The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum.The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study.     

Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae

CPR proteins are the largest cuticular protein family in arthropods. The whole genome sequence of Anopheles gambiae revealed 156 genes that code for proteins with the R&R Consensus and named CPRs. This protein family can be divided into RR-1 and RR-2 subgroups, postulated to contribute to different regions of the cuticle. We determined the temporal expression patterns of these genes throughout post-embryonic development by means of real-time qRT-PCR. Based on expression profiles, these genes were grouped into 21 clusters. Most of the genes were expressed with sharp peaks at single or multiple periods associated with molting. Genes coding for RR-1 and RR-2 proteins were found together in several co-expression clusters. Twenty-five genes were expressed exclusively at one metamorphic stage. Five out of six X-linked genes showed equal expression in males and females, supporting the presence of a gene dosage compensation system in A. gambiae. Many RR-2 genes are organized into sequence clusters whose members are extremely similar to each other and generally closely associated on a chromosome. Most genes in each sequence cluster are expressed with the same temporal expression pattern and at the same level, suggesting a shared mechanism to regulate their expression.     

Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae

The largest arthropod cuticular protein family, CPR, has the Rebers and Riddiford (R&R) Consensus that in an extended form confers chitin-binding properties. Two forms of the Consensus, RR-1 and RR-2, have been recognized and initial data suggested that the RR-1 and RR-2 proteins were present in different regions within the cuticle itself. Thus, RR-2 proteins would contribute to exocuticle that becomes sclerotized, while RR-1s would be found in endocuticle that remains soft. An alternative, and more common, suggestion is that RR-1 proteins are used for soft, flexible cuticles such as intersegmental membranes, while RR-2s are associated with hard cuticle such as sclerites and head capsules. We used TEM immunogold detection to localize the position of several RR-1 and RR-2 proteins in the cuticle of Anopheles gambiae. RR-1s were localized in the procuticle of the soft intersegmental membrane except for one protein found in the endocuticle of hard cuticle. RR-2s were consistently found in hard cuticle and not in flexible cuticle. All RR-2 antibodies localized to the exocuticle and four out of six were also found in the endocuticle. Hence the location of RR-1s and RR-2s depends more on properties of individual proteins than on either hypothesis.     

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

Bioinformatics-based identification of chemosensory proteins in African Malaria Mosquito, Anopheles gambiae

Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cys-teine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles     

Amino acid misincorporation in recombinant proteins

Proteins provide the molecular basis for cellular structure, catalytic activity, signal transduction, and molecular transport in biological systems. Recombinant protein expression is widely used to prepare and manufacture novel proteins that serve as the foundation of many biopharmaceutical products. However, protein translation bioprocesses are inherently prone to low-level errors. These sequence variants caused by amino acid misincorporation have been observed in both native and recombinant proteins. Protein sequence variants impact product quality, and their presence can be exacerbated through cellular stress, overexpression, and nutrient starvation. Therefore, the cell line selection process, which is used in the biopharmaceutical industry, is not only directed towards maximizing productivity, but also focuses on selecting clones which yield low sequence variant levels, thereby proactively avoiding potentially inauspicious patient safety and efficacy outcomes. Here, we summarize a number of hallmark studies aimed at understanding the mechanisms of amino acid misincorporation, as well as exacerbating factors, and mitigation strategies. We also describe key advances in analytical technologies in the identification and quantification of sequence variants, and some practical considerations when using LC-MS/MS for detecting sequence variants.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos     

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.     

Distribution and spread of pyrethroid and DDT resistance among the Anopheles gambiae complex in Tanzania

The development of insecticide resistance is a threat to the control of malaria in Africa. We report the findings of a national survey carried out in Tanzania in 2011 to monitor the susceptibility of malaria vectors to pyrethroid, organophosphate, carbamate and DDT insecticides, and compare these findings with those identified in 2004 and 2010. Standard World Health Organization (WHO) methods were used to detect knock‐down and mortality rates in wild female Anopheles gambiae s.l. (Diptera: Culicidae) collected from 14 sentinel districts. Diagnostic doses of the pyrethroids deltamethrin, lambdacyhalothrin and permethrin, the carbamate propoxur, the organophosphate fenitrothion and the organochlorine DDT were used. Anopheles gambiae s.l. was resistant to permethrin in Muleba, where a mortality rate of 11% [95% confidence interval (CI) 6–19%] was recorded, Muheza (mortality rate of 75%, 95% CI 66–83%), Moshi and Arumeru (mortality rates of 74% in both). Similarly, resistance was reported to lambdacyhalothrin in Muleba, Muheza, Moshi and Arumeru (mortality rates of 31–82%), and to deltamethrin in Muleba, Moshi and Muheza (mortality rates of 28–75%). Resistance to DDT was reported in Muleba. No resistance to the carbamate propoxur or the organophosphate fenitrothion was observed. Anopheles gambiae s.l. is becoming resistant to pyrethoids and DDT in several parts of Tanzania. This has coincided with the scaling up of vector control measures. Resistance may impair the effectiveness of these interventions and therefore demands close monitoring and the adoption of a resistance management strategy.     

