• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.97-97
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• 2002

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1371-1379
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• 2015

The Ca_v1.2 Ca²⁺ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca_v1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca_v1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca_v1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca_v1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca_v1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca_v1.2/WT. Tunicamycin treatment of oocytes expressing Ca_v1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Ca_v1.2.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.495-495
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• 2003

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.1
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• pp.40-43
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• 1998

The glycosylation pattern of Erythropoietin (EPO), produced by recombinant CHO cells, was studied using the simple and rapid technique of 'Lectin-blotting'. In this experiment we used three different kinds of lectins, MAA(Maackia amurensis agglutinine), RCA(Ricinus communis agglutinine), and DSA(Datura stramonium agglutinine), which bind to the terminal sialic acid, galactose, and the N-acetyllactosamine chain respectively. The lectin-blotting technique was used to analyze the carbohydrate structure of EPO produced in the presence of two physiologically active chemical compounds, ammonium and chloroquine. The effect of the ammonium ion on the glycosylation of EPO was studied because it accumulated in the medium mainly as a by-product of glutamine matabolism. Ammonium chloride significantly inhibited the sialylation of the terminal galactose residue at concentrations of 8mM or more. Chloroquine, a potent inhibitor of glycosylation, inhibited terminal sialylation at concentrations of 100 and 200 $\mu$M, and at a concentration of 300 $\mu$M, also inhibited Nacetyllactosamine chain synthesis.

• Proceedings of the Ginseng society Conference
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• 1993.09a
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• pp.74-83
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• 1993

In an attempt toward the synthesis of the difficulty accessible ginseng saponins the four dammarane glycosides identical to the natural $ginsenosides-Rh_2,$ - F2, compound K and chikusetsusaponin - LT8 have been prepared from betulafolienetriol(=dammar-24-ene-$3{\alpha},12{\beta}\;20(S)-triol).\;3-O-{\beta}-D-Glucopyranoside$ of 20(S) - protopanaxadiol $(=ginsenoside-Rh_2)$ have been obtained by the regio - and stereoselective glycosylation of the $12-O-acetyldammar-24-ene-3{\beta},\;12{\beta},$ 20(S)-triol. The 12-ketoderivative of 20(S)-protopanaxadiol has been used as aglycon in synthesis of chikusetsusaponin - LT8. Attempted regio - and stereoselective glycosylation of the less reactive tertiary C - 20 - hydroxyl group in order to synthesize the $20-O-{\beta}-D-glucopyranoside$ of 20(S)-protopanaxadiol(=compound K) using 3, 12 - di - O - acetyldammar - 24 - ene - $3{\beta},12{\beta},20(S)$-trial as aglycon was unsuccessful. Glycosylation of 3, 12 - diketone of betulafolienetriol followed by $NaBH_4$ reduction yielded the $20-O-{\beta}-D-glucopyranoside\;of\;dammar-24-ene-3{\beta},12{\alpha},$ 20(S)-triol, the $12{\alpha}-epimer$ of 20(S) - protopanaxadiol. Moreover, a number of semisynthetic ocotillol - type glucosides, analogs of natural pseudoginsenosides, have been prepared.

• Bulletin of the Korean Chemical Society
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• v.25 no.12
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• pp.1791-1800
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• 2004

Angiotensin I, a model decapeptide, was glycosylated and partially hydrolyzed with HCl (6 N, 80 $^{\circ}C$, 4 h), aminopeptidase, and carboxypeptidase Y. A single peptide mass map obtained from truncated peptides in the partial acid hydrolysate of angiotensin and its glycosylation product mixture by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry enabled sequencing of angiotensin by a combinatorial procedure. MALDI-TOF and electrospray ionization (ESI) tandem mass spectrometric results indicate that both the N-terminal amino group of aspartic acid and the guanidinium group of the second residue arginine are glycosylated.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.143-144
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• 2005

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2005.10a
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• pp.143-144
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• 2005

• BMB Reports
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• v.44 no.10
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• pp.653-658
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• 2011

Sclerotinia sclerotiorum fungus has three endoxylanases induced by wheat bran. In the first part, a partial xylanase sequence gene (90 bp) was isolated by PCR corresponding to catalytic domains (${\beta}5$ and ${\beta}6$ strands of this protein). The high homology of this sequence with xylanase of Botryotinia fuckeliana has permitted in the second part to amplify the XYN1 gene. Sequence analysis of DNA and cDNA revealed an ORF of 746 bp interrupted by a 65 bp intron, thus encoding a predicted protein of 226 amino acids. The mature enzyme (20.06 kDa), is coded by 188 amino acid (pI 9.26). XYN1 belongs to G/11 glycosyl hydrolases family with a conserved catalytic domain containing $E_{86}$ and $E_{178}$ residues. Bioinformatics analysis revealed that there was no Asn-X-Ser/Thr motif required for N-linked glycosylation in the deduced sequence however, five O-glycosylation sites could intervene in the different folding of xylanses isoforms and in their secretary pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10875-10878
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• 2015

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.