Earticle

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4IgP)reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4IgM). However, limitations of hCTLA4IgP include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing b1,2-xylose and a1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4IgP (hCTLA4IgP-Gal)was expressed through the coexpression of human β 1,4-galactosyltransferase (hGalT)and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4IgP-Galindicated that not only were the two glycans containing β1,4-galactose newly extended, but also glycans containing both b1,2-xylose and a1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4IgP,respectively. Unlike hCTLA4IgP, hCTLA4IgP-Gal was effective as an acceptor via β1,4-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4IgP-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4IgP, and the clearance pattern of hCTLA4IgP-Gal was close to that for hCTLA4IgM.These results indicate that the coexpression with hGalT and hCTLA4IgP is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.

Mushroom lectins harboring carbohydrate binding specificity are powerful tools to detect glycoconjugates with N-linked or O-linked glycans. In this study, we purified a novel core 1 O-linked glycan-specific lectin from the fruiting body extract of a mushroom Helicium erinaceus by ion-exchange chromatography, affinity chromatography on fetuin-agarose, gel filtration chromatography. The purified lectin was designed as HEL1 (H. erinaceus lectin 1). Tricine-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicated that the native lectin has an identical form with a molecular weight of approximate 15 kDa and a lectin-like structure sequence. Isoelectric focusing of the lectin showed bands near pI 5.4. Agglutination assay displayed HEL1 was more effective toward porcine’s erythrocyte rather than other animals red blood cells. Glycan microarray analysis showed that HEL1 interacts with Galβ1-3GalNAc present in Thomsen-Friedenreich antigen or core 1 O-linked glycan structure such as Peanut agglutinain (PNA). Comparing the glycan binding affinities in glycan microarray, both HEL1 and PNA bound core 1 O-linked glycan. Interestingly, HEL1 can bind a broad range of glycan structures with an extension on the β1-3Gal linked to the GalNAc as well as a terminal α(2-3) linked sialic acid which is not bound by PNA. These HEL1 binding affinities could be useful to apply the cancer research and diagnostic assay involving core 1 and extended core 1 glycan structures.

Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body ɤ-irraiation of mice increased caspase-3+ apoptotic lymphocyte numbers in secondary lymphoid organs. Following ɤ-irradiation, SIGN-R1 and complement (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively acrivated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b+ cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after ɤ-irradiation.

Cryptococcus neoformans is the opportunistic human-pathogenic fungus causing life-threatening meningoencephalitis in immunocompromised patients, especially patients with AIDS. Several C. neoformans mannoproteins, such as chitin deacetylase 2 (MP98), are reported as key antigens stimulating host T-cell response. Here, we analyzed the glycosylation pattern of protein , MP98(H), in the wild-type and various glycosylation mutant strains of C. neoformans. The apparent molecular weight (MW) of MP98(H)wassignificantly decreasedin the alg3Δ strain, which has defects in biosynthesis of N-glycans, but not in the O-glycosylation mutants. The MW of MP98(H) secreted from wild type and glycosylation mutant strains was converted to its predicted size after N-glycosidase treatment, supporting that MP98(H) is highly modified by N-glycosylation. We further investigated the function of in vitro adhesion assayMP98(H) proteins from the wild type and alg3Δ strains. Unexpectedly, the alg3Δ/MP98(H) protein, bearing short N glycans, adhered more intensely to the lung cells than the WT/MP98(H) protein, bearing high-mannose type N-glycans, indicating that truncated N-glycans rather enhanced adhesion of MP98(H) to epithelial lung cells. These results strongly indicate that N-glycan structure of MP98 affects the adhesion efficiency of C. neoformans to host cells.

Panax ginseng Meyer, belonging to the genus Panax of the family Araliaceae, is known for its human immune system-related effects such as immune-boosting effects. Ginseng polysaccharides (GPs) are the responsible ingredient of ginseng in immunomodulation, which are classified as acidic and neutral GPs. Although GPs participate in various immune reactions including the stimulation of immune cells and production of cytokines, the precise function of GPs together with its potential receptor(s) and their signal transduction pathways have remained largely unknown. Animal lectins are carbohydrate-binding proteins that are highly specific for sugar moieties. Among many different biological functions in vivo, animal lectins especially play important roles in the immune system by recognizing carbohydrates that are found exclusively on pathogens or that are inaccessible on host cells. This review summarizes the immunological activities of GPs and the diverse roles of animal lectins in the immune system, suggesting the possibility of animal lectins as the potential receptor candidates of GPs and giving insights into the development of GPs as therapeutic biomaterials for many immunological diseases.

O-GlcNAcylation is highly involved in cellular stress responses including the endoplasmic reticulum (ER) stress response. However, it is largely unknown which component(s) of the unfolded protein response (UPR) is directly regulated by O-GlcNAcylation. In this study, eukaryotic translation initiation factor 2α (eIF2α), a major branch of the UPR, was O-GlcNAcylated at Ser 219, Thr 239, and Thr 241. Upon ER stress, eIF2α is phosphorylated at Ser 51 by PERK and this inhibits global translation initiation, except for that of specific mRNAs that induce stress-responsive genes such as CHOP. Hyper-O-GlcNAcylation hindered phosphorylation of eIF2α at Ser 51. The level of O-GlcNAcylation of eIF2α was changed by DTT dependent on its phosphorylation at Ser 51. Point mutation of the O-GlcNAcylation sites of eIF2α increased its phosphorylation at Ser 51 and CHOP expression and resulted in increased apoptosis upon ER stress. This O-GlcNAcylation of eIF2α was reproduced in thiamet-G-injected mouse liver. In conclusion, proper regulation of O-GlcNAcylation and phosphorylation of eIF2α is important to maintain cellular homeostasis upon ER stress.

O-GlcNAc is a carbohydrate post-translational modification occurs on hydroxyl groups of serine or threonine residues of cytosolic and nuclear proteins. It is known that O-GlcNAc addition and removal regulate many biological events like transcription, protein degradation, proliferation. Epithelial-mesenchymal transition(EMT) is a process that epithelial cells lose its polarity and cell-cell contact, therefore get migratory capabilities to become mesenchymal stem cells. There are several reports that O-GlcNAc is associated with cancer EMT and metastasis. But there are still much to discover the role of O-GlcNAc on TGF-β -induced EMT. TGF-β is a powerful EMT inducer, and is primarily dependent on TGF-β /Smad signaling. Here we found that Smad protein expression is regulated by global O-GlcNAc change, consequentially controls TGF-β-induced EMT. Now we’re planning to verify how O-GlcNAc controls Smad expression.

Fast and accurate screening of illegal compounds in food and drugs are crucial for the public safety and health. Mass spectrometric analysis coupled with liquid chromatography enable the fast analysis of compounds and return the vast information for the identification of molecules. Structural information can be obtained from the fragmentation pattern from MS/MS data of compounds at different energy level. For the fast screening of illegal compounds, reference library of MS/MS spectral data has been built using 252 chemicals which frequently observed in food and drugs. In addition to the parent mass and retention time, MS/MS data at different energy levels have been collectively stored to compare the structural characteristics. Each MS/MS data at different energy level were converted to a single representative but artificial mass data sets to compare the fragmentation characteristics. Using this mass spectrometric big data of small compounds, its potential power to segregate the similar compounds to an adjacent cluster and ability to identify the unknown molecules.. For the general acceptability to the public, the cross platform study has been performed by four independent institutes with two different types of mass spectrometry form three companies. Results showed that the reference library are highly reproducible and applicable to identify the compounds. The compounds of structural similarity exhibited the higher similarity of MS/MS dataset that other compounds indicating unique fragmentation patterns. Despite the high similarity value, unique peaks were found as well enabling to distinguishing exact compounds from similar ones.

