• Title/Summary/Keyword: N-glycoprotein

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Structural Analysis of Oligosaccharides of a Plant Glycoprotein (식물 유래 당단백질의 당질 구조 분석)

  • Bae, Jae-Woo;Park, Byung-Tae;Yoon, Doo-Chun;Kim, Joo-Young;Hwang, Hye-Sung;Park, Hyun-Joo;Na, Jong-Chun;Kim, Ha-Hyung
    • YAKHAK HOEJI
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    • v.54 no.6
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    • pp.449-454
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    • 2010
  • The glycosylation of glycoproteins from mammalian or plants can affect their efficacy, stability, solubility, and half-life. In the present study, we investigated plant glycosylation and their relative intensity (%) in a plant carbohydratebinding protein with the hemagglutination and antiproliferative activities. The hemagglutination activity on the deglycosylated protein was decreased as a 16-fold than that of intact glycoprotein. Using the HPLC with fluorescence detector and mass spectrometer, the major eight bi- or triantennary oligosaccharides containing xylose, fucose, mannose, galactose, and N-acetylglucosamine were identified and structurally characterized. The present results indicate that the oligosaccharides on this plant glycoprotein is necessary for their own property.

Analysis of the spike glycoprotein gene and nonstructural protein gene of transmissible gastroenteritis virus using PCR and RFLP analysis (PCR과 RFLP분석을 이용한 transmissible gastroenteritis virus의 spike glycoprotein gene과 nonstructural protein gene의 분석)

  • Kwon, Hyuk-moo
    • Korean Journal of Veterinary Research
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    • v.36 no.3
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    • pp.627-633
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    • 1996
  • To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

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Cloning of cDNA Encoding PAS-4 Glycoprotein, an Integral Glycoprotein of Bovine Mammary Epithelial Cell Membrane

  • Hwangbo, Sik;Lee, Soo-Won;Kanno, Chouemon
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.4
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    • pp.576-584
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    • 2002
  • Bovine PAS-4 is an integral membrane glycoprotein expressed in mammary epithelial cells. Complementary DNA (cDNA) cloning of PAS-4 was performed by reverse-transcriptase polymerase chain reaction (RT-PCR) with oligonucleotide probes based on it's amino terminal and internal tryptic-peptides. The cloned PAS-4 cDNA was 1,852 nucleotides (nt) long and its open reading frame (ORF) was encoded 1,413 base long. The deduced amino acid sequence indicated that PAS-4 consisted of 471 amino acid residues with molecular weight of 52,796, bearing 8 potential N-glycosylation sites and 9 cysteine residues. Partial bovine CD36 cDNA from liver also was sequenced and the homology of both nucleotide sequence was 94%. Most of the identical amino acid residues were in the luminal/extracellular domains. Contrary to PAS-4, bovine liver CD36 displays 6 potential N-glycosylation sites, which were located, except for those at positions 101 and 171, at same positions as PAS-4 cDNA. Cysteine residues of PAS-4 and CD36 were same at position and in numbers. Northern blot analysis showed that PAS-4 was widely expressed, although its mRNA steady-state levels vary considerably among the analyzed cell types. PAS-4 possessed hydrophobic amino acid segments near the amino- and carboxyl-termini. Two short cytoplasmic tails of the amino- and carboxyl-terminal ends constituted of a 5-7 and 8-11 amino acid residues, respectively.

Glyco-engineering of Biotherapeutic Proteins in Plants

  • Ko, Kisung;Ahn, Mi-Hyun;Song, Mira;Choo, Young-Kug;Kim, Hyun Soon;Ko, Kinarm;Joung, Hyouk
    • Molecules and Cells
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    • v.25 no.4
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    • pp.494-503
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    • 2008
  • Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.

