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Sequential mannose trimming of N-glycan (Man 9 GlcNAc 2 -> Man 8 GlcNAc 2 -> Man 7 GlcNAc 2 ) facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). Our gene knockout experiments in human HCT116 cells have revealed that EDEM2 is required for the first step. However, it was previously shown that purified EDEM2 exhibited no α1,2-mannosidase activity toward Man 9 GlcNAc 2 in vitro. Here, we found that EDEM2 was stably disulfide-bonded to TXNDC11, an endoplasmic reticulum protein containing five thioredoxin (Trx)-like domains. C558 present outside of the mannosidase homology domain of EDEM2 was linked to C692 in Trx5, which solely contains the CXXC motif in TXNDC11. This covalent bonding was essential for mannose trimming and subsequent gpERAD in HCT116 cells. Furthermore, EDEM2-TXNDC11 complex purified from transfected HCT116 cells converted Man 9 GlcNAc 2 to Man 8 GlcNAc 2 (isomerB) in vitro. Our results establish the role of EDEM2 as an initiator of gpERAD, and represent the first clear demonstration of in vitro mannosidase activity of EDEM family proteins., Competing Interests: GG, SN, HY, TS, TI, TS, TY, KI, YI, KK, TO, KM No competing interests declared, (© 2020, George et al.)

While multiple α 1-2-mannosidases are necessary for glycoprotein N-glycan maturation in vertebrates, a single bacterial α1-2-mannosidase can be sufficient to cleave all α1-2-linked mannose residues in host glycoprotein N-glycans. We report here the characterization and crystal structure of a new α1-2-mannosidase (EfMan-I) from Enterococcus faecalis, a Gram-positive opportunistic human pathogen. EfMan-I catalyzes the cleavage of α1-2-mannose from not only oligomannoses but also high-mannose-type N-glycans on glycoproteins. Its 2.15 Å resolution crystal structure reveals a two-domain enzyme fold similar to other CAZy GH92 mannosidases. An unexpected potassium ion was observed bridging two domains near the active site. These findings support EfMan-I as an effective catalyst for in vitro N-glycan modification of glycoproteins with high-mannose-type N-glycans., (© 2019 Federation of European Biochemical Societies.)

