Immunoproteomic analysis of human serological antibody responses to vaccination with whole-cell pertussis vaccine (WCV)

Background

Pertussis (whooping cough) caused by Bordetella pertussis (B.p), continues to be a serious public health threat. Vaccination is the most economical and effective strategy for preventing and controlling pertussis. However, few systematic investigations of actual human immune responses to pertussis vaccines have been performed. Therefore, we utilized a combination of two-dimensional electrophoresis (2-DE), immunoblotting, and mass spectrometry to reveal the entire antigenic proteome of whole-cell pertussis vaccine (WCV) targeted by the human immune system as a first step toward evaluating the repertoire of human humoral immune responses against WCV.

Methodology/Principal Findings

Immunoproteomic profiling of total membrane enriched proteins and extracellular proteins of Chinese WCV strain 58003 identified a total of 30 immunoreactive proteins. Seven are known pertussis antigens including Pertactin, Serum resistance protein, chaperonin GroEL and two OMP porins. Sixteen have been documented to be immunogenic in other pathogens but not in B.p, and the immunogenicity of the last seven proteins was found for the first time. Furthermore, by comparison of the human and murine immunoproteomes of B.p, with the exception of four human immunoreactive proteins that were also reactive with mouse immune sera, a unique group of antigens including more than 20 novel immunoreactive proteins that uniquely reacted with human immune serum was confirmed.

Conclusions/Significance

This study is the first time that the repertoire of human serum antibody responses against WCV was comprehensively investigated, and a small number of previously unidentified antigens of WCV were also found by means of the classic immunoproteomic strategy. Further research on these newly identified predominant antigens of B.p exclusively against humans will not only remarkably accelerate the development of diagnostic biomarkers and subunit vaccines but also provide detailed insight into human immunity mechanisms against WCV. In particular, this work highlights the heterogeneity of the B.p immunoreactivity patterns of the mouse model and the human host.     

BMP9经Smad信号通路调控iSCAP成骨/成牙本质分化

目的：研究BMP9是否能够激活 iSCAP细胞中的Smad信号通路,以及Smad信号通路在BMP9诱导iSCAP细胞成骨/成牙本质向分化过程中的作用。方法：首先,采用Western印迹实验检测Ad-BMP9转染iSCAP后Smad1/5/8蛋白的磷酸化水平。随后,利用dnALK1重组腺病毒和BMP9条件培养基作用于iSCAP,Western印迹实验检测Smad1/5/8蛋白磷酸化水平;采用碱性磷酸酶（ALP）活性检测和染色方法分析早期成骨/成牙本质指标变化,茜素红染色法检测钙盐沉积程度;RT-PCR成骨/成牙本质相关基因Runx2、OCN、OPN和DMP1表达的影响。结果：BMP9可上调iSCAP中Smad1/5/8的磷酸化水平;dnALK1抑制BMP9条件培养基作用后,可抑制Smad1/5/8的磷酸化,iSCAP细胞中早期成骨/成牙本质标志物ALP活性和晚期成骨/成牙本质标志钙盐结节减少,重要成骨转录因子Runx2基因表达减少,成骨/成牙本质相关基因OCN、OPN、DMP1的表达也受到了抑制。结论：Smad信号通路在BMP9诱导iSCAP成骨/成牙本质过程中存在并起着重要作用。     

Single-cysteine substitution mutants at amino acid positions 55-75, the sequence connecting the cytoplasmic ends of helices I and II in rhodopsin: reactivity of the sulfhydryl groups and their derivatives identifies a tertiary structure that changes upon light-activation.

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.     

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

     

Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.     

Microfluidic formation of single cell array for parallel analysis of Ca release-activated Ca (CRAC) channel activation and inhibition

High-throughput single cell analysis is required for understanding and predicting the complex stochastic responses of individual cells in changing environments. We have designed a microfluidic device consisting of parallel, independent channels with cell-docking structures for the formation of an array of individual cells. The microfluidic cell array was used to quantify single cell responses and the distribution of response patterns of calcium channels among a population of individual cells. In this device, 15 cell-docking units in each channel were fabricated with each unit containing 5 sandbag structures, such that an array of individual cells was formed in 8 independent channels. Single cell responses to different treatments in different channels were monitored in parallel to study the effects of the specific activator and inhibitor of the Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Multichannel detection was performed to obtain the response patterns of the population of cells within this single cell array. The results demonstrate that it is possible to acquire single cell features in multichannels simultaneously with passive structural control, which provides an opportunity for high-throughput single cell response analysis in a microfluidic chip.     

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.     

Site-specific incorporation of unnatural amino acids into urate oxidase in Escherichia coli

Urate oxidase catalyzes the oxidation of uric acid with poor solubility to produce 5-hydroxyisourate and allantoin. Since allantoin is excreted in vivo, urate oxidase has the potential to be a therapeutic target for the treatment of gout. However, its severe immunogenicity limits its clinical application. Furthermore, studies on the structure-function relationships of urate oxidase have proven difficult. We developed a method for genetically incorporating p-azido-L-phenylalanine into target protein in Escherichia coli in a site-specific manner utilizing a tyrosyl suppressor tRNA/aminoacyl-tRNA synthetase system. We substituted p-azido-L-phenylalanine for Phe(170) or Phe(281) in urate oxidase. The products were purified and their enzyme activities were analyzed. In addition, we optimized the system by adding a "Shine-Dalgarno (SD) sequence" and tandem suppressor tRNA. This method has the benefit of site-specifically modifying urate oxidase with homogeneous glycosyl and PEG derivates, which can provide new insights into structure-function relationships and improve pharmacological properties of urate oxidase.     

Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases

The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries.