A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens.
Biochem Biophys Rep
; 26: 100940, 2021 Jul.
Article
en En
| MEDLINE
| ID: mdl-33732900
ABSTRACT
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 a protein 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. GENERALSIGNIFICANCE:
Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.
4 MU-NANA, 2'-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid; BCA, Bicinchonninic acid; CDAP-BF4, 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate; ECA, Erythrina cristagalli lectin; ELISA-type assay, Enzyme-linked Immuno Sorbent assay; Enzymatic desialylation; FBS, Fetal bovine serum; Gal, Galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Glycomic analysis; Immobilization; Neu5Ac, N-Acetyl neuraminic acid; Neura-agarose, Neuraminidase immobilized onto agarose; Neuraminidase; OPD, ortho-Phenylendiamine; OSM, Ovine submaxilar mucin; PBS, Phosphate saline buffer; PE, Phycoerythrin; PNA, Arachis hipogaea lectin; SNA, Sambucus nigra lectin; Sialic acid; Sialyl-Tn antigen, Neu 5 Ac-2,6 GalNAc
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Colección:
01-internacional
Banco de datos:
MEDLINE
Idioma:
En
Revista:
Biochem Biophys Rep
Año:
2021
Tipo del documento:
Article
País de afiliación:
Uruguay