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Bak is a critical executor of apoptosis belonging to the Bcl-2 protein family. Bak contains a hydrophobic groove where the BH3 domain of proapoptotic Bcl-2 family members can be accommodated, which initiates its activation. Once activated, Bak undergoes a conformational change to oligomerize, which leads to mitochondrial destabilization and the release of cytochrome c into the cytosol and eventual apoptotic cell death. In this study, we investigated the molecular aspects and functional consequences of the interaction between Bak and peroxisomal testis-specific 1 (Pxt1), a noncanonical BH3-only protein exclusively expressed in the testis. Together with various biochemical approaches, this interaction was verified and analyzed at the atomic level by determining the crystal structure of the Bak-Pxt1 BH3 complex. In-depth biochemical and cellular analyses demonstrated that Pxt1 functions as a Bak-activating proapoptotic factor, and its BH3 domain, which mediates direct intermolecular interaction with Bak, plays a critical role in triggering apoptosis. Therefore, this study provides a molecular basis for the Pxt1-mediated novel pathway for the activation of apoptosis and expands our understanding of the cell death signaling coordinated by diverse BH3 domain-containing proteins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Published by Elsevier Ltd.)

Cryptococcus neoformans is an opportunistic human fungal pathogen causing lethal meningoencephalitis. It has several cell wall mannoproteins (MPs) identified as immunoreactive antigens. To investigate the structure and function of N-glycans assembled on cryptococcal cell wall MPs in host cell interactions, we purified MP98 (Cda2) and MP84 (Cda3) expressed in wild-type (WT) and N-glycosylation-defective alg3 mutant (alg3Δ) strains. HPLC and MALDI-TOF analysis of the MP proteins from the WT revealed protein-specific glycan structures with different extents of hypermannosylation and xylose/xylose phosphate addition. In alg3Δ, MP98 and MP84 had truncated core N-glycans, containing mostly five and seven mannoses (M5 and M7 forms), respectively. In vitro adhesion and uptake assays indicated that the altered core N-glycans did not affect adhesion affinities to host cells although the capacity to induce the immune response of bone-marrow derived dendritic cells (BMDCs) decreased. Intriguingly, the removal of all N-glycosylation sites on MP84 increased adhesion to host cells and enhanced the induction of cytokine secretion from BMDCs compared with that on MP84 carrying WT N-glycans. Therefore, the structure-dependent effects of N-glycans suggested their complex roles in modulating the interaction of MPs with host cells to avoid nonspecific adherence to host cells and host immune response hyperactivation., (© 2023. The Author(s).)

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Bone marrow-derived mesenchymal stem cells (BMSCs) and bone morphogenetic protein-2 (BMP-2) have been studied for bone repair because they have regenerative potential to differentiate into osteoblasts. The development of injectable and in situ three-dimensional (3D) scaffolds to proliferate and differentiate BMSCs and deliver BMP-2 is a crucial technology in BMSC-based tissue engineering., Methods: The proliferation of mouse BMSCs (mBMSCs) in collagen/poly-γ-glutamic acid (Col/γ-PGA) hydrogel was evaluated using LIVE/DEAD and acridine orange and propidium iodide assays. In vitro osteogenic differentiation and the gene expression level of Col/γ-PGA(mBMSC/BMP-2) were assessed by alizarin red S staining and quantitative reverse-transcription polymerase chain reaction. The bone regeneration effect of Col/γ-PGA(mBMSC/BMP-2) was evaluated in a mouse calvarial bone defect model. The cranial bones of the mice were monitored by micro-computed tomography and histological analysis., Results: The developed Col/γ-PGA hydrogel showed low viscosity below ambient temperature, while it provided a high elastic modulus and viscous modulus at body temperature. After gelation, the Col/γ-PGA hydrogel showed a 3D and interconnected porous structure, which helped the effective proliferation of BMSCs with BMP-2. The Col/γ-PGA (mBMSC/BMP-2) expressed more osteogenic genes and showed effective orthotopic bone formation in a mouse model with a critical-sized bone defect in only 3-4 weeks., Conclusion: The Col/γ-PGA(mBMSC/BMP-2) hydrogel was suggested to be a promising platform by combining collagen as a major component of the extracellular matrix and γ-PGA as a viscosity reducer for easy handling at room temperature in BMSC-based bone tissue engineering scaffolds., (© 2022. Korean Tissue Engineering and Regenerative Medicine Society.)

The production of pharmacological vaccines in plants has been an important goal in the field of plant biotechnology. GA733-2, the protein that is also known as colorectal carcinoma (CRC)-associated antigen, is a strong candidate to produce a colorectal cancer vaccine. Tomato is the one of the major targets for production of an edible vaccine, as tomato is a fruit consumed in fresh form. It also contains high content of vitamins that aid activation of immune response. In order to develop an edible colorectal cancer vaccine, the transgene rGA733-Fc that encodes a fusion protein of GA733-2, the fragment crystallizable (Fc) domain, and the ER retention motif (rGA733-Fc) was introduced into tomato plants (Solanum lycopersicum cv. Micro-Tom). The transgenic plants producing rGA733-Fc (rGA733-Fc OX ) protein were screened based on stable integration of transgene expression cassette and expression level of rGA733-Fc protein. Further glycosylation pattern analysis revealed that plant derived rGA733-Fc protein contains an oligomannose glycan structure, which is a typical glycosylation pattern found on ER-processing proteins. The red fruits of rGA733-Fc OX transgenic tomato plants containing approximately 270 ng/g FW of rGA733-Fc protein were orally administered to C57BL/6 mice. Oral administration of tomato fruits of the rGA733-Fc expressing transgenic plants delayed colorectal cancer growth and stimulated immune responses compared to oral administration of tomato fruits of the h-Fc expressing transgenic plants in the C57BL/6J mice. This is the first study showing the possibility of producing an edible colorectal cancer vaccine using tomato plants. This research would be helpful for development of plant-derived cancer edible vaccines., (© 2022. The Author(s).)

A lysosome, an acidic membrane-bound organelle, contains hydrolytic enzymes to digest macromolecules for recycling. Many lysosomal enzymes (LEs) traffic to the lysosome through the mannose-6-phosphate (M6P)-dependent pathway. Some mannose residues of high-mannose type N -glycans on LEs can be phosphorylated in the Golgi apparatus through two-step enzyme reactions. The consequent M6P moiety is recognized by M6P receptors (MPRs) on the trans -Golgi network membrane and delivered through the endo-lysosomal pathway. On the other hand, secreted LEs containing M6P glycans can be recaptured by MPRs on the plasma membrane and targeted to the lysosome. Enzyme replacement therapy (ERT) for lysosomal storage diseases exploits this M6P-MPR-dependent endocytosis to deliver recombinant enzymes to lysosomes. This review discusses various engineering and application technologies using M6P's lysosomal targeting. Glyco-engineering for increasing M6P contents developed 'Bio-better' ERT enzymes with enhanced therapeutic efficacy. M6P-decorated peptides, proteins, liposomes, and nanoparticles have been developed for drug delivery and subcellular imaging. A recently developed lysosome-targeting chimera uses an M6P-based bifunctional binder to degrade specific extracellular and membrane proteins. The success and efficiency of M6P-based lysosomal targeting will boost further technological developments with new applications in the biomedical field., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

An invertebrate sialyltransferase, cST3Gal-I, identified from the sea squirt Ciona savignyi, was functionally characterized in vitro using recombinant enzyme expressed in yeast strains. cST3Gal-I was localized to the Golgi membrane when expressed in Saccharomyces cerevisiae. Enzymatic characterization for substrate specificity and kinetic property indicate that cST3Gal-I prefers O-glycans, rather than N-glycan, of asialoglycoproteins as substrates. Interestingly, C. savignyi sialyltransferase exhibited effectively Neu5Ac transfer to core 1 O-glycan, Gal β(1,3)GalNAc, compared to orthologous human glycosyltransferase. Further, it is shown that cST3Gal-I catalyzes the formation of α(2,3)-linkage, through lectin blot analysis with Maackia amurensis lectin and by linkage-specific sialidase treatments. The putative active sites of cST3Gal-I for putative acid/base catalysts and sialic acid acceptor/donor substrate bindings were also identical to the counterpart residues of a mammalian enzyme, porcine ST3Gal-I, as predicted through homologous structure modeling. These results could imply that an ancestral tunicate ST3Gal-I in C. savignyi would prefer O-glycan onto glycoproteins as its sialic acid acceptor than vertebrate enzymes., (Copyright © 2021. Published by Elsevier B.V.)

Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein-protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis.

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 , rMnn14 77-935 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 rMnn14 77-935 were determined through enzyme assays with a high-mannose type N -glycan (Man 8 GlcNAc 2 ) as a substrate. In addition, rMnn14 77-935 was shown to mannosyl-phosphorylate high-mannose type Nglycans (Man 7-9 GlcNAc 2 ) 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 rMnn14 77-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

The colorectal carcinoma-associated protein GA733-2 is one of the representative candidate protein for the development of plant-derived colorectal cancer vaccine. Despite of its significant importance for colorectal vaccine development, low efficiency of GA733-2 production limits its wide applications. To improve productivity of GA733-2 in plants, we here tested multiple factors that affect expression of recombinant GA733-2 (rGA733-2) and rGA733 fused to fragment crystallizable (Fc) domain (rGA733-Fc) protein. The rGA733-2 and rGA733-Fc proteins were highly expressed when the pBINPLUS vector system was used for transient expression in tobacco plants. In addition, the length of interval between rGA733-2 and left border of T-DNA affected the expression of rGA733 protein. Transient expression analysis using various combinations of Agrobacterium tumefaciens strains (C58C1, LBA4404, and GV3101) and tobacco species ( Nicotiana tabacum cv. Xanthi nc and Nicotiana benthamiana ) revealed that higher accumulation of rGA733-2 and rGA733-Fc proteins were obtained by combination of A. tumefaciens LBA4404 and Nicotiana benthamiana . Transgenic plants generated by introduction of the rGA733-2 and rGA733-Fc expression cassettes also significantly accumulated corresponding recombinant proteins. Bioactivity and stability of the plant-derived rGA733 and rGA733-Fc were evaluated by further in vitro assay, western blot and N-glycosylation analysis. Collectively, we here suggest the optimal condition for efficient production of functional rGA733-2 protein in tobacco system., (© Korean Society for Plant Biotechnology 2021.)