Effect of ${\alpha}$-Glycosidase Inhibitor in Multidrug Resistant Cell Lines

  • Paek, Nam-Soo;Namgung, Jun;Lee, Jung-Joon;Choi, Yong-Jin;Kim, Tae-Han;Kim, Kee-Won
    • BMB Reports
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    • v.31 no.3
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    • pp.269-273
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    • 1998
  • The objective of this study was to evaluate the reversal of multi drug resistance of human cell lines by specific inhibitors of ${\alpha}-glycosidase$ and mannosidases that had been reported to be involved in N-linked oligosaccharide processing of glycoproteins. N-methyldeoxynojirimycin, I-deoxynojirimycin, and castanospermine, which were known to be potent inhibitors of both ${\alpha}-glycosidase$ I and II, showed no activity against the multidrug resistant phenotype of the cell lines of SNU1DOX, KB-V1, and MCF-7/ADR. In contrast, I-deoxymannojirimycin, an inhibitor of mannosidase I, resulted in a slight reversal for the vinblastine resistance of the KB-V1 cell line, but did not show any activity toward the other cell lines. Parallel experiments with tunicamycin, an inhibitor of N-linked glycosylation, also resulted in no significant changes in multidrug resistant (MDR) phenotype of the cell lines tested in this work. These observations suggest that the unglycosylation of P-glycoprotein associated with the inhibitor treatments might not be correlated with the reversal of multidrug resistance of the cell lines tested in this study.

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Antimutagenic and Anticancer Effects of Glycoprotein and Chondroitin Sulfates from Sea Cucumber(Stichopus japonicus) (해산 극피동물 중의 당단백질의 특성과 이용 II. 해삼당단백질과 황산콘드로이친의 항돌연변이 및 항암효과)

  • 류홍수;문정혜;양훈석;서재수
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.27 no.2
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    • pp.350-358
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    • 1998
  • The antimutagenic and anticancer activities of glycoprotein(GP) and chondroitin sulfate(CS) from sea cucumbers were studied using Ames mutagenicity test and human cancer cells culture test. The GP's inhibitory effect toward aflatoxin B1(AFB1) and 3, 2'-dimethyl-4-amino-biphenyl(DMAB) increased with the higher added concentrations up to 5% level(w/w) regardless fractionation methods. The GP from sea cucumbers through DEAE-cellulose column chromatography showed an inhibitory effect ranged from 84 to 98%, and the maximum antimutagenicities resulted in red sea cucumber with 98% (AFB1) and 95% (DMAB). But 5% level of CS from various sea cucumbers had an inhibitory effect toward those both indirect mutagens ranged from 79% to 85%. However, in case of direct mutagens(N-methyl-N'-nitro-N-nitrosoguanidine, MNNG and 4-nitroquinoline-1-oxide, 4-NQO), the GP's inhibitory effect was 55∼78% and the CS had a low inhibitory effect(58∼70%) at the added level of 5%. The GP from sea cucumbers exhibited the strong inhibitory effects with 89∼95% and 82∼92% on the growth of HT-29 human crcinoma cells and AZ-521 human gastric cancer cells (at 5% level).

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Synthesis and Biological Evaluation of Phenoxy-N-phenylacetamide Derivatives as Novel P-glycoprotein Inhibitors

  • Lee, Kyeong;Roh, Sang-Hee;Xia, Yan;Kang, Keon-Wook
    • Bulletin of the Korean Chemical Society
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    • v.32 no.10
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    • pp.3666-3674
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    • 2011
  • Overexpression of P-glycoprotein (Pgp) is associated with multidrug resistance (MDR) of tumor cells to a number of chemotherapeutic drugs. Pgp inhibitors have been shown to effectively reverse Pgp-mediated MDR. We prepared a series of phenoxy-N-phenylacetamide derivatives and tested for their ability to inhibit Pgp as potential MDR reversing agents, using a Pgp over-expressing MCF-7/ADR cell line. Some of the synthesized compounds exhibited moderate to potent reversal activity. Of note, compound 4o showed a 3.0-fold increased inhibition compared with verapamil, a well-known Pgp inhibitor. In addition, co-treatment of the representative compound 4o and a substrate anticancer agent doxorubicin resulted in a remarkable increase in doxorubicin's antitumor effect and inhibition of DNA synthesis in the MCF-7/ADR cell line. Taken together, these findings suggest that compound 4o could be a useful lead for development of a novel Pgp inhibitor for treatment of MDR.

