Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes.
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.
Chatterjee
Mitali
M
Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta 700032, India. cmandal@iicb.res.in
Chava
Anil Kumar
AK
Kohla
Guido
G
Pal
Santanu
S
Merling
Anette
A
Hinderlich
Stephan
S
Unger
Ulrike
U
Strasser
Peter
P
Gerwig
Gerrit J
GJ
Kamerling
Johannis P
JP
Vlasak
Reinhard
R
Crocker
Paul R
PR
Schauer
Roland
R
Schwartz-Albiez
Reinhard
R
Mandal
Chitra
C
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*
D003470:Culture Media; D006001:Glycoconjugates; D037102:Lectins; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012794:Sialic Acids; D000115:Acetylesterase; D009439:Neuraminidase