Earticle

Capsosiphon fulvescens (commonly called Maesaengyi in Korea), a green algae, has been traditionally used in Korea for centuries as a functuinal food. Polysaccahrides (SPS-CF, 385 kDa) isolated from Capsosiphon fulvescens have been shown to exhibit various biological activities, including anti-coagulanting, immune modulating and anti-cancer properties. However, anti-osteoporosis activity of SPS-CF has been rarely explored. In this study, we investigated its anti-osteoporotic effects using RAW 264.7 osteoclastic cells. The results showed that SPS-CF have been dose-dependently reduced the TRAP activity in the RAW 264.7 cells, with an approximately 59 % reduction at 50 μg/ml, while SPS-CF did not reduced cell viability. The SPS-CF treatment reduced actin organization in RAW 264.7 cells and significantly decreased protein level of Carbonic anhydraseⅡ. The inhibition of osteoclast differentiation shown to be mediated by suppression of RANKL signaling pathway, as evidenced by decreased TRAF6, gelsolin and phosphorylation of Src. These results SPS-CF demonstrated for the first time that SPS-CF inhibits the osteoclast differentiation, suggestubg that SPS-CF can be a functional ingredient for health beneficial foods or therapeutic agents to treat osteoporosis.

Gastric cancer has one of the highest cancer mortality rates worldwide, largely because of di fficulties in early-stage detection. Aberrant glycosylation in serum proteins is related with many human diseases including inflammation and various types of cancer. Aberrant glycosylation is desirable in order to improve the specificity and sensitivity for clinical use. Here, we combined protein-specific immunoaffinity purification, glycan release, and MS analysis to examine haptoglobin glycosylation of gastric cancer patients for glyco-markers. Interestingly, abundances of several tri- and tetra-antennary fucosylated N-glycans were increased in gastric cancer patients. Additionally, structural analysis via LC/MS/MS demonstrated that the fucosylated complex type N-glycans were mainly decorated with antenna fucose. In this study, we developed a targeted glycoproteomic approach using chip-based nano LC-QTOF MS and MS/MS following antibody-assisted targeted purification to discover glycan signatures of serum haptoglobin for gastric cancer. We could further obtain a specific structure of fucosylated molecules that are potential glyco-markers for gastric cancer via LC/MS/MS. The current study demonstrates that glycomic profiling of targeted serum haptoglobin via LC/MS and LC/MS/MS may be used as a powerful platform to monitor the specific glycosylation associated with gastric cancer.

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was codon optimesed and expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. Among various polyols, GoSLDH exhibited highest activity towards D-sorbitol with Km and kcat values of 38.9 mM and 3820 S-1, respectively. The enzyme showed strong preference for NADP+ (only 2.5% relative activity with NAD+). Sequence and structure analysis of GoSLDH along with the biochemical properties confirmed that GoSLDH belongs to the family of NADP+ dependent polyol specific long-chain sorbitol dehydrogenase. Isothermal titration calorimetry showed that the protein binds strongly to D-sorbitol compared with other L-sorbose producing enzymes and substrate docking analysis confirmed the higher turnover rate. High oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of the GoSLDH was significantly improved up to 6-folds after immobilization on the silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, the immobilized GoSLDH should be useful for industrial production of L-sorbose from D-sorbitol.

Therapeutic proteins play an increasingly important role in the pharmaceutical industry. Glycosylation is a very critical modification of therapeutic proteins, known to significantly modulate yield, bioactivity, solubility, immunogenicity and efficacy. Most biotherapeutic proteins developed to date have been produced using the mammalian model. Although mammalian system can produce glycosylation similar to human, they also produce non-human glycosylation that can cause immune responses and safety issue. Firstly, we explored difference of glycosylation between human and mouse to design glycan-humanized mouse model. We selectively enriched glycans from blood cell (red blood cell and white blood cell) membrane and analyzed them by porous graphitized carbon (PGC) based nano-LC/MS which enables to confirm not only glycan composition but also structure isomer. Hundreds of glycan structures were directly detected by accurate mass and structurally elucidated by MS/MS, revealing the species-specific structure. These results could be targets for new signatures distinguishing the species and the beginning for design of glycan-humanized mouse model.

