High Molecular Mass Protein Research Articles

Pru p 10: the polygalacturonase described as a new peach tree pollen allergen

Peach tree pollen (PTP) is responsible for the induction of respiratory and ocular symptoms in a population exposed to peach cultivars, with great relevance in occupational allergy. In its allergenic profile, several high molecular mass proteins with significant IgE recognition were described. In order to deepen into these allergens, the main objective was the purification, identification and verification of the allergenic capacity of the 46 kDa band protein.

DIGE Analysis of Clinical Specimens.

Two-dimensional difference gel electrophoresis (2D-DIGE) is an elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2D-GE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The use of an internal pooled standard makes 2D-DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. Technical limitations of this technique (i.e., underrating of low abundant, high molecular mass and integral membrane proteins) are counterbalanced by the incomparable separation power which allows proteoforms and unknown PTM (posttranslational modification) identification. Moreover, the image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

Analyzing the influence of age and sex in Bothrops pauloensis snake venom

Considerable heterogeneity and ontogenetic changes in venom composition have already been observed in different species of snakes within the Viperidae family. Since the venom of young and adult can cause distinct pathological effects and because the antivenom may be less effective in neutralizing envenoming by young snakes compared to adults, it is of paramount importance to understand the ontogenetic variation of snake venom. Thus, the present study aimed to analyze and compare the venom of Bothrops pauloensis snakes, searching for possible influences of ontogeny and sex in their biochemical and biological aspects. The venom of younger individuals was more complex in relation to high molecular mass proteins, with a greater abundance of metalloproteinases, while adults showed a greater abundance of medium and low molecular mass proteins, such as phospholipases A2 (PLA2), C-type lectins and serine proteases. The antivenom showed better immunorecognition towards the venom of adult snakes than younger ones, in addition to a deficiency in the recognition of medium molecular mass proteins, suggesting the need for an improvement in the antivenom. Younger snakes showed higher coagulant, caseinolytic, and hemorrhagic activity, while adult snakes showed higher L-amino acid oxidase (LAAO) activity and acted faster in lethality. Differences between males and females were observed mainly in the rate of loss of coagulant activity, change in PLA2 activity and lethality action time. Furthermore, considering only the adult groups, males showed a higher LAAO and thrombin-like activity, while females showed a higher caseinolytic and hyaluronidase activity. With the results obtained in this work, it was possible to conclude that there is an ontogenetic variation in the composition and some activities of the B. pauloensis snake venom, in addition to differences between the venom of males and females, reinforcing that there is an intraspecific variation that may result in different symptoms in their envenoming and, consequently, differences in the response to treatment with the antivenom.

Fuzzy RNA recognition by the Trypanosoma brucei editosome.

The assembly of high molecular mass ribonucleoprotein complexes typically relies on the binary interaction of defined RNA sequences or precisely folded RNA motifs with dedicated RNA-binding domains on the protein side. Here we describe a new molecular recognition principle of RNA molecules by a high molecular mass protein complex. By chemically probing the solvent accessibility of mitochondrial pre-mRNAs when bound to the Trypanosoma brucei editosome, we identified multiple similar but non-identical RNA motifs as editosome contact sites. However, by treating the different motifs as mathematical graph objects we demonstrate that they fit a consensus 2D-graph consisting of 4 vertices (V) and 3 edges (E) with a Laplacian eigenvalue of 0.5477 (λ2). We establish that synthetic 4V(3E)-RNAs are sufficient to compete for the editosomal pre-mRNA binding site and that they inhibit RNA editing in vitro. Furthermore, we demonstrate that only two topological indices are necessary to predict the binding of any RNA motif to the editosome with a high level of confidence. Our analysis corroborates that the editosome has adapted to the structural multiplicity of the mitochondrial mRNA folding space by recognizing a fuzzy continuum of RNA folds that fit a consensus graph descriptor.