Levels of Viral Glycoprotein Provide a Measure of Modulated Chemotherapeutic Effect

  • Shin, Jaeyong;Yoon, Yeon-Sook;Pyo, Suhkneung
    • Biomolecules & Therapeutics
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    • v.7 no.3
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    • pp.216-220
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    • 1999
  • A chemosensitivity assay with small replicate Mm5mt/cl C3H mammary tumor cell cultures was developed to determine whether changes in viral antigen expression and release into culture fluids could be utilized as an in vitro measure of modulating drug effect. The 52,000 MW viral envelope glycoprotein (gp52) of the mouse mammary tumor virus (MMTV) was measured in culture fluids of control and drug-treated cultures while cell density was simultaneously determined by cell staining and OD 664 nm determination. While extra-cellular gp52 levels and cell density progressively increased over 72 hours for control cultures, declines in both parameters provided dual measures of effect for combination [N(phophonacetyl-L-aspartic acid)+5-fluorouracil], combination 〔N(phophonacetyl-L-aspartic acid )+5-fluoro-5'-deoxyuridine〕and single component treatment of this combination. At each treated time point, thesecombinations begin to produce a greater decline in both cell density and gp52 levels as compared to single drug treatments. These results indicate that N(phopho-nacetyl-L-aspartic acid) in combination can enhance the effectiveness of single drug.

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Analysis of Sugar Chain Structure of PAS-7 Glycoprotein from Bovine Milk Fat Globule Membrane by US RAAM 2000 (OGS RAAM2000을 이용한 유지방구막 PAS-7 당단백질의 당쇄구조 해석)

  • 석진석
    • Food Science of Animal Resources
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    • v.21 no.4
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    • pp.367-373
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    • 2001
  • Glycoproteins PAS-6(50 kDa) and -7(47 kDa) from the bovine milk fat globule membrane share a common protein core but differ in their carbohydrate moiety. We have analyzed and proposed the structures of the N-linked sugar chains of PAS-7 by Oxford Glyco System(OGS) RAAM2000. The N-linked sugar chains were liberated from PAS-7 by hydrazinolysis and, after modifying the reducing ends with 2-aminobenzamide(2-AB), were separated into one neutral(7N, 55%) and two acidic(7M, mono-, 43%; 7D, di-, 2%) sugar chain groups. 7N was finally separated into 5 chains(a, b, c, d, and e), respectively. The structure of this 2AB-neutral sugar chain was determined by sugar analysis, exoglycosidase digestion with OGS glycosidase Kit and OGS RAAM2000 system. The results show that fraction e was the same of reported 7N1A, the biantennary complex type with a fucose on reducing end and two N-acetyllactosamine branch on non-reducing end. Therefore, it was proved that OGS RAAM2000 method is in conformity with conventional analysis of sugar chain structure from bovine PAS-7.

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Isolation of cDNAs for Gonadotropin-II of Flounder (Paralichthys olivaceus) and Its Expressions in Adult Tissues

  • Lee, Jae-Hyung;Nam, Soo-Wan;Kim, Young-Tae
    • Journal of Microbiology and Biotechnology
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    • v.13 no.5
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    • pp.710-716
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    • 2003
  • Gonadotropin (GTH) is a pituitary glycoprotein hormone that regulates gonadal development in vertebrates. In teleosts, two types of gonadotropins, GTH-I and GTH-II, are produced in the pituitary, and they comprised of common ${\alpha}$ and distinct ${\beta}$ subunits. In the present study, the cDNAs encoding GTH ${\alpha}\;and\;GTH-II{\beta}$ subunits were cloned and sequenced from flounder (Paralichthys olivaceus) pituitary cDNA library. The nucleotide sequence of the a subunit was 619 bp long, encoding 124 amino acids, and that of the $GTH-II{\beta}$ subunit was 538 bp long, encoding 145 amino acids. GTH subunits had well conserved cysteines, when aligned with other members of the glycoprotein family. The ${\beta}$ subunit of gonadotropin II ($GTH-II{\beta}$) had a different N-linked glycosylation site. RT-PCR analysis showed an increase of GTH II mRNA levels in association with gonadal development, and also showed that the mRNA expression of the ${\alpha}$ subunit was detected only in tissues from pituitary glands.