Necroptosis is a form of regulated cell death caused by formation of the necrosome complex. However, the factors modulating this process and the systemic pathophysiological effects of necroptosis are yet to be understood. Here, we identified that Beclin 1 functions as an anti-necroptosis factor by being recruited into the necrosome complex upon treatment with TNFα, Smac mimetic, and pan-caspase inhibitor and by repressing MLKL oligomerisation, thus preventing the disruption of the plasma membrane. Cells ablated or knocked-out for Beclin 1 become sensitised to necroptosis in an autophagy-independent manner without affecting the necrosome formation itself. Interestingly, the recruitment of Beclin 1 into the necrosome complex is dependent on the activation and phosphorylation of MLKL. Biochemically, the coiled-coil domain (CCD) of Beclin 1 binds to the CCD of MLKL, which restrains the oligomerisation of phosphorylated MLKL. Finally, Beclin 1 depletion was found to promote necroptosis in leukaemia cells and enhance regression of xenografted-tumour upon treatment with Smac mimetics and caspase inhibitors. These results suggest that Beclin 1 functions as a negative regulator in the execution of necroptosis by suppressing MLKL oligomerisation.

Tumorigenesis can be induced by various stresses that cause aberrant DNA mutations and unhindered cell proliferation. Under such conditions, normal cells autonomously induce defense mechanisms, thereby stimulating tumor suppressor activation. ARF, encoded by the CDKN2a locus, is one of the most frequently mutated or deleted tumor suppressors in human cancer. The safeguard roles of ARF in tumorigenesis are mainly mediated via the MDM2-p53 axis, which plays a prominent role in tumor suppression. Under normal conditions, low p53 expression is stringently regulated by its target gene, MDM2 E3 ligase, which induces p53 degradation in a ubiquitin-proteasome-dependent manner. Oncogenic signals induced by MYC, RAS, and E2Fs trap MDM2 in the inhibited state by inducing ARF expression as a safeguard measure, thereby activating the tumor-suppressive function of p53. In addition to the MDM2-p53 axis, ARF can also interact with diverse proteins and regulate various cellular functions, such as cellular senescence, apoptosis, and anoikis, in a p53-independent manner. As the evidence indicating ARF as a key tumor suppressor has been accumulated, there is growing evidence that ARF is sophisticatedly fine-tuned by the diverse factors through transcriptional and post-translational regulatory mechanisms. In this review, we mainly focused on how cancer cells employ transcriptional and post-translational regulatory mechanisms to manipulate ARF activities to circumvent the tumor-suppressive function of ARF. We further discussed the clinical implications of ARF in human cancer.

The non-viral delivery of genes into macrophages, known as hard-to-transfect cells, is a challenge. In this study, the microporation of a CpG-free and small plasmid (pCGfd-GFP) showed high transfection efficiency, sustainable transgene expression, and good cell viability in the transfections of Raw 264.7 and primary bone marrow-derived macrophages. The non-viral method using the pCGfd vector encoding anti-EGFR single-chain Fv fused with Fc (scFv-Fc) generated the macrophages secreting anti-EGFR scFv-Fc. These macrophages effectively phagocytized tumor cells expressing EGFR through the antibody-dependent mechanism, as was proved by experiments using EGFR-knockout tumor cells. Finally, peri-tumoral injections of anti-EGFR scFv-Fc-secreting macrophages were shown to inhibit tumor growth in the xenograft mouse model. [BMB Reports 2020; 53(8): 442-447].

Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N -glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N -glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 ( alg3 Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N -glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro , the alg3 Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12 , encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N -glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from Asc - and Gsdmd -deficient mice infected with the alg3 Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N -glycans. Our results demonstrated N -glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformans IMPORTANCE We previously reported that the outer mannose chains of N -glycans are dispensable for the virulence of C. neoformans , which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans Here, we present evidence that an intact core N -glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ , and alg12Δ strains that have defects in lipid-linked N- glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N -glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans , thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N -glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents., (Copyright © 2020 Thak et al.)