Quercetin is suggested to be potentially beneficial for the prevention of various diseases. Enzymatically modified isoquercitrin was prepared by glycosylation of quercetin and the subsequent of oligoglucosylation of quercetin 3-O-β-glucoside (isoquercitrin) produced from quercetin. To enhance the production of isoquercitrin with high specificity, yield, and purity, an enzymatic bioconversion method was developed enzyme UGT78K1 from glycine max, which has quercetin transforming ability. UGT78K1 could effectively transform the quercetin into isoquercitrin. Enzymatically modified isoquercitrin was glucosylated to its oligoglucosyl quercetin 3-O-β-Glucoside by levansucrase of a genetically modified strain of Leuconostoc mesenteroides. levansucrase industrially important glycansucrase from L. mesenteroides.

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.

Pathogen recognition is a fundamental step in host immune response and survives against infections. Pathogen recognition happens by host cell surface receptor which induces downstream immune responses such as opsonization, phagocytosis. Among that glycan-binding receptor in C-type lectin superfamily form recognition molecules which are crucial to innate immunity. Many C-type lectins found in pathogen bind to glycan array, but host cell doesn't have so it makes distinguish self and non-self. However, some pathogen uses this selective recognition for primary infection and subsequent proliferation in a host. DC-SIGN(CD209) initially dendritic cell-specific ICAM-3-grabbing nonintegrin is one of the C-type lectin family dendritic cell surface receptor which binds with pathogen glycan. DC-SIGN can bind with two classes of carbohydrate structures: N-linked high mannose oligosaccharides, fucosylated oligosaccharides. Crystal structure of CRD from DC-SIGN with oligosaccharide reveal CRD forms 1-to-1 complex with high-mannose oligosaccharides. Surface force apparatus(SFA) is used to quantify interaction force of cell surface receptor and glycans on the target membrane. This kind of force measurements can quantify variations in the adhesion energy between DC-SIGN with ligand density which further reveal the role of ligand spacing in the multivalent interactions of CRDs with ligand. By SFA, binding-site geometry and flexibility in DC-SIGN demonstrated and calcium contributions for Dendritic cell receptors on carbohydrate recognition proved.

Most therapeutic proteins are glycosylated. These functional sugar moieties of proteins, so called glycan, are very important factor which determine pharmacological properties of the protein therapeutics. Therefore, regulation of glycosylation of protein is significant for producing Biotherapeutics. Polylactosamine is a basic structure of glycans having repeated structure of N-acetyllactosamine(LacNAc) which composed of Galactose(Gal) and N-acetylglucosamine(GlcNAc). Erythropoietin(EPO) is a heavily glycosylated cytokine that stimulates hematopoiesis. The EPO is known to be cleared much faster in vivo when containing polylactosamine than without it and in vivo activity of the EPO is proportional to the level of GlcNAc branching of its N-glycan. In this point of view, we tried to decrease the expression of polylactosamine structure as well as increase the ratio of the tetra antennary-structure on EPO as a model glycoprotein. We inhibited polylactosamine biosynthesis of recombinant human EPO in CHO cells by using siRNA which targets β3gnt2 gene that synthesize β1,4-Gal and β1,3-GlcNAc branch. As a result, the polylactosamine ratio of EPO was decreased to 2% compared with the 21% of wild type. In addition, the ratio of tetra-antennary structure was up-regulated to 46% compared with the 32% of wild type. This might be promising result for producing therapeutic glycoproteins with high in-vivo stability and activity.

