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

Abstract

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 125 I-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.

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

“…Previous studies on the biological roles of Leishmania glycosphingolipid (GSL) antigens demonstrated that monoclonal antibodies (mAbs) ST-3, ST-4 and ST-5, specifically directed at amastigote L. (L.) amazonensis GSLs, inhibited macrophage invasion by amastigotes by as much as 80%, suggesting that the epitope recognized by these mAbs present in surface GSLs is important in mediating binding of L. (L.) amazonensis amastigotes to macrophages (8,9).…”

The Immunologically Protective P-4 Antigen ofLeishmania Amastigotes

“…Metabolic differences are known to exist between the promastigote and amastigote stages (11)(12)(13)(14); in addition, several leishmanial molecules have been demonstrated to be up-regulated or specifically associated with the amastigote stage. These include specific glycosphingolipids, parasite lysosomal enzymes (cysteine proteinase(s); arylsulfatase), the Leishmania donovani A2 gene, superoxide dismutase, and the proteophosphoglycan molecule(s) (15)(16)(17)(18)(19). The biological functions of these stage-specific molecules are of interest in terms of their potential role(s) in parasite virulence, pathogenicity, and intracellular survival.…”

Effect of Glycosphingolipids Purified fromLeishmania (Leishmania) amazonensisAmastigotes on Human Peripheral Lymphocytes

“…Leishmania (Leishmania) amazonensis is responsible for most cases of human cutaneous leishmaniasis in the Amazon region of Brazil.A family of glycosphingolipids (GSLs) was identified in amastigotes of L. (L.) amazonensis (1,18,19). In an earlier report we demonstrated that GSLs purified from L. (L.) amazonensis amastigotes inhibited the proliferation of murine T and B cells (2), suggesting that parasite glycosphingolipids may play a relevant role in leishmaniasis.…”

“…The neutral GSL fraction purity was analyzed by high-performance thinlayer chromatography using staining with orcinol-H 2 SO 4 and Dittmer-Lester reagent, which detect carbohydrates and phosphodiester linkages, respectively. No contamination with phospholipids or peptides was detected, as described previously (2,18,19). The effect of amastigote GSLs on T-cell responses induced by mitogen was analyzed in lymphocytes purified from human peripheral blood by Ficoll-Hypaque density gradient centrifugation.…”

“…Concentrations of 12.5, 25, and 50 g/ml were able to inhibit the phosphorylation of the Ac-MBP(4-14) substrate in 48, 54, and 71%, respectively. The PKC inhibitor used, peptide PKC (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), is based on the pseudosubstrate region common to the ␣-, ␤-, and ␥-isozymes of PKC and acts as a potent and specific inhibitor for the Ac-MBP(4-14) substrate (7). This inhibitor at a concentration of 25 M reduced the PKC activity 77%.…”