Fluorometric coupled enzyme assay for N-sulfotransferase activity of N-deacetylase/N-sulfotransferase (NDST).

N-Deacetylase/N-sulfotransferases (NDSTs) are critical enzymes in heparan sulfate (HS) biosynthesis. Radioactive labeling assays are the preferred methods to determine the N-sulfotransferase activity of NDST. In this study, we developed a fluorometric coupled enzyme assay that is suitable for the study of enzyme kinetics and inhibitory properties of drug candidates derived from a large-scale in silico screening targeting the sulfotransferase moiety of NDST1. The assay measures recombinant mouse NDST1 (mNDST1) sulfotransferase activity by employing its natural substrate adenosine 3'-phophoadenosine-5'-phosphosulfate (PAPS), a bacterial analog of desulphated human HS, Escherichia coli K5 capsular polysaccharide (K5), the fluorogenic substrate 4-methylumbelliferylsulfate and a double mutant of rat phenol sulfotransferase SULT1A1 K56ER68G. Enzyme kinetic analysis of mNDST1 performed with the coupled assay under steady state conditions at pH 6.8 and 37°C revealed Km (K5) 34.8 µM, Km (PAPS) 10.7 µM, Vmax (K5) 0.53 ± 0.13 nmol/min/µg enzyme, Vmax (PAPS) 0.69 ± 0.05 nmol/min/µg enzyme and the specific enzyme activity of 394 pmol/min/µg enzyme. The pH optimum of mNDST1 is pH 8.2. Our data indicate that mNDST1 is specific for K5 substrate. Finally, we showed that the mNDST1 coupled assay can be utilized to assess potential enzyme inhibitors for drug development.

A C19MC-LIN28A-MYCN Oncogenic Circuit Driven by Hijacked Super-enhancers Is a Distinct Therapeutic Vulnerability in ETMRs: A Lethal Brain Tumor.

Embryonal tumors with multilayered rosettes (ETMRs) are highly lethal infant brain cancers with characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a C19MC-LIN28A-MYCN circuit fueled by multiple complex regulatory loops including an MYCN core transcriptional network and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. Our data show that this powerful oncogenic circuit, which entraps an early neural lineage network, is potently abrogated by bromodomain inhibitor JQ1, leading to ETMR cell death.

NDST1 Preferred Promoter Confirmation and Identification of Corresponding Transcriptional Inhibitors as Substrate Reduction Agents for Multiple Mucopolysaccharidosis Disorders.

The stepwise degradation of glycosaminoglycans (GAGs) is accomplished by twelve lysosomal enzymes. Deficiency in any of these enzymes will result in the accumulation of the intermediate substrates on the pathway to the complete turnover of GAGs. The accumulation of these undegraded substrates in almost any tissue is a hallmark of all Mucopolysaccharidoses (MPS). Present therapeutics based on enzyme replacement therapy and bone marrow transplantation have low effectiveness for the treatment of MPS with neurological complications since enzymes used in these therapies are unable to cross the blood brain barrier. Small molecule-based approaches are more promising in addressing neurological manifestations. In this report we identify a target for developing a substrate reduction therapy (SRT) for six MPS resulting from the abnormal degradation of heparan sulfate (HS). Using the minimal promoter of NDST1, one of the first modifying enzymes of HS precursors, we established a luciferase based reporter gene assay capable of identifying small molecules that could potentially reduce HS maturation and therefore lessen HS accumulation in certain MPS. From the screen of 1,200 compounds comprising the Prestwick Chemical library we identified SAHA, a histone deacetylase inhibitor, as the drug that produced the highest inhibitory effects in the reporter assay. More importantly SAHA treated fibroblasts expressed lower levels of endogenous NDST1 and accumulated less 35S GAGs in patient cells. Thus, by using our simple reporter gene assay we have demonstrated that by inhibiting the transcription of NDST1 with small molecules, identified by high throughput screening, we can also reduce the level of sulfated HS substrate in MPS patient cells, potentially leading to SRT.

Characterization of the biosynthesis, processing and kinetic mechanism of action of the enzyme deficient in mucopolysaccharidosis IIIC.

Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino-terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N-acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction.

Purification and proteomic analysis of lysosomal integral membrane proteins.

Lysosomes are essential for normal function of cells. This is best illustrated by the occurrence of greater than 40 lysosomal storage diseases. While the enzymes of the luminal compartment have been widely studied usually in the context of these diseases, the composition of the enveloping membrane has received scant attention. Advances in mass spectrometry and proteomics have laid the necessary groundwork to facilitate investigation of membranes such as those of lysosomes, mitochondria, and other organelles to find novel proteins and novel functions. Pure lysosomes are a prerequisite, and we have successfully identified an abundance of membrane proteins from lysosomes of rat liver. Here, we describe two comparable and easy methods to isolate lysosomes from mouse or rat liver in sufficient quantities for proteomics studies. Also included is a comparison of the soluble, luminal proteins obtained from each of the two preparations separated by 2D immobilized pH gradient (IPG) sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Lysosomal membranes from beige mice contain higher than normal levels of endoplasmic reticulum proteins.

