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

Developing a Potential Substrate Reduction Therapy for Six Mucopolysaccharidoses by Decreasing NDST1 Activity

Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.MAS

Western blot analysis of the endogenous N-acetyltransferase protein in three human fibroblast cell lines.

<p>Fibroblast from an unaffected individual (WT), a patient with I-Cell disease, and a MPS IIIC patient were extracted with 1% DDM, 20 µg each of extracted protein were separated by SDS-PAGE, and the N-acetyltransferase proteins visualized using the N-terminal antibody. The blot was reprobed with anti GAPDH as a loading control. The specific activity of each extract is shown at the bottom, ND is not detected.</p

Immobilized [3H]acetylated N-acetyltransferase-His8Flag intermediate cannot be detected in immunoprecipitation experiments with anti-Flag M2 beads.

1<p>Anti-Flag beads were incubated with extracts from HeLa cell expressing (Transfected) or not expressing (Untransfected) N-acetyltransferase-His8Flag, washed and then incubated with [3H]acetyl-CoA for the given time and pH, at room temperature and with or without 1.3 mM of lipids containing 20% PI.</p>2<p>N-acetyltransferase activity in nmoles of MU produced/hour, and the concentration of protein (pmoles), calculated based on the specific activity of the purified transferase, are given.</p>3<p>The data represent three independent beads binding experiments and assays.</p>4<p>Not detectable.</p

Kinetic parameters of the N-acetyltransferase-His8Flag.

1<p>Concentrations were varied between 0.01 and 0.5 mM while the AcCoA concentration was fixed at 2 mM.</p>2<p>Concentrations were varied between 0.083 and 2 mM while the MU-GlcNH₂ concentration was fixed at 1 mM.</p

The protein expressed by a construct encoding myc-N-acetyltransferase-His8Flag was analyzed by Western blotting.

<p>(A) Extracts from HeLa cells permanently expressing the singly-tagged N-acetyltransferase-His8Flag (lane 1) were compared with HeLa cell transiently expressing myc-N-acetyltransferase-His8Flag (lane 2) using the N-terminal N-acetyltransferase antibody. The levels of transferase activity in the extracts are given below each lane. (B) The oligosaccharides present on the doubly-tagged myc-N-acetyltransferase-His8Flag protein (lane 1) were analyzed based on endo-H (lane 2) and/or PNGase (lane 3) sensitivities by Western blotting using a myc antibody.</p

Effects of lipids on N-acetyltransferase activity are illustrated.

<p>Each set of data points represents the average of triplicate determinations of N-acetyltransferase activity with their standard deviations (SD) shown as error bars (A) DDM extracts from permanently transfected HeLa cells were serially diluted in CP buffer, pH 5.5 (filled diamonds), CP buffer containing 0.25% HSA (filled squares) or CP buffer containing 1.3 mM 20% PI (filled circles with the error bars representing SD too small to see, connected by the best fit line, R = 1) (X-axis, total extracted protein; Y-axis, transferase activity in nmol/h). (B) Equal amounts of anti-Flag-column purified N-acetyltransferase were assayed in the presence of increasing concentrations of lipids (X-axis, mM) with decreasing mole% of negatively charged PI (40-0% PI+40–80% PC+20% CH); 40% PI, darkly shaded bars; 20% PI, grey hatched bars; or PC (0% PI), white bars. (C) Stability of identical amounts of purified N-acetyltransferase in CP buffer pH 5.5, containing either 0.25% (w/v) HSA (white bars) or 1.3 mM, 20% PI, lipids (shaded bars) (X-axis, hours at 37°C; Y-axis transferase activity in nmol/h).</p

Purification of N-acetyltransferase by anti-Flag affinity chromatography.

<p>(A) Lane 1- SDS-PAGE separation of the purified enzyme under reducing conditions detected with fluorescent SYPRO Ruby protein stain. Also shown is a dilution series of BSA used to calculate the protein levels in the transferase sample and the specific activity of the purified enzyme; and (B) Western blotting using either an antibody against the Gln53-Asn156 epitope (N-term), or against the C-terminal Flag tag (Flag). Lane-1 contains the transferase that was bound, eluted with Flag peptides and then denatured, while lane 2 contains enzyme that was directly released and denatured from the anti-Flag column with SDS sample buffer (containing a reducing agent). Aliquots of the initial 1% DDM extract (Ex) were also examined by Western blotting. Bands marked “X” in the Ex lanes are non-specific proteins detected by the antibodies (the ∼30 kDa X band is only seen with the rabbit Flag antibody). (C) The effects of incubating the purified protein at 37°C for 0–2.5 h without added lipids were examined by Western blotting using the N-terminal antibody.</p

Western blot analyses of the N-acetyltransferase-His8Flag protein from the “Lys” fraction of <b>Figure 6</b> (lane 1) treated with either endo-H (lane 2) or PNGase (lane 3).

<p>Proteins were detected with either, (A) the N-terminal N-acetyltransferase antibody or (B) the Flag M2 antibody.</p