At present, only little is known about the enzymatic machinery required for N -glycosylation in Chlamydomonas reinhardtii , leading to the formation of N -glycans harboring Xyl and methylated Man. This machinery possesses new enzymatic features, as C. reinhardtii N -glycans are independent of β1,2- N -acetylglucosaminyltransferase I. Here we have performed comparative N -glycoproteomic analyses of insertional mutants of mannosidase 1A (IM Man1A ) and xylosyltransferase 1A (IM XylT1A ). The disruption of man1A affected methylation of Man and the addition of terminal Xyl. The absence of XylT1A led to shorter N -glycans compared to the wild type. The use of a IM Man1A xIM XylT1A double mutant revealed that the absence of Man1A suppressed the IM XylT1A phenotype, indicating that the increased N -glycan trimming is regulated by core β1,2-Xyl and is dependent on Man1A activity. These data point toward an enzymatic cascade in the N -glycosylation pathway of C. reinhardtii with interlinked roles of Man1A and XylT1A. The results described herein represent the first step toward a functional characterization of the enzymatic N -glycosylation machinery in C. reinhardtii ., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Within the endoplasmic reticulum, immature glycoproteins are sorted into secretion and degradation pathways through the sequential trimming of mannose residues from Man 9 GlcNAc 2 to Man 5 GlcNAc 2 by the combined actions of assorted α-1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it is unclear whether the specific signal glycoforms are produced by random mannosidase action or are produced regioselectively in a sequenced manner by specific α-1,2-mannosidases. Here, we report the identification of a set of selective mannosidase inhibitors and development of conditions for their use that enable production of distinct pools of Man 8 GlcNAc 2 isomers from a structurally defined synthetic Man 9 GlcNAc 2 substrate in an endoplasmic reticulum fraction. Glycan processing analysis with these inhibitors provides the first biochemical evidence for selective production of the signal glycoforms contributing to traffic control in glycoprotein quality control., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glycoprotein N-linked oligosaccharides in the endoplasmic reticulum function as tags to regulate glycoprotein folding, sorting, secretion and degradation. Since the N-glycan structure of a glycoprotein should reflect the folding state, N-glycan processing may be affected by the aglycone state. In this study, we examined the influence of aglycone structures on N-glycan processing using synthetic substrates. We prepared (Glc 1 )Man 9 GlcNAc 2 linked to hydrophobic BODIPY-dye with a systematic series of different linker lengths. With these compounds, glucose transfer, glucose trimming and mannose trimming reactions of an endoplasmic reticulum fraction were examined. The results showed that substrates with shorter linkers between the N-glycan and hydrophobic patch had higher activities for both the glucose transfer and the mannose trimming reactions. In contrast, the glucose trimming reaction showed lower activity when substrates had shorter linkers. Thus, the reactivity for N-linked oligosaccharide processing of glycoproteins in the endoplasmic reticulum might be tunable by the aglycone structure, e.g., protein portion of glycoproteins., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3-4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man9GlcNAc2 down to Man5GlcNAc2, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The methylotrophic yeast Pichia pastoris is an attractive expression system due to its ability to secrete large amounts of recombinant protein, with the potential for glycosylation. Advances in glycoengineering of P. pastoris have successfully demonstrated the humanization of both the N- and O-linked glycosylation pathways in this organism. However, in certain cases, the presence of O-linked glycans on a therapeutic protein may not be desirable. Recently, we have reported the in vitro utility of jack bean α-1,2/3/6-mannosidase to remove O-linked mannose from intact undenatured glycoproteins produced in glycoengineered P. pastoris. However, one caveat of this strategy is that jack bean mannosidase has yet to be cloned and as such is only available as crude cellular extracts. This raises several concerns for using this reagent to treat large preparations of therapeutic proteins generated in P. pastoris. Therefore, we postulated that lysosomal mannosidases which have been cloned and demonstrated to have similar activities to jack bean mannosidase on N-linked glycans would also process O-linked glycans in a similar fashion. To this end, we screened a panel of recombinant lysosomal mannosidases from different organisms and identified several which cannot only reduce extended O-linked mannose chains but which can also hydrolyze the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, not only do we show for the first time the utility of lysosomal mannosidase for O-linked mannose processing, but since this is a recombinant enzyme, it has several benefits over the use of crude jack bean mannosidase extracts.

Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern., (© 2015 Benyair et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.

Compared with in vitro conditions, the intracellular environment is highly crowded with biomolecules; this has numerous effects on protein functions, including enzymatic activity. We examined the effects of macromolecular crowding on glycan processing of N-glycoprotein in the endoplasmic reticulum as a model sequential metabolic pathway. Experiments with synthetic substrates of physiological glycan structure clearly showed that the first half of the pathway (glucose trimming) was accelerated, whereas the second (mannose trimming) was decelerated under molecular crowding conditions. Furthermore, calreticulin, a lectin-like molecular chaperone, bound more strongly to a glycan-processing intermediate under these conditions. This study demonstrates the diverse effects of molecular crowding on sequential enzymatic processing, and the importance of the effects of macromolecular crowding on in vitro assays for understanding sequential metabolic pathways., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation., Results: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man₈GlcNAc₂ or Man₅GlcNAc₂. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man₈GlcNAc₂ glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man₅GlcNAc₂. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme., Conclusions: We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man₈GlcNAc₂ or Man₅GlcNAc₂ N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc(2) structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5)GlcNAc(2) and GlcMan(5)GlcNAc(2) glycans, and to a lesser extent with Glc(2)Man(5)GlcNAc(2) glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man(3)GlcNAc(2) structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.