O-acetylated sialic acid (SA) attached to the N-glycans of therapeutic glycoproteins reportedly inhibit sialidase activity, increase protein half-life, decrease protein antigenicity, and stabilize protein conformation. Recombinant human acid α-glucosidase (Myozyme) is the only drug approved by the United States Food and Drug Administration for the treatment of Pompe disease. In this study, unreported N-glycans containing O-acetylated SA in Myozyme and the relative quantities of total glycans were investigated using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The 17 N-glycans (6.4% of total glycans) containing mono-, di-, mono/di-, and di/di-O-acetylated N-acetylneuraminic acid (Neu5Ac) were identified with mass accuracy, glycan-generated fragment ions, and the retention time on an LC column. The analysis of peptides containing mono- and/or di-O-acetylated Neu5Ac ions sorted from all peptides using nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites (Asn 140, Asn 233, Asn 390, Asn 470, Asn 652, and Asn 882), at least five of which (Asn 140, Asn 233, Asn 390, Asn 470, and Asn 652) could contribute to the drug efficacy or cellular uptake of Myozyme. This is the first study to identify N-glycans containing O-acetylated Neu5Ac and O-acetylation sites in Myozyme., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Many therapeutic enzymes for lysosomal storage diseases require a high content of mannose-6-phosphate (M6P) glycan, which is important for cellular uptake and lysosomal targeting. We constructed glyco-engineered yeast harboring a high content of mannosylphosphorylated glycans, which can be converted to M6P glycans by uncapping of the outer mannose residue. In this study, the cell wall of this yeast was employed as a natural M6P glycan source for conjugation to therapeutic enzymes. The extracted cell wall mannoproteins were digested by pronase to generate short glycopeptides, which were further elaborated by uncapping and α(1,2)-mannosidase digestion steps. The resulting glycopeptides containing M6P glycans (M6PgPs) showed proper cellular uptake and lysosome targeting. The purified M6PgPs were successfully conjugated to a recombinant acid α-glucosidase (rGAA), used for the treatment of Pompe disease, by two-step reactions using two hetero-bifunctional crosslinkers. First, rGAA and M6PgPs were modified with crosslinkers containing azide and dibenzocyclooctyne, respectively. In the second reaction using copper-free click chemistry, the azide-functionalized rGAA was conjugated with dibenzocyclooctyne-functionalized M6PgPs without the loss of enzyme activity. The M6PgP-conjugated rGAA had a 16-fold higher content of M6P glycan than rGAA, which resulted in greatly increased cellular uptake and efficient digestion of glycogen accumulated in Pompe disease patient fibroblasts.

Myozyme is a recombinant human acid alpha-glucosidase (rhGAA) that is currently the only drug approved for treating Pompe disease, and its low efficacy means that a high dose is required. Mannose-6-phosphate (M6P) glycosylation on rhGAA is a key factor influencing lysosomal enzyme targeting and the efficacy of enzyme replacement therapy (ERT); however, its complex structure and relatively small quantity still remain to be characterized. This study investigated M6P glycosylation on rhGAA using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The glycans released from rhGAA were labeled with procainamide to improve mass ionization efficiency and the sensitivity of MS/MS. The relative quantities (%) of 78 glycans were obtained, and 1.0% of them were glycans containing M6P (M6P glycans). These were categorized according to their structure into 4 types: 3 newly found ones, comprising high-mannose-type M6P glycans capped with N-acetylglucosamine (GlcNAc) (2 variants, 17.5%), hybrid-type M6P glycans (2 variants, 11.2%), and hybrid-type M6P glycans capped with GlcNAc (3 variants, 6.9%), as well as high-mannose-type M6P glycans (3 variants, 64.4%). HCD-MS/MS spectra identified six distinctive M6P-derived oxonium ions. The glycopeptides obtained from protease-digested rhGAA were analyzed using nano-LC-ESI-HCD-MS/MS, and the extracted-ion chromatograms of M6P-derived oxonium ions confirmed three M6P glycosylation sites comprising Asn 140, Asn 233 (newly found), and Asn 470 attached heterogeneously to nine M6P glycans (two types), eight M6P glycans (four types), and seven M6P glycans (two types), respectively. This is the first study of rhGAA to differentiate M6P glycans and identify their attachment sites, despite rhGAA already being an approved drug for Pompe disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In this study, synthetic vaccine nanoparticles (SVNPs) that efficiently targeted lymph nodes, where immune responses against foreign antigens are primed, were developed to enhance antitumor immunity. The size (20-70 nm) and surface character (amination) of poly(γ-glutamic acid)-based SVNPs were selected for effective loading and delivery (i.e., migration and retention) of model tumor antigen (OVA) and toll-like receptor 3 agonist (poly (I:C)) to immune cells in lymph nodes. Antigen-presenting cells treated with SVNP-OVA and SVNP-IC showed higher uptake of OVA and poly (I:C) and higher secretion of inflammatory cytokines (TNF-α, IL-6) and type I interferon (IFN-α, IFN-β) than those treated with OVA and poly (I:C) alone. In vivo analysis revealed higher levels of activation markers, inflammatory cytokines, and type I IFNs in the lymph nodes of mice immunized with SVNP-IC compared to those of mice in other groups. SVNP-IC-treated mice showed significantly greater in vivo natural killer cell expansion/activation (NK1.1 + cells) and CD8 + T cell response (CD8 + INF-γ + cells) in innate and adaptive immunity, respectively. Both preventive and therapeutic vaccination of EG7-OVA tumor-bearing mice using the simultaneous injection of both SVNP-OVA and SVNP-IC induced higher antitumor immunity and inhibited tumor growth., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mannosylphosphorylated glycans are found only in fungi, including yeast, and the elimination of mannosylphosphates from glycans is a prerequisite for yeast glyco-engineering to produce human-compatible glycoproteins. In Saccharomyces cerevisiae, MNN4 and MNN6 genes are known to play roles in mannosylphosphorylation, but disruption of these genes does not completely remove the mannosylphosphates in N-glycans. This study was performed to find unknown key gene(s) involved in N-glycan mannosylphosphorylation in S. cerevisiae. For this purpose, each of one MNN4 and five MNN6 homologous genes were deleted from the och1Δmnn1Δmnn4Δmnn6Δ strain, which lacks yeast-specific hyper-mannosylation and the immunogenic α(1,3)-mannose structure. N-glycan profile analysis of cell wall mannoproteins and a secretory recombinant protein produced in mutants showed that the MNN14 gene, an MNN4 paralog with unknown function, is essential for N-glycan mannosylphosphorylation. Double disruption of MNN4 and MNN14 genes was enough to eliminate N-glycan mannosylphosphorylation. Our results suggest that the S. cerevisiae och1Δmnn1Δmnn4Δmnn14Δ strain, in which all yeast-specific N-glycan structures including mannosylphosphorylation are abolished, may have promise as a useful platform for glyco-engineering to produce therapeutic glycoproteins with human-compatible N-glycans.

