title: Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes.
abstract: Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmaniasis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid-binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both alpha2,3- and alpha2,6-linked sialic acids. No endogenous biosynthetic machinery for Neu5Ac could be demonstrated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demonstrated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite membrane and culture medium showed three analogous sialoglycans corresponding to 130, 117, and 70 kDa, indicating that alpha2,3- and alpha2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in alpha2-->6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytometry, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialoglycans. Our results indicate that sialic acids (alpha2-->6 and alpha2-->3 linked) and 9-O-acetyl derivatives constitute components of the parasite cell surface.
authors.affiliation: Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta 700032, India. cmandal@iicb.res.in
authors.forename: Mitali
authors.initials: M
authors.lastname: Chatterjee
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Chava
authors.forename: Guido
authors.initials: G
authors.lastname: Kohla
authors.forename: Santanu
authors.initials: S
authors.lastname: Pal
authors.forename: Anette
authors.initials: A
authors.lastname: Merling
authors.forename: Stephan
authors.initials: S
authors.lastname: Hinderlich
authors.forename: Ulrike
authors.initials: U
authors.lastname: Unger
authors.forename: Peter
authors.initials: P
authors.lastname: Strasser
authors.forename: Gerrit J
authors.initials: GJ
authors.lastname: Gerwig
authors.forename: Johannis P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: Reinhard
authors.initials: R
authors.lastname: Vlasak
authors.forename: Paul R
authors.initials: PR
authors.lastname: Crocker
authors.forename: Roland
authors.initials: R
authors.lastname: Schauer
authors.forename: Reinhard
authors.initials: R
authors.lastname: Schwartz-Albiez
authors.forename: Chitra
authors.initials: C
authors.lastname: Mandal
mesh_terms: D000115:Acetylesterase; D000372:Agglutination Tests; D000818:Animals; D001769:Blood; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D008401:Gas Chromatography-Mass Spectrometry; D006001:Glycoconjugates / Q000032:analysis*; D006790:Host-Parasite Interactions; D037102:Lectins; D007893:Leishmania donovani / Q000737:chemistry / Q000502:physiology*; D009439:Neuraminidase; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012794:Sialic Acids / Q000032:analysis*
chemical_list: D003470:Culture Media; D006001:Glycoconjugates; D037102:Lectins; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012794:Sialic Acids; D000115:Acetylesterase; D009439:Neuraminidase