Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells

Background

N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway.

Scope of review

This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins.

Major conclusions

Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway.

General significance

Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.     

Intracellular lectins associated with N-linked glycoprotein traffic

The vectorial intracellular transport of N-glycan-linked glycoproteins is indispensable for biological functions. In order to sort these glycoproteins to the correct destination, animal intracellular lectins play important roles as sorting receptors. The roles of such lectins in the biosynthetic pathway from the endoplasmic reticulum (ER) to the cell surface are addressed in this review. Calnexin and calreticulin function via specific carbohydrates in quality control of newly synthesized glycoproteins in the ER, and ERGIC-53 seems to function in the transport of glycoproteins from ER to the Golgi complex. In addition to the well-understood role of mannose 6-phosphate receptor in lysosomal protein sorting, the vesicular integral protein of 36 kDa (VIP36) functions as a sorting receptor by recognizing high-mannose type glycans containing alpha1-->2Man residues for transport from Golgi to the cell surface in polarized epithelial cells.     

pH-induced conversion of the transport lectin ERGIC-53 triggers glycoprotein release

The recycling mannose lectin ERGIC-53 operates as a transport receptor by mediating efficient endoplasmic reticulum (ER) export of some secretory glycoproteins. Binding of cargo to ERGIC-53 in the ER requires Ca2+. Cargo release occurs in the ERGIC, but the molecular mechanism is unknown. Here we report efficient binding of purified ERGIC-53 to immobilized mannose at pH 7.4, the pH of the ER, but not at slightly lower pH. pH sensitivity of the lectin was more prominent when Ca2+ concentrations were low. A conserved histidine in the center of the carbohydrate recognition domain was required for lectin activity suggesting it may serve as a molecular pH/Ca2+ sensor. Acidification of cells inhibited the association of ERGIC-53 with the known cargo cathepsin Z-related protein and dissociation of this glycoprotein in the ERGIC was impaired by organelle neutralization that did not impair the transport of a control protein. The results elucidate the molecular mechanism underlying reversible lectin/cargo interaction and establish the ERGIC as the earliest low pH site of the secretory pathway.     

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.     

Role of the Lectin VIP36 in Post‐ER Quality Control of Human α1‐Antitrypsin

The leguminous‐type (L‐type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high‐mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar‐binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein α1‐antitrypsin (α1‐AT) as a cargo of VIP36. The VIP36/α1‐AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high‐mannose form of α1‐AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of α1‐AT were inactivated by mutagenesis. Silencing VIP36 accelerated α1‐AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and α1‐AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post‐ER quality control of α1‐AT.     

Clusters of VIP-36-positive vesicles between endoplasmic reticulum and Golgi apparatus in GH3 cells

The vesicular integral membrane protein VIP36 belongs to the family of animal lectins and may act as a cargo receptor trafficking certain glycoproteins in the secretory pathway. Immunoelectron microscopy of GH3 cells provided evidence that endogenous VIP36 is localized mainly in 70-100-nm-diameter uncoated transport vesicles between the exit site on the ER and the neighboring cis-Golgi cisterna. The thyrotrophin-releasing hormone (TRH) stimulation and treatment with actin filament-perturbing agents, cytochalasin D or B or latrunculin-B, caused marked aggregation of the VIP36-positive vesicles and the appearance of a VIP36-positive clustering structure located near the cis-Golgi cisterna. The size of this structure, which comprised conspicuous clusters of VIP36, depended on the TRH concentration. Confocal laser scanning microscopy confirmed the electron microscopically demonstrated distribution and redistribution of VIP36 in these cells. Furthermore, VIP36 colocalized with filamentous actin in the paranuclear Golgi area and its vicinity. This is the first study to show the ultrastructural distribution of VIP36 in the early secretory pathway in GH3 cells. It suggests that actin filaments are involved in glycoprotein transport between the ER and cis-Golgi cisterna by using the lectin VIP36.     

Lectin control of protein folding and sorting in the secretory pathway

Glycan moieties are essential for folding, sorting and targeting of glycoproteins through the secretory pathway to various cellular compartments. The molecular mechanisms that underlie these processes, however, are only now coming to light. Recent crystallographic and NMR studies of proteins located in the endoplasmic reticulum (ER), Golgi complex and ER-Golgi intermediate compartment have illuminated their roles in glycoprotein folding and secretion. Calnexin and calreticulin, both ER-resident proteins, have lectin domains that are crucial for their function as chaperones. The crystal structure of the carbohydrate-recognition domain of ER-Golgi intermediate compartment (ERGIC)-53 complements the biochemical and functional characterization of the protein, confirming that a lectin domain is essential for the role of this protein in sorting and transfer of glycoproteins from the ER to the Golgi complex. The lectin domains of calnexin and ERGIC-53 are structurally similar, although there is little primary sequence similarity. By contrast, sequence similarity between ERGIC-53 and vesicular integral membrane protein (VIP36), a Golgi-resident protein, leaves little doubt that a similar lectin domain is central to the transport and/or sorting functions of VIP36. The theme emerging from these studies is that carbohydrate recognition and modification are central to mediation of glycoprotein folding and secretion.     

