• Mass Spectrometry Letters
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• 제9권3호
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• pp.67-72
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• 2018

Alkaline phosphatase (AP) is a membrane-bound glycoprotein that is widely distributed in the plasma membrane of cells of various organs and also found in many organisms from bacteria to humans. The complete amino acid sequence and three-dimensional structure of human placental alkaline phosphatase have been reported. Based on the literature data, AP consists of two presumptive glycosylation sites, at Asn-144 and Asn-271. However, it only contains a single occupied N-linked glycosylation site and no occupied O-linked glycosylation sites. Hydrophilic interaction chromatography (HILIC) has been primarily employed for the characterization of the glycan structures derived from glycoproteins. N-glycan structures from human placental alkaline phosphatase (PLAP) were investigated using HILIC-Orbitrap MS, and subsequent data processing and glycan assignment software. 16 structures including 10 sialylated N-glycans were identified from PLAP.

• 한국발생생물학회지:발생과생식
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• 제25권4호
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• pp.199-211
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• 2021

Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGβ/αΔ56, substitution of α-subunit⁵⁶ N-linked glycosylation site; eCGβ-D/α, deletion of the O-linked glycosylation sites at the β-subunit, and eCGβ-D/αΔ56, double mutant. We produced recombinant eCG (rec-eCG) proteins in Chinese hamster ovary suspension (CHO-S) cells. We examined the biological activity of rec-eCG proteins in CHO-K1 cells expressing the eLH/CG receptor and found that signal transduction activities of deglycosylated mutants remarkably decreased. The EC₅₀ levels of eCGβ/αΔ56, eCGβ-D/α, and eCGβ-D/αΔ56 mutants decreased by 2.1-, 5.6-, and 3.4-fold, respectively, compared to that of wild-type eCG. The Rmax values of the mutants were 56%-80% those of wild-type eCG (141.9 nmol/10⁴ cells). Our results indicate that the biological activity of eCG is greatly affected by the removal of N- and O-linked glycosylation sites in cells expressing eLH/CGR. These results provide important information on rec-eCG in the regulation of specific glycosylation sites and improve our understanding of the specific biological activity of rec-eCG glycosylation sites in equidaes.

• Journal of Microbiology
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• 제45권1호
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• pp.41-47
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• 2007

The Hantaan virus (HTNV) is an enveloped virus that is capable of inducing low pH-dependent cell fusion. We molecularly cloned the viral glycoprotein (GP) and nucleocapsid (NP) cDNA of HTNV and expressed them in Vero E6 cells under the control of a CMV promoter. The viral gene expression was assessed using an indirect immunofluorescence assay and immunoprecipitation. The transfected Vero E6 cells expressing GPs, but not those expressing NP, fused and formed a syncytium following exposure to a low pH. Monoclonal antibodies (MAbs) against envelope GPs inhibited cell fusion, whereas MAbs against NP did not. We also investigated the N-linked glycosylation of HTNV GPs and its role in cell fusion. The envelope GPs of HTNV are modified by N-linked glycosylation at five sites: four sites on G1 (N134, N235, N347, and N399) and one site on G2 (N928). Site-directed mutagenesis was used to construct eight GP gene mutants, including five single N-glycosylation site mutants and three double-site mutants, which were then expressed in Vero E6 cells. The oligosaccharide chain on residue N928 of G2 was found to be crucial for cell fusion after exposure to a low pH. These results suggest that G2 is likely to be the fusion protein of HTNV.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.35-35
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• 2005

Recombinant human $interferon-{\beta}-1a(rIFN-{\beta})$ is a single glycosylated protein (at N80, 1N) with anti-viral activity. However, present drugs have a relatively short serum half-life of $rIFN-{\beta}$, thus patients suffer from frequent $infections.^{1)}$ To improve its half-life, eight glycosylation analogs were prepared, which have additional N-linked glycosylation consensus sequences (N-X-T/S) within the $IFN-{\beta}$ molecule and/or at C-terminal. Each $rIFN-{\beta}$ analog was examined for the presence of additional N-linked glycosylation and the maintenance of anti-viral activity in CHO cells. The molecular weights of five analogs were not changed. However, two analogs, R27T within $rIFN-{\beta}$ (27 kDa, 2N) and GNITVNITV at C-terminal (29kDa, 2N), showed a clear increase in molecular weights, compared to native $rIFN-{\beta}$ (23 kDa, 1N). And another combined analog of R27T+GNITVNITV showed increased molecular weight (33 kDa, 3N). It was confimed that the molecular weight increment of analogs was caued by the N-linked glycosylation with the treatment of N-glycansae. In the case of anti-viral activity, the analog GNITVNITV showed a reduction in activity compared to native $IFN-{\beta}$, whereas the analogs R27T and R27T+GNITVNITV were found to have distinctly increased activities. Pharmacokinetic study in rats also disclosed that the analogs R27T and R27T+GNITVNITV had 2 3 fold increased serum half-life, respectively. In conclusion, the addition of N-linked glycosylation in $rIFN-{\beta}$ increased serum half-life, thereby its less frequent administration will be expected.

