VE-cadherin-derived cell-penetrating peptide, pVEC, with carrier functions

Cell-penetrating peptides, CPPs, have been shown to translocate into living cells by a receptor-independent mechanism and to carry macromolecules over the plasma membrane. This article reports studies of the internalization of pVEC, an 18-amino acid-long peptide derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin, amino acids 615-632. Fluorophore-labeled pVEC entered four different cell lines tested: human aortic endothelial cells, brain capillary endothelial cells, Bowes melanoma cells, and murine brain endothelial cells. In order to evaluate the translocation efficiency of pVEC, we performed a side-by-side comparison with penetratin, a well-characterized CPP. The cellular uptake of pVEC was highest for murine brain endothelial cells. All cell lines tested contained equal or slightly higher concentrations of pVEC than penetratin. pVEC mainly accumulated in nuclear structures but was also found throughout the cells. Furthermore, pVEC functioned as a transporter of both a hexameric peptide nucleic acid molecule of 1.7 kDa and a 67-kDa protein, streptavidin-FITC, and cellular uptake of fluorophore-labeled pVEC took place at 4 degrees C, suggesting a nonendocytotic mechanism of translocation. In conclusion, our results indicate that pVEC is efficiently and rapidly taken up into cells and functions as a potent carrier peptide.     

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests

A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.     

Antitumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms

The effects of two cationic synthetic peptides, derived from the C-terminal region of Lys49 phospholipase A2 homologues from snake venoms, upon various murine tumor cell lines (B16 melanoma, EMT6 mammary carcinoma, S-180 sarcoma, P3X myeloma, tEnd endothelial cells) were evaluated. The peptides are 13-mers derived from Agkistrodon piscivorus piscivorus Lys49 PLA2 (p-AppK: KKYKAYFKLKCKK) and Bothrops asper Lys49 myotoxin II (pEM-2[D]: KKWRWWLKALAKK), respectively, in the latter case with slight modifications and with all-D amino acids. All tumor cells tested were susceptible to the lytic action of the peptides. The susceptibility of tumor cell lines was not higher than that of C2C12 skeletal muscle myoblasts, utilized as a non-transformed cell line control. However, in a murine model of subcutaneous solid tumor growth of EMT6 mammary carcinoma, the intraperitoneal administration of pEM-2[D] caused a tumor mass reduction of 36% (p<0.05), which was of similar magnitude to that achieved by the administration of paclitaxel, an antitumor drug in clinical use. Thus, the C-terminal peptides of Lys49 phospholipase A2 homologues present antitumor effects that might be of interest in developing therapeutic strategies against cancer.     

Enzymatic degradation studies of endomorphin-2 and its analogs containing N-methylated amino acids

In this paper, we describe the synthesis of novel endomorphin-2 analogs, containing N-methylated amino acids, consecutively in each position. The receptor-binding profile of the new analogs and their stability against enzymatic cleavage by commercially available peptidases, carboxypeptidase Y and aminopeptidase M, and a rat brain homogenate are reported. The best analog of this series, [Sar2]endomorphin-2, was almost equipotent with the parent peptide in the mu-receptor-binding assay and was also highly resistant to enzymatic degradation. This analog may be a suitable candidate for the in vivo antinociceptive studies.     

Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo

Cell-penetrating peptides (CPPs) are a group of peptides, which have the ability to cross cell membrane bilayers. CPPs themselves can exert biological activity and can be formed endogenously. Fragmentary studies demonstrate their ability to enhance transport of different cargoes across the blood-brain barrier (BBB). However, comparative, quantitative data on the BBB permeability of different CPPs are currently lacking. Therefore, the in vivo BBB transport characteristics of five chemically diverse CPPs, i.e. pVEC, SynB3, Tat 47–57, transportan 10 (TP10) and TP10-2, were determined. The results of the multiple time regression (MTR) analysis revealed that CPPs show divergent BBB influx properties: Tat 47–57, SynB3, and especially pVEC showed very high unidirectional influx rates of 4.73 μl/(g × min), 5.63 μl/(g × min) and 6.02 μl/(g × min), respectively, while the transportan analogs showed a negligible to low brain influx. Using capillary depletion, it was found that 80% of the influxed peptides effectively reached the brain parenchyma. Except for pVEC, all peptides showed a significant efflux out of the brain. Co-injection of pVEC with radioiodinated bovine serum albumin (BSA) did not enhance the brain influx of radiodionated BSA, indicating that pVEC does not itself significantly alter the BBB properties. A saturable mechanism could not be demonstrated by co-injecting an excess dose of non-radiolabeled CPP. No significant regional differences in brain influx were observed, with the exception for pVEC, for which the regional variations were only marginal. The observed BBB influx transport properties cannot be correlated with their cell-penetrating ability, and therefore, good CPP properties do not imply efficient brain influx.     

