Identification of phosphorylated oligosaccharides in cells of patients with a congenital disorders of glycosylation (CDG-I)

Protein N-glycosylation is initiated by the dolichol cycle in which the oligosaccharide precursor Glc₃Man₉GlcNAc₂-PP-dolichol is assembled in the endoplasmic reticulum (ER). One critical step in the dolichol cycle concerns the availability of Dol-P at the cytosolic face of the ER membrane. In RFT1 cells, the lipid-linked oligosaccharide (LLO) intermediate Man₅GlcNAc₂-PP-Dol accumulates at the cytosolic face of the ER membrane. Since Dol-P is a rate-limiting intermediate during protein N-glycosylation, continuous accumulation of Man₅GlcNAc₂-PP-Dol would block the dolichol cycle. Hence, we investigated the molecular mechanisms by which accumulating Man₅GlcNAc₂-PP-Dol could be catabolized in RFT1 cells. On the basis of metabolic labeling experiments and in comparison to human control cells, we identified phosphorylated oligosaccharides (POS), not found in human control cells and present evidence that they originate from the accumulating LLO intermediates. In addition, POS were also detected in other CDG patients’ cells accumulating specific LLO intermediates at different cellular locations. Moreover, the enzymatic activity that hydrolyses oligosaccharide-PP-Dol into POS was identified in human microsomal membranes and required Mn²⁺ for optimal activity. In CDG patients’ cells, we thus identified and characterized POS that could result from the catabolism of accumulating LLO intermediates.     

Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

Background

During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo β-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells.

Methods/Principal Findings

During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Δ deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man₈GlcNAc₂, corresponding to ∼70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc₁Man₉GlcNAc₂ and Man₉GlcNAc₂ that corresponds to ∼30% of total fOS.

Conclusions/Significance

As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.     

Isolation of the ALG6 locus of Saccharomyces cerevisiae required for glucosylation in the N-linked glycosylation pathway

N-Linked protein glycosylation in most eukaryotic cells initiateswith the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ fromthe lipid carrier dolichyl pyrophosphate to selected asparagineresidues. In the yeast Saccharomyces cerevisiae, alg mutationswhich affect the assembly of the lipid-linked oligosaccharideat the membrane of the endoplasmic reticulum result in the accumulationof lipid-linked oligosaccharide intermediates and a hypoglycosylationof proteins. Exploiting the synthetic growth defect of alg mutationsin combination with mutations affecting oligosaccharyl transferaseactivity (Stagljar et al., 1994), we have isolated the ALG6locus. alg6 mutants accumulate lipid-linked Man₉GlcNAc₂, suggestingthat this locus encodes an endoplasmic glucosyltransferase.Alg6p has sequence similarity to Alg8p, a protein required forglucosylation of Glc₁Man⁹GlcNAc². Saccharomyces cerevisiae endoplasmic reticulum glycosyltransferase dolichol     

Regulation of dolichol-linked glycosylation

In the majority of congenital disorders of glycosylation, the assembly of the glycan precursor GlcNAc₂Man₉Glc₃ on the polyprenol carrier dolichyl-pyrophosphate is compromised. Because N-linked glycosylation is essential to life, most types of congenital disorders of glycosylation represent partial losses of enzymatic activity. Consequently, increased availability of substrates along the glycosylation pathway can be beneficial to increase product formation by the compromised enzymes. Recently, we showed that increased dolichol availability and improved N-linked glycosylation can be achieved by inhibition of squalene biosynthesis. This review summarizes the current knowledge on the biosynthesis of dolichol-linked glycans with respect to deficiencies in N-linked glycosylation. Additionally, perspectives on therapeutic treatments targeting dolichol and dolichol-linked glycan biosynthesis are examined.     

Rapid detection of the alternativeN-glycosylation pathway using high pH anion exchange chromatography

An alternativeN-glycosylation pathway using Glc_1–3Man₅GlcNAc₂ as a donor to be transferred to a protein acceptor is found either in Man-P-Dol synthase deficient cells or in wild type CHO cells grown in energy deprivation conditions. Discrimination between oligomannosides of this alternative pathway and oligomannosides of the major one containing the same number of sugar residues Man_6–8GlcNAc₂ required structural studies. Taking advantage of the specific chromatographic behaviour of glucosylated oligomannosides, in pellicular high pH anion exchange chromatography, we developed a one-step method for the identification of the alternativeN-glycosylation pathway compounds differing from those of the major one.Abbreviations HPAEC high pH anion exchange chromatography - endo H endo betaN-acetylglucosaminidase H - PNGaseF peptideN-glycosidase F - M2 Man₂GlcNAc₂ - M4 Man₄GlcNAc₂ - M5 Man₅GlcNAc₂ - G1M5 Glc₁Man₅GlcNAc₂ - G2M5 Glc₂Man₅GlcNAc₂ - G3M5 Glc₃Man₅GlcNAc₂ - M6 Man₆GlcNAc₂ - M8 Man₈GlcNAc₂ - M9 Man₉GlcNAc₂ - G1M9 Glc₁Man₉GlcNAc₂ - G2M9 Glc₂Man₉GlcNAc₂ - G3M9 Glc₃Man₉GlcNAc₂ To whom correspondence should be addressed.     

