Intracellular Lectin Research Articles

The diverse dependence of galectin-1 and -8 on multivalency for the modulation of FGFR1 endocytosis

Fibroblast growth factor receptor 1 (FGFR1) is a N-glycosylated cell surface receptor tyrosine kinase, which upon recognition of specific extracellular ligands, fibroblast growth factors (FGFs), initiates an intracellular signaling. FGFR1 signaling ensures homeostasis of cells by fine-tuning essential cellular processes, like differentiation, division, motility and death. FGFR1 activity is coordinated at multiple steps and unbalanced FGFR1 signaling contributes to developmental diseases and cancers. One of the crucial control mechanisms over FGFR1 signaling is receptor endocytosis, which allows for rapid targeting of FGF-activated FGFR1 to lysosomes for degradation and the signal termination. We have recently demonstrated that N-glycans of FGFR1 are recognized by a precise set of extracellular galectins, secreted and intracellular multivalent lectins implicated in a plethora of cellular processes and altered in immune responses and cancers. Specific galectins trigger FGFR1 clustering, resulting in activation of the receptor and in initiation of intracellular signaling cascades that shape the cell physiology. Although some of galectin family members emerged recently as key players in the clathrin-independent endocytosis of specific cargoes, their impact on endocytosis of FGFR1 was largely unknown.Here we assessed the contribution of extracellular galectins to the cellular uptake of FGFR1. We demonstrate that only galectin-1 induces internalization of FGFR1, whereas the majority of galectins predominantly inhibit endocytosis of the receptor. We focused on three representative galectins: galectin-1, -7 and -8 and we demonstrate that although all these galectins directly activate FGFR1 by the receptor crosslinking mechanism, they exert different effects on FGFR1 endocytosis. Galectin-1-mediated internalization of FGFR1 doesn’t require galectin-1 multivalency and occurs via clathrin-mediated endocytosis, resembling in this way the uptake of FGF/FGFR1 complex. In contrast galectin-7 and -8 impede FGFR1 endocytosis, causing stabilization of the receptor on the cell surface and prolonged propagation of the signals. Furthermore, using protein engineering approaches we demonstrate that it is possible to modulate or even fully reverse the endocytic potential of galectins.

Galectin-9 in Gastroenterological Cancer

Immunochemotherapy has become popular in recent years. The detailed mechanisms of cancer immunity are being elucidated, and new developments are expected in the future. Apoptosis allows tissues to maintain their form, quantity, and function by eliminating excess or abnormal cells. When apoptosis is inhibited, the balance between cell division and death is disrupted and tissue homeostasis is impaired. This leads to dysfunction and the accumulation of genetically abnormal cells, which can contribute to carcinogenesis. Lectins are neither enzymes nor antibodies but proteins that bind sugar chains. Among soluble endogenous lectins, galectins interact with cell surface sugar chains outside the cell to regulate signal transduction and cell growth. On the other hand, intracellular lectins are present at the plasma membrane and regulate signal transduction by regulating receptor-ligand interactions. Galectin-9 expressed on the surface of thymocytes induces apoptosis of T lymphocytes and plays an essential role in immune self-tolerance by negative selection in the thymus. Furthermore, the administration of extracellular galectin-9 induces apoptosis of human cancer and immunodeficient cells. However, the detailed pharmacokinetics of galectin-9 in vivo have not been elucidated. In addition, the cell surface receptors involved in galectin-9-induced apoptosis of cancer cells have not been identified, and the intracellular pathways involved in apoptosis have not been fully investigated. We have previously reported that galectin-9 induces apoptosis in various gastrointestinal cancers and suppresses tumor growth. However, the mechanism of galectin-9 and apoptosis induction in gastrointestinal cancers and the detailed mechanisms involved in tumor growth inhibition remain unknown. In this article, we review the effects of galectin-9 on gastrointestinal cancers and its mechanisms.