Inhibition of enzymatic degradation of angiotensin II in membrane binding assays: utility of 1,10-phenanthroline

Studies of 125I-labelled angiotensin II binding to membranes from bovine uterus and aorta are rendered impossible because enzymatic degradation of 90% of the added ligand occurs within 30 min at 23 degrees C. High pressure liquid chromatographic analysis has demonstrated that a single iodinated product, which is more hydrophilic than intact angiotensin II, is produced by enzymatic cleavage. Investigation of a series of enzyme inhibitors has shown that the carboxypeptidase inhibitor 1,10-phenanthroline completely inhibits degradation of angiotensin II without disrupting ligand binding.     

The effect of glycation on the chemical and enzymatic stability of the endogenous opioid peptide, leucine-enkephalin, and related fragments

Nonenzymatic glycation is a posttranslational modification of peptides and proteins by sugars, which, after a cascade of reactions, leads to the formation of a complex family of irreversibly changed adducts implicated in the pathogenesis of human diseases. The stability of the Amadori compounds, the last reversible intermediates, determines the further course of the reaction. To provide information concerning the fate of glycated opioid peptides introduced into human circulation, the enzymatic (80% human serum) and chemical (phosphate buffer) stability of three Amadori compounds related to the endogenous opioid pentapeptide, leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu), and to its N-terminal fragments: N-(1-deoxy-D-fructos-1-yl)-l-tyrosyl-glycyl-glycyl-L-phenylalanyl-L-leucine, N-(1-deoxy-D-fructos-1-yl)-L-tyrosyl-glycyl-glycine, and N-(1-deoxy-D-fructos-1-yl)-L-tyrosine were investigated. The results obtained in human serum indicate that N-terminal glycation of leucine-enkephalin significantly enhances proteolytic stability. While leucine-enkephalin itself was rapidly degraded (t1/2 = 14.8 min), the glycated-derivative was slowly converted (t1/2 = 14 h) to the corresponding Amadori /compound of Tyr-Gly-Gly and Phe-Leu. In phosphate buffer, the rate of hydrolysis of the Amadori compounds depends on the structure and length of the peptide moiety as well as on the concentration of the phosphate buffer. The hydrolysis patterns for the Amadori compounds in phosphate buffer and in human serum were not the same and appear to be specific for each substrate.     

Lysosomal carboxypeptidase A

Lysosomal carboxypeptidase A (cathepsin A) is synthetized in the form of preproenzyme, which undergoes to active enzyme as a result of post-translational modification. It splits off C-terminal amino acid residues from peptides and proteins and synergizes with other proteases in degradation of cellular proteins in lysosomes. Lysosomal carboxypeptidase A has an effect on peptide hormones and peptides of biological activity of tissues and body fluids as well. It forms complexes with some glycosidases that protects them against proteolytic degradation. Deficiency of this enzyme induces storage diseases. Lysosomal carboxypeptidase A as multifunctional enzyme plays an important regulatory role in organismal metabolism.     

Purification and analysis of the major components of chum salmon protamine contained in insulin formulations using high-performance liquid chromatography

A simple high-performance liquid chromatographic method has been developed for the rapid purification and analysis of protamine components contained in insulin formulations. Only a single step is needed to separate peptides whose compositions, sizes, and unusual isoelectric points (pI 13.8) are nearly identical. The method involves their isocratic separation on a reversed-phase column using a pH 2 phosphate buffer and a low acetonitrile content as an eluant. The purified chum salmon components were analyzed by amino acid analysis, solid-phase amino acid sequencing, carboxypeptidase B digests, insulin complexation analysis, and a mass spectrophotometric procedure which gives an accurate mass of the intact peptides. This HPLC purification technique may also be applicable to protamines and other highly basic peptides isolated from other sources.     

CHARACTERIZATION OF A RAT BRAIN CATHEPTIC CARBOXYPEPTIDASE (CATHEPSIN A) INACTIVATING ANGIOTENSIN-II

—Catheptic carboxypeptidase (cathepsin A) is present in lysosomal-enriched fractions of rat brain at levels approximately 5-fold that of cathepsin B1 and of the classical carboxypeptidase A but lower than that of cathepsin C and carboxypeptidase B. Cathepsin A was purified 40-fold by extraction of calf brain with an acetate buffer containing 0.5% desoxycholate followed by heat treatment, salt precipitation and chromatography on DEAE-Sephadex. Purification revealed the presence of two distinct isoenzymatic forms of high mol. wt that were very stable when frozen in the presence of sucrose and KCl. Among N-protected dipeptides used as substrates the highest activity was given by Z-Glu-Tyr and Z-Phe-Tyr at a pH optimum of 5.5, K_m1.1 mm and V_max 0.5 μmol (Tyr)/mg protein per min. Brain cathepsin A was completely inhibited by low concentrations of DFP and PCMB but unaffected by thiols, EDTA and specific inhibitors of other cathepsins (pepstatin and chymostatin). The carboxypeptidase A-like specificity of cathepsin A was confirmed by breakdown of Ile⁵-angiotensin with release of C-terminal Phe. Cathepsin A may play a role in the turnover of selected hormonal peptides containing C-terminal neutral amino acids and in the sequential breakdown of proteins associated with degenerative conditions such as demyelination.     

