ALBERT

Description:    High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans ( e.g. , ConA), but not to other lectins capable of binding to other carbohydrates ( e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans. Content Type Journal Article Pages 1-12 DOI 10.1007/s10719-012-9449-3 Authors Seon-Joo Yoon, Division of Biomembrane Research, Pacific Northwest Research Institute, and Department of Global Health, University of Washington, Seattle, WA 98122, USA Natalia Utkina, Division of Biomembrane Research, Pacific Northwest Research Institute, and Department of Global Health, University of Washington, Seattle, WA 98122, USA Martin Sadilek, Depart of Chemistry, University of Washington, Seattle, WA 98195, USA Hirokazu Yagi, Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori 3-1, Mizuho-ku, Nagoya, 467-8603 Japan Koichi Kato, Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori 3-1, Mizuho-ku, Nagoya, 467-8603 Japan Sen-itiroh Hakomori, Division of Biomembrane Research, Pacific Northwest Research Institute, and Department of Global Health, University of Washington, Seattle, WA 98122, USA Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    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. Content Type Journal Article Pages 1-11 DOI 10.1007/s10719-012-9453-7 Authors Kyung Jin Lee, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806 Korea Sang Mee Lee, Alteogen Inc., Bioventure town, Daejeon, 305-812 Korea Jin Young Gil, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806 Korea Ohsuk Kwon, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806 Korea Jin Young Kim, Korea Basic Science Institute, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Korea Soon Jae Park, Alteogen Inc., Bioventure town, Daejeon, 305-812 Korea Hye-Shin Chung, Alteogen Inc., Bioventure town, Daejeon, 305-812 Korea Doo-Byoung Oh, Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806 Korea Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    As one of several biologically active compounds in milk, glycoproteins have been indicated to be involved in the protection of newborns from bacterial infection. As much of the physical and immune development of the tammar wallaby ( Macropus eugenii ) young occurs during the early phases of lactation and not in utero , the tammar is a model species for the characterization of potential developmental support agents provided by maternal milk. In the present study, the N - and O -linked glycans from tammar wallaby milk glycoproteins from six individuals at different lactation time points were subjected to glycomics analyses using porous graphitized carbon liquid chromatography electrospray ionization mass spectrometry. Structural characterization identified a diverse range of glycan structures on wallaby milk glycoproteins including sialylated, sulphated, core fucosylated and O -fucosylated structures. 30 % of N -linked structures contained a core (α1-6) fucose. Several of these structures may play roles in development, and exhibit statistically significant temporal changes over the lactation period. The N -glycome was found to contain structures with NeuGc residues, while in contrast the O -glycome did not. O -fucosylated structures were identified in the early stages of lactation indicating a potential role in the early stages of development of the pouch young. Overall the results suggest that wallaby milk contains structures known to have developmental and immunological significance in human milk and reproduction in other animals, highlighting the importance of glycoproteins in milk. Content Type Journal Article Pages 1-14 DOI 10.1007/s10719-012-9452-8 Authors Katherine Wongtrakul-Kish, Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Building E8C Room 307, North Ryde, NSW 2109, Australia Daniel Kolarich, Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Building E8C Room 307, North Ryde, NSW 2109, Australia Dana Pascovici, Australian Proteome Analysis Facility, Macquarie University, North Ryde, NSW 2109, Australia Janice L. Joss, Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia Elizabeth Deane, Department of Biological Sciences, Macquarie University, North Ryde, NSW 2109, Australia Nicolle H. Packer, Biomolecular Frontiers Research Centre, Department of Chemistry and Biomolecular Sciences, Macquarie University, Building E8C Room 307, North Ryde, NSW 2109, Australia Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description: Glycosylation effects on cancer development Content Type Journal Article Pages 1-2 DOI 10.1007/s10719-012-9448-4 Authors Sen-itiroh Hakomori, Division of Biomembrane Research, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA Richard D. Cummings, Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N -acetylneuraminic acid (Neu5Ac)-containing complex-type N -glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N -glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N -glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker. Content Type Journal Article Pages 1-10 DOI 10.1007/s10719-012-9435-9 Authors Masahiko Yabu, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, 537-8511 Japan Hiroaki