Hirokazu Yagi

Comparison of Methods for Profiling O-Glycosylation HUMAN PROTEOME ORGANISATION HUMAN DISEASE GLYCOMICS/PROTEOME INITIATIVE MULTI-INSTITUTIONAL STUDY OF IgA1

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiatives activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Edible bird's nest extract inhibits influenza virus infection

Abstract Edible birds nest (EBN) is the nest of the swift that is made from its saliva. Although EBN has been widely used for enhancing immunocompetence, its antiviral efficacy has not been studied in detail. We found that EBN extract could strongly inhibit infection with influenza viruses in a host range-independent manner when it was hydrolyzed with Pancreatin F. Western blotting assay showed that the EBN extract bound to influenza virus. Furthermore, EBN extract could neutralize the infection of MDCK cells with influenza viruses and inhibit hemagglutination of influenza viruses to erythrocytes, but it could not inhibit the activity of influenza virus sialidase. Fluorometric HPLC indicated that the major molecular species of sialic acid in EBN is N-acetylneuraminic acid. The results suggest that EBN is a safe and valid natural source for the prevention of influenza viruses.

Alterations in receptor-binding properties of swine influenza viruses of the H1 subtype after isolation in embryonated chicken eggs

Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptor-binding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) alpha2,6galactose (Gal) or a Neu5Acalpha2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Acalpha2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Acalpha2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galbeta1,3/4GlcNAcalpha2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The alpha2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Acalpha2,3Gal more abundantly than Neu5Acalpha2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) alpha2,6Gal and Neu5Acalpha2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.

Lewis X-carrying N-Glycans Regulate the Proliferation of Mouse Embryonic Neural Stem Cells via the Notch Signaling Pathway

Background: Glycosylation is known to be altered upon differentiation of neural stem cells (NSCs). Results: NSC-specific Lewis X-carrying N-glycans up-regulated the expression of Musashi-1 and the consequent cell proliferation. Conclusion: Lewis X operates as an activator of the Notch signaling pathway for the maintenance of NSC stemness. Significance: The well known undifferentiation glycomarker of NSCs is actively involved in their fate determination. Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. Several signaling pathways have been shown to be involved in the fate determination process of NSCs, but the molecular mechanisms underlying the maintenance of neural cell stemness remain largely unknown. Our previous study showed that human natural killer carbohydrate epitopes expressed specifically by mouse NSCs modulate the Ras-MAPK pathway, raising the possibility of regulatory roles of glycoprotein glycans in the specific signaling pathways involved in NSC fate determination. To address this issue, we performed comparative N-glycosylation profiling of NSCs before and after differentiation in a comprehensive and quantitative manner. We found that Lewis X-carrying N-glycans were specifically displayed on undifferentiated cells, whereas pauci-mannose-type N-glycans were predominantly expressed on differentiated cells. Furthermore, by knocking down a fucosyltransferase 9 with short interfering RNA, we demonstrated that the Lewis X-carrying N-glycans were actively involved in the proliferation of NSCs via modulation of the expression level of Musashi-1, which is an activator of the Notch signaling pathway. Our findings suggest that Lewis X carbohydrates, which have so far been characterized as undifferentiation markers, actually operate as activators of the Notch signaling pathway for the maintenance of NSC stemness during brain development.

A non-canonical UBA-UBL interaction forms the linear-ubiquitin-chain assembly complex

HOIL‐1L and its binding partner HOIP are essential components of the E3‐ligase complex that generates linear ubiquitin (Ub) chains, which are critical regulators of NF‐κB activation. Using crystallographic and mutational approaches, we characterize the unexpected structural basis for the specific interaction between the Ub‐like domain (UBL) of HOIL‐1L and the Ub‐associated domain (UBA) of HOIP. Our data indicate the functional significance of this non‐canonical mode of UBA–UBL interaction in E3 complex formation and subsequent NF‐κB activation. This study highlights the versatility and specificity of protein–protein interactions involving Ub/UBLs and their cognate proteins.