Demonstration that endoplasmic reticulum-associated degradation of glycoproteins can occur downstream of processing by endomannosidase

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.     

Protein quality control in the ER: The recognition of misfolded proteins

The mechanism, in molecular terms of protein quality control, specifically of how the cell recognizes and discriminates misfolded proteins, remains a challenge. In the secretory pathway the folding status of glycoproteins passing through the endoplasmic reticulum is marked by the composition of the N-glycan. The different glycoforms are recognized by specialized lectins. The folding sensor UGGT acts as an unusual molecular chaperone and covalently modifies the Man9 N-glycan of a misfolded protein by adding a glucose moiety and converts it to Glc1Man9 that rebinds the lectin calnexin. However, further links between the folding status of a glycoprotein and the composition of the N-glycan are unclear. There is little unequivocal evidence for other proteins in the ER recognizing the N-glycan and also acting as molecular chaperones. Nevertheless, based upon a few examples, we suggest that this function is carried out by individual proteins in several different complexes. Thus, calnexin binds the protein disulfide isomerase ERp57, that acts upon Glc1Man9 glycoproteins. In another example the protein disulfide isomerase ERdj5 binds specifically to EDEM (which is probably a mannosidase) and a lectin OS9, and reduces the disulfide bonds of bound glycoproteins destined for ERAD. Thus the glycan recognition is performed by a lectin and the chaperone function performed by a specific partner protein that can recognize misfolded proteins. We predict that this will be a common arrangement of proteins in the ER and that members of protein foldase families such as PDI and PPI will bind specifically to lectins in the ER. Molecular chaperones BiP and GRp94 will assist in the folding of proteins bound in these complexes as well as in the folding of non-glycoproteins.     

Reglucosylation of glycoproteins and quality control of glycoprotein folding in the endoplasmic reticulum of yeast cells

Proteins entering the secretory pathway may be glycosylated upon transfer of an oligosaccharide (Glc₃Man₉GlcNAc₂) from a dolichol-P-P derivative to nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). Oligosaccharides are then deglucosylated by glucosidases I and II (GII). Also in the ER, glycoproteins acquire their final tertiary structures, and species that fail to fold properly are retained and eventually degraded in the proteasome. It has been proposed that in mammalian cells the monoglucosylated oligosaccharides generated either by partial deglucosylation of the transferred compound or by reglucosylation of glucose-free oligosaccharides by the UDP-Glc:glycoprotein glucosyltransferase (GT) are recognized by ER resident lectins (calnexin and/or calreticulin). GT is a sensor of glycoprotein conformation as it only glucosylates misfolded species. The lectin-monoglucosylated oligosaccharide interaction would retain glycoproteins in the ER until correctly folded, and also facilitate their acquisition of proper tertiary structures by preventing aggregation. GII would liberate glycoproteins from the calnexin/calreticulin anchor, but species not properly folded would be reglucosylated by GT, and so continue to be retained by the lectins. Only when the protein becomes properly folded would it cease to be retained by the lectins. This review presents evidence suggesting that a similar quality control mechanism of glycoprotein folding is operative in Schizosaccharomyces pombe and that the mechanism in Saccharomyces cerevisiae probably differs substantially from that occurring in mammalian and Sch. pombe cells.     

Relationship between Golgi architecture and glycoprotein biosynthesis and transport in Chinese hamster ovary cells

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.     

Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans.     

Crystal structure of the carbohydrate recognition domain of p58/ERGIC-53, a protein involved in glycoprotein export from the endoplasmic reticulum

p58/ERGIC-53 is an animal calcium-dependent lectin that cycles between the endoplasmic reticulum (ER) and the Golgi complex and appears to act as a cargo receptor for a subset of soluble glycoproteins exported from the ER. We have determined the crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, to 1.46 A resolution. The fold and ligand binding site are most similar to those of leguminous lectins. The structure also resembles that of the CRD of the ER folding chaperone calnexin and the neurexins, a family of non-lectin proteins expressed on neurons. The CRD comprises one concave and one convex beta-sheet packed into a beta-sandwich. The ligand binding site resides in a negatively charged cleft formed by conserved residues. A large surface patch of conserved residues with a putative role in protein-protein interactions and oligomerization lies on the opposite side of the ligand binding site. Together with previous functional data, the structure defines a new and expanding class of calcium-dependent animal lectins and provides a starting point for the understanding of glycoprotein sorting between the ER and the Golgi.     

