Amino acid chalcogen analogues as tools in peptide and protein research

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur‐containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl‐tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl‐ and phenylalanyl‐tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl‐tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.     

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion: Analysis of N‐ and O‐linked glycans and identification and elimination of a xylose‐based O‐linked tetrasaccharide core in the linker region

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T₄₀₇ and S₄₀₉. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S₄₁₈. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E₄₁₆ (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E₄₁₆GS₄₁₈G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.     

Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.     

Immunohistochemical study of localization of gamma-glutamyl transpeptidase in the rat brain

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.     

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1β promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.     

Synthesis,Structure, and Biological Activities of a New μ‐Oxamido‐Bridged Dicopper(II) Complex: The Influence of Hydrophobicity of Bridging Ligand on DNA Binding and Cytotoxic Activities

A new μ‐oxamido‐bridged dicopper(II) complex, [Cu₂(papo)(H₂O)‐ (phen)]Cl·CH₃OH·H₂O, where H₃papo and phen represent N‐(2‐hydroxyphenyl)‐N'‐(3‐aminopropyl)oxamide and 1,10‐phenanthroline, respectively, has been synthesized and characterized by elemental analysis, molar conductivity measurement, infrared and electronic spectra studies, and single‐crystal X‐ray diffraction. The complex crystallizes in the triclinic space group P‐1. Each copper(II) ion is located in a slightly distorted square‐pyramidal environment. The Cu···Cu distance through the oxamide bridge is 5.1848(7) Å. The three‐dimensional supramolecular structure is built‐up by hydrogen bonds and π–π stacking interactions. The dicopper(II) complex exhibits cytotoxic activity against the SMMC‐7721 and A549 cell lines. The reactivity toward herring sperm DNA and protein bovine serum albumin (BSA) reveals that the dicopper(II) complex can interact with the DNA by the intercalation mode, and effectively quench the intrinsic fluorescence of BSA via a static mechanism. The influence of hydrophobicity of the bridging ligand on DNA‐binding properties and in vitro cytotoxic activities of this kind of dicopper(II) complexes was investigated.     

Enantiopure 4‐oxazolin‐2‐ones and 4‐methylene‐2‐oxazolidinones as chiral building blocks in a divergent asymmetric synthesis of heterocycles

Enantiopure 3‐((R)‐ and 3‐((S)‐1‐phenylethyl)‐4‐oxazoline‐2‐ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N‐(R)‐ or N‐(S)‐1‐phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4‐ and 5‐positions of the 4‐oxazolin‐2‐one ring through thermal and MW‐promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six‐membered carbo‐ and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4‐methylene‐2‐oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero‐Diels‐Alder cycloaddition.     

Biochemical characteristics of primary and passaged cultures of primate brain microvessel endothelial cells

Rhesus macaque monkey brain microvessel endothelial cells (BMECs) were isolated and grown in culture in an effort to establish an appropriate primate in vitro model of the endothelial component of the blood-brain barrier. The presence of Factor VIII antigen, alkaline phosphatase, -glutamyl transpeptidase, lactate dehydrogenanse, total protein, and the passive permeability properties was documented for both primary and passaged cultures. Primate BMECs were shown to exhibit similar morphological and biochemical properties described for other BMEC culture systems derived from other species. In addition, the passaged primate BMECs were particularly notable for the changes in enzyme activities and total protein that parallel age-dependent changes in brain capillary endothelia. This study provides further support for the possible application of BMEC culture systems in investigations of blood-brain barrier functions under normal, aging, and diseased conditions.To whom to address reprint requests. Phone (913)864-3609; FAX (913)864-3578.