Expression proteomics identifies biochemical adaptations and defense responses in transgenic plants with perturbed polyamine metabolism

Soluble proteins from leaves of transgenic tobacco plants with perturbed polyamine metabolism, caused by S-adenosylmethionine decarboxylase overexpression, were analysed by comparative proteomics. A group of proteins was found to be increasingly repressed, in parallel with the degree of polyamine perturbation, in each of the three independent transgenic lines. These were identified as isoforms of chloroplast ribonucleoproteins, known to be involved in chloroplast mRNA stability, processing and translation. Another group of eight proteins strongly induced in the most metabolically perturbed line was identified as multiple, uncharacterised isoforms of the defense protein PR-1, a known marker for systemic acquired resistance.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.     

Identification of a novel, highly variable amino-terminal amino acid sequence element in the nuclear intermediate filament protein lamin B(2) from higher vertebrates

By comparing newly available cDNA sequences of the human intermediate filament protein lamin B(2) with published sequences, we have identified an additional translation initiation codon 60 nucleotides upstream of the previously assumed translation start. In addition, corresponding sequences were identified in the chimpanzee, mouse, rat and bovine genes and cDNAs, respectively. Therefore, we generated antibodies against these potential 20 new amino acids of the human sequence. By immunoblot analysis and immunofluorescence microscopy we show that human lamin B(2) is indeed synthesized as a longer version than previously reported, because it contains these additional 20 amino acids. Notably, the sequence homology to mouse, rat and bovine lamin B(2) is significantly lower in this segment than in that between the second methionine codon and the start of the alpha-helical rod indicating that the tip of the "head" is engaged in more species-specific functions. Forced expression of the GFP-tagged authentic "long" and the 20 amino acid shorter version of lamin B(2) in human cultured SW-13 cells demonstrated that both the longer and the shorter version are properly integrated into the nuclear lamina, although the shorter version exhibited a tendency to disturb envelope architecture at higher expression levels.     

Establishment of a quantitative, qualitative, and high-throughput analysis of sulfatides from small amounts of sera by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Based on our previous measurements of sulfatides, we further developed a quantitative, qualitative, and high-throughput analytical method for serum sulfatides as forms of lysosulfatides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Using 0.1N NaOH in 90% MeOH for saponification instead of absolute MeOH, as previously used, we succeeded in eliminating the formation of lysosulfatide artifacts, facilitating much more sensitive detection. The use of MonoTip C18 allowed quantitation of serum sulfatides from 100 50-mul serum specimens within 1 working day. Purification of lysosulfatides with MonoTip C18 also gave rise to clear MALDI-TOF MS spectra, allowing overall analysis of sphingoid molecular species of sulfatides in serum. The composition was as follows: d18:1 (61.3+/-2.8%), d18:2 (13.3+/-1.7%), t18:0 (11.8+/-1.5%), d18:0 (7.6+/-0.8%), d20:0 (3.0+/-1.2%), t20:0 (2.3+/-0.8%), and d20:1 (1.6+/-0.5%). This is also the first detailed report on sphingoid molecular species of sulfatides in human serum. We believe that this method is suitable for daily clinical analysis of sulfatides in various clinical samples such as blood, urine, cerebrospinal fluid, and specimens from biopsies.     

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis

Background

Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.

Methods

HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.

Results

HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.

Conclusions

These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.

General significance

The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.     

ATP synthase b subunit dimerization domain: a right-handed coiled coil with offset helices

The dimerization domain of Escherichia coli ATP synthase b subunit forms an atypical parallel two-stranded coiled coil. Sequence analysis reveals an 11-residue abcdefghijk repeat characteristic of right-handed coiled coils, but no other naturally occurring parallel dimeric structure of this class has been identified. The arrangement of the helices was studied by their propensity to form interhelix disulfide linkages and analysis of the stability and shape of disulfide-linked dimers. Disulfides formed preferentially between cysteine residues in an a position of one helix and either of the adjacent h positions of the partner. Such heterodimers were far more stable to thermal denaturation than homodimers and, on the basis of gel-filtration chromatography studies, were similar in shape to both non-covalent dimers and dimers linked through flexible Gly(1-3)Cys C-terminal extensions. The results indicate a right-handed coiled-coil structure with intrinsic asymmetry, the two helices being offset rather than in register. A function for the right-handed coiled coil in rotational catalysis is proposed.     