High quality purification of German cockroach vitellin/vitellogenin from whole body acetone defatted powder

German cockroach (Blattella germanica; GCr) is a source of indoor aeroallergens associated with allergic rhinitis and asthma. Vitellin/vitellogenin, a high molecular mass and abundant protein in GCr eggs and egg cases, has been identified as a possible allergen serologically. To better study its allergenic properties we purified vitellin/vitellogenin from whole body defatted GCr powder, a readily available source material.

Pharmacologically Active Vanadium Species: Distribution in Biological Media and Interaction with Molecular Targets.

Biospeciation of some of the most studied vanadium (symbol V) complexes with biological or medicinal activity is discussed in this review in order to emphasize the importance of the distribution of V species in biological media. The exact knowledge of the chemical species present in blood or cells may provide essential information regarding the biological effect of V potential drugs. In blood serum, vanadium species can interact with low (citrate, lactate, oxalate, amino acids, etc., indicated with bL) and high molecular mass (proteins like transferrin, albumin, immunoglobulins, etc.) components, while the interaction with red blood cells can interfere with the transport of these drugs towards the target cells. The interaction of bLs and proteins is discussed through the analysis of instrumental and computational data. The fate of the active V species, when these are in the real serum samples and when they reach and cross cell membranes, is also discussed. The differences in the V complexes selected in this review (donor atoms, stability, coordination geometry, electric charge, hydrolipophilicity balance, substituents and redox properties) cover all the possible modes of interaction with bLs and proteins, allowing for the biodistribution of the studied compounds to be predicted. This approach could be applied to newly synthesized potential V drugs.

Antioxidant and Cytotoxic Effects and Identification of Ophiocordyceps sinensis Bioactive Proteins Using Shotgun Proteomic Analysis.

SUMMARYResearch backgroundOphiocordyceps sinensis, a highly valued medicinal fungus, is close to extinction due to overexploitation. Successful cultivation of O. sinensis fruiting body (OCS02®) shows that the cultivar has a promising nutritional value and numerous bioactive compounds. Antioxidant and antiproliferative properties and biologically active proteins of the OCS02® are investigated for possible development into nutraceuticals.Experimental approachThe chemical composition of the OCS02® cold water extract was determined, and the antioxidant activities were examined using ferric reducing, DPPH• and O2•- scavenging assays. Tetrazolium dye (MTT) cytotoxic assay was performed to assess the antiproliferative activity of the extract. Bioactive proteins in the active fraction of the extract were identified using liquid chromatography (LC) and tandem-mass spectrometry (MS/MS).Results and conclusionsThe OCS02® extract exhibited strong O2•- scavenging (expressed as Trolox equivalents (18.4±1.1) mol/g) and potent cytotoxic activities against adenocarcinomic human alveolar basal epithelial (A549) cells (IC50=(58.2±6.8) µg/mL). High molecular mass polysaccharides, proteins and protein-polysaccharide complexes could have contributed to the antioxidant and cytotoxic selectivity of the OCS02®. LC-MS/MS analysis identified several potential cytotoxic proteases and an oxalate decarboxylase protein which may exhibit protection effects on kidneys.Novelty and scientific contributionsThe findings demonstrate the potential of OCS02® to be developed into functional food due to its promising superoxide anion radical scavenging capacity, cytotoxic effect and presence of biopharmaceutically active proteins.

Purification and initial studies on vitellogenin, an allergen in German cockroach

German cockroach (Blattella germanica) is a source of important urban indoor aeroallergens, associated with allergic rhinitis and asthma. Vitellogenin, a high molecular mass and abundant protein in B. germanica eggs and egg cases (oothecae), was purified and assessed for its allergenicity.

2-Cyano-3-(2-thienyl)acrylic Acid as a New MALDI Matrix for the Analysis of a Broad Spectrum of Analytes.

A low-cost synthetic 2-cyano-3-(2-thienyl)acrylic acid (CTA) is developed as a new MALDI matrix for the analysis of various classes of compounds such as lipids (e.g., fatty acids), peptides, proteins, saccharides, natural products (i.e., iridoids), PEGs, and organometallics in the positive-ion mode. The difficulty in the analysis of high molecular mass PEGs was overcome by using CTA as matrix even at low concentrations. Both high molecular mass proteins and peptides were successfully analyzed using CTA. The mass spectra of all of the studied analytes with CTA showed high signal-to-noise (S/N) ratios and spectral resolutions when compared to other conventional matrices such as SA, DHB, DT, and HCCA. However, in the case of peptide analysis with CTA, the resulting mass spectra are found to be similar to that of the well-established HCCA matrix. On the basis of the physicochemical properties of the analytes, the CTA works as a proton/cation or electron-transfer matrix. It proves that the CTA can be used as a common matrix for the analysis of majority classes of analytes instead of using a specific matrix for the particular class of analytes. Further, the CTA provides an advantage in the analysis of unknown samples as it rules out ambiguity in the selection of particular matrix and it may also offer a complete profile of the tissue surface in the MALDI-imaging experiments.

Mechanism and Function of the Catch State in Molluscan Smooth Muscle: A Historical Perspective.

Molluscan smooth muscles exhibit the catch state, in which both tension and resistance to stretch are maintained with very low rates of energy consumption. The catch state is studied mainly on the anterior byssus retractor muscle (ABRM) of a bivalve molluscan animal, Mytilus, which can easily be split into small bundles consisting of parallel fibers. The ABRM contracts actively with an increase in the intracellular free Ca ion concentration, [Ca2+]i, as with all other types of muscle. Meanwhile, the catch state is established after the reduction of [Ca2+]i to the resting level. Despite extensive studies, the mechanism underlying the catch state is not yet fully understood. This article briefly deals with (1) anatomical and ultrastructural aspects of the ABRM, (2) mechanical studies on the transition from the active to the catch state in the isotonic condition, (3) electron microscopic and histochemical studies on the intracellular translocation of Ca ions during the transition from the active to the catch state, and (4) biochemical studies on the catch state, with special reference to a high molecular mass protein, twitchin, which is known to occur in molluscan catch muscles.

Exploring the Extracellular Macromolecular Composition of Crude Extracts of Penicillium rubens Strain 212 for Elucidation Its Mode of Action as a Biocontrol Agent.

Penicillium rubens strain 212 (PO212) acts as an inducer of systemic resistance in tomato plants. The effect of crude extracellular extracts of PO212 on the soil-borne pathogen Fusarium oxysporum f. sp. lycopersici has been evaluated. Evidence of the involvement of soluble, thermo-labile, and proteinase-inactivated macromolecules present in PO212 crude extracts in the control of Fusarium vascular disease in tomato plants was found. Proteomic techniques and the availability of the access to the PO212 genome database have allowed the identification of glycosyl hydrolases, oxidases, and peptidases in these extracellular extracts. Furthermore, a bioassay-guided fractionation of PO212 crude extracellular extracts using an integrated membrane/solid phase extraction process was set up. This method enabled the separation of a PO212 crude extracellular extract of seven days of growth into four fractions of different molecular sizes and polarities: high molecular mass protein fraction >5 kDa, middle molecular mass protein fraction 5–1 kDa, low molecular mass metabolite fraction, and nutrients from culture medium (mainly glucose and minerals). The high and middle molecular mass protein fractions retained disease control activity in a way similar to that of the control extracts. Proteomic techniques have allowed the identification of nine putatively secreted proteins in the high molecular mass protein fraction matching those identified in the total crude extracts. Therefore, these enzymes are considered to be potentially responsible of the crude extracellular extract-induced resistance in tomato plants against F. oxysporum f. sp. lycopersici. Further studies are required to establish which of the identified proteins participate in the PO212’s action mode as a biocontrol agent.

Bile acid metabolism in fish: disturbances caused by fishmeal alternatives and some mitigating effects from dietary bile inclusions

AbstractBile is a yellow‐green liquid produced in the liver from cholesterol and stored in the gallbladder of vertebrates. Bile improves the efficacy of lipid digestion by acting as an emulsifier and is essential in activating bile salt lipase, an enzyme that has broad substrate specificity. Bile improves the absorption of lipid soluble nutrients while also facilitating the excretion of cholesterol and toxic metabolites, particularly bilirubin. Dietary fishmeal alternatives often disturb bile acid status in fish resulting in either increased excretion/decreased intestinal reabsorption and/or decreased bile acid synthesis. Saponins and high molecular mass proteins are believed to contribute to altered bile acid status, which may reduce fish productivity. This situation can worsen with increased incorporations of plant‐based proteins in aquafeeds, but also may be mitigated by processing of fishmeal alternatives as well as the dietary inclusion of some bile acids/salts. This area of research will likely increase due to the roles dietary bile acids/salts can have on protecting digestive organs and improving nutrient utilization. This, however, depends on the bile type, level and fish species. This review discusses these aspects in regard to fish nutrition to help increase the inclusion of dietary fishmeal alternatives and thus enhance aquaculture sustainability.

Получение и определение концентрации в культуральной жидкости фаэцина – пептидного бактериоцина Enterococcus faecium

Phaecin, a peptide bacteriocin with a molecular mass of ~5 kDa, was obtained and purified to an electrophoretically pure state with a yield of ~70% of the total activity in the culture fluid. The method of time-of-flight mass spectrometry (MALDI) showed that bacteriocin consists of two components A and B with similar molecular mass. In previously published works, the yield of purified bacteriocins, usually did not exceed 4–5% of the total activity in the culture fluid. The authors believe that the increase in yield is due to the fact that not only the similarity of bacteriocins with high molecular mass proteins was taken into account, but also differences associated with low molecular mass, the ability to hydrophobic interaction at slightly alkaline pH values and resistance to denaturation. Using the Scopes method, it was possible to determine the concentration of phaecin in the final stage of purification and in the culture fluid in absolute units (mg/l). The methods of the purification with high yield described in this work are probably applicable to other bacteriocins due to the proximity of their physicochemical properties. Key words: Enterococcus faecium, Listeria monocytogenes, bacteriocin, phaecin, culture liquid, adsorption chromatography, Scopes equation

The role of chitin-rich skeletal organic matrix on the crystallization of calcium carbonate in the crustose coralline alga Leptophytum foecundum

The organic matrix (OM) contained in marine calcifiers has a key role in the regulation of crystal deposition, such as crystalline structure, initiation of mineralization, inhibition, and biological/environmental control. However, the functional properties of the chitin-rich skeletal organic matrix on the biological aspect of crystallization in crustose coralline algae have not yet been investigated. Hence, the characterization of organic matrices in the biomineralization process of this species was studied to understand the functions of these key components for structural formation and mineralization of calcium carbonate crystals. We purified skeletal organic matrix proteins from this species and explored how these components are involved in the mineralization of calcium carbonate crystals and environmental control. Intriguingly, the analytical investigation of the skeletal OM revealed the presence of chitin in the crustose coralline alga Leptophytum foecundum. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the OM revealed a high molecular mass protein as 300-kDa. Analysis of glycosylation activity exposed two strong glycoproteins as 300-kDa and 240-kDa. Our study of the biominerals of live collected specimens found that in addition to Mg-calcite up to 30% aragonite were present in the skeleton. Our experiment demonstrated that the chitin-rich skeletal OM of coralline algae plays a key role in the biocalcification process by enabling the formation of Mg-calcite. In addition, this OM did not inhibit the formation of aragonite suggesting there is an as yet unidentified process in the living coralline that prevents the formation of aragonite in the living skeletal cell walls.

Progesterone induced Warburg effect in HEK293 cells is associated with post-translational modifications and proteasomal degradation of progesterone receptor membrane component 1

Progesterone (P4) is a major steroid hormone that has important effects on metabolism. The progesterone receptor membrane component 1 (PGRMC1) is a non-canonical P4 binding protein. The biological functions affected by PGRMC1 include cholesterol/steroid biosynthesis and metabolism, iron homeostasis and heme trafficking, autophagy, regulation of cell cycle and proliferation, cell migration and invasion. PGRMC1 has been an attractive target for therapeutic intervention in cancer and neurodegenerative disorders due to its biological role in promoting cell survival. P4 has been used in a number of clinical applications and is considered neuroprotective. The involvement of PGRMC1 in P4-mediated regulation of cellular glucose metabolism is not well studied. PGRMC1 is a 21 kDa protein but complex post-translational modifications (PTMs) lead to the existence of several high molecular mass proteins whose molecular function, intracellular distribution, and physiological relevancies are not fully known. Therefore, in this study, P4-PGRMC1-mediated cellular glucose metabolism and PTMs of PGRMC1 were studied using wild-type and CRISPR/Cas9 mediated PGRMC1 knockout (KO) human embryonic kidney-derived (HEK293) cell lines. A 70 kDa (p70) and 100 kDa (p100) PGRMC1 proteins were identified that are predominantly associated with endoplasmic reticulum/mitochondria and nuclear fractions in the cells, respectively. Phosphorylation, acetylation, ubiquitination, and sumoylation of native PGRMC1 under serum starvation were identified which provided an explanation for the higher molecular masses. This study indicates that P4-PGRMC1 signaling caused a rapid increase in glycolysis in the presence of oxygen (aerobic glycolysis) and a corresponding decrease in cellular respiration, known as the Warburg effect. Further, it was demonstrated that the P4-induced increase in glycolysis is associated with rapid proteasomal degradation of the p70 and reduction of the nuclear p100 protein level. P4 treatment also caused significant alteration in the dynamics of PGRMC1 PTMs and its association with potential interacting proteins. Overall, this study provides a hitherto unknown aspect of P4-PGRMC1 mediated signaling that changes basic cellular metabolism in HEK293 cells.

Consequences of impaired 1-MDa TIC complex assembly for the abundance and composition of chloroplast high-molecular mass protein complexes.

We report a systematic analysis of chloroplast high-molecular mass protein complexes using a combination of native gel electrophoresis and absolute protein quantification by MSE. With this experimental setup, we characterized the effect of the tic56-3 mutation in the 1-MDa inner envelope translocase (TIC) on the assembly of the chloroplast proteome. We show that the tic56-3 mutation results in a reduction of the 1-MDa TIC complex to approximately 10% of wildtype levels. Hierarchical clustering confirmed the association of malate dehydrogenase (MDH) with an envelope-associated FtsH/FtsHi complex and suggested the association of a glycine-rich protein with the 1-MDa TIC complex. Depletion of this complex leads to a reduction of chloroplast ATPase to approx. 75% of wildtype levels, while the abundance of the FtsH/FtsHi complex is increased to approx. 140% of wildtype. The accumulation of the major photosynthetic complexes is not affected by the mutation, suggesting that tic56-3 plants can sustain a functional photosynthetic machinery despite a significant reduction of the 1-MDa TIC complex. Together our analysis expands recent efforts to catalogue the native molecular masses of chloroplast proteins and provides information on the consequences of impaired accumulation of the 1-MDa TIC translocase for chloroplast proteome assembly.

Biochemical and Biological Profile of Parotoid Secretion of the Amazonian Rhinella marina (Anura: Bufonidae).

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Comparative analysis of the high molecular mass subproteomes of eight Bothrops snake venoms

Snake venoms are extremely active biological secretions composed primarily of various classes of enzymes. The genus Bothrops comprises various pit viper species that represent the most medically significant taxa in Central and South America, accounting for more human envenomations and fatalities than any other snakes in the region. Venom proteomes of many Bothrops species have been well-characterized but investigations have focused almost exclusively on proteins smaller than 100 kDa despite expression of larger components being documented in several Bothrops venoms. This study sought to achieve detailed identification of major components in the high molecular mass subproteome of venoms from eight Bothrops species (B. brazili, B. cotiara, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. moojeni and B. neuwiedi). Enzymes such as metalloproteinases and L-amino acid oxidases were the most prominent components identified in the first size-exclusion chromatography fractions of these venoms. Minor components also identified in the first peaks included 5′-nucleotidase, aminopeptidase, phosphodiesterase, and phospholipases A2 and B. Most of these components disappeared in electrophoretic profiles under reducing conditions, suggesting that they may be composed of more than one polypeptide chain. A significant shift in the molecular masses of these protein bands was observed following enzymatic N-deglycosylation, indicating that they may contain N-glycans. Furthermore, none of the identified high molecular mass proteins were shared by all eight species, revealing a high level of interspecific variability among these venom components.

Cucumber Golgi protein CsMTP5 forms a Zn-transporting heterodimer with high molecular mass protein CsMTP12

Heterodimeric complexes formed by members of the cation facilitator (CDF) family catalyse the import of Zn into the secretory pathway of yeast and vertebrate cells. Orthologous proteins AtMTP5 and AtMTP12 from Arabidopsis have also been shown to form a heterodimeric complex at the Golgi compartment of plant cells that possibly transport Zn. In this study we show that cucumber proteins CsMTP5 and CsMTP12 form a functional heterodimer that is involved in the loading of Zn into the ER lumen under low Zn, and not in the detoxification of yeast from Zn excess through vesicle-mediated exocytosis. Using specific antibodies, we demonstrate that CsMTP5 is localized at the Golgi compartment of cucumber cells and is markedly up-regulated upon Zn deficiency. The level of CsMTP5 transcript in cucumber is also significantly elevated in Zn-limiting conditions, whereas the expression of CsMTP12 is independent of the availability of Zn. Therefore we propose that the cucumber heterodimeric complex CsMTP5-CsMTP12 functions to deliver Zn to Zn-dependent proteins of the Golgi compartment and is regulated by zinc at the level of CsMTP5 transcription.

Transcytosis of Junonia coenia densovirus VP4 across the gut epithelium of Spodoptera frugiperda (Lepidoptera: Noctuidae).

The Junonia coenia densovirus rapidly traverses the gut epithelium of the host lepidopteran without replicating in the gut cells. The ability of this virus to transcytose across the gut epithelium is of interest for the potential use of virus structural proteins as delivery vehicles for insecticidal peptides that act within the insect hemocoel, rather than in the gut. In this study, we used fall armyworm, Spodoptera frugiperda to examine the binding of the virus to brush border membrane vesicle proteins by two-dimensional ligand blot analysis. We also assessed the rate of flux of the primary viral structural protein, VP4 fused to eGFP with a proline-rich linker (VP4-P-eGFP) through the gut epithelium ex vivo in an Ussing chamber. The mechanisms involved with transcytosis of VP4-P-eGFP were assessed by use of inhibitors. Bovine serum albumin (BSA) and eGFP were used as positive and negative control proteins, respectively. In contrast to BSA, which binds to multiple proteins on the brush border membrane, VP4-P-eGFP binding was specific to a protein of high molecular mass. Protein flux was significantly higher for VP4-P-eGFP after 2 h than for albumin or eGFP, with rapid transcytosis of VP4-P-eGFP within the first 30 min. In contrast to BSA which transcytosed following clathrin-mediated endocytosis, the movement of VP4-P-eGFP was vesicle-mediated but clathrin-independent. The specificity of binding combined with the efficiency of transport across the gut epithelium suggest that VP4 will provide a useful carrier for insecticidal peptides active within the hemocoel of key lepidopteran pests including S. frugiperda.