Brian Arbogast

Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection

The ability to infect macrophages is a common characteristic shared among many mycobacterial species. Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium kansasii enter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identify M. avium genes and host cell pathways involved in the bacterial uptake by macrophages, we screened a M. avium transposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of six M. avium clones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to the M. avium subsp. paratuberculosis MAP2446c gene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in the M. avium genome. The region is unique for M. avium and is not present in M. tuberculosis or M. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understand M. avium interaction with two evolutionarily distinct hosts, the mutants were evaluated for Acanthamoeba castellanii invasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting that M. avium might have evolved mechanisms that are used to enter amoebas and human macrophages.

Coordinated Regulation of Transcription Factor Bcl11b Activity in Thymocytes by the Mitogen-activated Protein Kinase (MAPK) Pathways and Protein Sumoylation

Background: The transcription factor Bcl11b plays essential roles during T-cell development. Results: Bcl11b activity in thymocytes is regulated by MAPK-mediated phosphorylation and subsequent sumoylation and ubiquitination. Conclusion: A regulatory pathway links thymocyte stimulation, MAPK activation, and Bcl11b-dependent regulation of gene expression during late T-cell development. Significance: This work has implications for the role of Bcl11b in T-cell development and leukemogenesis. The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.

Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis

Spleen is a prime organ in which immuno-stimulation takes place in mammalians. Proteome analysis was used to investigate the elicited effects on mouse splenocytes upon exposure to holothurian triterpene glycosides. Cucumarioside A(2)-2, and Frondoside A, respectively, have been used to in-vitro stimulate primary splenocyte cultures. Differential protein expression was monitored by 2D gel analysis and proteins in spots of interest were identified by MALDI ToF MS and nano LC-ESI Q-ToF MS/MS, respectively. Differential image analysis of gels from control vs. gels from stimulated primary splenocyte cultures showed that approximately thirty protein spots were differentially expressed. Prime examples of differentially expressed proteins are NSFL1 cofactor p47 and hnRNP K (down-regulated), as well as Septin-2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and GRB2-related adaptor protein 2 (up-regulated). Immuno-analytical assays confirmed differential protein expression. Together with results from proliferation and cell adhesion assays, our results show that cellular proliferation is stimulated by holothurian triterpene glycosides. In conclusion, holothurian triterpene glycosides are thought to express their immuno-stimulatory effects by enhancing the natural cellular defense barrier that is necessary to fight pathogens and for which lymphocytes and splenocytes have to be recruited constantly due to limited lifetimes of B-cells and T-cells in the body.

Measuring copper and zinc superoxide dismutase from spinal cord tissue using electrospray mass spectrometry.

Metals are key cofactors for many proteins, yet quantifying the metals bound to specific proteins is a persistent challenge in vivo. We have developed a rapid and sensitive method using electrospray ionization mass spectrometry to measure Cu,Zn superoxide dismutase (SOD1) directly from the spinal cord of SOD1-overexpressing transgenic rats. Metal dyshomeostasis has been implicated in motor neuron death in amyotrophic lateral sclerosis (ALS). Using the assay, SOD1 was directly measured from 100 μg of spinal cord, allowing for anatomical quantitation of apo, metal-deficient, and holo SOD1. SOD1 was bound on a C(4) Ziptip that served as a disposable column, removing interference by physiological salts and lipids. SOD1 was eluted with 30% acetonitrile plus 100 μM formic acid to provide sufficient hydrogen ions to ionize the protein without dislodging metals. SOD1 was quantified by including bovine SOD1 as an internal standard. SOD1 could be measured in subpicomole amounts and resolved to within 2 Da of the predicted parent mass. The methods can be adapted to quantify modifications to other proteins in vivo that can be resolved by mass spectrometry.

Characterization of disulfide linkages and disulfide bond scrambling in recombinant human macrophage colony stimulating factor by fast-atom bombardment mass spectrometry of enzymatic digests

Fast-atom bombardment mass spectrometry was used to study disulfide bonding patterns in heat-denatured human recombinant macrophage colony stimulating factor (rhM-CSF). The heat-denaturated protein was studied by analysis of the pattern of peptides in the proteolytic digests. Native rhM-CSF is a homodimer with intramolecular disulfide linkages between Cys7–Cys90, Cys48–Cys139, and Cys102–Cys146 and intermolecular linkages between Cys31-Cys31, and the pairs Cys157 and Cys159. Brief heating for 1 min leads to partial disulfide bond scrambling. In addition to the native disulfide bonds between Cys7–Cys90, Cys48–Cys139, and Cys31-Cys31, nonnative disulfide bonds were detected between Cys48–Cys90 and Cys48–Cys102. When heated for 5 min the disulfide bonds of rhM-CSF are completely scrambled and lead to nonnative intramolecular disulfide bonds between Cys48–Cys102 and Cys90–Cys102 and one intermolecular disulfide bond between Cys102–Cys102.

The determination of polychlorodibenzo-p-dioxins in pentachlorophenol and wood treatment solutions

Analytical methods have been implemented to measure levels of polychlorodibenzo-p-dioxins, and polychlorodibenzofurans in pentachlorophenol solutions used in the Boulton wood treatment process. When normalized against the pentachlorophenol (PCP) content, the octachlorodibenzo-p-dioxin level was 34% higher in the recirculating PCP solution than in the fresh PCP solution, and in the sludge it was 90% higher. A smaller concentration increase was observed for the heptachlorodibenzo-p-dioxin in the recirculating solution, but a similar increase was observed in the sludge.

Solvent accessibility of βB2-crystallin and local structural changes due to deamidation at the dimer interface

In the lens of the eye the ordered arrangement of the major proteins, the crystallins, contributes to lens transparency. Members of the beta/gamma-crystallin family share common beta-sheet rich domains and hydrophobic regions at the monomer-monomer or domain-domain interfaces. Disruption of these interfaces, due to post-translational modifications, such as deamidation, decreases the stability of the crystallins. Previous experiments have failed to define the structural changes associated with this decreased stability. Using hydrogen/deuterium exchange with mass spectrometry (HDMS), deamidation-induced local structural changes in betaB2-crystallin were identified. Deamidation was mimicked by replacing glutamines with glutamic acids at homologous residues 70 and 162 in the monomer-monomer interface of the betaB2-crystallin dimer. The exchange-in of deuterium was determined from 15 s to 24 h and the global and local changes in solvent accessibility were measured. In the wild type betaB2-crystallin (WT), only about 20% of the backbone amide hydrogen was exchanged, suggesting an overall low accessibility of betaB2-crystallin in solution. This is consistent with a tightly packed domain structure observed in the crystal structure. Deuterium levels were initially greater in N-terminal domain (N-td) peptides than in homologous peptides in the C-terminal domain (C-td). The more rapid incorporation suggests a greater solvent accessibility of the N-td. In the betaB2-crystallin crystal structure, interface Gln are oriented towards their opposite domain. When deamidation was mimicked at Gln70 in the N-td, deuterium levels increased at the interface peptide in the C-td. A similar effect in the N-td was not observed when deamidation was mimicked at the homologous residue, Gln162, in the C-td. This difference in the mutants can be explained by deamidation at Gln70 disrupting the more compact C-td and increasing the solvent accessibility in the C-td interface peptides. When deamidation was mimicked at both interface Gln, deuterium incorporation increased in the C-td, similar to deamidation at Gln70 alone. In addition, deuterium incorporation was decreased in the N-td in an outside loop peptide adjacent to the mutation site. This decreased accessibility may be due to newly exposed charge groups facilitating ionic interactions or to peptides becoming more buried when other regions became more exposed. The highly sensitive HDMS methods used here detected local structural changes in solution that had not been previously identified and provide a mechanism for the associated decrease in stability due to deamidation. Changes in accessibility due to deamidation at the interface led to structural perturbations elsewhere in the protein. The cumulative effects of multiple deamidation sites perturbing the structure both locally and distant from the site of deamidation may contribute to aggregation and precipitation during aging and cataractogenesis in the lens.

