Biochemical Characterization of Human Heparan Sulfate 6-O-Endosulfatase

Heparan sulfate (HS) is a complex and diverse polysaccharide abundantly found on the cell surface of cells from several different species. The HS biosynthetic machinery creates very heterogeneous structures that interact with a variety of proteins that result in important biological functions. In addition, HS can be remodeled in the extracellular matrix by a novel class of extracellular sulfatases (Sulfs) which selectively remove 6-O-sulfo groups from glucosamine residues within HS. The activities of Sulfs have been correlated with various biological activities relating to embryonic development and cancer. Therefore, understanding and utilizing Sulf activity can aid in understanding the structure-function relationship of HS as well as aid in developing and tailoring HS based therapies. The goals of this work were to investigate the substrate specificity of human 6-O-endosulfatase isoform 2 (HSulf-2) as well as to utilize HSulf-2 editing activity to tailor anticoagulant HS structures. The use of synthetic polysaccharides along with active HSulf-2 found in the conditioned medium of mammalian cells has allowed for the investigation of the substrate specificity of HSulf-2. Results demonstrated that HSulf-2 is selective toward 6-O-sulfo groups on glucosamine residues that are found in moderately and highly sulfated domains of HS. In addition, 2-O-sulfation of uronic acid and N-sulfation of glucosamine are necessary for serving as a substrate. Results also showed HSulf-2 can tailor HS anticoagulant structures and is capable of maintaining anticoagulant properties while reducing the binding of HS to other proteins. The biochemical characterization of a human heparan sulfate 6-O-endosulfatase as described herein provides a novel method for determining the substrate specificity of HSulf-2. Because Sulf treated HS maintains excellent anticoagulant activity, results of my work open up a new approach to prepare anticoagulant HS with reduced side effects. The future development of this project could elucidate important structure-function relationships as well as become a valuable tool in drug discovery

Substrate specificity of 6-O-endosulfatase (Sulf-2) and its implications in synthesizing anticoagulant heparan sulfate

Heparan sulfate (HS) 6-O-endosulfatase (Sulf) catalyzes the hydrolysis of 6-O-sulfo groups from HS polysaccharides. The resultant HS has reduced sulfation levels and displays altered biological activities. The Sulfs have been associated with several cancers and developmental problems and could function as a tool for editing specific HS structures. Here, we characterize the substrate specificity of human Sulf-2 using site-specifically radiolabeled synthetic polysaccharides. The enzyme was expressed and harvested from the conditioned medium of Chinese hamster ovary cells transfected with Sulf-2 expression plasmids. The uniquely [35S]sulfated polysaccharides were prepared using purified recombinant HS biosynthetic enzymes. We found that Sulf-2 is particularly effective in removing the 6-O-sulfo group residing in the trisulfated disaccharide repeating unit comprising 2-O-sulfated uronic acid and N-sulfated 6-O-sulfo glucosamine, but can also hydrolyze sulfo groups from N- and 6-O-sulfated disaccharides. In addition, we found that Sulf-2 treatment significantly decreases HS's ability to bind to platelet factor 4 (PF4), a chemokine, while binding to antithrombin is maintained. Because HS–PF4 complexes are the initiating cause of heparin-induced thrombocytopenia, this finding provides a promising strategy for developing heparin therapies with reduced side effects. Further understanding of Sulf-2 activity will help elucidate HS structure–function relationships and provide a valuable tool in tailoring HS-based anticoagulant drugs

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Variable metastatic potentials correlate with differential plectin and vimentin expression in syngeneic androgen independent prostate cancer cells.

Prostate cancer is a clinically heterogeneous disease, ranging from indolent asymptomatic disease to very aggressive metastatic and life threatening forms of the disease. Distant metastasis represents the major lethal cause of prostate cancer. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. To understand the molecular mechanisms of prostate cancer metastasis and identify markers with metastatic potential, we have analyzed protein expression in two syngeneic prostate cancer cells lines PC3-N2 and PC3-ML2 using isobaric tags for relative and absolute quantitation labeling and multi-dimensional protein identification technology liquid chromatography matrix assisted laser desorption ionization tandem mass spectrometry. PC3-N2 is lowly metastatic while PC3-ML2 highly metastatic. A total of 1,756 proteins were identified in the analyses with 130 proteins showing different expression levels (p<0.01) in the two cell lines. Out of these, 68 proteins were found to be significantly up-regulated while 62 are significantly down-regulated in PC3-ML2 cells compared with PC3-N2 cells. The upregulation of plectin and vimentin which were the most significantly differentially expressed were validated by Western blot and their functional relevance with respect to invasion and migration was determined by siRNA gene silencing. To our knowledge, this study is the first to demonstrate that up-regulation of vimentin and plectin expression positively correlates with the invasion and metastasis of androgen-independent PCA

Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes

Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT2 PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions

Comparative Metabolomic and Lipidomic Analysis of Phenotype Stratified Prostate Cells.

