Earticle

Mesenchymal stem cells (MSCs), also known as multipotent stromal cells, has a possibility to differentiate into the neurallike cells as well as other type of cells such as osteoblast and adipocyte. Moreover, ganglioside, a sialic acidcontaining glycosphingolipids, plays a significantly critical role in the neuronal differentiation of MSCs. Meanwhile, macrophages (MPs), which secrets many type of proteins including human nucleoside diphosphate kinase A (hNME1) in the body, has been considered to involve closely in the neuronal differentiation of MSCs. Herein, we investigated and analyzed the correlation between the inhibitory effect of MP secretomes and the expression of ganglioside in the neuronal differentiation. Our results show that hNME1 strongly and specifically binds pST8SIA1, which is a ganglioside GD3 synthase, suggesting that hNME1 is a key factor in the inhibitory. Consequently, we produced nanobodies (NBs) named NB-hNME1 that effectively blocked the binding of hNME1, and thereby recovered the expression of ganglioside GD3 and the neuronal differentiation of MSCs.

Glycosylation on therapeutic glycoproteins is a critical quality attribute (CQA) that impacts the quality, safety, and potency of drugs. Therefore, glycosylation should be characterized during the whole drug development by adequate analytical techniques. Since the multiple levels of glycan heterogeneity pose a daunting analytical challenge, Glycan analysis usually involves the use of complementary analytical techniques methods, which include HPLC and MS. Since it is very time-consuming to assessing glycans for each approach, an analytical method that can be flexibly applied to both LC and MS is highly demanded. Therefore, InstantPC that provides not only chromophore for analysis on LC-FLD but also improved MS ionization efficiency by attaching a tertiary amine is emerging as a novel glycan labeling. Thus, we optimized MS-based sample preparation and analytical method for InstantPC labeled N-glycans of therapeutic proteins. N-glycans labeled with InstantPC were profiled using hydrophilic interaction chromatography (HILIC) coupled with MS. InstantPC labeled N-glycans provide increased mass spectrometric sensitivity and tandem MS efficiency. In addition, we found that the results in LC-FLD and LC/MS show high similarity, suggesting our approach could be utilized for glycan quality assessment. This platform provides a flexibility for MS-based rapid and in-depth characterization of N-glycans as well as screening applications by robust fluorescence-based quantitation.

Agalsidase beta, recombinant enzyme therapeutic, which is a glycoprotein that treats lysosome accumulation disorders caused by deficiency of the enzyme alpha galactosidase A has 3 glycosylation sites(N108, N161, N184). In this protein, there are various glycans from acidic glycans to high mannose, so accurate monitoring is required for proper afficacy and function of drug. Recently, intact analysis is heavily used due to the advantage of simple preparation, short MS analysis time, glycan occupancy and micro-heterogeneity confirmation. However, numerous glycosylation sites, high molecular weight and glycan heterogeneity complicate the mass spectrum, making data interpretation and glycan assignment difficult. Here, we obtained the mass value of intact agalsidase beta, which eased the complexity of the mass spectrum by lowering the high molecular weight of the glycoprotein through stepwise enzymatic treatment. To facilitate the assignment of sialic acid and phosphorylated glycans at each glycosylation sites, deconvoluted spectra of sialidase and phosphatase-treated and untreated agalsidase beta were compared. Possible glycoforms could be assigned for each peak in the deconvoluted spectra within the accepted mass tolerance of ±5 Da. In the monomer and dimer structures of agalsidase beta, a glycosylation pattern was confirmed in which HexNAcNeuAc residues(494.1748 Da) and 2HexNAc2NeuAc residues (988.3496 Da) were increased, respectively. Our approach can be a efficient tool to confirm glycosylation pattern of therapeutic glycoproteins in biosimilar development.

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β 1,2-xylose and α1,3-fucose residues (XylMan3FucGlcNAc2). Here, we investigated the physiological significance in Arabidopsis thaliana of α-1,6-mannosyl-glycoprotein 2--N-acetylglucosaminyltransferase (EC 2.4.1.143, N-acetylglucosaminyltransferase II [GnTII]), which transfers N-acetylglucosamine (GlcNAc) residue from the nucleotide sugar donor UDP-GlcNAc to the α1,6-mannose residue of the di-antennary N-glycan acceptor in the Golgi apparatus. We assessed the phenotypic effects of a loss-of-function mutation (gnt2-1) in GnTII, and used quantitative PCR (qPCR) to examine gene expression and callose deposition assays under stress conditions. Prolonged stress from tunicamycin or NaCl treatment enhanced GnTII expression in the wild type and caused severely disrupted development and abnormal callose deposition in root tissues in the gnt2-1 mutant. Lack of the 6-arm β1,2-linked GlcNAc residue in the N-glycans promoted the unfolded protein response (UPR) upon prolonged endoplasmic reticulum (ER) stress in gnt2-1. Thus, GnTII's promotion of the 6-arm GlcNAc addition to N-glycans is important for plant growth and development under stress conditions.

