Distinctive amino acid residue periodicities in terminal sequences of type III and type I secreted proteins from proteobacteria

The Fourier transform (FT) method was applied to specify the distribution of 14 predefined groups of amino acids (64 residues) at both termini of annotated type III and type I secreted proteins from proteobacteria. Type I proteins displayed a higher occurrence of significant periodicities at both C-and N-termini, indicating potent features to discriminate between secretion types, particularly by the use of variables selected from the full periodicity profiles at 19 orders of FT. The Fishers linear discriminant analysis, together with the stepwise selection of variables throughout equal pairs of combinations for all predefined groups of residues, revealed the C-terminal harmonics of aromatic (HFWY) and aliphatic (VLIA) residues as a set of strong predictor variables to classify both types of secreted proteins with an accuracy of 100% for original grouped cases and 96.4% for cross-validated grouped cases. The prediction accuracy of proposed discriminant function was estimated by repeated k-fold cross-validation procedures where the original data set was randomly divided into k subsets, with one of the k-subsets serving as the test set and the remaining data forming the training set. The average error rate computed across all k-trials and repeats did not exceed that of leave-one-out procedure. The proposed set of predictor variables could be used to assess the compatibility between secretion pathways and secretion substrates of proteobacteria by means of discriminant analysis.     

Distinguishable codon usage and amino acid composition patterns among substrates of leaderless secretory pathways from proteobacteria

The combined set of codon usage frequencies (61 sense codons) from the 111 annotated sequences of leaderless secreted type I, type III, type IV, and type VI proteins from proteobacteria were subjected to the forward and backward selection to obtain a combination of most effective predictor variables for classification/prediction purposes. The group of 24 codon frequencies displayed a strong discriminatory power with an accuracy of 100% for originally grouped and 97.3 ± 1.6% for cross-validated (LOOCV) cases and an acceptable error rate (0.062 ± 0.012) in k-fold (k = 6) cross-validation (KCV). The summary frequencies of synonymous codons for ten amino acids as the alternative predictor variables revealed a comparable discriminatory power (92.8 ± 2.5% for LOOCV), however at somewhat lower levels of prediction accuracy (0.106 ± 0.015 of KCV). A number of significant (p < 0.001) differences were found among indices of codon usage and amino acid composition depending on a definite secretion type. About 60% of secretion substrates were characterized as apparently originated from horizontal gene transfer events or putative alien genes and found to be unequally allocated in respect of groups. The proposed prediction approaches could be used to specify secretome proteins from genomic sequences as well as to assess the compatibility between bacterial secretion pathways and secretion substrates.     

Analysing the outer membrane subproteome of Methylococcus capsulatus (Bath) using proteomics and novel biocomputing tools

High-resolution two-dimensional gel electrophoresis and mass spectrometry has been used to identify the outer membrane (OM) subproteome of the Gram-negative bacterium Methylococcus capsulatus (Bath). Twenty-eight unique polypeptide sequences were identified from protein samples enriched in OMs. Only six of these polypeptides had previously been identified. The predictions from novel bioinformatic methods predicting β-barrel outer membrane proteins (OMPs) and OM lipoproteins were compared to proteins identified experimentally. BOMP () predicted 43 β-barrel OMPs (1.45%) from the 2,959 annotated open reading frames. This was a lower percentage than predicted from other Gram-negative proteomes (1.8–3%). More than half of the predicted BOMPs in M. capsulatus were annotated as (conserved) hypothetical proteins with significant similarity to very few sequences in Swiss-Prot or TrEMBL. The experimental data and the computer predictions indicated that the protein composition of the M. capsulatus OM subproteome was different from that of other Gram-negative bacteria studied in a similar manner. A new program, Lipo, was developed that can analyse entire predicted proteomes and give a list of recognised lipoproteins categorised according to their lipo-box similarity to known Gram-negative lipoproteins (). This report is the first using a proteomics and bioinformatics approach to identify the OM subproteome of an obligate methanotroph.     

