Adaptive Regulation at the Cell Surface by N-Glycosylation

The association of receptors and solute transporters with components of the endocytic machinery regulates their surface levels, and thereby cellular sensitivity to cytokines, ligands and nutrients in the extracellular environment. Most transmembrane receptors and solute transporters are glycoproteins, and the Asn ( N )-linked oligosaccharides ( N -glycans) can bind animal lectins, forming multivalent lattices or microdomains that regulate glycoprotein mobility in the plane of membrane. The N -glycan number (sequence-encoded NXS/T) and context-dependent Golgi N -glycan branching cooperate to regulate glycoprotein affinities for the galectin family of lectins. Galectin-3 binding reduces EGF receptor trafficking into clathrin-coated pits and caveolae lipid rafts, decreases ligand-independent receptor activation and promotes α5β1 integrin remodelling in focal adhesions. N -glycan branching in the medial Golgi increases glycan affinity for galectins, and the Golgi pathway is sensitive to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) supply, in turn hexosamine pathway metabolites (fructose-6-P, glutamine and acetyl-CoA). Thus, lattice avidity and cellular responsiveness to extracellular cues are regulated in an adaptive manner by metabolism and Golgi modification to glycoproteins. Computational modelling of the hexosamine/Golgi/lattice has provided new insight on cell surface adaptation in cancer and autoimmune disease.     

Lectins and traffic in the secretory pathway

Evidence is accumulating that intracellular animal lectins play important roles in quality control and glycoprotein sorting along the secretory pathway. Calnexin and calreticulin in conjunction with associated chaperones promote correct folding and oligomerization of many glycoproteins in the endoplasmic reticulum (ER). The mannose lectin ERGIC-53 operates as a cargo receptor in transport of glycoproteins from ER to Golgi and the homologous lectin VIP36 may operate in quality control of glycosylation in the Golgi. Exit from the Golgi of lysosomal hydrolases to endosomes requires mannose 6-phosphate receptors and exit to the apical plasma membrane may also involve traffic lectins. Here we discuss the features of these lectins and their role in glycoprotein traffic in the secretory pathway.     

Glycosylation, galectins and cellular signaling

Glycosylation is a common posttranslational modification of proteins and lipids of the secretory pathway that generates binding sites for galactose-specific lectins or galectins. Branching of Asn-linked (N-)glycans by the N-acetylglucosaminyltransferases (Mgat genes) increases affinity for galectins. Both tissue-specific expression of the enzymes and the metabolic supply of sugar-nucleotides to the ER and Golgi regulate glycan distribution while protein sequences specify NXS/T site multiplicity, providing metabolic and genetic contributions to galectin-glycoprotein interactions. Galectins cross-link glycoproteins forming dynamic microdomains or lattices that regulate various mediators of cell adhesion, migration, proliferation, survival and differentiation. There are a similar number of galactose-specific galectins in C. elegans and humans, but expression of higher-affinity branched N-glycans are a more recent feature of vertebrate evolution. Galectins might be considered a reading code for repetition of the minimal units of binding [Gal(NAc)β1-3/4GlcNAc] and NXS/T site multiplicity in proteins. The rapidly evolving and structurally complex Golgi modifications to surface receptors are interpreted through affinity for the lattice, which regulates receptor levels as a function of the cellular environment, and thereby the probability of various cell fates. Many important questions remain concerning the regulation of the galectins, the glycan ligands and lattice interaction with other membrane domains and endocytic pathways.     

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.     