Bioactivity and efficacy of therapeutic protein are dependent on structure-specific glycan attached to the therapeutic proteins. Therefore, precise glycomic characterization and monitoring are essential in the manufacture of therapeutic glycoproteins. However, the inherent structural diversity of glycosylation significantly hinders analysis. Thus, it is challenging to comprehensively characterize structure–specific glycans. We propose isomer-specific LC/MS and MS/MS screening as a method for rapid identification and structural elucidation of biopharmaceutical glycosylation. Biotherapeutic glycoproteins including mAbs, and EPOs were prepared and analyzed. After removing salts and detergents, N-glycans were enzymatically released and purified by graphitized carbon solid phase extraction. PGC chip nano-LC/Q-TOF provided chromatographic isomer separation profiling and structural characterization. Multiple structure isomers including antennary, polylactosamine, alpha galactose epitope, and linkage iosomers were identified within isomeric compositions. A database of accurate mass, RT, and diagnostic MS/MS fragments was developed and applied to identify and structurally characterize biopharmaceutical glycans. The approach is fast, does not need for labeling or derivatisation and is applicable for highly accurate identification of biotherapeutic glycosylation.

Kaempferol is a natural flavonol. It reduces the risk of various cancers and also has skin-lightening effect. Due to these efficacies, the possible application in food, medicine and cosmetics industry are increasing. But these flavonols has limited application due to water solubility and stability. Considering these demerits, synthesized Astragalin (kaempferol-3-O-β-D-glucopyranoside) from kaempferol using uridine 5′-diphosphate (UDP)-3-O-glycosyltransferase. (UDP)-3-O-glycosyltransferase (UGT78K1) was expressed in E. coli BL21 (DE3) which utilize the bacterial indigenous UDP-glucose as sugar donor to catalyze glycosylation reaction. During biotransformation reaction kaempferol was exogenously fed and the product was isolated as Astragalin. To use UDP-glucose efficiently, previously constructed recombinant mono-cistronic vector (piBR181) was used which contained UDP-glucose biosynthetic pathway genes (pgm2: phosphoglucomutase and galU: glucose 1-phosphate uridylyltransferase) along with D-glucose facilitator proteins (glf: glucose facilitator diffusion protein), glucokinase (glk: glucokinase) and glycosyltransferases (UGT78K1) in mono-cistronic fashion. Luria-Bertani (LB) media was used for biotransformation reaction while cell induction was done using Lactose. Highest product conversion was achieved and were confirmed by HPLC-PDA analyses comparing with previous result.

Glycosylation is one of the important post-translational modification of proteins, which have a variety of functions in a wide range of biological processes such as inflammation and immunization. Especially N-glycoproteins contain glycans attached to asparagine (N) residues. Dried Blood Spot (DBS) have a number of advantages which are easy to collection, transportation, and storage at room temperature compared with previous conventional sampling methods that store the blood in the liquid state at low temperature (-80 ℃). In this study, we performed direct analysis of the N-glycoproteins through optimization of sample preparation from human serum without any depletion and enrichment processes. Denaturing and desalting condition brfore MS analysis were tested and optimized conditions were applied to DBS to identify N-glycoproteins. We first found total 54 glycopeptides from 23 major glycoproteins in DBS directly. Among them, 17 site-specific N-glycopeptides from 11 major glycoproteins quantified by LC-MS/MS with GPA system [1]. The quantified N-glycopeptides have a similar retention time and CV% for quantitation was less than 30%. We were able to quantify neutral fucosylated and sialylated non-fucosylated glycan structures in DBS. Through these experiments, we confirmed that N-glycoproteins and N-glycopeptides analysis can be performed in dried blood spot directly. Further it is expected that N-glycoprotein analysis using dried blood spot can be applied to disease sample.

Doxorubicin (DXN) and daunorubicin (DNR) are anthracycline-type antitumor drugs. The glycosyltransferase dnrS responsible for glycosylation of DXN aglycone was disrupted from Streptomyces peucetius ATCC 27952. The mutant strain complementation with glycosyltransferases from Streptomyces galilaeus (aknS/aknT) and dimethyltransferase (desVI) from Streptomyces venezuelae caused modification in ε-rhodomycinone by transferring TDP-rhodosamine to produce an Epelmycin-D. A methyltransferase (dnrK) in S. peucetius changed that compound to 7-O-L-rhodosaminyl-4-O-methyl-e-rhodomycinone. The products were confirmed by high performance liquid chromatography-photodiode array (HPLC-PDA) and high resolution liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (HRLC-ESI-QTOF -MS) analyses.

