Search PubMed⌕ Search

Biomedical subjects

Ken-ichi Kasai

Publications and source records attributed to Ken-ichi Kasai.

17 recordsLinked to original sources
PubMed2006

Crosslinking of low-affinity glycoprotein ligands to galectin LEC-1 using a photoactivatable sulfhydryl reagent.

The N-terminal lectin domain (Nh) of the tandem repeat-type nematode galectin LEC-1 has a lower affinity for sugars than the C-terminal lectin domain. To confirm that LEC-1 forms a complex with N-acetyllactosamine-containing glycoproteins, we used several mutants of LEC-1 in which a unique cysteine residue was introduced into the Nh domain and examined their binding to bovine asialofetuin with a photoactivatable sulfhydryl crosslinking reagent. A crosslinked product was formed with the Q38C mutant, strongly suggesting the low-affinity interaction of Nh with the glycoprotein could be detected with this system.

Animals↗
PubMed2006

Analysis of protein-protein interactions with a multi-capillary electrophoresis instrument.

Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.35) containing 0.1% Tween 20 was used as a sample buffer. The dual buffer system allowed fast and reproducible analyses of interactions at a physiological pH using uncoated fused-silica capillaries. The change in the mobility moment, the first statistical moment of an electropherogram on the mobility axis (Shimura, K., Uchiyama, N., Enomoto, M., Matsumoto, H., Kasai, K., Anal. Chem. 2005, 77, 564-572), of the labeled proteins were analyzed as a function of the concentration of unlabeled proteins. The dissociation constants for seven antibodies ranging from sub nanomolar to micromolar was determined based on the results of one cycle of parallel electrophoresis runs, which completed in 30 min using 20 pmol (120 ng) of labeled insulin and 5 pmol (750 ng) each of the mAb.

Animals↗
PubMed2005

Xenopus galectin-VIIa binds N-glycans of members of the cortical granule lectin family (xCGL and xCGL2).

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.

Amino Acid Sequence↗
PubMed2005

Mobility moment analysis of molecular interactions by capillary electrophoresis.

A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility. After baseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. This moment represents the average mobility of a labeled component. The change in the mobility moment in the presence and absence of the unlabeled component is used to evaluate the degree of saturation of the binding site of a labeled molecule with an unlabeled molecule. Mixtures of fluorescence-labeled protein (Fab' fragment of antibody or concanavalin A) and its unlabeled interacting partner (alpha(1)-antitrypsin or succinylated ovalbumin, respectively) at various concentrations were injected into a bare-silica capillary, and zone electrophoresis was carried out. The change in the mobility moment of the fluorescence-labeled molecules was used to determine the dissociation constants of the complexes. The determined constants are comparable to those obtained by a well-established method, that is, an analysis based on the peak height of the complex. Since the mobility moment analysis is not affected by the total intensity of the signals, it should be advantageous in analyses in which multiple capillaries are used, in which the injection volume and the sensitivity of detection might be more difficult to control at constant values. The mobility moment analysis also has advantages for the analysis of heterogeneous samples, since the identification of peaks is not necessarily required.

Antigen-Antibody Reactions↗
PubMed2004

Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding.

Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.

Amino Acid Sequence↗
PubMed2004

Crystallization and preliminary X-ray crystallographic studies of the C-terminal domain of galactose-binding lectin EW29 from the earthworm Lumbricus terrestris.

The galactose-binding lectin EW29 from the earthworm Lumbricus terrestris is composed of two homologous domains, both of which are members of the R-type lectin family. The truncated mutant rC-half comprising the C-terminal domain was crystallized by the hanging-drop vapour-diffusion method. The crystal belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 61.2, c = 175.6 A, and diffracted to beyond 1.9 A resolution. Matthews coefficient calculations suggested that this crystal contained two molecules per asymmetric unit.