Allium hookeri is a wild herb found mainly in the Himalayas, growing at altitudes of 1400-4200 m. A. hookeri is widely consumed as a vegetable and herbal medicine in Asia, but its effects on bone health have not been reported previously. This study investigated the effects of a hot-water extract of A. hookeri roots on bone formation. The hot-water extract significantly increased the proliferation of in vitro human osteoblast-like MG-63 cells and the stimulatory effects on osteoblast differentiation were noticeably greater for the hot-water extract than for daidzein (a positive control), as reflected by alkaline phosphatase activity, collagen content, and mineral deposition. Expression of the bone-remodeling marker osteocalcin production and bone microstructural parameters were significantly improved in Sprague-Dawley rats in vivo after oral treatment with the hot-water extract compared with their control (saline-administered) counterparts. The chemical compounds of the hot-water extract were characterized by liquid chromatography-mass spectrometry, and alliin, sinapic acid, and ferulic acid, which exert beneficial effects on bone health, were identified. These findings indicate that A. hookeri can be used as a natural resource for increasing bone formation. This is the first report of the anabolic effects of A. hookeri extracts on bone formation in vitro and in vivo ., (Georg Thieme Verlag KG Stuttgart · New York.)

In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Genetic engineering approaches to improve the therapeutic potential of mesenchymal stem cells (MSCs) have been made by viral and non-viral gene delivery methods. Viral methods have severe limitations in clinical application because of potential oncogenic, pathogenic, and immunogenic risks, while non-viral methods have suffered from low transfection efficiency and transient weak expression as MSCs are hard-to-transfect cells. In this study, minicircle, which is a minimal expression vector free of bacterial sequences, was employed for MSC transfection as a non-viral gene delivery method. The conventional cationic liposome method was not effective for MSC transfection as it resulted in very low transfection efficiency (less than 5%). Microporation, a new electroporation method, greatly improved the transfection efficiency of minicircles by up to 66% in MSCs without any significant loss of cell viability. Furthermore, minicircle microporation generated much stronger and prolonged transgene expression compared with plasmid microporation. When MSCs microporated with minicircle harboring firefly luciferase gene were subcutaneously injected to mice, the bioluminescence continued for more than a week, whereas the bioluminescence of the MSCs induced by plasmid microporation rapidly decreased and disappeared in mice within three days. By minicircle microporation as a non-viral gene delivery, MSCs engineered to overexpress CXCR4 showed greatly increased homing ability toward an injury site as confirmed through in vivo bioluminescence imaging in mice. In summary, the engineering of MSCs through minicircle microporation is expected to enhance the therapeutic potential of MSCs in clinical applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans" (Kang et al., 2016 [1]).

Aggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular protective response to ER stress, the unfolded protein response (UPR), which is mediated by a basic leucine zipper (bZIP) transcription factor, Hac1p/Xbp1. In this study, we identified and studied the molecular functions of a HAC1 homolog from the thermotolerant yeast Hansenula polymorpha (HpHAC1). We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose interaction with the 5' untranslated region is responsible for the translational inhibition of the HpHAC1 mRNA. The H. polymorpha hac1-null (Hphac1Δ) mutant strain grew slowly, even under normal growth conditions, and was less thermotolerant than the wild-type (WT) strain. The mutant strain was also more sensitive to cell wall-perturbing agents and to the UPR-inducing agents dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the WT and Hphac1Δ strains treated with DTT and TM, we identified HpHAC1-dependent core UPR targets, which included genes involved in protein secretion and processing, particularly those required for N-linked protein glycosylation. Notably, different glycosylation and processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the WT and Hphac1Δ strains. Moreover, overexpression of active HpHac1p significantly increased the N-linked glycosylation efficiency and TM resistance. Collectively, our results suggest that the function of HpHac1p is important not only for UPR induction but also for efficient glycosylation in H. polymorpha., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

