engleski

NEW METHOD FOR ANALYSING PROTEIN GLYCOSYLATION

Glycans are the most abundant and most diverse biopolymers in nature and participate in many crucial biological processes. They are involved in genesis, development and processing of many diseases, and many diseases are associated with changes in glycan structures. Because of all that glycans are now in the focus of modern medicine, biotechnology and pharmacy through the improvement of diagnostics, disease monitoring and development of biotherapeutics that are mainly (glyco)proteins. Glycan structures are very complex and methods to analyze them are very demanding and time-consuming, thus the availability of simple techniques for the analysis of oligosaccharide structures and their changes could be a very helpful tool. Aiming to establish such a method, we are developing a lectin based ELISA method for the analysis of transferrin sialylation. Transferrin is a serum glycoprotein that has two N-linked oligosaccharide chains consisting of bi- or three- antennary structures, each possibly terminating in sialic acid. SNA (Sambucus nigra agglutinin) lectin specifically recognizes Neu5Ac( 2-6)Gal/GalNAc sequence present on transferrin and this lectin was used to develop the assay. Our method allows us to determine the level of sialic acid on the protein tested, having the protein mixture (serum) as a sample by using immobilized anti-transferrin antibody and to compare the results acquired on different multiwell plates by using standard curve of pure transferrin on each plate. If glycosylation results of differently sialylated controls obtained by lectin ELISA are put in correlation with their HPAE-PAD analysis even more detailed information could be obtained. Since many studies suggested changes of transferrin glycosylation in various diseases, this is a very useful diagnostic method. Similar method could be developed for glycosylation analysis of other proteins. This would be particularly important for glycoprotein therapeutics, since glycosylation changes can significantly influence function of these molecules.

glicoproteins; ELISA

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