30 results on '"Sz-Wei Wu"'
Search Results
2. A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance
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Beverly Dyas, Onur Mudanyali, Neven Karlovac, Sz-Wei Wu, Mohan Natesan, Stig M. R. Jensen, Robert G. Ulrich, and Chieh-I Chen
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0301 basic medicine ,Microbiological Techniques ,Computer science ,Protein Array Analysis ,Flow cell ,Bioengineering ,Cloud computing ,Smartphone application ,Antibodies, Viral ,Rapid detection ,Proof of Concept Study ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Viral Envelope Proteins ,Animals ,Humans ,030212 general & internal medicine ,Instrumentation ,Point of care ,Fluid Flow and Transfer Processes ,Immunoassay ,business.industry ,Process Chemistry and Technology ,telemonitoring ,Microfluidic Analytical Techniques ,Ebolavirus ,Specific antibody ,Macaca fascicularis ,030104 developmental biology ,Blood ,Nucleoproteins ,Marburgvirus ,Point-of-Care Testing ,point-of-care ,Marburg Disease ,Ebola ,smartphone reader ,Rabbits ,Smartphone ,disease surveillance ,User interface ,business ,Computer hardware - Abstract
We have developed a digital and multiplexed platform for the rapid detection and telemonitoring of infections caused by Ebola and Marburg filoviruses. The system includes a flow cell assay cartridge that captures specific antibodies with microarrayed recombinant antigens from all six species of filovirus, and a smartphone fluorescent reader for high-performance interpretation of test results. Multiplexed viral proteins, which are expandable to include greater numbers of probes, were incorporated to obtain highest confidence results by cross-correlation, and a custom smartphone application was developed for data analysis, interpretation, and communication. The smartphone reader utilizes an opto-electro-mechanical hardware attachment that snaps at the back of a Motorola smartphone and provides a user interface to manage the operation, acquire test results, and communicate with cloud service. The application controls the hardware attachment to turn on LEDs and digitally record the optically enhanced images. Assay processing time is approximately 20 min for microliter amounts of blood, and test results are digitally processed and displayed within 15 s. Furthermore, a secure cloud service was developed for the telemonitoring of test results generated by the smartphone readers in the field. Assay system results were tested with sera from nonhuman primates that received a live attenuated EBOV vaccine. This integrated system will provide a rapid, reliable, and digital solution to prevent the rapid overwhelming of medical systems and resources during EVD or MVD outbreaks. Further, this disease-monitoring system will be useful in resource-limited countries where there is a need for dispersed laboratory analysis of recent or active infections.
- Published
- 2018
3. Novel LC-MS2 Product Dependent Parallel Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact Glycopeptides
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Tsung-Hsien Pu, Kay-Hooi Khoo, Rosa Viner, and Sz-Wei Wu
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Glycan ,Chromatography ,Collision-induced dissociation ,biology ,Chemistry ,Computational biology ,Orbitrap ,Dissociation (chemistry) ,Analytical Chemistry ,law.invention ,Electron-transfer dissociation ,law ,biology.protein ,Database search engine ,Monoisotopic mass ,Ion trap - Abstract
Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS2 followed by electron transfer dissociation (ETD)-MS2 upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS2 analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS2 data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the pep...
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- 2014
4. Sweet-Heart — An integrated suite of enabling computational tools for automated MS2/MS3 sequencing and identification of glycopeptides
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Fang-Yu Chang, Kay-Hooi Khoo, Suh-Yuen Liang, Sz-Wei Wu, and Tsung-Hsien Pu
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Proteomics ,Glycosylation ,Computer science ,Biophysics ,Computational biology ,computer.software_genre ,Bioinformatics ,Biochemistry ,Glycoproteomics ,Mice ,Identification (information) ,chemistry.chemical_compound ,chemistry ,Sequence Analysis, Protein ,Protein methods ,False positive paradox ,Animals ,Cattle ,Database search engine ,Databases, Protein ,computer ,Glycoproteins ,Data integration - Abstract
High efficiency identification of intact glycopeptides from a shotgun glycoproteomic LC-MS2 dataset remains problematic. The prevalent mode of identifying the de-N-glycosylated peptides is littered with false positives and addresses only the issue of site occupancy. Here, we present Sweet-Heart, a computational tool set developed to tackle the heart of the problems in MS2 sequencing of glycopeptide. It accepts low resolution and low accuracy ion trap MS2 data, filters for glycopeptides, couples knowledge-based de novo interpretation of glycosylation-dependent fragmentation pattern with protein database search, and uses machine-learning algorithm to score the computed glyco and peptide combinations. Higher ranking candidates are then compiled into a list of MS2/MS3 entries to drive subsequent rounds of targeted MS3 sequencing of putative peptide backbone, allowing its validation by database search in a fully automated fashion. With additional fishing out of all related glycoforms and final data integration, the platform proves to be sufficiently sensitive and selective, conducive to novel glycosylation discovery, and robust enough to discriminate, among others, N-glycolyl neuraminic acid/fucose from N-acetyl neuraminic acid/hexose. A critical appraisal of its computing performance shows that Sweet-Heart allows high sensitivity comprehensive mapping of site-specific glycosylation for isolated glycoproteins and facilitates analysis of glycoproteomic data. Biological significance The biological relevance of protein site-specific glycosylation cannot be meaningfully addressed without first defining its pattern by direct analysis of glycopeptides. Sweet-Heart is a novel suite of computational tools allowing for automated analysis of mass spectrometry-based glycopeptide sequencing data. It is developed to accept ion trap MS2/MS3 data and uses a machine learning algorithm to score and rank the candidate peptide core and glycosyl substituent combinations. By eliminating the need for manual, labor-intensive, and subjective data interpretation, it facilitates high throughput shotgun glycoproteomic data analysis and is conducive to identification of unanticipated glycosylation, as demonstrated here with a recombinant EGFR.
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- 2013
5. In Vivo Regulation of Steroid Hormones by the Chst10 Sulfotransferase in Mouse*
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Misa Suzuki-Anekoji, Sz-Wei Wu, Michiko N. Fukuda, Minoru Fukuda, Kay-Hooi Khoo, Kazuhiro Sugihara, Atsushi Suzuki, Keith K. Murai, Jun Nakayama, Kiyohiko Angata, and Tomoya O. Akama
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medicine.medical_specialty ,Sulfotransferase ,medicine.drug_class ,medicine.medical_treatment ,Genetic Vectors ,Glycobiology and Extracellular Matrices ,Mice, Transgenic ,Biology ,Biochemistry ,Steroid ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Sulfation ,Glucuronic Acid ,Internal medicine ,medicine ,Animals ,Humans ,Testosterone ,Receptor ,Homologous Recombination ,Molecular Biology ,030304 developmental biology ,Neurons ,Recombination, Genetic ,0303 health sciences ,Models, Genetic ,Estrogens ,Cell Biology ,Estrogen ,Hormones ,Killer Cells, Natural ,Steroid hormone ,Endocrinology ,HEK293 Cells ,Gene Expression Regulation ,Female ,Steroids ,Glycolipids ,Sulfotransferases ,030217 neurology & neurosurgery ,Hormone - Abstract
Background: Chst10 transfers sulfate to glucuronic acid to form the HNK-1 antigen carried by glycoproteins and glycolipids in neurons and NK cells. Results: Chst10 transfers sulfate to glucuronidated steroid hormones, and Chst10-deficient mice exhibited subfertility. Conclusion: Subfertility in Chst10 null females is caused by a loss of steroid hormone dysregulation. Significance: This study identified a new regulatory mechanism mediated by sulfation of glucuronidated steroid., Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10−/− mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10−/− females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10−/− females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo.
- Published
- 2012
6. Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue
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Yoann Rombouts, Emeline Fabre, Kay-Hooi Khoo, Elisabeth Elass-Rochard, Colette Brassart, Yann Guérardel, Adeline Burguière, Sz-Wei Wu, Bernadette Coddeville, and Laurent Kremer
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Antigens, Bacterial ,Glycosylation ,biology ,Biophysics ,Mannose ,Mycobacterium tuberculosis ,biology.organism_classification ,Biochemistry ,Microbiology ,chemistry.chemical_compound ,Bacterial Proteins ,chemistry ,Antigen ,Concanavalin A ,Mycobacterium marinum ,biology.protein ,Amino Acid Sequence ,Pathogen ,Peptide sequence ,Glycoproteins - Abstract
The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.
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- 2012
7. MS-based glycomic strategies for probing the structural details of polylactosaminoglycan chain on N-glycans and glycoproteomic identification of its protein carriers
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Sz-Wei Wu, Shui-Hua Wang, and Kay-Hooi Khoo
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Proteomics ,Glycan ,Glycoside Hydrolases ,Molecular Sequence Data ,Biology ,Biochemistry ,Umbilical vein ,Cell Line ,chemistry.chemical_compound ,Polysaccharides ,Carbohydrate Conformation ,Humans ,Glycosyl ,Glycomics ,Molecular Biology ,Fucosylation ,Glycoproteins ,Glycopeptides ,Endothelial Cells ,Amino Sugars ,Glycopeptide ,Carbohydrate Sequence ,Membrane protein ,chemistry ,Cell culture ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Tomato lectin ,biology.protein ,Plant Lectins ,Chromatography, Liquid - Abstract
Most MS-based glycomic and glycoproteomic analyses focus on identifying changes in terminal glyco-epitopes represented by sialylation and fucosylation at specific positions of the terminal N-acetyllactosamine units. Much less attention was accorded to the underlying linear or branched poly-N-acetyllactosamine extension from the N-glycan trimannosyl core other than a simple inference of its presence due to mass data and hence glycosyl compositional assignment. Using the EA.hy926 cell line derived from human umbilical vein endothelial cells (HUVEC), we have systematically investigated the MALDI- and ESI-MS-based methodologies for probing the structural details of endothelial polylactosaminoglycans at both MS and MS(2) levels in conjunction with the use of endo-β-galactosidase to identify branching motifs and initiation sites. We showed that the polylactosaminoglycan chains on the N-glycans of EA.hy926 were less sialylated and fucosylated but more extended and branched than those of human umbilical vein endothelial cells, thus demonstrating a fundamental glycomic difference. For EA.hy926 that was investigated in more details, its polylactosaminoglycan chains were shown to be not restricted to extending from a specific antenna including the biologically important 6-arm position. Finally, experimental conditions for glycopeptide enrichment by tomato lectin were further optimized, which led to identification of over 40 candidate endothelial membrane protein carriers of polylactosaminoglycans by proteomic analysis.
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- 2011
8. Characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis
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Jian Zhang, Kay-Hooi Khoo, Sz-Wei Wu, and Chatterjee, Delphi
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Mycobacteria -- Physiological aspects ,Mycobacterium -- Physiological aspects ,Bacterial cell walls -- Research ,Transferases -- Research ,Chemistry - Abstract
The study presents the characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis, which is then used to explain the biosynthesis of cell wall arabinan. These transferases are found to be very helpful for the assembly of very complex structures.
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- 2007
9. Selective Extraction and Effective Separation of Galactosylsphingosine (Psychosine) and Glucosylsphingosine from Other Glycosphingolipids in Pathological Tissue Samples
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Ellen Sidransky, Sz-Wei Wu, Bruce A. Bunnell, Su Chen Li, Yu Teh Li, Mark E. Haskins, Wayne R. Buck, and Kay-Hooi Khoo
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Adult ,Brain chemistry ,Adolescent ,Biochemistry ,Glycosphingolipids ,Article ,Mice ,Cellular and Molecular Neuroscience ,Dogs ,medicine ,Psychosine ,Animals ,Humans ,Chemical pathology ,Child ,Chromatography, High Pressure Liquid ,Brain Chemistry ,Gaucher Disease ,Chromatography ,Chemistry ,Extramural ,Extraction (chemistry) ,Leukodystrophy ,Infant ,General Medicine ,medicine.disease ,Macaca mulatta ,Mice, Mutant Strains ,Leukodystrophy, Globoid Cell ,Child, Preschool ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Krabbe disease ,lipids (amino acids, peptides, and proteins) ,Chromatography, Thin Layer ,Spleen - Abstract
To facilitate the study of the chemical pathology of galactosylsphingosine (psychosine, GalSph) in Krabbe disease and glucosylsphingosine (GlcSph) in Gaucher disease, we have devised a facile method for the effective separation of these two glycosylsphingosines from other glycosphingolipids (GSLs) in Krabbe brain and Gaucher spleen samples. The procedure involves the use of acetone to selectively extract GalSph and GlcSph, respectively, from Krabbe brain and Gaucher spleen samples. Since acetone does not extract other GSLs except modest amounts of galactosylceramide, sulfatide, and glucosylceramide, the positively charged GalSph or GlcSph in the acetone extract can be readily separated from other GSLs by batchwise cation-exchange chromatography using a Waters Accell Plus CM Cartridge. GalSph or GlcSph enriched by this simple procedure can be readily analyzed by thin-layer chromatography or high-performance liquid chromatography.
