In-Depth Characterization of Korean HMOs by Mass Spectrometry

Nari Seo,Jae Han Kim,Hyun Joo An 한국당과학회 2016 한국당과학회 학술대회 Vol.2016 No.01

Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that are highly abundant in human milk. HMOs consisted of 3 to 22 monosaccharides can either be linear or branched. HMOs play an essential role in biological functions such as immune maturation, organ development, and healthy microbial colonization. The biosynthesis and the structure–specific biological role of HMOs is still conundrum. To advance the functional understanding of HMOs, the identification and quantification of them is of great importance. Characterization of Caucasian HMOs has reported, however, structure-specific characterization of Korean HMOs has not been investigated. In this study, we have performed in-depth characterization of Korean-specific HMOs from 22 mothers. By combining rapid MALDI-MS profiling with detailed structural interrogation by nano-LC-MS and MS/MS, Korean HMOs were analyzed. More than sixty HMOs were identified, corresponding to over thirty glycan compositions. Major twenty HMOs were structurally elucidated by CID tandem MS. Interestingly, we found that a few HMOs are presented in only Korean HMOs. Our study is first approach for Korean-specific HMOs and can serve as a recommendation for the artificial nutrition industry for baby formula. Furthermore, creation of the Korean HMOs database would support promoting health.

Development of Analytical Platform to Determine the Quantity of Glycopeptides on mAbs using LC/MRM-MS

Nari Seo,Unyong Kim,Myungjin Oh,Ye Rin Jin,Hyun Joo An 한국당과학회 2016 한국당과학회 학술대회 Vol.2016 No.07

Glycosylation of monoclonal antibody (mAb) therapeutics has critical roles such as biological activity, stability, plasma half-life, and immunogenicity. Therefore, detailed glycomic analyses are important to assess quality, safety, and potency of mAbs. Although mass spectrometry (MS) has emerged as a powerful tool for qualitative glycan analysis, MS-based quantitative analysis for glycome is still limited due to the absence of analytical platform and authentic standards. This study aims to develop the method for quantitative analysis of glycan and site-specific glycoforms directly from mAbs for QA/QC assessment using multiple reaction monitoring (MRM). Here, tryptic glycopeptide on trastuzumab, representative mAb, was selected as a standard for optimization of MRM transitions and instrument parameters of C18-UHPLC/triple quadrupole (QqQ). Three major tryptic glycopeptides having the same peptide moiety (EEQYNSTYR) with three different glycans (G0F, G1F, and G2F) on mAbs were targeted and quantified using MRM. In addition, we measured the absolute amounts of major glycans (G0F, G1F, and G2F) on each mAb (adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab) using FLD response of 2-aminobenzamide (2-AB) labeled glycan standards. Consequently, we found that the abundance of glycopeptide calculated by MRM has highly correlated with absolute amounts of 2-AB labeled glycan by LC-FLD. The developed MRM method can be applied to evaluate mAbs variants and/or biosimilars for both developmental and regulatory purposes.

Low-frequency pulsed electromagnetic field pretreated bone marrow-derived mesenchymal stem cells promote the regeneration of crush-injured rat mental nerve

Seo, NaRi,Lee, Sung-Ho,Ju, Kyung Won,Woo, JaeMan,Kim, BongJu,Kim, SoungMin,Jahng, Jeong Won,Lee, Jong-Ho Medknow PublicationsMedia Pvt Ltd 2018 Neural regeneration research Vol.13 No.1

<P>Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to promote the regeneration of injured peripheral nerves. Pulsed electromagnetic field (PEMF) reportedly promotes the proliferation and neuronal differentiation of BMSCs. Low-frequency PEMF can induce the neuronal differentiation of BMSCs in the absence of nerve growth factors. This study was designed to investigate the effects of low-frequency PEMF pretreatment on the proliferation and function of BMSCs and the effects of low-frequency PEMF pre-treated BMSCs on the regeneration of injured peripheral nerve using <I>in vitro</I> and <I>in vivo</I> experiments. In <I>in vitro</I> experiments, quantitative DNA analysis was performed to determine the proliferation of BMSCs, and reverse transcription-polymerase chain reaction was performed to detect S100 (Schwann cell marker), glial fibrillary acidic protein (astrocyte marker), and brain-derived neurotrophic factor and nerve growth factor (neurotrophic factors) mRNA expression. In the <I>in vivo</I> experiments, rat models of crush-injured mental nerve established using clamp method were randomly injected with low-frequency PEMF pretreated BMSCs, unpretreated BMSCs or PBS at the injury site (1 × 10<SUP>6</SUP> cells). DiI-labeled BMSCs injected at the injury site were counted under the fluorescence microscope to determine cell survival. One or two weeks after cell injection, functional recovery of the injured nerve was assessed using the sensory test with von Frey filaments. Two weeks after cell injection, axonal regeneration was evaluated using histomorphometric analysis and retrograde labeling of trigeminal ganglion neurons. <I>In vitro</I> experiment results revealed that low-frequency PEMF pretreated BMSCs proliferated faster and had greater mRNA expression of growth factors than unpretreated BMSCs. <I>In vivo</I> experiment results revealed that compared with injection of unpretreated BMSCs, injection of low-frequency PEMF pretreated BMSCs led to higher myelinated axon count and axon density and more DiI-labeled neurons in the trigeminal ganglia, contributing to rapider functional recovery of injured mental nerve. These findings suggest that low-frequency PEMF pretreatment is a promising approach to enhance the efficacy of cell therapy for peripheral nerve injury repair.</P>

