Identification of sialic acids on Leishmania donovani amastigotes

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Abstract
The presence of Neu5Ac on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, has been reported recently. Here we report the occurrence of Neu5Ac as a major component on amastigotes, as well as Neu5Gc, Neu5,9Ac2 and Neu9Ac5Gc as indicated by fluorimetric high performance liquid chromatography and gas liquid chromatography/electron impact mass spectrometry. Furthermore, binding studies with Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and various Siglecs, showed the presence of both (alpha2-6) and (alpha2-3)linked sialic acids; their binding was reduced after sialidase pretreatment. Western blotting of amastigote membrane glycoproteins with SNA demonstrated the presence of two sialoglycoconjugates of Mr values of 164 000 and 150 000. Similarly, binding of MAA demonstrated the presence of five distinct sialoglycans corresponding to molecular masses of 188, 162, 136, 137 and 124 kDa. Achatinin-H, a lectin that preferentially identifies 9-Oacetylated sialic acid (alpha2-6)linked to GalNAc, demonstrated the occurrence of two 9-Oacetylated sialoglycans with Mr 158 000 and 150 000, and was corroborated by flow cytometry; this binding was abolished by recombinant 9-Oacetylesterase pretreatment. Our results indicate that Neu5Ac w(alpha2-6) and (alpha2-3)linked, as well as Neu5Gc and their 9-Oacetyl derivatives, constitute components of the amastigote cell surface of L. donovani.