencarbonate, N-acetylation occurred at the amino groups of both neuraminosyl and sphingosyl residues, yielding D5. The structures of these derivatives were verified by 1H-n.m.r. spectroscopy and mass spectrometry.

Synthesis and characterization of ganglioside GM3 derivatives: lyso-GM3, de-N-acetyl-GM3, and other compounds.

Publisher Summary This chapter discusses various methods developed for the synthesis of five G M3 derivatives, as based on (l) preferential hydrolysis of the N-acetyl group of sialic acid, (2) de-N-acylation of both N-acetylsialic acid and N-acylsphingosine, followed by selective N-acetylation of sialic acid by protection of the amino group of sphingosine in dpPC liposomes, and (3) preferential N-acetylation of the sphingosine amino group by catalytic N-acetylation in an aqueous micellar solution. Thus, the desired derivatives of G M3 (D l - D 5 ) can be prepared and their structures verified by proton magnetic resonance ( I HNMR) and mass spectrometry. The functional role of membrane gangliosides has been thought to be modulation of membrane proteins such as receptors and transporters, although the gangliosides themselves have been implicated as receptors for cell-cell and cell-microbe recognition. The procedures described in this chapter for G M3 can be applied to any ganglioside to give derivatives that may be useful for the study of the cell physiological and pharmacological effects of membrane gangliosides.

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of anti- glycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.