The characterization of site-specific micro-heterogeneity in glycoprotein is very important for understanding cell biology and disease processes. Vitronectin is well known to be a multi-functional glycoprotein in blood and the extracellular matrix, which is related to hepatocellular carcinoma (HCC). Here, we systematically analyzed the site-specific N-glycopeptides of vitronectin in human plasma by tandem mass spectrometry combined with immunoprecipitation and HILIC enrichment. Vitronectin was purified with immunoprecipitation by monoclonal antibody from plasma and digested to tryptic N-glycopeptides. Then, enrichment with HILIC materials was used, and followed by analysis with nano-LC/MS/MS. The sequences of N-glycopeptides were identified from the mass spectra by high-energy C-trap dissociation (HCD) and collision-induced dissociation (CID). In HCD mode, oxonium ions were used for recognizing glycopeptides and y ions for sequencing the peptide backbone. In CID mode, Y ions were used for characterizing their glycoforms. As a result, total 17 site-specific N-glycopeptides were completely identified at all of three N-glycosylation sites of vitronectin in human plasma, including 12 N-glycopeptides first reported. Finally, we specifically found that three hybrid and four complex glycopeptides of tri-antennary forms with outer-fucosylation increased in HCC human plasma.

Polysaccharides of Korean pine nut were isolated, identified and their bioactivity was evaluated. The water-soluble polysaccharides were extracted from the defatted Korean pine nut by boiling for 3 h in water and purified by ethanol precipitation and DEAE-Cellulose column chromatography. The monosaccharide composition analysis showed that purified polysaccharide(s) (PNE-P1) consisted of xylose (7.2, molar ratio), arabinose (8.8), and galacturonic acid (4.4) as major sugars and other minor sugars including glucose, galactose, rhamnose, and glucuronic acid. Their molecular weights were determined to be 1,213 kDa, 634 kDa and 331 kDa by size-exclusion HPLC. FT-IR and proton NMR analyses showed that PNE-P1 is a mixture of pectin-type polysaccharides containing arabinan, homogalacturonan (HG), rhamnogalacturonan type 1 (RG-1) and heteroxylan. The immunostimulating activity of PNE-P1 was investigated in murine macrophage RAW264.7 cells. PNE-P1 did not show any detectable level of cytotoxicity to RAW264.7 cells in the concentration range tested (10-200 μg/ml) and rather, it promoted the cell proliferation. PNE-P1 induced the production of pro-inflammatory cytokines (TNF-α, Interleukin-6 and Interleukin-10) and chemokines (RANTES and MIP-1α). These results indicate that PNE-P1 stimulated the RAW264.7 cells to increase cytokine secretions. Taken together, these results suggest that PNE-P1 can be considered as a potential immunopotentiator.

Glycosylation is one of the most important post-translational modifications of protein, which plays a key role on the stability and immunogenicity of therapeutic monoclonal antibodies (mAbs). Alteration in N-glycoforms may significantly modify the biological activity of mAbs. The unexpected and heterogeneous N-glycoforms are often reported, because it is affected by the culture medium, the efficiency of protein expression, and the physiological status of the host cells. Thus, accurate identification and quantification of their N-glycoform are necessary in order to control the production processes. In this study, IgG (Human serum), two NIST (National institute of standards and technology), and three unknown therapeutic mAb samples were analyzed by LC-MS/MS and GPA (GlycoProteome Analyzer) system1. As a result, the complex types of N-glycoforms were identified in IgG and unknown sample A, whereas different high-mannose and hybrid types of N-glycoforms were identified in two NIST samples and unknown sample B and C. The quantitative patterns of N-glycoform were similar between IgG and unknown sample A. The hybrid type and high-mannose type of N-glycoform were quantitatively increased in two NIST samples and unknown sample B and C, respectively. This analytical method will be useful for verification of safety and efficacy in various biopharmaceutical products such as therapeutic monoclonal antibodies.

