Novel opioid peptides derived from casein (beta-casomorphins). II. Structure of active components from bovine casein peptone

Material with opioid activity had been isolated from an enzymatic casein digest. It was shown to contain a pure heptapeptide with the sequence Tyr-Pro-Phe-Pro-Gly-Pro-Ile. The identity between the opioid principle and the peptide was proven by the fact that chemical reagents or enzymes effecting one would effect the other. After carboxypeptidase Y digestion a pentapeptide, Tyr-Pro-Phe-Pro-Gly, could be isolated; this peptide showed a higher opioid activity than the heptapeptide. The opioid peptides were highly resistant towards proteolysis, even by pronase. The sequence of the hepatapeptide identified it as a fragment of bovine beta-casein. Therefore it was named beta-casomorphin.     

Novel opioid peptides derived from casein (beta-casomorphins). I. Isolation from bovine casein peptone

A material which displayed opioid activity in the guinea pig ileum longitudinal muscle-myenteric plexus preparation was extracted from an enzymatic casein digest into chloroform/methanol. The extract was roughly purified by adsorption/desorption procedures using charcoal and Amberlite XAD-2 resin as adsorbents. A high degree of purity was achieved by high-pressure liquid chromatography of the material on muBondapak C18 and mu-Porasil columns and finally by gel filtration chromatography on a Bio-Gel P-2 column. Several pronase-resistant compounds with opioid activity were obtained.     

Isolation and characterization of free radical scavenging activities peptides derived from casein

A peptide having the strong free radical scavenging activities was separated from casein protein hydrolysate by chromatographic analyses such as ion-exchange and gel filtration. SP-II fraction obtained by SP-Sephadex C-25 chromatography showed the most potent superoxide anion scavenging activity (SOSA), and it was further separated into a peptide using an octadecylsilano-high performance liquid chromatography. The amino acid sequence of the peptide was Tyr-Phe-Tyr-Pro-Glu-Leu (YFYPEL). The concentration of the test compound required to reduce the produced superoxide anion to one-half (IC(50)) value for SOSA was 79.2 microM using tetrazolium salt 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate method. The IC50 value for the 1,1-diphenyl-2-picrylhydrazyl radical and hydroxyl radical scavenging activities were 98 and 251 microM, respectively, based on the electron spin resonance method. We characterized SOSA of the C-terminal sequence using EL, PEL, YPEL, and FYPEL. The activities preferred sequences were EL>YFYPEL>FYPEL>YPEL>PEL, suggesting that the Glu-Leu sequence is important for the activity.     

The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2.

Two series of synthetic peptides that reproduce the amino- and carboxyl-terminal segments of the beta-subunit of casein kinase-2, including the sites phosphorylated by CK2 and cdc2 kinase, respectively, have been used as model substrates for these enzymes. The N-terminal peptide beta(1-9), MSSSEEVSW, is readily phosphorylated by CK2 but not all by cdc2. The opposite is true of the C-terminal peptide beta(206-215), NFKSPVKTIR, whose Ser-4 is a good target for cdc2 while being unaffected by CK2. The individual substitutions of Pro-5 and Lys-7 in the latter peptide with Gly and Ala (or Glu), respectively, prevent its phosphorylation by cdc2, whereas the substitution of Lys-3 with Ala is well tolerated and the substitution of the target Ser with Thr actually improves phosphorylation. Thus the consensus sequence for cdc2 is shown to be X-S-P-X-K. Such a requirement for a basic residue at position +3 is opposite to that of CK2 whose consensus sequence (S-X-X-E/D/Yp/Sp) includes an acidic residue at the same position. Moreover the motif Ser-Pro is detrimental for CK2, preventing the phosphorylation of otherwise suitable peptides. These observations would rule out the possibility that the site specificity of CK2 might overlap with that of cdc2 and possibly of other Pro-directed protein kinases.     

Atriopeptins: a family of potent biologically active peptides derived from mammalian atria

Extracts of rat atria are potent stimulators of sodium and urine excretion, and relax vascular and intestinal smooth muscle preparations. The structures of six biologically active peptides obtained from atrial extracts are reported here. Ion exchange chromatography of a low molecular weight fraction obtained by gel filtration of atrial extracts produced two natriuretic fractions: the first induced relaxation of intestinal smooth muscle strips only, whereas the second also relaxed vascular strips as well. From the first fraction four pure biologically active peptides obtained by reverse phase HPLC have been sequenced: the 21 amino acid peptide, designated atriopeptin I, and three homologs (des- ser1 -, des- ser1 -ser2-, and des- ser21 - atriopeptin I). From the second fraction two pure biologically active peptides were obtained, which had C-terminal extensions of atriopeptin I: atriopeptins II (23 amino acid residues) and III (24 residues), having respectively phe-arg and phe-arg-tyr C-termini. These results suggest that this family of six peptides, sharing the same 17 membered ring formed by an internal cystine disulfide, is derived from a common high molecular weight precursor.     

Antiestrogenic and anticancer activities of peptides derived from the active site of alpha‐fetoprotein

Cyclo[EKTOVNOGN] (AFPep), a cyclic 9‐amino acid peptide derived from the active site of alpha‐fetoprotein, has been shown to prevent carcinogen‐induced mammary cancer in rats and inhibit the growth of ER⁺ human breast cancer xenografts in mice. Recently, studies using replica exchange molecular dynamics predicted that the TOVN region of AFPep might form a dynamically stable putative Type I beta‐turn, and thus be biologically active without additional amino acids. The studies presented in this paper were performed to determine whether TOVN and other small analogs of AFPep would inhibit estrogen‐stimulated cancer growth and exhibit a broad effective‐dose range. These peptides contained nine or fewer amino acids, and were designed to bracket or include the putative pharmacophoric region (TOVN) of AFPep. Biological activities of these peptides were evaluated using an immature mouse uterine growth inhibition assay, a T47D breast cancer cell proliferation assay, and an MCF‐7 breast cancer xenograft assay. TOVN had very weak antiestrogenic activity in comparison to AFPep's activity, whereas TOVNO had antiestrogenic and anticancer activities similar to AFPep. OVNO, which does not form a putative Type I beta‐turn, had virtually no antiestrogenic and anticancer activities. A putative proteolytic cleavage product of AFPep, TOVNOGNEK, significantly inhibited E₂‐stimulated growth in vivo and in vitro over a wider dose range than AFPep or TOVNO. We conclude that TOVNO has anticancer potential, that TOVNOGNEK is as effective as AFPep in suppressing growth of human breast cancer cells, and that it does so over a broader effective‐dose range. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Human salivary peptides derived from histatins

Human saliva from a healthy donor was subjected to fractionation by gel chromatography and six pools were collected and analysed by MALDI-TOF-MS and HPLC-ESI-MS. Three peptides, corresponding to 888.3, 687.3, and 524.1 amu and SNYLYDN, YLYDN, and LYDN sequences (determined by automated Edman sequencing), were isolated from pool 4. YLYDN was not previously described in human saliva. The peptides show the common C-terminal sequence of histatin 3 and histatin 1. To exclude the possibility that the three peptides were an artifact of the purification procedure, nine samples of human saliva were collected from healthy donors, immediately acidified with 0.2% TFA, and analysed by RP-HPLC-ESI-MS. The three peptides were detected in all the analyzed samples. SNYLYDN was always found at a concentration higher than that of YLYDN and LYDN. A correlation analysis performed on quantitative data indicated that the three peptides derive only from histatin 3. Other already known histatins also were searched for in the chromatogram. Histatins 1, 2, 3, 5, 6, 7, 8, and 10 were observed, although not in all samples analyzed, whereas other minor histatins were not detected.     

乳酪蛋白源生物活性肽的研究进展

酪蛋白是生物活性肽的重要来源,可以通过体内的胃肠消化和食品加工过程中的酶解将其释放出来。乳酪蛋白源的生物活性肽的生物学意义、对人类健康的影响和其在新型功能性食品加工中的作用具有重要的研究价值。     

Inflammatory peptides derived from adipose tissue

The low-grade inflammation seen with aging is noted particularly in subjects with the metabolic syndrome of aging. Insulin resistance, obesity/abdominal obesity, and risks for many age-related diseases characterize this common syndrome. It is becoming clear that this increased adipose tissue is not simply a reservoir for excess nutrients, but rather an active and dynamic organ capable of expressing several cytokines and other fat-derived peptides (FDP). Some, but not all, FDP may have a role in development of the metabolic syndrome but there is no evidence that these FDP are causing inflammation directly. We suggest that high levels of inflammatory peptides are markers for obesity/abdominal obesity seen with aging, but some may not necessarily have a causative role in the development of inflammation.     

Production of active angiotensin-I converting enzyme inhibitory peptides derived from bovine beta-casein by recombinant DNA technologies

A wide range of anti-hypertensive peptides potentially able to lower blood pressure through the inhibition of vasoactive enzymes such as angiotensin-I converting enzyme (ACE) are known. Currently, ACE-inhibitory peptides can be produced from precursor proteins via enzymatic hydrolysis by proteolytic enzymes, or food fermentation with proteolytic starter cultures. These approaches are neither selective nor easy. In this study a novel procedure has been developed, based on recombinant DNA technologies, for the production of highly purified fractions of three polypeptides derived from bovine beta-casein active as ACE inhibitors in vitro. The procedure includes peptide expression in Escherichia coli cells as recombinant fusion proteins, purification by affinity chromatography, cleavage by proteinase from a selected strain of Lactobacillus helveticus and isolation of bioactive peptides (BPs). The reported concentration of inhibitor needed to reduce at 50% ACE activity (IC(50)) values for single BP calculated in inhibiting the ACE enzyme gave results in agreement with the same parameters available in literature for other milk-derived BPs. This procedure could be used to obtain quantities of pure peptides to determine their interactions with ACE, with the aim of designing peptides that have stronger inhibitory properties and exhibit new pharmacological profiles. Moreover, its scale up would be of commercial interest for the production of functional foods, e.g., milk beverages with blood pressure-lowering effects.     

Cathepsin B inhibitory peptides derived from beta-casein

Two cathepsin B inhibitory peptides were isolated from a commercial pancreatic digest of casein. The peptides were identified as the Pro-Phe-Pro-Gly-Pro-Ile and the Gly-Pro-Phe-Pro-Ile corresponding to the sequence 61-66 and 203-207 of bovine beta-casein. These peptides showed competitive inhibition for cathepsin B with the K(i) values of 2.31 and 3.30 mM, respectively. Two related analogues, Tyr-Pro-Phe-Pro-Gly-Pro-Ile and Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile, were synthesized but their cathepsin B inhibitory activity was not detected.     

Caseins of various origins and biologically active casein peptides and oligosaccharides: Structural and physiological aspects

Summary The first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (_sl-, _s2- - and ). The calcium-sensitive casein fractions are always phosphorylated whereas -caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca²⁺. In -caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine -casein and up to 10 in human -casein. The structures of the known -casein carbohydate moieties are described. Finally the milk clotting process (interaction -casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena.The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A strategic zone containing immunostimulating and opioid peptides could be located in cow and human -caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the -caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities.In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components.If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.     

Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides

The specificity of casein kinase II has been further defined by analyzing the kinetics of phosphorylation reactions using a number of different synthetic peptides as substrates. The best peptide substrates are those in which multiple acidic amino acids are present on both sides of the phosphorylatable serine or threonine. Acidic residues on the NH2-terminal side of the serine (threonine) greatly enhance the kinetic constants but are not absolutely required. Acidic residues on the COOH-terminal side of the serine (threonine) are absolutely required. One position for which the occupation of an acidic residue is especially critical is the position located 3 residues to the COOH terminus of the phosphate acceptor site, although the presence of an acidic amino acid in the positions that are 4 or 5 residues removed may also provide an appropriate structure that will serve as a substrate for the kinase. Aspartate serves as a better amino acid determinant than glutamate. A relatively short sequence of amino acids surrounding the phosphate acceptor site appears to serve as the basis for the specificity of casein kinase II. The peptides in this study were also assayed with casein kinase I and the casein kinase from the mammary gland so that the specificities of these kinases could be compared to that of casein kinase II.     

Protective peptides derived from novel glial proteins

In studying the mediators of VIP neurotrophism in the central nervous system, two glial proteins have been discovered. Both of these proteins contain short peptides that exhibit femtomolar potency in preventing neuronal cell death from a wide variety of neurotoxic substances. Extension of these peptides to models of oxidative stress or neurodegeneration in vivo have indicated significant efficacy in protection. These peptides, both as individual agents and in combination, have promise as possible protective agents in the treatment of human neurodegenerative disease and in pathologies involving oxidative stress.     

Cell-penetrating peptides derived from viral capsid proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl(2) changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.     

A new group of antifungal and antibacterial lipopeptides derived from non-membrane active peptides conjugated to palmitic acid

We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K(4)X(7)W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections.     

Design of peptides derived from anti-IgE antibody for allergic treatment.

We have designed and synthesized peptides derived from an anti-IgE antibody which has a potential for the treatment of allergy. It was indicated that conformational restriction of peptide via an intramolecular disulfide bond improved the binding affinity for IgE and that the peptide might have an ability to inhibit the IgE-receptor interaction.     

An overview of antifungal peptides derived from insect

Fungi are not classified as plants or animals. They resemble plants in many ways but do not produce chlorophyll or make their own food photosynthetically like plants. Fungi are useful for the production of beer, bread, medicine, etc. More complex than viruses or bacteria; fungi can be destructive human pathogens responsible for various diseases in humans. Most people have a strong natural immunity against fungal infection. However, fungi can cause diseases when this immunity breaks down. In the last few years, fungal infection has increased strikingly and has been accompanied by a rise in the number of deaths of cancer patients, transplant recipients, and acquired immunodeficiency syndrome (AIDS) patients owing to fungal infections. The growth rate of fungi is very slow and quite difficult to identify. A series of molecules with antifungal activity against different strains of fungi have been found in insects, which can be of great importance to tackle human diseases. Insects secrete such compounds, which can be peptides, as a part of their immune defense reactions. Active antifungal peptides developed by insects to rapidly eliminate infectious pathogens are considered a component of the defense munitions. This review focuses on naturally occurring antifungal peptides from insects and their challenges to be used as armaments against human diseases.