N-linked glycosylation of Kv1.2 voltage-gated potassium channel facilitates cell surface expression and enhances the stability of internalized channels.

KEY POINTS: Kv1.2 and related voltage-gated potassium channels have a highly conserved N-linked glycosylation site in the first extracellular loop, with complex glycosylation in COS-7 cells similar to endogenous Kv1.2 glycosylation in hippocampal neurons. COS-7 cells expressing Kv1.2 show a crucial role of this N-linked glycosylation in the forward trafficking of Kv1.2 to the cell membrane. Although both wild-type and non-glycosylated mutant Kv1.2 channels that have reached the cell membrane are internalized at a comparable rate, mutant channels are degraded at a faster rate. Treatment of wild-type Kv1.2 channels on the cell surface with glycosidase to remove sialic acids also results in the faster degradation of internalized channels. Glycosylation of Kv1.2 is important with respect to facilitating trafficking to the cell membrane and enhancing the stability of channels that have reached the cell membrane. ABSTRACT: Studies in cultured hippocampal neurons and the COS-7 cell line demonstrate important roles for N-linked glycosylation of Kv1.2 channels in forward trafficking and protein degradation. Kv1.2 channels can contain complex N-linked glycans, which facilitate cell surface expression of the channels. Additionally, the protein stability of cell surface-expressed Kv1.2 channels is affected by glycosylation via differences in the degradation of internalized channels. The present study reveals the importance of N-linked complex glycosylation in boosting Kv1.2 channel density. Notably, sialic acids at the terminal sugar branches play an important role in dampening the degradation of Kv1.2 internalized from the cell membrane to promote its stability.

Increased neuronal activity fragments the Golgi complex.

The Golgi complex is essential for many aspects of cellular function, including trafficking and sorting of membrane and secretory proteins and posttranslational modification by glycosylation. We observed reversible fragmentation of the Golgi complex in cultured hippocampal neurons cultured in hyperexcitable conditions. In addition, Golgi fragmentation was found in cultured neurons with hyperactivity due to prolonged blockade of GABA(A)-mediated inhibition or withdrawal of NMDA receptor antagonism. The interplay between neuronal hyperactivity and Golgi structure established in this study thus reveals a previously uncharacterized impact of neuronal activity on organelle structure. This finding may have important roles in protein processing and trafficking in the Golgi as well as effects on neuronal signaling.

Structural and Mechanistic Regulation of the Pro-degenerative NAD Hydrolase SARM1.

The NADase SARM1 is a central switch in injury-activated axon degeneration, an early hallmark of many neurological diseases. Here, we present cryo-electron microscopy (cryo-EM) structures of autoinhibited (3.3 Å) and active SARM1 (6.8 Å) and provide mechanistic insight into the tight regulation of SARM1's function by the local metabolic environment. Although both states retain an octameric core, the defining feature of the autoinhibited state is a lock between the autoinhibitory Armadillo/HEAT motif (ARM) and catalytic Toll/interleukin-1 receptor (TIR) domains, which traps SARM1 in an inactive state. Mutations that break this lock activate SARM1, resulting in catastrophic neuronal death. Notably, the mutants cannot be further activated by the endogenous activator nicotinamide mononucleotide (NMN), and active SARM1 is product inhibited by Nicotinamide (NAM), highlighting SARM1's functional dependence on key metabolites in the NAD salvage pathway. Our studies provide a molecular understanding of SARM1's transition from an autoinhibited to an injury-activated state and lay the foundation for future SARM1-based therapies to treat axonopathies.

Antibacterial cyclic D,L-alpha-glycopeptides.

We report the design, synthesis, membrane activity, biophysical characterization, and in vitro antibacterial activities of cationic cyclic D,L-alpha-glycopeptides bearing d-glucosamine (GlcNH(2)), D-galactose (Gal), or D-mannose (Man) glycosyl side chains.

Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase.

The 35 kDa thioesterase (TE) domain excised from the megadalton tyrocidine synthetase (Tyc Syn) retains autonomous capacity to macrocyclize peptidyl thioesters to D-Phe1-L-Leu10-macrolactams. Since a number of nonribosomal peptides undergo O-glycosylation events during tailoring to gain biological activity, the Tyc Syn TE domain was evaluated for cyclization capacity with glycosylated peptidyl-S-NAC substrates. First, Tyr7 was replaced with Tyr(beta-D-Gal) and Tyr(beta-D-Glc) as well as with Ser-containing beta-linked D-Gal, D-Glc, D-GlcNAc, and D-GlcNH2, and these new analogs were shown to be cyclized with comparable kcat/Km catalytic efficiency. Similarly, Gal- or tetra-O-acetyl-Gal-Ser could also be substituted at residues 5, 6, and 8 in the linear decapeptidyl-S-NAC sequences and cyclized without substantial loss in catalytic efficiency by Tyc Syn TE. The cyclic glycopeptides retained antibiotic activity as membrane perturbants in MIC assays, opening the possibility for library construction of cyclic glycopeptides by enzymatic macrocyclization.

An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging.

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.

Mechanisms of distribution and targeting of neuronal ion channels.

The discovery and development of pharmaceutical drugs targeting ion channels is important for treating a variety of medical conditions and diseases. Ion channels are expressed ubiquitously throughout the body, and are involved in many basic physiological processes. Neuronal ion channels are particularly appealing drug targets, and recent advances in screening ion channel function using optical-based and electrophysiological technologies have improved drug development in this field. Moreover, methods for the discovery of peptide-based neurotoxins and other natural products have proven useful in the pharmacological assessment of ion channel structure and function, while also contributing to the identification of lead molecules for drug development.

Vancomycin analogues containing monosaccharides exhibit improved antibiotic activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy.

Many natural products contain carbohydrate moieties that contribute to their biological activity. Manipulation of the carbohydrate domain of natural products through multiple glycosylations to identify new derivatives with novel biological activities has been a difficult and impractical process. We report a practical one-pot enzymatic approach with regeneration of cosubstrates to synthesize analogues of vancomycin that contain an N-alkyl glucosamine, which exhibited marked improvement in antibiotic activity against a vancomycin-resistant strain of Enterococcus.

Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen.

Understanding the specificity of cell-surface carbohydrates interaction with antibodies and receptors is important for the development of new therapeutics and high-sensitivity diagnostics. This approach is, however, limited to the availability of natural and truncated sequences of the oligosaccharides and the sensitivity of the assay system. Reported here is the synthesis of the cancer antigen Globo H hexasaccharide, an epitope found on the cell surface of breast, prostate, and ovarian cancers, and its truncated sequences by using the programmable one-pot synthesis strategy. The saccharides were then arrayed covalently on glass slides with different density and used for the fluorencense-based binding analysis of two monoclonal antibodies against Globo H and the serum from breast cancer patients, to define the specificity of these antibodies. It was shown that the terminal tetrasaccharide binds the monoclonal antibodies equally well as does the hexasaccharide and the fucose residue is required for effective binding. The serum binds both the defucosylated pentasaccharide and the fucosylated hexasaccharide without a significant difference, perhaps because of the polyclonal nature of the serum or the presence of diverse immune responses to different sugar epitopes at various stages. This method requires very small amounts of materials and is more effective and sensitive than the traditional ELISA method, and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment.

The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit.

The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtA(C) protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtA(C) does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtA(C) to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtA(C). Assays of epitope-tagged wild-type and mutant variants of CgtA(C) indicate that the C terminus of CgtA(C) is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtA(C) alleles to function in vivo. Depletion of CgtA(C) led to perturbations in the polysome profile, raising the possibility that CgtA(C) is involved in ribosome assembly or stability.

DNA detection and signal amplification via an engineered allosteric enzyme.

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.