Fucosylation of N-glycoproteins has been implicated in various diseases, such as hepatocellular carcinoma (HCC). However, few studies have performed site-specific analysis of fucosylation in liver-secreted proteins. In this study, we characterized the fucosylation patterns of liversecreted proteins in HCC plasma using a workflow to identify site-specific N-glycoproteins, where characteristic B- and/or Y-ion series with and without fucose in collision-induced dissociation were used in tandem mass spectrometry. In total, 71 fucosylated N-glycopeptides from 13 major liver-secreted proteins in human plasma were globally identified by LCMS/MS. Of the fucosylated N-glycopeptides, bi- and tri-antennary glycoforms were the most common ones identified in liver-secreted proteins from HCC plasma. Therefore, we suggest that this analytical method is effective for characterizing fucosylation in liver-secreted proteins.

Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC−MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycancleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

Xylose is the most abundant carbohydrate in biomass hemicellulose hydrolysate. However, most living organisms are unable to uptake this five-carbon sugar. A few microbes including Candida tropicalis and Pichia stipitis can metabolize this monosaccharide. We focused on genetically engineering C. tropicalis to utilize xylose rapidly. Xylitol reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) are responsible for xylose utilization in yeast. But xylitol is accumulated during xylose metabolism as byproduct. Hence, xylose isomerase (XI) gene in fungal xylose pathway was introduced not to produce xylitol. Glyceraldehyde 3-phosphate dehyderogenase (GAPDH) promoter was used throughout the experiments to transcribe genes constantly without glucose repression, and the fungal XI gene was codon-optimized for correct amino acids in alternative codon usage system. In addition, the composition of media was modified to minimize xylitol production. To increase xylose consumption rate more fast, additional three genes were overexpressed which encode transketolase (TKL), transaldolase (TAL) and xylulokinase (XK) gene. Also, pho13 gene was disrupted. For overexpression and disruption harboring genes, above four genes were identified by polymerase chain reaction with specific-designed primers based on sequence homology. The resulting genetically engineered organism consumed 50g xylose/L in 72 hours from chemically defined media with 20g glucose/L similar to biomass hemicellulose hydrolysate. By using this strain, we can use biomass hemicellulose hydrolysate more effectively.

A water-soluble polysaccharide named CF-P1 was extracted and purified from the Capsosiphon fulvescens (green alga, CF) mainly by acidic water extraction and DEAE-cellulose column chromatography. Previously, we reported that CF-P1 is a glucuronorhamnoxylan, an ulvan type polysaccharide typical for green algae, having rhamnoxylan backbone with glucuronic acid and sulfated glucuronic acid units as the side chains. In the present study, we examined anti-cancer effects of CF-P1 on HT-29 human colon cancer cells in vitro and in vivo. This polysaccharide significantly inhibited the growth of HT-29 cells up to 40% at 500 μg/ml and dose dependantly induced DNA fragmentation and cleaveage of caspase 8, 9, 3 and PARP. Enhanced apoptosis by this polysaccharide was dependent on caspase activation. In addition, we examined whether CF-P1 is equally active against HT-29 colon cancer cell xenograft tumors in a mouse model. Administration of CF-P1 through intraperitoneal injection to BALB/c-nu mice bearing HT-29 cells dose-dependently reduced both tumor volume and weight. The histological analysis of tumor tissues with H&E staining showed that CF-P1 decreased the cell numbers in tumor tissues and TUNEL assay showed DNA fragmentation of tumor cells. Taken collectively, these results clearly demonstrated that a water-soluble polysaccharide isolated from C. fulvescens effectively inhibits the tumor growth by induction of apoptotic cell death in vitro and in vivo, suggesting that it can be a good candidate as a potent anti-cancer agent against human colon cancer.