Cargo selection in the early secretory pathway of African trypanosomes

Membrane and secretory proteins are synthesized by ribosomes and then enter the endoplasmic reticulum (ER) where they undergo glycosylation and quality control for proper folding. Subsequently, proteins are transported to the Golgi apparatus and then sorted to the plasma membrane or intracellular organelles. Transport vesicles are formed at ER-exit sites (ERES) on the ER with several coat protein complexes. Cargo proteins loaded into the vesicles are selected by specific interactions with cargo receptors and/or adaptors during vesicle formation. p24 family and intracellular lectin ERGIC-53-membrane proteins are the known cargo receptors acting in the early secretory pathway (ER-Golgi). Oligomerization of the cargo receptors have been suggested to play an important role in cargo selection and sorting via posttranslational modifications in fungi and metazoans. On the other hand, the mechanisms involved in the early secretory pathway in protozoa remain unclear. In this review, we focus on Trypanosoma brucei as a representative of protozoan and discuss differences and commonalities in the molecular mechanisms of its early secretory pathway compared with other organisms.

Crystallographic snapshots of the EF-hand protein MCFD2 complexed with the intracellular lectin ERGIC-53 involved in glycoprotein transport.

The transmembrane intracellular lectin ER-Golgi intermediate compartment protein 53 (ERGIC-53) and the soluble EF-hand multiple coagulation factor deficiency protein 2 (MCFD2) form a complex that functions as a cargo receptor, trafficking various glycoproteins between the endoplasmic reticulum (ER) and the Golgi apparatus. It has been demonstrated that the carbohydrate-recognition domain (CRD) of ERGIC-53 (ERGIC-53CRD) interacts with N-linked glycans on cargo glycoproteins, whereas MCFD2 recognizes polypeptide segments of cargo glycoproteins. Crystal structures of ERGIC-53CRD complexed with MCFD2 and mannosyl oligosaccharides have revealed protein-protein and protein-sugar binding modes. In contrast, the polypeptide-recognition mechanism of MCFD2 remains largely unknown. Here, a 1.60 Å resolution crystal structure of the ERGIC-53CRD-MCFD2 complex is reported, along with three other crystal forms. Comparison of these structures with those previously reported reveal that MCFD2, but not ERGIC-53-CRD, exhibits significant conformational plasticity that may be relevant to its accommodation of various polypeptide ligands.

One-Step Enrichment of Intact Glycopeptides From Glycoengineered Chinese Hamster Ovary Cells.

Recently, the glycoproteomic analysis of intact glycopeptides has emerged as an effective approach to decipher the glycan modifications of glycoproteins at the site-specific level. A rapid method to enrich intact glycopeptides is essential for the analysis of glycoproteins, especially for biopharmaceutical proteins. In this study, we established a one-step method for the rapid capture of intact glycopeptides for analysis by mass spectrometry. Compared to the conventional sequential enrichment method, the one-step intact glycopeptide enrichment method reduced the sample preparation time and improved the detection of intact glycopeptides with long sequences or non-polar amino acids. Moreover, an increased number of glycosite-containing peptides was identified by the one-step method compared with the sequential method. When we applied this method to the glycoproteomic analysis of glycoengineered Chinese hamster ovary (CHO)-K1 cells with α1,6-fucosyltransferase (FUT8) knockout, the results showed that the knockout of FUT8 altered the overall glycosylation profile of CHO-K1 cells with the elimination of core fucosylation and together with increases in high-mannose and sialylated N-glycans. Interestingly, the knockout of the FUT8 also appeared to regulate the expression of glycoproteins involved in several functions and pathways in CHO-K1 cells, such as the down-regulation of an intracellular lectin LMAN2 showing cellular adaptation to the alterations in FUT8 knockout cells. These findings indicate that the site-specific characterization of glycoproteins from glycoengineered CHO-K1 cells can be achieved rapidly using the one-step intact glycopeptide enrichment method, which could provide insights for bio-analysts and biotechnologists to better tailor therapeutic drugs.

