N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc 2 -PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc 2 -PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.Asparagine (N)-glycosylation is an essential modification that regulates protein folding and stability. Prior to its attachment to protein, the oligosaccharide Glu 3 Man 9 GlcNAc 2 is assembled on the lipid carrier, dolichyl pyrophosphate (dol-PP), in the ER 2 (see Refs. 1-4 for review). The earliest steps of this lipid-linked oligosaccharide (LLO) synthesis begin on the cytoplasmic face of the ER. Seven sugars, (two N-acetylglucosamines and five mannoses) are sequentially added to dol-P to form Man 5 GlcNAc 2 -PP-dol by enzymes that have their catalytic domain on the cytosolic side of the ER membrane and use sugar nucleotide substrates (2, 5, 6). The enzymes that catalyze addition of the next seven sugars (four mannoses and three glucoses) do so within the lumen of the ER and use dolichol-linked sugar substrates (see Ref. 1 for review). Once assembled, this core oligosaccharide is transferred to protein by oligosaccharyltransferase through an N-glycosidic bond to an asparagine that is part of the Asn-X-(Ser/Thr) consensus sequence (7). Proteinlinked oligosaccharide is immediately modified by the removal of glucoses and mannose by ER glucosidases and mannosidases. Failure to properly synthesize, transfer, or modify the N-linked glycan results in glycoproteins that are recognized by the quality control systems that restrict these abe...
Glycosylation is the most widespread posttranslational modification in eukaryotes; however, the role of oligosaccharides attached to proteins has been little studied because of the lack of a sensitive and easy analytical method for oligosaccharide structures. Recently, tandem mass spectrometric techniques have been revealing that oligosaccharides might have characteristic signal intensity profiles. We describe here a strategy for the rapid and accurate identification of the oligosaccharide structures on glycoproteins using only mass spectrometry. It is based on a comparison of the signal intensity profiles of multistage tandem mass (MSn) spectra between the analyte and a library of observational mass spectra acquired from structurally defined oligosaccharides prepared using glycosyltransferases. To smartly identify the oligosaccharides released from biological materials, a computer suggests which ion among the fragment ions in the MS/MS spectrum should yield the most informative MS3 spectrum to distinguish similar oligosaccharides. Using this strategy, we were able to identify the structure of N-linked oligosaccharides in immunoglobulin G as an example.
A new member of the UDP-N-acetylglucosamine: bgalactose b1,3-N-acetylglucosaminyltransferase (b3Gn-T) family having the b3-glycosyltransferase motifs was identified using an in silico method. This novel b3Gn-T was cloned from a human colon cancer cell line and named b3Gn-T8 based on its position in a phylogenetic tree and enzymatic activity. b3Gn-T8 transfers GlcNAc to the non-reducing terminus of the Galb1-4GlcNAc of tetraantennary N-glycan in vitro. HCT15 cells transfected with b3Gn-T8 cDNA showed an increase in reactivity to both LEA and PHA-L4 in a flow cytometric analysis. These results indicated that b3Gn-T8 is involved in the biosynthesis of poly-Nacetyllactosamine chains on tetraantennary (b1,6-branched) N-glycan. In most of the colorectal cancer tissues examined, the level of b3Gn-T8 transcript was significantly higher than in normal tissue. b3Gn-T8 could be an enzyme involved in the synthesis of poly-N-acetyllactosamine on b1-6 branched N-glycans in colon cancer.
The multiple use of lectin-assisted glycan profiling enabled us to construct a reliable sandwich assay kit for monitoring liver fibrosis in patients with viral hepatitis. This will assist in the development pipeline for other glycodiagnostic agents.
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