Glycan plays numerous roles in the nervous system during development, regeneration and synaptic plasticity, forming of a complex meshwork at the cell surface. We newly established an analytical platform for exploring two molecules- glycan attached to proteins and glycan attached to lipids- from human brain using mass spectrometry. 69 brain samples were obtained from the University of Cambridge. Glycan extraction method from previous study, and new ganglioside extraction method were paired with MS. Briefly, each brain tissues were grinded by sonication. Then for glycans, the membrane was pelleted by ultracentrifugation. N-glycan release and associated processing steps were performed by previously optimized procedures. For sphingolipids, homogenized brain is mixed with chloroform/methanol/water and centrifuged. Total lipids extracts were partitioned again with chloroform/methanol to collect gangliosides. After further enrichment by SPE, N-glycans and gangliosides were profiled by positive and negative ion mode nano-LC/MS respectively. Stucture information was obtained by nano-LC/MS/MS. Brain samples were divided into the 7 groups: neonates, infants, toddlers, school-ages, teenagers, young adults. We successfully profiled brain glycome and sphingolipids, identifying approximately 100 N-glycan compositions and 120 ganglioside compounds with several unique “brain type” characters. This study promise insights into involvement of glycans to massive flow of information that underpins brain, with providing meaningful understanding to neuropathology related with aging.

Gastric cancer has one of the highest cancer mortality rates worldwide, largely because of di fficulties in early-stage detection. Aberrant glycosylation in serum proteins is related with many human diseases including inflammation and various types of cancer. Aberrant glycosylation is desirable in order to improve the specificity and sensitivity for clinical use. Here, we combined protein-specific immunoaffinity purification, glycan release, and MS analysis to examine haptoglobin glycosylation of gastric cancer patients for glyco-markers. Interestingly, abundances of several tri- and tetra-antennary fucosylated N-glycans were increased in gastric cancer patients. Additionally, structural analysis via LC/MS/MS demonstrated that the fucosylated complex type N-glycans were mainly decorated with antenna fucose. In this study, we developed a targeted glycoproteomic approach using chip-based nano LC-QTOF MS and MS/MS following antibody-assisted targeted purification to discover glycan signatures of serum haptoglobin for gastric cancer. We could further obtain a specific structure of fucosylated molecules that are potential glyco-markers for gastric cancer via LC/MS/MS. The current study demonstrates that glycomic profiling of targeted serum haptoglobin via LC/MS and LC/MS/MS may be used as a powerful platform to monitor the specific glycosylation associated with gastric cancer.

Glycan modifications of therapeutic glycoproteins influence on pharmacological functions including efficacy, safety, and biological activity. MS-based analytical tools for N-glycan chracterization have been intensively developed and now N-glycans including various variants (O-Acetylation, mannose-phosphates, and polylactosamines) on biotherapeutics are readily identified. However, there are few studies for the characterization of O-glycans because of the absence of analytical platform. Here, we have characterized novel O-glycosylation on therapeutic factor IX using nanoLC/MS and tandem MS. Interestingly, we could determine O-fucosylation and O-glucosylation as well as typical O-glycosylation having a core 1. Using tandem MS, we found that O-fucosylated glycans consisted of Fuc, GlcNAc, and Gal w/wo NeuAc while O-glucosylated glycans included Glc, Xyl, and Hex w/wo NeuAc. In addition the O-glycopeptide analysis showed that O-glucosylation and O-fucosylation were present at specific O-glycosylation sites of growth factor domain, respectively 53Ser and 61Ser. In previous studies the O-fucosylation of growth factor domain was known as an important factor to modulate signal transduction by the interaction with other proteins. Thus this study on the identification of O-glycan modifications could be useful for proving the correlation between glycosylation and drug efficacy.

Genome editing technology promises to provide versatile tools for the generation of various model cell lines, plants and animals as well as for gene therapy by gene-editing in a target-specific manner. To date, three distinct modes of genome editing technologies have been introduced and extensively investigated in experimental settings, and attempted for use in clinical settings. Despite the revolutionized efficiency and sophistication in gene editing owing to development of CRISPR/Cas9, there remains technical limitations for currently available programmable nucleases in the routine use in experimental and clinical settings. Here, we introduce a universal genome editing technology (UGET) that relies on gene targeting through simple base pairings between a 20-28 nt nucleotide probe and a stretch of single-strand target DNA at the replication fork. UGET has no target limitations, shows lower-than-expected off-target incidence, is straightforward to use, and is compatible to gene correction via HDR. In particular, an overall construct size is less than 2.3kb, which means a high flexibility for use in gene therapy using viral vectors including adenovirus-associated viruses as well as for agrobacterium-based transformation in plants.