Insights into the catalytic mechanism of a retaining xylanase from Cellulomonas fimi

The family 10 xylanase from Cellulomonas fimi (Cex) is an important model enzyme on which numerous mechanistic studies have been performed. This enzyme catalyzes the hydrolysis of β-glycosidic linkages via a double-displacement mechanism involving the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate with net retention of stereochemistry at the centre undergoing substitution. The finer details of the mechanism of this enzyme were investigated in three studies in order to gain a better understanding of this family of enzymes. In the first study presented in Chapter 2, the roles of key active-site residues in the catalytic mechanism of Cex were investigated by utilizing site-directed mutagenesis in combination with steady state kinetic analyses and pH-rate dependencies. The rate-determining step for the aryl substrates tested remains deglycosylation for many of the enzymes, while the altered pH profiles demonstrate a role for these highly conserved residues in the hydrogen-bond network responsible for maintaining the ionization state of the two catalytic residues. In Chapter 3, a second study addresses a fundamental enquiry of mechanistic enzymology; that is, how distal and proximal substrate interactions influence catalysis. By systematically removing hydrogen-bonding interactions through modification, individually, of substrate and enzyme, deep insight is gained into the effects of these modifications on each step of the hydrolysis reaction catalyzed by Cex and a family 11 xylanase (Bcx). The data obtained provide significant insight into the contributions of hydrogen-bonding interactions at the distal and proximal sites. The strongest bond energies were measured in the proximal site, suggesting that these interactions are critical for substrate binding and bond hydrolysis. A particularly important finding of this study is that both 'uniform' and 'differential' binding interactions are recruited in the active site of a single enzyme. The third study, presented in Chapter 4, examines how well a series of five high affinity inhibitors mimic the transition state of Cex as a function of the sp²- or sp³ -hybridization state of the "anomeric carbon". Kinetic parameters for o-nitrophenyl β-xylobioside were determined, and very good correlations were observed in logarithmic plots relating the K[sub i] value for the sp² -hybridized class of inhibitor with 10 mutants and k[sub cat]/K[sub m] for the hydrolysis of the substrate by the corresponding mutants. The dependence was significantly less in the plot of log(and k[sub cat]/K[sub m]) versus log(1/K[sub i]) for the sp³-hybridized class of inhibitor, indicating that the sp²-hybridized class of inhibitors more closely mimics the geometry of the transition state than does the sp³-hybridized class of inhibitors.Science, Faculty ofChemistry, Department ofGraduat

Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy

<div><p>The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI) as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic <i>mdx^4cv</i> mice were systemically treated with a recombinant adeno-associated viral (AAV) vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus) when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in histological sections. T2 relaxation, alongside diffusion and magnetization transfer effects provides useful data towards the goal of non-invasively monitoring the treatment of muscular dystrophy.</p></div

Tissue Sample Analysis.

<p>Histology was done on the left hindlimbs of all mice involved in the study. Representative T2 images are present (A) and (B). (C) Representative H&E images of an untreated <i>mdx</i> mouse at 1.6x and (E) is a 20x magnification of the gastrocnemius (GA) muscle (boxed area in (C)). (I) Representative Masson trichrome staining from an untreated <i>mdx</i> mouse at 1.6x and (F) is a 20x magnification of the GA muscle (boxed area in (C)). (D) Representative H&E images of a treated <i>mdx</i> mouse at 1.6x magnification and (G) is a 20x magnification of the tibialis anterior (TA) muscle (boxed area in (D)). (J) Representative Masson trichrome staining from a treated <i>mdx</i> mouse at 1.6x magnification and (H) is a 20x magnification of the TA muscle (boxed area in (J)).</p

Systemic administration of rAAV6-μDys enhances the structural properties of EDL muscles in dystrophic mice.

<p>(A) Treatment restored expression of dystrophin in the majority of EDL myofibers of treated mice, in contrast with no expression in the muscles of untreated mice and complete expression in the muscles of wild-type mice. (B) Dystrophin-positive myofibers of treated EDL muscles (mdx T Dys(+)) were larger in diameter (median myofiber diameter; wild-type, 54 μm; dystrophic, 43 μm; treated dystrophin-positive fibers, 52 μm). Shown is the mean and distribution (25th and 75th percentile are represented by the box, and the whiskers represent the farthest diameter of muscle fibers). (C) There was no change in central nucleation compared with the myofibers in the muscles of untreated mice. (D) EDL muscle mass was restored to wild-type values in treated dystrophic mice. <i>n</i> = 3 for WT; <i>n</i> = 3 for <i>mdx</i>; and <i>n</i> = 4 for <i>mdx</i> T. Error bars represent SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ***<i>P</i> < 0.001.</p

T2 values analyzed for TA, GA and SOL muscles.

<p>Graphs for the quantified image values taken from the T2 modality for different muscle types of TA, GA and SOL muscles—all images were reviewed and measured. The graphs on the left column depict average T2% change values for both treated and untreated <i>mdx</i> mice at each time point for each muscle type while the graphs on the right display averaged T2 values for all mice including the normal (the normal mice had both pre and post time point data values averaged). *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 and ****<i>P</i> < 0.0001.</p

ADC values analyzed for TA, GA and SOL muscles.

<p>Graphs for the quantified image values taken from the ADC modality for different muscle types of TA, GA and SOL muscles—all images were reviewed and measured. The graphs on the left column depict average ADC % change values for both treated and untreated <i>mdx</i> mice at each time point for each muscle type while the graphs on the right display averaged ADC values for all mice including the normal (the normal mice had both pre and post time point data values averaged).</p

Immunofluorescent staining of 9 month EDL muscle for laminin (red), dystrophin (green), and nuclei (blue).

<p>(A) A composite cross-sectional image of an EDL muscle from a treated <i>mdx</i> mouse and a detail of the boxed area. (B) Control C57BL/6 EDL muscle sections demonstrate normal sarcolemmal localization of dystrophin and normal morphology. (C) Staining of EDL sections from <i>mdx</i> mice reveals the absence of dystrophin and demonstrates morphological characteristics of dystrophy, including variation in fiber size and abundant centrally located myonuclei. Scale bars: 100 μm (A-C); 1 mm (composite image).</p

Whole-body micro-dystrophin gene transfer to the musculature using rAAV6.

<p>AAV6.CK8.H3μDys was administered by retro-orbital injection of 3-month-old <i>mdx</i> mice at a dose of 10¹³ vg, and tissues were analyzed 6 months later. All striated muscles were found to express widespread levels of the human micro-dystrophin. Shown are representative cross-sections of the TA, EDL, soleus, gastrocnemius, quadriceps, and diaphragm muscles immunostained with a rabbit polyclonal antibody against the N-terminal domain of dystrophin. Top row, muscles from wild-type mice; middle row, muscles from <i>mdx</i> mice; bottom row, muscles from <i>mdx</i> mice injected with rAAV6.CK8.H3μDys. Scale bar: 100 μm.</p

Anti-dystrophin staining for TA, GA and SOL muscles.

<p>Anti-dystrophin sAnti-dystrophin staining (green) shows clusters of transduced myofibers in representative sections of (A) TA, (B) GA, and (C) SOL muscles. Negative fibers (red) revealed morphological features of necrosis. (D) Between 50 to 65% of the total cross-sectional myofiber area of the selected muscles stained positive for dystrophin (<i>n</i> = 4). Scale bar: 500 μm.</p