The Conformational Properties of the Glc3Man Unit Suggest Conformational Biasing within the Chaperone-assisted Glycoprotein Folding Pathway

A major puzzle is: are all glycoproteins routed through the ER calnexin pathway irrespective of whether this is required for their correct folding? Calnexin recognizes the terminal Glcα1-3Manα linkage, formed by trimming of the Glcα1-2Glcα1-3Glcα1-3Manα (Glc₃Man) unit in Glc₃Man₉GlcNAc₂. Different conformations of this unit have been reported. We have addressed this problem by studying the conformation of a series of N-glycans; i.e. Glc₃ManOMe, Glc₃Man₄,₅,₇GlcNAc₂ and Glc₁Man₉GlcNAc₂ using 2D NMR NOESY, ROESY, T-ROESY and residual dipolar coupling experiments in a range of solvents, along with solution molecular dynamics simulations of Glc₃ManOMe. Our results show a single conformation for the Glcα1-2Glcα and Glcα1-3Glcα linkages, and a major (65%) and a minor (30%) conformer for the Glcα1-3Manα linkage. Modeling of the binding of Glc₁Man₉GlcNAc₂ to calnexin suggests that it is the minor conformer that is recognized by calnexin. This may be one of the mechanisms for controlling the rate of recruitment of proteins into the calnexin/calreticulin chaperone system and enabling proteins that do not require such assistance for folding to bypass the system. This is the first time evidence has been presented on glycoprotein folding that suggests the process may be optimized to balance the chaperone-assisted and chaperone-independent pathways.     

Glucosidase II β Subunit Modulates N-Glycan Trimming in Fission Yeasts and Mammals

Glucosidase II (GII) plays a key role in glycoprotein biogenesis in the endoplasmic reticulum (ER). It is responsible for the sequential removal of the two innermost glucose residues from the glycan (Glc₃Man₉GlcNAc₂) transferred to Asn residues in proteins. GII participates in the calnexin/calreticulin cycle; it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase. GII is a heterodimer whose α subunit (GIIα) bears the glycosyl hydrolase active site, whereas its β subunit (GIIβ) role is controversial and has been reported to be involved in GIIα ER retention and folding. Here, we report that in the absence of GIIβ, the catalytic subunit GIIα of the fission yeast Schizosaccharomyces pombe (an organism displaying a glycoprotein folding quality control mechanism similar to that occurring in mammalian cells) folds to an active conformation able to hydrolyze p-nitrophenyl α-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc₂Man₉GlcNAc₂ (G2M9) and Glc₁Man₉GlcNAc₂ (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIβ and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIβ modulates G2M9 and G1M9 trimming.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

Structure of the lipid-linked oligosaccharides that accumulate in class E Thy-1-negative mutant lymphomas.

Studies reported in the preceding paper (Trowbridge and Hyman, 1979) have demonstrated that Thy-1^? mutant lymphoma cells of the class E complementation group lack the normal high molecular weight lipid-linked oligosaccharide, but instead accumulate two smaller species termed I and II. This paper reports studies which elucidate the structures of lipid-linked oligosaccharides I and II. By subjecting oligosaccharides radiolabeled with ³H-mannose, ³H-glucose or ³H-glucosamine to methylation, acetolysis, periodate oxidation and exoglycosidase digestion, the structures were shown to be: where R = GlcNac B1,4(3) GlcNAc. A comparison of I and II with lipid-linked oligosaccharides from normal Chinese hamster ovary cells indicates that both I and II are normal biosynthetic intermediates. On the basis of these data we suggest that the defect in the class E mutant cells is the lack of an α1,3 mannosyltransferase involved in the conversion of the Man₅GlcNAc₂ lipid-linked oligosaccharide to the Man₆GlcNAc₂ intermediate. It is also impossible that the same enzyme is involved in conversion of the Glc₃Man₅GlcNAc₂ lipid-linked oligosaccharide to Glc₃Man₆GlcNAc₂. The latter reaction, however, has not yet been demonstrated in normal cells.     

