Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins

Antibiotic Permeation across the OmpF Channel: Modulation of the Affinity Site in the Presence of Magnesium

We characterize the rate-limiting interaction of the antibiotic 10 enrofloxacin with OmpF, a channel from the outer cell wall of Escherichia coli. 11 Reconstitution of a single OmpF trimer into planar lipid membranes allows 12 measurement of the ion current through the channel. Penetration of antibiotics causes 13 ion current blockages, and their frequency allows a conclusion on the kinetics of 14 channel entry and exit. In contrast to other antibiotics, enrofloxacin is able to block the 15 OmpF channel for several milliseconds, reflecting high affinities comparable to 16 substrate-specific channels such as the maltodextrin-specific maltoporin. Surprisingly, 17 the presence of a divalent ion such as Mg2+ leads to fast flickering with an increase in 18 the rates of association and dissociation. All-atom computer modeling provides the 19 most probable pathway able to identify the relevant rate-limiting interaction during antibiotic permeation. Mg2+ has a high affinity 20 for the aspartic acid at the 113 position (D113) in the center of the OmpF intracellular binding site. Therefore, the presence of Mg2+ 21 reverses the charge and enrofloxacin may cross the constriction region in its favorable orientation with the carboxylic group first

Structural basis for nutrient acquisition by dominant members of the human gut microbiota

The human large intestine is populated by a high density of microorganisms, collectively termed the colonic microbiota, which has an important role in human health and nutrition. The survival of microbiota members from the dominant Gram-negative phylum Bacteroidetes depends on their ability to degrade dietary glycans that cannot be metabolized by the host. The genes encoding proteins involved in the degradation of specific glycans are organized into co-regulated polysaccharide utilization loci, with the archetypal locus sus (for starch utilisation system) encoding seven proteins, SusA-SusG. Glycan degradation mainly occurs intracellularly and depends on the import of oligosaccharides by an outer membrane protein complex composed of an extracellular SusD-like lipoprotein and an integral membrane SusC-like TonB-dependent transporter. The presence of the partner SusD-like lipoprotein is the major feature that distinguishes SusC-like proteins from previously characterized TonB-dependent transporters. Many sequenced gut Bacteroides spp. encode over 100 SusCD pairs, of which the majority have unknown functions and substrate specificities. The mechanism by which extracellular substrate binding by SusD proteins is coupled to outer membrane passage through their cognate SusC transporter is unknown. Here we present X-ray crystal structures of two functionally distinct SusCD complexes purified from Bacteroides thetaiotaomicron and derive a general model for substrate translocation. The SusC transporters form homodimers, with each β-barrel protomer tightly capped by SusD. Ligands are bound at the SusC-SusD interface in a large solvent-excluded cavity. Molecular dynamics simulations and single-channel electrophysiology reveal a 'pedal bin' mechanism, in which SusD moves away from SusC in a hinge-like fashion in the absence of ligand to expose the substrate-binding site to the extracellular milieu. These data provide mechanistic insights into outer membrane nutrient import by members of the microbiota, an area of major importance for understanding human-microbiota symbiosis