Multidimensional Analysis of 16 Glucose Isomers by Ion Mobility Spectrometry.

Diastereomeric adducts comprising an enantiomerically pure monosaccharide analyte, a peptide, and/or an amino acid and a divalent metal ion (for 16 different monosaccharide isomers) are generated by electrospray ionization and analyzed by combined ion mobility spectrometry-mass spectrometry (IMS-MS) techniques. Mobility distributions of [l-Ser + M + H](+) (where l-Ser is l-serine and M is a given monosaccharide), [l-Phe-Gly + M + H](+) (where l-Phe-Gly is l-phenylalanine-glycine), and [Mn(II) + (l-Phe-Gly - H) + M](+) complex ions are used to determine collision cross sections (ccs in Å(2)), and groups of cross sections for different clusters are proposed as means of identifying the sugar isomers. Within one type of complex, variations in ccs do not always allow delineation between the 16 glucose isomers, but interestingly, when ccs of three different ions are combined as a spatial vector, enantiomers are partially resolved. As a result of this analysis, l-glucose, d-glucose, l-allose, d-allose, d-gulose, d-galactose, and l-mannose are delineated, and for all eight enantiomeric pairs, d and l entities display different coordinates. In addition, different combinations of amino acids, peptide, and metal ions are surveyed, and the potential for yielding unique coordinates for the generated diastereomeric complexes is assessed.

Carbohydrate-functionalized nanovaccines preserve HIV-1 antigen stability and activate antigen presenting cells.

The functionalization of polymeric nanoparticles with ligands that target specific receptors on immune cells offers the opportunity to tailor adjuvant properties by conferring pathogen mimicking attributes to the particles. Polyanhydride nanoparticles are promising vaccine adjuvants with desirable characteristics such as immunomodulation, sustained antigen release, activation of antigen presenting cells (APCs), and stabilization of protein antigens. These capabilities can be exploited to design nanovaccines against viral pathogens, such as HIV-1, due to the important role of dendritic cells (DCs) and macrophages in viral spread. In this work, an optimized process was developed for carbohydrate functionalization of HIV-1 antigen-loaded polyanhydride nanoparticles. The carbohydrate-functionalized nanoparticles preserved antigenic properties upon release and also enabled sustained antigen release kinetics. Particle internalization was observed to be chemistry-dependent with positively charged nanoparticles being taken up more efficiently by DCs. Up-regulation of the activation makers CD40 and CD206 was demonstrated with carboxymethyl-α-d-mannopyranosyl-(1,2)-d-mannopyranoside functionalized nanoparticles. The secretion of the cytokines IL-6 and TNF-α was shown to be chemistry-dependent upon stimulation with carbohydrate-functionalized nanoparticles. These results offer important new insights upon the interactions between carbohydrate-functionalized nanoparticles and APCs and provide foundational information for the rational design of targeted nanovaccines against HIV-1.

Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual priming mechanism.

R1128 substances are anthraquinone natural products that were previously reported as non-steroidal estrogen receptor antagonists with in vitro and in vivo potency approaching that of tamoxifen. From a biosynthetic viewpoint, these polyketides possess structurally interesting features such as an unusual primer unit that are absent in the well studied anthracyclic and tetracyclic natural products. The entire R1128 gene cluster was cloned and expressed in Streptomyces lividans, a genetically well developed heterologous host. In addition to R1128C, a novel optically active natural product, designated HU235, was isolated. Nucleotide sequence analysis of the biosynthetic gene cluster revealed genes encoding two ketosynthases, a chain length factor, an acyl transferase, three acetyl-CoA carboxylase subunits, two cyclases, two oxygenases, an amidase, and remarkably, two acyl carrier proteins. Feeding studies indicate that the unusual 4-methylvaleryl side chain of R1128C is derived from valine. Together with the absence of a dedicated ketoreductase, dehydratase, or enoylreductase within the R1128 gene cluster, this suggests a functional link between fatty acid biosynthesis and R1128 biosynthesis in the engineered host. Specifically, we propose that the R1128 synthase recruits four subunits from the endogenous fatty acid synthase during the biosynthesis of this family of pharmacologically significant natural products.

Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase.

BACKGROUND: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system. RESULTS: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate. CONCLUSIONS: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.

Strength in numbers: non-natural polyvalent carbohydrate derivatives.

Many processes mediated by protein-carbohydrate interactions involve multivalent low-affinity binding, which is inherently difficult to study. New structural templates for the generation of multivalent carbohydrate displays have recently been developed, and tailored multivalent saccharide derivatives can now be used to study and modulate a wide variety of biological recognition events.