• Proceedings of the Korean Society of Life Science Conference
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• 2002.12a
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• pp.13-19
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• 2002

To elucidate the regulatory mechanism for expression of sialyl-glycoconjugates and their biological functions, ninetheen sialyltransferase cDNAs including eleven by our group or co-works have been cloned and characterized so far. The cloned sialyltransferases are classified into four families according to the carbohydrate linkages they synthesize: ${\alpha}2,3-sialyltransferase$ (ST3Gal I-VI), ${\alpha}$ 2,6-sialyltransferase (ST6Gal I), GalNAc ${\alpha}$ 2,6-sialyltransferase (ST6GalNAc I-VI), and ${\alpha}2,8-sialyltransferase$ (ST8Sia I-VI). Each of the sialyltransferase genes is differentially expressed in a tissue-, cell type-, and stage-specific manner. These enzymes differ in their substrate specificity and various biochemical parameters. However, enzymatic analysis conducted in vitro with recombinant enzyme revealed that one linkage can be synthesized by multiple enzymes. We present here an overview of structure and function of sialyltransferases performed by our group and co-works. Genomic structures and transcriptional regulation of two kinds of human sialyltransferase gene are also presented.

• Journal of Life Science
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• v.14 no.6 s.67
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• pp.1009-1017
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• 2004

Sialyltransferases cloned so far show the remarkable tissue-specific expression, which is correlated with the existence of cell type-specific sialylated sugar structure in glycoconjugates. In the previous studies, we found various mRNA isoforms of human sialyltransferases generated by alternative splicing and alternative promoter utilization. To understand the regulatory mechanisms for specific expression of human sialyltransferase genes and for production of their mRNA isoforms, in this study, we have isolated and characterized five kinds of human sialyltransferase genes: hST3Gal II, hST8Sia II, hST8Sia III, hST8Sia IV, and hST8Sia V. The hST3Gal II gene is composed of six exons, which span over 17kb, with exons ranging in size from 46 to over 1017 bp. The hST8Sia III gene comprises over 10 kb, and consists of only four exons, which is much smaller and simpler than other human sialyltransferase genes. In contrast, three genes (hST8Sia II, hST8Sia IV and hST8Sia V) span more than 70 kb, and comprise five or more exons. All exon-intron boundaries follow the GT-AG rule. In particular, the sialylmotif L, which is a highly conserved region in all cloned sialyltransferases, was found in one exon of hST8Sia III, whereas this motif is encoded by discrete exons in the other human sialyltransferases. Exon structures of these sialyltransferase genes show the structural diversity, as found in other human sialyltransferase genes reported so far. We determined the transcription start site of hST3Gal II gene by the 5'-RACE and cap site hunting experiments.

• BMB Reports
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• v.30 no.2
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• pp.95-100
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• 1997

Two kinds of cDNA encoding mouse $Gal{\beta}$1,3(4)GlcNAc ${\alpha}$2,3-sialyltransferase (mST3Gal III) and $Gal{\beta}$1,4(3)GlcNAc ${\alpha}$2,3-sialyltransferase (mST3Gal IV) were isolated from mouse brain cDNA library by means of a PCR-based approach. The cDNA sequences included an open reading frame coding for proteins of 374 and 333 amino acids, respectively, and the primary structure of these enzymes suggested a putative domain structure consisting of four regions, like that in other glycosyltransferases. The deduced amino acid sequences of mST3GaI III and IV showed a 98% and 89% identity with rat ST3GaI III and human ST3Gal IV, respectively. Northern analysis indicated that the expression of mST3Gal III mRNA was abundant in heart, liver and adult brain, while that of mST3GaI IV mRNA was detected in all tissues tested except for testis, but the level was the highest in liver. Soluble forms of mST3GaI III and IV transiently expressed in COS cells exhibited enzyme activity toward acceptor substrates containing the terminal either $Gal{\beta}$1,3GlcNAc or $Gal{\beta}$1,4GlcNAc sequences. The substrate preferences of both enzymes were stronger for tetrasaccharides than for disaccharides.

• KSBB Journal
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• v.32 no.3
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• BMB Reports
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• v.41 no.1
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• pp.48-54
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• 2008

PM0188 is a newly identified sialyltransferase from P. multocida which transfers sialic acid from cytidine 5'-monophosphonuraminic acid (CMP-NeuAc) to an acceptor sugar. Although sialyltransferases are involved in important biological functions like cell-cell recognition, cell differentiation and receptor-ligand interactions, little is known about their catalytic mechanism. Here, we report the X-ray crystal structures of PM0188 in the presence of an acceptor sugar and a donor sugar analogue, revealing the precise mechanism of sialic acid transfer. Site-directed mutagenesis, kinetic assays, and structural analysis show that Asp141, His311, Glu338, Ser355 and Ser356 are important catalytic residues; Asp141 is especially crucial as it acts as a general base. These complex structures provide insights into the mechanism of sialyltransferases and the structure-based design of specific inhibitors.

