Polyphenolic disaccharides endow proteins with unusual resistance to aggregation

Protein aggregation is a common problem during the purification and formulation of therapeutic proteins. Here we report that polyphenolic disaccharides are unusually effective at preventing protein aggregation. We find that two polyphenolic glycosides-naringin and rutin-endow diverse proteins with the ability to unfold without aggregating when heated, as well as the ability to refold without aggregating when cooled at low glycoside concentrations (<5 mM). This extreme solubilizing activity is a synergistic combination of the glycone and aglycone moieties, as combinations of polyphenols and sugars fail to suppress aggregation. Moreover, the activity of polyphenolic disaccharides is remarkably specific since their monosaccharide counterparts (as well as other common excipients such as arginine, trehalose, and cyclodextrin) fail to prevent aggregation at similar concentrations (<25 mM). We expect that polyphenolic disaccharides will be valuable additives for enhancing the solubility of proteins in applications plagued by protein aggregation.     

Location and analysis of byssal structural proteins of Mytilus edulis

The acellular attachment organ (byssus) of the marine mussel Mytilus edulis L. is composed of threads that emanate from the body of the mussel to adhesive discs that anchor the threads to rocks, sand and other mussels. Three proteins have been purified by immunohistological methods and located to specific regions of the byssus. A collagenous protein with subunit molecular weights of 53,000, 55,000 and 65,000 is found in the matrix of the elastic thread region. Its 73,000-MW precursor was extracted from foot glands in the area proximal to the animal body and was identified by immune cross-reactivity. A cystine-rich, acidic protein was found in all regions of the byssus associated with a third protein, the polyphenolic protein. The L-dopa-containing polyphenolic protein appears in the cortex of the entire thread and adhesive plaque and at the substrate-plaque interface. Antiserum to this protein stains spherical vesicles in the phenol gland of the foot. Using immuno-electrophoretic methods, the polyphenolic protein and the cystine-rich protein were shown to form high molecular weight aggregates with aging of the byssus.     

ADHESION IN BYSSALLY ATTACHED BIVALVES

The byssus is a structure produced by marine bivalve molluscs to adhere, usually permanently, to substrata under water. As the adhesion of synthetic polymers to surfaces is predictably compromised by the presence of water, particularly bulk water, it is of particular interest to discover the mechanism of byssal adhesion. In most species, the byssus consists of at least four essential components: acid mucopolysaccharides, adhesive protein, fibrous proteins, and an oxidative enzyme, polyphenoloxidase. The function of the mucopolysaccharide component is still uncertain, but it can conceivably be used by the animal as a temporary adhesive, a surface modifying agent, and/or a stabilizing filler for the permanent adhesive. The adhesive protein known as the polyphenolic protein in Mytilus is but a thin plaque applied to the substrate surface by the foot of the animal. The molecular and physical properties of this adhesive protein conform remarkably well to what one expects of an ideal synthetic polymer, i.e. high molecular weight, abundance of large and polar side chains, near-zero surface contact angle, and total water-insolubility after setting. The fibrous proteins constitute the major portion of the thread or ribbon-like material connecting the animal to the adhesive plaque on the substrate surface. These proteins are packed in ordered crystalline arrays, e.g. β-pleated sheet and collagen helix (in mytilids) as is to be expected from structural tensile elements of Nature. The enzyme polyphenoloxidase is presumed to induce intermolecular cross-linking of proteins in the fibrous and adhesive portions of the byssus. In Mytilus the natural substrates of the enzymc may be the dopa-containing polyphenolic protein and accessory gland protein.     

Purification of adhesive proteins from mussels

The adhesive polyphenolic proteins from the mussels Mytilus chilensis and Choromytilus chorus have been purified based on their solubility in dilute perchloric acid and on differential precipitation with acetone containing about 0.3 N HCl. The specific activity of the proteins obtained was 0.16 mg of 3,4-dihydroxyphenylalanine per milligram of protein, or higher. The proteins have an apparent molecular weight of about 100,000 and they contain a high proportion of 3,4-dihydroxyphenylalanine, lysine, and proline.     

Structural characteristics of green tea catechins for formation of protein carbonyl in human serum albumin

Catechins are polyphenolic antioxidants found in green tea leaves. Recent studies have reported that various polyphenolic compounds, including catechins, cause protein carbonyl formation in proteins via their pro-oxidant actions. In this study, we evaluate the formation of protein carbonyl in human serum albumin (HSA) by tea catechins and investigate the relationship between catechin chemical structure and its pro-oxidant property. To assess the formation of protein carbonyl in HSA, HSA was incubated with four individual catechins under physiological conditions to generate biotin-LC-hydrazide labeled protein carbonyls. Comparison of catechins using Western blotting revealed that the formation of protein carbonyl in HSA was higher for pyrogallol-type catechins than the corresponding catechol-type catechins. In addition, the formation of protein carbonyl was also found to be higher for the catechins having a galloyl group than the corresponding catechins lacking a galloyl group. The importance of the pyrogallol structural motif in the B-ring and the galloyl group was confirmed using methylated catechins and phenolic acids. These results indicate that the most important structural element contributing to the formation of protein carbonyl in HSA by tea catechins is the pyrogallol structural motif in the B-ring, followed by the galloyl group. The oxidation stability and binding affinity of tea catechins with proteins are responsible for the formation of protein carbonyl, and consequently the difference in these properties of each catechin may contribute to the magnitude of their biological activities.     

The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein

The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure.     

In vitro polymerization of mussel polyphenolic proteins catalyzed by mushroom tyrosinase

The in vitro enzymatic polymerization of the polyphenolic protein purified from the mussels Aulacomya ater, Mytilus edulis chilensis and Choromytilus chorus was studied. Mushroom tyrosinase was used to oxidize the dopa residues present in these proteins, and polymerization was monitored by acid-urea polyacrylamide gel electrophoresis. The protein from A. ater polymerized at a faster rate than the other two. Amino acid analysis of the crosslinked protein showed a notable decrease in the content of dopa, but no significant change of other amino acids. This suggests that crosslink formation may be limited to the oxidized dopa derivatives of the protein molecules.     

Prolyl 4-hydroxylase in the foot of the marine mussel Mytilus edulis L.: purification and characterization

The mussel foot secretes a variety of unusual hydroxyproline-containing collagenous and noncollagenous proteins. Prolyl 4-hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (alpha 2 beta 2) composed of two subunits with Mr of 60,000 (alpha) and 57,000 (beta) as estimated by HPLC gel filtration and SDS-PAGE. The enzyme utilized (Pro-Pro-Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8-8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20 degrees C. No detectable hydroxylation occurred with poly-L-proline or the unhydroxylated decapeptide analog (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4-hydroxylase was competitively inhibited by poly-L-proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzyme-substrate i.e. (Pro-Pro-Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.     

Hemicellulosic complexes from the cell walls of runner bean (Phaseolus coccineus).

The 1 M-KOH extract from the depectinated cell walls of parenchymatous tissues of mature runner bean (Phaseolus coccineus) on neutralization, dialysis and concentration gave insoluble (hemicellulose A) and soluble (hemicellulose B) carbohydrate complexes in the weight ratio 2:1. Both fractions contained polysaccharide, protein and polyphenolic material. The structural features of the carbohydrates were examined by methylation analysis. Hemicellulose A contained mainly pectic arabinogalactan, with lesser amounts of arabinoxylan and glucan. Sequential fractionation of hemicellulose B by anion-exchange and hydroxyapatite chromatography gave a range of polysaccharide-protein-polyphenolic complexes. The main polysaccharides in these complexes were (acidic) arabinoxylans, galactans, arabinogalactans 1 and 2 and xyloglucans. The proteins contained small amounts of hydroxyproline, but were rich in aspartic acid and glutamic acid. Attempts to determine the nature of the polyphenolic material were unsuccessful. The structural features of the polysaccharide-protein-polyphenolic complexes are discussed in relation to the structure of the cell walls of parenchymatous tissues.     