Korekane, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Hidenori Takahashi, Department of Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, 537-8511 Japan Hiroaki Ohigashi, Department of Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, 537-8511 Japan Osamu Ishikawa, Department of Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, 537-8511 Japan Yasuhide Miyamoto, Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka, 537-8511 Japan Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    Sulfatides, 3- O -sulfogalactosylceramides, are known to have multifunctional properties. These molecules are distributed in various tissues of mammals, where they are synthesized from galactosylceramides by sulfation at C3 of the galactosyl residue. Although this reaction is specifically catalyzed by cerebroside sulfotransferase (CST), the mechanisms underlying the transcriptional regulation of this enzyme are not understood. With respect to this issue, we previously found potential sequences of peroxisome proliferator-activated receptor (PPAR) response element on upstream regions of the mouse CST gene and presumed the possible regulation by the nuclear receptor PPARα. To confirm this hypothesis, we treated wild-type and Ppara -null mice with the specific PPARα agonist fenofibrate and examined the amounts of sulfatides and CST gene expression in various tissues. Fenofibrate treatment increased sulfatides and CST mRNA levels in the kidney, heart, liver, and small intestine in a PPARα-dependent manner. However, these effects of fenofibrate were absent in the brain or colon. Fenofibrate treatment did not affect the mRNA level of arylsulfatase A, which is the key enzyme for catalyzing desulfation of sulfatides, in any of these six tissues. Analyses of the DNA-binding activity and conventional gene expression targets of PPARα has demonstrated that fenofibrate treatment activated PPARα in the kidney, heart, liver, and small intestine but did not affect the brain or colon. These findings suggest that PPARα activation induces CST gene expression and enhances sulfatide synthesis in mice, which suggests that PPARα is a possible transcriptional regulator for the mouse CST gene. Content Type Journal Article Pages 1-8 DOI 10.1007/s10719-012-9454-6 Authors Takero Nakajima, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Yuji Kamijo, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Huang Yuzhe, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Takefumi Kimura, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Naoki Tanaka, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Eiko Sugiyama, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Kozo Nakamura, Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Minami-Minowa, Kami-Ina, Nagano, Japan Mamoru Kyogashima, Division of Microbiology and Molecular Cell Biology, Nihon Pharmaceutical University, Ina, Kita-Adachi, Saitama, Japan Atsushi Hara, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Toshifumi Aoyama, Department of Metabolic Regulation, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    The bisecting GlcNAc is transferred to the core mannose residue of complex or hybrid N-glycans on glycoproteins by the β1,4- N -acetylglucosaminyltransferase III (GlcNAcT-III) or MGAT3. The addition of the bisecting GlcNAc confers unique lectin recognition properties to N-glycans. Thus, LEC10 gain-of-function Chinese hamster ovary (CHO) cells selected for the acquisition of ricin resistance, carry N-glycans with a bisecting GlcNAc, which enhances the binding of the erythroagglutinin E-PHA, but reduces the binding of ricin and galectins-1, -3 and -8. The altered interaction with galactose-binding lectins suggests that the bisecting GlcNAc affects N-glycan conformation. LEC10 mutants expressing polyoma middle T antigen (PyMT) exhibit reduced growth factor signaling. Furthermore, PyMT-induced mammary tumors lacking MGAT3, progress more rapidly than tumors with the bisecting GlcNAc on N-glycans of cell surface glycoproteins. In recent years, evidence for a new paradigm of cell growth control has emerged involving regulation of cell surface residency of growth factor and cytokine receptors via interactions and cross-linking of their branched N-glycans with a lattice of galectin(s). Specific cross-linking of glycoprotein receptors in the lattice regulates their endocytosis, leading to effects on growth factor-induced signaling. This review will describe evidence that the bisecting GlcNAc of N-glycans regulates cellular signaling and tumor progression, apparently through modulating N-glycan/galectin interactions. Content Type Journal Article Pages 1-10 DOI 10.1007/s10719-012-9373-6 Authors Hazuki E. Miwa, Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA Yinghui Song, Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA Richard Alvarez, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Richard D. Cummings, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Pamela Stanley, Department of Cell Biology, Albert Einstein College of Medicine, New York, NY 10461, USA Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    The past 25 years have seen