Analysis of Lipid-linked Oligosaccharides Synthesized in vivo in Schizosaccharomyces pombe.

Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N -glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [ 14 C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms. Graphical abstract: Workflow for the labeling, extraction, separation, and identification of LLO species in S. pombe . (A) Radioactive pulse of S. pombe cells with [ 14 C]-glucose for 15 min at 28 °C. (B) Organic extraction of LLOs from labeled yeasts sequentially using methanol, chloroform, H 2 O, chloroform:methanol:H 2 O (1:1:0.3), 0.02 M HCl (to separate glycans from dolichol), and chloroform:methanol:H 2 O (1:16:16). (C) Preparation of the sample for chromatography on paper: drying by airflow and radioactivity check. (D) Loading of samples in chromatographic paper and descendent chromatography in a glass chamber. The obtained plots (CPM versus running distance) need to be analyzed to identify single glycan species.

Abrogation of glucosidase I-mediated glycoprotein deglucosylation results in a sick phenotype in fission yeasts: Model for the human MOGS-CDG disorder.

Glucosidase I (GI) removes the outermost glucose from protein-linked Glc3Man9GlcNAc2 (G3M9) in the endoplasmic reticulum (ER). Individuals with congenital disorders of glycosylation MOGS-CDG bear mutations in the GI-encoding gene (gls1). Although GI absence has been reported to produce lethality in Schizosaccharomyces pombe yeasts, here we obtained two viable Δgls1 mutants, one with a very sick but not lethal phenotype (Δgls1-S) and the other with a healthier one (Δgls1-H). The sick strain displayed only G3M9 as an ER protein-linked oligosaccharide, whereas the healthier strain had both G3M9 and Man9GlcNAc2 The lipid-linked oligosaccharide patterns of the two strains revealed that the most abundantly formed glycans were G3M9 in Δgls1-S and Glc2Man9GlcNAc2 in Δgls1-H, suggesting reduced Alg10p glucosyltransferase activity in the Δgls1-H strain. A mutation in the alg10+ gene was indeed observed in this strain. Our results indicated that abrogated G3M9 deglucosylation was responsible for the severe defects observed in Δgls1-S cells. Further studies disclosed that the defects could not be ascribed to disruption of glycoprotein entrance into calnexin-folding cycles, inhibition of the oligosaccharyltransferase by transfer reaction products, or reduced proteasomal degradation of misfolded glycoproteins. Lack of triglucosylated glycoprotein deglucosylation neither significantly prevented glycan elongation in the Golgi nor modified the overall cell wall monosaccharide composition. Nevertheless, it resulted in a distorted cell wall and in the absence of underlying ER membranes. Furthermore, Golgi expression of human endomannosidase partially restored normal growth in Δgls1-S cells. We propose that accumulation of G3M9-bearing glycoproteins is toxic and at least partially responsible for defects observed in MOGS-CDG.

The conundrum of UDP-Glc entrance into the yeast ER lumen.

UDP-Glc entrance into the endoplasmic reticulum (ER) of eukaryotic cells is a key step in the quality control of glycoprotein folding, a mechanism requiring transfer of a Glc residue from the nucleotide sugar (NS) to glycoprotein folding intermediates by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). According to a bioinformatics search there are only eight genes in the Schizosaccharomyces pombe genome belonging to the three Pfam families to which all known nucleotide-sugar transporters (NSTs) of the secretory pathway belong. The protein products of two of them (hut1+ and yea4+) localize to the ER, those of genes gms1+, vrg4+, pet1+, pet2+ and pet3+ to the Golgi, whereas that of gms2+ has an unknown location. Here we demonstrate that (1) Δhut1 and Δgpt1 (UGGT null) mutants share several phenotypic features; (2) Δhut1 mutants show a 50% reduction in UDP-Glc transport into ER-derived membranes; (3) in vivo UDP-Glc ER entrance occurred in Δhut1Δyea4Δgms2 mutants and in cells in which Δhut1 disruption was combined with that of each of four of the genes encoding Golgi-located proteins. Therefore, disruption of all genes whose products localize to the ER or have an unknown location did not obliterate UDP-Glc ER entrance. We conclude that the hut1+ gene product is involved in UDP-Glc entrance into the ER, but that at least another as yet unknown NST displaying an unconventional sequence operates in the yeast secretory pathway. This conclusion agrees with our previous results showing that UDP-Glc entrance into the yeast ER does not follow the classical NST antiport mechanism.

A sweet code for glycoprotein folding.

Glycoprotein synthesis is initiated in the endoplasmic reticulum (ER) lumen upon transfer of a glycan (Glc3Man9GlcNAc2) from a lipid derivative to Asn residues (N-glycosylation). N-Glycan-dependent quality control of glycoprotein folding in the ER prevents exit to Golgi of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones (calnexin and calreticulin) that recognize monoglucosylated polymannose protein-linked glycans, lectin-associated oxidoreductase acting on monoglucosylated glycoproteins (ERp57), a glucosyltransferase that creates monoglucosylated epitopes in protein-linked glycans (UGGT) and a glucosidase (GII) that removes the glucose units added by UGGT. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded glycoproteins or in not completely assembled multimeric glycoprotein complexes. Glycoproteins that fail to properly fold are eventually driven to proteasomal degradation in the cytosol following the ER-associated degradation pathway, in which the extent of N-glycan demannosylation by ER mannosidases play a relevant role in the identification of irreparably misfolded glycoproteins.