- Published
- 2010
10. Terminal disialylated multiantennary complex-type N-glycans carried on acutobin define the glycosylation characteristics of the Deinagkistrodon acutus venom
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Sz-Wei Wu, Ying-Ming Wang, Inn-Ho Tsai, Kay-Hooi Khoo, Chia-Wei Lin, and Jin-Mei Chen
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Oligosaccharides, Branched-Chain ,Glycan ,Glycosylation ,Molecular Sequence Data ,Oligosaccharides ,Venom ,Biochemistry ,chemistry.chemical_compound ,Polysaccharides ,Tandem Mass Spectrometry ,Crotalid Venoms ,Animals ,Humans ,Amino Acid Sequence ,chemistry.chemical_classification ,biology ,Deinagkistrodon acutus ,Polysialic acid ,Crotalus ,Thrombin ,Amino Sugars ,biology.organism_classification ,Sialic acid ,carbohydrates (lipids) ,Carbohydrate Sequence ,chemistry ,Snake venom ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Sialic Acids ,biology.protein ,Glycoprotein ,Protein Processing, Post-Translational - Abstract
Glycosylation analysis of nonmammalian sources often springs surprises and conjures up intriguing views of evolutionary adaptation. Many of the constituents of snake venoms are known to be glycosylated and yet very few were fully characterized and accorded specific functions. In the process of glycomic screening through the venoms from Asian pit vipers, a partially O-acetylated NeuAcα2-8NeuAcα2-3Galβ1-4GlcNAcβ1-terminal epitope was found to be the predominant glycosylation characteristic of the snake venom produced by the monotypic Deinagkistrodon acutus, with acutobin, a highly specific fibrinogenase, being identified as a primary protein carrier. Full structural definition and glycosylation site mapping were completed through advanced mass spectrometry analyses at both the glycan and glycopeptide levels in conjunction with chemical and enzymatic cleavages. Although similar occurrence of such terminal disialyl cap on the N-glycans of several mammalian glycoproteins has been implicated, most of these correspond to only minor constituents of the full glycomic heterogeneity and remain poorly characterized. In contrast, each antennae of the hybrid- and complex-type N-glycans derived from acutobin was found to be rather homogeneously disialylated. With up to eight sialic acids evenly distributed on nonextended tetraantennary core structure, these unusual N-glycans are among those of highest sialic acid density ever identified without actually carrying polysialic acid chains. It remains to be tested whether they may serve as multivalent disialyl ligands for several of the human Siglecs and thus meddle with the natural immuno-recognition systems of snakebite victims, apart from affecting the general efficacy of acutobin as anticoagulant in biomedical applications.
- Published
- 2010
11. Sequencing of oligoarabinosyl units released from mycobacterial arabinogalactan by endogenous arabinanase: Identification of distinctive and novel structural motifs
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Lee, Arwen, Sz-Wei Wu, Scherman, Michael S., Torrelles, Jordi B., Chatterjee, Delphi, McNeil, Michael R., and Kay-Hooi Khoo
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Mycobacterium tuberculosis -- Research ,Nucleotide sequence -- Research ,Linkage (Genetics) -- Analysis ,Biological sciences ,Chemistry - Abstract
A study presents opportunities for the better understanding of the structural level by mapping and sequencing of the digestion products, which were dominated by distinctive [Ara.sub.18] and [Ara.sub.19] structural units, together with [Ara.sub.7] and lesser amount of [Ara.sub.11] and [Ara.sub.12]. The establishment of linkage-specific MS/MS fragmentation characteristics led to the identification of a galactosamine substituent of the C2 position of a portion of the internal 3,5-branched Ara residue of the arabinogalactan (AG) of Mycobacterium tuberculosis.
- Published
- 2006
12. Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans
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Kay-Hooi Khoo, Shin-Yi Yu, He-Hsuan Hsiao, and Sz-Wei Wu
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Glycan ,Collision-induced dissociation ,Stereochemistry ,Molecular Sequence Data ,Mass spectrometry ,Methylation ,Biochemistry ,Mice ,chemistry.chemical_compound ,Sulfation ,Fragmentation (mass spectrometry) ,Polysaccharides ,Tandem Mass Spectrometry ,Animals ,Humans ,Moiety ,Derivatization ,Glycomics ,chemistry.chemical_classification ,Chromatography ,biology ,Sulfates ,Chemistry ,Glycosidic bond ,Carbohydrate Sequence ,biology.protein ,Lymph Nodes - Abstract
Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed.