Characterization of synthetic peptide byproducts from cyclization reactions using on-line HPLC-ion spray and tandem mass spectrometry

The cyclization of a linear dynorphin A (Dyn A) analogue to give the lactam derivative cyclo[d-Asp2, Dap5]Dyn A(1–13)NH2 (where Dap=α,β-diaminopropionic acid) was studied to evaluate the usefulness of different coupling reagents for side chain to side chain lactam formation. This cyclization proved to be difficult and yielded substantial byproducts that varied depending upon the activating reagent used. On-line HPLC-ion spray mass spectrometry was more practical and useful than conventional HPLC alone for characterizing the products of these cyclization reactions. Peptide byproducts could be identified from the series of multiply charged ions observed, even when some of these peptides eluted from the HPLC with similar retention times. In addition to the desired cyclic peptide, the peptide byproducts observed following the cyclization using BOP (benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate) were the linear peptide, the cyclic dimeric peptide and the linear peptide resulting from aspartimide rearrangement. The peptide byproducts obtained following cyclization using HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and HAPyU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate) were predominantly linear tetramethylguanidinium (Tmg) and dipyrrolidinylguanidinium (Dpg) derivatives resulting from alkylation of the side chain of Dap by HATU and HAPyU, respectively; in addition to monomeric guanidinium derivatives, dimeric and aspartimide-containing peptides were also produced. Peptide sequencing by ion spray tandem mass spectrometry was performed to confirm the structure of both pure peptides and peptide byproducts in the crude samples. A unique fragmentation for the β,γ-bond of the Dap side chain was demonstrated and could be used to identify linear peptide byproducts. The distinctive fragment ions from this cleavage were also observed for the peptides containing the Tmg and Dpg functionalities on the Dap side chain.

A mass spectrometric study of the heterogeneity of the monomer subunit of Lumbricus terrestris hemoglobin

The subunits of the hemoglobin of Lumbricus terrestris consist of heme-binding globin chains, designated a, b, c, and d, and linker chains. The sequence of chain d, which is also referred to as a monomer subunit, has been reported by Shishikura et al. (Biol. Chem.1987, 262, 3123–3131). This subunit has been found to be heterogeneous and in this study three fractions were separated by C18 reverse-phase high-performance liquid chromatography. The major chain d1 with molecular weight 15,993. 5 ± 2. 1 u possesses S7 instead of G7, the partial sequence RDIIDD (33–38) instead of KGILRE, E instead of Q in positions 23 and 58, and T84 instead of A84. The C-terminus is K140 instead of D141I142. Chain d3 with molecular weight 15,963. 2 ± 1. 6 u showed high sequence homology with chain d1, differing apparently only in residue 84 where A84 replaces T84. Analysis of the third chromatographie fraction revealed two additional chains with molecular weights of 15,996. 1 ± 1. 4 and 15,937. 6 ± 1. 2 u. These are designated chains d2 and d4, respectively, but their sequence assignments are not yet certain.

Liquid secondary ion mass spectrometry analysis of permethylated, n-Hexylamine derivatized oligosaccharides. Application to baculovirus expressed mouse interleukin-3

Reductive amination with n-hexylamine followed by permethylation was used as a procedure for the liquid secondary ion mass spectrometry (LSIMS) analysis of Asn-linked oligosaccharides. Initial experiments with this procedure were performed on maltoheptaose. These experiments show that exhaustive methylation at the newly formed secondary nitrogen forms a quaternary ammonium salt. When this is subjected to positive ion LSIMS, an abundant M+ ion is observed. This procedure was applied to the Asn-linked oligosaccharides released from human transferrin and ribonuclease-B. The reductively aminated, permethylated mixture of oligosaccharides from ribonuclease-B afforded a positive ion LSI mass spectrum in which M+ ions for Mans5–9GlcNAc2 could be assigned. The positive ion LSI mass spectrum obtained from the mixture of oligosaccharides isolated from human transferrin showed M+ ions that could be assigned to both monosialylated and disialylated biantennary complex type oligosaccharides. Reductive amination followed by permethylation of the Asn-linked oligosaccharides isolated from baculovirus expressed mouse interleukin-3 produced in Bombyx mori gave a positive ion LSI mass spectrum in which the oligosaccharides could be assigned the monosaccharide composition Man2–4[Fuc]GlcNAc2 and Man2GlcNAc2. These are believed to be dimannose, trimannose, and tetramannose chitobiose core oligosaccharides, three of which are fucosylated.