Production of a recombinant peroxidase in different glyco-engineered <Emphasis Type="Italic">Pichia pastoris</Emphasis> strains: a morphological and physiological comparison

Background

The methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce.

Results

A Man₅GlcNAc₂ glycosylating and a Man_8–10GlcNAc₂ glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man₅GlcNAc₂ glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man_8–10GlcNAc₂ glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man₅GlcNAc₂ glycosylating strain is best at a temperature of 30 °C.

Conclusion

This study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man₅GlcNAc₂ glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man₅GlcNAc₂ glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.

     

Processing of asparagine-linked saccharides in Mucor rouxii

Asparagine-linked Glc₁Man₉GlcNAc₂, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂ were detected in mycelial-form cells of the dimorphic fungus Mucor rouxii inculbated with [U-¹⁴C]glucose for 3 min. The oligosaccharides were absent from glycoproteins isolated from cells chased for 15 min with the unlabed monosaccharide. This was due to deglucosylation of the oligosaccharides and not to further addition of mannose residues to them. The half-lives of the glucosylated compounds were much shorter, therefore, in M. rouxii than in other eucaryotic cells. Further processing of N-linked saccharides led to the synthesis of mannan-like glycoproteins, some of whch contained methyl groups in position 3 or the mannose residues. Methylation occurred only at the non-reducing ends and prevented further elongation of the branches.     

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional ��1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc₃Man₉ GlcNAc₂-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the α1,3- and the α1,6-mannose-linked residue from GDP-mannose to Man₁GlcNAc₂-PP-Dol forming the Man₃GlcNAc₂-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX₇E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys²³⁰, as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1–4), and several features of this pathway even occur in prokaryotes (5–7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc₃Man₉ GlcNAc₂-PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8–10). The initial steps of the dolichol pathway up to Man₅GlcNAc₂-PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER),² using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14–17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18–20).One of the temperature-sensitive alg mutants, alg2, was shown to accumulate lipid-linked Man₂GlcNAc₂ at the restrictive temperature (15), indicating that alg2 might have a defect in the glycosyltransferase catalyzing the transfer of the third, α1,6-linked mannose, i.e. in the formation of the branched pentasaccharide Man₃GlcNAc₂-PP-Dol (see Fig. 8). On the other hand, biochemical studies in human fibroblasts from a patient with a defect in the human ALG2 ortholog, causing congenital disorder of glycosylation type CDG1i, pointed to a role in the transfer of the second, α1,3-linked mannose residue, because no elongation of Man(1,6)ManGlcNAc₂-PP-Dol occurred (21). In contrast, control fibroblasts were able to do so, albeit with reduced efficiency when compared with Man(1,3)ManGlcNAc₂-PP-Dol as glycosyl acceptor. Because a bioinformatic approach of the yeast data base did not reveal an unknown open reading frame that might encode an additional putative mannosyltransferase being involved in LLO synthesis, we reasoned that ALG2 may have a dual function, i.e. synthesizing both Man₂GlcNAc₂-PP-Dol and Man₃GlcNAc₂-PP-Dol. While the current study was in progress, evidence was presented that a membrane fraction from Escherichia coli, expressing ALG2 from yeast, is able to carry out an α1,3- and α1,6-mannosylation to form the branched pentasaccharide intermediate (22). However, the contribution of native E. coli enzymes could not entirely be ruled out. So far Alg2 has not been studied biochemically in yeast. Here, we confirm and extent this finding by investigating Alg2 in yeast. We first established a radioactive in vitro assay and demonstrate that Alg2, immunoprecipitated from detergent extracts of yeast microsomal membranes, is indeed sufficient to catalyze both elongation of Man₁GlcNAc₂-PP-Dol to Man₂GlcNAc₂-PP-Dol and subsequently to Man₃GlcNAc₂-PP-Dol. Furthermore we investigated the membrane topology of Alg2 mannosyltransferase. Evidence will be presented that Alg2 is composed only of the two N-terminal of four predicted transmembrane domains (TMDs), whereas the C-terminal hydrophobic sequences contribute to ER localization merely in a nontransmembrane manner. Surprisingly, none of the four domains is essential for Alg2 activity because deletion of either the two N-terminal or C-terminal domains gives rise to an active transferase. Finally, we perform a mutational analysis of Alg2 and identify amino acids required for its activity.Open in a separate windowFIGURE 8.Early steps of lipid-linked oligosaccharide formation on the cytosolic side of the ER membrane. Biosynthesis starts with the transfer of a GlcNAc-phosphate to dolichol phosphate with formation of the pyrophosphate bond, catalyzed by Alg7. The second step is catalyzed be the dimeric Alg14/Alg13 complex, whereby membrane-bound Alg14 recruits cytosolic Alg13 to the membrane with formation of the active GlcNAc transferase. Following the addition of the β1,4-linked mannose by Alg1, Alg2 catalyzes, as demonstrated here, both the transfer of the α1,3- and α1,6-linked mannose. The two final α1,2-mannose residues are transferred by Alg11, before the Man₅GlcNAc₂-PP heptasaccharide is translocated across the ER membrane to the lumen, where further elongation takes place to the full-length core saccharide. All of the sugar residues are donated by sugar nucleotides.     

