Isolation and characterization of a novel perivitellin from the eggs of Pomacea scalaris (Mollusca, Ampullariidae)

Perivitellins are important components of the perivitelline fluid (PVF) that surrounds gastropod embryos. The glyco-lipo-carotenoprotein ovorubin (OR) from eggs of the snail Pomacea canaliculata has been the most studied to date. Here we report the characterization of scalarin (SC), a glyco-lipo-carotenoprotein from the PVF of P. scalaris. SC was purified by ultracentrifugation and exclusion chromatography. It is the major egg protein, representing 64% of the total soluble protein. The particle has a hydration density of 1.26 g/ml, an apparent molecular mass of 380 kDa and it is an elongated compact protein as estimated by small angle X-ray scattering (SAXS). It is composed of three subunits of ca. 35, 28, and 24 kDa noncovalently bonded. SC is highly glycosylated (carbohydrate content 20.1%, by wt.), with a low lipid content (0.7%), being esterified sterols, pigments and polar lipids the most abundant lipid classes. HPTLC and spectrophotometric analysis of the carotenoid fraction revealed the presence of free astaxanthin (ASX; 62.0%), and an unidentified carotenoid (38.0%). The carotenoid-apoprotein interaction was studied by spectrophotometry. Carotenoids do not seem to affect the structural characteristics of the oligomer. However, the carotenoid-protein association protected ASX against oxidation. The cross-reactivity between SC and perivitellins of P. canaliculata was tested using polyclonal antibodies (PAb) against SC, OR, and perivitellin PV2. The PAbs failed to cross-react with any egg proteins of either the same or other species. SC, among other functional similarities with OR, would be an antioxidant carrier, protecting at the same time carotenoids from oxidation in the perivitellin fluid of the egg.     

Characterization of the major egg glycolipoproteins from the perivitellin fluid of the apple snail Pomacea canaliculata

Ovorubin and PV2 are the major lipoglycocarotenoproteins present in the perivitellus of the freshwater snail eggs of Pomacea canaliculata, a rapidly expanding rice field pest. We have previously characterized these two particles regarding their lipid and protein compositions, their synthesis and tissular distribution, and their contributions of energy and structural precursors for the developing embryo. In the present study, we have characterized the glycosidic moieties associated to these perivitellines. Both proteins were isolated from egg homogenates by ultracentrifugation, and high performance liquid chromatography (HPLC) using anionic exchange and size exclusion columns. Total carbohydrates accounted for 17.8% and 2.5% (w/w) of the apparent molecular mass of ovorubin and PV2, respectively. Analysis by size exclusion chromatography showed that the amount of O-linked oligosaccharides is higher than that of the N-linked species (59% and 67% w/w of total carbohydrates of ovorubin and PV2, respectively). Glycosylation patterns were determined by a set of biotinilated lectins onto blotted purified proteins. Lectin affinities confirmed the presence of aspargine-linked carbohydrates, probably of hybrid and high mannose types. Jacaline affinity suggested the presence of O-linked residues derived from the T-antigen. Total carbohydrate composition determined by gas liquid chromatography (GLC) showed that mannose was the major monosaccharide in both perivitellins followed by GlcNAc and Gal in ovorubin, and Gal and GlcNAc in PV2. Only one fatty acid (22:1 n-9) accounted for 46% and 56% of the fatty acids present in ovorubin and PV2, respectively. Carbohydrate role on these reserve proteins during embryogenesis of the apple snail is discussed.     

Analysis of albumen gland proteins suggests survival strategies of developing embryos of Pomacea canaliculata

The albumen gland of Pomacea canaliculata secretes the perivitelline fluid (PVF) surrounding the developing embryo which gives its eggs a highly effective defence against environmental factors and predators. Although previous studies have determined the functions of some PVF proteins extracted from P. canaliculata eggs, knowledge of the protein composition of PVF is still limited. In this study we use LC-MS/MS to identify 87 proteins from the albumen gland of P. canaliculata, 53 of which (60.9%) were annotated using the KEGG database. These classified into five functional groups: metabolism (54.7%); genetic information processing (9.4%); environmental information processing (3.8%); cellular processes (11.3%); and organismal systems (20.8%). The analyses found 12 proteins related to innate immunity and three proteins linked to defence against predation, providing important information for studies on embryo survival strategies and the invasive capabilities of P. canaliculata.     

Antioxidant defense system in the apple snail eggs, the role of ovorubin

A novel role of ovorubin as a protection system against oxidative damage in eggs from Pomacea canaliculata was investigated. Carotenoid composition, and their antioxidant capacity, as well as the carotenoid-apoprotein interaction, were studied for this lipoglycocarotenoprotein. Carotenoid extracts from ovorubin were analysed by TLC and spectrophotometry. The major carotenoid was astaxanthin in its free (40%), monoester (24%), and diester (35%) forms, mainly esterified with 16:0 fatty acid. The antioxidant capacity of ovorubin carotenoids was studied by the inhibition of microsomal oxidation in a non-enzymatic system, showing strong protection against oxidative damage (IC50=3.9 nmol/mg protein). The carotenoid-apoprotein interaction was studied by spectrophotometry and electrophoresis using reconstituted ovorubin. Astaxanthin does not seem to affect the structural characteristics of ovorubin, however the carotenoid-protein association significantly protected astaxanthin against oxidation. Ovorubin therefore, besides its role in providing energy and structural precursors during embryogenesis, would be an antioxidant carrier, protecting at the same time this pigment from oxidation in the perivitellin fluid environment of the egg.     

Metabolism of ovorubin,the major egg lipoprotein from the apple snail

The site of synthesis of molluscs lipoproteins is little known and was investigated for the egg lipoprotein perivitellin 1 (PV1) or ovorubin in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad–digestive gland complex and muscle) of vitellogenic females were incubated in vitro at 25°C for 12 h with ¹⁴C Leucine. After that, soluble proteins from tissue homogenates and medium samples were analysed for de novo protein synthesis by electrophoresis and HPLC, and radiolabelled proteins quantified by liquid scintillation. Gonad–digestive gland complex did not synthesise ovorubin, in spite its high protein synthesis levels. Three albumen gland radiolabelled proteins (35, 32 and 28 kDa) comigrated with the subunits of ovorubin and represented 1.3% of the total labelled protein of that tissue. Western blot analysis with polyclonal antibodies confirmed that these were ovorubin subunits. In vivo experiments where vitellogenic females were injected with ³H Leucine, revealed that ovorubin was not present in hemolymph. ELISA analysis confirmed ovorubin presence only in albumen gland and developing eggs with levels of 800 and 582 mg/g protein, which represent 30.3 and 28.4 mg ovorubin/g of tissue, respectively. Therefore, albumen gland is the single site of ovorubin synthesis as no extragland synthesis, circulation or accumulation could be detected in the apple snail.     

The role of the proteinase inhibitor ovorubin in apple snail eggs resembles plant embryo defense against predation

Background

Fieldwork has thoroughly established that most eggs are intensely predated. Among the few exceptions are the aerial egg clutches from the aquatic snail Pomacea canaliculata which have virtually no predators. Its defenses are advertised by the pigmented ovorubin perivitellin providing a conspicuous reddish coloration. The nature of the defense however, was not clear, except for a screening for defenses that identified a neurotoxic perivitellin with lethal effect on rodents.Ovorubin is a proteinase inhibitor (PI) whose role to protect against pathogens was taken for granted, according to the prevailing assumption. Through biochemical, biophysical and feeding experiments we studied the proteinase inhibitor function of ovorubin in egg defenses.

Methodology/Principal Findings

Mass spectrometry sequencing indicated ovorubin belongs to the Kunitz-type serine proteinase inhibitor family. It specifically binds trypsin as determined by small angle X-ray scattering (SAXS) and cross-linking studies but, in contrast to the classical assumption, it does not prevent bacterial growth. Ovorubin was found extremely resistant to in vitro gastrointestinal proteolysis. Moreover feeding studies showed that ovorubin ingestion diminishes growth rate in rats indicating that this highly stable PI is capable of surviving passage through the gastrointestinal tract in a biologically active form.

Conclusions

To our knowledge, this is the first direct evidence of the interaction of an egg PI with a digestive protease of potential predators, limiting predator''s ability to digest egg nutrients. This role has not been reported in the animal kingdom but it is similar to plant defenses against herbivory. Further, this would be the only defense model with no trade-offs between conspicuousness and noxiousness by encoding into the same molecule both the aposematic warning signal and an antinutritive/antidigestive defense. These defenses, combined with a neurotoxin and probably unpalatable factors would explain the near absence of predators, opening new perspectives in the study of the evolution and ecology of egg defensive strategies.     

Global shape and pH stability of ovorubin, an oligomeric protein from the eggs of Pomacea canaliculata

Ovorubin, a 300-kDa thermostable oligomer, is the major egg protein from the perivitellin fluid that surrounds the embryos of the apple snail Pomacea canaliculata. It plays essential roles in embryo development, including transport and protection of carotenoids, protease inhibition, photoprotection, storage, and nourishment. Here, we report ovorubin dimensions and global shape, and test the role of electrostatic interactions in conformational stability by analyzing the effects of pH, using small-angle X-ray scattering (SAXS), transmission electron microscopy, CD, and fluorescence and absorption spectroscopy. Analysis of SAXS data shows that ovorubin is an anisometric particle with a major axis of 130 A and a minor one varying between 63 and 76 A. The particle shape was not significantly affected by the absence of the cofactor astaxanthin. The 3D model presented here is the first for an invertebrate egg carotenoprotein. The quaternary structure is stable over a wide pH range (4.5-12.0). At a pH between 2.0 and 4.0, a reduction in the gyration radius and a loss of tertiary structure are observed, although astaxanthin binding is not affected and only minor alterations in secondary structure are observed. In vitro pepsin digestion indicates that ovorubin is resistant to this protease action. The high stability over a considerable pH range and against pepsin, together with the capacity to bear temperatures > 95 degrees C, reinforces the idea that ovorubin is tailored to withstand a wide variety of conditions in order to play its key role in embryo protection during development.     

Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan

Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two‐dimensional gel electrophoresis and identified by matrix‐assisted laser desorption/ionization of time‐of‐flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co‐occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen‐related and energy‐production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies.     

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.     

Proteomics of ionically bound and soluble extracellular proteins in Medicago truncatula leaves

A large proportion of the apoplast proteome resides in the intercellular fluid (IF) or is ionically bound (IB) to the wall matrix. A combined analysis of IF and IB proteins of the Medicago truncatula leaf apoplast was performed. 2-DE analyses demonstrated the reproducible presence of 220 IF and 84 IB proteins in the apoplast. These two protein populations were largely distinct; 22 proteins could be spatially matched, but MALDI-TOF/TOF analyses suggested a considerably smaller number had common identities. MALDI-TOF/TOF characterisation identified 81 distinct proteins. Analyses of selected IF proteins (45) indicated 17 distinct proteins with mainly defence-related functions, whereas analyses of IB proteins (70) identified 63 distinct proteins of diverse natures, including proteins of non-canonical natures. The presence of non-canonical proteins in IB extracts is discussed in the light of evidence supporting a low level of contamination of purified walls from symplastic proteins. This work indicates that IB and IF proteins are functionally distinct fractions of the apoplast. The data obtained complements earlier studies of the Medicago proteome and therefore will be useful in future studies investigating the role of apoplastic proteins in plant processes.     

Differential recruitment of nuclear coregulators directs the isoform-dependent action of mutant thyroid hormone receptors

Studies using mice deficient in thyroid hormone receptors (TR) indicate that the two TR isoforms, TRα1 and TRβ1, in addition to mediating overlapping biological activities of the thyroid hormone, T3, also mediate distinct functions. Mice harboring an identical dominant negative mutation (denoted PV) at the C terminus of TRα1 (Thra1(PV) mice) or β1 (Thrb(PV) mice) also exhibit distinct phenotypes. These knockin mutant mice provide an opportunity to understand the molecular basis of isoform-dependent functions in vivo. Here we tested the hypothesis that the distinct functions of TR mutant isoforms are directed by a subset of nuclear regulatory proteins. Tandem-affinity chromatography of HeLa nuclear extracts showed that distinct 33 nuclear proteins including nuclear receptor corepressor (NCoR1) and six other proteins preferentially associated with TRα1PV or TRβ1PV, respectively. These results indicate that recruitment of nuclear regulatory proteins by TR mutants is subtype dependent. The involvement of NCoR1 in mediating the distinct liver phenotype of Thra1(PV) and Thrb(PV) mice was further explored. NCoR1 preferentially interacted with TRα1PV rather than with TRβ1PV. NCoR1 was recruited more avidly to the thyroid hormone response element-bound TRα1PV than to TRβ1PV in the promoter of the CCAAT/enhancer-binding protein α gene to repress its expression in the liver of Thra1(PV) mice, but not in Thrb(PV) mice. This preferential recruitment of NCoR1 by mutant isoforms could contribute, at least in part, to the distinct liver lipid phenotype of these mutant mice. The present study highlights a novel mechanism by which TR isoforms direct their selective functions via preferential recruitment of a subset of nuclear coregulatory proteins.     

Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two‐dimensional gel electrophoresis and separation by ion‐exchange and reverse‐phase high‐performance liquid chromatography followed by mass spectrometry using tanden matrix‐assisted laser desorption/ionization with time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry and electrospray ionization‐quadrupole with time‐of‐flight (ESI‐Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10‐ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.     

Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae

Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate‐rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF‐MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density‐gradient ultracentrifugation. The vacuole‐containing fraction was subjected to SDS–PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non‐lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o‐methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin‐tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.     

Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans

The hydatid cyst fluid (HCF) of Echinococcus granulosus is a complex biological mixture containing a wide range of proteins of both parasite and host origin. Using a combination of in- and off-gel protein fractionation techniques and tandem mass spectrometry 130 HCF proteins were identified from fertile cysts of sheep and human origin and infertile cysts from cattle. Forty-eight proteins were of parasite origin including Antigen 5 and Antigen B--the most abundant parasite proteins, thioredoxin, low-density lipoprotein receptors, cyclophilin and ferritin. Across the three host species the identified HCF proteins were broadly similar although, based on spectral counts, three proteins, including an antigen B isoform, were more abundant in sheep HCF compared with the fluids of cattle and human origin. Eighty-two host proteins were identified in HCF from the three species. Host plasma proteins were the most abundant, although approximately thirty of the host proteins that were identified are not considered constituents of plasma. The identification of parasite heat shock proteins and annexin A13 exclusively in infertile cysts, along with an increased spectral count for cathepsin B, supports the hypothesis of increased cellular stress and apoptosis as the cause of their infertility.     

Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases

We have identified and cloned 22 human cDNAs encoding novel members of the ubiquitin-specific protease (USP) family. Eighteen of the identified proteins contain all structural features characteristic of these cysteine proteinases, whereas four of them have been classified as non-peptidase homologues. Northern blot analysis demonstrated that the identified USPs are broadly and differentially distributed in human tissues, some of them being especially abundant in skeletal muscle or testis. Enzymatic studies performed with the identified USPs revealed that at least twelve of them are deubiquitylating enzymes based on their ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein. These results provide additional evidence of the extreme complexity and diversity of the USP proteolytic system in human tissues and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in normal and pathological functions.     

A proteomic fingerprint of dissolved organic carbon and of soil particles

Mass spectrometry-based proteomics was applied to analyze proteins isolated from dissolved organic matter (DOM). The focal question was to identify the type and biological origin of proteins in DOM, and to describe diversity of protein origin at the level of higher taxonomic units, as well as to detect extracellular enzymes possibly important in the carbon cycle. Identified proteins were classified according to their phylogenetic origin and metabolic function using the National Center for Biotechnology Information (NCBI) protein and taxonomy database. Seventy-eight percent of the proteins in DOM from the lake but less than 50% in forest soil DOM originated from bacteria. In a deciduous forest, the number of identified proteins decreased from 75 to 28 with increasing soil depth and decreasing total soil organic carbon content. The number of identified proteins and taxonomic groups was 50% higher in winter than in summer. In spruce forest, number of proteins and taxonomic groups decreased by 50% on a plot where trees had been girdled a year before and carbohydrate transport to roots was terminated. After girdling, proteins from four taxonomic groups remained as compared to nine taxonomic groups in healthy forest. Enzymes involved in degradation of organic matter were not identified in free soil DOM. However, cellulases and laccases were found among proteins extracted from soil particles, indicating that degradation of soil organic matter takes place in biofilms on particle surfaces. These results demonstrate a novel application of proteomics to obtain a proteomic fingerprint of presence and activity of organisms in an ecosystem.This revised version was published online in December 2004 with corrections to Figs.1-4     

MafT, a new member of the small Maf protein family in zebrafish

Small Maf proteins play critical roles on morphogenesis and homeostasis through associating with CNC proteins. To date, three small Maf proteins, MafF, MafG, and MafK, have been reported in vertebrates, which share redundant functions. In this study, we tried to identify and characterize small Maf proteins in zebrafish to elucidate their conservation and diversity in the fish kingdom. We identified homolog genes of MafG and MafK but not MafF in zebrafish, indicating the former two are conserved among vertebrates. In addition, a novel type of small Maf protein MafT was identified. MafT protein bound MARE sequence as a homodimer or heterodimers with zebrafish Nrf2 or p45 Nfe2. Co-overexpression of MafT and Nrf2 synergistically activated MARE-mediated gene expression in zebrafish embryos. These results indicated that MafT is a new member of small Maf proteins and involved in the Nrf2-dependent gene regulation in cellular defense system.