• Mass Spectrometry Letters
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• 제13권4호
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• pp.139-145
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• 2022

Protein glycosylation is a common post-translational modification by non-template-based biosynthesis. In fungal biotechnology, which has great applications in pharmaceuticals and industries, the importance of research on fungal glycoproteins and glycans is accelerating. In particular, the importance of quantitative analysis of fungal glycans is emerging in research on the production of filamentous fungal proteins by genetic modification. Reliable mass spectrometry-based techniques for quantitative glycomics have evolved into chemical, enzymatic, and metabolic stable isotope labeling methods. In this study, we intend to expand quantitative glycomics by metabolic isotope labeling of glycans in Aspergillus niger, a filamentous fungus model, by the MILPIG method. We demonstrate that incubation of filamentous fungi in a culture medium with carbon-13 labeled glucose (1-¹³C₁) efficiently incorporates carbon-13 into N-linked glycans. In addition, for quantitative validation of this method, light and heavy glycans are mixed 1:1 to show the performance of quantitative analysis of various N-linked glycans simultaneously. We have successfully quantified fungal glycans by MILPIG and expect it to be widely applicable to glycan expression levels under various biological conditions in fungi.

• BMB Reports
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• 제44권12호
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• pp.772-781
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• 2011

Branched N-glycans are produced by a series of glycosyltransferases including N-acetylglucosaminyltransferases and fucosyltransferases and their corresponding genes. Glycans on specific glycoproteins, which are attached via the action of glycosyltransferases, play key roles in cell adhesion and signaling. Examples of this are adhesion molecules or signaling molecules such as integrin and E-cadherin, as well as membrane receptors such as the EGF and TGF-${\beta}$ receptors. These molecules also play pivotal roles in the underlying mechanism of a variety of disease such as cancer metastasis, diabetes, and chronic obstructive pulmonary disease (COPD). Alterations in the structures of branched N-glycans are also hall marks and are useful for cancer biomarkers and therapeutics against cancer. This mini-review describes some of our recent studies on a functional glycomics approach to the study of branched N-glycans produced by N-acetylglucosaminyltransferases III, IV, V and IX (Vb) (GnT-III, GnT-IV, V and IX (Vb)) and fucosyltransferase 8 (Fut8) and their pathophysiological significance, with emphasis on the importance of a systems glycobiology approach as a future perspective for glycobiology.

• Journal of Plant Biotechnology
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• 제37권3호
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• pp.292-304
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• 2010

High value-added therapeutic proteins have been leading the biologics industry and occupied major portion of the market. More than 60% of the currently available protein therapeutics are glycoproteins attached with glycans which play crucial roles for the protein folding, therapeutic efficacy, in vivo half-life and immunogenecity. This review introduces the process of glycosylation and the impacts of glycans in the aspects of therapeutics. The important glycan structures in therapeutic performances were also summarized focusing on three representative categories of glycoproteins, cytokines, therapeutic antibody and enzyme. Currently, mammalian expression systems such as Chinese hamster ovary cells are preferred for the production of therapeutic glycoproteins due to their ability to synthesize glycans having similar structures with human type glycans. However, recent advances of plant glycoengineering to overcome the limitation originating from different glycan structures will soon allow to develop more efficient and economic plant-based production systems for therapeutic glycoproteins.

• Mass Spectrometry Letters
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• 제8권1호
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• pp.14-17
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• 2017

The effect of cationization agent concentration on glycan detection via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was investigated using $Na^+$ ions in the form of NaCl as the cationization agent. NaCl solution concentrations ranging from 1 mM to 1 M were investigated. Glycans from ovalbumin were mixed with the cationization agent solution and the 2,5-dihydroxybenzoic acid (2,5-DHB) matrix solution in a volume ratio of 1:1:1. The resulting mixture was loaded onto the MALDI plate. Two MALDI-TOF MS instruments (Voyager DE-STR MALDI-TOF MS and Tinkerbell RT MALDI-TOF MS) were used for detection of glycans. The best detection, in terms of the number of identified glycans, the peak intensity, and the signal-to-noise (S/N) ratio, was obtained with NaCl concentrations of 0.01-0.1 M for both MALDI-TOF MS instruments.

• Molecules and Cells
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• 제24권3호
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• pp.416-423
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• 2007

Alterations in the glycan chains of cell surface glycoconjugates are frequently involved biological processes such as cell-cell interaction, cell migration, differentiation and development. Cultured embryonic (E18) rat cortical neurons underwent apoptosis in response to camptothecin, and lectin histochemistry showed that binding to apoptotic neurons of FITC-conjugated Maackia amurensis agglutinin (MAA), which is specific for terminal ${\alpha}2,3$-sialic acid residues, increased progressively with increasing concentrations of camptothecin. Analysis of the total proteins of apoptotic neurons by SDS-PAGE, and lectin blotting using HRP-labeled MAA, revealed that the expression of terminal ${\alpha}2,3$-sialic acid residues on an unknown protein with an apparent molecular mass of 25.6 kDa also increased in apoptotic neurons. NP-HPLC analysis of the total cellular N-glycans of normal and apoptotic neurons demonstrated that the expression of structurally simpler biantennary types of N-glycans fell by 49% during apoptosis whereas the more branched triantennary types of N-glycans with terminal sialic acid residues increased by up to 59%. These results suggest that increased surface expression of ${\alpha}2,3$-sialic acid residues and hyperglycosylation of N-glycans is a common feature of cellular responses to changes in cell physiology such as tumorigenesis and apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• 제17권2호
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• pp.691-695
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• 2016

Protein glycosylation is the most common posttranslational modification in mammalian cells. Aberrant protein glycosylation has been reported in various diseases, including cancer. We identified and quantified the glycan structures of O-linked glycoprotein from cholangiocarcinoma (CCA) cell lines from different histological types and compared their profiles by nanospray ionization-linear ion trap mass spectrometry (NSI-$MS^n$). Five human CCA cell lines, K100, M055, M139, M213 and M214 were characterized. The results showed that the O-linked glycans of the CCA cell lines comprised tri- to hexa-saccharides with terminal galactose and sialic acids: NeuAc1Gal1GalNAc1, Gal2GlcNAc1GalNAc1, NeuAc2Gal1GalNAc1 NeuAc1Gal2GlcNAc1GalNAc1 and NeuAc2Gal2GlcNAc1GalNAc1 All five CCA cell lines showed a similar glycan pattern, but with differences in their quantities. NeuAc1Gal1GalNAc1 proved to be the most abundant structure in poorly differentiated adenocarcinoma (K100; 57.1%), moderately differentiated adenocarcinoma (M055; 42.6%) and squamous cell carcinoma (M139; 43.0%), while moderately to poorly differentiated adenocarcinoma (M214; 40.1%) and adenosquamous cell carcinoma (M213; 34.7%) appeared dominated by $NeuA_{c2}Gal_1GalNA_{c1}$. These results demonstrate differential expression of the O-linked glycans in the different histological types of CCA. All five CCA cell lines have abundant terminal sialic acid (NeuAc) O-linked glycans, suggesting an important role for sialic acid in cancer cells. Our structural analyses of glycans may provide important information regarding physiology of disease-related glycoproteins in CCA.

• 약학회지
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• 제57권4호
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• pp.250-257
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• 2013

Biosimilars are important drugs in medicine and contain many glycosylated proteins. Thorough analysis of the glycosylated protein is a prerequisite for evaluation of biosimilar glycan drugs. A method to assess the diversity of N-glycosylation sites and N-glycans from biosimilar glycan drugs has been developed using two separate methods, LC-MS/MS and MALDI-TOF MS, respectively. Development of sensitive, accurate, and efficient methods for evaluation of glycoproteins is still needed. In this study, analysis of both N-glycosylation sites and N-glycans of glycoprotein was performed using the same LC-MS/MS with two different nano-LC columns, nano-C18 and nano-porous graphitized carbon (nano-PGC) columns. N-glycosylated proteins, including RNAse B (one N-glycosylation site), Fetuin (three sites), and ${\alpha}$-1 acid glycoprotein (four sites), were used, and small amounts of each protein were used for identification of N-glycosylation sites. In addition, high mannose N-glycans (one type of typical glycan structure), Mannose 5 and 9, eluted from RNAse B, were successfully identified using nano-PGC-LC MS/MS analysis, and the abundance of each glycan from the glycoprotein was calculated. This study demonstrated an accurate and efficient method for determination of N-glycosylation sites and N-glycans of glycoproteins based on high sensitive LC-MS/MS using two different nano-columns; this method could be applied for evaluation of the quality of various biosimilar drugs containing N-glycosylation groups.

• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Mass Spectrometry Letters
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• 제4권2호
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• pp.38-40
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• 2013

Glycans released from ovalbumin by PNGase F were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry using three different dihydroxybenzoic acid (DHB) matrix systems: 2,5-DHB, 2,6-DHB, and a 2,5-DHB/2,6-DHB binary matrix. Relative to the results obtained with the single-component matrices (2,5-DHB or 2,6-DHB), the 2,5-DHB/2,6-DHB binary matrix boasted lower background noise and higher sensitivity. A total of 16 glycan peaks were observed using the 2,5-DHB/2,6-DHB binary matrix, while only 10 and 9 glycan peaks were observed using the 2,5-DHB and 2,6-DHB matrices, respectively.

• KSBB Journal
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• 제22권4호
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• pp.234-238
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• 2007

본 연구에서는 전극분무 이온화-이온 포획 질량 분석기를 이용하여 인체 IgG의 N-연결 글라이칸 중 이중촉각 구조를 가지면서 비환원 말단의 갈락토오즈 개수가 0, 1, 2 개인 서로 다른 세 가지 글라이칸의 단일 쪼개짐 (MS/MS) 및 다중 쪼개짐 현상을 관찰하고 이를 구조 분석에 이용하였다. MS/MS 분석에서는 퓨코오즈가 결합된 환원 말단의 N-아세틸 글루코사민의 0,2-고리 쪼개짐으로 파생되는 조각 피크가 가장 높은 세기로 나타나는 것을 관찰할 수 있었고, 전구체 피크와 별개로 연속적인 당 단위체의 쪼개짐이 일어나는 것을 알 수 있었다. 또한 G1 글라이칸의 경우에서만 비환원 말단의 갈락토오즈와 N-아세틸글루코사민이 결합된 채 쪼개지는 현상이 일어나는 것을 관찰할 수 있었다. 다중 쪼개짐 질량 분석 기법을 이용하여 MS/MS 스펙트럼에서 나타나는 조각 피크들의 구조를 재확인할 수 있었고, 이를 트리 구조로 정리할 수 있었다. 또한 추가적인 2,4-고리 쪼개짐 현상이 환원 말단 하나 바깥쪽의 N-아세틸 글루코사민에서 공통적으로 일어나는 것을 관찰할 수 있었다. 이와 같은 다중 쪼개짐 질량 분석기법을 이용하여 보다 복잡한 구조의 글라이칸 구조 분석에 이용될 수 있을 것으로 기대된다.