Glycan binding avidity determines the systemic fate of adeno-associated virus type 9

Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions.     

Solution NMR of a 463-residue phosphohexomutase: domain 4 mobility, substates, and phosphoryl transfer defect

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. (13)C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k(cat). Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2-3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster (15)N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. (15)N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1-3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.     

The DOMON domains are involved in heme and sugar recognition

We expand the functionally uncharacterized DOMON domain superfamily to identify several novel families, including the first prokaryotic representatives. Using several computational tools we show that it is involved in ligand binding--either as heme- or sugar-binding domains. We present evidence that the DOMON domain along with the DM13 domain comprises a novel electron-transfer system potentially involved in oxidative modification of animal cell-surface proteins. Other novel versions might function as sugar sensors of histidine kinases of bacterial two component systems. Supplementary information: Supplementary data are available at Bioinformatics online and also at ftp://ftp.ncbi.nih.gov/pub/aravind/domon/.     

Magnetic resonance-guided, real-time targeted delivery and imaging of magnetocapsules immunoprotecting pancreatic islet cells

In type I diabetes mellitus, islet transplantation provides a moment-to-moment fine regulation of insulin. Success rates vary widely, however, necessitating suitable methods to monitor islet delivery, engraftment and survival. Here magnetic resonance-trackable magnetocapsules have been used simultaneously to immunoprotect pancreatic beta-cells and to monitor, non-invasively in real-time, hepatic delivery and engraftment by magnetic resonance imaging (MRI). Magnetocapsules were detected as single capsules with an altered magnetic resonance appearance on capsule rupture. Magnetocapsules were functional in vivo because mouse beta-cells restored normal glycemia in streptozotocin-induced diabetic mice and human islets induced sustained C-peptide levels in swine. In this large-animal model, magnetocapsules could be precisely targeted for infusion by using magnetic resonance fluoroscopy, whereas MRI facilitated monitoring of liver engraftment over time. These findings are directly applicable to ongoing improvements in islet cell transplantation for human diabetes, particularly because our magnetocapsules comprise clinically applicable materials.     

Spatiotemporal expression of ameloblastin isoforms during murine tooth development

Ameloblasts synthesize and secrete the enamel matrix proteins (amelogenin, ameloblastin, and enamelin). This investigation examined the profiles of ameloblastin in the ameloblasts and in the enamel matrix during different postnatal (PN) days (days 0-9) of development of mouse molar, using an antibody specific for C-terminal sequence of ameloblastin (Ct; GNKVHQPQVHNAWRF). Ameloblastin is found in three different molecular sizes (37, 55, and 66 kDa) in both ameloblasts and enamel matrix during PN development. In the ameloblasts, the sequence of expression of these fractions varied. The 37-kDa fraction was observed (even before the appearances of mRNA of the proteases, enamelysin and kallikrein-4) on days 0 and 1, persisted until day 3, and was not found thereafter. Other isoforms (55 and 66 kDa) distinctly appeared in ameloblasts after day 1, reached a peak on day 5, and remained thereafter. The Ct-positive granules appeared beaded in the ameloblasts on day 3. In the extracellular matrix, a 37-kDa (but not 66- or 55-kDa) fraction was detected on days 0 and 1 and remained in the matrix throughout the PN days. The larger isoforms (55 and 66 kDa) appeared in the enamel matrix from day 3 onward. On days 0-3, but not later, the 37-kDa isoform co-localizes with amelogenin in Tomes' process and formative enamel, as revealed by laser scan confocal microscopy. Autoradiography confirmed accumulation of 3H-labeled amelogenin trityrosyl motif peptide in the region of Tomes' process and formative enamel from day 0 to 3. These observations suggest that the 37-kDa isoform interacts with amelogenin during early tooth development.     

Zinc protects Ceratophyllum demersum L. (free-floating hydrophyte) against reactive oxygen species induced by cadmium

Evidence for Zn protection against Cd-induced reactive oxygen species in the free-floating hydrophyte Ceratophyllum demersum L. is presented in this paper. Metal treatments of 10 μmol/L Cd, 10 Cd μmol/L supplemented with Zn (10, 50, 100 and 200 μmol/L) and Zn-alone treatments of the same concentrations were used. Using 5,5 dimethyl pyrroline-N-oxide as the spin-probe, electron spin resonance spectra indicated a drastic increase in hydroxyl radicals (OH) in Cd-10 μmol/L treatments, which was closely correlating with the enhanced formation of hydrogen peroxide (H₂O₂) and generation of superoxide radical (O₂^?) triggered by the oxidation of NADPH. The supplementation of adding Zn (10–200 μmol/L) to the Cd-10 μmol/L treatments significantly decreased the production of free radicals especially by eliminating the precursors of OH through inhibition of NADPH oxidation. Cd-enhanced ROS production which substantially increased the oxidative products of proteins measured as carbonyls was effectively inhibited by Zn supplementation.