- Published
- 2009
13. Sequencing of Oligoarabinosyl Units Released from Mycobacterial Arabinogalactan by Endogenous Arabinanase: Identification of Distinctive and Novel Structural Motifs
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Delphi Chatterjee, Sz-Wei Wu, Kay-Hooi Khoo, Jordi B. Torrelles, Michael R. McNeil, Arwen Lee, and Michael S. Scherman
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Glycoside Hydrolases ,Stereochemistry ,Amino Acid Motifs ,Molecular Sequence Data ,Mycobacterium smegmatis ,Galactosamine ,Cleavage (embryo) ,Galactans ,Biochemistry ,Article ,Substrate Specificity ,Mycolic acid ,Cell wall ,chemistry.chemical_compound ,Cell Wall ,Polysaccharides ,Tandem Mass Spectrometry ,Arabinogalactan ,Carbohydrate Conformation ,Glycosyl ,Structural motif ,Cellulomonas ,chemistry.chemical_classification ,Lipomannan ,Polysaccharides, Bacterial ,Mycobacterium tuberculosis ,Arabinose ,Carbohydrate Sequence ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Peptidoglycan - Abstract
One of the most prominent macromolecular entities of all mycobacterial cell walls is the D-arabinofuran, a common constituent of both arabinogalactan (AG) and lipoarabinomannan (LAM) (1). In the chemical setting of AG, the central role of arabinan appears to be in maintaining the structural integrity of the cell wall proper by tethering the outer mycolic acid lipid barrier to the underlying peptidoglycan layer through the flexible glycosyl linkages of AG to form the mycolylarabinogalactan-peptidoglycan (mAGP) complex. Its most characteristic structural feature is a non-reducing terminal hexaarabinofuranosyl (Ara6) motif, Arafβ1→2Arafα1→5(Arafβ1→2Arafα1→3)Arafα1→5Arafα1→, where both the terminal β-sAraf and the penultimate 2-α-Araf serve as the anchoring points for the mycolic acids. In contrast, the non-reducing termini of the arabinan in LAM appear to be less strictly branched and a linear tetraarabinofuranosyl (Ara4) motif, Arafβ1→2Arafα1→5Arafα1→5Arafα1→, is known to coexist with the branched Ara6 termini. Through varying degree of mannose-capping at its non-reducing terminal β-Araf and attached at the reducing end to a lipomannan anchor, the arabinan in LAM plays instead pivotal immuno-modulatory functions in host-pathogen interactions and disease outcome (reviewed in (2, 3)). Despite the well recognized importance of the mycobacterial arabinans and more than a decade of investigation following their first description (4, 5), structural details beyond the aforementioned non-reducing terminal motifs are still lacking. This severely hampers our delineation of the arabinosylation process. The homo-polymeric nature of the arabinan with intricate branching pattern and extreme size heterogeneity presents a major technical difficulty defying detailed structural analysis intended to reconstruct the intact polymer out of the chemically derived and characterized structural motifs. To date, principal findings from two complementary analytical approaches essentially conceptualize and confine our current understanding of the mycobacterial arabinan. First, mass spectrometry (MS) analysis of the mild acid hydrolysates of the permethyl derivatives of arabinan from AG, has led to recognition of a speculative Ara22mer structural motif comprising two terminal Ara6 motifs (6). By virtue of the O-Me tag, it was possible to identify fragments from the non-reducing end formed by only a single arabinosyl cleavage. These included the expected single cleavages corresponding to Ara5, as well as Ara6, Ara7, Ara8 and Ara17-22 but not Ara9-16 (Fig. 1). In contrast, similar studies on LAM produced no evidence for the kind of branching and organization for AG-type arabinan. These seminal studies not only indicated some fundamental difference in the respective arabinan architecture but also gave rise to the current structural model in which the arabinan of AG was often depicted as comprising different alternative arrangement of three Ara22mers, to give a total of approximately 60–70 arabinosyl residues per galactan chain. Technically, this chemical strategy is powerful but degradative and tedious. Acid labile substituents could not be mapped and alternative arrangement superimposed on the Ara22mer framework cannot be delineated. Fig. 1 The Ara22mer structural model for AG. The proposed branching pattern was based primarily on the absence of Ara3,4 and Ara9-16 among the single chemical cleavage fragments detected by MS, as indicated above the 5-arabinan chain. Apart from the well established ... A second powerful tool is provided in the form of a crude arabinanase preparation from a Cellulomonas species which was consistently shown to be capable of releasing the non-reducing end Ara6 and/or Ara4 (along with their mannose caps if present) from the arabinan while digesting the remaining into mostly a dimeric Arafα1→5Araf (Ara2) unit (7, 8). Thus a hallmark of digestion with this enzyme preparation on AG is the characteristic production of Ara6 and Ara2 whereas when applied to LAM, would additionally produced Ara4. The released arabinosyl oligomers could be rapidly profiled by high pH anion exchange chromatography on a Dionex LC system, as well as rapidly mapped and sequenced by MS and MS/MS analysis. Since its first application on mannose-capped LAM from wild type M. tuberculosis (7), the developed assay has since been effectively used to probe a variety of arabinan structures derived from different mycobacterial species, from drug resistant and other genetic mutants (9–12). A major limitation though is that it proves equally difficult to derive a full picture of the intact arabinan beyond the facile mapping of its terminal structural motifs. Critical to gaining a better understanding at the structural level is the further development of analytical tools that would allow mapping and sequencing of larger arabinosyl motifs which retain all functionality including any additional non-arabinosyl substituents. A potential major breakthrough came when an endogenous M. smegmatis arabinanase activity that could release arabinan fragments of a size significantly larger than 10 oligoglucosyl units from AG was identified (13). The exact structures of the digestion products were then not reported although efforts have since been directed to purify and further characterize this novel arabinanase activity for both structural and functional studies. We report here how this enzyme has been effectively coupled to current generation of MALDI-MS instrumentation to provide new structural insight and direct evidence for the implicated Ara22 structural model of AG (Fig. 1) based on MALDI-MS mapping and MS/MS sequencing of the released intact oligoarabinosyl fragments. In the process, we defined the enzyme activity against AG and the precise location of a galactosamine substituent on the intact arabinan unit of AG from M. tuberculosis CSU20, which distinguishes it from that of M. smegmatis AG.