Evolutionary relationships among photosynthetic bacteria

To understand the evolution of photosynthetic bacteria it is necessary to understand how the main groups within Bacteria have evolved from a common ancestor, a critical issue that has not been resolved in the past. Recent analysis of shared conserved inserts or deletions (indels) in protein sequences has provided a powerful means to resolve this long-standing problem in microbiology. Based on a set of 25 indels in highly conserved and widely distributed proteins, all main groups within bacteria can now be defined in clear molecular terms and their relative branching orders logically deduced. For the 82 presently completed bacterial genomes, the presence or absence of these signatures in various proteins was found to be almost exactly as predicted by the indel model, with only 11 exceptions observed in 1842 observations. The branching order of different bacterial groups as deduced using this approach is as follows: low G+C Gram-positive (Heliobacterium chlorum) ↔ high G+C Gram-positive ↔ Clostridium–Fusobacterium–Thermotoga ↔ Deinococcus–Thermus ↔ green nonsulfur bacteria (Chloroflexus aurantiacus) ↔ Cyanobacteria ↔ Spirochetes ↔ Chlamydia–Cytophaga–Flavobacteria–green sulfur bacteria (Chlorobium tepidum) ↔ Aquifex ↔ Proteobacteria (δ and ∈) ↔ Proteobacteria (α) ↔ Proteobacteria (β) and ↔ Proteobacteria (γ). The Heliobacterium species, which contain an Fe–S type of reaction center (RC 1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm bacteria (i.e., bounded by only a single membrane), is indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative or diderm bacteria (containing both inner and outer cell membranes) the green nonsulfur bacteria, which contain a pheophytin-quinone type of reaction center (RC 2), are indicated to have evolved first. The later emerging photosynthetic groups which contain either one or both of these reaction centers could have acquired such genes from the earlier branching lineages by either direct descent or by means of lateral gene transfer. This revised version was published online in August 2006 with corrections to the Cover Date.     

Prediction of beta-turns at over 80% accuracy based on an ensemble of predicted secondary structures and multiple alignments

Background  

β-turn is a secondary protein structure type that plays significant role in protein folding, stability, and molecular recognition. To date, several methods for prediction of β-turns from protein sequences were developed, but they are characterized by relatively poor prediction quality. The novelty of the proposed sequence-based β-turn predictor stems from the usage of a window based information extracted from four predicted three-state secondary structures, which together with a selected set of position specific scoring matrix (PSSM) values serve as an input to the support vector machine (SVM) predictor.     

Identification and characterization of a novel legume-like lectin cDNA sequence from the red marine algae <Emphasis Type="Italic">Gracilaria fisheri</Emphasis>

A legume-type lectin (L-lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid sequence with NCBI-BLAST revealed a high homology (30–68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain (CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure prediction of GFL showed a β-sheet structure, connected with turn and coil. The most abundant structural element of GFL was the random coil, while the α-helixes were distributed at the N- and C-termini, and 21 β-sheets were distributed in the CRD. Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular shape that was composed of a β-sandwich of two anti-parallel β-sheets, monosaccharide binding sites, were on the top of the structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial GFL sequence revealed a hybridization signal of ~1.7 kb consistent with the length of the full-length GFL cDNA identified by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae.     

Cloning and expression of three peptide-linked β2-microglobulin molecules in <Emphasis Type="Italic">Escherichia coli</Emphasis> with an isocaudamer technique

The application of the peptide-linked β2-microglobulin (β2m) strategy is limited in some cases due to the incompatibility between the sequences of the peptides and the restriction sites of the plasmid vectors. An isocaudamer technique was adapted to overcome this restriction. Three peptide-linked β2m genes, HBc_18–27-hβ2m gene, OVA_257–264-mβ2m gene and HER2/neu_369–377-mβ2m gene, were inserted into the pET28a vectors with this technique. The corresponding proteins were expressed in Escherichia coli with yields of over 50 mg/l culture and purities of over 80%. This strategy facilitates the construction of peptide-linked β2m molecules and will simplify the preparation of major histocompatibility complex-peptide complexes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

Prominent collagen type VI expression in juvenile angiofibromas

The extracellular matrix component collagen type VI demonstrates potent growth-stimulatory effects and has been associated with aggressive tumour growth. Although, juvenile angiofibromas (JAs) often exhibit an aggressive growth pattern, the collagen type VI expression of this fibrovascular tumour has not been addressed so far. RT-PCR, Western blot analysis and immunohistochemistry were used in this study to analyse collagen type VI, type VI collagen receptor subunits (integrin α1, α2, α10, α11 and β1) and the type VI collagen receptor NG2 in JAs (N = 15) and nasal mucosa (NM, N = 8) samples. The mRNA expression of all three collagen type VI chains was found to be up-regulated significantly (P < 10⁻³–10⁻⁵, adjusted) in JAs compared to NM tissues. The Western blot analysis proved highly prominent collagen-type VI expression in JAs. The ApoTome technique revealed strong collagen-type VI signals in tumour endothelium. NG2 (P < 10⁻³, adjusted) and α11-integrin (P = 0.04, adjusted) showed a significantly higher mRNA expression levels in JAs than in NM samples. NG2, α1-, α2- and β1-intergin were located to tumour vessels, and additional stromal signals were observed for NG2 and α1-integrin in JAs. This study demonstrates a prominent collagen-type VI expression in JAs. The collagen-type VI may exert an important growth stimulus in this tumour.     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

Biomarkers of Alzheimer′s Disease in the Cerebrospinal Fluid of Spanish Patients With Mild Cognitive Impairment

The study of biomarkers in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) is a technique used with increasing frequency in the early diagnosis of Alzheimers disease (AD). Our objectiv was to gain an own experience while evaluating the reliability, sensitivity, and reproducibility of this technique in Spanish patients. Thirty-seven patients with MCI and twenty-four control subjects were studied by means of AD biomarker analysis in CSF. xMAP Luminex and INNO-BIA Alzbio3 reagents of Innogenetics were used. The study variables assessed were levels of Aβ_1–42, T-tau and P-tau_181p proteins as well as the ratios of T-tau/Aβ_1–42 and P-tau_181p/Aβ_1–42. Samples from nineteen patients were examined twice. Intra-class correlation coefficients for the three biomarkers used showed values higher than 0.95. We observed significant differences between the control group and the MCI groups. In the 6 months following lumbar puncture (LP), eleven (29%) patients with MCI developed AD. These patients showed significant lower levels in Aβ_1–42 protein (276.35 ± 78 vs. 367.13 ± 123.49, P < 0.03) and higher ratios (T-tau/Aβ_1–42 [0.38 ± 0.2 vs. 0.22 ± 0.14, P < 0.01] and P-tau_181p/Aβ_1–42 [0.27 ± 0.13 vs. 0.16 ± 0.1, P < 0.008]) to those in the same group who remained stable. We obtained similar results to those in the most recent reliable literature with our ROC curves, especially with our P-tau_181p values and T-tau/Aβ_1–42 ratio in order to differentiate between control and AD groups. Our experience showed that the analysis of CSF-AD biomarkers in patients with MCI is reliable, sensitive and reproducible. In our knowledge, this is the first experience in Spanish patients.     

Effects of hypericin on the structure and aggregation properties of β-amyloid peptides

We have determined the secondary structure of 1–40 β-amyloid peptides by Fourier-transform infrared spectroscopy (FTIR) and characterized the peptide photophysical properties before and after self-assembly by using intrinsic tyrosine steady-state and time-resolved fluorescence. All measurements were performed in the presence and absence of hypericin (Hyp), an exogenous natural polycyclic pigment that has been shown to inhibit fibril formation and has also been used as a fluorescent probe. We monitored the time course of the aggregation process measuring 405 nm light diffusion at 90° and used thioflavin T to reveal the presence of fibrils. FTIR quantitative analysis evidenced a prevalent random conformation at t = 0 with and without Hyp. Fibrils showed a predominant parallel β-sheet structure and a small percentage of α-helix. The results of fluorescence measurements showed that Hyp does significantly interact with peptides in β-sheet conformation. In conclusion, hypericin does hinder the formation of fibrils, but the percentages of parallel β-sheets were not significantly different from those found in samples not treated with Hyp.     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

β 2 -microglobulin in neotropical primates (Platyrrhini)

