• Mass Spectrometry Letters
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• 제15권1호
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• pp.1-25
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• 2024

Protein glycosylation, a highly significant and ubiquitous post-translational modification (PTM) in eukaryotic cells, has attracted considerable research interest due to its pivotal role in a wide array of essential biological processes. Conducting a comprehensive analysis of glycoproteins is imperative for understanding glycoprotein bio-functions and identifying glycosylated biomarkers. However, the complexity and heterogeneity of glycan structures, coupled with the low abundance and poor ionization efficiencies of glycopeptides have all contributed to making the analysis and subsequent identification of glycans and glycopeptides much more challenging than any other biopolymers. Nevertheless, the significant advancements in enrichment techniques, chromatographic separation, and mass spectrometric methodologies represent promising avenues for mitigating these challenges. Numerous substrates and multifunctional materials are being designed for glycopeptide enrichment, proving valuable in glycomics and glycoproteomics. Mass spectrometry (MS) is pivotal for probing protein glycosylation, offering sensitivity and structural insight into glycopeptides and glycans. Additionally, enhanced MS-based glycopeptide characterization employs various separation techniques like liquid chromatography, capillary electrophoresis, and ion mobility. In this review, we highlight recent advances in enrichment methods and MS-based separation techniques for analyzing different types of protein glycosylation. This review also discusses various approaches employed for glycan release that facilitate the investigation of the glycosylation sites of the identified glycoproteins. Furthermore, numerous bioinformatics tools aiding in accurately characterizing glycan and glycopeptides are covered.

• 한국가축번식학회지
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• 제27권4호
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• pp.299-307
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• 2003

The effects of additions/deletions in glycosylated residues of recombinant human EPO (rhEPO) produced in CHO-K1 on their secretion were examined. hEPO cDNA was amplified from human liver mRNA and cloned into the pCR2.1 TOPO. Using overlapping-extension site-directed mutagenesis method, glycosylation sites at 24th, 38th, 83rd, and 126th were respectively or accumulatively removed by substituting its asparagine (or serine) with glutamine. To add novel glycosylation sites, 69 and 105th leucine was mutated to asparagine. Mutant and wild type rhEPO constructs were cloned into the pcDNA3 expression vector with CMV promoter and transfected into CHO cell line, CHO-K1, to produce mutant rhEPO mutant rhEPO proteins. Enzyme-linked immunosorbant assay (ELISA) and Western analysis with monoclonal anti-EPO antibody were performed using supernatants of the cultures showing transient and stable expressions respectively. Addition of novel glycosylation reduced rhEPO secretion dramatically while deletion mutants had little effect except some double deletion mutants ($\Delta$24/83 and $\Delta$38/83) and triple mutant ($\Delta$24/38/83). This fact suggests that not single but combination of changes in glycosyl groups affect secretion of rhEPO in cell culture, possibly via changes in their conformations.

• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1639-1648
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• 2017

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, anti-inflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP-${\alpha}-{\text\tiny{D}}$-glucose or UDP-${\alpha}-{\text\tiny{D}}$-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'-O-${\beta}$-glucoside, curcumin 4',4"-di-O-${\beta}$-glucoside, curcumin 4'-O-${\beta}$-2-deoxyglucoside, and curcumin 4',4"-di-O-${\beta}$-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'-O-${\beta}$-glucoside and curcumin 4'-O-${\beta}$-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

• Mass Spectrometry Letters
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• 제12권3호
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• pp.66-75
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• 2021

Interleukin-4 (IL-4) and IL-13 are cytokines secreted by immune cells. Cytokines induce the proliferation of macrophages or promote the differentiation of secretory cells. The initiation and progression of allergic inflammatory diseases, such as asthma, are dependent on cytokines acting through related receptor complexes. IL-4 and IL-13 are N-glycoproteins. Glycan structures in glycoproteins play important roles in protein folding, protein stability, enzymatic function, inflammation, and cancer development. Therefore, the glycan structure of IL-4 and IL-13 needs to be elucidated in detail for the development of effective therapies. We report the first attempt to characterize the site-specific N-glycosylation of recombinant IL-4 and IL-13 via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The tandem mass spectra of intact N-glycopeptides were identified using the Integrated GlycoProteome Analyzer (I-GPA) platform, which can automatically and rapidly analyze multiple N-glycopeptides, including their glycan composition and amino acid sequences. The recombinant IL-4 and IL-13 were identified with amino acid sequence coverages of 84% and 96%, respectively. For IL-4, 52 glycoforms on one N-glycosylation site were identified and quantified. In IL-13, 232 N-glycopeptides from three N-glycosylation sites were characterized, with the site Asn52 being the most extensively glycosylated (~80%). The complex glycans were the most abundant glycan on IL-4 and IL-13 (~96% and 91%, respectively), and the biantennary glycans were the most abundant in both recombinant IL-4 and IL-13 proteins.