Evidence for a repeating 3,4-dihydroxyphenylalanine- and hydroxyproline-containing decapeptide in the adhesive protein of the mussel, Mytilus edulis L

Previous work has shown that the permanent adhesive of the marine mussel Mytilus edulis is a protein containing large amounts of hydroxyproline (13%) and 3,4-dihydroxyphenylalanine (Dopa, 11%). The protein also known as the polyphenolic protein is produced and stored in the exocrine phenol gland of the mussel and deposited onto marine surfaces by the animal's foot during the formation of new adhesive plaques. The adhesive protein has been purified by a combination of ion exchange on sulfonylpropyl-Sephadex and gel filtration on low surface energy chromatographic media. Polyacrylamide gel electrophoresis of the protein at acidic pH shows it to consist of two components having a molecular weight of about 130,000. Treatment of the protein with clostridial collagenase reduced the molecular weight by less than 10%. The collagenase-resistant fragment contains most or all of the Hyp and Dopa. Trypsin treatment of the polyphenolic protein results in extensive degradation. The major tryptic peptide (80%) contains 10 amino acids including Hyp and Dopa and was shown by sequence analysis to be H2N-Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Dopa-Lys-COOH. Calculations suggest that this and related sequences may be repeated as often as 75 times in the polyphenolic protein.     

Inhibition of proteasomal function by curcumin induces apoptosis through mitochondrial pathway

Curcumin is a natural polyphenolic compound having an antiproliferative property, which recent evidence suggests is due to its ability to induce apoptosis. However, the molecular mechanisms through which curcumin induces apoptosis are not fully understood. Here, we report that the curcumin-induced apoptosis is mediated through the impairment of the ubiquitin-proteasome system. Exposure of curcumin to the mouse neuro 2a cells causes a dose-dependent decrease in proteasome activity and an increase in ubiquitinated proteins. Curcumin exposure also decreases the turnover of the destabilized enhanced green fluorescence protein, a model substrate for proteasome and cellular p53 protein. Like other proteasome inhibitors, curcumin targets proliferative cells more efficiently than differentiated cells and induces apoptosis via mitochondrial pathways. Addition of curcumin to neuro 2a cells induces a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into cytosol, followed by activation of caspase-9 and caspase-3.     

Baicalin administration is effective in positive regulation of twenty-four ischemia/reperfusion-related proteins identified by a proteomic study

Baicalin is a major plant polyphenolic compound derived from the dried root of Scutellaria baicalensis Georgi, a Traditional Chinese Medicine material. The current study applied proteomics to investigate the different protein expression modes in mice brains after middle cerebral artery occlusion (MCAO) with or without administration of baicalin. Twenty-four proteins which had a 3-fold change in abundance compared to the sham control sample were selected to be identified. Statistical analysis demonstrated that there was no significant difference in expression between the twenty-four proteins baicalin-treated MCAO group and the sham-operation group (n = 24, p = 0.102). Gene Ontology analysis linked these proteins to fifteen biological processes, including cellular process, developmental process and biological regulation. Results indicated that baicalin performed well in regulating proteins in energy metabolism but had a relatively weak effect in the regulation of proteins in neurogenesis and apoptosis. In sum, our findings suggest baicalin may be a potential therapeutic agent in treating stroke and may also be a strong candidate for future research in its actions on individual proteins.     

Mussel glue fromMytilus californianus Conrad: a comparative study

Summary Marine mussels secrete a byssus in order to attach to solid surfaces in the sea. The polyphenolic protein is the glue in the adhesive plaques of the byssus. InMytilus californianus, the polyphenolic protein has an apparent molecular weight of 85,000±5,000 and is rich in the amino acids lysine, 3,4-dihydroxyphenylalanine, serine, threonine, and hydroxyproline. In composition it resembles the polyphenolic protein ofM. edulis (M _r=125,000), although theM. edulis protein contains significantly less isoleucine and more alanine. Tryptic digestion ofM. californianus polyphenolic protein revealed two types of repeating decapeptides (1) (Ser/Thr)-Thr-(Tyr/Dopa)-Hyp-Hyp-Thr-Dopa-Lys-Hyp-Lys and (2) Ile-(Thr/Ser)-(Tyr/Dopa)-Hyp-Hyp-Thr-Dopa-Lys-Hyp-Lys. Residues 2 to 8 are identical with residues 4–10 inM. edulis decapeptides.Abbreviation Dopa 3,4hydroxyphenylalanine     

Molecular diversity of marine glues: polyphenolic proteins from five mussel species.

Adhesive polyphenolic proteins have been purified and characterized from the feet of five marine mussels (Brachidontes exustus, Modiolus modiolus squamosus, Mytella guyanensis, Septifer bifurcatus, and Trichomya hirsuta). All five proteins contain high levels of 3,4-dihydroxyphenylalanine (DOPA), lysine, glycine, and serine or threonine. All but B. exustus also contain high levels (> or = 10%) of proline or 4-hydroxyproline. The polyphenolic proteins of all the mussels have repeated sequences of the motif X1-Y*-X2-Y*-X3-K, where Y* denotes tyrosine or DOPA. In two species (S. bifurcatus and B. exustus), X2 represents 3 amino acids (frequently glycine) and X3 is absent. M. guyanensis is similar except that X2 is reduced to 2 amino acids. In T. hirsuta and M. m. squamosus, however, X2 is absent and X3 occurs as alanine or hydroxyproline. All proteins share approximately equimolar proportions of tyrosyl- and lysyl-derived residues. Although all of the mussels examined thus far are adhesively opportunistic with respect to substratum type, a rigidly invariant sequence does not appear to be necessary for achieving this.     

Select polyphenolic fractions from dried plum enhance osteoblast activity through BMP-2 signaling

Dried plum supplementation has been shown to enhance bone formation while suppressing bone resorption. Evidence from previous studies has demonstrated that these responses can be attributed in part to the fruit's polyphenolic compounds. The purpose of this study was to identify the most bioactive polyphenolic fractions of dried plum with a focus on their osteogenic activity and to investigate their mechanisms of action under normal and inflammatory conditions. Utilizing chromatographic techniques, six fractions of polyphenolic compounds were prepared from a crude extract of dried plum. Initial screening assays revealed that two fractions (DP-FrA and DP-FrB) had the greatest osteogenic potential. Subsequent experiments using primary bone-marrow-derived osteoblast cultures demonstrated these two fractions enhanced extracellular alkaline phosphatase (ALP), an indicator of osteoblast activity, and mineralized nodule formation under normal conditions. Both fractions enhanced bone morphogenetic protein (BMP) signaling, as indicated by increased Bmp2 and Runx2 gene expression and protein levels of phosphorylated Smad1/5. DP-FrB was most effective at up-regulating Tak1 and Smad1, as well as protein levels of phospho-p38. Under inflammatory conditions, TNF-α suppressed ALP and tended to decrease nodule formation (P=.0674). This response coincided with suppressed gene expression of Bmp2 and the up-regulation of Smad6, an inhibitor of BMP signaling. DP-FrA and DP-FrB partially normalized these responses. Our results show that certain fractions of polyphenolic compounds in dried plum up-regulate osteoblast activity by enhancing BMP signaling, and when this pathway is inhibited by TNF-α, the osteogenic response is attenuated.     

Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways

Prostate cancer (PCA), the most frequently diagnosed malignancy in men, represents an excellent candidate disease for chemoprevention studies because of its particularly long latency period, high rate of mortality and morbidity. Infusion of black tea and its polyphenolic constituents have been shown to possess antineoplastic effects in androgen dependent PCA in both in vivo and in vitro models including transgenic animals. In the present study, we report that black tea polyphenol, Theaflavins (TF)-induced apoptosis in human prostate carcinoma, LNCaP cells is mediated via modulation of two related pathways: up-regulation of p53 and down-regulation of NF-kappa B activity, causing a change in the ratio of pro-and antiapoptotic proteins leading to apoptosis. The altered expression of Bcl-2 family member proteins triggered the release of cytochrome-C and activation of initiator capsase 9 followed by activation of effector caspase 3. Furthermore, TF also affected the protein expression of mitogen activated protein kinases (MAPK) pathways. Our results demonstrated that TF treatment resulted in down-regulation of phospho-extracellular signal-regulated protein kinase (Erk1/2) and phospho-p38 MAPK expressions. We conclude that TF induces apoptosis in LNCaP cells by shifting the balance between pro-and antiapoptotic proteins and down-regulation of cell survival pathways leading to apoptosis. Further extending this work, we also showed that TF induces apoptosis in androgen independent PCA cell line, PC-3 through caspases and MAPKs mediated pathways. Thus, effect of TF on PCA cell lines seems to be irrespective of their androgen status.     

Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrol

Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardioprotection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apoptosis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and preconditioning- related proteins were identified that were all upregulated by resveratrol: MAPKK, two different alphaB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxiredoxin- 2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -alphaB-crystallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofilament regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins.     

Down-regulation of fibroblast growth factor 2 and its co-receptors heparan sulfate proteoglycans by resveratrol underlies the improvement of cardiac dysfunction in experimental diabetes

Cardiac remodeling in diabetes involves cardiac hypertrophy and fibrosis, and fibroblast growth factor 2 (FGF2) is an important mediator of this process. Resveratrol, a polyphenolic antioxidant, reportedly promotes the improvement of cardiac dysfunction in diabetic rats. However, little information exists linking the amelioration of the cardiac function promoted by resveratrol and the expression of FGF2 and its co-receptors, heparan sulfate proteoglycans (HSPGs: Glypican-1 and Syndecan-4), in cardiac muscle of Type 2 diabetic rats. Diabetes was induced experimentally by the injection of streptozotocin and nicotinamide, and the rats were treated with resveratrol for 6 weeks. According to our results, there is an up-regulation of the expression of genes and/or proteins of Glypican-1, Syndecan-4, FGF2, peroxisome proliferator-activated receptor gamma and AMP-activated protein kinase in diabetic rats. On the other hand, resveratrol treatment promoted the attenuation of left ventricular diastolic dysfunction and the down-regulation of the expression of all proteins under study. The trigger for the changes in gene expression and protein synthesis promoted by resveratrol was the presence of diabetes. The negative modulation conducted by resveratrol on FGF2 and HSPGs expression, which are involved in cardiac remodeling, underlies the amelioration of cardiac function.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

Anti-glycation effect and the α-amylase,lipase, and α-glycosidase inhibition properties of a polyphenolic fraction derived from citrus wastes

Abstract

The advanced glycation end products (AGEs) constitute a wide variety of substances synthesized from interactions between amino groups of proteins and reducing sugars, which excess induces pathogenesis of chronic diseases. Brazil is the major producer of citrus, a low-cost source of hesperidin, which is a polyphenol recognized for its capacity to inhibit AGEs formation. This is the first work to evaluate the effects of a polyphenolic fraction derived from citrus wastes on the antiglycation and on the inhibition properties of digestive enzymes on the possibility to process these wastes in high value-added products. At concentrations of 10, 15 and 20?mg/mL inhibition of AGEs was higher than 60%. The extracts were able to inhibit by 76% the activity of pancreatic lipase and by 98% the activity of α-glucosidase. For the α-amylase the inhibition capacity was lower than 50%. Strong correlation was obtained among anti-glycation with polyphenolic content and antioxidant capacity.