• Archives of Pharmacal Research
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• v.8 no.2
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• pp.49-57
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• 1985

The effect of ginseng components on the potassium depleted cardiomyopathic rat heart was investigated. In the perfused heart experiment using Langendorff apparatus, the deterioration rate of contriactile force of potassium depleted rat heart (low potassium diet group) was faster than that of normal rat heart and ginseng components showed the ability to slow the deterioration rate of potassium depleted hearts. Both sialic acid contents in carcolemmal ghost and sialyltransferase activity of 40,000 * g subcellular fraction prepared from cardiac ventricular tissue of low potassium diet group were significantly decreased compared to those of normal group. The decrease of the sialic acid content and sialyltransferase activity in sarcolemma of low potassium diet group was inhibited when ginseng was concomitantly administered. Calcium uptake of sarcoplasmic reticulum prepared from low potassium diet group was significantly greaterthan that of normal group. Ginseng extract or total saponin showed the tendency to inhibit the increase of cacium uptake.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.699-706
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• 2013

We have previously reported that N-acetylcysteine (NAC) not only delayed apoptosis but also enhanced the production of recombinant erythropoietin (EPO) in Chinese hamster ovary (CHO) cell culture. To investigate the production enhancement mechanism, the effects of similar thiol-reducing agents were studied. Intriguingly, all mild reducing agents examined including mercaptoethanesulfonic acid (MESNA), thiolactic acid (TLA), and thioglycolate (TG) were shown to block apoptosis and increase EPO production. A pulse-chase study of EPO secretion revealed that all four thiol-reducing agents increased the EPO secretion rate; among them TLA showed the highest rate. In terms of product quality, the sialic acid content of the glycoprotein is one of the most important factors. It was reported that a number of glycoproteins produced by CHO cells often have incomplete sialylation, particularly under high-producing conditions. Human ${\alpha}2,3$-sialyltransferase (${\alpha}2,3$-ST) was introduced into EPO-producing CHO cells in order to compensate for the reduced sialylation during supplementation with NAC. When ${\alpha}2,3$-ST was expressed in the presence of NAC, reduced sialylation was restored and an even more sialylated EPO was produced. Thus, our study is significant in that it offers increased EPO production while still allowing the prevention of decreased sialylation of EPO.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.511-518
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• 2011

To avoid hyperacute rejection of xenografts, ${\alpha}1,3$-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of ${\alpha}1,3$-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that ${\alpha}2,3$-sialyltransferase (${\alpha}2,3ST$) and ${\alpha}2,6$-sialyltransferase (${\alpha}2,6ST$) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of ${\alpha}$-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of ${\alpha}2,6$-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.

• Archives of Pharmacal Research
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• v.22 no.3
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• pp.243-248
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• 1999

Sialic acids are key determinants for biological processes, such as cell-cell interaction and differentiation. Sialyltransferases contribute to the diversity in carbohydrate structure through their attachment of sialic acid in various terminal positions on glycolipid and glycoprotein (N-linked and O-linked) carbohydrate groups. Gal$\beta$ 1,3(4)GlcNAc $\alpha$2,3-sialyltransferase (ST3Gal III) is involved in the biosynthesis of $sLe^{X}$ and sLe^{a}$ known as selection ligands and tumor-associated carbohydrate structures. The appearance and differential distribution of ST3Gal III mRNA during mice embryogenesis [embryonic (E) days; E9, E11, E13, E15] were investigated by in situ hybridization with digoxigenin-labeled RNA probes coupled with alkaline phosphatase detection. On E9, all tissues were positive for ST3Gal III mRNA expression whereas ST3Gal III mRNA on E11 was not detected throughout all tissues. On E13, ST3GAl III mRNA was expressed in different manner in various tissues. In this stage, ST3Gal III mRNA was positive only in the liver, pancreas and bladder. On E15, specific signal for ST3GAl III was detected in the liver, lung and forebrain. These results indicate that ST3Gal III is differently expressed at developmental stages of mice embryo, and this may be importantly related with regulation of organogenesis in mice.

• Proceedings of the Zoological Society Korea Conference
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• 1998.10b
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• pp.224.1-224
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• 1998

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