Construction of an in vitro trans-sialylation system: surface display of Corynebacterium diphtheriae sialidase on Saccharomyces cerevisiae.
Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae 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.
Kim
Seonghun
S
Integrative Omics Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-dong, Yuseong-gu, Daejeon 305-333, South Korea.
Oh
Doo-Byoung
DB
Kwon
Ohsuk
O
Kang
Hyun Ah
HA
D001212:Asialoglycoproteins / Q000378:metabolism; D003353:Corynebacterium diphtheriae / Q000201:enzymology* / Q000235:genetics; D004800:Enzymes, Immobilized / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000378:metabolism; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism; D012795:Sialoglycoproteins / Q000378:metabolism*; D013499:Surface Properties
D001212:Asialoglycoproteins; D004800:Enzymes, Immobilized; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D012795:Sialoglycoproteins; D009439:Neuraminidase