• BMB Reports
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• 제45권11호
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• pp.623-628
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• 2012

Tissue inhibitor of metalloproteinases (TIMPs; TIMP-1, -2, -3 and -4) are endogenous inhibitor for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. Unlike under normal conditions, the imbalance between MMPs and TIMPs is associated with various diseased states. Among TIMPs, TIMP-1, a 184-residue protein, is the only N-linked glycoprotein with glycosylation sites at N30 and N78. The structural analysis of the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1 suggests new possibilities of the role of TIMP-1 glycan moieties as a tuner for the proteolytic activities by MMPs. Because the TIMP-1 glycosylation participate in the interaction, aberrant glycosylation of TIMP-1 presumably affects the interaction, thereby leading to pathogenic dysfunction in cancer cells. TIMP-1 has not only the cell proliferation activities but also anti-oncogenic properties. Cancer cells appear to utilize these bilateral aspects of TIMP-1 for cancer progression; an elevated TIMP-1 level exerts to cancer development via MMP-independent pathway during the early phase of tumor formation, whereas it is the aberrant glycosylation of TIMP-1 that overcome the high anti-proteolytic burden. The aberrant glycosylation of TIMP-1 can thus be used as staging and/or prognostic biomarker in colon cancer.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.143-144
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• 2005

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.143-144
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• 2005

• Applied Microscopy
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• 제23권2호
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• pp.84-96
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• 1993

This study was to investigate the effects of maternal diabetes on the lung tissue of the fetal rat using lectin histochemistry and electron microscope technique. Maternal diabetes was induced by intraperitoneal injection of streptozotocin (75 mg/kg the body weight) into pregnant Sprague-Dawley rats on the 7th day of gestation. Fetuses of streptozotocin induced diabetic rats exhibited delayed lung maturation and reduced air space. In lectin histochemistry, the binding of Maclura pomifera (MPA) to fetal lungs from diabetic mothers was reduced, but no significant changes in the bindings of Concanavalin A (Con A), Wheat germ agglutinin (WGA), Ricinus communis I (RCA I) and Griffonia simplicifolia (GSI-$B_4$) were noted. Because the MPA has affinity to terminal N-acetyl-D-galactosamine residues constantly linked O-glycosidically to serine or threonine, the present findings may indicates that maternal diabetes interfere with the processes of O-linked glycosylation in fetal rat lung.

• 대한화장품학회지
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• 제35권1호
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• pp.73-78
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• 2009

닭의장풀(Commelina communis L. Dayflower) 추출물의 미백효능을 확인하기 위하여 B16 melanoma 세포를 이용하여 melanin 생성에 관련된 다양한 실험을 실시하였다. 닭의장풀 추출물은 버섯 tyrosinase 활성 실험에서 저해 효과를 보이지 않았으나, melanin 생성 저해효과를 나타내었다. B16-F10 멜라노마 세포를 이용한 활성시험 결과에서, 닭의장풀 추출물은 1,000 ${\mu}g/mL$의 농도에서 약 32%의 멜라닌 생성을 억제하였으며, 세포내 티로시나제 활성 억제 능도 우수하여 닭의장풀 추출물 250 ${\mu}/mL$ 이상의 농도에서 50% 이상의 저해 효과를 보였다. 추출물의 생성 기작에 대한 영향을 조사한 결과, melanin 합성의 key protein 인 tyrosinase 발현의 우수한 저해 능력을 보였고, tyrosinase related protein-1 (TRP-1)의 발현 억제와 tyrosinase related protein-2 (TRP-2)의 N-glycosylatin 억제를 통해 melanin 합성이 억제되는 것으로 분석되었다. 따라서 닭의장풀 추출물은 melanin 합성에 필수적인 효소(tyrosinase, TRP-1)의 발현 저해 및 TRP-2의 N-glycosylation 억제를 통해 미백 효과를 나타내는 것으로 확인되었으며, 이에 따라 본 추출물은 melanin 합성의 효소 경로를 저해하는 미백 소재로 활용할 수 있을 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제26권7호
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• pp.1163-1172
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• 2016

The Ω-loop is a nonregular and flexible structure that plays an important role in molecular recognition, protein folding, and thermostability. In the present study, molecular dynamics simulation was carried out to assess the molecular stability and flexibility profile of the porcine trypsin structures. Two Ω-Loops (fragment 57-67 and fragment 78-91) were confirmed to represent the flexible region. Subsequently, glycosylation site-directed mutations (A73S, N84S, and R104S) were introduced within the Ω-loop region and its wing chain based on its potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences) and structure information to improve the thermostability of trypsin. The result demonstrated that the half-life of the N84S mutant at 50℃ increased by 177.89 min when compared with that of the wild-type enzyme. Furthermore, the significant increase in the thermal stability of the N84S mutant has also been proven by an increase in the T_m values determined by circular dichroism. Additionally, the optimum temperatures of the wild-type enzyme and the N84S mutant were 75℃ and 80℃, respectively. In conclusion, we obtained the thermostability-improved enzyme N84S mutant, and the strategy used to design this mutant based on its structural information and N-linked glycosylation modification could be applied to engineer other enzymes to meet the needs of the biotechnological industry.