In Saccharomyces cerevisiae, proteins with misfolded lumenal, membrane, and cytoplasmic domains are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L, -M, and -C, respectively. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man(7)GlcNAc(2) (d1) N-glycans with non-reducing α1,6-mannosyl residues. Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and then deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). Here, we examined yeast fOS metabolism as a function of cell growth in order to obtain quantitative and mechanistic insights into ERAD. We demonstrate that both Png1p-dependent generation of Man(7-10)GlcNAc(2) fOS and vacuolar α-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man(1)GlcNAc(2) are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. Gene deletions in the ams1Δ background revealed that, as anticipated, Mns1p and Mnl1p are required for efficient generation of the Man(7)GlcNAc(2) (d1) fOS, but for the first time, we demonstrate that small amounts of this fOS are generated in an Mnl1p-independent, Mns1p-dependent pathway and that a Man(8)GlcNAc(2) fOS that is known to bind Yos9p is generated in an Mnl1p-dependent, Mns1p-independent manner. This latter observation adds mechanistic insight into a recently described Mnl1p-dependent, Mns1p-independent ERAD pathway. Finally, we show that 50% of fOS generation is independent of ERAD-L, and because our data indicate that ERAD-M and ERAD-C contribute little to fOS levels, other important processes underlie fOS generation in S. cerevisiae.

The Golgi complex has been implicated as a possible component of endoplasmic reticulum (ER) glycoprotein quality control, although the elucidation of its exact role is lacking. ERManI, a putative ER resident mannosidase, plays a rate-limiting role in generating a signal that targets misfolded N-linked glycoproteins for ER-associated degradation (ERAD). Herein we demonstrate that the endogenous human homologue predominantly resides in the Golgi complex, where it is subjected to O-glycosylation. To distinguish the intracellular site where the glycoprotein ERAD signal is generated, a COPI-binding motif was appended to the N terminus of the recombinant protein to facilitate its retrograde translocation back to the ER. Partial redistribution of the modified ERManI was observed along with an accelerated rate at which N-linked glycans of misfolded α1-antitrypsin variant NHK were trimmed. Despite these observations, the rate of NHK degradation was not accelerated, implicating the Golgi complex as the site for glycoprotein ERAD substrate tagging. Taken together, these data provide a potential mechanistic explanation for the spatial separation by which glycoprotein quality control components operate in mammalian cells.

During quality control in the ER (endoplasmic reticulum), nascent glycoproteins are deglucosylated by ER glucosidases I and II. In the post-ER compartments, glycoprotein endo-α-mannosidase provides an alternative route for deglucosylation. Previous evidence suggests that endomannosidase non-selectively deglucosylates glycoproteins that escape quality control in the ER, facilitating secretion of aberrantly folded as well as normal glycoproteins. In the present study, we employed FOS (free oligosaccharides) released from degrading glycoproteins as biomarkers of ERAD (ER-associated degradation), allowing us to gain a global rather than single protein-centred view of ERAD. Glucosidase inhibition was used to discriminate between glucosidase- and endomannosidase-mediated ERAD pathways. Endomannosidase expression was manipulated in CHO (Chinese-hamster ovary)-K1 cells, naturally lacking a functional version of the enzyme, and HEK (human embryonic kidney)-293T cells. Endomannosidase was shown to decrease the levels of total FOS, suggesting decreased rates of ERAD. However, following pharmacological inhibition of ER glucosidases I and II, endomannosidase expression resulted in a partial switch between glucosylated FOS, released from ER-confined glycoproteins, to deglucosylated FOS, released from endomannosidase-processed glycoproteins transported from the Golgi/ERGIC (ER/Golgi intermediate compartment) to the ER. Using this approach, we have identified a previously unknown pathway of glycoprotein flow, undetectable by the commonly employed methods, in which secretory cargo is targeted back to the ER after being processed by endomannosidase., (© The Authors Journal compilation © 2011 Biochemical Society)

Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man(8)GlcNAc(2) carbohydrates bound to unfolded proteins, yielding Man(7)GlcNAc(2). This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathways and how the flux through these pathways is adjusted to stress conditions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although the trimming of α1,2-mannose residues from precursor N-linked oligosaccharides is an essential step in the delivery of misfolded glycoproteins to endoplasmic reticulum (ER)-associated degradation (ERAD), the exact role of this trimming is unclear. EDEM1 was initially suggested to bind N-glycans after mannose trimming, a role presently ascribed to the lectins OS9 and XTP3-B, because of their in vitro affinities for trimmed oligosaccharides. We have shown before that ER mannosidase I (ERManI) is required for the trimming and concentrates together with the ERAD substrate and ERAD machinery in the pericentriolar ER-derived quality control compartment (ERQC). Inhibition of mannose trimming prevents substrate accumulation in the ERQC. Here, we show that the mannosidase inhibitor kifunensine or ERManI knockdown do not affect binding of an ERAD substrate glycoprotein to EDEM1. In contrast, substrate association with XTP3-B and with the E3 ubiquitin ligases HRD1 and SCF(Fbs2) was inhibited. Consistently, whereas the ERAD substrate partially colocalized upon proteasomal inhibition with EDEM1, HRD1, and Fbs2 at the ERQC, colocalization was repressed by mannosidase inhibition in the case of the E3 ligases but not for EDEM1. Interestingly, association and colocalization of the substrate with Derlin-1 was independent of mannose trimming. The HRD1 adaptor protein SEL1L had been suggested to play a role in N-glycan-dependent substrate delivery to OS9 and XTP3-B. However, substrate association with XTP3-B was still dependent on mannose trimming upon SEL1L knockdown. Our results suggest that mannose trimming enables delivery of a substrate glycoprotein from EDEM1 to late ERAD steps through association with XTP3-B.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related alpha-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.

The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway.

The secretory pathway provides a physical route through which only correctly folded gene products are delivered to the eukaryotic cell surface. The efficiency of endoplasmic reticulum (ER)-associated degradation (ERAD), which orchestrates the clearance of structurally aberrant proteins under basal conditions, is boosted by the unfolded protein response (UPR) as one of several means to relieve ER stress. However, the underlying mechanism that links the two systems in higher eukaryotes has remained elusive. Herein, the results of transient expression, RNAi-mediated knockdown and functional studies demonstrate that the transcriptional elevation of EDEM1 boosts the efficiency of glycoprotein ERAD through the formation of a complex that suppresses the proteolytic downregulation of ER mannosidase I (ERManI). The results of site-directed mutagenesis indicate that this capacity does not require that EDEM1 possess inherent mannosidase activity. A model is proposed in which ERManI, by functioning as a downstream effector target of EDEM1, represents a checkpoint activation paradigm by which the mammalian UPR coordinates the boosting of ERAD.

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-dependent 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.

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative alpha-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man8GlcNAc2, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man8GlcNAc2 to Man12GlcNAc2. Digestion with alpha-1,2- and alpha-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first alpha-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.

High-mannose-type oligosaccharides have been shown to play important roles in protein quality control. Several intracellular proteins, such as lectins, chaperones and glycan-processing enzymes, are involved in this process. These include calnexin/calreticulin, UDP-glucose:glycoprotein glucosyltransferase (UGGT), cargo receptors (such as VIP36 and ERGIC-53), mannosidase-like proteins (e.g. EDEM and Htm1p) and ubiquitin ligase (Fbs). They are thought to recognize high-mannose-type glycans with subtly different structures, although the precise specificities are yet to be clarified. In order to gain a clear understanding of these protein-carbohydrate interactions, comprehensive synthesis of high-mannose-type glycans was conducted. In addition, two approaches to the synthesis of artificial glycoproteins with homogeneous oligosaccharides were investigated. Furthermore, a novel substrate of UGGT was discovered.

The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3-MDa elementary unit, consisting of six sets of a disulfide-linked heavy chain (H-chain)-light chain (L-chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H-L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal-level fibroin-producing breeds (J-139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27-kDa fhx/P25 was produced from the 30-kDa form by digestion with the bacterial alpha1,2-mannosidase in vitro. The elementary unit in the ER extract contained only the 30-kDa fhx/P25, whereas both 30- and 27-kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked-pupa mutants [Nd(2), Nd-s and Nd-sD], extremely small amounts of fibroin were produced and they consisted of one molecule of 27-kDa fhx/P25 and six molecules of H-chain but no L-chain. When the Nd-sD mutant was subjected to transgenesis with the normal L-chain gene, the (H-L)6fhx1-type elementary unit containing the 30-kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi alpha1,2-mannosidases when L-chains are present in the unit. Models suggesting a role of L-chain for the protection of alpha1,2-mannose residues of fhx/P25 are presented.

Endoplasmic reticulum-associated degradation of newly synthesized glycoproteins has been demonstrated previously using various mammalian cell lines. Depending on the cell type, glycoproteins bearing Man9 glycans and glycoproteins bearing Man5 glycans can be efficiently degraded. A wide variety of variables can lead to defective synthesis of lipid-linked oligosaccharides and, therefore, in mammalian cells, species derived from Man9GlcNAc2 or Man5GlcNAc2 are often recovered on newly synthesized glycoproteins. The degradation of glycoproteins bearing these two species has not been studied. We used a Chinese hamster ovary cell line lacking Glc-P-Dol-dependent glucosyltransferase I to generate various proportions of Man5GlcNAc2 and Man9GlcNAc2 on newly synthesized glycoproteins. By studying the structure of the soluble oligomannosides produced by degradation of these glycoproteins, we demonstrated the presence of a higher proportion of soluble oligomannosides originating from truncated glycans, showing that glycoproteins bearing Man5GlcNAc2 glycans are degraded preferentially.

We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5'-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine2-mannose3-N-acetylglucosamine2 (GlcNAc2Man3GlcNAc2). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.

Clusterin is a heterodimeric, disulfide-linked 70-80 kDa glycoprotein that is induced during regression of most, if not all, hormone-dependent epithelial tissues. These studies describe the biogenesis and intracellular trafficking of clusterin in MCF-7 cells before and after the initiation of apoptosis with antiestrogens and TNF alpha. Under physiological conditions, clusterin is modified in the endoplasmic reticulum (ER), and proteolytically cleaved in the Golgi to generate discrete alpha and beta chains prior to secretion. Treatment with TNFalpha or the antiestrogen, ICI 182,780, induces apoptosis in MCF-7 cells and leads to substantial changes in the activity of Golgi-resident enzymes, significantly altering the biogenesis of clusterin. This leads to the appearance of a 50-53 kDa uncleaved, nonglycosylated, disulfide-linked isoform of clusterin that accumulates in the nucleus. While clusterin contains a cryptic SV-40-like nuclear localization signal, mutation of this sequence does not affect the nuclear accumulation of the disulfide-linked nuclear isoform. Confocal microscopy demonstrates that the nuclear accumulation of clusterin is coincident with DNA fragmentation. These data suggest that, at least in secretory epithelial cells, retrograde transport from the Golgi to the ER of a nonglycosylated, uncleaved isoform and the subsequent translocation of clusterin to the nucleus occur in dying cells.

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.

In the early secretory pathway, asparagine-linked glycosylation facilitates the conformational maturation of diverse polypeptides by promoting their physical engagement with the glycoprotein-folding machinery. Misfolded glycoproteins are selectively eliminated from the endoplasmic reticulum by a stringent process of conformation-based quality control. Recent studies indicate that a small ensemble of oligosaccharide-processing enzymes and lectins use the asparagine-linked appendage to orchestrate the selective disposal of numerous transport-defective glycoproteins from the early secretory pathway. The glycan-based disposal system functions as an evolutionarily conserved terminal checkpoint in eukaryote genome expression. That the mechanisms by which glycoprotein substrates are recruited for degradation diverge at the level of signal recognition reflects a previously unappreciated component of cellular differentiation in higher eukaryotes.

Synapsin I is abundant in neural tissues. Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton. Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I. Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical (32)P-peptide maps after limited proteolysis. We also detected synapsin I mRNA in liver by northern blot analysis. These results indicate that the expression of synapsin I is more widespread than previously thought. Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident. We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways.

