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杜氏利什曼原虫前鞭毛体上吸附的血清唾液酸糖蛋白的鉴定与表征

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes.

作者信息

Chatterjee Mitali, Chava Anil Kumar, Kohla Guido, Pal Santanu, Merling Anette, Hinderlich Stephan, Unger Ulrike, Strasser Peter, Gerwig Gerrit J, Kamerling Johannis P, Vlasak Reinhard, Crocker Paul R, Schauer Roland, Schwartz-Albiez Reinhard, Mandal Chitra

机构信息

Immunobiology Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Calcutta 700032, India.

出版信息

Glycobiology. 2003 May;13(5):351-61. doi: 10.1093/glycob/cwg027. Epub 2002 Dec 17.

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.

摘要

唾液酸作为寡糖链的末端残基,在多种细胞识别事件中发挥着关键作用。通过荧光高效液相色谱法证明,印度内脏利什曼病的病原体杜氏利什曼原虫前鞭毛体上存在唾液酸,显示出Neu5Ac,在较小程度上还有Neu5,9Ac2。GC/MS分析证实了Neu5Ac的存在。此外,与唾液酸结合凝集素黑接骨木凝集素(SNA)、山嵛凝集素(MAA)和唾液酸免疫球蛋白超家族成员的结合表明存在α2,3-和α2,6-连接的唾液酸。在寄生虫中未发现Neu5Ac的内源性生物合成机制。用SNA对寄生虫膜和培养基进行同步蛋白质印迹分析表明存在常见的唾液酸糖缀合物(123、90和70 kDa)。同样,MAA与寄生虫膜和培养基的结合显示出三种类似的唾液酸聚糖,对应于130、117和70 kDa,表明α2,3-和α2,6-连接的唾液酸聚糖是从培养基中存在的胎牛血清中吸附而来的。杜氏利什曼原虫前鞭毛体也与玛瑙螺凝集素-H发生反应,玛瑙螺凝集素-H是一种优先识别α2→6 GalNAc连接中9-O-乙酰化唾液酸的凝集素。通过细胞凝集、ELISA和流式细胞术在寄生虫细胞表面证实了该决定簇,在用源自丙型流感病毒酯酶基因HE1区域的重组9-O-乙酰酯酶预处理细胞后,其结合被消除。此外,9-O-乙酰基GD3特异性单克隆抗体CD60b的结合证实了末端9-O-乙酰化二唾液酸聚糖的存在。我们的结果表明,唾液酸(α2→6和α2→3连接)和9-O-乙酰基衍生物构成了寄生虫细胞表面的成分。

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