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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.11.047 Byline: Catalina Ruiz-Canada (1), Daniel J. Kelleher (1), Reid Gilmore (1) Keywords: CELLBIO; PROTEINS Abstract: Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation. Author Affiliation: (1) Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School, Worcester, MA 01605, USA Article History: Received 23 June 2008; Revised 17 October 2008; Accepted 25 November 2008 Article Note: (miscellaneous) Published: January 22, 2009

Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an [alpha]-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing [alpha]-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-de+pendent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing [alpha]-mannosidase-like protein and Mns1 orthologs, respectively, each of which had [alpha]-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD. protein folding | protists | Trichomonas | Entamoeba

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-Glc[NAc.sub.2][Man.sub.9][Glc.cub.3]. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-Glc[Nac.sub.2][Man.sub.5] (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked Glc[Nac.sub.2] (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases. evolution | N-glycans | protist

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61[alpha] and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

Research has been conducted on the oligosaccharyltransferase which utilizes the fully assembled dolichol-linked oligosaccharide. The proteoliposome assay system that consist of the purified yeast enzyme has been used in investigating the steady-state kinetic properties of the oligosaccharyltransferase and the results are described.

DAD1, the defender against apoptotic cell death, was initially identified as a negative regulator of programmed cell death in the BHK21-derived tsBN7 cell line. Of interest, the 12.5-kDa DAD1 protein is 40% identical in sequence to Ost2p, the 16-kDa subunit of the yeast oligosaccharyltransferase (OST). Although the latter observation suggests that DAD1 may be a mammalian OST subunit, biochemical evidence to support this hypothesis has not been reported. Previously, we showed that canine OST activity is associated with an oligomeric complex of ribophorin I, ribophorin II, and OST48. Here, we demonstrate that DAD1 is a tightly associated subunit of the OST both in the intact membrane and in the purified enzyme. Sedimentation velocity analyses of detergent-solubilized WI38 cells and canine rough microsomes show that DAD1 cosediments precisely with OST activity and with the ribophorins and OST48. Radioiodination of the purified OST reveals that DAD1 is present in roughly equimolar amounts relative to the other subunits. DAD1 can be crosslinked to OST48 in intact microsomes with dithiobis(succinimidylpropionate). Crosslinked ribophorin II-OST48 heterotrimers, DAD1-ribophorin II-OST48 heterotrimers and DAD1-ribophorin I-ribophorin II-OST48 heterotetramers also were detected. The demonstration that DAD1 is a subunit of the OST suggests that induction of a cell death pathway upon loss of DAD1 in the tsBN7 cell line reflects the essential nature of N-linked glycosylation in eukaryotes.

The gene encoding the 16-kD subunit of the yeast enzyme oligosaccharyltransferase is related to the vertebrate DAD1 protein, and participates in in vivo and in vitro oligosaccharyltransferase functioning in a wbp1-2 strain. The temperature-dependent phenotype of the wbp1-2 allele is restricted during overexpression of the Ost2 protein. The mutant proteins are responsible for pleiotropic underglycosylation of soluble and membrane-associated glycoproteins.

The functional and structural characterization of the Saccharomyces cerevisiae oligosaccaryltransferase (OST), Ost3p corresponding to the 34-kD gamma-subunit of the shows that Ost3p may increase oligosaccaride transport to a subset of acceptor substrates in vivo. A loss of Ost3p is associated with considerable underglycosylation of membrane-bound and soluble glycoproteins in vivo.

Protein sequence analysis and mutational analysis help study the OST1 gene of Saccharomyces cerevisiae, which encodes the alpha-subunit of oligosaccharyltransferase. Vegetative growth of yeast requires the presence of the Ost1 protein, which is similar to mammalian ribophorin 1. Ost1 mutants exhibit pleiotropic undeglycosylation of glycoproteins that are free or bound to the membrane under different growth temperatures. The transfer of high mannose oligosaccharide to an acceptor tripeptide at the glycosylation site is reduced in microsomal membranes from ost1 mutants.