Purification and characterisation of a xylose-specific mitogenic lectin from Fusarium sambucinum

A xylose-specific intracellular lectin, showing hemagglutination only with rabbit erythrocytes was purified from mycelium of Fusarium sambucinum which was designated as FSL. An array of anion exchange chromatography on Q-Sepharose and gel-exclusion chromatography on Sephadex G-100 resulted in 84.21% yield and 53.99-fold purification of lectin with specific activity of 169.53 titre/mg. Molecular weight of FSL determined by SDS-PAGE was 70.7 kDa, which was further confirmed by gel-exclusion chromatography. Native-PAGE analysis of FSL showed its monomeric nature. FSL was observed to be a glycoprotein containing 2.9% carbohydrate. Hapten inhibition profile of FSL displayed its strong affinity towards D-xylose (MIC 1.562 mM), L-fucose (MIC 6.25 mM), D-mannose (MIC 3.125 mM), fetuin (MIC 15.62 μg/mL), asialofetuin (MIC 125 μg/mL) and BSM (MIC 3.125 μg/mL). Affinity of FSL towards xylose is rare. FSL was found stable over a pH range 6.0–7.5 and upto 40 °C temperature. Hemagglutination activity of FSL remained unaffected by divalent ions. Lectin concentration of 5 μg/mL was found sufficient to stimulate proliferation of murine spleen cells and its concentration 75 μg/mL exhibited highest mitogenic potential. FSL exhibited maximum mitogenic stimulatory index of 14.35. The purification, characterisation and mitogenicity of F. sambucinum lectin has been reported first time.

Recombinant Expression and Purification of Animal Intracellular L-Type Lectins.

Animal leguminous-type (L-type) lectins, including ERGIC-53 and VIP36 are responsible for intracellular transport and quality control of N-linked glycoproteins in the early secretory pathway. These lectins possess the carbohydrate recognition domain (CRD), which recognizes high-mannose-type glycans in a Ca2+-dependent manner. Here we describe the procedures involved in bacterial overproduction and purification of the CRDs of the animal L-type lectins.

Signaling through plant lectins: modulation of plant immunity and beyond.

Lectins constitute an abundant group of proteins that are present throughout the plant kingdom. Only recently, genome-wide screenings have unraveled the multitude of different lectin sequences within one plant species. It appears that plants employ a plurality of lectins, though relatively few lectins have already been studied and functionally characterized. Therefore, it is very likely that the full potential of lectin genes in plants is underrated. This review summarizes the knowledge of plasma membrane-bound lectins in different biological processes (such as recognition of pathogen-derived molecules and symbiosis) and illustrates the significance of soluble intracellular lectins and how they can contribute to plant signaling. Altogether, the family of plant lectins is highly complex with an enormous diversity in biochemical properties and activities.

Structural Aspects of ER Glycoprotein Quality-Control System Mediated by Glucose Tagging.

N-linked oligosaccharides attached to proteins act as tags for glycoprotein quality control, ensuring their appropriate folding and trafficking in cells. Interactions with a variety of intracellular lectins determine glycoprotein fates. Monoglucosylated glycoforms are the hallmarks of incompletely folded glycoproteins in the protein quality-control system, in which glucosidase II and UDP-glucose/glycoprotein glucosyltransferase are, respectively, responsible for glucose trimming and attachment. In this review, we summarize a recently emerging view of the structural basis of the functional mechanisms of these key enzymes as well as substrate N-linked oligosaccharides exhibiting flexible structures, as revealed by applying a series of biophysical techniques including small-angle X-ray scattering, X-ray crystallography, high-speed atomic force microscopy , electron microscopy , and computational simulation in conjunction with NMR spectroscopy.

Comparative study of two single CRD C-type lectins, CgCLec-4 and CgCLec-5, from pacific oyster Crassostrea gigas

C-type lectins (CTLs), a superfamily of Ca2+-dependent carbohydrate-recognition proteins, are involved in nonself-recognition and pathogen elimination, and play crucial roles in the innate immunity. In the present study, two single CRD C-type lectins, CgCLec-4 and CgCLec-5, were identified from oyster Crassostrea gigas. The open reading frame (ORF) of CgCLec-4 and CgCLec-5 encoded polypeptides of 152 and 150 amino acids, respectively. Both CgCLec-4 and CgCLec-5 contained one CRD with six conserved cysteines to form three disulfide bridges. The motif in Ca2+-binding site 2 of CgCLec-4 was QPE, while it was QYE, a non-a typical motif in CgCLec-5. CgCLec-4 was a secreted lectin with a signal peptide which was highly expressed in hepatopancreas, mantle and hemocytes. CgCLec-5 was an intracellular lectin which was mostly expressed in hemocytes. The lipopolysaccharide stimulation could induce the expressions of CgCLec-4 and CgCLec-5. The recombinant proteins of CgCLec-4 and CgCLec-5 (rCgCLec-4 and rCgCLec-5) could bind to various PAMPs including LPS, PGN, GLU and mannan, while the binding affinity of rCgCLec-5 was stronger than that of rCgCLec-4. Meanwhile, rCgCLec-4 and rCgCLec-5 could bind to different kinds of microorganisms, including Staphylococcus aureus, Escherichia coli and Vibro anguillarum and Yarrowia lipolytica, and the microbial agglutinating ability of rCgCLec-4 was stronger than that of CgCLec-5. Moreover, rCgCLec-4 exhibited anti-microbial activity against bacteria and fungi, but anti-microbial activity of CgCLec-5 was not obvious. All these results suggested that CgCLec-4 and CgCLec-5 could function as an important PRR involved in immune defense against invading pathogen in oyster, and the diversity and complexity of motifs in Ca2+ binding site 2 in CRDs determined their comprehensive recognition spectrum and multiple immune functions.

Structural basis for two-step glucose trimming by glucosidase II involved in ER glycoprotein quality control.

The endoplasmic reticulum (ER) has a sophisticated protein quality control system for the efficient folding of newly synthesized proteins. In this system, a variety of N-linked oligosaccharides displayed on proteins serve as signals recognized by series of intracellular lectins. Glucosidase II catalyzes two-step hydrolysis at α1,3-linked glucose–glucose and glucose–mannose residues of high-mannose-type glycans to generate a quality control protein tag that is transiently expressed on glycoproteins and recognized by ER chaperones. Here we determined the crystal structures of the catalytic α subunit of glucosidase II (GIIα) complexed with two different glucosyl ligands containing the scissile bonds of first- and second-step reactions. Our structural data revealed that the nonreducing terminal disaccharide moieties of the two kinds of substrates can be accommodated in a gourd-shaped bilocular pocket, thereby providing a structural basis for substrate-binding specificity in the two-step deglucosylation catalyzed by this enzyme.

Emerging Structural Insights into Glycoprotein Quality Control Coupled with N-Glycan Processing in the Endoplasmic Reticulum

In the endoplasmic reticulum (ER), the sugar chain is initially introduced onto newly synthesized proteins as a triantennary tetradecasaccharide (Glc3Man9GlcNAc2). The attached oligosaccharide chain is subjected to stepwise trimming by the actions of specific glucosidases and mannosidases. In these processes, the transiently expressed N-glycans, as processing intermediates, function as signals for the determination of glycoprotein fates, i.e., folding, transport, or degradation through interactions of a series of intracellular lectins. The monoglucosylated glycoforms are hallmarks of incompletely folded states of glycoproteins in this system, whereas the outer mannose trimming leads to ER-associated glycoprotein degradation. This review outlines the recently emerging evidence regarding the molecular and structural basis of this glycoprotein quality control system, which is regulated through dynamic interplay among intracellular lectins, glycosidases, and glycosyltransferase. Structural snapshots of carbohydrate-lectin interactions have been provided at the atomic level using X-ray crystallographic analyses. Conformational ensembles of uncomplexed triantennary high-mannose-type oligosaccharides have been characterized in a quantitative manner using molecular dynamics simulation in conjunction with nuclear magnetic resonance spectroscopy. These complementary views provide new insights into glycoprotein recognition in quality control coupled with N-glycan processing.

