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Isolation and characterisation of crocodile and python ovotransferrins

100%

Ciuraszkiewicz J., Olczak M., Wątorek W.

2007

vol. 54

issue 1

175-182

Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile (Crocodilus niloticus, C. rhombifer) and one snake (Python molurus bivittatus) ovotransferrins. The proteins were purified by anion exchange and hydrophobic chromatography, and their N-terminal amino-acid sequences, molecular mass and isoelectric points were determined. All three proteins are glycosylated and their N-glycan chromatographic profiles show the largest contribution of neutral oligosaccharides in crocodile and disialylated glycans in python ovotransferrin. The absorption spectra of iron-saturated transferrins were analysed. Iron release from these proteins is pH-dependent, showing a biphasic character in crocodile ovotransferrins and a monophasic type in the python protein. The reason for the different types of iron release is discussed.

Analysis of individual azurocidin N-glycosylation sites in regard to its secretion by insect cells, susceptibility to proteolysis and antibacterial activity

88%

Indyk K., Olczak T., Ciuraszkiewicz J., Wątorek W., Olczak M.

2007

vol. 54

issue 3

567-573

Azurocidin is an inactive serine protease homolog with primary sequence similarity to neutrophil elastase, cathepsin G, and proteinase 3. The aim of this study was to investigate possible consequences of differential glycosylation of azurocidin in regard to its secretion, protein stability as measured by susceptibility to proteolysis, and antibacterial activity. Site-directed mutagenesis was employed to generate mutant azurocidin variants lacking individual N-glycosylation sites. Our results show that N-linked glycans may play a role in proper azurocidin folding and subsequent secretion by insect cells. We also demonstrate that N-linked glycosylation contributes to azurocidin stability by protecting it from proteolysis. The lack of N-glycosylation at individual sites does not significantly influence the azurocidin antibacterial activity.

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2 Acta Biochimica Polonica

2 2007

2 Ciuraszkiewicz J.

2 Olczak M.

2 Wątorek W.

1 Indyk K.

1 Olczak T.

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Isolation and characterisation of crocodile and python ovotransferrins

Analysis of individual azurocidin N-glycosylation sites in regard to its secretion by insect cells, susceptibility to proteolysis and antibacterial activity