Prostate cancer (PCa) is the most prevalent cancer amongst men and the second most common cause of cancer related-deaths in the USA. Prostate cancer is a heterogeneous disease ranging from indolent asymptomatic cases to very aggressive life threatening forms. The goal of this study was to identify differentially expressed metabolites and lipids in prostate cells with different tumorigenic phenotypes. We have used mass spectrometry metabolomic profiling, lipidomic profiling, bioinformatic and statistical methods to identify, quantify and characterize differentially regulated molecules in five prostate derived cell lines. We have identified potentially interesting species of different lipid subclasses including phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), glycerophosphoinositols (PIs) and other metabolites that are significantly upregulated in prostate cancer cells derived from distant metastatic sites. Transcriptomic and biochemical analysis of key enzymes that are involved in lipid metabolism demonstrate the significant upregulation of choline kinase alpha in the metastatic cells compared to the non-malignant and non-metastatic cells. This suggests that different de novo lipogenesis and other specific signal transduction pathways are activated in aggressive metastatic cells as compared to normal and non-metastatic cells

Proteomic Analysis of Disease Stratified Human Pancreas Tissue Indicates Unique Signature of Type 1 Diabetes

<div><p>Type 1 diabetes (T1D) and type 2 diabetes (T2D) are associated with functional beta cell loss due to ongoing inflammation. Despite shared similarities, T1D is an autoimmune disease with evidence of autoantibody production, as well as a role for exocrine pancreas involvement. Our hypothesis is that differential protein expression occurs in disease stratified pancreas tissues and regulated proteins from endocrine and exocrine tissues are potential markers of disease and potential therapeutic targets. The study objective was to identify novel proteins that distinguish the pancreas from donors with T1D from the pancreas from patients with T2D, or autoantibody positive non-diabetic donors. Detailed quantitative comprehensive proteomic analysis was applied to snap frozen human pancreatic tissue lysates from organ donors without diabetes, with T1D-associated autoantibodies in the absence of diabetes, with T1D, or with T2D. These disease-stratified human pancreas tissues contain exocrine and endocrine tissues (with dysfunctional islets) in the same microenvironment. The expression profiles of several of the proteins were further verified by western blot. We identified protein panels that are significantly and uniquely upregulated in the three disease-stratified pancreas tissues compared to non-disease control tissues. These proteins are involved in inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to, and likely involved in, T1 and T2 diabetes pathogenesis. Several new proteins were differentially upregulated in prediabetic, T1D, and T2D pancreas. The results identify proteins that could serve as novel prognostic, diagnostic, and therapeutic tools to preserve functional islet mass in Type 1 Diabetes.</p></div

Functional categories and biological processes of all the proteins that are identified in ND, AAb+, T1D and T2D cases.

<p>Functional categories and biological processes of all the proteins that are identified in ND, AAb+, T1D and T2D cases.</p

Validation of differentially upregulated of specific proteins in pancreas tissue lysates.

<p>Normalized total protein lysates from pooled normal, Aab+, T1D and T2D samples were subjected Western blot analysis to detect for REGIIIα/γ (Panel A), Olfactomedin 4 (Panel B), and ENPP1 (Panel C),, . GAPDH detection was included as a loading control. ImageJ analysis was used to confirm the expression of the three proteins after normalization using GAPDH values. The bar graphs represent data from triplicate analyses.</p

(A) Venn diagram comparison of uniquely upregulated proteins in AAb+, T1D and T2D cases compared to ND.

<p>The cut-off for the threshold fold change differences were ≥ 2.0 for upregulation with p- values < 0.05. <b>(B).</b> Ingenuity pathway analysis showing top interaction network for uniquely upregulated proteins in AAb+ cases. Those highlighted with red color are upregulated genes and those with green are downregulated genes. The names of these genes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135663#pone.0135663.s018" target="_blank">S8 Table</a>.</p