Animals↗
PubMed2003

Galectin-1 induces astrocyte differentiation, which leads to production of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a neuroprotective polypeptide that is thought to be responsible for neuron proliferation, differentiation, and survival. An agent that enhances production of BDNF is expected to be useful for the treatment of neurodegenerative diseases. Here we report that galectin-1, a member of the family of beta-galactoside binding proteins, induces astrocyte differentiation and strongly inhibits astrocyte proliferation, and then the differentiated astrocytes greatly enhance their production of BDNF. Induction of astrocyte differentiation and BDNF production by an endogenous mammalian lectin may be a new mechanism for preventing neuronal loss after injury.

Animals↗
PubMed2003

Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.

We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18O, specifically into the N-glycosylation site. The 18O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.

Caenorhabditis elegans Proteins↗
PubMed2003

Stimulation of proliferation of rat hepatic stellate cells by galectin-1 and galectin-3 through different intracellular signaling pathways.

We found that the expression of galectin-1 and galectin-3 was significantly up-regulated in hepatic stellate cells (HSCs) both in the course of their transdifferentiation into myofibroblasts, a process of "self-activation," and in the fibrosis of liver tissues. Recombinant galectin-1 and galectin-3 stimulated the proliferation of cultured HSCs via the MEK1/2-ERK1/2 signaling pathway. However, galectin-3 utilized protein kinases C and A to induce this process, whereas galectin-1 did not. We also found that thiodigalactoside, a potent inhibitor of beta-galactoside binding, attenuated the effects of both galectins. In addition, galectin-1, but not galectin-3, promoted the migration of HSCs. Thus, it appears that galectin-1 and galectin-3, generated by activated HSCs, could participate in beta-galactoside binding and induce different intracellular signaling pathways leading to the proliferation of HSCs.

Animals↗
PubMed2002

Oligosaccharide specificity of galectins: a search by frontal affinity chromatography.

Galectins are widely distributed sugar-binding proteins whose basic specificity for beta-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67-87]. By using this system, we quantitatively analyzed the interactions at 20 degrees C between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3-OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Le(x), Le(a)). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for alpha1-2Fuc-, alpha1-3Gal-, alpha1-3GalNAc-, or alpha2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either "branched", "repeated", or "substituted" glycans, while conserving their ability to recognize basic disaccharide units, Galbeta1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type.

Animals↗
PubMed2002

Separation technologies for glycomics.

Progress in genome projects has provided us with fundamentals on genetic information; however, the functions of a large number of genes remain to be elucidated. To understand the in vivo functions of eukaryotic genes, it is essential to grasp the features of their post-translational modifications. Among them, protein glycosylation is a central issue to be discussed, considering the predominant roles of glycoproteins in cell-cell and cell-substratum recognition events in multicellular organisms. In this context, it is necessary to establish a core strategy for analyzing glycosylated proteins under the concept of the "glycome" [Trends Glycosci. Glycotechnol. 12 (2000) 1]. Though the term glycome should be defined, in analogy to the genome and proteome, as "a whole set of glycans produced in a single organism", here we propose a glycome project specifically focusing on glycoproteins. Principal objectives in the project are to identify: (1) which genes encode glycoproteins (i.e. genome information); (2) which sites among potential glycosylation sites are actually glycosylated (i.e. glycosylation site information); (3) what are the structures of glycans (i.e. structural information); and (4) what are the effects (functions) of glycosylation (functional information). For these purposes, two affinity technologies have been introduced. One is named the "glyco-catch method" to identify genes encoding glycoproteins [Proteomics 1 (2001) 295], and the other is the recently reinforced "frontal affinity chromatography" [J. Chromatogr. A 890 (2000) 261]. By the former method, genes that encode glycoproteins as well as glycosylation sites are systematically identified by the efficient combination of conventional lectin-affinity chromatography and contemporary in silico database searching. The following three actions have been devised for rapid and systematic characterization of glycans: (1) mass spectrometry to acquire exact mass information; (2) 2-D/3-D mapping to obtain refined chemical information; and (3) reinforced frontal affinity chromatography to determine affinity constants (K(a)-values) for a set of lectins. Pyridylaminated glycans are used throughout the characterization processes. In this review, the concept and strategy of glycomic approaches are described referring to the on-going glycome project focused on the nematode Caenorhabditis elegans.