Protein glycosylation with structural diversity is one of the most abundant post translational modifications. A wide range of functions for glycans has been described, from structural to regulatory roles in molecular and cellular level. Especially, sialic acid and its modifications are known to affect stability, efficacy, antigenicity, and half-life of biopharmaceuticals. Current glycosylation profiling is mainly focused on N-glycans due to availability of enzymes for their release. In contrast, there is no corresponding enzymes for O-glycan liberation. In general, O-glycans are liberated using alkaline β-elimination which conditions are highly destructive toward sialic acid and its modifications. For this reason, comprehensive O-glycosylation profiling method which preserves the sialic acid and its modifications is required to demonstrate in respect of quality, safety and efficacy of therapeutic glycoproteins. In this study, we developed a new analytical strategy for non-destructive O-glycosylation profiling of biopharmaceuticals using O-glycopeptides produced by non-specific protease. Erythropoietins (EPOs) were digested with pronase E for six hours, and then resulting glycopeptides were enriched using a solid phase extraction with porous graphitized carbon. The biopharmaceutical O-glycosylation profiling and structural characterization were conducted on nanoLC-chip/Q-TOF MS system. We successfully applied this method to analysis of batch-to-batch variation study of darbepoetin alfa, and O-glycosylation profiling of epoetin alfa and beta.

Maltohexaose (G6) is a maltodextrin consisted of six-glucose in α-1,6-glycosidic linkages and essential research tool in food, cosmetic and pharmaceutical. In previous study, we have proven that the possibility of large-scale continuous production of pure maltodextrins from cyclodextrins (CDs) using Pyrococcus furiosus thermostable amylase with immobilization in a packed-bed reactor (PBR). In this study, we used this PBR for G6 production from α-CD. After enzyme reaction, 1-decanol was added to G6 mixture as precipitating agent with a 1:1 ratio of 1-decanol to α-CD. In which, the unreacted α-CD was precipitated, then it was separated by centrifugation. Finally, the G6-product was obtained in homogeneous form and high-purity (95 %). Through this study, we successfully demonstrated the enzymatic synthesis method to produce pure G6 from α-CD.

양파(Allium cepa.)는 백합과에 속하는 두해살이풀로 다량의 퀘세틴이 함유되어있는 식품으로 알 려져 있다. 퀘세틴은 항산화능이 우수한 생리활성물질로 이상지질혈증과 비알콜성 지방간질환 개 선효과가 우수한 것으로 보고 되어있다. 그러나, 양파에 존재하는 퀘세틴은 배당체형으로 존재하 고 있어 생체이용률이 낮다. 본 연구에서는 양파즙내의 배당체 형태 퀘세틴을 상업용 섬유소 분해 효소를 이용하여 비배당체로 전환하여 비배당체 퀘세틴의 수율을 높이는 것을 목표로 하였다. 양 파즙의 비배당체 및 배당체 퀘세틴의 함유량을 분석하기 위하여 퀘세틴 표준물질 및 시료의 HPLC, LC-MS/MS를 실시하였다. 섬유소 분해효소는 Novozyme사의 ViscozymeTM과 CelluclastTM, C-TecTM, H-TecTM을 이용하였으며, 각각의 섬유소 분해효소 단일처리군과 복합처 리군으로 나누어 효소의 농도를 양파 파쇄액 무게 기준으로 0.05%⋅0.2%⋅1%로 설정하여 6시 간동안 효소반응을 실시한 이후 HPLC를 이용하여 비배당체 퀘세틴의 함량을 측정하였다. 비효소 처리 양파즙의 비배당체 퀘세틴의 함유량이 184 ppm 인 것에 비하여 효소복합처리를 실시한 양 파즙의 경우 554 ppm의 비배당체 퀘세틴을 함유한 것으로 확인되어 효소복합처리를 통하여 약 3 배의 비배당체 퀘세틴의 증가를 확인할 수 있었다. 배당체 퀘세틴의 함량은 대조군의 경우 총 퀘 세틴 함량중 96%였으나 복합효소처리 시료의 경우에는 11.9%로 현격히 감소하였다. 더불어 수용 성 당의 함량이 1.5배 증가하였는데, 비효소 처리 양파즙의 총 당함량이 7.7%인 것에 비해 효소 복합처리를 실시한 양파즙의 경우 13.9%로 나타났다. 이러한 실험결과를 통하여 섬유소 분해효소 에 의한 퀘세틴의 비배당체 전환을 확인할 수 있었다. 또한 양파즙 생산 부산물도 비효소처리 방 법에 비하여 50% 가량 감소하였다. 결론적으로 양파즙의 상업적 생산에 있어 섬유소 분해효소 첨가는 기능성성분강화는 물론 수율향상 등의 효과를 가져 올 것으로 기대된다.

Glycogen branching enzyme(GBE)은 α-1,4-glycosyl 결합을 새로운 α-1,6-glycosyl 결합으로 전이시키는 특성을 가지며, 생체내 glycogen의 생합성에 중요한 역할을 담당한다. 본 연구에서는 각각의 Escherichia coli(EBE), Deinococcus geothermalis(DgBE), Vibrio vulnificus(VvBE)의 branching enzyme 유전자를 동정, 클로닝 한 재조합 플라스미드를 Escherichia coli를 이용하여 단백질을 대량생산하고 정제하였다. 정제된 GBE 3종류를 이용하여 amylopectin을 기질로 변성전 분을 생산하였다. 이후 변성전분의 구조적, 이화학적 특성을 알아보기 위하여 가지결합분포, 수 용액상에서의 분자량 및 부피,점도, hydrolysis kinetics를 통한 소화속도 측정 등을 실시하였다. 생산된 변성전분은 EBE처리 시료의 경우 중합도 10~18정도의 chain을 다량으로 함유하는 특성 을 보였으며 DBE의 경우 중합도 5~8정도의 chain의 비율이 높았으며, VBE 처리 시료의 경우에 는 중합도 3~5정도의 짧은 chain의 비율이 상대적으로 매우 높은 특성을 보였다. 변성전분은 Rapid Visco Analyzer 측정결과 8% 농도에서 점도가 거의 나타나지 않았다. 나노 입도측정기를 통하여 변성전분의 입자 사이즈를 측정하였는데, VvBE변성전분의 경우 13.54(d.nm) EBE변성전 분의 경우 15.69(d.nm), DgBE변성전분의 경우 11.70(d.nm)의 크기를 나타내었다. Porcine pancreatic amylase와 human pancreatic amylase를 이용한 enzyme kinetics를 통한 소화속도 측 정시 VvBE변성전분의 경우 소화속도가 amylopectin보다 약 2배정도 감소하는 것으나 나타났으나 EBE변성전분은 경우 amylopectin보다 약간 빠르게 소화되는 것으나 측정되었다. DgBE변성전분 의 경우 VvBE와 DgBE의 중간정도의 소화속도를 확인할 수 있었다. 위와 같은 결과를 바탕으로 종합적으로 유추하였을 때 glycogen branching enzyme을 이용한 변성전분 제조 시 amylopectin 에 비하여 α-1,6-glycosyl결합이 많아지고 중합체의 구조가 컴팩한 구조의 클러스터 단위화 되 는 것으로 보인다. 이 때 변성전분은 분자크기가 1000배 이상 줄어들고 점도가 현격히 감소하며, 짧은 가지결합으로 인해 소화속도가 느려져 지소화성 전분소재로서의 식품산업 등에 응용이 가능 할 것으로 판단된다.

Cell surface glycans lie at the interface between the cell and its environment, playing an active role in mediating interactions with foreign substances. Although current lectin-based methods enable detection of select glycan epitopes present on the cell surface, detailed structural information of glycan heterogeneity is needed to better assess their involvement in cell-cell interactions, particularly with pathogens. Mass spectrometry-based glycomics has the potential to provide a single platform to monitor hundreds of native structures simultaneously. This study constitutes a comprehensive and quantitative analysis that defines the sequence of oligosaccharides on human cells that affect the nature of Salmonella Typhimurium infection. We first examined the glycan products remaining on the host cell surface after brief contact with bacteria, which included mainly, desialylated and high mannose glycans. We then describe a glycomic landscaping (glycoscaping) strategy to present predominantly high mannose glycans on host cells to better correlate phenotype with structure. A better understanding of the biological roles of glycosylation is achieved by structure-specific re-engineering towards specified glycosylation patterns. Changes in the glycan landscape were induced on live cells, using a mixture of glycosidases and glycosidase inhibitors. Following treatment, the exact changes were characterized by mass spectrometry. Such glycoscaping, when applied to intestinal epithelial cells, yielded significant increases in membrane enzyme activity, cell permeability, migration, and microbial infection.

Recombinant erythropoietin (EPO) as a regulator for formation of red blood cells is well known glycosylated therapeutic protein. Darbepoetin alfa (NESP) is a heavily heterogeneous glycoprotein biotherapeutic containing one O-glycosylation and five N-glycosylation sites. The type, extent, and location of glycosylation on NESP mediate biological activity, plasma half-life, immunogenicity, and stability. Thus, the determination of site-specific glycan microheterogeneity is important to assess biotherapeutic quality and establish the equivalency of biosimilar EPOs. However, there is no method to monitor site-specific glycosylation on NESP. Here, we have developed a highly reproducible analytical platform for characterization site-specific glycosylation with microheterogeneity, which combines molecular size fractionation, multiple proteolysis, PGC nanoLC separation, in silico screening by accurate mass, and tandem MS analysis. We were successful in identifying all N- and O-glycosylation sites and quantifying glycoforms including 61 different N-glycopeptides and 8 different O-glycopeptides on NESP. This is the first comprehensive study to determine site-specific glycosylation with extreme glycan heterogeneity on NESP.

Monoclonal antibodies (mAbs) are one of the most fast-growing classes of the biopharmaceuticals market. The quality, safety, and potency of mAbs are determined largely by their glycosylation. Thus, detailed glycomic analyses are important to assess biotherapeutic quality and establish the equivalency of biosimilar mAbs now coming to market. However, the inherent structural diversity of glycosylation significantly hinders analysis. Here, we created glycoform database for rapid identification and structural elucidation of mAbs glycans. Glycans were released by PNGase F digestion, then enriched by porous graphitized carbon solid phase extraction. Enriched glycans were separated and analyzed by nanoLC PGCchip/Q-TOF MS and MS/MS. Accurate mass MS was used to compositionally annotate and profile the N-glycans, while CID MS/MS was used for structural elucidation. We generate DB platform which combines RT, accurate mass, diagnostic fragment ion, and identified glycan structures. Up to now, glycans from five representative mAbs including adalimumab, bevacizumab, infliximab, rituximab and trastuzumab were analyzed and total 27 glycans were incorporated to database. The glycome DB removes the bias and user-dependency associated with manual data interpretation and enables an algorithmic approach towards biopharmaceutical glycan characterization.

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.

Gangliosides are anionic glycosphingolipids that contain one to several sialic acid residues. They participate in diverse biological processes such as cell-cell recognition, and the modulation of membrane protein function. Nonetheless of the biological importance of gangliosides, there are few studies on gangliosides due to their structural complexity and the lack of sensitive analytical tools. In this study, we have performed detailed profiling of gangliosides present on various cancer cell membrane to differentiate the cancer origins and subtype using negative ion detection mode nano LC/MS combined with membrane enrichment technique. Cell surface gangliosides were extracted from eight cancer cell lines originated from breast, lung, cervical, and lymphatic tumor and then analyzed by nano LC/MS. Extracted gangliosides were assigned based on accurate mass and the structures were further elucidated using tandem MS. GD1, GM1 and GM3 were commonly observed as major gangliosides in all cancer cell lines having various ceramide portion. Hierarchical clustering based on Pearson correlation coefficients were also used to compare and classify each cell line. Cancer origins and molecular subtypes could be distinguished based on distinctive ganglioside profiles. These results demonstrate that significant differences in cancer cell surface gangliosides can be used for potential application toward cancer diagnosis and treatment.

We synthesized dextran-chlorin e6 conjugates having disulfide linkage for photodynamic treatment (PDT) of colon cancer cells. Cystamine was conjugated to reductive end group of dextran and then amine end group of dextran-cystamine conjugates were conjugated with chlorin e6 (Ce6). Dextran-Ce6 conjugates having disulfide linkage (DEX6ss) has small particle sizes less than 100nm and spherical shapes. Ce6 was released from nanoparticles by responsive manner with glutathione concentration and dextrase. DEX6ss nanoparticles were efficiently delivered to HCT116 colon carcinoma cells compared to Ce6 itself. ROS production and phototoxicity of DEX6ss nanoparticles were significantly higher than Ce6 itself. In animal tumor xenograft model, tumor targetability of DEX6ss nanoparticles was significantly higher than Ce6 itself. These results indicating that DEX6ss nanoparticles have redox and colonic enzyme-responsiveness as a tumor targetable photosensitizer.