Effects of adult body size on fecundity and the pre-gravid rate of Anopheles gambiae females in Tanzania

Abstract. The influence of adult body size on the pre-gravid state and fecundity was studied in Anopheles gambiae Giles females hand-caught inside houses and virgin females collected as pupae in Tanzania. Blood-fed mosquitoes were kept for 2-3 days before dissection and examination for insemination and ovarian condition. Those females which did not develop eggs were classified as pre-gravid. The number of mature eggs in those mosquitoes which became gravid was counted. Virgin females were fed and kept for egg maturation in the laboratory. Wing-length of females was measured as an index of mosquito size. The overall pre-gravid rate in the resting An. gambiae population was found to be 21% and, of these, 66% had been inseminated. In the virgin females the pre-gravid rate was 92.6%. The mean wing-length of wild females which became gravid was significantly larger than those which remained pre-gravid. There was a positive correlation between fecundity and wing-length. Smaller females tended to require two or three bloodmeals to facilitate completion of the first gonotrophic cycle. The critical size permitting oviposition from the first blood-meal was a wing-length of 3 mm.     

Identification of electrophysiologically-active compounds for the malaria mosquito, Anopheles gambiae, in human sweat extracts

Abstract. Human sweat samples were chemically fractionated into acid and non-acid components. The most abundant volatile compounds present in the fractions were identified by linked gas chromatography mass spectrometry. The acid fractions were found to be composed of a range of twenty aliphatic and three aromatic carboxylic acids ranging, on average, from 0.02 to 20 ig per ml of sweat sampled. Non-acid fractions were found to contain: 6-methyl-5-hepten-2-one, l-octen-3-ol, decanal, benzyl alcohol, dimethylsulphone, phenylethanol, phenol and 4-mefhylphenol, collectively amounting to 0.1 and 3 |ig per ml of sweat. The major component of sweat was found to be L-lactic acid which constituted from 1 to 5 mg/ml.
Using the intact antennae of the anthropophilic malaria vector mosquito Anopheles gambiae Giles, the peripheral olfactory activities of compounds identified in the sweat fractions were investigated by electroantennography (EAG). Short-chain saturated carboxylic acids, methanoic, ethanoic, propanoic, butanoic, pentanoic and hexanoic acids were found to elicit significantly larger EAG responses than longer chain saturated carboxylic acids from female An.gambiae. For a given dose the largest amplitude EAG response was elicited by methanoic acid. Pentanoic acid elicited larger EAG responses than either butanoic or hexanoic acids. Two non-acidic compounds, l-octen-3-ol and 4-methylphenol, were found to elicit significant dose-dependent EAG responses from female An.gambiae. 1 -Octen-3-ol elicited larger EAG responses than 4-methylphenol for a given dose, but both compounds elicited smaller EAG responses than the same dose of C_]-C₆straight-chain aliphatic carboxylic acids. The possible behavioural significance of the EAG-active compounds identified in human sweat samples is discussed.     

Proteome analysis for the identification of in vivo estrogen-regulated proteins in bone

Estrogen deficiency results in a reduced bone mass, which can be prevented by treatment with estrogens. This study used a proteomic approach for the first time to obtain a global perspective of estrogens' effects on whole-bone proteins. Bone proteome profiles were examined in three groups of mice: (1) sham-operated with normal ovarian functions, (2) ovariectomised and (3) ovariectomised with estrogen replacement therapy. Bone proteins extracted from the humerus were separated by 2-DE and visualised by CBB colloidal staining. Spot detection and quantification was done by image analysis. Differentially expressed proteins were identified by MS and database search, using peptide mass fingerprint and peptide sequence analysis. Differential expression analysis in the three experimental groups showed significant changes for 14 proteins. These included proteins related to bone metabolism, cytoskeleton components and energy metabolic pathways. Our data suggest that some proteins related to cytoskeleton and to energy pathways, such as tropomyosins, aconitase 2 and enolase beta, might be new molecular targets responsive to the effects of estrogen. Differentially expressed proteins identified in this model may offer a useful starting point for elucidating novel aspects of the pleiotropic effects of estrogens on bone.     

Detection of glycation sites in proteins by high-resolution mass spectrometry combined with isotopic labeling

The products of nonenzymatic glycation of proteins are formed in a chemical reaction between reducing sugars and the free amino group located either at the N terminus of the polypeptide chain or in the lysine side chain. Glycated proteins and their fragments could be used as markers of the aging process as well as diabetes mellitus and Alzheimer’s disease, making them an object of interest in clinical chemistry. In this article, we propose a new method for the identification of peptide-derived Amadori products in the mixtures obtained by enzymatic hydrolysis of glycated proteins. Two proteins, ubiquitin and human serum albumin (HSA), were modified with an equimolar mixture of glucose and [¹³C₆]glucose and were subjected to enzymatic hydrolysis. The obtained enzymatic digests were analyzed by high-resolution mass spectrometry (HRMS), and the peptide-derived Amadori products were identified on the basis of specific isotopic patterns resulting from ¹³C substitution. The number of glycated peptides in the digest of HSA detected by our procedure was in agreement with the data recently reported in the literature.     

Proteomic analysis of the binding partners to enteropathogenic Escherichia coli virulence proteins expressed in Saccharomyces cerevisiae

Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much worldwide morbidity and mortality. EPEC uses a type III secretion system to inject bacterial proteins into the cytosol of intestinal epithelial cells to cause diarrheal disease. We are interested in determining the host proteins to which EPEC translocator and effector proteins bind during infection. To facilitate protein enrichment, we created fusions between GST and EPEC virulence proteins, and expressed these fusions individually in Saccharomyces cerevisiae. The biology of S. cerevisiae is well understood and often employed as a model eukaryote to study the function of bacterial virulence factors. We isolated the yeast proteins that interact with individual EPEC proteins by affinity purifying against the GST tag. These complexes were subjected to ICAT combined with ESI-MS/MS. Database searching of sequenced peptides provided a list of proteins that bound specifically to each EPEC virulence protein. The dataset suggests several potential mammalian targets of these proteins that may guide future experimentation.     

Distribution of alpha1-adrenoceptor subtype proteins in different tissues of neonatal and adult rats

Distribution of alpha(1)-adrenoceptor (alpha(1)AR) subtype (alpha(1A), alpha(1B), alpha(1D)) proteins in brain, heart, kidney, and liver of 1-week-old rats and in brain, heart, aorta, kidney, liver, vas deferens, prostate, and adrenal glands of adult rats was investigated by Western analysis, using receptor subtype specific polyclonal antibodies. High levels of immunoreactive alpha(1A)AR and alpha(1D)AR in brain and heart and of alpha(1B)AR in liver and heart of neonatal rats were detected. In adult rat tissues, the abundance of alpha(1A)AR protein was most marked in the brain, intermediate in heart, aorta, liver, vas deferens, and adrenals, and minimal in the kidney and prostate; relative to other tissues, the expression of alpha(1B)AR was higher in brain and heart and that of alpha(1D)AR in brain. All the three receptor subtypes increased with age in the brain cortex, whereas the abundance of alpha(1B)AR increased in the heart but decreased in the liver; alpha(1A)AR and alpha(1D)AR in liver, kidney, and heart were not affected by age. It is concluded that alpha(1)AR subtypes are widely expressed in different neonatal and adult rat tissues.     

冈比亚按蚊嗅觉结合蛋白候选基因cDNA的克隆、鉴定及其表达型分析

疟蚊主要依靠嗅觉发现寄主。非洲疟蚊冈比亚按蚊Anopheles gambiae是一种嗜吸人血的疟疾传播媒介昆虫。该文作者基于其全基因组序列,采用RT-PCR和标准分子克隆技术获得2个嗅觉结合蛋白候选基因agLZ3788和agLZ9988。测序分析结果表明,它们具有嗅觉结合蛋白的标志性结构域。进一步采用半定量RT-PCR技术研究了它们的空间表达型,结果发现它们不但在雌蚊触角中表达,也在其他部位(尤其是蚊虫足部)有强的表达。这一发现说明疟蚊嗅觉结合蛋白可能具有更广的功能,也为进一步重组表达和功能研究提供了重要依据。     

Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A.arabiensis

~~Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A. arabiensis1. Curtis, C. F., Introduction 1: An overview of mosquito biology, behaviour and importance, in Olfaction in Mosquito-Host Interactions (eds. Bock, G. R.. Cardew, G.), New York: Wiley, 1996, 3-7. 2. Nighom, A., Hildebrand. J. G.. Dissecting the molecular mechanisms of olfaction in a malaria-vector mosquito, PNAS, 2002, 99(3): 1113-…     

Identification of differentially expressed proteins in Ficedula flycatchers

This is the first report on the large-scale identification and comparison of proteins in non-model organisms, Ficedula flycatchers. It highlights the potential of proteomics approaches in both non-sequenced and non-model organisms for identification of differentially expressed proteins. Not surprisingly, more than 55% of the proteins failed to be identified even though the MS spectra were of high quality. Nevertheless, the protein information obtained in this study will serve as a valuable resource for continued research.