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Jeong, Jae Kap,Kwon, Ohsuk,Lee, Yun Mi,Oh, Doo-Byoung,Lee, Jung Mi,Kim, Seonghun,Kim, Eun-Hye,Le, Tu Nhat,Rhee, Dong-Kwon,Kang, Hyun Ah American Society for Microbiology 2009 Journal of Bacteriology Vol.191 No.9
<B>ABSTRACT</B><P><I>Streptococcus pneumoniae</I> is a causative agent of high morbidity and mortality. Although sugar moieties have been recognized as ligands for initial contact with the host, only a few exoglycosidases have been reported to occur in <I>S. pneumoniae</I>. In this study, a putative β-galactosidase, encoded by the <I>bgaC</I> gene of <I>S. pneumoniae</I>, was characterized for its enzymatic activity and virulence. The recombinant BgaC protein, expressed and purified from <I>Escherichia coli</I>, was found to have a highly regiospecific and sugar-specific hydrolysis activity for the Galβ1-3-GlcNAc moiety of oligosaccharides. Interestingly, the BgaC hydrolysis activity was localized at the cell surface of <I>S. pneumoniae</I>, indicating that BgaC is expressed as a surface protein although it does not have a typical signal sequence or membrane anchorage motif. The surface localization of BgaC was further supported by immunofluorescence microscopy analysis using an antibody raised against BgaC and by a reassociation assay with fluorescein isothiocyanate-labeled BgaC. Although the <I>bgaC</I> deletion mutation did not significantly attenuate the virulence of <I>S. pneumoniae</I> in vivo, the <I>bgaC</I> mutant strain showed relatively low numbers of viable cells compared to the wild type after 24 h of infection in vivo, whereas the mutant showed higher colonization levels at 6 and 24 h postinfection in vivo. Our data strongly indicate for the first time that <I>S. pneumoniae bgaC</I> encodes a surface β-galactosidase with high substrate specificity that is significantly associated with the infection activity of pneumococci.</P>
Kyung Jin Lee,Jin Young Kil,Ohsuk Kwon,Kisung Ko,Doo-Byoung Oh 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1
Several classes of peptide:N-glycanse (PNGase) enzymes have been identified and used for release of N-glycan from glycoproteins. PNGase F, derived from Flavobacterium meningosepticum, has been most widely used while an almond PNGase A has been employed specifically for deglycosylation of plant- orinsect-derived glycoproteins. In the present study, we identified a new PNGase enzyme from Yarrowia lipolytica, of which amino acid sequence shows weak homology with PNGase A. This gene was cloned and successfully expressed in Pichia pastrois using the commercially available vector for secretory expression. The prepared enzyme, designated as PNGase Y, was shown to release various types of N-glycans including high-mannose and complex type glycans. Especially, it could release the glycans containing core α (1,3)-fucosefound in plant and insect as well as bi-phosphorylated glycans for lysosomal proteins, which are not good substrates for PNGase F. Moreover, PNGase Y was shown to release N-glycans efficiently from denatured glycoprotein as well as glycopeptide. It is a great advantage compared to PNGas A which requires an additional step of protease digestion due to the preference for glycopeptides as a substrate. Taken together, we suggest that PNGase Y will be a good tool to release various types of N-glycans with broad range of substrate specificity under favorable conditions without the need of glycopeptides preparation.
Enhanced Sialylation by Knockout of Sialidase Genes in CHO Cells Using CRISPR/Cas9
Se-Jong Lim,Keun Koo Shin,Ohsuk Kwon,Doo-Byoung Oh 한국당과학회 2016 한국당과학회 학술대회 Vol.2016 No.01
Sialic acids present on non-reducing terminal of glycan is important for prolonged in vivo half-life of glycoproteins. Many efforts have been made to increase the sialic acid content in the manufacturing of therapeutic glycoproteins because glycoproteins containing non-sialylated glycans do not show effective in vivo efficacies owing to a short half-life. It was reported that knock down of sialidase expression using the RNA interference (RNAi) technology enhanced protein sialylation in CHO cells which are the most widely used for therapeutic glycoprotein production [1]. However, knock down strategy did not completely shut down the sialidase expression. In this study, we performed knockout of sialidase genes in CHO cells by using the CRISPR/Cas9 genome editing technology. Only three sialidases (Neu1, Neu2, and Neu3) were actively expressed in CHO cells when analyzing their expressions using real-time polymerase chain reaction. First, we disrupted Neu3 gene because it is located in the plasma membrane while Neu1 and Neu2 exist in lysosome and cytosol, respectively. After the transfection of vectors expressing Cas9 and sgRNAs, Neu1 gene-disrupted clones were selected by using the T7 endonucleases 1 (T7E1) assay and cell surface sialidase activity assay. Disruption of Neu1 gene did not affect the viability of CHO cells. The disruptions of Neu1 and Neu2 genes in CHO cells are currently in the progress.
Yun Mi Lee,Jae Kap Jeong,Ohsuk Kwon,Doo-Byoung Oh,Jung Mi Lee,Seonghun Kim,Eun-Hye Kim,Tu Nhat Le,Dong-Kwon Rhee,Hyun-Ah Kang 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Streptococcus pneumoniae is a causative agent for high morbidity and mortality. Although sugar moieties have been recognized as a ligand for initial contact with the host, only a few exoglycosidaseshave been reported in S. pneumoniae. In this study, a putative -galactosidase, encoded by the bgaC gene of S. pneumoniae, was characterized for its enzymatic activity and virulence. The recombinant BgaC protein, expressed and purified from Escherichia coli, was found to have a highly regiospecific and sugar specific hydrolysis activity for the Gal1-3-GlcNAc moiety of oligosaccharide. Interestingly, the BgaC hydrolysis activity was localized at the cell surface of S. pneumoniae, indicating that BgaC is expressed as a surface protein although it does not have a typical signal sequenceor membrane anchorage motif. The surface localization of BgaC was further supported by immunofluorescence microscopy analysis using an antibody raised against BgaC. Although the bgaC deletion mutation did not significantly attenuate the virulence of S. pneumoniae in vivo, the bgaC mutant strain showed relatively lower viable cell numbers compared to the wild type after 24 h infection in vivo, whereas it showed higher adherence and invasion at 6 and 12 h post- infection in vivo. Our data strongly indicate for the first time that S. pneumoniae bgaC encodes a surface β-galactosidase with high substrate specificity that is significantly associated with the infection activity of pneumococci.