Effects of selective cleavage of high-mannose-type glycans of Maackia amurensis leukoagglutinin on sialic acid-binding activity

Abstract

Background: Maackia amurensis leukoagglutinin (MAL) is a glycoprotein and sialic arid-binding lectin that is used widely in the detection and characterization of sialoglycoconjugates and human cancer cells. However, its N-linked glycan structure and role have yet to be determined. Methods: The N-linked glycans were analyzed using high-performance liquid chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and the secondary structure was investigated using circular dichroism analysis. A hemagglutination assay was performed. Furthermore, surface plasmon resonance analysis, and fluorescence microscopy and fluorescence-activated cell-sorting analysis were conducted to assess the sialoglycoprotein-binding ability and its usefulness in the detection of human breast cancer MCF-7 cells, respectively. Results: Analysis of the N-linked glycan structure of MAL confirmed the presence of eight glycans, comprising two alpha 1,3-fucosylated paucimannosidic-type and six high-mannose-type glycans. Glycan analysis of MAL that had been treated with peptide N-glycosidase F (de-M-MAL) revealed that while the two alpha 1,3-fucosylated paucimannosidic glycans remained attached following the treatment, the six high-mannose-type glycans had been completely cleaved from the original MAL There were almost no secondary structural changes between MAL and de-M-MAL; however, the lectin activities exhibited by MAL, such as hemagglutination and binding to a sialoglycoprotein, were completely absent in de-M-MAL, and the ability to detect human breast cancer MCF-7 cells was 77% lower in de-M-MAL than in MAL. Conclusion: The high-mannose-type glycans in intact MAL are closely associated with its lectin activities. General significance: This is the first report of the N-linked glycan structure of MAL and the effect of high-mannose-type glycans on lectin activities. (C) 2015 Elsevier B.V. All rights reserved.