Chediak-Higashi syndrome is characterized by dysfunctional giant organelles of common origin, that is, lysosomes, melanosomes, and platelet dense bodies. Its defective gene LYST encodes a large molecular weight protein whose function is unknown. The Beige mouse also defective in Lyst is a good model of the human disease. Purified lysosomes from Beige and normal black mouse livers were used to carry out a proteomics study. Two-dimensional gel electrophoretic separation of soluble lysosomal proteins of Beige and normal mice revealed no major differences. The cleavable isotope-coded affinity tag (cICAT) technique was used to compare the composition of Beige and normal lysosomal membrane proteins. While the levels of common proteins, that is, Lamp1, Lamp2, and Niemann-Pick type C1, were decreased in Beige mice, there was an increase in the levels of endoplasmic reticulum (ER) resident proteins, for example, cytochrome P450, NADPH-cytochrome P450 oxidoreductase, and flavin-containing monooxygenase. Confocal microscopy confirmed that another ER protein, calnexin, colocalizes with Lamp1 on membranes of giant lysosomes from fibroblasts of Chediak-Higashi syndrome patient. Our results suggest that LYST may play a role in either preventing inappropriate incorporation of proteins into the lysosomal membrane or in membrane recycling/maturation.

Identification of the gene encoding the enzyme deficient in mucopolysaccharidosis IIIC (Sanfilippo disease type C).

Mucopolysaccharidosis IIIC (MPS IIIC), or Sanfilippo C, represents the only MPS disorder in which the responsible gene has not been identified; however, the gene has been localized to the pericentromeric region of chromosome 8. In an ongoing proteomics study of mouse lysosomal membrane proteins, we identified an unknown protein whose human homolog, TMEM76, was encoded by a gene that maps to 8p11.1. A full-length mouse expressed sequence tag was expressed in human MPS IIIC fibroblasts, and its protein product localized to the lysosome and corrected the enzymatic defect. The mouse sequence was used to identify the full-length human homolog (HGSNAT), which encodes a protein with no homology to other proteins of known function but is highly conserved among plants and bacteria. Mutational analyses of two MPS IIIC cell lines identified a splice-junction mutation that accounted for three mutant alleles, and a single base-pair insertion accounted for the fourth.

Identification of the hydrophobic glycoproteins of Caenorhabditis elegans.

Hydrophobic proteins such as integral membrane proteins are difficult to separate, and therefore to study, at a proteomics level. However, the Asn-linked (N-linked) carbohydrates (N-glycans) contained in membrane glycoproteins are important in differentiation, embryogenesis, inflammation, cancer and metastasis, and other vital cellular processes. Thus, the identification of these proteins and their sites of glycosylation in a well-characterized model organism is the first step toward understanding the mechanisms by which N-glycans and their associated proteins function in vivo. In this report, a proteomics method recently developed by our group was applied to identify 117 hydrophobic N-glycosylated proteins of Caenorhabditis elegans extracts by analysis of 195 glycopeptides containing 199 Asn-linked oligosaccharides. Most of the proteins identified are involved in cell adhesion, metabolism, or the transport of small molecules. In addition, there are 18 proteins for which no function is known or predictable by sequence homologies and two proteins which were previously predicted to exist only on the basis of genomic sequences in the C. elegans database. Because N-glycosylation is initiated in the lumen of the endoplasmic reticulum (ER), our data can be used to reassess the previously predicted subcellular localizations of these proteins. As well, the identification of N-glycosylation sites helps establish the membrane topology of the associated glycoproteins. Caenorhabditis elegans strains are presently available with mutations in 17 of the genes we have identified. The powerful genetic tools available for C. elegans can be used to make other strains with mutations in genes encoding N-glycosylated proteins and thereby determine N-glycan function.

A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides.

Glycosylated proteins on the cell surface have been shown to be essential for cell-cell interactions in development and differentiation. Our ultimate goal is to identify Asn-linked oligosaccharides that are directly involved in these critical in vivo functions. Because such oligosaccharides would be expected to reside on the integral plasma membrane proteins, and conventional two-dimensional gel techniques are ineffective at separating such proteins, we have developed a new approach to their identification on a proteomics scale from Caenorhabditis elegans. Membrane proteins are solubilized in guanidine-HCl, precipitated, and digested with trypsin. The glycopeptides are then separated by lectin chromatography. Next, glycopeptidase F digestion removes the oligosaccharides from the peptides and converts to Asp each Asn to which one was attached. The peptides are then analyzed by matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) mass spectrometry. Thus, the membrane glycoproteins are identified through the sequence tags of these peptides and the conversion of at least one deduced Asn residue to Asp at the Asn-X-Ser/Thr consensus sequence. To validate the utility of this approach, we have identified 13 membrane-bound N-glycosylated proteins from the major peaks observed on MALDI-Q-TOF analysis of our total glycopeptide fraction.

Functional post-translational proteomics approach to study the role of N-glycans in the development of Caenorhabditis elegans.

Glycosylation is one of the most common post-translational protein modifications. Carbohydrate-mediated interactions between cells and their environment are important in differentiation, embryogenesis, inflammation, cancer and metastasis and other processes. Humans and mice with mutations that prevent normal N-glycosylation show multi-systemic defects in embryogenesis, thereby proving that these molecules are essential for normal development; however, a large number of proteins undergo defective glycosylation in these human and mouse mutants, and it is therefore difficult to determine the precise molecular roles of specific N-glycans on individual proteins. We describe here a 'functional post-translational proteomics' approach that is designed to determine the role of N-glycans on individual glycoproteins in the development of Caenorhabditis elegans.