[Show abstract][Hide abstract] ABSTRACT: We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units.
[Show abstract][Hide abstract] ABSTRACT: Leguminous lectins have a conserved carbohydrate recognition site comprising four loops (A-D). Here, we randomly mutated the sequence and length of loops C and D of peanut agglutinin (PNA) and expressed the proteins on the surface of mouse green fluorescent protein (GFP)-reporter cells. Flow cytometry, limiting dilution, and cDNA cloning were used to screen for several mutated PNAs with distinct properties. The mutated PNA clones obtained using NeuAcα2-6(Galβ1-3)GalNAc as a ligand showed preference for NeuAcα2-6(Galβ1-3)GalNAc rather than non-sialylated Galβ1-3GlcNAc, whereas wild-type PNA binds to Galβ1-3GlcNAc but not sialylated Galβ1-3GalNAc. Sequence analyses revealed that for all of the glycan-reactive mutated PNA clones, (i) loop C was eight amino acids in length, (ii) loop D was identical to that of wild-type PNA, (iii) residue 127 was asparagine, (iv) residue 125 was tryptophan, and (v) residue 130 was hydrophobic tyrosine, phenylalanine, or histidine. The sugar-binding ability of wild-type PNA was increased nine-fold when Tyr125 was mutated to tryptophan, and that of mutated clone C was increased more than 30-fold after His130 was changed to tyrosine. These results provide an insight into the relationship between the amino acid sequences of the carbohydrate recognition site and sugar-binding abilities of leguminous lectins.
[Show abstract][Hide abstract] ABSTRACT: In the post genomic era, glycomics - the systematic study of all glycan structures of a given cell or organism - has emerged as an indispensable technology in various fields of biology and medicine. Lectins are regarded as "decipherers of glycans", being useful reagents for their structural analysis, and have been widely used in glycomic studies. However, the inconsistent activity and availability associated with the plant-derived lectins that comprise most of the commercially available lectins, and the limit in the range of glycan structures covered, have necessitated the development of innovative tools via engineering of lectins on existing scaffolds. This review will summarize the current state of the art of lectin engineering and highlight recent technological advances in this field. The key issues associated with the strategy of lectin engineering including selection of template lectin, construction of a mutagenesis library, and high-throughput screening methods are discussed.
[Show abstract][Hide abstract] ABSTRACT: Although various molecular profiling technologies have the potential to predict specific tumor phenotypes, the comprehensive profiling of lectin-bound glycans in human cancer tissues has not yet been achieved.
We examined 242 advanced gastric cancer (AGC) patients without or with lymph node metastasis-N0 (n = 62) or N+ (n = 180)-by lectin microarray, and identified the specific lectins highly associated with AGC phenotypes.
In seven gastric cancer cell lines, in contrast to expressed-in-cancer lectins, not-expressed-in-cancer (NEC) lectins were tentatively designated by lectin microarray. Binding signals of the specific lectins were robustly reduced in AGC patients with N+ status as compared with those with N0 status. The receiver operating characteristic curve determined the optimal cutoff value to differentiate N0 status from N+ status, and subsequent profiling of NEC lectins identified Vicia villosa agglutinin (VVA) association with the significant other lectins involved in lymph node metastasis. VVA reaction was clearly found on cancer cells, suggesting that it may result from carcinoma-stroma interaction in primary AGC, because VVA is an NEC lectin. Most intriguingly, VVA reaction was remarkably attenuated in the tumor cells of the metastatic lymph nodes, even if it was recognized in primary AGC. In AGC, histological type was strongly associated with soybean agglutinin and Bauhinia purpurea lectin, whereas p53 mutation was the best correlated with Griffonia simplicifolia lectin II.
Lectin microarrays can be used to very accurately quantify the reaction of glycans with tumor tissues, and such profiles may represent the specific phenotypes, including N+ status, histological type, or p53 mutation of AGC.
[Show abstract][Hide abstract] ABSTRACT: The lectin microarray is an emerging technology for glycomics. It has already found maximum use in diverse fields of glycobiology by providing simple procedures for differential glycan profiling in a rapid and high-throughput manner. Since its first appearance in the literature in 2005, many application methods have been developed essentially on the same platform, comprising a series of glycan-binding proteins immobilized on an appropriate substrate such as a glass slide. Because the lectin microarray strategy does not require prior liberation of glycans from the core protein in glycoprotein analysis, it should encourage researchers not familiar with glycotechnology to use glycan analysis in future work. This feasibility should provide a broader range of experimental scientists with good opportunities to investigate novel aspects of glycoscience. Applications of the technology include not only basic sciences but also the growing fields of bio-industry. This chapter describes first the essence of glycan profiling and the basic fabrication of the lectin microarray for this purpose. In the latter part the focus is on diverse applications to both structural and functional glycomics, with emphasis on the wide applicability now available with this new technology. Finally, the importance of developing advanced lectin engineering is discussed.
