Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.
Background: Exosome, a type of extracellular vesicles, can associate with A in vitro. Results: Intracerebrally injected exosomes trapped A on surface glycosphingolipids and transported it into microglia in AD mouse brains, resulting in reductions in A pathology. Conclusion: Exogenous exosomes act as potent scavengers for A in mouse brains. Significance: The findings provide a novel therapeutic approach for AD.
a b s t r a c tElevated amyloid-b peptide (Ab) in brain contributes to Alzheimer's disease (AD) pathogenesis. We demonstrated the presence of exosome-associated Ab in the cerebrospinal fluid (CSF) of cynomolgus monkeys and APP transgenic mice. The levels of exosome-associated Ab notably decreased in the CSF of aging animals. We also determined that neuronal exosomes, but not glial exosomes, had abundant glycosphingolipids and could capture Ab. Infusion of neuronal exosomes into brains of APP transgenic mice decreased Ab and amyloid depositions, similarly to what reported previously on neuroblastoma-derived exosomes. These findings highlight the role of neuronal exosomes in Ab clearance, and suggest that their downregulation might relate to Ab accumulation and, ultimately, the development of AD pathology.
Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N-and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.omics-based biomarker discovery | stemness | interglycomic correlations | glycoblotting | β-elimination in the presence of pyrazolone
Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.
We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis x/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the b-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the b-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcb4(NeuAca3)Galb3(NeuAca6)GlcNAcbGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Monoand disialyl core Type 1 O-linked structures were identified after reductive b-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this Oglycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its b-chain are characterized by enhanced branching as well as antenna fucosylation. ' 2007 Wiley-Liss, Inc.
A rapid and quantitative method for solid-phase methyl esterification of carboxy groups of various sialylated oligosaccharides has been established. The method employed a triazene derivative, 3-methyl-1-p-tolyltriazene, for facile derivatization of oligosaccharides immobilized onto general solid supports such as Affi-Gel Hz and gold colloidal nanoparticles in a multiwell plate. The workflow protocol was optimized for the solid-phase processing of captured sialylated/unsialylated oligosaccharides separated from crude sample mixtures by chemical ligation. From tryptic and/or PNGase F-digest mixtures of glycoproteins, purification by chemoselective immobilization, esterification and recovery were achieved in the same well of the filter plate within three hours when used in conjunction with "glycoblotting technology" (S.-I. Nishimura, K. Niikura, M. Kurogochi, T. Matsushita, M. Fumoto, H. Hinou, R. Kamitani, H. Nakagawa, K. Deguchi, N. Miura, K. Monde, H. Kondo, High-throughput protein glycomics: Combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry: Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). The recovered materials were directly applicable to subsequent characterization by mass spectrometric techniques such as MALDI-TOF for large-scale glycomics of both neutral and sialylated oligosaccharides. On-bead/on-gold nanoparticle derivatization of glycans containing sialic acids allowed rapid and quantitative glycoform profiling by MALDI-TOF MS with reflector and positive ion mode. In addition to its simplicity and speed, the method eliminates the use of unfavorable halogenated solvents such as chloroform and dichloromethane or volatile solvents such as diethyl ether and hexane, resulting in a practical and green chemical method for automated robotic adaptation.
Clinical proteomics focusing on the identification and validation of biomarkers and the discovery of proteins as therapeutic targets is an emerging and highly important area of proteomics. Biomarkers are measurable indicators of a specific biological state (particularly one relevant to the risk of contraction) and the presence or the stage of disease, and are thus expected to be useful for the prediction, detection, and diagnosis of disease as well as to follow the efficacy, toxicology, and side effects of drug treatment, and to provide new functional insights into biological processes.At present, proteomics methods based on mass spectrometry (MS) have emerged as the preferred strategy for discovery of diagnostic, prognostic, and therapeutic protein biomarkers. Most biomarker discovery studies use unbiased, "identified-based" approaches that rely on high performance mass spectrometers and extensive sample processing. Semiquantitative comparisons of protein relative abundance between disease and control patient samples are used to identify proteins that are differentially expressed and, thus, to populate lists of potential biomarkers. De novo proteomics discovery experiments often result in tens to hundreds of candidate biomarkers that must be subsequently verified in serum. However, despite the large numbers of putative biomarkers, only a small number of them are passed through the development and validation process into clinical practice, and their rate of introduction is declining. The first non-standard abbreviation (MS above is standard) must be footnoted the same as the abbreviation footnote, and MRM must be the first abbreviation in the list because it is the one footnoted. After that the order does not matter.Targeted proteomics using multiple reaction monitoring (MRM) 1 is emerging as a technology that complements the discovery capabilities of shotgun strategies as well as an alternative powerful novel MS-based approach to measure a series of candidate biomarkers (1-7). Therefore, MRM is expected to provide a powerful high throughput platform for biomarker validation, although clinical validation of novel biomarkers has been traditionally relying on immunoassays (8, 9). MRM exploits the unique capabilities of triple quadrupoles (QQQ) MS for quantitative analysis. In MRM, the first and the third quadrupoles act as filters to specifically select predefined m/z values corresponding to the peptide precursor ion and specific fragment ion of the peptide, whereas the second quadrupole serves as collision cell. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with retention time and signal intensity for a specific transition as coordinates. These measurements have been multiplexed to provide 30 or From the
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