Voluntary stand-up physical activity enhances endurance exercise capacity in rats

Seo, Dae Yun,Lee, Sung Ryul,Kwak, Hyo-Bum,Seo, Kyo Won,McGregor, Robin A,Yeo, Ji Young,Ko, Tae Hee,Bolorerdene, Saranhuu,Kim, Nari,Ko, Kyung Soo,Rhee, Byoung Doo,Han, Jin The Korean Society of Pharmacology 2016 The Korean Journal of Physiology & Pharmacology Vol.20 No.3

Involuntary physical activity induced by the avoidance of electrical shock leads to improved endurance exercise capacity in animals. However, it remains unknown whether voluntary stand-up physical activity (SPA) without forced simulating factors improves endurance exercise capacity in animals. We examined the effects of SPA on body weight, cardiac function, and endurance exercise capacity for 12 weeks. Twelve male Sprague-Dawley rats (aged 8 weeks, n=6 per group) were randomly assigned to a control group (CON) or a voluntary SPA group. The rats were induced to perform voluntary SPA (lifting a load equal to their body weight), while the food height (18.0 cm) in cages was increased progressively by 3.5 every 4 weeks until it reached 28.5 cm for 12 weeks. The SPA group showed a lower body weight compared to the CON group, but voluntary SPA did not affect the skeletal muscle and heart weights, food intake, and echocardiography results. Although the SPA group showed higher grip strength, running time, and distance compared to the CON group, the level of irisin, corticosterone, genetic expression of mitochondrial biogenesis, and nuclei numbers were not affected. These findings show that voluntary SPA without any forced stimuli in rats can effectively reduce body weight and enhance endurance exercise capacity, suggesting that it may be an important alternative strategy to enhance endurance exercise capacity.

N-glycan Catalogue: Therapeutic Antibodies

Nari Seo,MyungJinOh,Youngsuk Seo,UnyoungKim,HyunJooAn 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.01

Glycosylation of monoclonal antibody (mAb) therapeutics plays vital roles such as biological activity, stability, plasma half-life, and immunogenicity. It has been known that the glycan variations in mAbs are caused by cell-line selection and changes in culture-medium parameters. These variations can lead to changes in drug potency of therapeutic glycoproteins. Therefore, monitoring of glycosylation on protein is required to access the quality, safety, and equivalence of therapeutic antibodies. Here, in order to develop the reference data for quality by design (QbD) in therapeutic antibodies’ production, we performed the absolute quantitation and evaluation of major glycan (G0F, G1F, and G2F) on human immunoglobulin G (IgG) and five representative recombinant mAb drugs including adalimumab, bevacizumab, infliximab, rituximab, and trastuzumab using FLD response of 2-aminobenzamide (2-AB) labeled glycan standards. N-glycans on mAbs were enzymatically released and then labeled by 2-AB reagent. Followed by 2-AB labeled glycans were purified by HILIC-SPE. 2-AB labeled glycans were analyzed by UHPLC-HILIC-FLD which can provide chromatographic glycan separation and quantitation. The calibration curves of glycan standard were linear over the range of about 10 fmole to 10 amoles (3 orders of magnitude) and its linear correlation coefficients were excess of 0.999 (R2). As a result, the concentration of the glycans on commercial mAbs measured 2 to 10 times higher than that of IgG. Additionally, we confirmed that quantitative difference of glycan in each sample. This result can be directly applied to evaluate mAbs variants and/or biosimilars for both developmental and regulatory purposes.

Abnormal variation of serum N-glycan on Bechet’s disease: tracing the structural isomeric distribution

Nari Seo,Nguyen Thi My Tuyen,Joong Kyong Ahn,Hoon-Suk Cha,Kyoung Heon Kim,Hyunjoo An,Jaehan Kim 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.01

Bechet’s disease (BD) is an immune disease which showed a chronic and relapsing systemic vasculitis of unknown etiology. It is characterized by recurrent ulcers and inflammations on oral, genital, uvea area and further vascular, central nervous system (CNS), and gastrointestinal tracks. Bechet’s disease has a wide spectrum of symptoms ranging from benign episodes to more serious complications such as blindness. Although many diagnostic criteria have been developed and revised by experts in the field, diagnosing BD is still complicated and challenging. To discover the diagnostic biomarkers, serum N-glycans from Bechet patient (n=100) and healthy control (n=100) have been compared. Instead of conventional global profiling of serum N-glycome, quantitative ratio between structural isomers were analyzed by UPLC-triple quadruple mass spectrometry. Three acidic glycans with the composition of 5:4:0:1, 5:4:0:2, and 5:4:1:1 (Hex:HexNAc:Fuc:NeuAc) were monitored in MRM mode, then six, two and three isomers were found, respectively. Among eleven bi-antennary glycan isomers, four of them showed high diagnostic efficacy having AUC of ROC curve over 0.98. When the sensitivity was fixed to 90%, corresponding specificities were over 92% to 96%. Another four isomers showed the AUC from 0.84 to 0.94 showing 64%~84% specificity with the sensitivity of 90%. Marker glycans exhibits unique correlations with biological characteristics of cohorts. Two isomers of 5:4:0:1 and one of 5:4:0:2 isomer were able to completely distinguish male and female groups in healthy control cohort. Glycan expression level in female groups were 6 to 10 times higher than those observed in male groups resulting 1.0 AUC in ROC curve. Expression level in patient cohort were 5 to 10-folds higher than those in control group showing complete segregation. Correlation between the glycans were also clear. Groups of isomers with each composition exhibits Pearson’s product over ±0.90 within or between the groups. This study present the novel approach for the disease marker discovery that tracing the isomeric structure of specific N-glycan. As results, the markers with undoubtable quantitative deviation and the correlations reflecting their biological characteristics have been found.

Digging into Human Milk Oligosaccharides by Milk Pasteurization and Freeze-Dry

Nari Seo,Tuyen Nguyen,Ye Rin Jin,Nam Mi Kang,Jae Han Kim,Hyun Joo An 한국당과학회 2017 한국당과학회 학술대회 Vol.2017 No.01

Human breast milk is frequently heated or frozen to restrain microbial growth. Nutrient compounds consisting of carbohydrates, proteins, and lipids are often affected by heating and freezing steps. In this study, we have monitored the change of human milk oligosaccharides (HMOs) after pasteurization and freeze-drying steps. HMOs are a family of structurally diverse unconjugated glycans that are highly abundant in human milk. It is well known that various structures of HMOs are associated with absorption, catabolism and biological functions such that contribute to immune maturation, organ development, and health microbial colonization. However, there is no previous study regarding chemical stability of HMOs during high temperature pasteurization and freeze-dried. Briefly, human milk samples from 3 mothers were pasteurized at 63℃ for 30minutes and freeze-dried at-83℃ for 5days, respectively. HMOs were enriched and profiled by mass spectrometry. Initially the composition and structure of HMOs was assigned by MALDI-TOF/TOF MS. Further, native HMOs were relatively quantified by PGC column-based LC/MS providing an enhanced separation of isomeric structure. More than 30 HMOs have been identified and quantified. We could confirm that pasteurization and freeze-drying of human milk does not influence chemical property of HMOs at all.

Semiglobal Robust Stabilization of Nonlinear Systems Without Vanishing Condition

Nari Bok,Hyungbo Shim,Young I. Son,Jin H. Seo 대한전기학회 2001 KIEE International Transactions on systems and Con Vol.11D No.3

Protective Effects of Chungkookjang Extract on High Glucose Induced Oxidative Stress in LLC-PK₁ Cells

Nari Yi,Kyoung-Chun Seo,Ji-Myung Choi,Eun-Ju Cho,Young-Ok Song,Ji-Sook Han 한국식품영양과학회 2008 Preventive Nutrition and Food Science Vol.13 No.2

This study was designed to investigate the protective effect of a methanol extract of Chungkookjang (CKJ) on high glucose induced oxidative stress in LLC-PK₁ cells (renal tubular epithelial cells), which are susceptible to oxidative stress. Freeze dried CKJ powder was extracted with methanol, and the extract solution was concentrated, and then used in this study. To determine the protective effect of CKJ extract, oxidative stress was induced by exposing of LLC-PK₁ cells to high glucose (30 mM) or normal glucose (5 mM) for 24 hr. Exposure of LLC-PK₁ cells to high glucose for 24 hr resulted in a significant (p<0.05) decrease in cell viability, catalase, SOD and GSH-px activity and a significant (p<0.05) increase in intracellular ROS level and thiobarbituric acid reactive substances (TBARS) formation in comparison to the cells treated with 5 mM glucose. CKJ extract treatment decreased intracellular ROS level and TBARS formation, and increased cell viability and activities of antioxidant enzymes including catalase, SOD and GSH-px in high glucose pretreated LLC-PK₁ cells. These results suggest that CKJ extract may be able to protect LLC-PK₁ cells from high glucose-induced oxidative stress, partially through the antioxidative defense systems.

Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth

Yi, Nari,Kim, Youn Shic,Jeong, Min-Ho,Oh, Se-Jun,Jeong, Jin Seo,Park, Su-Hyun,Jung, Harin,Choi, Yang Do,Kim, Ju-Kon Springer-Verlag 2010 Planta Vol.232 No.3