Bacterial α(2,6)-sialyltransferases (STs) from Photobacterium damsela, Photobacterium sp. JT-ISH-224, and P. leiognathi JT-SHIZ-145 were recombinantly expressed in Escherichia coli and their ST activities were compared directly using a galactosylated bi-antennary N-glycan as an acceptor substrate. In all ST reactions, there was an increase of sialylated glycans at shorter reaction times and later a decrease in prolonged reactions, which is related with the inherent sialidase activities of bacterial STs. These sialidase activities are greatly increased by free cytidine monophosphate (CMP) generated from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac) during the ST reactions. The decrease of sialylated glycans in prolonged ST reaction was prevented through an inhibition of sialidase activity by simple treatment of alkaline phosphatase (AP), which dephosphorylates CMP to cytidine. Through supplemental additions of AP and CMP-Neu5Ac to the reaction using the recombinant α(2,6)-ST from P. leiognathi JT-SHIZ-145 (P145-ST), the content of bi-sialylated N-glycan increased up to ~98% without any decrease in prolonged reactions. This optimized P145-ST reaction was applied successfully for α(2,6)-sialylation of asialofetuin, and this resulted in a large increase in the populations of multi-sialylated N-glycans compared with the reaction without addition of AP and CMP-Neu5Ac. These results suggest that the optimized reaction using the recombinant P145-ST readily expressed from E. coli has a promise for economic glycan synthesis and glyco-conjugate remodeling.

Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. In this study, we show that heterologous expression of YlMpo1p can perform and enhance mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, the level of mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants. When YlMpo1p overexpression is applied to glyco-engineered S. cerevisiae in which the synthesis of immunogenic glycans is abolished, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in yeast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig(P)) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig(M)). However, limitations of hCTLA4Ig(P) include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (β1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4Ig(P) (hCTLA4Ig(P)-Gal) was expressed through the coexpression of human β1,4-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 hCTLA4Ig(P)-Gal indicated that not only were the two glycans containing (β1-4)-galactose newly extended, but also glycans containing both β1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig(P), respectively. Unlike hCTLA4Ig(P), hCTLA4Ig(P)-Gal was effective as an acceptor via (β1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig(P)-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig(P), and the clearance pattern of hCTLA4Ig(P)-Gal was close to that for hCTLA4Ig(M). These results indicate that the coexpression with hGalT and hCTLA4Ig(P) 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.

The modular auxiliary activity (AA) family of proteins is believed to cause amorphogenesis in addition to oxidative cleavage of crystalline cellulose although the supporting evidence is limited. HcAA10-2 is a modular AA10 family protein (58 kDa) composed of a AA10 module and a family two carbohydrate binding module (CBM2), joined by a long stretch of 222 amino acids of unknown function. The protein was expressed in Escherichia coli and purified to homogeneity. Scanning electron microscopy and X-ray diffraction analysis of Avicel treated with HcAA10-2 provided evidence for the disruption of the cellulose microfibrils ("amorphogenesis") and reduction of the crystallinity index, resulting in a twofold increase of cellulase adsorption on the polysaccharide surface. HcAA10-2 exhibited weak endoglucanase-like activity toward soluble cellulose and cello-oligosaccharides with an optimum at pH 6.5 and 45 °C. HcAA10-2 catalyzed oxidative cleavage of crystalline cellulose released native and oxidized cello-oligosaccharides in the presence of copper and an electron donor such as ascorbic acid. Multiple sequence alignment indicated that His1, His109, and Phe197 in the AA10 module formed the conserved copper-binding site. The reducing sugar released from Avicel by the endoglucanase Cel5 and Celluclast accompanying HcAA10-2 was increased by four- and sixfold, respectively. Moreover, HcAA10-2 and Celluclast acted synergistically on pretreated wheat straw biomass resulting in a threefold increase in reducing sugar than Celluclast alone. Taken together, these results suggest that HcAA10-2 is a novel multifunctional modular AA10 protein possessing amorphogenesis, weak endoglucanase, and oxidative cleavage activities useful for efficient degradation of crystalline cellulose.

The influence of developmental stage and position (top, middle, and base) of leaves and stem tissues on the expression and glycosylation pattern of a recombinant therapeutic protein -GA733-FcK- was observed in transgenic seedlings during a 16-week growth period. RNA expression gradually increased with age in the middle and basal leaves and decreased in top leaves after 14 weeks. The protein expression level at all leaf positions increased until 14 weeks and slightly decreased at 16 weeks; it was lower in yellow leaves than in green leaves. In stem, protein expression gradually decreased from the top to the base. The glycosylation patterns of GA733-FcK were analyzed from 10 to 16 weeks. The plant-specific glycans increased in the top leaves at 14 weeks, but only slightly changed in the middle and basal leaves. The structure of glycans varied with tissue position. The glycosylation level in the top and middle leaves increased until 12 and 14 weeks, respectively, and decreased thereafter, whereas it decreased in basal leaves until 14 weeks and increased at 16 weeks. In stem, all three sections showed high-mannose type glycan structures. The area size of the glycans was significantly higher in the top stem than in both the middle and basal stems, and it was smaller in yellow leaves than in green leaves. The glycan profiles were similar between green and yellow leaves until 16 weeks. Thus, biomass-harvesting time should be optimized to obtain recombinant therapeutic proteins with ideal glycan structure profiles.

