Novel Poly-GalNAcβ1–4GlcNAc (LacdiNAc) and Fucosylated Poly-LacdiNAc N-Glycans from Mammalian Cells Expressing β1,4-N-Acetylgalactosaminyltransferase and α1,3-Fucosyltransferase

Abstract

Glycans containing the GalNAc␤1-4GlcNAc (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 Gal␤1-4GlcNAc (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 (-3GalNAc␤1-4Glc-NAc␤1-) n . These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Gal␤1-4GlcNAc␤1-) 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) GalNAc␤1-4(Fuc␣1-3)GlcNAc␤1-R as well as novel "poly-LDNF" structures (-3GalNAc␤1-4(Fuc␣ 1-3)GlcNAc␤1-) 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 Nglycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO 4 -4GalNAc␤1-4Glc-NAc-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.

Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells

“…The Lec8 loss-of-function mutant has a deletion mutation in the Golgi UDP-Gal transporter (Oelmann et al, 2001) and has few galactosylated glycoconjugates (Stanley et al, 1980;Kawar et al, 2005). The Lec2 mutant has a CMPsialic acid transporter mutation (Eckhardt et al, 1998) and has almost no sialylated glycoconjugates (Stanley et al, 1980).…”

Molecular Phylogeny and Functional Genomics of β-Galactoside α2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes

“…Although information on these enzymes is lacking for lower vertebrates, we postulate that these biochemical properties extend to all of the vertebrate ST6Gal enzymes. Interestingly, the distribution of LacdiNAc in mammals is very limited, and LacdiNAc might be substituted by 4-O-sulfated-, ␣1,3-fucosylated, or ␣2,6-sialylated derivatives (99). As indicated by these authors, the glycans bearing LacdiNAc are notably recorded in pituitary glycoprotein hormones and tenascin-R produced by oligodendrocytes and small interneurons in the hippocampus and cerebellum.…”

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

“…Although it was possible to detect ␤4GalNAc-T activity in vitro in CHO cells, no evidence of LacdiNAc addition to glycoproteins expressed in CHO cells was obtained (48). However, expression of a ␤1,4GalNAcT cloned from Caenorhabditis elegans in CHO Lec8 cells resulted in LacdiNAc synthesis on multiple endogenous glycoproteins as well as the glycoprotein hormone ␣ subunit (50). Until other ␤4GalNAc-Ts can be identified or cloned, it will be difficult to assess whether they are protein-specific; however, the approach we have taken using chimeric glycoprotein acceptors makes this a more approachable problem in the future.…”