An alternative mechanism for the catalysis of peptide bond formation by L/F transferase: substrate binding and orientation

Eubacterial leucyl/phenylalanyl tRNA protein transferase (L/F transferase) catalyzes the transfer of a leucine or a phenylalanine from an aminoacyl-tRNA to the N-terminus of a protein substrate. This N-terminal addition of an amino acid is analogous to that of peptide synthesis by ribosomes. A previously proposed catalytic mechanism for Escherichia coli L/F transferase identified the conserved aspartate 186 (D186) and glutamine 188 (Q188) as key catalytic residues. We have reassessed the role of D186 and Q188 by investigating the enzymatic reactions and kinetics of enzymes possessing mutations to these active-site residues. Additionally three other amino acids proposed to be involved in aminoacyl-tRNA substrate binding are investigated for comparison. By quantitatively measuring product formation using a quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay, our results clearly demonstrate that, despite significant reduction in enzymatic activity as a result of different point mutations introduced into the active site of L/F transferase, the formation of product is still observed upon extended incubations. Our kinetic data and existing X-ray crystal structures result in a proposal that the critical roles of D186 and Q188, like the other amino acids in the active site, are for substrate binding and orientation and do not directly participate in the chemistry of peptide bond formation. Overall, we propose that L/F transferase does not directly participate in the chemistry of peptide bond formation but catalyzes the reaction by binding and orientating the substrates for reaction in an analogous mechanism that has been described for ribosomes.     

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.     

N-labeling to determine chlorophyll synthesis and degradation in Synechocystis sp. PCC 6803 strains lacking one or both photosystems

Rates of chlorophyll synthesis and degradation were analyzed in Synechocystis sp. PCC 6803 wild type and mutants lacking one or both photosystems by labeling cells with (¹⁵NH₄)₂SO₄ and Na¹⁵NO₃. Pigments extracted from cells were separated by HPLC and incorporation of the ¹⁵N label into porphyrins was subsequently examined by MALDI-TOF mass spectrometry. The life time (τ) of chlorophyll in wild-type Synechocystis grown at a light intensity of 100 μmol photons m⁻² s⁻¹ was determined to be about 300 h, much longer than the cell doubling time of about 14 h. Slow chlorophyll degradation (τ ∼200-400 h) was also observed in Photosystem I-less and in Photosystem II-less Synechocystis mutants, whereas in a mutant lacking both Photosystem I and Photosystem II chlorophyll degradation was accelerated 4-5 fold (τ ∼50 h). Chlorophyllide and pheophorbide were identified as intermediates of chlorophyll degradation in the Photosystem I-less/Photosystem II-less mutant. In comparison with the wild type, the chlorophyll synthesis rate was five-fold slower in the Photosystem I-less strain and about eight-fold slower in the strain lacking both photosystems, resulting in different chlorophyll levels in the various mutants. The results presented in this paper demonstrate the presence of a regulation that adjusts the rate of chlorophyll synthesis according to the needs of chlorophyll-binding polypeptides associated with the photosystems.     

Detailed N-glycan analysis of mannose receptor purified from murine spleen indicates tissue specific sialylation

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognises both mannosylated and sulphated ligands through its C-type lectin domains (CTLDs) and cysteine-rich (CR) domain, respectively. It is widely expressed among different tissues and by certain cell types in vivo. Our previous study suggested that the glycosylation, especially terminal sialylation, regulated the functional specificities of MR. In the current investigation, the distribution of MR among various mouse tissues was studied and the N-linked glycosylation of spleen MR was analysed. Our results showed that spleen expressed the most abundant MR, consistent with its wide distribution in different cell types in this organ. Spleen MR was heterogeneously N-glycosylated. The majority of the glycans were sialylated in the α2 → 6-linkage and both Neu5Ac and Neu5Gc sialic acids were detected. Most glycans were bi-antennary (74%) with ∼22% tri-antennary and most were core fucosylated (68%). About 13% contained α-galactose. In the lung, MR exhibited more terminal sialic acids in the α2 → 3- rather than in the α2 → 6-configuration. Our study provides a profile of MR N-linked glycosylation that will facilitate our understanding of their physiological role on MR biology in vivo.     

CE-MS for proteomics: Advances in interface development and application

Capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a powerful technique for the analysis of proteins and peptides. Over the past few years, significant progress has been made in the development of novel and more effective interfaces for hyphenating CE to MS. This review provides an overview of these new interfacing techniques for coupling CE to MS, covering the scientific literature from January 2007 to December 2011. The potential of these new CE-MS interfacing techniques is demonstrated within the field of (clinical) proteomics, more specifically "bottom-up" proteomics, by showing examples of the analysis of various biological samples. The relevant papers on CE-MS for proteomics are comprehensively summarized in tables, including, e.g. information on sample type and pretreatment, interfacing and MS detection mode. Finally, general conclusions and future perspectives are provided.     