No preview · Article · Mar 2015 · Topics in current chemistry
[Show abstract][Hide abstract] ABSTRACT: Studies of β-glucans are often hampered by their structural diversity and complexity, which is problematic because interest in their effects on animal cells has increased in recent years. Herein, we present a comprehensive strategy for structural characterization of branched β-glucans, and as a proof-of-concept study, characterized laminarin and acid-soluble β-gluco-oligosaccharides (<4,000 Da, void volume elute fraction of gel filtration on Bio-gel P-2) from the brown algae, Ecklonia stolonifera. The strategy involves quantitative fluorescence detection-high performance liquid chromatography that enables the characterization of di- and oligosaccharides after acid hydrolysis of the glucan. We found that laminarin is composed of β1–3 (72% in mol) and β1–6 (28%) anomeric bonds, whereas the E. stolonifera glucan is composed of β1–3 (57%) and β1–6 (43%) anomeric bonds. This composition is distinct from that of other brown algae β-glucans, for which the β1–6 bond content is much smaller. We also performed a detailed structural analysis of the 11 major β-gluco-oligosaccharides prepared by mild acid hydrolysis and β1–3-specific laminarinase digestion. All 11 oligosaccharides contained branches joined to the backbone by β1–6 bonds. Five of the oligosaccharides had extended branches; in this regard, the E. stolonifera glucan is unlike other characterized β-glucans. Our strategy should enable structural characterizations of β-branched glucans, for which no practical approach has been available until now.
No preview · Article · Mar 2015 · Bioactive Carbohydrates and Dietary Fibre
[Show abstract][Hide abstract] ABSTRACT: Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience.
[Show abstract][Hide abstract] ABSTRACT: Lectin microarray is an emerging technology that has undergone significant development over the past decade. It facilitates the rapid and highly sensitive profiling of glycans in a high-throughput manner. Unlike conventional techniques, such as mass spectrometry and multidimensional high-performance liquid chromatography (HPLC) mapping, the lectin microarray method does not determine the precise structures or compositions of glycans, but rather it provides essential information concerning the complex features of glycans (e.g., glycoproteins) in terms of binding patterns to a panel of lectins with different carbohydrate-binding specificities. The method is highly advantageous for the analysis of crude samples, such as cell/tissue extracts and body fluids (e.g., sera, bile) containing glycoproteins and even intact cells. In contrast to other methods, it does not require the prior liberation of glycans from core proteins. Practical applications of the lectin microarray have also been developed for use with many target molecules/cells/tissues. The most advanced applications can be found in cancer-related biomarker investigation
, stem cell characterization and evaluation, and biological drug development. Here, a lectin microarray system developed in the author’s group is described; this system adopts a unique detection principle that provides for an easy-to-perform, sensitive, and reproducible glycan analysis approach undertaken with a fully commercialized instrument.
[Show abstract][Hide abstract] ABSTRACT: Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K
d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K
d’s for interactions between a series of standard glycans and various galectins.
No preview · Article · Jan 2015 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: Two jacalin-related lectins (JRLs) were purified by mannose-agarose and melibiose-agarose from seeds of Treculia africana. One is galactose-recognizing JRL (gJRL), named T. africana agglutinin-G (TAA-G), and another one is mannose-recognizing JRL (mJRL), TAA-M. The yields of the two lectins from the seed flour were approximately 7.0 mg/g for gJRL and 7.2 mg/g for mJRL. The primary structure of TAA-G was determined by protein sequencing of lysyl endopeptic peptides and chymotryptic peptides. The sequence identity of TAA-G to other gJRLs was around 70%. Two-residue insertion was found around the sugar-binding sites, compared with the sequences of other gJRLs. Crystallographic studies on other gJRLs have shown that the primary sugar-binding site of gJRLs can accommodate Gal, GalNAc, and GalNAc residue of T-antigen (Galβ1-3GalNAcα-). However, hemagglutination inhibition and glycan array showed that TAA-G did not recognize GalNAc itself and T-antigen. TAA-G preferred melibiose and core 3 O-glycan.
No preview · Article · Aug 2014 · Bioscience Biotechnology and Biochemistry
[Show abstract][Hide abstract] ABSTRACT: Recent progress in structural biology has elucidated the three-dimensional structures and carbohydrate-binding mechanisms of most lectin families. Lectins are classified into 48 families based on their three-dimensional structures. A ribbon drawing gallery of the crystal and solution structures of representative lectins or lectin-like proteins is appended and may help to convey the diversity of lectin families, the similarity and differences between lectin families, as well as the carbohydrate-binding architectures of lectins.
No preview · Article · Aug 2014 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: More than 100 years have passed since the first lectin ricin was discovered. Since then, a wide variety of lectins (lect means "select" in Latin) have been isolated from plants, animals, fungi, bacteria, as well as viruses, and their structures and properties have been characterized. At present, as many as 48 protein scaffolds have been identified as functional lectins from the viewpoint of three-dimensional structures as described in this chapter. In this chapter, representative 53 lectins are selected, and their major properties that include hemagglutinating activity, mitogen activity, blood group specificity, molecular weight, metal requirement, and sugar specificities are summarized as a comprehensive table. The list will provide a practically useful, comprehensive list for not only experienced lectin users but also many other non-expert researchers, who are not familiar to lectins and, therefore, have no access to advanced lectin biotechnologies described in other chapters.
No preview · Article · Aug 2014 · Methods in Molecular Biology