Novel Asn-linked oligosaccharides terminating in GaINAc (1-&gt;4)[Fuca(1-&gt;3)]GlcNAcB(1-&gt; ) are present in recombinant human Protein C expressed in human kidney 293 cells

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.

Section: Discussionmentioning

confidence: 99%

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

Kawar

Haslam

Morris³

et al. 2005

“…The levels of hormone-specific GalNAc-transferase changes in parallel to those of LH (56). Since the terminal GalNAc on the N-linked oligosaccharides of LH and other glycoproteins can also be modified with terminal ␣2,6-linked sialic acid (3,13,26,(57)(58)(59)(60)(61)(62), the levels of GalNAc-4-sulfotransferase as well as ␣2,6-sialyltransferase may determine which structure is ultimately present on LH and other glycoproteins bearing such oligosaccharides. This would have a profound impact on both the rate and the site of clearance for LH.…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning and Expression of the Pituitary Glycoprotein HormoneN-Acetylgalactosamine-4-O-sulfotransferase

Xia¹,

Evers²,

Kang³

et al. 2000

The pituitary hormones LH, 1 thyrotropin, and pro-opiomelanocortin bear unique N-linked oligosaccharides that terminate with the sequence SO 4 -4-GalNAc␤1,4GlcNAc␤1,2Man␣ (1-6).The terminal GalNAc-4-SO 4 is recognized by a receptor, the Man/GalNAc-4-SO 4 receptor (7-9), which is located in hepatic endothelial cells and controls the circulatory half-life of LH and other glycoproteins bearing this structure (10, 11). A short circulatory half-life in conjunction with the regulated release from storage granules in the gonadotroph produces the episodic rise and fall seen in circulating LH levels (12, 13). This is essential for efficient activation of the LH receptor in the ovary and testis due to the down-regulation and internalization of the activated receptor by ␤-arrestin (14). Sequential actions of a hormone-specific ␤1,4 GalNAc-transferase and a GalNAc-4-sulfotransferase are required for the synthesis of GalNAc-4-SO 4 on LH and other glycoproteins (13,(15)(16)(17)(18)(19). Three key functions have been conserved throughout vertebrate evolution: 1) the peptide recognition determinant utilized by the GalNAc-transferase, 2) the GalNAc-transferase itself, and 3) the GalNAc-4-sulfotransferase. As a result, Nlinked structures terminating with ␤1,4-linked GalNAc-4-SO 4 occur on glycoprotein hormones from all vertebrate species (20). GalNAc-transferase and GalNAc-4-sulfotransferase activities with the same properties as those expressed in the pituitary have been detected in other tissues and cells (21). Furthermore, a number of other glycoproteins bearing N-and O-linked structures terminating with ␤1,4-linked GalNAc-4-SO 4 have been described (2,5,(22)(23)(24)(25)(26). Thus, oligosaccharides terminating with GalNAc-4-SO 4 may fulfill a number of different roles in vivo.Recently the HNK-1 sulfotransferase (HNK-1 ST), a glucuronyl-3-sulfotransferase that mediates synthesis of the HNK-1 epitope SO 4 -3-GlcA␤1,3Gal␤1,4GlcNAc (27) was cloned (28, 29). The HNK-1 structure is found in the central and peripheral nervous systems on glycolipids and glycoproteins involved in neural recognition and synaptogenesis (Ref. 30; for reviews, see Refs. 31 and 32). Expression of the HNK-1 structure is spatially and temporally regulated, suggesting an important role in axon outgrowth (33). It is a predominant autoantigen in demyelinating diseases of the peripheral nervous system (34). Recently this glycan has been recognized to be a crucial player in synaptic plasticity involving inhibitory interneurons in the hippocampus (35). The sulfate group of the HNK-1 glycan is essential for its function (36, 37).

“…Human protein C (HPC) expressed in HEK293 cells shows stronger anticoagulant activity than plasma HPC. The HPC from HEK293 cells has the LacdiNAc structure in its N-glycans, whereas plasma HPC does not (34). LacdiNAc may contribute to the increased anticoagulant activity of the HPC produced by HEK293 cells (34).…”

mentioning

confidence: 99%

“…The HPC from HEK293 cells has the LacdiNAc structure in its N-glycans, whereas plasma HPC does not (34). LacdiNAc may contribute to the increased anticoagulant activity of the HPC produced by HEK293 cells (34). In HT-1080 fibrosarcoma cells, LacdiNAc, sialylated LacdiNAc, and fucosylated LacdiNAc have been found (36).…”

mentioning

confidence: 99%

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

Sato

Gotoh

Kiyohara

et al. 2003

We found a novel human glycosyltransferase gene carrying a hypothetical ␤1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5 -rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank TM accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc ␤-benzyl. The product was deduced to be GalNAc␤1-4GlcNAc␤-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme ␤1,4-N-acetylgalactosaminyltransferase-III (␤4GalNAc-T3). ␤4GalNAc-T3 effectively synthesized N,N -diacetylgalactosediamine, GalNAc␤1-4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N -diacetylgalactosediamine structures in their N-glycans, we examined the ability of ␤4GalNAc-T3 to synthesize N,N -diacetylgalactosediamine structures in N-glycans on a model protein.When fetal calf fetuin treated with neuraminidase and ␤1,4-galactosidase was utilized as an acceptor protein, ␤4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that ␤4GalNAc-T3 could transfer GalNAc residues, producing N,N -diacetylgalactosediamine structures at least in Nglycans and probably in both N-and O-glycans.