ChtVis-Tomato, a genetic reporter for in vivo visualization of chitin deposition in Drosophila

Chitin is a polymer of N-acetylglucosamine that is abundant and widely found in the biological world. It is an important constituent of the cuticular exoskeleton that plays a key role in the insect life cycle. To date, the study of chitin deposition during cuticle formation has been limited by the lack of a method to detect it in living organisms. To overcome this limitation, we have developed ChtVis-Tomato, an in vivo reporter for chitin in Drosophila. ChtVis-Tomato encodes a fusion protein that contains an apical secretion signal, a chitin-binding domain (CBD), a fluorescent protein and a cleavage site to release it from the plasma membrane. The chitin reporter allowed us to study chitin deposition in time lapse experiments and by using it we have identified unexpected deposits of chitin fibers in Drosophila pupae. ChtVis-Tomato should facilitate future studies on chitin in Drosophila and other insects

From 1,4-Disaccharide to 1,3-Glycosyl Carbasugar : Synthesis of a Bespoke Inhibitor of Family GH99 Endo-α-mannosidase

Understanding the enzyme reaction mechanism can lead to the design of enzyme inhibitors. A Claisen rearrangement was used to allow conversion of an α-1,4-disaccharide into an α-1,3-linked glycosyl carbasugar to target the endo-α-mannosidase from the GH99 glycosidase family, which, unusually, is believed to act through a 1,2-anhydrosugar "epoxide" intermediate. Using NMR and X-ray crystallography, it is shown that glucosyl carbasugar α-aziridines can act as reasonably potent endo-α-mannosidase inhibitors, likely by virtue of their shape mimicry and the interactions of the aziridine nitrogen with the conserved catalytic acid/base of the enzyme active site

An epoxide intermediate in glycosidase catalysis

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism

Structure of human endo-a-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

Mammalian protein N-linked glycosylation is critical for glycoprotein folding, quality control, trafficking, recognition, and function. N-linked glycans are synthesized from Glc3Man9GlcNAc2precursors that are trimmed and modified in the endoplasmic reticulum (ER) and Golgi apparatus by glycoside hydrolases and glycosyltransferases. Endo-a-1,2-mannosidase (MANEA) is the sole endoacting glycoside hydrolase involved in N-glycan trimming and is located within the Golgi, where it allows ER-escaped glycoproteins to bypass the classical N-glycosylation trimming pathway involving ER glucosidases I and II. There is considerable interest in the use of small molecules that disrupt N-linked glycosylation as therapeutic agents for diseases such as cancer and viral infection. Here we report the structure of the catalytic domain of human MANEA and complexes with substrate-derived inhibitors, which provide insight into dynamic loop movements that occur on substrate binding. We reveal structural features of the human enzyme that explain its substrate preference and the mechanistic basis for catalysis. These structures have inspired the development of new inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and dengue viruses in cellular models. These results may contribute to efforts aimed at developing broad-spectrum antiviral agents and help provide a more in-depth understanding of the biology of mammalian glycosylation

A family of dual-activity glycosyltransferasesphosphorylases mediates mannogen turnover and virulence in Leishmania parasites

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches

Phenotypes associated with 42 hr genes.

<p>A and B show the dorsal notum of Ore-R and <i>ap>CG8213</i>. <i>apterous-Gal4</i> drives expression in the cells that form the dorsal surface of the wing but not those that form the ventral surface. It also drives expression in the dorsal thorax (notum). Panels C-H show adult flies where a 42 hr gene was knocked down using <i>ap-Gal4</i>. Ore-R is shown (C) for comparison. I and J show a thoracic macrochaete from Ore-R and from <i>ap>CG8213</i>. Panels K-N show unmounted wings from Ore-R or knocked down 42 hr genes. Note the curved kd wings.</p

Heat maps for 4 groups of related genes.

<p>A. Annotated cuticle proteins. B. Genes with a known function in cuticle deposition or maturation. C. ZP domain protein encoding genes. D. Genes that regulate transcription.</p

Similar results are obtained by both RNAseq and RT-qPCR.

<p>A. The relative gene expression patterns for the two mRNAs encoded by the CG1005 gene. Note the similar pattern seen with both RNAseq and RT-qPCR. B. The expression pattern for <i>dyl</i> and two isoforms of <i>Cht6</i>. Note that Cht6-RG expression follows a similar pattern to <i>dyl</i>. C. The expression patterns for two isoforms of <i>mwh</i> and CG14257 show qualitatively similar changes when assayed by both RNAseq and RT-qPCR. Note that relative expression values as a function of time and not absolute expression values are shown in this figure.</p

Number of genes whose expression level changed between time points.

<p>Number of genes whose expression level changed between time points.</p

Models to explain the procuticle phenotypes of 42 hr genes.

<p>In the Instructive 1 model the 42 hr proteins form the envelope, which signals back to the cell to organize the cytoskeleton so that the epicuticle and procuticle is secreted in the proper pattern. In Instructive 2 the 42 hr proteins form the envelope and when the proteins that form the epicuticle are secreted they bind to and are patterned by envelope and 42 hr proteins. A similar situation could result in the epicuticle patterning the procuticle. In the Platform model the 42 hr proteins are not part of the envelope but they form a complex that is essential for the patterned secretion of the proteins that form the 3 cuticular layers. Our second model is that many of the 42 hr genes do not encode proteins that are part of the envelope. Rather they would form a “platform” or complex that in some way mediates the tight juxtaposition of the cuticle and the apical surface of the epithelial cells and that this platform is needed for the proper deposition of the envelope and other cuticular components [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006100#pgen.1006100.ref022" target="_blank">22</a>].</p