Glycoproteins↗
PubMed2002

Fluorescence-labeled peptide pI markers for capillary isoelectric focusing.

Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection. Tetra- to tridecapeptides containing one cysteine residue were designed to focus sharply at their respective isoelectric points by including amino acids that contain charged side chains, the pKa values of which are close to the corresponding pI values. An iodoacetylated derivative of tetramethylrhodamine was coupled to the thiol group of cysteine to yield fluorescent pI markers. The pI values of the labeled peptides were precisely determined after isoelectric focusing on polyacrylamide gel slabs by direct measurement of the pH of the focused bands. The markers were subjected to capillary isoelectric focusing for 10-15 min in coated capillaries under conditions of low electroosmosis and were detected by means of a scanning laser-induced fluorescence detector down to a level of subpicomolar range. The markers permitted the calibration of a wide-range pH gradient formed in a capillary by fluorescence detection for the first time and should facilitate the development of highly sensitive analytical methods based on a combination of capillary isoelectric focusing and laser-induced fluorescence detection.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Immunoassay of serum alpha(1)-antitrypsin by affinity-probe capillary isoelectric focusing using a fluorescence-labeled recombinant antibody fragment.

An immunoassay for human alpha(1)-antitrypsin based on affinity-probe capillary isoelectric focusing (AP-CIEF) is described. The method is based on the separation of free and bound labeled antibody fragments by CIEF with laser-induced fluorescence detection. A recombinant Fab' fragment of mouse immunoglobulin G1 (IgG1) against human alpha(1)-antitrypsin was labeled with tetramethylrhodamine on the single cysteine residue at the hinge region. A single pI isomer of the labeled Fab' was purified by IEF in a slab of agarose gel and was then used as the affinity probe for alpha(1)-antitrypsin. The use of recombinant Fab' considerably simplified the labeling process. Although there was some difficulty in the quantification of native alpha(1)-antitrypsin with the affinity probe, carbamylation of the antigen sample by heat treatment with urea made the complex peaks appear reproducibly and more distinct, thus facilitating the identification and quantification of the complex. The system provided an almost linear response to a pure sample of alpha(1)-antitrypsin over a concentration range of 5-1000 ng/mL and the detection limit extended down to around 2 ng/mL. Alpha(1)-antitrypsin in a serum sample was determined using this system to be 1.2 mg/mL which is comparable to the reported value for the protein.

Acrylamides↗
PubMed2002

Galectin-induced activation of the transcription factors NFAT and AP-1 in human Jurkat T-lymphocytes.

Galectin-mediated ligation of glycoepitopes on T-cell activation markers induces an increase in the cytosolic calcium concentration ([Ca(2+)](i)) originating from a transient Ca(2+) release of internal stores as well as a sustained influx across the plasma membrane. In transiently transfected Jurkat T-lymphocytes, galectins [galectin-1 (gal-1), recombinant human galectin-1 (rec gal-1), nematode 32-kDa galectin (LEC-1), nematode 16-kDa galectin (LEC-6)] differentially stimulate the expression of the luciferase reporter gene constructs pNFAT-TA-Luc and pAP1(PMA)-TA-Luc, which are activated by the nuclear factor of activated T-cells (NFAT) or the transcription factor, activator protein 1 (AP-1), respectively. The galectin-stimulated expression of the reporter constructs is completely inhibited by lactose (30 mM) and asialofetuin (30 microM) carrying Galbeta1-4GlcNAc sequences. Preincubation of pNFAT-TA-Luc-transfected cells with cyclosporine A (0.1 microM) and FK506 (0.01 microM) abrogated the gal-1-induced expression of the reporter luciferase (Luc). Electrophoretic mobility shift assays (EMSAs) provided evidence for gal-1-stimulated increase in the binding of nuclear extracts to a synthetic oligonucleotide with an AP-1 consensus sequence.

Asialoglycoproteins↗
PubMed2002

Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans.

Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.

Adsorption↗