The major Leishmania donovani chagasi surface glycoprotein in tunicamycin-resistant promastigotes.

Two populations of Leishmania donovani chagasi promastigotes resistant to the lethal effects of tunicamycin (TM), an inhibitor of N-linked glycosylation, were raised. These parasites exhibited altered patterns of glycosylation when compared to wild-type controls. In particular the major surface glycoprotein gp63 was present in membranes of one population of TM-resistant promastigotes (population 1) primarily in a deglycosylated form, which migrated at a lower Mr than wild-type gp63. The deglycosylated protein was proteolytically inactive on substrate-containing gels, in contrast to glycosylated gp63. Assays of promastigote attachment to human macrophages revealed that, despite a proposed role for gp63 protease activity in binding to macrophage receptors, population 1 TM-resistant promastigotes appeared to attach to the CR3 but not to the mannose-fucose receptor. Control promastigotes bound to both receptors. In contrast, a second population of TM-resistant promastigotes (population 2) did not produce gp63 that could be detected on immunoblots, either in a glycosylated or deglycosylated form. The latter TM-resistant promastigotes bound to neither CR3 nor the mannose-fucose receptor, suggesting that expression of gp63 might be important for promastigotes to bind to CR3. LPG was present in immunoblots of both TM-resistant and control populations suggesting this molecule might not account for the differences in attachment. Surprisingly both TM-resistant promastigote populations contained gp63 mRNA by Northern analysis in apparently equal amounts. We conclude that N-glycosylation is probably necessary for the protease function of gp63, but that neither N-glycosylation nor protease activity of gp63 is necessary for L. donovani chagasi promastigotes to bind to CR3. Furthermore the expression of gp63 protein by TM-resistant promastigotes is dependent upon postranscriptional events.

Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes.

Using both hamster and mouse models of infection, we documented that the virulence of Leishmania donovani chagasi promastigotes decreases over time, when parasites are maintained in long term culture after isolation from an infected animal. Concomitant with this loss of virulence is a marked decrease in amount of the major promastigote surface glycoprotein, gp63, present in promastigotes. The latter was shown by a decrease in binding of polyclonal anti-gp63 serum to attenuated (cultivated long term) as compared to virulent (recently isolated) promastigotes, using immunofluorescence and Western blot assays. Binding of Con A to promastigote glycoproteins, separated by SDS-PAGE, documented a similar decrease. An alteration in the mechanism of promastigote attachment to macrophages was also noted: purified gp63 inhibited attachment of virulent promastigotes to human monocyte-derived macrophages, but it did not affect the attachment of attenuated promastigotes. Northern blot analysis showed that, despite marked differences in the amount of gp63 protein, the quantity of gp63 RNA was comparable in attenuated and virulent promastigotes. However, virulent promastigotes contained two major gp63 RNA species of 3.0 and 2.7 kb, whereas attenuated promastigotes had one predominant gp63 RNA of 2.7 kb and only minor amounts of 3.0 kb RNA. Thus, the decrease in gp63 expression in attenuated, contrasted to virulent, promastigotes is associated with qualitative, but not quantitative, differences in the gp63 messenger RNA.

The major concanavalin A-binding surface glycoprotein of Leishmania donovani chagasi promastigotes is involved in attachment to human macrophages.

Leishmania donovani, the protozoan causing visceral leishmaniasis, is an obligate intracellular parasite of mammalian macrophages. Considerable evidence has suggested that the ingestion of L. donovani promastigotes by macrophages occurs via receptors on the surface of the phagocyte. During this study, a glycoconjugate that may be involved in the receptor-mediated ingestion of L. donovani chagasi promastigotes was isolated from the parasite membrane. Octyl glucoside-soluble extracts of promastigote membranes contained a predominant doublet migrating at 60 kDa, seen by SDS-PAGE. The 60-kDa molecule was the major externally disposed promastigote surface protein labeled by 125I, and it was the major Con A-binding protein on L. donovani chagasi, as determined by Con A binding to parasite proteins transferred to nitrocellulose. Attachment of promastigotes to human monocyte-derived macrophages was inhibited by varying concentrations of the membrane extract containing both proteins, and adsorption of extracts on Con A-Sepharose resulted in both removal of the 60,000 Mr glycoprotein and loss of the ability of extracts to inhibit promastigote attachment to human macrophages. After further purification of the 60-kDa glycoprotein by gel filtration, its inhibitory activity increased 45-fold over the unpurified membrane extract. Examination of Con A blots of stationary phase promastigotes isolated from an infected hamster revealed a marked loss in the major Con A-binding glycoprotein over 4 mo in in vitro culture after isolation from the rodent host, corresponding to a loss in infectivity of the promastigotes for hamsters. The results suggest that the major Con A-binding surface glycoprotein from L. donovani chagasi promastigotes is important in attachment to human macrophages, and may be a factor in parasite virulence for a mammalian host.