A soluble form of ribophorin I (RI(332)) is rapidly degraded in Hela and Chinese hamster ovary (CHO) cells by a cytosolic proteasomal pathway, and the N-linked glycan present on the protein may play an important role in this process. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoproteins to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glycoprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc(1)Man(5)GlcNAc(2) N-glycans. This oligomannoside structure interferes with protein maturation and folding, leading to an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribophorin I (RI(332)-3HA) expressed in MadIA214 cells was rapidly degraded, comparable to control cells with the complete Glc(3)Man(9)GlcNAc(2) N-glycan. ER-associated degradation (ERAD) of RI(332)-3HA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI(332)-3HA degradation with agents specifically blocking proteasomal activities. Two inhibitors of alpha1,2-mannosidase activity also stabilized RI(332)-3HA in the glycosylation-defective cell line. This is striking, because the major mannosidase activity in the ER is the one of mannosidase I, specific for a mannose alpha1,2-linkage that is absent from the truncated Man(5) structure. Interestingly, though the Man(5) derivative was present in large amounts in the total protein pool, the two major species linked to RI(332)-3HA shortly after synthesis consisted of Glc(1)Man(5 )and Man(4), being replaced by Man(4 )and Man(3) when proteasomal degradation was inhibited. In contrast, the untrimmed intermediate of RI(332)-3HA was detected in mutant cells treated with mannosidase inhibitors. Our results unambiguously demonstrate that an alpha1,2-mannosidase that is not ER mannosidase I is involved in ERAD of RI(332-)3HA in the glycosylation-defective cell line, MadIA214.

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.

The quality control mechanism in the endoplasmic reticulum (ER) discriminates correctly folded proteins from misfolded polypeptides and determines their fate. Terminally misfolded proteins are retrotranslocated from the ER and degraded by cytoplasmic proteasomes, a mechanism known as ER-associated degradation (ERAD). We report the cDNA cloning of Edem, a mouse gene encoding a putative type II ER transmembrane protein. Expression of Edem mRNA was induced by various types of ER stress. Although the luminal region of ER degradation enhancing alpha-mannosidase-like protein (EDEM) is similar to class I alpha1,2-mannosidases involved in N-glycan processing, EDEM did not have enzymatic activity. Overexpression of EDEM in human embryonic kidney 293 cells accelerated the degradation of misfolded alpha1-antitrypsin, and EDEM bound to this misfolded glycoprotein. The results suggest that EDEM is directly involved in ERAD, and targets misfolded glycoproteins for degradation in an N-glycan dependent manner.

Quality control in the endoplasmic reticulum must discriminate nascent proteins in their folding process from terminally unfolded molecules, selectively degrading the latter. Unassembled Ig-mu and J chains, two glycoproteins with five N-linked glycans and one N-linked glycan, respectively, are degraded by cytosolic proteasomes after a lag from synthesis, during which glycan trimming occurs. Inhibitors of mannosidase I (kifunensine), but not of mannosidase II (swainsonine), prevent the degradation of mu chains. Kifunensine also inhibits J chain dislocation and degradation, without inhibiting secretion of IgM polymers. In contrast, glucosidase inhibitors do not significantly affect the kinetics of mu and J degradation. These results suggest that removal of the terminal mannose from the central branch acts as a timer in dictating the degradation of transport-incompetent, glycosylated Ig subunits in a calnexin-independent way. Kifunensine does not inhibit the degradation of an unglycosylated substrate (lambda Ig light chains) or of chimeric mu chains extended with the transmembrane region of the alpha T cell receptor chain, implying the existence of additional pathways for extracting proteins from the endoplasmic reticulum lumen for proteasomal degradation.

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.

Secretory glycoproteins that fail to fold or assemble correctly are retained in the endoplasmic reticulum and eventually degraded. Recent evidence shows that trimming of their N-linked oligosaccharide chains plays a key role in targeting glycoproteins for destruction.

In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded alpha(1)-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed.

Synaptic glycoproteins are synthesized and glycosylated in the neuronal cell body, and conveyed to terminals by fast axonal transport. We used the alpha-mannosidase inhibitor, 2-deoxymannojirimycin (dMan), to investigate the effects of disrupting N-glycan processing on the axonal trafficking of proteins in vivo. dMan significantly reduced rapid axonal transport in retinal ganglion cells to about 34% of control values 4h after metabolic labeling; at 8 h post-labeling the inhibition was reversed. 2-D gel analysis showed that dMan completely inhibited the arrival of radiolabeled L1 and NCAM at axon terminals, and resulted in the appearance of two novel proteins of 230 kDa and 155 kDa. Our results show that disruption of the N-glycosylation pathway has an immediate inhibitory effect on total axonal transport and longer lasting effects on the trafficking of specific glycoproteins to axon terminals in vivo.

Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc(1-3)Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of mono-glucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-beta-N- acetylglucosaminidase H can distinguish between those terminating in a single N-acetylglucosamine or in a di-N-acetylchitobiose sequence.

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.

Mammalian spermatozoa undergo the acrosome reaction (AR) in response to the interaction of a carbohydrate-recognizing molecule(s) on the sperm plasma membrane (sperm surface receptor) and its complementary glycan (ligand) moiety(ies) on the zona pellucida (ZP). Previously, we demonstrated that a hexose (mannose) or two amino sugars (glucosaminyl or galactosaminyl residues) when covalently conjugated to a protein backbone (neoglycoproteins) mimicked the mouse ZP3 glycoprotein and induced the AR in capacitated mouse spermatozoa (Loeser and Tulsiani, Biol Reprod 1999; 60:94-101). To elucidate the mechanism underlying sperm-neoglycoprotein interaction and the induction of the AR, we have examined the effect of several AR blockers on neoglycoprotein-induced AR. Our data demonstrate that two known L-type Ca(2+) channel blockers prevented the induction of the AR by three neoglycoproteins (mannose-BSA, N-acetylglucosamine-BSA, and N-acetylgalactosamine-BSA). The fact that the L-type Ca(2+) channel blockers (verapamil, diltiazem) had no inhibitory effect on sperm surface galactosyltransferase or alpha-D-mannosidase, two carbohydrate-recognizing enzymes thought to be sperm surface receptors, suggests that the reagents block the AR by a mechanism other than binding to the active site of the enzymes.

Glycosylation is a cellular process accomplished by a series of sequential enzymatic processing steps through the endoplasmic reticulum and Golgi apparatus with vesicle transport between the membranous organelles. The capacity for complex glycosylation is considered to be conferred by cell genetics, while the roles of nongenetic factors in protein processing are often ignored. It was hypothesized that the glycosyltransferase reactions in the insect cell-baculovirus system were limited by the small supply of sugar donor cosubstrates. By adding mannosamine, the glycosylation of a human secreted alkaline phosphatase in Spodoptera frugiperda (Sf-21) cells was extended to include terminal N-acetylglucosamine structures which were not seen in control cultures, and in Trichoplusic ni (BTI-Tn5B1-4) cells the amount of terminal N-acetylglucosamine structures was increased.

Ribonuclease (RNase) A and the more stable glycosylated RNase B differ by a carbohydrate moiety (GlcNAc2Man5-9) attached to Asn34. As previously shown, the first proteolytic cleavage sites to appear on thermal denaturation of both enzymes are in the structural region around Asn34. To discriminate the contribution of the modifying moiety to the stabilization toward thermal unfolding, on the one hand, and proteolytic fragmentation, on the other hand, the carbohydrate chain of RNase B was shortened by treatment with glycosidases to obtain GlcNAc-RNase and (GlcNAc)2Man3 -RNase and extended by binding to concanavalin A or concanavalin A-agarose. The results show a saltatory increase of the thermal unfolding constants and transition temperatures of GlcNAc-RNase in comparison to RNase A, whereas the extension of the modification at Asn34 in the other RNase species does not further increase thermal stability. Therefore, the stability difference between RNase A and RNase B derivatives is attributed to the first carbohydrate unit. In contrast, the rate of proteolysis decreases gradually with increasing volume of the modifying moiety. As concluded from the analysis of the primary cleavage fragments, the main degradation pathway is shifted from the Asn34-Leu35 to the Thr45-Phe46 peptide bond due to increasing shielding effects.

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal alpha1,2 glucose and the first alpha1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for misfolded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N-acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1-deoxy)mannojirimycin, the conversion of the G protein into an endo H-resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.