Acidic Isoelectric Point Research Articles

The phytochelatin synthase from Nitella mucronata (Charophyta) plays a role in the homeostatic control of iron(II)/(III)

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata.

Mouse Antibody of IgM Class is Prone to Non-Enzymatic Cleavage between CH1 and CH2 Domains

IgM is a multivalent antibody which evolved as a first line defense of adaptive immunity. It consists of heavy and light chains assembled into a complex oligomer. In mouse serum there are two forms of IgM, a full-length and a truncated one. The latter contains μ’ chain, which lacks a variable region. Although μ’ chain was discovered many years ago, its origin has not yet been elucidated. Our results indicate that μ’ chain is generated from a full-length heavy chain by non-enzymatic cleavage of the protein backbone. The cleavage occurred specifically after Asn209 and is prevented by mutating this residue into any other amino acid. The process requires the presence of other proteins, preferentially with an acidic isoelectric point, and is facilitated by neutral or alkaline pH. This unique characteristic of the investigated phenomenon distinguishes it from other, already described, Asn-dependent protein reactions. A single IgM molecule is able to bind up to 12 epitopes via its antigen binding fragments (Fabs). The cleavage at Asn209 generates truncated IgM molecules and free Fabs, resulting in a reduced IgM valence and probably affecting IgM functionality in vivo.

Assessing the Impact of Charge Variants on Stability and Viscosity of a High Concentration Antibody Formulation

Characterizing molecular charge variants or isoforms is essential for understanding safety, potency, and bioavailability of antibody therapeutics. However, there is little information on how they influence stability and viscosity—properties governing immunogenicity and delivery. To bridge this gap, we studied antibody stability as a function of charge variant content generated via bioreactor process. We were able to systematically vary acidic variant levels as a function of bioreactor harvest time. Importantly, we do not observe any impact on aggregation behavior of a formulated antibody at high protein concentration as a function of acidic variant level. Furthermore, we confirm that acidic variants enriched using fractionation do not influence viscosity, colloidal or conformational stability. Interestingly, variants with the most acidic isoelectric points contribute disproportionately to formulation color. We discuss our findings in context of antibody manufacturing processes that may yield increased charge variant content.

Auxin Response Factor Genes Repertoire in Mulberry: Identification, and Structural, Functional and Evolutionary Analyses

Auxin Response Factors (ARFs) are at the core of the regulation mechanism for auxin-mediated responses, along with AUX/IAA proteins.They are critical in the auxin-mediated control of various biological responses including development and stress. A wild mulberry species genome has been sequenced and offers an opportunity to investigate this important gene family. A total of 17 ARFs have been identified from mulberry (Morus notabilis) which show a wide range of expression patterns. Of these 17 ARFs, 15 have strong acidic isoelectric point (pI) values and a molecular mass ranging from 52 kDa to 101 kDa. The putative promoters of these ARFs harbour cis motifs related to light-dependent responses, various stress responses and hormone regulations suggestive of their multifactorial regulation. The gene ontology terms for ARFs indicate their role in flower development, stress, root morphology and other such development and stress mitigation related activities. Conserved motif analysis showed the presence of all typical domains in all but four members that lack the PB1 domain and thus represent truncated ARFs. Expression analysis of these ARFs suggests their preferential expression in tissues ranging from leaf, root, winter bud, bark and male flowers. These ARFs showed differential expression in the leaf tissue of M. notabilis, Morus laevigata and Morus serrata. Insights gained from this analysis have implications in mulberry improvement programs.

Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome

Protein phosphorylation is involved in the regulation of most eukaryotic cells functions and mass spectrometry-based analysis has made major contributions to our understanding of this regulation. However, low abundance of phosphorylated species presents a major challenge in achieving comprehensive phosphoproteome coverage and robust quantification. In this study, we developed a workflow employing titanium dioxide phospho-enrichment coupled with isobaric labeling by Tandem Mass Tags (TMT) and high-resolution isoelectric focusing (HiRIEF) fractionation to perform in-depth quantitative phosphoproteomics starting with a low sample quantity. To benchmark the workflow, we analyzed HeLa cells upon pervanadate treatment or cell cycle arrest in mitosis. Analyzing 300 µg of peptides per sample, we identified 22,712 phosphorylation sites, of which 19,075 were localized with high confidence and 1,203 are phosphorylated tyrosine residues, representing 6.3% of all detected phospho-sites. HiRIEF fractions with the most acidic isoelectric points are enriched in multiply phosphorylated peptides, which represent 18% of all the phospho-peptides detected in the pH range 2.5–3.7. Cross-referencing with the PhosphoSitePlus database reveals 1,264 phosphorylation sites that have not been previously reported and kinase association analysis suggests that a subset of these may be functional during the mitotic phase.

β-1,4-endoglucanases from Talaromyces amestolkiae: Production of glucooligosaccharides from different β-glucans

This article presents the purification and characterization of two β-1,4-endoglucanases from Talaromyces amestolkiae. The cellulase activities secreted by this fungus were studied in the presence of different carbon sources, attaining the maximal levels in the presence of Avicel as carbon source. In these conditions, two glycosylated β-1,4-endoglucanases with molecular masses of 25,573 kDa (EG1) and 51,825 kDa (EG2), were purified. Both isoenzymes have acidic isoelectric points, 5.4 and 4.6, respectively. Their optimum pH and temperature, either in crudes or after purification, were in the range normally used for the simultaneous saccharification and fermentation in bioethanol production. In addition, the enzymatic hydrolysis of different β-glucans by both enzymes was studied. In the assayed conditions, both enzymes hydrolysed carboxymethylcellulose, a typical substrate for endoglucanases, although EG2 was much more efficient. However, EG1 was also able to hydrolyse lichenan and laminarin. These findings suggest the potential interest of EG2 for specific hydrolysis of cellulose, present in plant cell walls, to produce bioethanol, while the more promiscuous enzyme EG1 could be used for production of glucooligosaccharides.

Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A2, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom

Phospholipases A2 (PLA2s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA2s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA2s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic phospholipase A2 isoforms in snake venoms.

Isolation, Purification and Identification of Parvalbumin Allergen in <em>Cyprinus carpio</em>

Parvalbumin (PV) is a major fish allergen that is characterized by high water solubility, low molecular weight (i.e., ~12,000), acidic isoelectric point, heat stability and high phenylalanine content (compared with tyrosine and tryptophan). We used a fast, simple and efficient method to purify PV from <em>Cyprinus carpio</em> (carp). Heating, two runs of trichloroacetic acid precipitation and gel filtration chromatography were integrated in this method. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot showed that the purified protein was PV and the purity of carp PV obtained using this method was more than 90%. Moreover, the PV was characterized by MALDI-TOF-TOF MS and an unusually high 260 to 280 nm run absorbance ratio in the final purification stages. The rapid purification method used in this study resulted in better purification and identification of PV compared with other methods. Further studies on PV detection in freshwater fish can be performed based on the results of this experiment.

Development of a modular virus clearance package for anion exchange chromatography operated in weak partitioning mode.

Anion exchange chromatography (AEX) operated under weak partitioning mode has been proven to be a powerful polishing step as well as a robust viral clearance step in Pfizer's monoclonal antibody (mAb) platform purification process. A multivariate design of experiment (DoE) study was conducted to understand the impact of operating parameters and feedstream impurity levels on viral clearance by weak partitioning mode AEX. Bacteriophage was used initially as a surrogate for neutral and acidic isoelectric point mammalian viruses (e.g., retrovirus and parvovirus). Five different mAbs were used in the evaluation of process parameters such as load challenge (both product and impurities), load pH, load conductivity, and contact time (bed height and flow-rate). The operating ranges obtained from phage clearance studies and Pfizer's historical data were used to define an appropriate operating range for a subsequent clearance study with model retrovirus and parvovirus. Both phage and virus clearance evaluations included feedstreams containing different levels of impurities such as high molecular mass species (HMMS), host cell proteins (HCPs), and host cell DNA. For all the conditions tested, over 5 log10 of clearance for both retrovirus and parvovirus was achieved. The results demonstrated that weak partitioning mode AEX chromatography is a robust step for viral clearance and has the potential to be included as part of the modular viral clearance approach.

Influence of binding pH and protein solubility on the dynamic binding capacity in hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in biopharmaceutical industry. A major drawback of HIC, however, is the rather low dynamic binding capacity (DBC) obtained when compared to e.g. ion exchange chromatography (IEX). The typical purification procedure for HIC includes binding at neutral pH, independently of the proteins nature and isoelectric point. Most approaches to process intensification are based on resin and salt screenings.In this paper a combination of protein solubility data and varying binding pH leads to a clear enhancement of dynamic binding capacity. This is shown for three proteins of acidic, neutral, and alkaline isoelectric points. High-throughput solubility screenings as well as miniaturized and parallelized breakthrough curves on Media Scout RoboColumns (Atoll, Germany) were conducted at pH 3–10 on a fully automated robotic workstation. The screening results show a correlation between the DBC and the operational pH, the protein's isoelectric point and the overall solubility. Also, an inverse relationship of DBC in HIC and the binding kinetics was observed. By changing the operational pH, the DBC could be increased up to 30% compared to the standard purification procedure performed at neutral pH. As structural changes of the protein are reported during HIC processes, the applied samples and the elution fractions were proven not to be irreversibly unfolded.

Proteomic pathway analysis of the hippocampus in schizophrenia and bipolar affective disorder implicates 14-3-3 signaling, aryl hydrocarbon receptor signaling, and glucose metabolism: Potential roles in GABAergic interneuron pathology

Neuropathological changes of the hippocampus have been associated with psychotic disorders such as schizophrenia and bipolar disorder. Recent work has particularly implicated hippocampal GABAergic interneurons in the pathophysiology of these diseases. However, the molecular mechanisms underlying structural and cellular hippocampal pathology remain poorly understood. We used data from comprehensive difference-in-gel electrophoresis (2-D DIGE) investigations of postmortem human hippocampus of people with schizophrenia and bipolar disorder, covering the acidic (isoelectric point (pI) between pH4 and 7) and, separately, the basic (pI between pH6 and 11) sub-proteome, for Ingenuity Pathway Analysis (IPA) of implicated protein networks and pathways. Comparing disease and control cases, we identified 58 unique differentially expressed proteins in schizophrenia, and 70 differentially expressed proteins in bipolar disorder, using mass spectrometry. IPA implicated, most prominently, 14-3-3 and aryl hydrocarbon receptor signaling in schizophrenia, and gluconeogenesis/glycolysis in bipolar disorder. Both disorders were characterized by alterations of proteins involved in the oxidative stress response, mitochondrial function, and protein-endocytosis, -trafficking, -degradation, and -ubiquitination. These findings are interpreted with a focus on GABAergic interneuron pathology in the hippocampus.

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

BackgroundLegumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca2+ concentration, which is a fundamental prerequisite for any Ca2+-based signalling system, is accomplished by complex mechanisms including Ca2+ binding proteins acting as Ca2+ buffers. In this work we investigated the occurrence of Ca2+ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.ResultsA soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca2+-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca2+-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca2+ binding ability of the M. loti protein was demonstrated by 45Ca2+-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca2+ adducts.ConclusionsThe present data indicate that ferredoxin II is a major Ca2+ binding protein in M. loti that may participate in Ca2+ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca2+ regulatory systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.

Proteomic Analyses of Soybean Root Tips During Germination

Plant root systems form complex networks with the surrounding soil environment and are controlled by both internal and external factors. To better understand the function of root tips of soybean during germination, three proteomic techniques were used to analyze the protein profiles of root tip cells. Proteins were extracted from the root tips of 4-day-old soybean seedlings and analyzed using two-dimensional (2D) gel electrophoresis-based proteomics, SDS-gel based proteomics, and gel-free proteomics techniques. A total of 121, 862, and 341 proteins were identified in root tips using the 2D gel-based, SDS gel-based, and gel-free proteomic techniques, respectively. The proteins identified by 2D gel-based proteomic analysis were predominantly localized in the cytoplasm, whereas nuclear-localized proteins were most commonly identified by the SDS gel-based and gel-free proteomics techniques. Of the 862 proteins identified in the SDS gel-based proteomic analysis, 190 were protein synthesis-related proteins. Furthermore, 24 proteins identified using the 2D-gel based proteomic technique shifted between acidic and basic isoelectric points, and 2 proteins, heat shock protein 70.2 and AAA-type ATPase, displayed two different molecular weights at the same isoelectric point. Taken together, these results suggest that a number of proteins related to protein synthesis and modification are activated in the root tips of soybean seedlings during germination.

Factors Affecting Zeta Potential of Iron Oxides

This review examines the factors that affect the isoelectric point (IEP) and the magnitude of the Zeta potential for hematite. It addresses the various values that have been reported in the literature, with natural hematite typically having a more acidic IEP than does high-purity synthetic hematite. The key finding is that the IEP for natural hematite is very similar to that for silica. This is consistent with a coating of silica slimes forming on natural hematite, causing the hematite surfaces to behave as if they were silica surfaces. Adsorption of chemical reagents on the hematite surfaces causes changes in the magnitude of the Zeta potential, generally without changing the IEP.

Collagen-based layer-by-layer coating on electrospun polymer scaffolds

Preparation of microfibre constructs of collagen by electrospinning has been problematic due to the instability of collagen in volatile solvents, such as 1,1,1,3,3,3-hexafluoro-2-propanol, so that electrospinning leads to a substantial amount of gelatin fibres. In the present study we have demonstrated the production of collagen-based microfibre constructs by use of a layer-by-layer coating process onto a preformed synthetic polymer microfibre base. Soluble native collagen, which has a basic isoelectric point, has been used with modified triple-helical collagens that have acidic isoelectric points. These modified collagens have been prepared as deamidated, succinylated, maleylated and citraconylated derivatives. Together, the acidic and basic collagens have successfully coated polyacrylonitrile and poly(DL-lactide-co-glycolide) fibres, as shown by spectroscopy and microscopy. These coatings allow good cell attachment and spreading on the fibres. The native, triple helical form of the collagen has been confirmed through use of a conformation dependent monoclonal antibody.

Single pH buffer refolding screen for protein from inclusion bodies

We previously reported the set up of an automated test for screening the refolding of recombinant proteins expressed as inclusion bodies in Escherichia coli[1]. The screen used 96 refolding buffers and was validated with 24 proteins, 70% of which remained soluble in at least one buffer. In the present paper, we have analyzed in more detail these experimental data to see if the refolding process can be driven by general rules. Notably, we found that proteins with an acidic isoelectric point (pI) refolded in buffers the average pH of which was alkaline and conversely. In addition, the number of refolding buffers wherein a protein remained soluble increased with the difference between its pI and the average pH of the buffers in which it refolded. A trend analysis of the other variables (ionic strength, detergents, etc.) was also performed. On the basis of this analysis, we devised and validated a new refolding screen made of a single buffer for acidic proteins and a single buffer for alkaline proteins.

Novel GFP expression using a short N-terminal polypeptide through the defined twin-arginine translocation (Tat) pathway.

Escherichia coli is frequently used as a convenient host organism for soluble recombinant protein expression. However, additional strategies are needed for proteins with complex folding characteristics. Here, we suggested that the acidic, neutral, and alkaline isoelectric point (pI) range curves correspond to the channels of the E. coli type-II cytoplasmic membrane translocation (periplasmic translocation) pathways of twin-arginine translocation (Tat), Yid, and general secretory pathway (Sec), respectively, for unfolded and folded target proteins by examining the characteristic pI values of the N-termini of the signal sequences or the leader sequences, matching with the known diameter of the translocation channels, and analyzing the N-terminal pI value of the signal sequences of the Tat substrates. To confirm these proposed translocation pathways, we investigated the soluble expression of the folded green fluorescent protein (GFP) with short N-terminal polypeptides exhibiting pI and hydrophilicity separately or collectively. This, in turn, revealed the existence of an anchor function with a specific directionality based on the N-terminal pI value (termed as N-terminal pI-specific directionality) and distinguished the presence of the E. coli type-II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for the unfolded and folded target proteins. We concluded that the pI value and hydrophilicity of the short N-terminal polypeptide, and the total translational efficiency of the target proteins based on the ΔGRNA value of the N-terminal coding regions are important factors for promoting more efficient translocation (secretion) through the largest diameter of the Tat channel. These results show that the short N-terminal polypeptide could substitute for the Tat signal sequence with improved efficiency.

Oxidative stress-induced proteome alterations target different cellular pathways in human myoblasts

Although increased oxidative stress has been associated with the impairment of proliferation and function of adult human muscle stem cells, proteins either involved in the stress response or damaged by oxidation have not been identified. A parallel proteomics approach was performed for analyzing the protein expression profile as well as proteins preferentially oxidized upon hydrogen peroxide-induced oxidative stress. Fifteen proteins involved in the oxidative stress response were identified. Among them, protein spots identified as peroxiredoxins 1 and 6, glyceraldehyde-3-phosphate dehydrogenase, and α-enolase were shifted to a more acidic isoelectric point upon oxidative stress, indicating posttranslational modifications. Oxidized proteins were evidenced by immunodetection of derivatized carbonyl groups followed by identification by mass spectrometry. The carbonylated proteins identified are mainly cytosolic and involved in carbohydrate metabolism, cellular assembly, cellular homeostasis, and protein synthesis and degradation. Pathway analysis revealed skeletal and muscular disorders, cell death, and cancer-related as the main molecular networks altered. Interestingly, these pathways were focused on two distinct proteins: p53 for altered protein expression and huntingtin for increased protein carbonylation. This study emphasizes the importance of performing analysis addressing different aspects of the cellular proteome to have a more accurate view of their changes upon stress.

Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa

Hemolymph of Rapana venosa snails is a complex mixture of biochemically and pharmacologically active components such as peptides and proteins. Antimicrobial peptides are gaining attention as antimicrobial alternatives to chemical food preservatives and commonly used antibiotics. Therefore, for the first time we have explored the isolation, identification and characterisation of 11 novel antimicrobial peptides produced by the hemolymph of molluscs. The isolated peptides from the hemolymph applying ultrafiltration and reverse-phase high-performance liquid chromatography (RP-HPLC) have molecular weights between 3000 and 9500Da, determined by mass spectrometric analysis. The N-terminal sequences of the peptides identified by Edman degradation matched no peptides in the MASCOT search database, indicating novel proline-rich peptides. UV spectra revealed that these substances possessed the characteristics of protein peptides with acidic isoelectric points. However, no Cotton effects were observed between 190 and 280nm by circular dichroism spectroscopy. Four of the Pro-rich peptides also showed strong antimicrobial activities against tested microorganisms including Gram-positive and Gram-negative bacteria.

Responses of the photosynthetic electron transport system to excess light energy caused by water deficit in wild watermelon

In plants, drought stress coupled with high levels of illumination causes not only dehydration of tissues, but also oxidative damage resulting from excess absorbed light energy. In this study, we analyzed the regulation of electron transport under drought/high-light stress conditions in wild watermelon, a xerophyte that shows strong resistance to this type of stress. Under drought/high-light conditions that completely suppressed CO(2) fixation, the linear electron flow was diminished between photosystem (PS) II and PS I, there was no photoinhibitory damage to PS II and PS I and no decrease in the abundance of the two PSs. Proteome analyses revealed changes in the abundance of protein spots representing the Rieske-type iron-sulfur protein (ISP) and I and K subunits of NAD(P)H dehydrogenase in response to drought stress. Two-dimensional electrophoresis and immunoblot analyses revealed new ISP protein spots with more acidic isoelectric points in plants under drought stress. Our findings suggest that the modified ISPs depress the linear electron transport activity under stress conditions to protect PS I from photoinhibition. The qualitative changes in photosynthetic proteins may switch the photosynthetic electron transport from normal photosynthesis mode to stress-tolerance mode.