- Published
- 2006
14. Distinctive characteristics of MALDI-Q/TOF and TOF/TOF tandem mass spectrometry for sequencing of permethylated complex type N-glycans
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Sz-Wei Wu, Kay-Hooi Khoo, and Shin-Yi Yu
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Glycan ,Nitrogen ,Molecular Sequence Data ,Mass spectrometry ,Tandem mass spectrometry ,Methylation ,Biochemistry ,Glycomics ,Mice ,Fragmentation (mass spectrometry) ,Polysaccharides ,Tandem Mass Spectrometry ,Cell Line, Tumor ,Cricetinae ,Carbohydrate Conformation ,Animals ,Molecular Biology ,Zebrafish ,chemistry.chemical_classification ,biology ,Chemistry ,Glycosidic bond ,Cell Biology ,Combinatorial chemistry ,Matrix-assisted laser desorption/ionization ,Carbohydrate Sequence ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Rabbits ,Carbohydrate conformation - Abstract
Concerted MALDI-MS profiling and CID MS/MS sequencing of permethylated glycans is one of the most effective approaches for high throughput glycomics applications. In essence, the identification of larger complex type N-glycans necessitates an unambiguous definition of any modification on the trimannosyl core and the complement of non-reducing terminal sequences which constitute the respective antennary structures. Permethylation not only affords analyses of both neutral and sialylated glycans at comparable ease and sensitivity but also yields more sequence-informative fragmentation pattern. Facile glycosidic cleavages directed mostly at N-acetylglucosamine under low energy CID, as implemented on a quadrupole/time-of-flight (Q/TOF) instrument, often afford multiple losses of the attached antenna resulting in characteristic ions related to the number of antennary branches on the trimannosyl core. Non-reducing terminal epitopes can be easily deduced but information on the linkage specific substituent on the terminal units is often missing. The high energy CID MS/MS afforded by TOF/TOF instrument can fill in the gap by giving an array of additional cross-ring and satellite ions. Glycosidic cleavages occurring specifically in concert with loss of 2-linked or 3-linked substituents provide an effective way to identify the branch-specific antennary extension. These characteristics are shown here to be effective in deriving the sequences of additionally galactosylated, sialylated and fucosylated terminal N-acetyllactosamine units and their antennary location. Together, a highly reproducible fragmentation pattern can be formulated to simplify spectral assignment. This work also provides first real examples of sequencing multiply sialylated complex type N-glycans by high energy CID on a TOF/TOF instrument.
- Published
- 2006
15. A Smartphone-Based Rapid Telemonitoring System for Ebola and Marburg Disease Surveillance.
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Natesan, Mohan, Sz-Wei Wu, Chieh-I Chen, Jensen, Stig M. R., Karlovac, Neven, Dyas, Beverly K., Mudanyali, Onur, and Ulrich, Robert G.
- Published
- 2019
- Full Text
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16. A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate, Lipoarabinomannan*
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Delphi Chatterjee, Patrick J. Brennan, Michael R. McNeil, Mary Jackson, Shiva K. Angala, Sz-Wei Wu, Kay-Hooi Khoo, and Devinder Kaur
- Subjects
Lipopolysaccharides ,Glycosylation ,Molecular Sequence Data ,Mycobacterium smegmatis ,Mannose ,Glycobiology and Extracellular Matrices ,Phosphatidylinositols ,Biochemistry ,Mannans ,chemistry.chemical_compound ,Polysaccharides ,Tandem Mass Spectrometry ,Carbohydrate Conformation ,Phosphatidylinositol ,Molecular Biology ,Mannan ,Lipomannan ,Lipoarabinomannan ,Cell Biology ,chemistry ,Carbohydrate Sequence ,Mannosylation ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Carbohydrate conformation - Abstract
Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.
- Published
- 2014
17. An adaptive workflow coupled with Random Forest algorithm to identify intact N-glycopeptides detected from mass spectrometry
- Author
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Sz-Wei Wu, Suh-Yuen Liang, Fang-Yu Chang, Tsung Hsien Pu, and Kay-Hooi Khoo
- Subjects
Statistics and Probability ,Computer science ,Herpesvirus 2, Human ,Feature selection ,Mass spectrometry ,computer.software_genre ,Biochemistry ,Mass Spectrometry ,Workflow ,Mice ,Artificial Intelligence ,Animals ,Humans ,Sensitivity (control systems) ,Molecular Biology ,Chromatography, High Pressure Liquid ,Glycopeptides ,Sampling (statistics) ,Glycopeptide ,Computer Science Applications ,Random forest ,Computational Mathematics ,Identification (information) ,Computational Theory and Mathematics ,Sample size determination ,Data mining ,computer ,Algorithms - Abstract
Motivation: Despite many attempts for algorithm development in recent years, automated identification of intact glycopeptides from LC-MS 2 spectral data is still a challenge in both sensitivity and precision. Results: We implemented a supervised machine learning algorithm, Random Forest, in an automated workflow to identify N-glycopeptides using spectral features derived from ion trap-based LC-MS 2 data. The workflow streamlined high-confident N-glycopeptide spectral data and enabled adaptive model optimization with respect to different sampling strategies, training sample size and feature set. A critical evaluation of the features important for glycopeptide identification further facilitated effective feature selection for model improvement. Using split sample testing method from 577 high-confident N-glycopeptide spectral data, we demonstrated that an optimal true-positive rate, precision and false-positive rate of 73, 88 and 10%, respectively, can be attained for overall N-glycopeptide identification Availability and implementation: The workflow developed in this work and the application suite, Sweet-Heart, that the workflow supports for N-glycopeptide identification are available for download at http://sweet-heart.glycoproteomics.proteome.bc.sinica.edu.tw/ . Contact: syliang@gate.sinica.edu.tw or kkhoo@gate.sinica.edu.tw Supplementary information: Supplementary data are available at Bioinformatics online.
- Published
- 2014
18. AGO61-dependent GlcNAc modification primes the formation of functional glycans on α-dystroglycan
- Author
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Sz-Wei Wu, Shogo Oka, Naoki Nakagawa, Koichi Kato, Takaya Abe, Takuya Saito, Kay-Hooi Khoo, Hiroshi Kiyonari, Hirokazu Yagi, and Tatsushi Toda
- Subjects
Glycan ,Glycosylation ,α dystroglycan ,Glycobiology ,N-Acetylglucosaminyltransferases ,Article ,Cell Line ,Mice ,chemistry.chemical_compound ,Polysaccharides ,Tandem Mass Spectrometry ,Transferases ,Chlorocebus aethiops ,medicine ,Animals ,Phosphorylation ,Dystroglycans ,Mice, Knockout ,Glucosamine ,Multidisciplinary ,biology ,Glycosyltransferases ,medicine.disease ,Phenotype ,Recombinant Proteins ,In vitro ,Cell biology ,carbohydrates (lipids) ,medicine.anatomical_structure ,chemistry ,COS Cells ,Mutation ,Immunology ,Congenital muscular dystrophy ,biology.protein ,Basal lamina ,Laminin ,Peptides - Abstract
Dystroglycanopathy is a major class of congenital muscular dystrophy that is caused by a deficiency of functional glycans on α-dystroglycan (α-DG) with laminin-binding activity. A product of a recently identified causative gene for dystroglycanopathy, AGO61, acted in vitro as a protein O-mannose β-1, 4-N-acetylglucosaminyltransferase, although it was not functionally characterized. Here we show the phenotypes of AGO61-knockout mice and demonstrate that AGO61 is indispensable for the formation of laminin-binding glycans of α-DG. AGO61-knockout mouse brain exhibited abnormal basal lamina formation and a neuronal migration defect due to a lack of laminin-binding glycans. Furthermore, our results indicate that functional α-DG glycosylation was primed by AGO61-dependent GlcNAc modifications of specific threonine-linked mannosyl moieties of α-DG. These findings provide a key missing link for understanding how the physiologically critical glycan motif is displayed on α-DG and provides new insights on the pathological mechanisms of dystroglycanopathy.
