Glycoreplica Peptides.

"Glycoreplica peptides" are prepared using a phage display peptide library and monoclonal antibodies that recognize the carbohydrate epitopes of glycoconjugate antigens. The peptides obtained not only mimic the shapes of original glycoconjugate antigens but also have some of their functions. We herein describe how to identify the amino acid alignments of glycoreplica peptides using phage display selection against carbohydrate-binding proteins. Target-specific peptides and proteins may be selected from the large repertory of a peptide/protein library using phage display technology. Glycoreplica peptides have the potential to become alternatives to carbohydrate ligands such as mimotopes for vaccinations and carbohydrate-derived drugs for carbohydrate-related diseases.

Alzheimer's disease is associated with disordered localization of ganglioside GM1 molecular species in the human dentate gyrus.

Alzheimer's disease (AD) is a progressive dementia associated with loss of memory and cognitive dysfunction. In a previous study, we demonstrated a decrease in b-series gangliosides along with a change in ganglioside molecular species in the hippocampal grey matter of patients with AD. The present study demonstrates the use of imaging mass spectrometry for analyzing the spatial arrangement of ganglioside GM1 (GM1) molecular species in the hippocampus. In AD patients, we found a decrease in the ratio of GM1(d20:1/C18:0) to GM1 d18:1/C18:0) in the outer molecular layer (ML) of the dentate gyrus. Because the outer ML is the region of main input into the hippocampus, our findings may have a direct relationship to the mechanism of dysfunction in AD.

TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells.

We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MS(n) analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1-2Galß1-3GlcNAcß1-3Galß1-4Glc). A critical role of the terminal Fucα1-2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1-2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.

Bio-recognition and functional lipidomics by glycosphingolipid transfer technology.

Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced.(Communicated by Kunihiko SUZUKI, M.J.A.).

Attenuation of acetic acid-induced gastric ulcer formation in rats by glucosylceramide synthase inhibitors.

INTRODUCTION: Ceramide has been suggested to play a role in apoptosis during gastric ulcerogenesis. The present study is designed to investigate whether accumulated ceramide could serve as the effector molecules of ulcer formation in a rat model of acetic acid-induced gastric ulcer. METHODS: The effect of fumonisin B1, an inhibitor of ceramide synthase, and of d,l,-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP) and N-butyldeoxynojirimycin (NB-DNJ), both inhibitors of glucosylceramide synthase, on the accumulation of ceramide and formation of gastric ulcer were examined in the rat model of acetic acid-induced gastric ulcer. RESULTS: Fumonisin B1 attenuated acetic acid-induced gastric ulcer formation, associated with a decrease in the number of apoptotic cells. Our results showed that it is neither the C18- nor the C24-ceramide itself, but the respective metabolites that were ulcerogenic, because PPMP and NB-DNJ attenuated gastric mucosal apoptosis and the consequent mucosal damage in spite of their reducing the degradation of ceramide. CONCLUSION: The ceramide pathway, in particular, the metabolites of ceramide, significantly contributes to acetic acid-induced gastric damage, possibly via enhancing apoptosis. On the other hand, PPMP and NB-DNJ treatment attenuated gastric mucosal apoptosis and ulcer formation despite increasing the ceramide accumulation, suggesting that it was not the ceramides themselves, but their metabolites that contributed to the ulcer formation in the acetic acid-induced gastric ulcer model.

An approach to glycobiology from glycolipidomics: ganglioside molecular scanning in the brains of patients with Alzheimer's disease by TLC-blot/matrix assisted laser desorption/ionization-time of flight MS.