Detection of the lipid-linked precursor oligosaccharide of N-linked protein glycosylation in Drosophila melanogaster

The presence of a glycan of the same molecular size as the lipid linked precursor oligosaccharide (Glc₃Man₉GlcNAc₂) of the N-linked protein glycosylation pathway in mammalian cells has been detected in a glycolipid fraction of cultured Drosophila melanogaster cells. Oligosaccharide sequencing studies were consistent with the existence of a glucosylated high mannose containing structure, which may be the common precursor for N-linked protein glycosylation in insect cells.     

Characterization of the single-chain Fv-Fc antibody MBP10 produced in <Emphasis Type="Italic">Arabidopsis alg3</Emphasis> mutant seeds

ER resident glycoproteins, including ectopically expressed recombinant glycoproteins, carry so-called high-mannose type N-glycans, which can be at different stages of processing. The presence of heterogeneous high-mannose type glycans on ER-retained therapeutic proteins is undesirable for specific therapeutic applications. Previously, we described an Arabidopsis alg3-2 glycosylation mutant in which aberrant Man₅GlcNAc₂ mannose type N-glycans are transferred to proteins. Here we show that the alg3-2 mutation reduces the N-glycan heterogeneity on ER resident glycoproteins in seeds. We compared the properties of a scFv-Fc, with a KDEL ER retention tag (MBP10) that was expressed in seeds of wild type and alg3-2 plants. N-glycans on these antibodies from mutant seeds were predominantly of the intermediate Man₅GlcNAc₂ compared to Man₈GlcNAc₂ and Man₇GlcNAc₂ isoforms on MBP10 from wild-type seeds. The presence of aberrant N-glycans on MBP10 did not seem to affect MBP10 dimerisation nor binding of MBP10 to its antigen. In alg3-2 the fraction of underglycosylated MBP10 protein forms was higher than in wild type. Interestingly, the expression of MBP10 resulted also in underglycosylation of other, endogenous glycoproteins.     

Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux

Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc₃Man₉GlcNAc₂ (G₃M₉Gn₂) from the lipid-linked oligosaccharide (LLO) glucose₃mannose₉N-acetylglucosamine₂-P-P-dolichol (G₃M₉Gn₂-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2α (eIF2α) on Ser⁵¹ by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G₃M₉Gn₂-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2α Ser⁵¹ phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser⁵¹ kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. AfterN-acetylation, the oligosaccharides were labelled with a UV-absorbing compound,p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz¹H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc1-3Man_7–9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man_5–9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man1-6(±GlcNAc1-4)(Man1-3)Man1-4GlcNAc1-4(±Fuc1-6)GlcNAc. The glucosylated oligosaccharides, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂, have not previously been reported in mature glycoproteins from any source.Abbreviations IgG, IgM, IgD, IgE, and IgA immunoglobulin G, M, D, E, and A, respectively - IgY egg-yolk antibody - ABEE p-aminobenzoic acid ethyl ester - HPLC high performance liquid chromatography - FAB-MS fast atom bombardment mass spectrometry - Hex hexose - HexNAc N-acetylhexosamine - hCG human chorionic gonadotropsin     

Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe

The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc₂Man₃-Dansyl was faster than that of Glc₁Man₃-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc₂Man₉GlcNAc₂ and Glc₁Man₉GlcNAc₂) although the activity was low.     