N-Glycans from Porcine Trachea and Lung: Predominant NeuAcα2-6Gal Could Be a Selective Pressure for Influenza Variants in Favor of Human-Type Receptor

It is known that pigs acted as “mixing vessels” for genesis of a new reassortant influenza strain responsible for pandemic H1N1 2009. However, the host factors driving the evolution of a reassorted virus in pigs to ‘jump species’ resulting in a human outbreak remain unclear. N-glycans derived from the porcine respiratory tract were enzymatically released, fluorescent labeled with 2-aminopyridine, separated according to charge, size and hydrophobicity, and structurally identified by a two-dimensional (size and hydrophobicity) HPLC mapping technique and MALDI-TOF mass spectrometry before and after exo-glycosidase digestion. We found a 3-, 5-, and 13-fold increases in NeuAcα2-6, a preferable human influenza receptor, over NeuAcα2-3, an avian influenza receptor, from upper and lower parts of the porcine trachea towards the porcine lung, a major target organ for swine virus replication. The large proportion of NeuAcα2-6 may exert selective pressure for selection of influenza variants with altered receptor preference for this human-type α2-6 receptor, a crucial first step for generating a human pandemic.

The expression of sialylated high-antennary N-glycans in edible bird’s nest

Edible birds nest (EBN) is the nest made from the saliva of Collocalia swift. Recently, we have found that EBN extract could strongly inhibit infection of influenza viruses in a host-range-independent manner [Guo, C. T.; Takahashi, T.; Bukawa, W.; Takahashi, N.; Yagi, H.; Kato, K.; Hidari, K. I.; Miyamoto, D.; Suzuki, T.; Suzuki, Y. Antiviral Res.2006, 70, 140-146]. Although this antiviral activity might be attributed to O- or N-glycoconjugates, no N-glycan structures have so far been described for EBN. Here, we report the N-glycosylation profile of EBN, in which a tri-antennary N-glycan bearing the alpha2,3-N-acetylneuraminic acid residues is displayed as a major component. We suggest that the sialylated high-antennary N-glycans of EBN contribute to the inhibition of influenza viral infection.

Comparison of the N-linked glycosylation of human β1,3-N-acetylglucosaminyltransferase 2 expressed in insect cells and silkworm larvae

N-Glycosylation of human beta1,3N-acetylglucosaminyltransferase 2 (beta3GnT2) is essential for its biological function. beta3GnT2 fused to GFP(uv) (GFP(uv)-beta3GnT2) was produced by non-virus expression systems in stably transformed insect cells and silkworm larvae using a recombinant BmNPV bacmid, and purified for analysis of N-glycosylation. The N-glycan structure of beta3GnT2 was identified by glycoamidase A digestion, labeling with 2-aminopyridine (PA), and HPLC mapping. The paucimannosidic N-glycan structure (73.2%) was predominant in stably transformed Trichoplusia ni cells. In contrast, N-glycan with Gal (21.3%) and GlcNAc (16.2%) terminal residues linked to Manalpha(1,3) branch were detected on beta3GnT2 expressed in silkworm larvae. The presence of terminal Gal and bisecting GlcNAc residues such as Galbeta1, 4GlcNAcbeta1, 2Manalpha1,3(GlcNAcbeta1,4)(Manalpha1,6)Manbeta1, 4GlcNAc is not typical structure for lepidopteran insect N-glycosylation. Although allergenic alpha1,3-fucose residues have been found in T. ni cells, only alpha1,6-fucose residues were attached to the beta3GnT2 glycan in silkworm larvae. Therefore, silkworm larvae might be a useful host for producing human glycoproteins.

Crystal structures of human secretory proteins ZG16p and ZG16b reveal a Jacalin-related β-prism fold

ZG16p is a secretory protein that mediates condensation-sorting of pancreatic enzymes to the zymogen granule membrane in pancreatic acinar cells. ZG16p interacts with glycosaminoglycans and the binding is considered to be important for condensation-sorting of pancreatic enzymes. ZG16b/PAUF, a paralog of ZG16p, has recently been found to play a role in gene regulation and cancer metastasis. However, the detailed functions of ZG16p and ZG16b remain to be clarified. Here, in order to obtain insights into structure-function relationships, we conducted crystallographic studies of human ZG16p lectin as well as its paralog, ZG16b, and determined their crystal structures at 1.65 and 2.75 Å resolution, respectively. ZG16p has a Jacalin-related β-prism fold, the first to be reported among mammalian lectins. The putative sugar-binding site of ZG16p is occupied by a glycerol molecule, mimicking the mannose bound to Jacalin-related mannose-binding-type plant lectins such as Banlec. ZG16b also has a β-prism fold, but some amino acid residues of the putative sugar-binding site differ from those of the mannose-type binding site suggesting altered preference. A positively charged patch, which may bind sulfated groups of the glycosaminoglycans, is located around the putative sugar-binding site of ZG16p and ZG16b. Taken together, we suggest that the sugar-binding site and the adjacent basic patch of ZG16p and ZG16b cooperatively form a functional glycosaminoglycan-binding site.