Binding and cross-linking properties of galectins

The galectins are a family of animal lectins that possess similar carbohydrate binding specificities and conserved consensus sequences. The biological properties of mammalian galectins include the regulation of inflammation, cell adhesion, cell proliferation and cell death. Evidence suggests that the biological activities of the galectins are related to their multivalent binding properties since most galectins possess two carbohydrate recognition domains and are therefore bivalent. For example, galectin-1, which is dimeric, binds and cross-links specific glycoprotein counter-receptors on the surface of human T-cells leading to apoptosis [J. Immunol. 163 (1999) 3801]. Different galectin-1 counter-receptors associated with specific phosphatase or kinase activities formed separate clusters on the surface of the cells as a result of the lectin binding to the carbohydrate chains of the respective glycoproteins. Importantly, monovalent galectin-1 is inactive in this system. This indicates that the separation and organization of signaling molecules that result from galectin-1 binding is involved in the apoptotic signal. The separation of specific glycoprotein receptors induced by galectin-1 binding was modeled on the basis of molecular and structural studies of the binding of lectins to multivalent carbohydrates resulting in the formation of specific two- and three-dimensional cross-linked lattices [Biochemistry 36 (1997) 15073]. In this article, the binding and cross-linking properties of galectin-1 and other lectins are reviewed as a model for the biological signal transduction properties of the galectin family of animal lectins.     

Clusters,bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions

Multivalent protein-carbohydrate interactions regulate essential cellular events, including cell proliferation, adhesion and death. These multivalent interactions can create homogeneous complexes of lectins, such as the galectins, with their saccharide ligands. Lectin-saccharide complexes can concentrate specific glycoproteins or glycolipids within the lattice, while excluding other cell surface molecules. The formation of lectin-saccharide lattices on the cell surface can thus organize the plasma membrane into specialized domains that perform unique functions.     

Galectins as modulators of receptor tyrosine kinases signaling in health and disease

Receptor tyrosine kinases (RTKs) constitute a large group of cell surface proteins that mediate communication of cells with extracellular environment. RTKs recognize external signals and transfer information to the cell interior, modulating key cellular activities, like metabolism, proliferation, motility, or death. To ensure balanced stream of signals the activity of RTKs is tightly regulated by numerous mechanisms, including receptor expression and degradation, ligand specificity and availability, engagement of co-receptors, cellular trafficking of the receptors or their post-translational modifications. One of the most widespread post-translational modifications of RTKs is glycosylation of their extracellular domains. The sugar chains attached to RTKs form a new layer of information, so called glyco-code that is read by galectins, carbohydrate binding proteins. Galectins are family of fifteen lectins implicated in immune response, inflammation, cell division, motility and death. The versatility of cellular activities attributed to galectins is a result of their high abundance and diversity of their cellular targets. A various sugar specificity of galectins and the differential ability of galectin family members to form oligomers affect the spatial distribution and the function of their cellular targets. Importantly, galectins and RTKs are tightly linked to the development, progression and metastasis of various cancers. A growing number of studies points on the close cooperation between RTKs and galectins in eliciting specific cellular responses. This review focuses on the identified complexes between galectins and RTK members and discusses their relevance for the cell physiology both in healthy tissues and in cancer.     

Secretion of the galectin family of mammalian carbohydrate-binding proteins

Galectins are cytosolic proteins that lack any signal sequence for transport into the endoplasmic reticulum and are not glycosylated, although several galectins contain consensus sites for N-glycosylation, indicating that these proteins do not traverse the ER-Golgi network. However, there is abundant evidence for the extracellular localisation of some galectins at cell surfaces, in the extracellular matrix and in cell secretions consistent with other evidence for extracellular roles of galectins as modulators of cell adhesion and signalling. How then are galectins secreted if not through the classical secretory pathway? Do all galectins share the same secretory pathway? Can a particular galectin utilise more than one secretory pathway? If galectins play important extracellular roles how is their secretion regulated in relation to function? These are still largely unanswered questions but recent studies are beginning to give glimpses into some novel aspects of the secretion of these intriguing proteins.     

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.     

A Combined Molecular Modelling and CIDNP Study of Similarities in the Pattern of Ligand Binding in Mammalian and Avian Galectins

Galectins (Galactose binding lectins) from bacteria, plants and animals have been shown to possess tyrosine or tryptophan residues that form hydrophobic contacts with their ligands in the binding sites. At the present time, the X-ray structures of only two galectins from human and bovine tissues are known. In the present study we applied X-ray data of bovine heart galectin-1 as a template for homology modelling of a number of galectins from mammalian and avian tissues. The conservation of one tryptophan and at least one histidine in binding pocket can be observed from the comparison of the model structures. We also show that it is possible to obtain information of the architecture of the binding pocket of several galectins in solution using CIDNP (Chemically Induced Dynamic Nuclear Polarisation) techniques. The CIDNP approach offers a possibility to analyse these lectins in solution thereby providing supplementary information to the available X-ray data. All studied galectins show comparable alterations when they are recorded by CIDNP-technique in the absence and in the presence of their specific carbohydrate ligands.     