The aim of the present study is to prepare the chitosan hydrolysates and characterize their potential antibacterial activity. High molecular weight chitosan (CTSN-H) and medium molecular weight chitosan (MMWC, 2~10-kDa) were further degraded efficiently by using chitosanase from Bacillus subtilis JH-1122. Molecular weight (MW) of the final product was determined by MALDI-TOF MS, showed several peaks ranging from 0.8 to 2.5-kDa. Based on antibacterial activity of the hydrolysates, final products were named as active molecular chitosan (AMC-S1). AMC-S1 was evaluated as IC50 to 12.5 mg/mL of the concentration toward Candida albicans. Our present study demonstrates that AMC-S1 derived from the digestion of CTSN-H and MMWC can be selected as a potent factor affecting on antimicrobial activity against C. albicans for the industry and pharmaceutical applications.

Control of carotenoids production from the yeast Rhodotorula mucilaginosa AY-01, based on the results of 28S rDNA sequence (D1/D2 region) analysis and homology search, was evaluated under different carbon and nitrogen sources to establish the optimal condition. Herein, we report that the control of production facilitates the extraction of intracellular carotenoids from yeast R. mucilaginosa AY-01. The use of methanol only for carotenoids production was found to be the best approach when compared with other enzymatical, chemical or physical processes. Glucose partially purified from carotenoids using an immobilized metal affinity chromatography (IMAC) was identified as the major monosaccharide with minor unidentified sugars by high performance anion-exchange chromatography (HPAEC) analysis. The glucosidal carotenoids extracted from the yeast R. mucilaginosa AY-01 showed highly significant antioxidant activity. In addition, a significant antibacterial activity of glucosidal carotenoids toward antibiotic resistant bacteria (ARB) was elucidated with potential biological applications in various food, cosmetic and/or hygiene supplements industries. Collectively, our study describes the production and characterization of a red pigment with antioxidant and antibacterial activities from the yeast R. mucilaginosa AY-01.

Understanding sulfated polysaccharides from pants or seaweed are well known for antioxidant, anticoagulant activity or other biological activities. The aim of the present study is to optimize the condition for the isolation of water soluble polysaccharides (WSP) and evaluate the biological activity. WSP was isolated from defatted microalgae B. braunii using Chlorosulfonic acid or Sulfuric acid. Also the preliminary structural analysis of WSP was performed by FT-IR spectroscopy analysis. Among WSP isolated using sulfuric acid showed the lowest the content of total carbohydrates (g/L), whereas showed the highest antioxidant activity. High production of WSP may allow further exploration of microalga B. braunii as a potential biomass provider for biological and industrial exploitation of bioactive substrates.

An extracellular alkaline α-D-mannosidase produced by a strain named as MA-01 was produced and its preliminary enzyme activity was characterized. Upon determining the 16S rDNA sequence and its homology search, the strain was identified to be one of species of the Bacillus safensis. Localization of enzyme was elucidated that α-D-mannosidase can be found in culture medium as an extracellular enzyme. In addition, partial enzyme activity of 63% compared with the extracellular enzyme activity was observed in membrane protein. The optimal pH and temperature of the α-D-mannosidase were pH 7.5 and 37℃, respectively. The Km and Vmax values of the α-D-mannosidase in crude enzyme toward p-nitrophenyl-α-D-mannopyranoside were determined to be 455.6 μM and 10.8 μ mole/min/mg of protein, respectively. To the best of our knowledge, this is the first report described the alkaline α-D-mannosidase from the family of B. safensis.

Most of the therapeutic enzymes, which is used for treatment of lysosomal storage diseases, require the glycans containing mannose-6-phosphate (M-6-P). It has been shown that the increased content of M-6-P glycan enhances lysosomal targeting and, therefore, the efficacy of therapeutic enzymes [1]. We have constructed the glyco-engineered yeast harboring very high content of mannosylphosphorylated N-glycans, which can be converted to M-6-P glycan through an uncapping process [2]. In this study, cell wall mannoproteins of the glyco-engineered yeast were extracted by using hot citrate buffer and then digested with pronase, which generated mannosylphosphorylated glycans as attached to asparagine amino acid (Asn). Through the mild acid hydrolysis for uncapping and α(1,2)-mannosidase treatment for trimming, these Asn-linked glycans were converted to Asn-linked M-6-P glycans, which were specifically purified by titanium dioxide resin. The purified Asn-linked M-6-P glycans could be easily conjugated with the crosslinkers containing an amine-reactive group such as a N- hydroxysuccinimide esters which attacks amine group of Asn. Using a chemical group (maleimide, azide or dibenzo-bicyclo-octyne) at the opposite site of the attached cross-linkers, M-6-P glycans were successfully conjugated to target proteins. Proper conjugation of M-6-P glycan to target protein was confirmed by increase of molecular weight in SDS-PAGE and lectin blot using domain 9 of cation-independent M-6-P receptor. Further, M-6-P glycan-conjugated protein was shown to be internalized and targeted to lysosomes of cells.