We have established a new approach to glyco- and lipidomics using a thin layer chromatography (TLC)-Blot/matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system. This new approach consists of a combination of a method for transferring lipids separated on a TLC-plate to a poly-vinylidene difluoride (PVDF) membrane and direct mass spectrometric analysis of the individual lipids on the membrane by ion trap-type MALDI-TOF MS. This technology was applied to the analysis of individual lipids from the human brain. Then, based on the results of this analysis, ganglioside molecular species in neural diseases were analyzed. The levels of gangliosides GD1b and GT1b were lower in the hippocampal gray matter of patients with Alzheimer's disease than in the hippocampal gray matter of patients with Parkinson's disease or the control patients. The molecular scanning of individual ganglioside molecular species showed a significant reduction of d20 : 1/C18 : 0 ceramide-containing gangliosides in patients with Alzheimer's disease. These findings suggest that Alzheimer's disease is a kind of ganglioside metabolic disease affecting the hippocampal area. A new approach to glycobiology by the TLC-Blot/MALDI-TOF MS system is proposed.

Thymic involution and corticosterone level in Sandhoff disease model mice: new aspects the pathogenesis of GM2 gangliosidosis.

Sandhoff disease (SD) is a lysosomal disease caused by a mutation of the HEXB gene associated with excessive accumulation of GM2 ganglioside (GM2) in lysosomes and neurological manifestations. Production of autoantibodies against the accumulated gangliosides has been reported to be involved in the progressive pathogenesis of GM2 gangliosidosis, although the underlying mechanism has not been fully elucidated. The thymus is the key organ in the acquired immune system including the development of autoantibodies. We showed here that thymic involution and an increase in cell death in the organ occur in SD model mice at a late stage of the pathogenesis. Dramatic increases in the populations of Annexin-V(+) cells and terminal deoxynucletidyl transferase dUTP nick end labeling (TUNEL) (+) cells were observed throughout the thymuses of 15-week old SD mice. Enhanced caspase-3/7 activation, but not that of caspase-1/4, -6 ,-8, or -9, was also demonstrated. Furthermore, the serum level of corticosterone, a potent inducer of apoptosis of thymocytes, was elevated during the same period of apoptosis. Our studies suggested that an increase in endocrine corticosterone may be one of the causes that accelerate the apoptosis of thymocytes leading to thymic involution in GM2 gangliosidosis, and thus can be used as a disease marker for evaluation of the thymic condition and disease progression.

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

OBJECTIVE: Novel recombinant human lysosomal ß-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. METHODS: A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. RESULTS: The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. INTERPRETATION: This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases.

GalNAcß1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121.

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with ß-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcß-nLc4Cer² sequence.

New approach for glyco- and lipidomics--molecular scanning of human brain gangliosides by TLC-Blot and MALDI-QIT-TOF MS.

We have developed a TLC-Blot system that makes possible the direct analysis of blotted glycosphingolipids on a polyvinylidene difluoride membrane from a high-performance TLC plate by immunological staining, chemical staining, enzymatic treatment and mass spectrometric (MS) analysis. An ion trap type matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF) MS apparatus improved not only the molecular identification but also the analysis of molecular species of lipids on the polyvinylidene difluoride membrane. A new approach for glyco- and lipidomics, molecular scanning technology by a combination of TLC-Blot and MALDI-QIT-TOF MS, was developed and applied to human brain gangliosides separated from the tissues of patients with neural diseases and control patients. The results clearly showed a change of ganglioside composition, in addition to identifying individual ganglioside molecular species, in the hippocampus gray matter of patients with Alzheimer's disease. The results strongly suggested that metabolic changes of gangliosides played an important role in the progression of this disease. The present technology with molecular imaging should provide valuable information for elucidating the significance of molecular species in neuronal functions such as neural transmission, memory, and learning.

Sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy.

Influenza is an infectious disease caused by the influenza virus, and each year many people suffer from this disease. Hemagglutinin (HA) in the membrane of type A influenza viruses recognizes sialylglycoconjugate receptors on the host cell surface at an initial step in the infection process; consequently, HA inhibitors are considered potential candidates for antiviral drugs. We identified peptides that bind to receptor-binding sites through a multiple serial selection from phage-displayed random peptide libraries. Using the HA of the H1 and H3 strains as target proteins, we obtained peptides that bind to both HAs. The binding affinities of peptides for these HAs were improved by secondary and tertiary selections from the corresponding sublibraries. A docking simulation suggested that, similar to sialic acid, the peptides are recognized by the receptor-binding site in HA, which indicates that these peptides mimic the sialic acid structure. N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus. Such HA-inhibitors are promising candidates for novel antiviral drugs.