significant advances in understanding the diversity and functions of glycoprotein glycans in Drosophila melanogaster . Genetic screens have captured mutations that reveal important biological activities modulated by glycans, including protein folding and trafficking, as well as cell signaling, tissue morphogenesis, fertility, and viability. Many of these glycan functions have parallels in vertebrate development and disease, providing increasing opportunities to dissect pathologic mechanisms using Drosophila genetics. Advances in the sensitivity of structural analytic techniques have allowed the glycan profiles of wild-type and mutant tissues to be assessed, revealing novel glycan structures that may be functionally analogous to vertebrate glycans. This review describes a selected set of recent advances in understanding the functions of N-linked and O-linked (non-glycosaminoglycan) glycoprotein glycans in Drosophila with emphasis on their relatedness to vertebrate organisms. Content Type Journal Article Pages 1-10 DOI 10.1007/s10719-012-9442-x Authors Toshihiko Katoh, The Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA Michael Tiemeyer, The Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    In this study, we purified and characterized the β-xylosidase involved in the turnover of plant complex type N -glycans to homogeneity from mature red tomatoes. Purified β-xylosidase (β-Xyl’ase Le-1) gave a single band with molecular masses of 67 kDa on SDS-PAGE under a reducing condition and 60 kDa on gelfiltration, indicating that β-Xyl’ase Le-1 has a monomeric structure in plant cells. The N- terminal amino acid could not be identified owing to a chemical modification. When pyridylaminated (PA-) N- glycans were used as substrates, β-Xyl’ase Le-1 showed optimum activity at about pH 5 at 40 °C, suggesting that the enzyme functions in a rather acidic circumstance such as in the vacuole or cell wall. β-Xyl’ase Le-1 hydrolyzed the β1-2 xylosyl residue from Man 1 Xyl 1 GlcNAc 2 -PA, Man 1 Xyl 1 Fuc 1 GlcNAc 2 -PA, and Man 2 Xyl 1 Fuc 1 GlcNAc 2 -PA, but not that from Man 3 Xyl 1 GlcNAc 2 -PA or Man 3 Xyl 1 Fuc 1 GlcNAc 2 -PA, indicating that the α1-3 arm mannosyl residue exerts significant steric hindrance for the access of β-Xyl’ase Le-1 to the xylosyl residue, whereas the α1-3 fucosyl residue exerts little effect. These results suggest that the release of the β1-2 xylosyl residue by β-Xyl’ase Le-1 occurs at least after the removal the α-1,3-mannosyl residue in the core trimannosyl unit. Content Type Journal Article Pages 1-10 DOI 10.1007/s10719-012-9441-y Authors Daisuke Yokouchi, Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka 1-1-1, Okayama, 700-8530 Japan Natsuko Ono, Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka 1-1-1, Okayama, 700-8530 Japan Kosuke Nakamura, Kagome Research Institute, Kagome Co., Ltd., 17 Nishitomiyama, Nasushiobara, Tochigi 329-2762, Japan Megumi Maeda, Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka 1-1-1, Okayama, 700-8530 Japan Yoshinobu Kimura, Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Tsushima-Naka 1-1-1, Okayama, 700-8530 Japan Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080

Description:    Many post-translational modifications, including glycosylation, are pivotal for the structural integrity, location and functional activity of glycoproteins. Sub-populations of proteins that are relocated or functionally changed by such modifications can change resting proteins into active ones, mediating specific effector functions, as in the case of monoclonal antibodies. To ensure safe and efficacious drugs it is essential to employ appropriate robust, quantitative analytical strategies that can (i) perform detailed glycan structural analysis, (ii) characterise specific subsets of glycans to assess known critical features of therapeutic activities (iii) rapidly profile glycan pools for at-line monitoring or high level batch to batch screening. Here we focus on these aspects of glycan analysis, showing how state-of-the-art technologies are required at all stages during the production of recombinant glycotherapeutics. These data can provide insights into processing pathways and suggest markers for intervention at critical control points in bioprocessing and also critical decision points in disease and drug monitoring in patients. Importantly, these tools are now enabling the first glycome/genome studies in large populations, allowing the integration of glycomics into other ‘omics platforms in a systems biology context. Content Type Journal Article Pages 1-10 DOI 10.1007/s10719-012-9443-9 Authors Tharmala Tharmalingam, NIBRT Glycobiology Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland Barbara Adamczyk, NIBRT Glycobiology Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland Margaret A. Doherty, NIBRT Glycobiology Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland Louise Royle, Ludger Ltd., Culham Science Centre, Oxfordshire, OX14 3EB UK Pauline M. Rudd, NIBRT Glycobiology Laboratory, NIBRT - The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland Journal Glycoconjugate Journal Online ISSN 1573-4986 Print ISSN 0282-0080