Novel Bacterial Sialidase Mutants for Site Specific Sialylation of Biologically Important Glycoproteins
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Sialylation is a unique modification of glycans existing in different glycoproteins on cell surfaces, which significantly modulates many important physiological and pathological processes, such as anti-inflammatory and tumor metastasis. Despite tremendous efforts on the synthesis of this crucial glycan decoration using chemical and enzymatic methods, site-specific attachment of sialic acid moieties onto biologically important glycopeptides or glycoproteins is still challenging. In the last two decades, converting glycosidases into their transfer mutants has become a fascinating approach in the synthesis of glycoconjugates. In this work, a bacterial α2,6-sialidase (MvNA) from Micromonospora viridifaciens was selected as candidate for efficient transfer of sialic acid in a regio- and stereo-controlled manner due to its inherent trans-sialylation activity. First, wild-type MvNA was cloned and overexpressed in E. coli. Two types of sialidase mutants with mutation site at the nucleophile (Y370) and the general acid/base (D92) residues were then created by site-directed mutagenesis and successfully expressed. In the preliminary activity tests, the wild-type MvNA showed strong hydrolytic activity towards a natural sialylated complex-type N-glycopeptide, while terminal sialic acids from the same glycopeptide can’t be removed by created MvNA mutants, including Y370A, Y370G and D92A. The potential trans-sialylation activities of those sialidase mutants will be systematically tested using various synthetic activated substrate donors, such as sialyl fluorides and nitrophenyl sialosides. We are also aiming to create sialidase mutants derived from other types of sialidases with distinct specificities, e.g. α2,3 and α2,8-linkages. The potential sialidase transfer mutants could provide a new avenue to access the structurally well-defined sialylated glycopeptides and glycoproteins for sialylation bio-function study and therapeutic applications.