A new lipidomics approach by thin-layer chromatography-blot-matrix-assisted laser desorption/ionization imaging mass spectrometry for analyzing detailed patterns of phospholipid molecular species.

Thin-layer chromatography (TLC) is a highly established convenient technique for lipid separation and partial characterization of neutral and acidic glycosphingolipids (GSLs) and phospholipids, in mixtures. Meanwhile, imaging mass spectrometry (IMS) is a promising tool for lipidomics. However, some lipid classes are detected more sensitively than others, which can lead to suppression effects when complex mixtures are analyzed. Therefore to analyze complex lipid mixtures, a precise separation into the individual lipid classes is necessary. Here we present our highly sensitive and convenient analytical technology that combines TLC and IMS, namely the TLC-Blot-MALDI-IMS method, to visualize whole lipids and individual molecular species with high sensitivity compared with common staining methods. This method allows for easy visualization of all lipids with a linear range of approximately one order of magnitude and precision <16% RSD, making it useful for differential display analysis of lipids.

Gangliosides determine the amyloid pathology of Alzheimer's disease.

Gangliosides, GM3 and GM1, are suggested to accelerate the deposition of the amyloid beta-protein as amyloid angiopathy and senile plaques, respectively, in the Alzheimer brain. We investigated the profile of amyloid deposition in the brains of transgenic mice expressing a mutant amyloid precursor protein with a disrupted GM2 synthase gene, in which GM3 accumulates whereas GM1 is lacking. These mice showed a significantly increased level of deposited amyloid beta-protein in the vascular tissues. Furthermore, formation of severe dyshoric-form amyloid angiopathy, in which amyloid extended from the blood vessel walls deeply into the surrounding parenchyma was observed. Our results indicate that the expression of gangliosides is a critical determinant for the amyloid pathology in the Alzheimer brain.

Inhibition of influenza virus infections by sialylgalactose-binding peptides selected from a phage library.

Influenza virus hemagglutinin recognizes sialyloligosaccharides of glycoproteins and glycolipids as cell surface receptors in the initial stage of the infection process. We demonstrate that pentadecapeptides that bind to a sialylgalactose structure (Neu5Ac-Gal) inhibited the infection of cells by influenza virus. The pentadecapeptides were identified through affinity selection from a phage-displayed random peptide library using a monolayer of the ganglioside Neu5Acalpha2-3Galbeta1-4Glcbeta1-1'Cer (GM3). The peptides were found to have affinity for GM3, and alanine scanning showed seven amino acid residues that contribute to carbohydrate recognition. The binding of peptides to the cell surface was significantly inhibited in the presence of sialic acid or by the digestion of cell surface sialyl residues by neuraminidase. Plaque assays indicated that a molecular assembly of alkylated peptides inhibited the infection of Madin-Darby canine kidney cells by influenza virus. Carbohydrate-binding peptides that inhibit carbohydrate-virus interaction showed inhibitory activity. These results may lead to a new approach to the design of antiviral drugs.

TLC blot (far-eastern blot) and its applications.

A simple method for transfer of lipids including phospholipids, glycolipids, and neutral lipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, called TLC blot (far-eastern blot), is presented. Lipids separated on a HPTLC plate are blotted quantitatively. This procedure made it possible to purify individual lipids from a blotted membrane in a short time. Binding study, immunodetection, and mass spectrometric analysis are available for PVDF membrane. Furthermore, the world of molecular species imaging is opened by a scanning analysis with a combination of TLC blot and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (TLC-Blot/MALDI-TOF MS).