 Nucleotide sequences for the three exons of the β₂-microglobulin (β₂m) gene (B2m) were determined for 135 animals representing 37 species and all 16 genera of neotropical primates (Platyrrhini). Twenty-eight different nucleotide sequences, encoding for 26 different proteins, were obtained. In comparison with those of other primate species, the β₂-microglobulins of the Platyrrhini form a distinct clade. Individual genera of neotropical primates have distinctive B2m sequences, but within a genera species can have either the same or different B2m sequences. B2m polymorphism was found within three of the species sampled: Callicebus personatus, Saguinus midas, and Aotus azarae. Of these only the polymorphism in A. azarae has an effect upon the mature, functional β₂m protein: residue 4 being either alanine or threonine. The A. azarae B2m allele encoding alanine at position 4 is shared with another species of Aotus (A. infulatus). In pairwise comparison the mature β₂m proteins of neotropical primates differ by 1–9 amino acid substitutions which can occur at 18 positions within the sequence. The substitutions are distributed throughout the primary structure but are more commonly found in loops rather than β strands of the tertiary structure. Of 17 residues of β₂m which hydrogen-bond with the class I heavy chain in human MHC class I molecules, 13 are conserved in the neotropical primates. The overall pattern of sequence variation in the B2m genes of the Platyrrhini is consistent with an evolution by successive selectively neutral events. Received: 13 November 1997 / Revised: 12 January 1998     

Differential Proteomic Analysis of Temperature-Induced Autolysis in Mycelium of <Emphasis Type="Italic">Pleurotus tuber</Emphasis>-<Emphasis Type="Italic">regium</Emphasis>

Autolysis is an important physiological process found in fungal cultivation. However, there is hitherto no report on the autolysis of Pleurotus tuber-regium. We have investigated the enzymes secreted by temperature-induced (40°C as treatment versus 10°C as control) autolysis of the mycelium of P. tuber-regium grown in submerged cultivation. A comparison between the intracellular proteins (inside the mycelium) and the extracellular proteins (in the culture medium) of the treatment and control by proteomic analysis involving 2D PAGE and MALDI–TOF–MS was made. Twenty-two up-regulated protein spots were detected and eight proteins were identified. They included proteasome which participates in the ubiquitin–proteasome pathway; β-1,3-glucanosyltransferase and tubulin which are involved in the renewal and repair of cell wall; protease and endoglucanase which promote the natural degradation of cell wall and cytoplasm; 14-3-3 protein which takes part in cell signal transduction; and two putative proteins presumably relate to the autolysis process. These identified proteins suggest partially the metabolic processes of the autolysis in the P. tuber-regium mycelium.     

Structural elements of the C-terminal domain of subunit E (E<Subscript>133-222</Subscript>) from the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> V<Subscript>1</Subscript>V<Subscript>O</Subscript> ATPase determined by solution NMR spectroscopy

Subunit E of the vacuolar ATPase (V-ATPase) contains an N-terminal extended α helix (Rishikesan et al. J Bioenerg Biomembr 43:187–193, 2011) and a globular C-terminal part that is predicted to consist of a mixture of α-helices and β-sheets (Grüber et al. Biochem Biophys Res Comm 298:383–391, 2002). Here we describe the production, purification and 2D structure of the C-terminal segment E_133-222 of subunit E from Saccharamyces cerevisiae V-ATPase in solution based on the secondary structure calculation from NMR spectroscopy studies. E_133-222 consists of four β-strands, formed by the amino acids from K136-V139, E170-V173, G186-V189, D195-E198 and two α-helices, composed of the residues from R144-A164 and T202-I218. The sheets and helices are arranged as β1:α1:β2:β3:β4:α2, which are connected by flexible loop regions. These new structural details of subunit E are discussed in the light of the structural arrangements of this subunit inside the V₁- and V₁V_O ATPase.     

The β-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">sensu stricto</Emphasis> complex

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and β-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the β-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that β-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the β-tubulin gene. The β-tubulin gene sequence data reported in this paper appear in the GenBank nucleotide sequence database with the following accession numbers: FJ238316–FJ238341.     

Extracellular β-1,3-glucanases are induced during early somatic embryogenesis in Cichorium

In leaf tissues of the Cichorium hybrid clone `474' (C. intybus L. var. sativum × C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer et al., 1993, Plant Sci 93: 41–53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36–57% homologous with plant β-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related tobacco glucanase PR2a and their β-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are β-1,3-glucanases. Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic `474' line than in the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation during somatic embryogenesis is discussed. Received: 5 May 1997 / Accepted: 25 September 1997     

Identification of pig liver esterase variants by tandem mass spectroscopy analysis and their characterization

Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (α-, β- and γ-PLE). The functional expression of the γ-isoenzyme was already shown and differences in the enantioselectivity compared to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the α- and β-PLE are still unknown. In this work, putative sequences of the α-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis, digestion with proteases and analysis using Matrix-assisted laser desorption/ionization–time of flight (TOF) and electrospray ionisation quadrupole–TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene encoding γ-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity. No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G.