In this study, we characterized the CpxRA two-component signal transduction system of the rumen bacterium Mannheimia succiniciproducens. The truncated form of the CpxA sensor kinase protein without its transmembrane domain was able to autophosphorylate and transphosphorylate the CpxR response regulator protein in vitro. We identified 152 putative target genes for the Cpx system in M. succiniciproducens, which were differentially expressed by more than twofold upon overexpression of the CpxR protein. Genes of a putative 16-gene operon related to the cell wall and lipopolysaccharide biosynthesis were induced strongly upon CpxR overexpression. The promoter region of the first gene of this operon, wecC encoding UDP-N-acetyl-D-mannosaminuronate dehydrogenase, was analyzed and found to contain a sequence homologous to the CpxR box of Escherichia coli. An electrophoretic mobility shift assay showed that the phosphorylated CpxR proteins were able to bind specifically to PCR-amplified DNA fragments containing the promoter sequence of wecC. Furthermore, a cpxR-disrupted mutant strain exhibited increased envelope permeability compared with a wild-type strain. These results suggest that the Cpx system of M. succiniciproducens is involved in the maintenance of the integrity of the cell envelope.

Since the advent of nanoparticle technology, novel and versatile properties of nanomaterials have been introduced, which has constantly expanded their applications in therapeutics. Introduction of nanomaterials for immunomodulation has opened up new avenues with tremendous potential. Interesting properties of nanoparticles, such as adjuvanticity, capability to enhance cross-presentation, polyvalent presentation, siRNA delivery for silencing of immunesuppressive gene, targeting and imaging of immune cells have been known to have immense utility in vaccination and immunotherapy. A thorough understanding of the merits associated with nanomaterials is crucial for designing of modular and versatile nanovaccines, for improved immune response. With the emerging prerequisites of vaccination, nanomaterial-based immune stimulation, seems to be capable of taking the field of immunization to a next higher level.

Peptide:N-glycanase (PNGase) A is used preferentially to cleave the glycans from plant and insect glycopeptides. Although many putative PNGase A homologous genes have been found in the plant and fungus kingdoms through sequence similarity analyses, only several PNGases from plants and one from a filamentous fungus have been characterized. In this study, we identified and characterized a PNGase A-like enzyme, PNGase Yl, in the dimorphic yeast Yarrowia lipolytica. The corresponding gene was cloned and recombinantly expressed in Pichia pastoris. The purified enzyme cleaved glycans from glycopeptides with the maximum activity at pH 5. No metal ions were required for full activity, and rather it was repressed by three metal ions (Fe(3+), Cu(2+) and Zn(2+)). Using glycopeptide substrates, PNGase Yl was shown to release various types of N-glycans including high-mannose and complex-type glycans as well as glycans containing core-linked α(1,3)-fucose that are frequently found in plants and insects. Moreover, in comparison with PNGase A, PNGase Yl was able to cleave with higher efficiency the glycans from some denatured glycoproteins. Taken together, our results suggest that PNGase Yl, the first biochemically characterized yeast PNGase A homologue, can be developed through protein engineering as a useful deglycosylation tool for N-glycosylation study., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-β-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley β-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at 50°C, with Azo-barley-glucan as the substrate. The metal ions Mn(2+), Mg(2+), Ca(2+), and Fe(2+) enhanced the enzymatic activity, whereas the Cu(2+) and Zn(2+) ions inhibited the enzymatic activity. The Km and Vmax values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

Bioinformatic analysis of the genome of the methylotrophic yeast Hansenula polymorpha revealed 39 putative glycosylphosphatidylinositol-anchored proteins (GPI-proteins). Notably, dibasic motifs in the proximal ω-site, that has been reported as a plasma membrane retention signal in Saccharomyces cerevisiae GPI-proteins, were not found in any of the predicted GPI-proteins of H. polymorpha. To evaluate the in silico prediction, C-terminal peptides of 40 amino acids derived from ten H. polymorpha GPI-proteins were fused to the Aspergillus saitoi α-1,2-mannosidase (msdS). Cell wall fraction analysis showed that nine of the ten msdS-GPI fusion proteins were mostly localized at the cell wall. Surface expression of functional msdS was further confirmed by in vitro enzyme activity assay and by glycan structure analysis of cell wall mannoproteins. The recombinant H. polymorpha strains expressing surface-displayed msdS have the potential as useful hosts to produce glycoproteins with decreased mannosylation.

In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species.