- Published
- 2013
- Full Text
- View/download PDF
19. Glycoproteomics analysis to identify a glycoform on haptoglobin associated with lung cancer
- Author
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Sz-Wei Wu, Kay-Hooi Khoo, Supawadee Sriyam, Kongsak Boonyapranai, Hsien-Yu Tsai, Suree Phutrakul, Shui-Tein Chen, and Chong-Jen Yu
- Subjects
Adult ,Proteomics ,Glycan ,Glycosylation ,Lung Neoplasms ,Clinical Biochemistry ,Blotting, Western ,Biochemistry ,Fucose ,chemistry.chemical_compound ,Polysaccharides ,Tandem Mass Spectrometry ,medicine ,Biomarkers, Tumor ,Carbohydrate Conformation ,Humans ,Trypsin ,Lung cancer ,Molecular Biology ,Fucosylation ,Aged ,Glycoproteins ,biology ,Haptoglobins ,Haptoglobin ,Cancer ,Middle Aged ,medicine.disease ,N-Acetylneuraminic Acid ,Peptide Fragments ,Glycoproteomics ,chemistry ,Case-Control Studies ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Cancer research ,biology.protein - Abstract
Glycosylation is a common protein modification that is of interest in current cancer research because altered carbohydrate moieties are often found during cancer progress. A search for biomarkers in human lung cancer serum samples using glycoproteomic approaches identified fucosylated haptoglobin (Hp) significantly increased in serum of each subtype of lung cancer compared to normal donors. In addition, MS provided evidence of an increase of Hp fucosylation; the glycan structure was determined to be an α 2,6-linked tri-sialylated triantennary glycan containing α1,3-linked fucose attached to the four-linked position of the three-arm mannose of N-linked core pentasaccharide. These preliminary findings suggest that the specific glycoform of Hp may be useful as a marker to monitor lung cancer progression.
- Published
- 2010
20. Characterization of a distinct arabinofuranosyltransferase in Mycobacterium smegmatis
- Author
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Kay-Hooi Khoo, Sz-Wei Wu, Jian Zhang, and Delphi Chatterjee
- Subjects
Glycosylation ,biology ,Mycobacterium smegmatis ,General Chemistry ,Tandem mass spectrometry ,biology.organism_classification ,Biochemistry ,Catalysis ,Cell wall ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Biosynthesis ,chemistry ,Membrane protein ,Tandem Mass Spectrometry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Mutation ,Chromatography, Thin Layer ,Pentosyltransferases ,Arabinosyltransferase activity ,Nuclear Magnetic Resonance, Biomolecular ,Mycobacterium - Abstract
The D-arabinans in Mycobacterium are essential, extraordinarily complex entity comprised of d-arabinofuranose residues which are rarely found in nature. Despite the well-recognized importance of the mycobacterial arabinan, delineation of the arabinosylation process has been severely hampered due to lack of positively identified arabinosyltransferases. Identification of genes involved in arabinan biosynthesis entailed the use of ethambutol (EMB), a first-line antituberculosis agent that is known to inhibit cell wall arabinan synthesis. The three genes (embA, embB, and embC) encode novel membrane proteins, implicated as the only known mycobacterial arabinosyltransferases to this date. We have now adapted a multifaceted approach involving development of convenient arabinosyltransferase assay using novel synthetic acceptors to identify arabinosyltransferase/s that will be distinct from the Emb proteins. In our present work, Mycobacterium smegmatis mc(2) 155 (WTMsm) was used as a model to study the biosynthesis of cell wall arabinan. In an in vitro assay, we demonstrate that transfer of only alpha-Araf had occurred from decaprenylphosphoryl-D-arabinofuranose (DPA) on a newly synthesized branched acceptor [alpha-D-Araf](2)-3,5-alpha-D-Araf-(1-->5)-alpha-d-Araf-(1-->5)-alpha-D-Araf with an octyl aglycon. Higher molecular weight (up to Ara(10)) oligomers were also detected in a parallel reaction using cold phosphoribosepyrophosphate (pRpp). Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) analysis of these products revealed that isomeric products were formed and initiation and elongation of arabinan can occur either on the 5-arm or 3-arm of the branched 3,5-alpha-D-Araf. Individual embA, embB, and embC knockout strains retained this alpha-1,5 arabinosyltransferase activity, and the activity was partially inhibited by ethambutol. This particular enzyme function is distinct from the function of the Emb proteins.
- Published
- 2007
21. Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system
- Author
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Sz-Wei Wu, Manish S. Patankar, Richard L. Easton, Anne Dell, Frank A. Lattanzio, Howard R. Morris, Nyet Kui Wong, Kay-Hooi Khoo, Shin-Yi Yu, Gary F. Clark, and Mark Sutton-Smith
- Subjects
Male ,Glycosylation ,Erythrocytes ,Somatic cell ,Mice, Inbred Strains ,Biology ,Biochemistry ,Extracellular matrix ,chemistry.chemical_compound ,Mice ,Polysaccharides ,medicine ,Carbohydrate Conformation ,Cell Adhesion ,Animals ,Cell adhesion ,Zona pellucida ,Zona Pellucida ,Sperm-Ovum Interactions ,Periodic Acid ,Proteins ,Oxidants ,Sperm ,Spermatozoa ,Sialic acid ,medicine.anatomical_structure ,chemistry ,Carbohydrate Sequence ,Rabbits ,Oxidation-Reduction ,Germ cell - Abstract
Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type.