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons

Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.

Intracellular lectins are involved in quality control of glycoproteins

Glycoprotein quality control is categorized into three kinds of reactions; the folding of nascent glycoproteins, ER-associated degradation of misfolded or unassembled glycoproteins, and transport and sorting of correctly folded glycoproteins. In all three processes, N-glycans on the glycoproteins are used as tags that are recognized by intracellular lectins. We analyzed the functions of these intracellular lectins and their sugar-binding specificities. The results clearly showed that the A, B, and C-arms of high mannose-type glycans participate in the folding, transport and sorting, and degradation, respectively, of newly synthesized peptides. After correctly folded glycoproteins are transported to the Golgi apparatus, N-glycans are trimmed into Man3GlcNAc2 and then rebuilt into various complex-type glycans in the Golgi, resulting in the addition of diverse sugar structures that allow glycoproteins to play various roles outside of the cells.

Parallel quantification of lectin–glycan interaction using ultrafiltration

Using ultrafiltration membrane, a simple method for screening protein–ligand interaction was developed. The procedure comprises three steps: mixing ligand with protein, ultrafiltration of the solution, and quantification of unbound ligands by HPLC. By conducting analysis with variable protein concentrations, affinity constants were easily obtained. Multiple ligands can be analyzed simultaneously as a mixture, when concentration of ligands was controlled. Feasibility of this method for lectin–glycan interaction analysis was examined using fluorescently labeled high-mannose-type glycans and recombinant intracellular lectins or endo-α-mannosidase mutants. Estimated Ka values of malectin and VIP36 were in good agreement indeed with those evaluated by conventional methods such as isothermal titration calorimetry (ITC) or frontal affinity chromatography (FAC). Finally, several mutants of endo-α-mannosidase were produced and their affinities to monoglucosylated glycans were evaluated.

Application of Metabolic 13C Labeling in Conjunction with High-Field Nuclear Magnetic Resonance Spectroscopy for Comparative Conformational Analysis of High Mannose-Type Oligosaccharides.

High mannose-type oligosaccharides are enzymatically trimmed in the endoplasmic reticulum, resulting in various processing intermediates with exposed glycotopes that are recognized by a series of lectins involved in glycoprotein fate determination in cells. Although recent crystallographic data have provided the structural basis for the carbohydrate recognition of intracellular lectins, atomic information of dynamic oligosaccharide conformations is essential for a quantitative understanding of the energetics of carbohydrate–lectin interactions. Carbohydrate NMR spectroscopy is useful for characterizing such conformational dynamics, but often hampered by poor spectral resolution and lack of recombinant techniques required to produce homogeneous glycoforms. To overcome these difficulties, we have recently developed a methodology for the preparation of a homogeneous high mannose-type oligosaccharide with 13C labeling using a genetically engineered yeast strain. We herein successfully extended this method to result in the overexpression of 13C-labeled Man9GlcNAc2 (M9) with a newly engineered yeast strain with the deletion of four genes involved in N-glycan processing. This enabled high-field NMR analyses of 13C-labeled M9 in comparison with its processing product lacking the terminal mannose residue ManD2. Long-range NOE data indicated that the outer branches interact with the core in both glycoforms, and such foldback conformations are enhanced upon the removal of ManD2. The observed conformational variabilities might be significantly associated with lectins and glycan-trimming enzymes.