植物表达分泌蛋白的运输及定位

分泌途径主要由内膜系统构成,内质网和高尔基体对于分泌蛋白的运输及定位具有重要作用。分泌蛋白的运输包括顺行途径和逆行途径。蛋白质通过质流和受体介导的途径运输到小泡中。在植物中,分泌蛋白的运输主要通过小泡和相连的小管来完成。分子伴侣和质量控制不仅能优化新合成蛋白的折叠和组装,而且去除了有折叠缺陷的蛋白。分泌蛋白的定位需要特定的信号肽,而高尔基体固有蛋白以依赖跨膜长度的方式,沿着分泌途径的细胞器分布。本文对植物表达分泌蛋白的分泌途径及定位、相关的分子伴侣和质量控制进行了综述。     

Dissecting glycoprotein quality control in the secretory pathway

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.     

Specific glycan elements determine differential binding of individual egg glycoproteins of the human parasite Schistosoma mansoni by host C-type lectin receptors

During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite’s eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the immune system. However, it is not yet known which individual glycoproteins interact with the different C-type lectin receptors, and which structural components are involved. Here we investigated the structural basis of the binding of two abundant egg antigens, kappa-5 and IPSE/α1, by the C-type lectin receptor dendritic cell-specific ICAM3-grabbing non-integrin, macrophage galactose-type lectin and mannose receptor. In the natural soluble form, the secretory egg glycoprotein IPSE/α1 interacts with dendritic cells mainly via mannose receptors. Surprisingly, in plate-based assays mannose receptors preferentially bound to mannose conjugates, while in cell-based assays, IPSE/α1 is bound via the fucosylated Galβ1-4(Fucα1-3)GlcNAc (LeX) motif on diantennary N-glycans. Kappa-5, in contrast, is bound by dendritic cells via all three C-type lectin receptors studied and for a minor part also via other, non-C-type lectin receptors. Kappa-5 interacts with macrophage galactose-type lectins via the GalNAcβ1-4GlcNAc antenna present on its triantennary N-glycans, as well as the GalNAcβ1-4(Fucα1-3)GlcNAc antennae present on a minor N-glycan subset. Dendritic cell-specific ICAM3-grabbing non-integrin binding of kappa-5 was mediated via the GalNAcβ1-4(Fucα1-3)GlcNAc antennae, whereas binding of mannose receptors may involve either GalNAcβ1-4(Fucα1-3)GlcNAc antennae or the fucosylated and xylosylated chitobiose core. This study provides a molecular and structural basis for future studies of the interaction between C-type lectin receptors and other soluble egg antigen glycoproteins and their effects on the host immune response.     

Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.     

Sar1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments

Two new members (Sar1a and Sar1b) of the SAR1 gene family have been identified in mammalian cells. Using immunoelectron microscopy, Sar1 was found to be restricted to the transitional region where the protein was enriched 20-40-fold in vesicular carriers mediating ER to Golgi traffic. Biochemical analysis revealed that Sar1 was essential for an early step in vesicle budding. A Sar1-specific antibody potently inhibited export of vesicular stomatitis virus glycoprotein (VSV-G) from the ER in vitro. Consistent with the role of guanine nucleotide exchange in Sar1 function, a trans-dominant mutant (Sar1a[T39N]) with a preferential affinity for GDP also strongly inhibited vesicle budding from the ER. In contrast, Sar1 was not found to be required for the transport of VSV-G between sequential Golgi compartments, suggesting that components active in formation of vesicular carriers mediating ER to Golgi traffic may differ, at least in part, from those involved in intra-Golgi transport. The requirement for novel components at different stages of the secretory pathway may reflect the recently recognized differences in protein transport between the Golgi stacks as opposed to the selective sorting and concentration of protein during export from the ER.     

Absence of nucleoside diphosphatase activities in the yeast secretory pathway does not abolish nucleotide sugar-dependent protein glycosylation

It is accepted that glycosyltransferase-generated nucleoside diphosphates are converted to monophosphates in the secretory pathway by nucleoside diphosphatases (NDPases) to provide substrates for antiport transport systems by which entrance of nucleotide sugars from the cytosol into the lumen is coupled to exit of nucleoside monophosphates. Working with Saccharomyces cerevisiae mutants affected in anterograde and/or retrograde endoplasmic reticulum (ER)-Golgi vesicular traffic and/or defective in one or both secretory pathway (Golgi) NDPases, we show that UDP-Glc: glycoprotein glucosyltransferase-mediated glucosylation is not dependent on the presence of NDPases or on ER-Golgi vesicular traffic and that GDP-Man-dependent N- and O-mannosylations are reduced but not abolished in the absence of NDPases in the secretory pathway. Further, the absence of the main Man-1-P transferase (a Golgi GMP-generating enzyme) does not modify the limited mannosylation observed in the absence of NDPases. Based on these results and on available additional information, we suggest that in the absence of NDPases, the already characterized nucleotide sugar transporters allow entrance of nucleotide sugars into the luminal compartments and that resulting nucleoside diphosphates exit to the cytosol by a still unknown mechanism. Further, an unexpected side result suggests that formation of Ser/Thr-Man(2) may occur in the ER and not exclusively in the Golgi.