• Clinical and Experimental Pediatrics
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• 제49권4호
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• pp.431-438
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• 2006

목 적 : 새로운 암억제 유전자로 알려진 IGFBP-3의 주요 기능은 IGF-I과 IGF-II와 결합을 하여 IGF의 기능을 조절하는 IGF dependent mechanism과 IGFBP-3 자체가 IGF와 결합과는 무관하게 세포의 apoptosis를 유도하는 IGF independent mechanism이 보고되었다. 암억제 유전자 p53의 대표적인 항암 기전의 하나는 직접 IGFBP-3의 발현을 증가시키며, 발현된 IGFBP-3는 암세포의 apoptosis를 유도시킨다. IGFBP-3의 항암작용은 보고되었지만, IGFBP-3의 변역 후 변형에 의한 항암기전은 전혀 밝혀져 있지 않다. 본 연구에서는 p53의 항암기전과 관련하여 IGFBP-3의 당화에 관련된 기전을 밝히고, IGFBP-3 당화의 의미를 규명하였다. 방 법 : 실험 세포주로는 p53의 변이를 보이며 p53의 발현이 일반세포에 비교하여 낮은 특징을 갖고 있는 사람의 유방암세포인 MDA-MB-231를 사용하였으며, Ad/p53과 Ad/IGFBP-3 아데노바이러스를 감염시킨 후 IGFBP-3의 발현 변화와 apoptosis 기전을 분석하였다. glycosylation 억제제로 알려져 있는 tunicamycin을 처리하여 당화의 정도를 조사하였다. 결 과 : 실험 세포에 Ad/p53을 감염시켜 p53을 발현시킨 결과 성장의 억제와 apoptosis가 유도되었고, IGFBP-3의 발현이 현저하게 증가되었으며, 특히 IGFBP-3의 당화 형태를 증가시켰다. 당화된 IGFBP-3의 증가는 세포의 apoptosis의 유도가 촉진되었으며, 이러한 IGFBP-3의 당화는 p53과 IGFBP-3의 발현을 동시에 유도시킨 결과 더욱 항진되었다. 결 론 : 이상의 연구에서 IGFBP-3의 암억제 능력의 향상은 p53에 의한 IGFBP-3의 당화의 증가를 통하여 안정화됨으로서 나타나고 있음을 알 수 있었다. 이는 p53과 IGFBP-3를 이용한 혼합유전자 치료가 가능할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1949-1954
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• 2007

A simple way to prevent protein hyperglycosylation in Hansenula polymorpha was found. When glucose oxidase from Aspergillus niger and carboxymethyl cellulase from Bacillus subtilis were expressed under the control of an inducible methanol oxidase (MOX) promoter using methanol as a carbon source, hyperglycosylated forms occurred. In contrast, MOX-repressing carbon sources (e.g., glucose, sorbitol, and glycerol) greatly reduced the extent of hyperglycosylation. Carbon source starvation of the cells also reduced the level of glycosylation, which was reversed to hyperglycosylation by the resumption of cell growth. It was concluded that the proteins expressed under actively growing conditions are produced as hyperglycosylated forms, whereas those under slow or nongrowing conditions are as short-glycosylated forms. The prevention of hyperglycosylation in the Hansenula polymorpha expression system constitutes an additional advantage over the traditional Saccharomyces cerevisiae system in recombinant production of glycosylated proteins.

• Journal of Microbiology and Biotechnology
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• 제30권1호
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• pp.11-20
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• 2020

Quercetin and its derivatives are important metabolites that belong to the flavonol class of flavonoids. Quercetin and some of the conjugates have been approved by the FDA for human use. They are widely distributed among plants and have various biological activities, such as being anticancer, antiviral, and antioxidant. Hence, the biosynthesis of novel derivatives is an important field of research. Glycosylation and methylation are two important modification strategies that have long been used and have resulted in many novel metabolites that are not present in natural sources. A strategy for modifying quercetin in E. coli by means of glycosylation, for example, involves overexpressing respective glycosyltransferases (GTs) in the host and metabolic engineering for increasing nucleoside diphosphate sugar (NDP-sugar). Still others have used microorganisms other than E. coli, such as Streptomyces sp., for the biotransformation process. The overall study of the structural activity relationship has revealed that modification of some residues in quercetin decreased one activity but increased others. This review summarizes all of the information mentioned above.

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권1호
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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.