排序方式: 共有101条查询结果,搜索用时 46 毫秒
1.
Jae-Min Lim Edith E Wollaston-Hayden Chin Fen Teo Dorothy Hausman Lance Wells 《Clinical proteomics》2014,11(1):20
Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes. 相似文献
2.
Dong-Jik Lee Yong-Sun Bahn Hong-Jin Kim Seung-Yeon Chung Hyun Ah Kang 《The Journal of biological chemistry》2015,290(3):1861-1873
Cryptococcus neoformans is an encapsulated basidiomycete causing cryptococcosis in immunocompromised humans. The cell surface mannoproteins of C. neoformans were reported to stimulate the host T-cell response and to be involved in fungal pathogenicity; however, their O-glycan structure is uncharacterized. In this study, we performed a detailed structural analysis of the O-glycans attached to cryptococcal mannoproteins using HPLC combined with exoglycosidase treatment and showed that the major C. neoformans O-glycans were short manno-oligosaccharides that were connected mostly by α1,2-linkages but connected by an α1,6-linkage at the third mannose residue. Comparison of the O-glycan profiles from wild-type and uxs1Δ mutant strains strongly supports the presence of minor O-glycans carrying a xylose residue. Further analyses of C. neoformans mutant strains identified three mannosyltransferase genes involved in O-glycan extensions in the Golgi. C. neoformans KTR3, the only homolog of the Saccharomyces cerevisiae KRE2/MNT1 family genes, was shown to encode an α1,2-mannosyltransferase responsible for the addition of the second mannose residue via an α1,2-linkage to the major O-glycans. C. neoformans HOC1 and HOC3, homologs of the Saccharomyces cerevisiae OCH1 family genes, were shown to encode α1,6-mannosyltransferases that can transfer the third mannose residue, via an α1,6-linkage, to minor O-glycans containing xylose and to major O-glycans without xylose, respectively. Moreover, the C. neoformans ktr3Δ mutant strain, which displayed increased sensitivity to SDS, high salt, and high temperature, showed attenuated virulence in a mouse model of cryptococcosis, suggesting that the extended structure of O-glycans is required for cell integrity and full pathogenicity of C. neoformans. 相似文献
3.
《Molecular & cellular proteomics : MCP》2020,19(11):1767-1776
Highlights
- •N-glycan patterns are distinct in pediatric and adult urine.
- •Sex differences of N-glycans are much larger in adults.
- •Pediatric urine has almost no sex differences in N-glycan levels.
- •In adults, the majority of N-glycans were more abundant in males.
4.
糖组学研究策略及前沿技术研究进展 总被引:2,自引:0,他引:2
糖组学是继基因组学和蛋白质组学后的新兴研究领域,主要研究聚糖结构与功能.通过与蛋白质组数据库结合,糖捕捉法能系统鉴定糖蛋白和糖基化位点.糖微阵列技术可以对生物个体产生的全部蛋白聚糖结构进行鉴定与表征,提高了聚糖分析通量.而化学选择糖印迹技术简化了聚糖纯化步骤并提高了糖基化分析的灵敏度.双消化并串联柱法通过双酶消化双柱分离,在分析聚糖结构的同时也鉴定蛋白质的序列,并与蛋白质组学研究兼容. 相似文献
5.
Simone Kurz Chunsheng Jin ) Alba Hykollari Daniel Gregorich Barbara Giomarelli Gerardo R. Vasta Iain B. H. Wilson Katharina Paschinger 《The Journal of biological chemistry》2013,288(34):24410-24428
The eastern oyster (Crassostrea virginica) has become a useful model system for glycan-dependent host-parasite interactions due to the hijacking of the oyster galectin CvGal1 for host entry by the protozoan parasite Perkinsus marinus, the causative agent of Dermo disease. In this study, we examined the N-glycans of both the hemocytes, which via CvGal1 are the target of the parasite, and the plasma of the oyster. In combination with HPLC fractionation, exoglycosidase digestion, and fragmentation of the glycans, mass spectrometry revealed that the major N-glycans of plasma are simple hybrid structures, sometimes methylated and core α1,6-fucosylated, with terminal β1,3-linked galactose; a remarkable high degree of sulfation of such glycans was observed. Hemocytes express a larger range of glycans, including core-difucosylated paucimannosidic forms, whereas bi- and triantennary glycans were found in both sources, including structures carrying sulfated and methylated variants of the histo-blood group A epitope. The primary features of the oyster whole hemocyte N-glycome were also found in dominin, the major plasma glycoprotein, which had also been identified as a CvGal1 glycoprotein ligand associated with hemocytes. The occurrence of terminal blood group moieties on oyster dominin and on hemocyte surfaces can account in part for their affinity for the endogenous CvGal1. 相似文献
6.
Margaret Doherty Jonathan Bones Niaobh McLoughlin Jayne E. Telford Bryan Harmon Michael R. DeFelippis Pauline M. Rudd 《Analytical biochemistry》2013
Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5-h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies. 相似文献
7.
Gregory O. Staples Michael J. Bowman Catherine E. Costello Alicia M. Hitchcock James M. Lau Nancy Leymarie Christine Miller Hicham Naimy Xiaofeng Shi Joseph Zaia Professor 《Proteomics》2009,9(3):686-695
A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable, and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation, and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple MS with an on‐line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide–silica hydrophilic interaction chromatography (HILIC) in a chip‐based format for LC/MS of heparin, heparan sulfate (HS) GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built‐in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate (Ac) groups and acidic sulfate groups. In summary, chip based amide‐HILIC LC/MS is an enabling technology for GAG glycomics profiling. 相似文献
8.
Scott R. Kronewitter Hyun Joo An Maria Lorna de Leoz Carlito B. Lebrilla Suzanne Miyamoto Gary S. Leiserowitz 《Proteomics》2009,9(11):2986-2994
Annotation of the human serum N‐linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state‐transition networks. We find that at least 331 compositions are possible in the serum N‐linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high‐resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N‐linked glycan mass library that was used for accurate high‐throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan‐based markers. 相似文献
9.
The relative amount of high mannose structures within an N‐glycomic pool differs from one source to another, but quite often it predominates over the larger size complex type structures carrying biologically important glyco‐epitopes. An efficient method to separate these two classes of N‐glycans would significantly aid in detecting the lower abundant components by MS. Capitalizing on an initial observation that only high mannose type structures were recovered in the flow‐through fraction when peptide‐N‐glycosidase F digested peptides were passed through a C18 cartridge in 0.1% formic acid, we demonstrated here that native complex type N‐glycans can be retained by C18 cartridge and to be efficiently separated from both the smaller high mannose type structures, as well as de‐N‐glycosylated peptides by stepwise elution with increasing ACN concentration. The weak retention of the largely hydrophilic N‐glycans on C18 resin is dependent not only on size but also increased by the presence of α6‐fucosylation. This was shown by comparing the resulting N‐glycomic profiles of the washed and low‐ACN eluted fractions derived from both a human cancer cell line and an insect cell line. 相似文献
10.
Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0 Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs. 相似文献