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J. Biol. Chem., Vol. 283, Issue 24, 16455-16468, June 13, 2008

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Identification of Further Elongation and Branching of Dimeric Type 1 Chain on Lactosylceramides from Colonic Adenocarcinoma by Tandem Mass Spectrometry Sequencing Analyses^*

Yao-Yun Fan, Shin-Yi Yu, Hiromi Ito^¶, Akihiko Kameyama^¶, Takashi Sato^¶, Chi-Hung Lin^||, Lung-Chih Yu, Hisashi Narimatsu^¶, and Kay-Hooi Khoo¹

From the Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, ^¶Glycogene Function Team of Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan, and ^||CBMB, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan and Institute of Bioinformatics and Structural Biology, National Tsing-Hua University, Hsinchu 30013, Taiwan

Mammalian glycan chain elongation is mostly based on extending the type 2 chain, Galβ1–4GlcNAc, whereas the corresponding type 1 chain, Galβ1–3GlcNAc, is not normally extended. In a broader context of developing high sensitivity mass spectrometry methodologies for glycomic identification of Le^a versus Le^x and linear versus branched poly-N-acetyllactosamine (polyLacNAc), we have now shown that the dimeric type 1 glycan chain, as carried on the lactosylceramides of a human colonic adenocarcinoma cell line, Colo205, not only can be further extended linearly but can likewise be branched at C6 of 3-linked Gal in a manner similar to polyLacNAc. A combination of chemical and enzymatic derivatization coupled with advanced mass spectrometry analyses afforded unambiguous identification of a complex mixture of type 1 and 2 hybrids as well as those fucosylated variants founded exclusively on linear and branched trimeric type 1 chain. We further showed by in vitro enzymatic synthesis that extended type 1 and the hybrid chains can be branched by all three forms of the human I branching enzymes (IGnT) currently identified but with lower efficiency and stringency with respect to branching site preference. Importantly, it was found that a better substrate is one that carries a Gal site for branching that is extended at the non-reducing end by a type 2 and not a type 1 unit, whereas the IGnTs are less discriminative with respect to whether the targeted Gal site is itself β3- or β4-linked to GlcNAc at the reducing end.

Received for publication, August 30, 2007 , and in revised form, February 20, 2008.

^* This work was supported by an Academia Sinica Program Project grant (to K.-H. K.) and was performed as a part of the R&D Project of the Industrial Science and Technology Frontier Program supported by the New Energy and Industrial Technology Development Organization (to H. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ To whom correspondence should be addressed: Institute of Biological Chemistry, Academia Sinica, 128, Academia Rd. Sec 2, Nankang, Taipei 11529, Taiwan. Fax: 886-2-27889759; E-mail: kkhoo{at}gate.sinica.edu.tw.

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