Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.     

Structural analysis of <Emphasis Type="Italic">N</Emphasis>-/<Emphasis Type="Italic">O</Emphasis>-glycans assembled on proteins in yeasts

Protein glycosylation, the most universal and diverse post-translational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species-and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N-and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.     

Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8

ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.     

Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.     

N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop: A CRITICAL DETERMINANT FOR LIGAND-INDUCED RECEPTOR ACTIVATION AND INTERNALIZATION*

Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling.     

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.     

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.     

Misfolding and aggregation of vacuolar glycoproteins in plant cells

Phaseolin and lectin-related polypeptides, the abundant oligomeric glycoproteins of bean seeds, are synthesized on the endoplasmic reticulum (ER) and then transported to the storage vacuole via the Golgi apparatus. Glycosylation and folding are among the major modifications these proteins undergo in the ER. Although a recurrent role of N-glycosylation is on protein folding, in previous studies on common bean (Phaseolus vulgaris) seeds we demonstrated that the oligosaccharide side-chains are not required for folding, intracellular transport and activity of storage glycoproteins. We show here that in lima bean (Phaseolus lunatus), incubation of the developing cotyledon with tunicamycin to prevent glycosylation has a dramatic effect on the intracellular transport of the storage glycoproteins. When lacking their glycans, phaseolin and lectin-related polypeptides misfold and are retained in the ER as mixed aggregates to which the chaperone BiP irreversibly associates. The lumen of the ER becomes enlarged to accommodate the aggregated polypeptides. Intracellular transport of legumin, a naturally unglycosylated storage protein, is mostly unaffected by the inhibitor, indicating that the observed phenomenon specifically occurs on glycoproteins. Furthermore, recombinant lima bean phaseolin synthesized in tobacco protoplasts is also correctly folded and matured in the presence of tunicamycin. To our knowledge, this is the first report that describes in detail the block of intracellular transport of vacuolar glycoproteins in plant cells due to aggregation following glycosylation inhibition.     

The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin

Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA– calnexin–calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.     

Glycomics Profiling of Chinese Hamster Ovary Cell Glycosylation Mutants Reveals N-Glycans of a Novel Size and Complexity

Identifying biological roles for mammalian glycans and the pathways by which they are synthesized has been greatly facilitated by investigations of glycosylation mutants of cultured cell lines and model organisms. Chinese hamster ovary (CHO) glycosylation mutants isolated on the basis of their lectin resistance have been particularly useful for glycosylation engineering of recombinant glycoproteins. To further enhance the application of these mutants, and to obtain insights into the effects of altering one specific glycosyltransferase or glycosylation activity on the overall expression of cellular glycans, an analysis of the N-glycans and major O-glycans of a panel of CHO mutants was performed using glycomic analyses anchored by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry. We report here the complement of the major N-glycans and O-glycans present in nine distinct CHO glycosylation mutants. Parent CHO cells grown in monolayer versus suspension culture had similar profiles of N- and O-GalNAc glycans, although the profiles of glycosylation mutants Lec1, Lec2, Lec3.2.8.1, Lec4, LEC10, LEC11, LEC12, Lec13, and LEC30 were consistent with available genetic and biochemical data. However, the complexity of the range of N-glycans observed was unexpected. Several of the complex N-glycan profiles contained structures of m/z ∼13,000 representing complex N-glycans with a total of 26 N-acetyllactosamine (Galβ1–4GlcNAc)_n units. Importantly, the LEC11, LEC12, and LEC30 CHO mutants exhibited unique complements of fucosylated complex N-glycans terminating in Lewis^x and sialyl-Lewis^x determinants. This analysis reveals the larger-than-expected complexity of N-glycans in CHO cell mutants that may be used in a broad variety of functional glycomics studies and for making recombinant glycoproteins.     

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

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

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.     

Combined biochemical and cytological analysis of membrane trafficking using lectins

We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz–sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein β-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER–Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.     

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.     

A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans

Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVN^Lec1. We found that Sf-RVN and Sf-RVN^Lec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVN^Lec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVN^Lec1 cells were Endo H-cleavable Man₅GlcNAc₂ structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.     

Role for Calnexin and N-Linked Glycosylation in the Assembly and Secretion of Hepatitis B Virus Middle Envelope Protein Particles

Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly of HBV. Beyond being modified by N glycosylation, M is modified by O glycosylation occurring within its amino acid sequence at positions 27 to 47. The O glycans, however, were found to be dispensable for secretion of M but may rather support viral infectivity. Surprisingly, nonglycosylated M localizes exclusively to the cytosol, either for degradation or for a yet-unknown function.     

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 N‐glycan on Asn54 affects the atypical N‐glycan composition of plant‐produced interleukin‐22, but does not influence its activity

Human interleukin‐22 (IL‐22) is a member of the IL‐10 cytokine family that has recently been shown to have major therapeutic potential. IL‐22 is an unusual cytokine as it does not act directly on immune cells. Instead, IL‐22 controls the differentiation, proliferation and antimicrobial protein expression of epithelial cells, thereby maintaining epithelial barrier function. In this study, we transiently expressed human IL‐22 in Nicotiana benthamiana plants and investigated the role of N‐glycosylation on protein folding and biological activity. Expression levels of IL‐22 were up to 5.4 μg/mg TSP, and N‐glycan analysis revealed the presence of the atypical Lewis A structure. Surprisingly, upon engineering of human‐like N‐glycans on IL‐22 by co‐expressing mouse FUT8 in ΔXT/FT plants a strong reduction in Lewis A was observed. Also, core α1,6‐fucoylation did not improve the biological activity of IL‐22. The combination of site‐directed mutagenesis of Asn54 and in vivo deglycosylation with PNGase F also revealed that N‐glycosylation at this position is not required for proper protein folding. However, we do show that the presence of a N‐glycan on Asn54 contributes to the atypical N‐glycan composition of plant‐produced IL‐22 and influences the N‐glycan composition of N‐glycans on other positions. Altogether, our data demonstrate that plants offer an excellent tool to investigate the role of N‐glycosylation on folding and activity of recombinant glycoproteins, such as IL‐22.