The transmembrane domain of the respiratory syncytial virus F protein is an orientation-independent apical plasma membrane sorting sequence

The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.     

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.     

Correspondence of gradual developmental increases of expression of galectin-reactive glycoconjugates with alterations of the total contents of the two differentially regulated galectins in chicken intestine and liver as indication for overlapping functions.

The duplication of genes for recognition molecules and the ensuing diversification of the members of such families generate complex groups of homologous proteins. One example are galactoside-specific lectins whose sequences display constant features related to sugar binding, the galectins. Based on the inverse abundance of the chicken galectins CG-14 and CG-16 in adult intestine and liver, these two lectins represent a model to comparatively study expression of the related proteins and the galectin-reactive sites (glycoproteins and glycolipids) biochemically and histochemically. Functional overlap and/or acquisition of distinct functions would be reflected in qualitative and/or quantitative aspects of ligand display. Using five different stages of embryogenesis, differential regulation of the two galectins was detected in liver and intestine. The clear preference for one galectin (CG-14) was observed in intestine already at rather early stages, whereas equivalence for both proteins was noted in liver from day 12 to day 18 prior to hatching, as seen by ELISA assays and Western blot analysis. Presentation of galectin-reactive glycoproteins showed a tendency for gradual increase in both organs. Galectin-blotting analysis revealed primarily very similar patterns of positive bands at the different stages of development and only few quantitative and qualitative changes. The reactivity of glycolipids in a solid-phase assay was more variable, even surpassing the response of extracts of the adult organ at several embryonic stages. While the localization patterns of the galectins and galectin-reactive sites were nearly indistinguishable in the liver, intestinal tissue differed with respect to the placement and accessibility of binding sites. Thus, the results suggest a differential regulation of galectin activities in the two organs. As a sum they resemble the course of development of availability of glycoprotein ligands in vitro. These findings support the notion for a partial functional redundancy in this family. The described approach to employ galectin-specific antibodies and the labeled galectins as tools to assess presentation of ligands is suggested to be of general relevance to address the question of distinct vs. overlapping functions of related recognition molecules.     

Genetic evidence that the steroid-regulated trafficking of cell surface glycoproteins in rat hepatoma cells is mediated by glucocorticoid-inducible cellular components

The biological control of posttranslational maturation and compartmentalization reactions that operate upon proteins during transport to their final cellular destinations is crucial for normal cellular function. Using the expression of mouse mammary tumor virus (MMTV) glycoproteins as sensitive probes in the viral-infected rat hepatoma cell line M1.54, we have discovered and documented a novel glucocorticoid-regulated trafficking pathway that controls the cell surface localization of MMTV glycoproteins. One complement-selected derivative of M1.54 cells, CR4, failed to compartmentalize cell surface MMTV glycoproteins in the presence of dexamethasone. To test genetically if this glycoprotein trafficking pathway is mediated by cellular or viral gene products, CR4 cells were fused with uninfected Fu5 rat hepatoma cells. Indirect immunofluorescence of CR4 X Fu5 heterokaryons revealed that Fu5 complemented the defect in CR4 only after exposure to 1 microM dexamethasone. The glucocorticoid inhibition of Fu5 proliferation was exploited to recover the receptor-deficient uninfected derivative EDR3 that expressed a 100-fold lower level of [3H]dexamethasone binding activity. Analysis of CR4 X EDR3 cell fusions by indirect immunofluorescence revealed that EDR4 cells complemented CR4 in a dexamethasone-dependent manner, suggesting that EDR3 supplied a functinal trafficking component while CR4 provided a functional glucocorticoid receptor to the heterokaryons. Taken together, our results demonstrate that cellular-encoded glucocorticoid-inducible components mediate the regulated trafficking of cell surface MMTV glycoproteins.     