Gene expression analysis during platelet-like particle production in phorbol myristate acetate-treated MEG-01 cells.

A comprehensive gene-expression analysis during platelet (PLT) production from megakaryocytes may give important information on genes involved in the PLT production process. However, the low abundance of primary megakaryocytes makes the gene expression analysis difficult. Therefore, we employed MEG-01 cells, a human megakaryocytic cell line, and confirmed that the cell line produces PLT-like particles by treatment with phorbol myristate acetate (PMA). After treatment of MEG-01 cells with PMA for 8 or 24 h, comprehensive gene expression analysis was carried out using a microarray and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). From the microarray analysis, 141 genes were up-regulated (>2-fold) and 164 genes were down-regulated (<1/2-fold). However, known PLT-related genes were not included in the up- or down-regulated genes. On the other hand, RT-PCR analysis detected increased expression of beta1-tubulin, CD62P, gpIbalpha and gpIII, which are related to PLT function and megakaryocyte differentiation, following PMA treatment for 24 h. These results indicate that the MEG-01 cell may be an alternative model system to study the process of human PLT production from megakaryocytes. The gene-expression analysis might be a powerful tool for identifying genes related to PLT production, if the experimental conditions are optimized.

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes.

Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).

Lactoferrin inhibits platelet production from human megakaryocytes in vitro.

The mechanism of megakaryopoiesis, proplatelet formation (PPF) and platelet (PLT) production is not fully elucidated. Lactoferrin (LF) has been reported to have many biological functions including cell proliferation and differentiation, and the LF receptor is present on megakaryocytic cells. In the present study, we examined the effect of human LF (hLF) on PLT production from primary megakaryocytes (MKs). At first, we developed a PLT production system derived from human CD34+ cells by thrombopoietin (TPO) stimulation. Because the number of proplatelets, PLTs and CD41+ MKs was remarkably increased after day 5, we employed the TPO-induced CD34+ cells on day 5. Then, the effect of hLF on PLT production from human primary MKs was examined. In the range of 3-30 micrg/ml, hLF significantly inhibited PLT production up to about 60%. However, it did not significantly change the intensity of CD41 expression in MKs and the ploidy of MKs. In addition, it did not inhibit MK progenitors. These results suggest that LF directly inhibits PLT production from matured MKs, but does not inhibit megakaryopoiesis, including proliferation/maturation processes.

A new approach for drug discovery from glycobiology and phage-displayed peptide library technology.

Peptides which mimic functional activities of glycosphingolipids were prepared by a technology of phage-displayed peptide library using monoclonal antibodies against glycosphingolipids. These peptides were named glyco-replica peptides. Peptides prepared with anti-GD1alpha antibody by this technology were found to contain WHW as common motif, and they showed suppressive activity not only on adhesion between hepatic sinusoidal endothelial cells and lymphosarcoma RAW117-H10 cells, but also on metastasis of the tumor cell to the liver and lung. The WHW motif seems to be important to mimic the functional activity of the ganglioside GD1alpha. Next, we prepared GD3-replica peptides using a monoclonal antibody against GD3 (4F6). A peptide, GD3-P4 with highest affinity to 4F6 was used to immunize mice to examine if the mice show their immune response to raise antibodies against GD3. We confirmed the immune response and succeeded in the production of a monoclonal antibody (3D2) against GD3. The monoclonal antibody 3D2 showed specific binding to GD3 on a thin-layer chromatography plate and also melanoma tissues. Interestingly, the amino acid sequence of the CDR regions of light and heavy chains showed high similarity with those of the original GD3 monoclonal antibody (4F6) used for the preparation of GD3-replica peptide. The technology of the phage-displayed peptide library was applied to in vivo bio-panning study using an angiogenesis experimental model. The obtained peptides were found to show strong binding property to the neo-vasculature system and to be quite useful to carry an anti-tumor drug to the tumor tissue. Based on these experimental results, we discuss about some applications of this method to drug discovery.