An aluminum silicate binding assay for quantitation of degradation of cholecystokinin octapeptide and other short peptides.

Most available techniques for the quantitation of enzymatic degradation of peptide hormones are time-consuming and require expensive equipment and/or novel reagents. Our aim here was to develop a rapid and sensitive assay for the measurement of degradation of cholecystokinin octapeptide (CCK-8) as well as other short, hydrophobic peptides. The proposed technique is based on our novel observation that intact CCK-8, but not its degradation product(s), binds to Lloyd reagent, a form of aluminum silicate. When radiolabeled CCK-8 was exposed to rat liver cytosol containing endogenous CCK-degrading activity, there was a time-dependent decrease in the binding of radiolabel to aluminum silicate [from 86 to 8% over 60 min at 37 degrees C]. The decrease in binding closely paralleled the extent of CCK-8 degradation over time as assessed by high-performance liquid chromatography and immunoprecipitation with specific polyclonal antibodies to CCK-8. While aluminum silicate did not efficiently bind to C-terminal and N-terminal CCK tetrapeptides, magnesium silicate bound to both tetrapeptides (> 82%), but not to their radiolabeled degradation products. Both aluminum and magnesium silicate also extensively bound (> 82%) to other peptide hormones including Met-enkephalin, somatostatin, and secretin, but did not bind their degradation products. These binding assays will be useful in studies of peptidases which degrade cholecystokinin or other small, hydrophobic peptides.     

Uptake of cell-penetrating peptides in yeasts

The uptake of different cell-penetrating peptides (CPPs) in two yeast species, Saccharomyces cerevisiae and Candida albicans, was studied using fluorescence HPLC-analyses of cell content. Comparison of the ability of penetratin, pVEC and (KFF)(3)K to traverse the yeast cell envelope shows that the cellular uptake of the peptides varies widely. Moreover, the intracellular degradation of the CPPs studied varies from complete stability to complete degradation. We show that intracellular degradation into membrane impermeable products can significantly contribute to the fluorescence signal. pVEC displayed highest internalizing capacity, and considering its stability in both yeast species, it is an attractive candidate for further studies.     

Purification and characterization of a small (7.3 kDa) putative lipid transfer protein from maize seeds

The present study reports, for the first time in literature, the purification and biochemical characterization of a small basic protein from maize seeds similar to plant lipid transfer proteins-2, named mLTP2. The mLTP2 consists of 70 amino acid residues and has an M(r) of 7303.83, determined by electrospray ionization mass spectrometry. The primary structure of mLTP2 was determined by automated Edman degradation of the intact protein and peptides obtained from digestions with trypsin and by C-terminal sequencing using carboxypeptidase Y. The mLTP2 exhibits high sequence similarity (51-44% identical positions) with other plant LTP2s previously described.     

Identification of the C-terminal portion of a protein by comparative peptide mapping

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Cellular uptake of cell-penetrating peptides pVEC and transportan in plants.

Internalization of fluorescently labeled CPPs, pVEC, transportan and scrambled pVEC, in a range of plant cells was investigated. Cellular uptake of the peptides was found to be tissue dependent. pVEC and transportan were distinctly internalized in triticale mesophyll protoplasts, onion epidermal cells, leaf bases and root tips of seven-day old triticale seedlings but showed negligible florescence in coleoptile and leaf tips as observed under a fluorescence microscope. Further, pVEC and transportan uptake studies were focused on mesophyll protoplasts as a system of investigation. In fluorimetric studies transportan showed 2.3 times higher cellular internalization than pVEC in protoplasts, whereas scrambled pVEC failed to show any significant fluorescence. Effect of various factors on cellular internalization of pVEC and transportan in protoplasts was also investigated. The cellular uptake of both the peptides was concentration dependent and nonsaturable. The cellular uptake of pVEC and transportan was enhanced at low temperature (4 degrees C). The presence of endocytic/macropinocytosis inhibitors did not reduce the cellular uptake of the peptides, suggesting direct cell penetration, receptor-independent internalization of pVEC and transportan into the plant cells.     