Mannosylphosphorylated glycans, which confers negative charges on the cell surface, are only found in yeasts and fungi groups. Both MNN4 and MNN6 genes are required for mannosylphosphorylation in the traditional yeast Saccharomyces cerevisiae. Mnn4 protein has been known to be a positive regulator of Mnn6p, a real enzyme for mannosylphosphorylation. We constructed the quadruple-disrupted och1Δ mnn1 Δmnn4Δmnn6Δ strain by disrupting MNN4 and MNN6 genes from the och1Δmnn1Δ strain in which yeast-specific hyper-mannosylated glycan and immunogenic α(1,3)-mannose structures were already abolished [1]. However, yeast-specific mannosylphosphorylated N-glycan structures were still observed in the och1Δmnn1Δmnn4Δmnn6Δ strain, which suggests that there is another gene involved in mannosylphosphorylation. From the och1Δmnn1Δmnn4Δmnn6Δ strain, we further disrupted the genes (MNN14, YUR1, KTR2, KTR4, KTR5, or KTR7) having homology with MNN4 or MNN6 genes. Analyses of N-glycans obtained from cell wall mannoproteins of six quintuple-disrupted strains indicated that mannosylphosphorylated glycan structure was completely abolished only in the och1Δmnn1Δmnn4Δ mnn6Δmnn14Δ strain. Since MNN14 has the homology with MNN4, the och1Δmnn1Δmnn14Δ and och1 Δmnn1Δmnn4Δmnn14Δ strains were constructed. While mannosylphoshorylated N-glycans were observed in och1Δmnn1Δmnn14Δ strain, there was no mannosylphosphorylation in the och1Δmnn1Δmnn4Δmnn14Δ strain, which clearly shows that disruption of both MNN4 and MNN14 genes was required to remove mannosylphosphorylated glycan structures. Complementation of one gene (either MNN4 or MNN14) restored mannosylphosphorylation in the och1Δmnn1Δmnn4Δmnn14Δ strain, which supports that MNN4 and MNN14 can play a redundant role on the deficiency of one gene. The och1Δmnn1Δmnn4Δmnn14 Δ strain without any yeast-specific glycan structure will serve as an useful platform for production of glycoproteins with human-compatible N-glycans.

Sialic acids present on non-reducing terminal of glycan is important for prolonged in vivo half-life of glycoproteins. Many efforts have been made to increase the sialic acid content in the manufacturing of therapeutic glycoproteins because glycoproteins containing non-sialylated glycans do not show effective in vivo efficacies owing to a short half-life. It was reported that knock down of sialidase expression using the RNA interference (RNAi) technology enhanced protein sialylation in CHO cells which are the most widely used for therapeutic glycoprotein production [1]. However, knock down strategy did not completely shut down the sialidase expression. In this study, we performed knockout of sialidase genes in CHO cells by using the CRISPR/Cas9 genome editing technology. Only three sialidases (Neu1, Neu2, and Neu3) were actively expressed in CHO cells when analyzing their expressions using real-time polymerase chain reaction. First, we disrupted Neu3 gene because it is located in the plasma membrane while Neu1 and Neu2 exist in lysosome and cytosol, respectively. After the transfection of vectors expressing Cas9 and sgRNAs, Neu1 gene-disrupted clones were selected by using the T7 endonucleases 1 (T7E1) assay and cell surface sialidase activity assay. Disruption of Neu1 gene did not affect the viability of CHO cells. The disruptions of Neu1 and Neu2 genes in CHO cells are currently in the progress.