Identification and Characterization of an Intracellular Lectin,Calnexin, from Aspergillus oryzae Using N-Glycan-Conjugated Beads

Recently, asparagine-linked oligosaccharides (N-glycans) have been found to play a pivotal role in glycoprotein quality control in the endoplasmic reticulum (ER). In order to screen proteins interacting with N-glycans, we developed affinity chromatography by conjugating synthetic N-glycans on sepharose beads. Using the affinity beads with the dodecasaccharide Glc₁Man₉GlcNAc₂, one structure of the N-glycans, a 75-kDa protein, was isolated from the membranous fraction including the ER in Aspergillus oryzae. By LC-MS/MS analysis using the A. oryzae genome database, the protein was identified as one (AO090009000313) sharing similarities with calnexin. Further affinity chromatographic experiments suggested that the protein specifically bound to Glc₁Man₉GlcNAc₂, similarly to mammalian calnexins. We designated the gene AoclxA and expressed it as a fusion gene with egfp, revealing the ER localization of the AoClxA protein. Our results suggest that our affinity chromatography with synthetic N-glycans might help in biological analysis of glycoprotein quality control in the ER.     

Suppression of Rft1 Expression Does Not Impair the Transbilayer Movement of Man5GlcNAc2-P-P-Dolichol in Sealed Microsomes from Yeast

To further evaluate the role of Rft1 in the transbilayer movement of Man₅GlcNAc₂-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc₃Man₉GlcNAc₂-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[³H]mannose or UDP-[³H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [³H]Man₉GlcNAc₂-P-P-dolichol or Man₉[³H]GlcNAc₂-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc₂-P-P-Dol₁₅, a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [³H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [³H]GlcNAc₂-P-P-Dol₁₅ in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.The lipid-linked oligosaccharyl donor, Glc₃Man₉GlcNAc₂-P-P-dolichol (mature DLO²), in protein N-glycosylation is formed in two stages in the endoplasmic reticulum (ER) (1–4). In the first stage the lipid intermediates Man-P-dolichol (Man-P-Dol), Glc-P-dolichol (Glc-P-Dol), and Man₅GlcNAc₂-P-P-dolichol (M5-DLO) are formed on the cytoplasmic leaflet of the ER with GDP-Man, UDP-Glc, and UDP-GlcNAc, serving as the glycosyl donors. The biosynthesis of the mature DLO is completed with the addition of four more mannosyl units and the formation of the triglucosyl cap in the second stage after the transbilayer movement of Man-P-Dol, Glc-P-Dol, and M5-DLO to the lumenal monolayer. Although many details about the genetics, enzymology, and regulation of these 14 glycosylation reactions are known, there is virtually nothing known about the ER proteins that are presumably required to allow the lipid-bound hydrophilic glycosyl groups to traverse the hydrophobic core of the ER bilayer.The PER5/RFT1 gene was originally identified by Walter and coworkers (5) as a gene that was up-regulated by the unfolded protein response and required for efficient protein N-glycosylation in yeast. In a related study (6), the rft1 mutation was shown to be inscrutably suppressed by p53, a soluble protein that has not been found in yeast.Helenius et al. (7) have reported evidence from metabolic labeling experiments indicating that the RFT1 gene in Saccharomyces cerevisiae encodes a protein that is involved in the flipping of M5-DLO in vivo. More recently, a point mutation in the human orthologue of the RFT1 gene has been shown to result in the accumulation of M5-DLO in fibroblasts from a patient containing an R67C amino acid substitution (8). Although these results implicate Rft1 in the transverse diffusion of M5-DLO, the topological orientation of the accumulated intermediate in the mutant cells and the precise function of the protein in the transbilayer movement of the glycolipid intermediate remain to be defined.Two reports (9, 10) have demonstrated that Rft1 is not required for the “flipping” of M5-DLO in a reconstituted proteoliposomal system, raising questions about the precise relationship between Rft1 and the M5-DLO flippase. A more recent corroborative study further characterizing the reconstituted flippase activity indicates that the in vitro assay exhibits an impressive specificity for M5-DLO (11).The current study was conducted to further explore the possible role of Rft1 in the transbilayer movement of M5-DLO in the ER. Our results establish the accumulation of chemical amounts of M5-DLO in the Rft1-depleted cells by FACE analysis, supporting the results obtained by metabolic labeling in the yeast (7) and human (8) mutant cells. However, a series of experiments conducted with sealed microsomal vesicles indicate that, although Rft1 may be required to overcome a biophysical constraint for the flipping of M5-DLO in vivo, its depletion does not hinder the flipping of M5-DLO in sealed microsomal preparations in vitro. The resemblance of these results to the loss of the requirement for the Lec35 gene (12) in the transverse diffusion and/or utilization of Man-P-Dol and Glc-P-Dol for lipid intermediate biosynthesis during disruption of intact cells is discussed.