pVEC hydrophobic N‐terminus is critical for antibacterial activity

Cell‐penetrating peptides (CPPs) are commonly defined by their shared ability to be internalized into eukaryotic cells, without inducing permanent membrane damage, and to improve cargo delivery. Many CPPs also possess antimicrobial action strong enough to selectively lyse microbes in infected mammalian cultures. pVEC, a CPP derived from cadherin, is able to translocate into mammalian cells, and it is also antimicrobial. Structure‐activity relationship and sequence alignment studies have suggested that the hydrophobic N‐terminus (LLIIL) of pVEC is essential for this peptide's uptake into eukaryotic cells. In this study, our aim was to examine the contribution of these residues to the antimicrobial action and the translocation mechanism of pVEC. We performed antimicrobial activity and microscopy experiments with pVEC and with del5 pVEC (N‐terminal truncated variant of pVEC) and showed that pVEC loses its antimicrobial effect upon deletion of the LLIIL residues, even though both peptides induce membrane permeability. We also calculated the free energy of the transport process using steered molecular dynamic simulations and replica exchange umbrella sampling simulations to compare the difference in uptake mechanism of the 2 peptides in atomistic detail. Despite the difference in experimentally observed antimicrobial activity, the simulations on the 2 peptides showed similar characteristics and the energetic cost of translocation of pVEC was higher than that of del5 pVEC, suggesting that pVEC uptake mechanism cannot be explained by simple passive transport. Our results suggest that LLIIL residues are key contributors to pVEC antibacterial activity because of irreversible membrane disruption.     

Mass spectrometric strategy for primary structure determination of N-terminally blocked peptides

The mass spectrometric strategy including three steps is presented for primary structure determination of the N-terminally blocked peptides. First, the C-terminal sequencing is performed by using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry coupled with carboxypeptidase Y digestion. Then, the peptide is cleaved according to the obtained C-terminal sequence information and the resulting peptides are identified by mass spectrometry and Edman degradation after fractionation by reverse-phase chromatography. Finally, the N-terminal fragment is sequenced by tandem mass spectrometry. The strategy was successfully applied to the sequence determination of two novel N-terminally blocked peptides named EAFP1 and EAFP2.     

Derepression of bacteriophage mu transposition functions by truncated forms of the immunity repressor

To trigger bacteriophage Mu transposition and replication in response to physiological signals, its immunity repressor must be synchronously inactivated. Two repressor mutants (Vir), which have an altered C-terminal domain and are highly susceptible to degradation by ClpXP protease, confer a dominant negative phenotype by promoting degradation of the wild-type repressor. To search for other modified repressors that can induce Mu derepression in vivo and to determine what part of the inducing repressor molecules are needed to target the unmodified repressor population, repressor peptides with nested deletions starting at the C-terminal end were constructed. Such peptides with a C-terminal ssrA degradation tag promoted a sharp reduction in cellular levels of full-length unmodified repressor, a process largely dependent upon the clpP protease function. Only the repressor DNA-binding domain, located at the N-terminal end, was required in tagged peptides to target unmodified repressor. In addition, some repressor peptides containing the DNA-binding domain promoted derepression without the clpP function, being able to promote repressor inactivation without promoting its degradation. None of the modified repressors could promote derepression if immunity was established by a mutant repressor lacking 18 residues at its C-terminal end. The results indicate that inducing repressor peptides influence the function of the C-terminal domain of the intact repressor, a domain that regulates its degradation and DNA binding. They suggest the possibility that tagged repressor molecules, produced by stalled ribosomes, can be inducers of transposition under starvation conditions.     

Structure-activity relationship study of the cell-penetrating peptide pVEC

The peptide pVEC is a recently described cell-penetrating peptide, derived from the murine vascular endothelial-cadherin protein. In order to define which part of this 18-amino acid long peptide is important for the cellular translocation, we performed a structure-activity relationship study of pVEC. Together with the l-alanine substituted peptides, the retro-pVEC, D-pVEC and the scramble pVEC are studied for comparison. The peptide analogues are labeled with carboxyfluorescein at the N-terminus for monitoring the cellular uptake into human Bowes melanoma cells with different efficacy. We show that all the Fl-pVEC analogues internalize in live Bowes melanoma cells. l-Alanine substitution of the five respective N-terminal hydrophobic amino acids significantly decreases the translocation property, while replacing of Arg(6), Arg(8) or Ser(17) by alanine enhances the uptake. The uptake of pVEC is significantly reduced by treatment with an endocytosis inhibitor wortmannin. Treatment with heparinase III, nystatin and EIPA had no effect on the peptide uptake. The data presented here show that the N-terminal hydrophobic part of pVEC is crucial for efficient cellular translocation.