Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

A comparative study of eggshell proteins in lepidoptera

As a first step in the study of chorion composition, biochemical development and morphogenesis, we have studied the proteins of moth chorions (eggshells). We draw attention to the extensive similarities of these proteins in a variety of species. We also report that the eggshell proteins are deposited in succession, each with its characteristic time table. This phenomenon may be related to the morphogenesis of chorion.     

Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB

Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.     

Minor proteins and enzymes of the Drosophila eggshell matrix

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.     

Biosynthetic relationship between the major matrix proteins of adrenal chromaffin granules

The matrix of the chromaffin granule contains a family of acidic proteins, collectively known as the chromogranins. It has been suggested that this family results from protease action on the major component, chromogranin A. Evidence for this has now been obtained from in vitro translation of adrenal medullary messenger RNA and immunoprecipitation of translation products using an antiserum directed against chromogranin A, but which also recognises other chromogranins.     

Ostrich (Struthio camelus) eggshell matrix contains two different C-type lectin-like proteins. Isolation, amino acid sequence, and posttranslational modifications

Comparative analysis of the human and mouse genomic sequences downstream of the apolipoprotein E gene (APOE) revealed a highly conserved element with previously undefined function. In reporter gene transfection studies, this element which is located approximately 42 kb distal to APOE was found to have silencer activity in a subset of cell lines examined. Analysis of transgenic mice containing a fusion construct linking this distal 631 bp conserved element to a reporter gene comprised of the human APOE gene with its proximal promoter resulted in robust brain expression of the transgenic human apoE mRNA in three independent transgenic lines, supporting the identification of a novel brain controlling region (BCR). Further studies using immunohistochemistry revealed widespread human apoE localization throughout the brains of the BCR-apoE transgenic mice with prominent expression in the cortex and diencephalon. In addition, double-label immunofluorescence performed on brain sections and cultures of primary cortical cells localized human apoE protein to cortical neurons and microglia. These studies demonstrate that comparative sequence analysis is a successful strategy to predict candidate regulatory regions in vivo, although they do not imply that this element controls apoE expression physiologically.     

Structure-function relationship of lapemis toxin: a synthetic approach

The synthetic approach to the structure-function relationship of lapemis toxin has been very useful in clarifying the important binding regions. To identify the neurotoxic binding domain(s) of lapemis toxin, several peptides were synthesized using the 9-fluorenylmethoxycarbonyl protocols. These peptides were based on the sequence of lapemis toxin, a 60-amino-acid, short-chain postsynaptic neurotoxin found in sea snake (Lapemis hardwickii) venom. The peptides were purified using high-performance liquid chromatography and sequenced to verify the correct synthesis, isolation, and purity. The synthetic peptide names and single letter sequences were Peptide A1 (15 mer) CCNQQSSQPKTTTNC Peptide B1 (18 mer) CYKKTWSDHRGTRIERGC Peptide B2 (16 mer) YKKTWSDHRGTRIERG Peptide C1 (12 mer) CPQVKPGIKLEC Peptide NS (20 mer) EACDFGHIKLMNPQRSTVWY. The peptide NS (nonsense peptide) sequence was arbitrarily determined and used as a control peptide. Biological activities of the synthetic peptides were determined by in vivo as well as by in vitro assay methods. For the in vivo assay, lethality was determined by intravenous injection in mice (Swiss Webster). For the in vitro assay, peptide binding to the Torpedo californica nicotinic acetylcholine receptor was determined. The peptides were found to be nontoxic at approximately 114 times the known LD50 of lapemis toxin. Binding studies with 125I-radiolabeled lapemis toxin and tyrosine-containing peptides indicated that lapemis toxin and peptide B1 bound the receptor, while the other peptides had no detectable binding. The central loop domain of lapemis toxin (peptide B1) plays a dominate role in the toxin's binding ability to the receptor. These results and the hydrophilicity analysis predict peptide B1 may serve as an antagonist or antigen to neutralize the neurotoxin effects in vivo.     

Crystal structure of ovocleidin-17, a major protein of the calcified Gallus gallus eggshell: implications in the calcite mineral growth pattern

Ovocleidin-17 (OC17) from Gallus gallus is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. Here, the crystal structure of monomeric OC17, determined at a resolution of 1.5 A, was refined to a crystallographic R-factor of 20.1%. This is the first protein directly involved in a non-pathological biomineralization process resolved by x-ray diffraction to date. The protein has a mixed alpha/beta structure containing a single C-type lectin-like domain. However, although OC17 shares the conserved scaffold of the C-type lectins, it does not bind carbohydrates. Nevertheless, in vitro OC17 modifies the crystalline habit of calcium carbonate (CaCO3) and the pattern of crystal growth at intervals of 5-200 microg/ml. Determining the three-dimensional structure of OC17 contributes to a better understanding of the biological behavior of structurally related biomolecules and of the mechanisms involved in eggshell and other mineralization processes.     

Crystallization and preliminary x-ray analysis of ovocleidin-17 a major protein of the gallus gallus eggshell calcified layer

In this work, we report the crystallization of ovocleidin-17, the major protein of the avian eggshell calcified layer and the preliminary X-ray characterization of this soluble protein which is implied into the CaCO(3) formation of the eggshell in avians. Crystals belong to one of the trigonal space group P3 with cell dimensions a= b= 59.53 A and c = 83.33 A, and alpha=beta= 90 degrees and gamma=120 degrees. Crystals diffract up to 3.0 A.     

Aeroallergens: a comparative study of two monitoring methods

Olive and grass pollen grains are the major causes of hay fever in the Mediterranean region. A number of samplers and methods have been developed in recent years in order to obtain reliable data regarding airborne allergen concentrations. This paper reports on a detailed comparison of two samplers—Cyclone and ChemVol—and on the parameters that could influence their efficiency. Airborne concentrations of two key olive and grass allergens, Ole e 1 and Phl p 5, respectively, were monitored over two years with different weather patterns, 2012 and 2014. Allergenic particles were quantified by ELISA assay, and results were compared with pollen concentrations monitored using a Hirst-type volumetric spore trap over the same study periods. The influence of weather-related parameters on local airborne pollen and allergen concentrations was also analysed. Although a positive correlation was detected between results obtained using the two samplers during the pollen season, results for the cumulative annual Allergen Index varied considerably. The two samplers revealed a positive correlation between pollen concentrations and both minimum temperature during the warmer year (2012) and maximum temperature during the cooler year (2014); a negative significant correlation was observed in both cases with rainfall and relative humidity. In summary, although some differences were observed between the two samplers studied, both may be regarded as suitable for allergen detection.     

Interaction between two major outer membrane proteins of Escherichia coli: the matrix protein and the lipoprotein.

The affinity to the matrix protein, one of the major outer membrane proteins of Escherichia coli, for the peptidoglycan was examined of extracting the cell envelope complex at 55 degrees C and 2% sodium dodecyl sulfate containing different amounts of NaCl. It was found that the matrix protein was extracted from the peptidoglycan of a mutant strain (lpo) that lacks another major membrane protein, the lipoprotein, at a lower NaCl concentration than was the matrix protein of the wild-type cell (lpo+). When the envelope fraction of the wild-type strain was treated with trypsin, which is known to cleave the bound-form lipoprotein from the peptidoglycan, the affinity of the matrix protein for the peptidoglycan decreased to the same level as that of the affinity of the matrix protein for the peptidoglycan of the mutant strain. It was further shown that the free-form lipoprotein was also retained in the matrix protein-peptidoglycan complex, although the extent of retention of the free form of the lipoprotein was less than that of the matrix protein. These results indicate that both the free and the bound forms of the lipoprotein are closely associated with the matrix protein and that the bound form of the lipoprotein plays and important role in the association between the matrix protein and the peptidoglycan.     

Microsatellites and single nucleotide polymorphisms in avian hybrid identification: a comparative case study

The correct identification of hybrids is essential in avian hybridisation studies, but selection of the appropriate set of genetic markers for this purpose is at times complicated. Microsatellites and single nucleotide polymorphisms (SNPs) are currently the most commonly used markers in this field. We compare the efficiency of these two marker types, and their combination, in the identification of the threatened avian species, the greater spotted eagle and the lesser spotted eagle, as well as hybrids between the two species. We developed novel SNP markers from genome-wide distributed 122 candidate introns using only sympatric samples, and tested these markers successfully in 60 sympatric and allopatric spotted eagles using Bayesian model-based approaches. Comparatively, only one out of twelve previously described avian nuclear intron markers showed significant species-specific allele frequency difference, thus stressing the importance of selecting the proper markers. Twenty microsatellites outperformed selected nine SNPs in species identification, but were poorer in hybrid detection, whereas the resolution power of ten microsatellites remained too low for correct assignment. A combination of SNPs and microsatellites resulted in the most efficient and accurate identification of all individuals. Our study shows that the use of various sets of markers could lead to strikingly different assignment results, hybridisation studies may have been affected by too low a resolution power of used markers, and that an appropriate set of markers is essential for successful hybrid identification.     

Soluble organic matrix of two Scleractinian corals: partial and comparative analysis

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.     

Lipid-protein interactions of integral membrane proteins: a comparative simulation study

The interactions between membrane proteins and their lipid bilayer environment play important roles in the stability and function of such proteins. Extended (15-20 ns) molecular dynamics simulations have been used to explore the interactions of two membrane proteins with phosphatidylcholine bilayers. One protein (KcsA) is an alpha-helix bundle and embedded in a palmitoyl oleoyl phosphatidylcholine bilayer; the other (OmpA) is a beta-barrel outer-membrane protein and is in a dimyristoyl phosphatidylcholine bilayer. The simulations enable analysis in detail of a number of aspects of lipid-protein interactions. In particular, the interactions of aromatic amphipathic side chains (i.e., Trp, Tyr) with lipid headgroups, and "snorkeling" interactions of basic side chains (i.e., Lys, Arg) with phosphate groups are explored. Analysis of the number of contacts and of H-bonds reveal fluctuations on an approximately 1- to 5-ns timescale. There are two clear bands of interacting residues on the surface of KcsA, whereas there are three such bands on OmpA. A large number of Arg-phosphate interactions are seen for KcsA; for OmpA, the number of basic-phosphate interactions is smaller and shows more marked fluctuations with respect to time. Both classes of interaction occur in clearly defined interfacial regions of width approximately 1 nm. Analysis of lateral diffusion of lipid molecules reveals that "boundary" lipid molecules diffuse at about half the rate of bulk lipid. Overall, these simulations present a dynamic picture of lipid-protein interactions: there are a number of more specific interactions but even these fluctuate on an approximately 1- to 5-ns timescale.     

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.     

Heat-shock responses in two leguminous plants: a comparative study

Relative growth rates, basal and acclimated thermotolerance, membrane damage, fluorescence emission, and relative levels of free and conjugated ubiquitin and HSP70 were compared after 2 h of treatment at different temperatures between Prosopis chilensis and Glycine max (soybean), cv. McCall, to evaluate if the thermotolerance of these two plants was related to levels of accumulation of heat shock proteins. Seedlings of P. chilensis germinated at 25 degrees C and at 35 degrees C and grown at temperatures above germination temperature showed higher relative growth than soybean seedlings treated under the same conditions. The lethal temperature of both species was 50 degrees C after germination at 25 degrees C. However, they were able to grow at 50 degrees C after germination at 35 degrees C. Membrane damage determinations in leaves showed that P. chilensis has an LT(50) 6 degrees C higher than that of soybean. There were no differences in the quantum yield of photosynthesis (F(v)/F(m)), between both plants when the temperatures were raised. P. chilensis showed higher relative levels of free ubiquitin, conjugated ubiquitin and HSP70 than soybean seedlings when the temperatures were raised. Time-course studies of accumulation of these proteins performed at 40 degrees C showed that the relative accumulation rates of ubiquitin, conjugated ubiquitin and HSP70 were higher in P. chilensis than in soybean. In both plants, free ubiquitin decreased during the first 5 min and increased after 30 min of heat shock, conjugated ubiquitin increased after 30 min and HSP70 began to increase dramatically after 20 min of heat shock. From these data it is concluded that P. chilensis is more tolerant to acute heat stress than soybean.     

Structure-function relationship of Atg12, a ubiquitin-like modifier essential for autophagy

Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation.