Novel polyLacdiNAc and fucosylated polyLacdiNAc N-glycans from mammalian cells expressing 1,4-GalNAcT and 1,3-FucT
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 C. elegans 1,4N-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 "polyLDN" 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 (FucT 9) 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 "polyLDNF" structures [-3GalNAc1-4(Fuc1-3)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.
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