- Published
- 2007
22. Design and characterization of a multimeric DNA binding protein using Sac7d and GCN4 as templates
- Author
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Tzu-Ping Ko, Sz-Wei Wu, Chia-Cheng Chou, and Andrew H.-J. Wang
- Subjects
Sulfolobus acidocaldarius ,Circular dichroism ,Saccharomyces cerevisiae Proteins ,HMG-box ,Stereochemistry ,Macromolecular Substances ,Dimer ,Archaeal Proteins ,Saccharomyces cerevisiae ,Biology ,Antiparallel (biochemistry) ,Crystallography, X-Ray ,Biochemistry ,law.invention ,chemistry.chemical_compound ,Tetramer ,X-Ray Diffraction ,Structural Biology ,law ,Molecular Biology ,Leucine Zippers ,Binding Sites ,Base Sequence ,Peptide Fragments ,DNA-Binding Proteins ,Crystallography ,Basic-Leucine Zipper Transcription Factors ,DNA, Archaeal ,chemistry ,Recombinant DNA ,Thermodynamics ,DNA ,Transcription Factors - Abstract
The protein Sac7d belongs to a class of small chromosomal proteins from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Sac7d is extremely stable to heat, acid, and chemical agents. This protein is a monomer and it binds DNA without any particular sequence preference, while inducing a sharp kink in the DNA. By appending a leucine-zipper-like helical peptide derived from the yeast transcriptional activator GCN4 to the C-terminal end of Sac7d, the modified monomers (denoted S7dLZ) are expected to interact with each other via hydrophobic force to form a parallel dimer. The recombinant S7dLZ was expressed in Escherichia coli and purified by heating and ion-exchange chromatography. The formation of dimer was detected by gel-filtration chromatography and chemical cross-link. The results of surface plasmon resonance and circular dichroism experiments showed that the DNA-binding capacity was retained. Furthermore, X-ray diffraction analysis of single crystals of S7dLZ in complex with DNA decamer CCTATATAGG showed that the leucine-zipper segments of S7dLZ were associated into an antiparallel four-helix bundle. There are two DNA fragments bound to each S7dLZ tetramer in the crystal. This model works as a successful template that endows protein a new function without losing original properties. Proteins 2005. © 2005 Wiley-Liss, Inc.
- Published
- 2005
23. Novel LC-MS2 Product Dependent Parallel Data Acquisition Function and Data Analysis Workflow for Sequencing and Identification of Intact Glycopeptides.
- Author
-
Sz-Wei Wu, Tsung-Hsien Pu, Viner, Rosa, and Kay-Hooi Khoo
- Subjects
- *
DATA acquisition systems , *DATA analysis , *GLYCOPEPTIDES , *DISSOCIATION (Chemistry) , *OXONIUM ions , *MOIETIES (Chemistry) - Abstract
Data dependent acquisition (DDA) of higher collision energy dissociation (HCD)-MS2 followed by electron transfer dissociation (ETD)-MS2 upon detection of glycan-specific oxonium is one of the better approaches in current LC-MS2 analysis of intact glycopeptides. Although impressive numbers of glycopeptide identification by a direct database search have been reported, false positives remained high and difficult to determine. Even in cases when the peptide backbones were correctly identified, the exact glycan moieties were often erroneously assigned. Any attempt to fit the best glycosyl composition match by mass only is problematic particularly when the correct monoisotopic precursor cannot be determined unambiguously. Taking advantage of a new trihybrid Orbitrap configuration, we experimented with adding in a parallel ion trap collision induced dissociation (CID)-MS2 data acquisition to the original HCD-product dependent (pd)-ETD function. We demonstrated the feasibility and advantage of identifying the peptide core ion directly from edited HCD-MS2 data as an easy way to reduce false positives without compromising much sensitivity in intact glycopeptide positive spectrum matches. Importantly, the additional CID-MS2 data allows one to validate the glycan assignment and provides insight into possible glycan modifications. Moreover, it is a viable alternative to deduce the glycopeptide backbone particularly in cases when the peptide backbone cannot be identified by ETD/HCD. The novel HCD-pd-CID/ETD workflow combines the best possible decision tree dependent MS2 data acquisition modes currently available for glycoproteomics within a rapid Top Speed DDA duty cycle. Additional informatics can conceivably be developed to mine and integrate the rich information contained within for simultaneous N- and O-glycopeptide analysis. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
24. AGO61-dependent GlcNAc modification primes the formation of functional glycans on α-dystroglycan.
- Author
-
Hirokazu Yagi, Naoki Nakagawa, Takuya Saito, Hiroshi Kiyonari, Takaya Abe, Tatsushi Toda, Sz-Wei Wu, Kay-Hooi Khoo, Shogo Oka, and Koichi Kato
- Subjects
GLYCANS ,MUSCULAR dystrophy ,LAMININS ,MANNOSE ,PHENOTYPES - Abstract
Dystroglycanopathy is a major class of congenital muscular dystrophy that is caused by a deficiency of functional glycans on α-dystroglycan (α-DG) with laminin-binding activity. A product of a recently identified causative gene for dystroglycanopathy, AGO61, acted in vitro as a protein O-mannose β-1, 4-N-acetylglucosaminyltransferase, although it was not functionally characterized. Here we show the phenotypes of AGO61-knockout mice and demonstrate that AGO61 is indispensable for the formation of laminin-binding glycans of α-DG. AGO61-knockout mouse brain exhibited abnormal basal lamina formation and a neuronal migration defect due to a lack of laminin-binding glycans. Furthermore, our results indicate that functional α-DG glycosylation was primed by AGO61-dependent GlcNAc modifications of specific threonine-linked mannosyl moieties of α-DG. These findings provide a key missing link for understanding how the physiologically critical glycan motif is displayed on α-DG and provides new insights on the pathological mechanisms of dystroglycanopathy. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
25. BAD-Lectins: Boronic Acid-Decorated Lectins with Enhanced Binding Affinity for the Selective Enrichment of Glycoproteins.