Macrophages foam cell formation is characterized by lipid accumulation and plays a key role in early atherogenesis. In this study, we showed that glucosamine dose dependently stimulated lipid accumulation in RAW 264.7 cells. In addition, glucosamine synergistically promoted oleic acid-induced lipid accumulation. Sterol regulatory element-binding proteins (SREBPs) have been described as key transcription factors for lipogenic gene induction. Glucosamine enhanced DNA binding activity on SRE/E-box of SREBP-1 and mRNA expression of this target genes Acetyl-CoA carboxylase (ACC) and Fatty acid synthas (FAS). Since SREBP transcription activity is regulated by mammalian target of rapamycin (mTOR), we examined the effect of mTORC1-specific inhibitor, rapamycin on glucosamine-induced lipid accumulation; Glucosamine-induced lipid accumulation was suppressed by rapamycin. Although glucosamine promoted the endoplasmic reticulum stress marker, glucose-regulated protein-78 (GRP78) protein and mRNA level, glucosamine-induced lipid accumulation dose not suppressed by ER stress inhibitor 4-phenylbutyric acid (PBA). This suggest that glucosamine-induced lipid accumulation were not likely mediated with ER stress.

T cells play a key role in the chronic inflammation and immune responses. We investigated the immuno-suppressive effects of glucosamine in both mice naive splenocyte and Jurkat T cell. Glucosamine inhibited T cell proliferation as well as IL-2, IFN-gamma, TNF-alpha induction in response to anti-CD3 or PMA/Ionomycin in splenocyte and Jurkat cell, respectively. MAP Kinase pathways play a critical role T cell activation and IL-2 production in T cell. Our data show that glucosamine inhibited PMA/Ionomycin induced JNK phosphorylation and anti-CD3 induced ERK1/2 phosphorylation in Jurkat cell. In addition, glucosamine suppressed nuclear factor of activated T cells (NFAT) transcription activity but not nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) of activated Jurkat cell. These results suggest that glucosamine might inhibit expressions of T cell-specific cytokines such as IL-2, IFN-gamma, and TNF-alpha via the NFAT pathway in activated T cells. These results demonstrate that glucosamine may be an important modulator of T-cell mediated immune response.

Amylosucrase (ASase, EC. 2. 4. 1. 4) forms an insoluble α-1,4-glucan polymer by transferring glucose molecules liberated from sucrose to its appropriate substrates, such as maltooligosaccharides, arbutin, and salicin. It was found that ASase could be attached into the insoluble α-1,4-glucan polymer after its catalytic work was done. In this study, therefore, ASase, a glucan-polymerization enzyme, was employed as a fusion partner for foreign proteins to make the foreign proteins be easily purified. Green fluorescent protein (GFP) and β-glucosidase were used as model fusion partner proteins. ASase-foreign gene cassettes were constructed and transformed into E. coli (BL21 DE3). As a result, it was found that ASase-fusion proteins were easily purified by a simple centrifugation since the majority of ASase-fusion proteins were present in the insoluble glucan polymer. In this study, optimal condition of polymer production was characterized.

The trehalose phosphorylase (TPase, EC 2.4.1.64) and kojibiose phosphorylase (KPase, EC 2.4.1.230) catalyze the phospholytic activities on the α1,1glucosidic linkage and α 1,2glucosidic linkage in each substrates (trehalose and kojibiose), respectively. Both enzymes also perform the reverse reaction of the phosphorylase, with which α1,1 and α 1,2- glucosidic linkages are formed with glucose and glucose-6-phosphate as substrates. In this study, the KPase and TPase genes were expressed in E. coli. Two phosphorylases, TPase and KPase from each Anaerocellum thermophilum DSM 6725 and Pyrococcus sp. ST04 were incorporated into E. coli BL21, respectively. The presence of catalytic activities of both recombinant enzymes were confirmed. The continuous reaction with TPase and KPase was carried out with trehalose as a sole substrate. The thin layer chromatography (TLC) and high performance anion exchange chromatography (HPAEC) analysis revealed the formation of an unusual trisaccharide in a reaction mixture. The structural analysis of an unusual trisaccharide showed that the final product was a rare tripolysaccharide, selaginose. The result indicated that a rare tripolysaccharide, selaginose, can be synthesized by one-step enzymatic reaction system with KPase and TPase.