We report the complete sequence of the 4,274,642-bp genome of Haloarcula marismortui, a halophilic archaeal isolate from the Dead Sea. The genome is organized into nine circular replicons of varying G+C compositions ranging from 54% to 62%. Comparison of the genome architectures of Halobacterium sp. NRC-1 and H. marismortui suggests a common ancestor for the two organisms and a genome of significantly reduced size in the former. Both of these halophilic archaea use the same strategy of high surface negative charge of folded proteins as means to circumvent the salting-out phenomenon in a hypersaline cytoplasm. A multitiered annotation approach, including primary sequence similarities, protein family signatures, structure prediction, and a protein function association network, has assigned putative functions for at least 58% of the 4242 predicted proteins, a far larger number than is usually achieved in most newly sequenced microorganisms. Among these assigned functions were genes encoding six opsins, 19 MCP and/or HAMP domain signal transducers, and an unusually large number of environmental response regulators-nearly five times as many as those encoded in Halobacterium sp. NRC-1-suggesting H. marismortui is significantly more physiologically capable of exploiting diverse environments. In comparing the physiologies of the two halophilic archaea, in addition to the expected extensive similarity, we discovered several differences in their metabolic strategies and physiological responses such as distinct pathways for arginine breakdown in each halophile. Finally, as expected from the larger genome, H. marismortui encodes many more functions and seems to have fewer nutritional requirements for survival than does Halobacterium sp. NRC-1.

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.

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An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography–high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure–activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.

Here, we describe the first sequencing method of a complex mixture of heparan sulfate tetrasaccharides by LC-MS/MS. Heparin and heparan sulfate (HS) are linear polysaccharides that are modified in a complex manner by N- and O-sulfation, N-acetylation, and epimerization of the uronic acid. Heparin and HS are involved in various essential cellular communication processes. The structural analysis of these glycosaminoglycans is challenging due to the lability of their sulfate groups, the high heterogeneity of modifications, and the epimerization of the uronic acids. While advances in liquid chromatography (LC) and mass spectrometry (MS) have enabled compositional profiling of HS oligosaccharide mixtures, online separation and detailed structural analysis of isomeric and epimeric HS mixtures has not been achieved. Here, we report the development and evaluation of a chemical derivatization and tandem mass spectrometry method that can separate and identify isomeric and epimeric structures from complex mixtures. A series of well-defined synthetic HS tetrasaccharides varying in sulfation patterns and uronic acid epimerization were analyzed by chemical derivatization and LC-MS/MS. These synthetic compounds made it possible to establish relationships between HS structure, chromatographic behavior and MS/MS fragmentation characteristics. Using the analytical characteristics determined through the analysis of the synthetic HS tetrasaccharide standards, an HS tetrasacharide mixture derived from natural sources was successfully sequenced. This method represents the first sequencing of complex mixtures of HS oligosaccharides, an essential milestone in the analysis of structure-function relationships of these carbohydrates.