A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens.

Background: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation.

Methods: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for
desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry.

Results: Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was
of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of aprotein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles.

Conclusions: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic
desialylation purposes.

General significance: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.

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