The involvement of mnn4 and mnn6 mutations in mannosylphosphorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae

The structure of O-linked acidic oligosaccharide from Saccharomyces cerevisiae was analyzed. The chitinase, exclusively O-glycosylated extracelluar protein, was purified from strains mnn1, mnn1 mnn4, mnn1 mnn6 and Δkre2 and the oligosaccharides were hydrolyzed by O-linked sugar chain specific hydrazinolysis. The mannosylphosphorylated mannotriose (M3-P-M) was detected in strain mnn1, but not in the other three strains (mnn1 mnn4, mnn1 mnn6 and Δkre2). α-Mannosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of mannosylphosphorylated mannotriose revealed that mannosylphosphate was attached to a middle mannose of α-1,2-linked mannotriose. This result indicates that the mnn4 and mnn6 mutations affect the mannosylphosphorylation of O-linked oligosaccharide, together with that of N-linked oligosaccharide. The amount of mannosylphosphorylated mannotriose was 7% of total O-linked oligosaccharides (20% of neutral mannotriose) of chitinase in strain mnn1.     

Structural investigation of PsbO from plant and cyanobacterial photosystem II

The manganese-stabilizing protein PsbO is associated with the luminal side of thylakoids close to the redox-active Mn₄Ca cluster at the catalytically active site of photosystem II (PSII). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn₄Ca cluster against photoinhibition. Using small-angle X-ray scattering, we investigated the low-resolution structure of wild-type spinach PsbO and that of chimeric spinach PsbO fused with maltose-binding protein. Small-angle X-ray scattering data revealed that both proteins are monomeric in solution, and that plant and cyanobacterial PsbO have similar structures. We show a highly efficient expression system that allows recombinant production of the active wild type and the chimeric PsbO from spinach and cyanobacteria, with yields compatible with biophysical and structural studies. The binding of spinach PsbO fused with maltose-binding protein to PSII depleted of extrinsic subunits (PSII-ΔpsbO,P,Q) was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The reconstituted PSII was shown to have similar oxygen evolution rates as obtained with wild-type spinach PsbO.     

Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry

Thermal-assisted partial acid hydrolysis of the carbohydrate moieties of N-glycosylated peptides of horseradish peroxidase (HRP) is used to generate oligosaccharide cleavage ladders. These ladders allow direct reading of components of the oligosaccharides by mass spectrometry. Acid hydrolysis performed with 1.4, 3.1, 4.5, or 6.7M trifluoroacetic acid at 37, 65, or 95 degrees C for 30min to 24h hydrolyzed mainly the oligosaccharide units of glycopeptides with least peptide bond or amino acid side chain hydrolysis. Tryptic N-glycosylated peptides from HRP with molecular weights of 2533, 2612, 3355, 3673, and 5647Da were used as test systems in these experiments. Data showed that the most labile group of oligosaccharides is the fucose (Fuc) and the majority of the end cleavage products are peptides with one or no N-acetylglucosamine (GlcNAc) residue linked to Asparagine (Asn). Additionally, the data agree with previous reports that glycopeptides 3355 and 3673Da carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, glycopeptide 5647Da carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and glycopeptides 2612 and 2533Da carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the 2612Da peptide at Asn286 is partially occupied. This method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.     

Characterization of Inhibitor-Bound α-Synuclein Dimer: Role of α-Synuclein N-Terminal Region in Dimerization and Inhibitor Binding

α-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other α-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit α-synuclein assembly with low micromolar IC₅₀ values, and that SDS-stable, noncytotoxic soluble α-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound α-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound α-synucleins in detail, we have prepared α-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated α-synuclein monomer and dimer were oxidized at all four methionine residues of α-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of α-synuclein. Ultra-high-field NMR analysis of inhibitor-bound α-synuclein dimers showed that the signals derived from the N-terminal region of α-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric α-synuclein. We propose that the N-terminal region of α-synuclein plays a key role in the formation of α-synuclein assemblies.     

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.     