Caenorhabditis elegans galectins LEC-6 and LEC-10 interact with similar glycoconjugates in the intestine

Galectins are a family of metazoan proteins that show binding to various β-galactoside-containing glycans. Because of a lack of proper tools, the interaction of galectins with their specific glycan ligands in the cells and tissues are largely unknown. We have investigated the localization of galectin ligands in Caenorhabditis elegans using a novel technology that relies on the high binding specificity between galectins and their endogenous ligands. Fluorescently labeled recombinant galectin fusions are found to bind to ligands located in diverse tissues including the intestine, pharynx, and the rectal valve. Consistent with their role as galactoside-binding proteins, the interaction with their ligands is inhibited by galactose or lactose. Two of the galectins, LEC-6 and LEC-10, recognize ligands that co-localize along the intestinal lumen. The ligands for LEC-6 and LEC-10 are absent in three glycosylation mutants bre-1, fut-8, and galt-1, which have been shown to be required to synthesize the Gal-β1,4-Fuc modifications of the core N-glycans unique to C. elegans and several other invertebrates. Both galectins pull down the same set of glycoproteins in a manner dependent on the presence of these carbohydrate modifications. Endogenous LEC-6 and LEC-10 are expressed in the intestinal cells, but they are localized to different subcellular compartments that do not appear to overlap with each other or with the location of their glycan targets. An altered subcellular distribution of these ligands is found in mutants lacking both galectins. These results suggest a model where LEC-6 and LEC-10 interact with glycoproteins through specific glycans to regulate their cellular fate.     

The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags

The majority of proteins that traverse the secretory pathway receive asparagine (Asn)-linked glycosylations. Glycans are bulky hydrophilic modifications that serve a variety of structural and functional roles within the cell. Here, we review the recent growing knowledge on the role of Asn-linked glycans as maturation and quality-control protein tags in the early secretory pathway. The carbohydrate composition encodes crucial information about the structure, localization and age of glycoproteins. The "glycan code" is encoded by a series of glycosidases and carbohydrate transferases that line the secretory pathway. This code is deciphered by carbohydrate-binding proteins that possess distinct carbohydrate binding properties and act as molecular chaperones or sorting receptors. These glycosidases and transferases work in concert with resident secretory pathway carbohydrate-binding proteins to form a network that assists in the maturation and trafficking of both native and aberrant glycoproteins within the cell.     

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.     

Molecular basis of sugar recognition by the human L-type lectins ERGIC-53, VIPL, and VIP36

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.     

Axonal transport and neuronal transcytosis of trophic factors, tracers, and pathogens

Neurons can specifically internalize macromolecules, such as trophic factors, lectins, toxins, and other pathogens. Upon internalization in terminals, proteins can move retrogradely along axons, or, upon internalization at somatodendritic domains, they can move into an anterograde axonal transport pathway. Release of internalized proteins from neurons after either retrograde or anterograde axonal transport results in transcytosis and trafficking of proteins across multiple synapses. Recent studies of binding properties of several such proteins suggest that pathogens and lectins may utilize existing transport machineries designed for trafficking of trophic factors. Specific pathways may protect trophic factors, pathogens, and toxins from degradation after internalization and may target the trophic or pathogenic cargo for transcytosis after either retrograde or anterograde transport along axons. Elucidating the molecular mechanisms of sorting steps and transport pathways will further our understanding of trophic signaling and could be relevant for an understanding and possible treatment of neurological diseases such as rabies, Alzheimer's disease, and prion encephalopathies. At present, our knowledge is remarkably sparse about the types of receptors used by pathogens for trafficking, the signals that sort trophins or pathogens into recycling or degradation pathways, and the mechanisms that regulate their release from somatodendritic domains or axon terminals. This review intends to draw attention to potential convergences and parallels in trafficking of trophic and pathogenic proteins. It discusses axonal transport/trafficking mechanisms that may help to understand and eventually treat neurological diseases by targeted drug delivery.     

Localization of VIP36 in the post-Golgi secretory pathway also of rat parotid acinar cells.

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.