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Báo cáo Y học: Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(b1–6)Man-motifs

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymati-cally liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC | Eur. J. Biochem. 269 5459-5473 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03244.x Hemocyanin from the keyhole limpet Megathura crenulata KLH carries a novel type of N-glycans with Gal pi-6 Man-motifs Tomofumi Kurokawa1 2 Manfred Wuhrer2 Gunter Lochnit2 Hildeaard Gever2. Jiiraen Markl3 and Rudolf Geyer2 4 1 Pharmaceutical Discovery Center Pharmaceutical Research Division Takeda Chemical Industries Ltd Osaka Japan 2Institute of Biochemistry University of Giessen Giessen and 3Institute of Zoology Johannes-Gutenberg University of Mainz Mainz Germany Keyhole limpet Megathura crenulata hemocyanin KLH an extracellular respiratory protein is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH knowledge of its carbohydrate structure is still limited. Therefore we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses MALDI- TOF-MS ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature a number of the studied N-glycans contained a Gal b1-6 Man-unit which has not been found in glycoprotein-N-glycans so far. Hence our studies demonstrate that this marine molluskglycoprotein is characterized by a unique oligosaccharide pattern comprising in part novel structural elements. Keywords keyhole limpet hemocyanin carbohydrate structure analysis mass spectrometry N-glycans. Hemocyanins are oxygen-transporting proteins found in many arthropod and mollusc species 1 . Binding of oxygen is mediated by binuclear copper-binding sites resulting in the .