Dataset Information

Oligosaccharide Substrate Preferences of Human Extracellular Sulfatase Sulf2

ABSTRACT: In this study, we characterized the substrate preferences of human HSulf2 using HS oligosaccharides with various lengths and sulfation degrees from several naturally occurring HS sources by applying liquid chromatography mass spectrometry based glycomics methods.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Small Intestine Mucosa

SUBMITTER: Yu Huang

LAB HEAD: Joseph Zaia

PROVIDER: PXD001157 | Pride | 2014-07-22

REPOSITORIES: Pride

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Publications

Oligosaccharide substrate preferences of human extracellular sulfatase Sulf2 using liquid chromatography-mass spectrometry based glycomics approaches.

Huang Yu Y Mao Yang Y Buczek-Thomas Jo Ann JA Nugent Matthew A MA Zaia Joseph J

PloS one 20140815 8

Sulfs are extracellular endosulfatases that selectively remove the 6-O-sulfate groups from cell surface heparan sulfate (HS) chain. By altering the sulfation at these particular sites, Sulfs function to remodel HS chains. As a result of the remodeling activity, HSulf2 regulates a multitude of cell-signaling events that depend on interactions between proteins and HS. Previous efforts to characterize the substrate specificity of human Sulfs (HSulfs) focused on the analysis of HS disaccharides and ...[more]

PMID: 25127119

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Project description:Gene expression of adherent cells in a coculture of the human bone marrow stromal cell line HS-27a and peripheral blood monocytes (CD14+ cells) was compared to HS-27a cultured alone. Microarray experiments were conducted using CD14+ cells from 7 healthy donors over a period of >7 months. Monocytes were isolated by ficoll separation of whole blood, followed by labelling cells with CD14 monoclonal antibody Tuek4 and magnetic bead selection (Milteny rat anti mouse IgG2a+2b beads) on an AutoMacs. HS27a cells were plated in T75 flasks at approximately 80% confluence. The next day 1.5x10E6 freshly isloated human CD14+ cells were added to the culture. At 3 days the cultures were washed 3x with Hankâ??s buffer to remove nonadherent monocytes. Co-cultures and HS27a cultured alone were harvested by brief trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42ÂºC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS27a and HS27a-monocyte cocultures was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 ul of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were mean centered and linear transformed to obtain the log and linear values given in the data tables.

Project description:Fast-growing Eucalyptus grandis trees are one of the most efficient producers of wood in South Africa. It is essential to maximize the effectiveness of these plantations by increasing their productivity, the quality and value of their products. We used microarray-based DNA-amplified fragment length polymorphism (AFLP) analysis in combination with expression profiling to develop fingerprints and profile gene expression of wood-forming tissue of seven individual E. grandis trees. A 1532-probe cDNA microarray was constructed by arraying 768 cDNA-AFLP fragments and 810 cDNA library clones from seven individual E. grandis trees onto silanised slides. The results revealed that 32% of the spotted fragments showed distinct expression patterns (with a fold change of at least 1.4 or -1.4 and a p value of 0.01) and could be grouped into clusters representing co-expressed genes. Evaluation of the binary distribution of cDNA-AFLP fragments on the array showed that the individual genotypes could be discriminated. A simple, yet general method was developed for genotyping and expression profiling of wood-forming tissue of E. grandis trees differing in their splitting characteristics and in their lignin contents. Evaluation of gene expression profiles and the binary distribution of cDNA-AFLP fragments on the chip suggest that the prototype chip developed could be useful for transcript profiling and for the identification of Eucalyptus trees with preferred wood quality traits in commercial breeding programmes. Transcriptional profiling and genotyping of seven E. grandis trees , namely (741-H (high lignin), 108-L (low lignin), 243-L (low lignin), 1/23/4-HS (high splitting), 1/71/6-HS (high splitting), 1/91/7-LS (low splitting) and 1/92/7-LS (low splitting), were performed with an in-house spotted cDNA microarray. All trees were characterized for their splitting qualities and lignin content and only the trees best corresponding to the selected traits were used for further analysis (Verryn and Turner 2000). RNA was isolated and cDNA synthesis was performed. A reference design microarray experiment was conducted and tree 1/23/4-HS served as a reference. Samples were fluorescently labelled with Cy5 or Cy3 dye and co-hybridized overnight. Two biological and one technical replicate (using independent labelling reactions) was performed, each replication consisting of a reverse labelling experiment.

Dataset Information

Oligosaccharide Substrate Preferences of Human Extracellular Sulfatase Sulf2

Publications

Oligosaccharide substrate preferences of human extracellular sulfatase Sulf2 using liquid chromatography-mass spectrometry based glycomics approaches.

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