Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors.

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.     

Structure of the F‐spondin domain of mindin,an integrin ligand and pattern recognition molecule

Mindin (spondin‐2) is an extracellular matrix protein of unknown structure that is required for efficient T‐cell priming by dendritic cells. Additionally, mindin functions as a pattern recognition molecule for initiating innate immune responses. These dual functions are mediated by interactions with integrins and microbial pathogens, respectively. Mindin comprises an N‐terminal F‐spondin (FS) domain and C‐terminal thrombospondin type 1 repeat (TSR). We determined the structure of the FS domain at 1.8‐Å resolution. The structure revealed an eight‐stranded antiparallel β‐sandwich motif resembling that of membrane‐targeting C2 domains, including a bound calcium ion. We demonstrated that the FS domain mediates integrin binding and identified the binding site by mutagenesis. The mindin FS domain therefore represents a new integrin ligand. We further showed that mindin recognizes lipopolysaccharide (LPS) through its TSR domain, and obtained evidence that C‐mannosylation of the TSR influences LPS binding. Through these dual interactions, the FS and TSR domains of mindin promote activation of both adaptive and innate immune responses.     

Impregnation of the Bacterial Cellulose Membrane with Biologically Produced Silver Nanoparticles

Different wound dressings with antibacterial property have been surveyed and one among them is bacterial cellulose (BC). Since the BC does not have antibacterial property, the biologically produced silver nanoparticles (SNPs) were impregnated into the BC. For the BC production, Hestrin–Schramm broth was used. Formation of the BC was proven by enzymatic hydrolysis. For SNPs production, the bacterial supernatant was treated with AgNO₃ and formation of SNPs was monitored through spectrophotometer, TEM and XRD. For impregnation of SNPs into the BC, the cleaned membrane was placed in the bacterial supernatant that contained 1 mmol of AgNO₃. The antibacterial assay was done for the BC/SNPs. Enzymatic hydrolysis proved the presence of the BC. Spectrophotometer and XRD results showed the formation of SNPs. TEM analysis revealed the presence of SNPs with sizes around 5–100 nm. SEM micrographs showed the impregnation of SNPs into the BC. Antibacterial test exhibited the antibacterial activity of the BC/SNPs.     

Voltammetric and mathematical evidence for dual transport mediation of serotonin clearance in vivo

The neurotransmitter serotonin underlies many of the brain's functions. Understanding serotonin neurochemistry is important for improving treatments for neuropsychiatric disorders such as depression. Antidepressants commonly target serotonin clearance via serotonin transporters and have variable clinical effects. Adjunctive therapies, targeting other systems including serotonin autoreceptors, also vary clinically and carry adverse consequences. Fast scan cyclic voltammetry is particularly well suited for studying antidepressant effects on serotonin clearance and autoreceptors by providing real‐time chemical information on serotonin kinetics in vivo. However, the complex nature of in vivo serotonin responses makes it difficult to interpret experimental data with established kinetic models. Here, we electrically stimulated the mouse medial forebrain bundle to provoke and detect terminal serotonin in the substantia nigra reticulata. In response to medial forebrain bundle stimulation we found three dynamically distinct serotonin signals. To interpret these signals we developed a computational model that supports two independent serotonin reuptake mechanisms (high affinity, low efficiency reuptake mechanism, and low affinity, high efficiency reuptake system) and bolsters an important inhibitory role for the serotonin autoreceptors. Our data and analysis, afforded by the powerful combination of voltammetric and theoretical methods, gives new understanding of the chemical heterogeneity of serotonin dynamics in the brain. This diverse serotonergic matrix likely contributes to clinical variability of antidepressants.

     

Comparison of analytical methods for profiling <Emphasis Type="Italic">N-</Emphasis> and <Emphasis Type="Italic">O-</Emphasis>linked glycans from cultured cell lines

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.     

Structure of a capsular polysaccharide isolated from Salmonella enteritidis

Salmonella enteritidis is a food-borne enteric human pathogen that can form a complex protective extracellular matrix. We describe here a component of this matrix which is distinct from other known salmonella extracellular polysaccharides such as cellulose and colanic acid. We have used glycosyl composition and linkage analysis, as well as 1D and 2D NMR spectroscopy to determine the structure of this polysaccharide. We propose that the primary saccharide in the S. enteritidis capsule has a branched tetrasaccharide repeating unit having the following structure: -->3)-alpha-D-Galp-(1-->2)-[alpha-Tyvp-(1-->3)]-alpha-D-Manp-(1-->4)-alpha-L-Rhap-(1-->. This structure is partially substituted on both tyvelose and galactose with a glucose-containing side chain. It further bears considerable similarity to the O antigen from this organism, a feature found in a number of other capsules from Gram-negative bacteria. In addition, we have detected fatty acids at levels that indicate the presence of a lipid anchor.     

Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

Integrin-growth factor receptor cross-talk plays a role in growth factor signaling, but the specifics are unclear. In a current model, integrins and growth factor receptors independently bind to their ligands (extracellular matrix and growth factors, respectively). We discovered that neuregulin-1 (NRG1), either as an isolated EGF-like domain or as a native multi-domain form, binds to integrins αvβ3 (with a K_D of 1.36 × 10⁻⁷ m) and α6β4. Docking simulation predicted that three Lys residues at positions 180, 184, and 186 of the EGF-like domain are involved in integrin binding. Mutating these residues to Glu individually or in combination markedly suppressed integrin binding and ErbB3 phosphorylation. Mutating all three Lys residues to Glu (the 3KE mutation) did not affect the ability of NRG1 to bind to ErbB3 but markedly reduced the ability of NRG1 to induce ErbB3 phosphorylation and AKT and Erk1/2 activation in MCF-7 and T47D human breast cancer cells. This suggests that direct integrin binding to NRG1 is critical for NRG1/ErbB signaling. Notably, stimulation of cells with WT NRG1 induced co-precipitation of ErbB3 with α6β4 and with αvβ3 to a much lower extent. This suggests that WT NRG1 induces integrin-NRG1-ErbB3 ternary complex formation. In contrast, the 3KE mutant was much less effective in inducing ternary complex formation than WT NRG1, suggesting that this process depends on the ability of NRG1 to bind to integrins. These results suggest that direct NRG1-integrin interaction mediates integrin-ErbB cross-talk and that α6β4 plays a major role in NRG-ErbB signaling in these cancer cells.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015