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Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases

100%

Grodecka M., Czerwiński M., Duk M., Lisowska E., Waśniowska K.

Acta Biochimica Polonica

2010

vol. 57

issue 1

49-53

Duffy antigen is a glycosylated blood group protein acting as a malarial and chemokine receptor. Using glycosylation mutants we have previously demonstrated, that all three potential glycosylation sites of the Duffy antigen are occupied by N-linked oligosaccharide chains. In this study, wild-type Duffy glycoprotein and three mutants, each containing a single N-glycan, were used to characterize the oligosaccharide chains by lectin blotting and endoglycosidase digestion. The positive reaction of all the recombinant Duffy forms with Datura stramonium and Sambucus nigra lectins showed that each Duffy N-linked glycan contains Galβ1-4GlcNAc units terminated by (α2-6)-linked sialic acid residues, typical of complex oligosaccharides. The reactivity with Aleuria aurantia and Lens culinaris lectins suggested the presence of (α1-6)-linked fucose at the N-glycan chitobiose core. The failure of the Galanthus nivalis and Canavalia ensiformis lectins to bind to any of the Duffy mutants or to the wild-type antigen indicated that none of the three Duffy N-glycosylation sites carries detectable levels of high-mannose oligosaccharide chains. Digestion of Duffy samples with peptide N-glycosidase F and endoglycosidase H confirmed the presence of N-linked complex oligosaccharides. Our results indicate that Duffy antigen N-glycans are mostly core-fucosylated complex type oligosaccharides rich in N-acetyllactosamine and terminated by (α2-6)-linked sialic acid residues.

Bacterially expressed truncated F2 domain of Plasmodium falciparum EBA-140 antigen can bind to human erythrocytes

86%

Rydzak J., Kryńska K., Suchanowska A., Kaczmarek R., Łukasiewicz J., Czerwiński M., Jaśkiewicz E.

Acta Biochimica Polonica

2012

vol. 59

issue 4

685-691

The recently identified erythrocyte binding antigen-140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 antigen. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte interaction during invasion. It was shown that the F2 domain of EBA-175 antigen seems to be more important for erythrocyte binding. In order to study activity and immunogenicity of EBA-140 antigen F2 domain, it is necessary to obtain recombinant protein of high purity and in a sufficient amount, which used to pose a challenge due to the high content of disulphide bridges. Here, we present a new method for expression and purification of Plasmodium falciparum EBA-140 antigen F2 domain in E. coli Rosetta-gami strain in fusion with the maltose binding protein (MBP). The truncated F2 domain formed by spontaneous proteolytic degradation of the fusion protein was purified by affinity chromatography on Ni-NTA resin followed by size exclusion chromatography. Molecular mass of this protein was confirmed by mass spectrometry. Its N-terminal amino acid sequencing revealed a proteolytic cleavage site within the F2 domain. The proper folding of the recombinant, truncated F2 domain of EBA-140 antigen was confirmed by circular dichroism analysis. The truncated F2 domain can specifically bind to human erythrocytes but its binding is not as efficient as that of full Region II. This confirms that both the F1 and F2 domains of EBA-140 antigen are required for effective erythrocyte binding.

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2 Acta Biochimica Polonica

1 2012

1 2010

2 Czerwiński M.

1 Duk M.

1 Grodecka M.

1 Jaśkiewicz E.

1 Kaczmarek R.

1 Kryńska K.

1 Lisowska E.

1 Rydzak J.

1 Suchanowska A.

1 Waśniowska K.

1 Łukasiewicz J.

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Analysis of recombinant Duffy protein-linked N-glycans using lectins and glycosidases

Bacterially expressed truncated F2 domain of Plasmodium falciparum EBA-140 antigen can bind to human erythrocytes