- Author
-
Ying-Wei Lu, Chih-Wei Chien, Po-Chiao Lin, Li-De Huang, Chang-Yang Chen, Sz-Wei Wu, Chia-Li Han, Kay-Hooi Khoo, Chun-Cheng Lin, and Yu-Ju Chen
- Published
- 2013
- Full Text
- View/download PDF
26. Enabling techniques and strategic workflow for sulfoglycomics based on mass spectrometry mapping and sequencing of permethylated sulfated glycans.
- Author
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Shin-Yi Yu, Sz-Wei Wu, He-Hsuan Hsiao, and Kay-Hooi Khoo
- Subjects
- *
MASS spectrometry , *SULFATES , *PHOSPHORYLATION , *DISSOCIATION (Chemistry) , *SCISSION (Chemistry) - Abstract
Sulfate modifications on terminal epitopes of N- and O-glycans have increasingly been implicated as critical determinants mediating a diverse range of biological recognition functions. To address these low abundance but important sulfated glycans, and the sulfoglycome in general, further development of enrichment strategies and enabling mass spectrometry (MS)-based mapping techniques are needed. In this report, we demonstrate that the sulfated glycans, with and without additional sialylation, can be successfully permethylated by the sodium hydroxide slurry method and be distinguished from phosphorylated glycans by virtue of this derivatization. In conjunction with simple microscale postderivatization fractionation steps, permethyl derivatives fully retaining the negatively charged sulfate moiety and separated from the nonsulfated ones, can be efficiently detected and sequenced de novo by advanced MS/MS in the positive-ion mode. In particular, we show that the highly sequence and linkage informative high energy collision induced dissociation (CID) MS/MS afforded by MALDI-TOF/TOF can be extended to sulfoglycomic applications. The sulfated parent ion selected for CID MS/MS was found to mostly retain the sulfate moiety and therefore allow efficient fragmentation via the usual array of glycosidic, cross ring, and concerted double cleavages. Collectively, the optimized strategy enables a high sensitivity detection and critical mapping of the sulfoglycome such as the one derived from lymph node tissues or cell lines in both negative and positive-ion modes. Novel sulfated epitopes were identified from a crude mouse lymph node preparation, which fully attested to the practical utility of the methodology developed. [ABSTRACT FROM PUBLISHER]
- Published
- 2009
- Full Text
- View/download PDF
27. Sequencing of Oligoarabinosyl Units Released from Mycobacterial Arabinogalactan by Endogenous Arabinanase: Identification of Distinctive and Novel Structural Motifs.
- Author
-
Arwen Lee, Sz-Wei Wu, Scherman, Michael S., Torrelles, Jordi B., Chatterjee, Delphi, McNeill, Michael R., and Kay-Hooi Khoo
- Subjects
- *
MYCOBACTERIAL diseases , *MYCOBACTERIUM , *SCISSION (Chemistry) , *MYCOBACTERIUM tuberculosis , *ARABINOGALACTAN , *BIOCHEMISTRY - Abstract
The mycobacterial D-arabinofuran is a common constituent of both cell wall mycolyl-arabinogalactan (AG) and the associated lipoarabinomannan (LAM), and is thus accorded critical structural and immunological roles. Despite a well-recognized importance, progress in understanding its full structural characteristics beyond the nonreducing terminal motifs has hitherto been limited by available analytical tools. An endogenous arabinanase activity recently isolated from Mycobacterium smegmatis was previously shown to be capable of releasing large oligoarabinosyl units from AG. Advanced tandem mass spectrometry utilizing both low and high energy collision induced dissociation now afforded a facile way to map and directly sequence the digestion products which were dominated by distinctive Ara18 and Aral9 structural units, together with Ara7 and lesser amount of Ara11 and Ara12. Significantly, evidence was obtained for the first time which validated the linkages and branching pattern of the previously inferred Ara22 structural motif of AG, on which the preferred cleavage sites of the novel arabinanase could be localized. The established linkage-specific MS/MS fragmentation characteristics further led to identification of a galactosamine substituent on the C2 position of a portion of the internal 3,5-branched Ara residue of the AG of Mycobacterium tuberculosis, but not that of the nonpathogenic, fast growing M. smegmatis. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
28. Analysis of protein-linked glycosylation in a sperm-somatic cell adhesion system.
- Author
-
Mark Sutton-Smith, Nyet Kui Wong, Kay-Hooi Khoo, Sz-Wei Wu, Shin-Yi Yu, Manish S Patankar, Richard Easton, Frank A Lattanzio, Howard R Morris, Anne Dell, and Gary F Clark
- Subjects
GLYCOSYLATION ,SOMATIC cells ,CELL adhesion ,SPERM-ovum interactions - Abstract
Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell (“sperm–somatic cell adhesion system”) is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galα1–3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galα1–3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with β1–6-linked N-acetyllactosamine or its α1–3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
29. A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate, Lipoarabinomannan.
- Author
-
Kaur, Devinder, Angala, Shiva K., Sz-Wei Wu, Kay-Hooi Khoo, Chatterjee, Delphi, Brennan, Patrick J., Jackson, Mary, and McNeil, Michael R.
- Subjects
- *
PHOSPHATIDYLINOSITOLS , *IMMUNOREGULATION , *CELL envelope (Biology) , *GLYCOCONJUGATES , *LIPOARABINOMANNANS - Abstract
Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation,we showed that the reducing end region of the mannan that is attached to inositolhas 5-7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two othermutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ~10-12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl "primer." Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present inmature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
30. In Vivo Regulation of Steroid Hormones by the Chst10 Sulfotransferase in Mouse.
- Author
-
Suzuki-Anekoji, Misa, Suzuki, Atsushi, Sz-Wei Wu, Angata, Kiyohiko, Murai, Keith K., Sugihara, Kazuhiro, Akama, Tomoya O., Kay-Hooi Khoo, Nakayama, Jun, Fukuda, Michiko N., and Fukuda, Minoru
- Subjects
- *
STEROID hormones , *SULFOTRANSFERASES , *ESTROGEN regulation , *ENZYME activation , *HIGH performance liquid chromatography , *ESTRADIOL , *MASS spectrometry - Abstract
Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10-/- mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10-/- females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10-/- females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones.Wealso identified an HPLCpeak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
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