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J. Biol. Chem., Vol. 280, Issue 13, 12810-12819, April 1, 2005

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Novel Poly-GalNAc1–4GlcNAc (LacdiNAc) and Fucosylated Poly-LacdiNAc N-Glycans from Mammalian Cells Expressing 1,4-N-Acetylgalactosaminyltransferase and 1,3-Fucosyltransferase^*

Ziad S. Kawar, Stuart M. Haslam, Howard R. Morris¶, Anne Dell||, and Richard D. Cummings**

From the Department of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, the Department of Biological Sciences, Imperial College London, London SW7 2AZ, United Kingdom, and ^¶M-SCAN Mass Spectrometry Research and Training Centre, Silwood Park, Ascot SL5 7PZ, United Kingdom

Glycans containing the GalNAc1–4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Gal1–4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans 1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAc1–4GlcNAc1-)_n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Gal1–4GlcNAc1-)_n. We also stably expressed human 1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with 1,3-fucosylated LDN (LDNF) GalNAc1–4(Fuc1–3)GlcNAc1-R as well as novel "poly-LDNF" structures (-3GalNAc1–4(Fuc 1–3)GlcNAc1-)_n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common -subunit in L8-GalNAcT cells. The -subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO₄-4GalNAc1–4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.

Received for publication, December 20, 2004 , and in revised form, January 14, 2005.

^* This work was supported by National Institutes of Health Grant RO1 CH/HD54832-01 (to R. D. C.) and by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust (to A. D. and H. R. M.). 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.

^|| A Biotechnology and Biological Sciences Research Council Professorial Fellow.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, OK Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 N.E. 10th St., BRC Rm. 417, Oklahoma City, OK 73104. Tel.: 405-271-2481; Fax: 405-271-3910; E-mail: richard-cummings{at}ouhsc.edu.

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