Analysis of N-glycosylation in maize cytokinin oxidase/dehydrogenase 1 using a manual microgradient chromatographic separation coupled offline to MALDI-TOF/TOF mass spectrometry

Cytokinin oxidase/dehydrogenase (CKO; EC 1.5.99.12) irreversibly degrades the plant hormones cytokinins. A recombinant maize isoenzyme 1 (ZmCKO1) produced in the yeast Yarrowia lipolytica was subjected to enzymatic deglycosylation by endoglycosidase H. Spectrophotometric assays showed that both activity and thermostability of the enzyme decreased after the treatment at non-denaturing conditions indicating the biological importance of ZmCKO1 glycosylation. The released N-glycans were purified with graphitized carbon sorbent and analyzed by MALDI-TOF MS. The structure of the measured high-mannose type N-glycans was confirmed by tandem mass spectrometry (MS/MS) on a Q-TOF instrument with electrospray ionization. Further experiments were focused on direct analysis of sugar binding. Peptides and glycopeptides purified from tryptic digests of recombinant ZmCKO1 were separated by reversed-phase chromatography using a manual microgradient device; the latter were then subjected to offline-coupled analysis on a MALDI-TOF/TOF instrument. Glycopeptide sequencing by MALDI-TOF/TOF MS/MS demonstrated N-glycosylation at Asn52, 63, 134, 294, 323 and 338. The bound glycans contained 3-14 mannose residues. Interestingly, Asn134 was found only partially glycosylated. Asn338 was the sole site to carry large glycan chains exceeding 25 mannose residues. This observation demonstrates that contrary to a previous belief, the heterologous expression in Y. lipolytica may lead to locally hyperglycosylated proteins.     

The fellowship of the RING: the RING-B-box linker region interacts with the RING in TRIM21/Ro52, contains a native autoantigenic epitope in Sjögren syndrome, and is an integral and conserved region in TRIM proteins

Ro52 is a major autoantigen that is targeted in the autoimmune disease Sjögren syndrome and belongs to the tripartite motif (TRIM) protein family. Disease-related antigenic epitopes are mainly found in the coiled-coil domain of Ro52, but one such epitope is located in the Zn^2 +-binding region, which comprises an N-terminal RING followed by a B-box, separated by a ∼ 40-residue linker peptide. In the present study, we extend the structural, biophysical, and immunological knowledge of this RING-B-box linker (RBL) by employing an array of methods. Our bioinformatic investigations show that the RBL sequence motif is unique to TRIM proteins and can be classified into three distinct subtypes. The RBL regions of all three subtypes are as conserved as their known flanking domains, and all are predicted to comprise an amphipathic helix. This helix formation is confirmed by circular dichroism spectroscopy and is dependent on the presence of the RING. Immunological studies show that the RBL is part of a conformation-dependent epitope, and its antigenicity is likewise dependent on a structured RING domain. Recombinant Ro52 RING-RBL exists as a monomer in vitro, and binding of two Zn^2 + increases its stability. Regions stabilized by Zn^2 + binding are identified by limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry. Furthermore, the residues of the RING and linker that interact with each other are identified by analysis of protection patterns, which, together with bioinformatic and biophysical data, enabled us to propose a structural model of the RING-RBL based on modeling and docking experiments. Sequence similarities and evolutionary sequence patterns suggest that the results obtained from Ro52 are extendable to the entire TRIM protein family.     

Schistosome biology and proteomics: progress and challenges

The recent availability of schistosomal genome-sequence information allows protein identification in schistosome-derived samples by mass spectrometry (proteomics). Over the last few years, several proteome studies have been performed that addressed important questions in schistosome biology. This review summarizes the applied experimental approaches that have been used so far, it provides an overview of the most important conclusions that can be drawn from the performed studies and finally discusses future challenges in this research area.     

Analysis of lysozyme in cheese by immunocapture mass spectrometry

The enzyme lysozyme is used as a preservative to prevent late blowing of ripened cheese, caused by Clostridium tyrobutyricum. Since the enzyme is extracted from hen egg white, lysozyme has to be declared on food product labels as a potential allergen. Here, a method is reported that combines immunocapture purification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis for the detection of lysozyme in cheese samples. Cheese extracts were treated with magnetic particles coated with a monoclonal antibody directed against lysozyme. After immunocapture purification, lysozyme was detected by MALDI-TOF-MS. The limit of detection of the assay was about 5 mg/kg lysozyme in cheese. The method reliably distinguished between cheese samples which had been produced with and without lysozyme. Thus, the novel assay allows the reliable, sensitive, and specific detection of lysozyme in a food matrix. The assay could be easily adapted to other target peptides and proteins in complex food matrices and, therefore, has a broad application potential, e.g. for the analysis of allergens.