English Journal

Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan.

Kakizaki I1, Mineta T2, Sasaki M3, Tatara Y4, Makino E3, Kato Y5.Author information 1Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Electronic address: kaki@cc.hirosaki-u.ac.jp.2Department of Intelligent Machines and System Engineering, Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan; Department of Mechanical Systems Engineering, Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa 992-8510, Japan.3Department of Intelligent Machines and System Engineering, Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan.4Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.5Laboratory of Food Science, Faculty of Education, Hirosaki University, 1 Bunkyo-cho, Hirosaki 036-8560, Japan.AbstractBiological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilage aggrecan purified from 4M guanidine hydrochloride extract were analyzed using HPLCs and atomic force microscopy with bovine tracheal cartilage aggrecan as a control. The estimated numbers of sulfates per disaccharide unit of chondroitin sulfate chains of salmon and bovine aggrecans were similar (approximately 0.85). However, the disaccharide composition showed a higher proportion of chondroitin 6-sulfate units in salmon aggrecan, 60%, compared to 40% in bovine. Gel filtration HPLC and monosaccharide analysis showed the salmon aggrecan had a lower number (approximately one-third), but 1.5-3.3 times longer chondroitin sulfate chains than the bovine aggrecan. Atomic force microscopic molecular images of aggrecan supported the images predicted by biochemical analyses.
Carbohydrate polymers.Carbohydr Polym.2014 Mar 15;103:538-49. doi: 10.1016/j.carbpol.2013.12.083. Epub 2014 Jan 7.
Biological activities of salmon nasal cartilage proteoglycan fractions are known, however, structural information is lacking. Recently, the major proteoglycan of this cartilage was identified as aggrecan. In this study, global molecular images and glycosaminoglycan structure of salmon nasal cartilag
PMID 24528764

One set system for the synthesis and purification of glycosaminoglycan oligosaccharides reconstructed using a hyaluronidase-immobilized column.

Suto S1, Kakizaki I, Nakamura T, Endo M.Author information 1Department of Glycobiochemistry, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan; Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, 036-8562, Japan.AbstractUsing the transglycosylation reaction as a reverse reaction for the hydrolysis of hyaluronidase, new artificial oligosaccharides may be synthesized by reconstructing natural glycosaminoglycans (GAGs) according to preliminary planned arrangements. However, as some problems have been associated with the method, including the low yields of reaction products and complicated processes of separation and purification, improvements in this method were investigated. Transglycosylation reactions were carried out using bovine testicular hyaluronidase-immobilized resin packed in a column. For the transglycosylation reaction, pyridylaminated (PA) GAG hexasaccharides, which were the minimum size for hydrolysis sensitivity and the transglycosylation reaction, were used as acceptors, whereas large size GAGs were used as donors. The reaction mixture was pooled after incubation in the hyaluronidase-immobilized resin column and was then introduced into continuously joined HPLC columns constructed from three steps: the first step of ion-exchange HPLC for concentrating newly synthesized GAG oligosaccharides as reaction products, the second step of reverse phase HPLC for separating PA oligosaccharides from non-PA oligosaccharides, and the third step of size fractionation HPLC for fractionating newly synthesized oligosaccharides. Newly synthesized oligosaccharides were obtained by one complete cycle of the transglycosylation reaction and separation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 189-196, 2014.
Biopolymers.Biopolymers.2014 Mar;101(3):189-96. doi: 10.1002/bip.22300.
Using the transglycosylation reaction as a reverse reaction for the hydrolysis of hyaluronidase, new artificial oligosaccharides may be synthesized by reconstructing natural glycosaminoglycans (GAGs) according to preliminary planned arrangements. However, as some problems have been associated with t
PMID 23754563

Effect of a cholesterol-rich lipid environment on the enzymatic activity of reconstituted hyaluronan synthase.

Ontong P1, Hatada Y2, Taniguchi S2, Kakizaki I3, Itano N4.Author information 1Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan.2Department of Molecular Oncology, Division of Molecular and Cellular Biology, Institute of Pathogenesis and Disease Prevention, Shinshu University Graduate School of Medicine, Matsumoto, Nagano 390-8621, Japan.3Department of Glycotechnology, Center for Advanced Medical Research, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.4Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan; Department of Molecular Biosciences, Faculty of Life Sciences, Kyoto Sangyo University, Kita-ku, Kyoto 603-8555, Japan. Electronic address: itanon@cc.kyoto-su.ac.jp.AbstractHyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-environment. The culture of human dermal fibroblasts in lipoprotein-depleted medium attenuated the synthesis of hyaluronan. The sequestration of cellular cholesterol by methyl-ß-cyclodextrin also decreased the hyaluronan production of fibroblasts, as well as the HAS activity. To directly evaluate the effects of cholesterol on HAS activity, a recombinant human HAS2 protein with a histidine-tag was expressed as a membrane protein by using a baculovirus system, then successfully solubilized, and isolated by affinity chromatography. When the recombinant HAS2 proteins were reconstituted into liposomes composed of both saturated phosphatidylcholine and cholesterol, this provided a higher enzyme activity as compared with the liposomes formed by phosphatidylcholine alone. Cholesterol regulates HAS2 activity in a biphasic manner, depending on the molar ratio of phosphatidylcholine to cholesterol. Furthermore, the activation profiles of different lipid compositions were determined in the presence or absence of cholesterol. Cholesterol had the opposite effect on the HAS2 activity in liposomes composed of phosphatidylethanolamine or phosphatidylserine. Taken together, the present data suggests a clear functional association between HAS activity and cholesterol-dependent alterations in the physical and chemical properties of cell membranes.
Biochemical and biophysical research communications.Biochem Biophys Res Commun.2014 Jan 10;443(2):666-71. doi: 10.1016/j.bbrc.2013.12.028. Epub 2013 Dec 11.
Hyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-e
PMID 24333423

Japanese Journal

Photocrosslinking Sugars for Capturing Glycan-dependent Interactions

Fujita Akiko,Kohler Jennifer J.
Trends in Glycoscience and Glycotechnology
Photocrosslinking approaches are an important strategy to covalently capture biomolecular interactions. Covalent capture by photocrosslinking is particularly powerful for glycan-dependent interactions …
NAID 130005089114

光架橋糖による糖鎖依存的相互作用の検出

藤田明子,Kohler Jennifer J.
Trends in Glycoscience and Glycotechnology
光架橋法は、生体分子の相互作用を紫外線照射により共有結合を形成し固定化する方法である。糖鎖を介した相互作用は、親和性が弱く通常の精製方法では検出できないことが多い。それゆえ、特に糖鎖を介した相互作用を検出するために光架橋法は強力なツールである。光架橋を行うには、まず紫外線照射により活性化される反応基を糖鎖含有分子に導入する。紫外線照射により光感受性の反応基は活性化され、近隣の分子と共有結合を形成す …
NAID 130005089113