Polysaccharides from marine algae are known to exhibit various biological and pharmacological activities, including anticancer, immunostimulatory, anti-inflammatory, anti-coagulant, antioxidant, antibacterial, and antiviral activities, offering a variety of potential applications in the fields of foods, cosmetics, drug-delivery, tissue engineering and regenerative medicine, vaccines, and pharmaceutical industries. Sargassum horneri, which is a brown alga and abundantly grows along the coasts of Jeju island in South Korea, has anti-inflammatory, antioxidant and immnomodulatory activities. However, the anticancer effect against human colon cancer has not been revealed yet. We isolated polysaccharides from S. horneri (termed as SHP), analyzed the monosaccharides composition and molecular weight, and investigated the anticancer activity using human colon cancer cell line, HT-29 cells. Bio-LC and HPLC analysis showed that SHP mainly consists of fucose, galactose, and glucose with the molar ratios of 73.37, 17.61 and 9.02 mol%, and has molecular weight of approximately 122,153 and 14,170 kDa. SHP dose-dependently reduced cell viability on HT-29 and increased the cleavage of poly (ADP-ribose) polymerase(PARP) by approximately 10-fold compared to the untreated control group. Furthermore, SHP treatment decreased the procaspase-9, procaspase-8 and Bcl-2 protein expression by about 0.4, 0.7, and 0.3-fold compared to the untreated control group in HT-29 cells. Taken collectively, SHP is a mixture of high-molecular weight glucogalactofucan type polysaccharides and induces apoptotic cell death of human colon cancer cells via upregulation of the expression of apoptosis-related factors, suggesting that S. horneri-derived polysaccharides can be potent ingredients for health-beneficial foods or anti-cancer agents against human colon cancer.

The opportunistic human fungal pathogen Cryptococcus neoformans is the main causative agent of fungal meningoencephalitis in immunocompromised patients. The mannoprotein MP84 of C. neoformans, reported as an adhesion protein and as an immunoreactive antigen to host CD4(+) T cell, is highly mannosylated cell wall protein. In this study, we investigated the structure of N-glycans attached on MP84 and the effect of altered N-glycan structures on its interaction with host cells. We expressed the His tagged MP84 as a secretory form in WT and alg3Δ mutant, a N-glycosylation defective strain, to generate MP84 with the truncated core N-glycans. In in-vitro adhesion and uptake assay, the purified MP84 proteins carrying the truncated N-glycans did not show significant difference in host cell interactions compared to MP84 carrying the WT N-glycans. Moreover, we expressed a secretory MP84 lacking N-glycosylation site, constructed by protein engineering, to investigate the effect of full-deglycosylation on the interaction with MP84 with host cells. Aglycosylated MP84 revealed much higher adhesion affinity to host cells than MP84 carrying the WT and truncated N-glycans. On contrast, aglycosylated MP84 still retains the cytokine induction activity comparable to WT, although MP84 with truncated N-glycans showed a decreased immunostimulatory activity. In conclusion, the mixed effects of N-glycan structures of MP84 on the interaction with host cells strongly suggest the modulatory role of N-glycans to avoid non-specific interaction with host cells.

The human fungal pathogen Cryptococcus neoformans assembles N-/O-linked glycans on its proteins in two types with and without xylose (1, 2). In this study, the CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to the minor O-glycans with xylose, was identified and functionally analyzed. The CAP6 deletion in the ktr3Δ strain, in which the α1,2-mannose addition to the major O-glycans is defected, resulted in almost complete blockage of O-glycan extension. Notably, the putative two cell surface sensor proteins, Wml (Wsc/Mid2-Like)1p and Wml2p, were shown to be subjected to minor and major O-mannosylation by Cap6 and Ktr3, respectively. The proteins levels of Wml1 and Wml2 were remarkably decreased in the ktr3Δ cap6Δ mutant, indicating that proper O-mannosylation is essential for their stability. The phosphorylation of Mpk1, induced by tunicamycin, was greatly decreased in the ktr3Δcap6Δ and the wml1Δwml2Δ, supporting an essential role of O-glycans on cell surface sensors in cell wall integrity signaling. As reflecting its defective growth under several stress conditions, the ktr3Δcap6Δ strain showed fully attenuated virulence in a mouse model of cryptococcosis. The delineation of the roles of protein glycosylation in fungal pathogenesis will not only provide a deep insight into the glycan-based fungal infection mechanism but also aid in the development of novel antifungal agents.

Glycosylation contributes to various molecular and cellular functions such as protein folding, localization, secretion, cell signaling, and communication by transferring glycans to proteins or lipids. Glycoproteins are expected to play essential roles for various biological processes including pathogenicity and reproductions in fungi; however, little is known about glycosylation process in filamentous fungi, especially in plant pathogenic fungi. The plant pathogenic fungus Fusarium graminearum causes a devastating disease called Fusarium head blight (FHB) on cereal crops. In addition to grain yield losses, infection of this pathogen causes severe contamination of mycotoxins, which are harmful to human and animal health. To understand molecular mechanisms underlying development and pathogenesis of this fungus, putative 78 N or O-glycosylation-related gene (GLY) knockout mutant library is being generated and we have successfully obtained 53 deletion mutations till now. Phenotypic changes, such as vegetative growth, sexual and asexual reproduction, and virulence of the mutants are being analyzed. Some mutants showed severe phenotypic defects suggesting that individual GLY gene plays important roles in this fungus. The ongoing works are focusing on the elucidation of the potential roles of GLY genes in diverse biological processes, such as fungal virulence, mycotoxin production and we are also analyzing glycan structure of glycoproteins in F. graminearum. This study is the first comprehensive functional analysis of GLY gene in plant pathogenic fungi, and it will be the fungal glycobiome study initiative.