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca(2+) greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to grow at elevated temperature up to 48 °C as one of a few yeast strains which are naturally capable of alcoholic fermentation of xylose, a pentose sugar abundant in lignocellulosic biomass. However, the current level of ethanol production from xylose by H. polymorpha is still very low compared to those of other xylose-fermenting strains. Therefore, it is necessary to analyze and remodel the xylose metabolism in H. polymorpha at the whole genome level to identify and overcome these limits. In the present study, the transcriptomes of H. polymorpha grown on xylose were compared with those of glucose-grown cells under both aerobic and microaerobic conditions. Approximately, two percent of H. polymorpha genes were either up- or down-regulated by more than two-fold during the growth on xylose. The majority of the up-regulated genes were involved in metabolism. Some genes involved in xylose metabolism, such as XYL1, XYL2, and TAL1 were also up-regulated, despite the fact that the differences in their induction level were only about three-fold. On the other hand, the majority of the down-regulated genes were involved in metabolism and cellular transport. Interestingly, some genes involved in glycolysis and ethanol fermentation were also repressed during growth on xylose, suggesting that these genes are good targets for engineering H. polymorpha to improve xylose fermentation.

The genome of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologues (HpPMT1, HpPMT2, HpPMT4, HpPMT5, and HpPMT6) encoding protein O-mannosyltransferases. Here, we report on the systematic characterization of HpPMT5 and HpPMT6, encoding novel PMT1 and PMT2 subfamily members, respectively. Although no apparent growth defects were detected in the Hppmt5Δ and Hppmt6Δ single mutants, the single mutants showed dramatic sensitivity to the Pmt1p inhibitor, and the Hppmt1pmt5Δ and Hppmt1pmt6Δ double mutants displayed increased susceptibility to cell wall-disturbing reagents. Activation of the cell wall integrity signaling pathway in the double mutant strains was further indicated by the markedly induced phosphorylation of MAP kinases, such as HpMpk1p and HpHog1p. Noticeably, O-mannosylation of the surface glycoproteins HpWsc1p and HpMid2p became severely defective only in the double mutants, supporting the involvement of HpPmt5p and HpPmt6p in O-mannosylation of these sensor proteins. On the other hand, co-immunoprecipitation experiments revealed only marginal interaction between HpPmt5p and HpPmt2p, even in the absence of HpPmt1p. Taken together, our results suggest that the functions of HpPmt5p and HpPmt6p are minor but become crucial upon the loss of HpPmt1p for protein O-mannosylation, which is essential for cell growth, cell wall integrity, and stress resistance in H. polymorpha., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(P)s), provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P) SO57 with or without an endoplasmic reticulum (ER)-retention peptide signal (Lys-Asp-Glu-Leu; KDEL) in transgenic tobacco plants (Nicotiana tabacum) were analyzed. The expression levels of mAb(P) SO57 with KDEL (mAb(P)K) were significantly higher than those of mAb(P) SO57 without KDEL (mAb(P)) regardless of the transcription level. The Fc domains of both purified mAb(P) and mAb(P)K and hybridoma-derived mAb (mAb(H)) had similar levels of binding activity to the FcγRI receptor (CD64). The mAb(P)K had glycan profiles of both oligomannose (OM) type (91.7%) and Golgi type (8.3%), whereas the mAb(P) had mainly Golgi type glycans (96.8%) similar to those seen with mAb(H). Confocal analysis showed that the mAb(P)K was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P) with KDEL in the ER. Both mAb(P) and mAb(P)K disappeared with similar trends to mAb(H) in BALB/c mice. In addition, mAb(P)K was as effective as mAb(H) at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P) by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.

Human α-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version.

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase δ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'-->5' exonuclease activity. The resulting HpPOL3* gene encoding the error-prone proofreading-deficient DNA polymerase δ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a URA3(-) mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of HpPOL3*. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

To explore the physiological significance of N-glycan maturation in the plant Golgi apparatus, gnt1, a mutant with loss of N-acetylglucosaminyltransferase I (GnTI) function, was isolated in Oryza sativa. gnt1 exhibited complete inhibition of N-glycan maturation and accumulated high-mannose N-glycans. Phenotypic analyses revealed that gnt1 shows defective post-seedling development and incomplete cell wall biosynthesis, leading to symptoms such as failure in tiller formation, brittle leaves, reduced cell wall thickness, and decreased cellulose content. The developmental defects of gnt1 ultimately resulted in early lethality without transition to the reproductive stage. However, callus induced from gnt1 seeds could be maintained for periods, although it exhibited a low proliferation rate, small size, and hypersensitivity to salt stress. Shoot regeneration and dark-induced leaf senescence assays indicated that the loss of GnTI function results in reduced sensitivity to cytokinin in rice. Reduced expression of A-type O. sativa response regulators that are rapidly induced by cytokinins in gnt1 confirmed that cytokinin signaling is impaired in the mutant. These results strongly support the proposed involvement of N-glycan maturation in transport as well as in the function of membrane proteins that are synthesized via the endomembrane system., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)