Biomedical subjects

Ken-ichi Kasai

Publications and source records attributed to Ken-ichi Kasai.

8 recordsLinked to original sources

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↗