Crystal Structure and Functional Analyses of the Lectin Domain of Glucosidase II: Insights into Oligomannose Recognition.

N-Glycans are modified as part of a quality control mechanism during glycoprotein folding in the endoplasmic reticulum (ER). Glucosidase II (GII) plays a critical role by generating monoglucosylated glycans that are recognized by lectin chaperones, calnexin and calreticulin. To understand how the hydrolytic activity of GIIα is enhanced by the mannose 6-phosphate receptor (MPR) homology domain (MRH domain) of its ß subunit, we now report a 1.6 Å resolution crystal structure of the MRH domain of GIIß bound to mannose. A comparison of ligand-bound and unbound structures reveals no major difference in their overall fold, but rather a repositioning of side chains throughout the binding pocket, including Y372. Mutation of Y372 inhibits GII activity, demonstrating an important role for Y372 in regulating GII activity. Comparison of the MRH domains of GIIß, MPRs, and the ER lectin OS-9 identified conserved residues that are critical for the structural integrity and architecture of the carbohydrate binding pocket. As shown by nuclear magnetic resonance spectroscopy, mutations of the primary binding pocket residues and adjacent W409, all of which inhibit the activity of GII both in vitro and in vivo, do not cause a significant change in the overall fold of the GIIß MRH domain but impact locally the stability of the binding pocket. W409 does not directly contact mannose; rather, its indole ring is stabilized by binding into a hydrophobic pocket of an adjacent crystallographic neighbor. This suggests that W409 interacts with a hydrophobic region of the GIIß or GIIα subunit to modulate its effect on GII activity.

Structure of the lectin mannose 6-phosphate receptor homology (MRH) domain of glucosidase II, an enzyme that regulates glycoprotein folding quality control in the endoplasmic reticulum.

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIß subunit. GIIß participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIß MRH domain by NMR spectroscopy. It adopts a ß-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIß but not in other MRHs that influences GII glucose trimming activity.

The two Caenorhabditis elegans UDP-glucose:glycoprotein glucosyltransferase homologues have distinct biological functions.

The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.

Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo.

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the ß subunit (GIIß) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIß MRH domain and that the N-terminal GIIß G2B domain is involved in the GIIα-GIIß interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIß MRH domains with a higher specificity for glycans with high mannose content.

Functional cooperation between BiP and calreticulin in the folding maturation of a glycoprotein in Trypanosoma cruzi.

Proteins may adopt diverse conformations during their folding in vivo, ranging from extended chains when they emerge from the ribosome to compact intermediates near the end of the folding process. Accordingly, a variety of chaperones and folding assisting enzymes have evolved to deal with this diversity. Chaperone selection by a particular substrate depends on the structural features of its folding intermediates. In addition, this process may be modulated by competitive effects between chaperones. Here we address this issue by using TcrCATL as model substrate. TcrCATL is an abundant Trypanosoma cruzi lysosomal protease and it was the first identified endogenous UDP-Glc:glycoprotein glucosyltransferase (UGGT) substrate. We found that TcrCATL associated sequentially with BiP and calreticulin (CRT) during its folding process. Early, extended conformations were bound to BiP, while more advanced and compact folding intermediates associated to CRT. The interaction between TcrCATL and CRT was impeded by deletion of the UGGT-encoding gene but, similarly to what was observed in wild type cells, in mutant cells TcrCATL associated to BiP only when displaying extended conformations. The absence of TcrCATL-CRT interactions in UGGT null cells resulted in a drastic reduction of TcrCATL folding efficiency and triggered the aggregation of TcrCATL through intermolecular disulfide bonds. These observations show that BiP and CRT activities complement each other to supervise a complete and efficient TcrCATL folding process. The present report provides further evidence on the early evolutionary acquisition of the basic tenets of the N-glycan dependent quality control mechanism of glycoprotein folding.

Endoplasmic reticulum calcium regulates the retrotranslocation of Trypanosoma cruzi calreticulin to the cytosol.

For most secretory pathway proteins, crossing the endoplasmic reticulum (ER) membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD), is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT) being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C), since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.

UDP-GlC:glycoprotein glucosyltransferase-glucosidase II, the ying-yang of the ER quality control.

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.

The structure of calreticulin C-terminal domain is modulated by physiological variations of calcium concentration.

