earticle

영어

Corynebacterium diphtheriae is a Gram-positive pathogenic bacterium that causes diphtheria. It can decorate its cell surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using its surface-localized sialidase related enzymes. In the present study, we have identified a putative sialidase gene, nanH, from C. diphtheriae KCTC3075 and characterized its product for enzyme activity. C. diphtheriae sialidase NanH can cleave α(2,3)- and α(2,6)-linked sialic acid from sialic acid-containing substrates. Even though the efficiency was low, the sialidase was able to catalyse the transfer of sialic acid using several sialoconjugates as donor. In addition, using the reversible reaction mechanism of C. diphtheriae NanH, we also constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. C. diphtheriae nanH gene was cloned into the yeast surface display vector pYD1 based on the Aga1p-Aga2p platform to immobilize the enzyme on the surface of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids from pNP- α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage. The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis of N-glycans containing α (2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling.