Molecular cloning and characterization of β1,4‐N‐acetylgalactosaminyltransferases IV synthesizing N,N′‐diacetyllactosediamine1

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

“…Recombinant ␤1,4-N-Acetylgalactosamine Transferases Modify Oligosaccharides on SorLA/LR11 with GalNAc-Three ␤1.4-N-acetylgalactosamine transferases (␤1,4GalNAcT) have been described that are able to modify N-linked oligosaccharides with ␤1,4-linked GalNAc: a ␤1,4GalNAcT cloned from Caenorhabditis elegans (39) (␤1,4GalNAcT-CE), and two ␤1,4GalNAcTs, ␤1,4GalNAcTIII and ␤1,4GalNAcTIV, cloned from human cDNA libraries (40,41). We cloned the mouse orthologues of ␤1,4GalNAcTIII and ␤1,4GalNAcTIV and established that they are active using both in vitro and in vivo assays.…”

Section: Recombinant Sorla/lr11 Is Modified With Terminal Galnac-4-somentioning

confidence: 99%

N-Linked Oligosaccharides on the Low Density Lipoprotein Receptor Homolog SorLA/LR11 Are Modified with Terminal GalNAc-4-SO4 in Kidney and Brain

Fiete

Oats

et al. 2007

Sorting protein-related receptor (SorLA/LR11) is a highly conserved mosaic receptor that is expressed by cells in a number of different tissues including principal cells of the collecting ducts in the kidney and neurons in the central and peripheral nervous systems. SorLA/LR11 has features that indicate it serves as a sorting receptor shuttling between the plasma membrane, endosomes, and the Golgi. We have found that a fraction of SorLA/LR11 that is synthesized in the kidney and the brain bears N-linked oligosaccharides that are modified with terminal ␤1,4-linked GalNAc-4-SO 4 . Oligosaccharides located in the vacuolar sorting (Vps) 10p domain (Vps10p domain) are modified with ␤1,4-linked GalNAc when the Vps10p domain is expressed in cells along with either of two recently cloned protein-specific ␤1,4GalNAc-transferases, GalNAcTIII and GalNAcTIV. Either of two sequences with basic amino acids located within the Vps10p domain is able to mediate recognition by these ␤1,4GalNAc-transferases. The highly specific modification of oligosaccharides in the Vps10p domain of SorLA/LR11 with terminal GalNAc-4-SO 4 suggests that this unusual modification may modulate the interaction of SorLA/LR11 with proteins and influence their trafficking.Sorting protein-related receptor (SorLA), 2 also known as LR11, is a highly conserved mosaic, type 1 receptor. The luminal/extracellular domain consists of a short NH 2 -terminal sequence preceding a consensus site for furin cleavage, a vacuolar sorting (Vps) 10p domain, 5 F/YWTD/␤-propeller modules, an epidermal growth factor precursor sequence, 11 low density lipoprotein receptor (LDLR) class A (LDLR-A) repeats, and 6 fibronectin type III repeats (1, 2). The single transmembrane domain is followed by a 54-amino acid cytosolic domain containing a motif that mediates interaction with GGA (Golgilocalized, ␥-ear containing, ARF binding) proteins involved in Golgi-endosome sorting (3). The presence of these varied domains places SorLA/LR11 in a number of different families and underscores its ability to interact with a range of different ligands. A Vps10p domain is also present in the receptors sortilin-1 (4) and SorCS (5). The Vps10p domain of SorLA/ LR11 mediates the endogenous binding of the propeptide released from the N terminus of SorLA/LR11 by furin cleavage. The peptides neurotensin and hydra head activator also bind to the Vps10p domain (6). The YWTD repeats, epidermal growth factor repeat, and the LDLR class A repeats are characteristic of LDLR family members (7) and likely mediate apolipoprotein E and receptor-associated protein binding by SorLA/LR11 (6). In addition, the 6 fibronectin type III repeats are characteristic of a number of neural adhesion molecules (2).The peptide portion of SorLA/LR11 consists of 2215 amino acids with a calculated molecular weight of 247,000. The receptor migrates with M r of over 300,000 when examined by SDS-PAGE indicating that a large fraction of the 20 potential Asn-glycosylation sites present on the luminal domain are utilized. We have determin...

“…Note Added in Proof-It should be noted that Narimatsu and coworkers have recently reported the isolation of two human ␤1,4-N-acetylgalactosaminyltransferase genes (105,106). Futhermore, they experimentally verified that both gene products were ␤1,4-N-acetylgalactosaminyltransferases involved in the biosynthesis of LacDiNAc.…”

mentioning

confidence: 99%

Molecular Cloning and Functional Characterization of a Lepidopteran Insect β4-N-Acetylgalactosaminyltransferase with Broad Substrate Specificity, a Functional Role in Glycoprotein Biosynthesis, and a Potential Functional Role in Glycolipid Biosynthesis

Vadaie

Jarvis

2004

A degenerate PCR approach was used to isolate a lepidopteran insect cDNA encoding a ␤4-galactosyltransferase family member. The isolation and initial identification of this cDNA was based on bioinformatics, but its identification as a ␤4-galactosyltransferase family member was experimentally confirmed. The newly identified ␤4-galactosyltransferase family member had unusually broad donor and acceptor substrate specificities in vitro, as transfered galactose, N-acetylglucosamine, and N-acetylgalactosamine to carbohydrate, glycoprotein, and glycolipid acceptors. However, the enzyme preferentially utilized N-acetylgalactosamine as the donor for all three acceptors, and its derived amino acid sequence was closely related to a known N-acetylgalactosaminyltransferase. These data suggested that the newly isolated cDNA encodes a ␤4-N-acetylgalactosaminyltransferase that functions in insect cell glycoprotein biosynthesis, glycolipid biosynthesis, or both. The remainder of this study focused on the role of this enzyme in N-glycoprotein biosynthesis. The results showed that the purified enzyme transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to a synthetic N-glycan in vitro. The structure of the reaction product was confirmed by chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses. Co-expression of the new cDNA product in insect cells with an N-glycoprotein reporter showed that it transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to this N-glycoprotein in vivo. Confocal microscopy showed that a GFP-tagged version of the enzyme was localized in the insect cell Golgi apparatus. In summary, this study demonstrated that lepidopteran insect cells encode and express a ␤4-N-acetylgalactosaminyltransferase that functions in N-glycoprotein biosynthesis and perhaps in glycolipid biosynthesis, as well. The isolation and characterization of this gene and its product contribute to our basic understanding of insect protein N-glycosylation pathways and to the growing body of evidence that insects can produce glycoproteins with complex N-glycans.