Calreticulin is an abundant endoplasmic reticulum resident protein that fulfills at least two basic functions. Firstly, due to its ability to bind monoglucosylated high mannose oligosaccharides, calreticulin is a central component of the folding quality control system of glycoproteins. On the other hand, thanks to its capacity to bind high amounts of calcium, calreticulin is one of the main calcium buffers in the endoplasmic reticulum. This last activity resides on a highly negatively charged domain located at the C terminus. Interestingly, this domain has been proposed to regulate the intracellular localization of calreticulin. Structural information for this domain is currently scarce. Here we address this issue by employing a combination of biophysical techniques and molecular dynamics simulation. We found that calreticulin C-terminal domain at low calcium concentration displays a disordered structure, whereas calcium addition induces a more rigid and compact conformation. Remarkably, this change develops when calcium concentration varies within a range similar to that taking place in the endoplasmic reticulum upon physiological fluctuations. In addition, a much higher calcium concentration is necessary to attain similar responses in a peptide displaying a randomized sequence of calreticulin C-terminal domain, illustrating the sequence specificity of this effect. Molecular dynamics simulation reveals that this ordering effect is a consequence of the ability of calcium to bring into close proximity residues that lie apart in the primary structure. These results place calreticulin in a new setting in which the protein behaves not only as a calcium-binding protein but as a finely tuned calcium sensor.

Glucosidase II beta 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 (Glc(3)Man(9)GlcNAc(2)) 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 alpha subunit (GIIalpha) bears the glycosyl hydrolase active site, whereas its beta subunit (GIIbeta) role is controversial and has been reported to be involved in GIIalpha ER retention and folding. Here, we report that in the absence of GIIbeta, the catalytic subunit GIIalpha 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 alpha-d-glucopyranoside. However, the heterodimer is required to efficiently deglucosylate the physiological substrates Glc(2)Man(9)GlcNAc(2) (G2M9) and Glc(1)Man(9)GlcNAc(2) (G1M9). The interaction of the mannose 6-phosphate receptor homologous domain present in GIIbeta and mannoses in the B and/or C arms of the glycans mediates glycan hydrolysis enhancement. We present evidence that also in mammalian cells GIIbeta modulates G2M9 and G1M9 trimming.

N-glycan trimming by glucosidase II is essential for Arabidopsis development.

Glucosidase II, one of the early N-glycan processing enzymes and a major player in the glycoprotein folding quality control, has been described as a soluble heterodimer composed of alpha and beta subunits. Here we present the first characterization of a plant glucosidase II alpha subunit at the molecular level. Expression of the Arabidopsis alpha subunit restored N-glycan maturation capacity in Schizosaccharomyces pombe alpha- or alphabeta-deficient mutants, but with a lower efficiency in the last case. Inactivation of the alpha subunit in a temperature sensitive Arabidopsis mutant blocked N-glycan processing after a first trimming by glucosidase I and strongly affected seedling development.

All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae.

The transfer of lipid-linked oligosaccharide to asparagine residues of polypeptide chains is catalyzed by oligosaccharyltransferase (OTase). In most eukaryotes, OTase is a hetero-oligomeric complex composed of eight different proteins, in which the STT3 component is believed to be the catalytic subunit. In the parasitic protozoa Leishmania major, four STT3 paralogues, but no homologues to the other OTase components seem to be encoded in the genome. We expressed each of the four L. major STT3 proteins individually in Saccharomyces cerevisiae and found that three of them, LmSTT3A, LmSTT3B, and LmSTT3D, were able to complement a deletion of the yeast STT3 locus. Furthermore, LmSTT3D expression suppressed the lethal phenotype of single and double deletions in genes encoding other essential OTase subunits. LmSTT3 proteins did not incorporate into the yeast OTase complex but formed a homodimeric enzyme, capable of replacing the endogenous, multimeric enzyme of the yeast cell. Therefore, these protozoan OTases resemble the prokaryotic enzymes with respect to their architecture, but they used substrates typical for eukaryotic cells: N-X-S/T sequons in proteins and dolicholpyrophosphate-linked high mannose oligosaccharides.

How sugars convey information on protein conformation in the endoplasmic reticulum.

The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic reticulum to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and not completely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectin-associated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase and a glucosidase that creates monoglucosylated epitopes in glycans transferred in protein N-glycosylation or removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The purpose of the review is to describe the most significant recent findings on the mechanism of glycoprotein folding and assembly quality control and to discuss the main still unanswered questions.

The interplay between calcium and the in vitro lectin and chaperone activities of calreticulin.

The ER resident protein calreticulin fulfills at least two important roles. It works as a chaperone preventing Golgi exit of non-native protein species and enhancing protein folding efficiency in either N-glycan-dependent, lectin chaperone, or classical chaperone, N-glycan-independent, modes and is one of the main calcium buffers in the cell. This last feature is independent from the lectin chaperone properties of the protein as this last activity is also observed in a CRT fragment lacking calcium buffer capacity. Here we study the interplay between calcium and the lectin and chaperone activities of CRT. The affinity of CRT for monoglucosylated glycans measured in solution by equilibrium dialysis and fluorescence anisotropy was not affected by the absence of calcium. Binding of CRT to monoglucosylated neoglycoproteins displaying either native or molten globule-like conformations was also independent of calcium concentration. Moreover, calcium and monoglucosylated glycans stabilized the CRT structure in an apparent additive, independent manner when the protein was subjected to thermal or urea-induced denaturation. In addition, the ability of CRT to decrease the level of aggregation of a chemically denatured monoglucosylated and nonglycosylated protein was also independent of calcium level.