Role of malectin in Glc2Man9GlcNAc2-dependent quality control of α1-antitrypsin

Malectin was first discovered as a novel endoplasmic reticulum (ER)–resident lectin from Xenopus laevis that exhibits structural similarity to bacterial glycosylhydrolases. Like other intracellular lectins involved in glycoprotein quality control, malectin is highly conserved in animals. Here results from in vitro membrane-based binding assays and frontal affinity chromatography confirm that human malectin binds specifically to Glc2Man9GlcNAc2(G2M9) N-glycan, with a Kaof 1.97 × 105M−1, whereas binding to Glc1Man9GlcNAc2(G1M9), Glc3Man9GlcNAc2(G3M9), and other N-glycans is barely detectable. Metabolic labeling and immunoprecipitation experiments demonstrate that before entering the calnexin cycle, the folding-defective human α1-antitrypsin variant null Hong Kong (ATNHK) stably associates with malectin, whereas wild-type α1-antitrypsin (AT) or N-glycan–truncated variant of ATNHK(ATNHK-Q3) dose not. Moreover, malectin overexpression dramatically inhibits the secretion of ATNHKthrough a mechanism that involves enhanced ER-associated protein degradation; by comparison, the secretion of AT and ATNHK-Q3 is only slightly affected by malectin overexpression. ER-stress induced by tunicamycin results in significantly elevated mRNA transcription of malectin. These observations suggest a possible role of malectin in regulating newly synthesized glycoproteins via G2M9 recognition.

Abstract 1495: Tyrosine phosphorylation of galectin-3 regulated its cleavage by PSA

Abstract Galectin-3, one of 15 animal lectins, is an intracellular and extracellular lectin which interacts with intracellular glycoproteins, cell surface molecules and extracellular matrix proteins. Recent research revealed that galectin-3 (gal-3) is involved in various biological phenomena including cell growth, adhesion, angiogenesis, apoptosis, motility and metastasis. This protein is widely expressed in various tumor cells and its expression is correlated with tumorigenesis, tumor progression and metastasis. The functions of gal-3 are dependent on its binding partners, its localization and can change as a result of post-translational modifications such as cleavage and phosphorylation. Recently we demonstrated that gal-3 can be phosphorylated by c-Abl at Tyr 79, 107 and 118; Tyr107 is the major phosphorylation site. In a recent report Saraswati et al revealed that gal-3 can be cleaved by prostate specific antigen after Tyr107. Our research confirms their data and demonstrates that phosphorylation of Tyr107 blocks this cleavage. This cleavage removes the collagen like sequence from carbohydrate binding domain of gal-3 and reduces the ability of gal-3 to create oligomers, thus blocking its ability to create lattice and promote cancer behavior of the cells. These results indicate an important role of post-translational modifications of galectin-3 in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1495. doi:10.1158/1538-7445.AM2011-1495

Structural and Molecular Basis for Intracellular Glycoprotein-Fate Determination through Sugar Recognition

Growing evidence indicates that the processing of asparagine-linked oligosaccharides is coupled with the quality control of glycoproteins in cells. Namely, the intracellular lectins operating as molecular chaperones, cargo receptors, and ubiquitin ligases recognize partially trimmed intermediates of high-mannose-type glycans and thereby govern the intracellular processes of folding, transport, and degradation of carrier polypeptides. These intracellular lectins have unique mechanisms to check folding states of glycoproteins and to sense the subcellular environmental conditions. This review provides an overview on our current knowledge of the structural and molecular basis for glycoprotein-fate determination in cells through sugar recognition by the lectins.

Intracellular Lectins Involved in Folding and Transport in the Endoplasmic Reticulum

Glycosylation is a highly sophisticated post-translational modification of proteins that occurs in the endoplasmic reticulum (ER) and the Golgi apparatus. The vital biological functions served by protein glycosylation can be separated into two types. The first is the role of glycosylation in the extracellular environment, where a variety of sugar chains participate in protein secretion and stability, cell-cell adhesion and signaling, innate immunity, embryogenesis, and morphogenesis. Another role is in quality control of glycoproteins in the ER, a topic that has received recent attention. Quality control of glycoproteins is categorized into three kinds of reactions; the first is folding of newly synthesized glycoproteins, the second is ER-associated degradation of terminally misfolded or unassembled glycoproteins, and the last is sorting and transport of glycoproteins between organelles. In all three processes, N-glycans on the glycoproteins are used as tags to initiate the reactions. The process of glycosylation is conserved in yeast, plants, invertebrates and vertebrates, including mammals. Here, I focus on the intracellular lectins that participate in these processes and discuss the role of glycosylation based on the structural differences of N-glycans.