Biomedical subjects

M Meldal

Publications and source records attributed to M Meldal.

At least 19 recordsLinked to original sources

PubMed2000

Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.

Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Alanine↗

PubMed2000

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana.

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.

Amino Acid Sequence↗

PubMed2000

Glycopeptide and Oligosaccharide Libraries.

Despite the burgeoning interest in the various biological functions and consequent therapeutic potential of the vast number of oligosaccharides found in nature on glycoproteins and cell surfaces, the development of combinatorial carbohydrate chemistry has not progressed as rapidly as expected. The reason for this imbalance is rooted in the difficulty of oligosaccharide assembly and analysis that renders synthesis a rather cumbersome endeavor. Parallel approaches that generate series of analogous compounds rather than real libraries have therefore typically been used. Since generally low affinity is obtained for interactions between carbohydrate receptors and modified oligosaccharides designed as mimetics of natural carbohydrate ligands, glycopeptides have been explored as alternative mimics. Glycopeptides have been proven in many cases to be superior ligands with higher affinity for a receptor than the natural carbohydrate ligand. High-affinity glycopeptide ligands have been found for several types of receptors including the E-, P-, and L-selectins, toxins, glycohydrolases, bacterial adhesins, and the mannose-6-phosphate receptor. Furthermore, the assembly of glycopeptides is considerably more facile than that of oligosaccharides and the process can be adapted to combinatorial synthesis with either glycosylated amino acid building blocks or by direct glycosylation of peptide templates. The application of the split and combine approach using ladder synthesis has allowed the generation of very large numbers of compounds which could be analyzed and screened for binding of receptors on solid phase. This powerful technique can be used generally for the identification and analysis of the complex interaction between the carbohydrates and their receptors.

Journal Article↗

PubMed2000

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.

Amino Acid Sequence↗

PubMed2000

Hydrolysis by cathepsin B of fluorescent peptides derived from human prorenin.

Cathepsin B is a lysosomal thiolprotease that, because of its colocalization with renin and its ability to activate prorenin, has been proposed as a prorenin processing enzyme. To characterize the biochemical aspect of this potential cathepsin B activity in more detail, we synthesized and assayed with human cathepsin B the internally quenched fluorescent peptide Abz-FSQPMKRLTLGNTTQ-EDDnp (Abz, ortho-aminobenzoic acid fluorescent group and EDDnp, N-¿2, 4-dinitrophenyl-ethylenediamine quencher group) that contains 7 amino acids for each side of the R-L bond that is the processing site of human prorenin. Human cathepsin B hydrolyzed this peptide at the correct site (R-L bond), with k(cat)/K(m)=75 mmol/L(-1) s(-1). Analogues of this peptide obtained by Ala scanning at positions P(5) to P(5)' were also synthesized and assayed as substrates for human cathepsin B. The obtained specificity constant (k(cat)/K(m)) values have a significant parallel with the previous data of prorenin activation by AtT-20 cells and in vitro by cathepsin B. In addition, we demonstrated the presence of cathepsin B-like activity in rat mesangial cells and the ability of its whole soluble fraction lysates, as well as that of purified cloned rat cathepsin B, to hydrolyze Abz-IKKSSF-EDDnp at the K-S bond, which contains 6 amino acids of rat prorenin processing site. The specificity data of cathepsin B toward peptides derived from prorenin processing site support the view that human or rodent cathepsin B could be involved in the intracellular processing of prorenin that is locally synthesized or taken up from the extracellular compartment.

Animals↗

PubMed1999

Shared structural motifs in TCR of glycopeptide-recognizing T cell hybridomas.

The TCR structure of T cell hybridomas recognizing a tumor glycan-defined epitope has been studied using reverse transcriptase-PCR and gene sequencing. The hybridomas had been raised against a glycopeptide, T72(Tn), consisting of the mouse hemoglobin-derived decapeptide Hb(67 - 76), O-glycoslated in position 72 with alpha-D-GalNAc. The glycan-specific hybridomas varied widely in their use of Valpha genes although Valpha4 was predominant, being present in one third of them. The Vbeta gene usage was more restricted and dominated by Vbeta1 and Vbeta15. There was no correlation between Valpha and Vbeta usage and antigen fine specificity of the hybridomas. The overall amino acid composition of the complementarity-determining region (CDR) 3 of the hybridomas was dominated by small polar residues such as Gly, Asn, Ser, Glu and Ala, amino acids reported in the literature to be frequent in glycan-recognizing proteins. Furthermore, the CDR3 of most hybridomas also contained an aromatic residue with preference for Tyr. A few of the hybridomas raised against the T72(Tn) glycopeptide were peptide specific, i. e. they responded to the unglycosylated peptide only. The amino acid usage of their CDR3 regions was not radically different from that of the glycopeptide specific hybridomas. They also preferentially used Valpha4. However, Vbeta4 and Vbeta8 were the dominating beta chains.

Amino Acid Sequence↗

PubMed1998

Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides.

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.

Amino Acid Sequence↗

PubMed1998

Analysis of organic reactions by thin layer chromatography combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The products of a wide variety of organic reactions were rapidly identified by their masses through a combination of thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Crude mixtures of peptides and glycopeptides, complex carbohydrate reactions, and a classical organic reaction were analysed using the following standard protocol. The components of the reaction mixtures were first separated on the TLC plate, scraped off, extracted and analysed by MALDI-TOFMS. The technique used is easy and applicable to most organic reactions, becoming a very powerful technique in reaction optimization once composition and identity of TLC spots have been established by MS. Moreover, the TLC/MALDI-TOFMS method was used to identify low molecular weight compounds with masses within the matrix region (100-500 u), a goal which is normally difficult to achieve. We successfully detected low molecular weight compounds by suppression of the matrix peaks using a relatively low matrix:analyte ratio (15:1 or lower). Doping both matrix and analyte solutions with [Cs]+ ions resulted in suppression of both [Na]+ and [K]+ peaks, thus enhancing the spectral signals and making identification of low molecular weight compounds more facile.

Carbohydrates↗

PubMed1998

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases.

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluorescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluorescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA-beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead-linked library and a peptide containing the quenching chromophore (Tyr(NO2)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluorescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized.

Cystine↗

PubMed1998

Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor library assay. D-amino acid inhibitors for cruzipain, cathepsin B and cathepsin L.

A PEGA-resin was derivatized with a 3:1 mixture of hydroxymethyl benzoic acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO2)KLRFSKQK(Abz)-PEGA was assembled on the lysine using the active ester approach. Following esterification of the hydroxymethyl benzoic acid with Fmoc-Val-OH a library XXX-k/r-XXXV containing approximately 200,000 beads was assembled by split synthesis. The resulting 'one bead, two peptides' library was subjected to extensive hydrolysis with cruzipain. One hundred darker beads were isolated and the 14 most persistently dark beads were collected and sequenced. The putative inhibitor peptides and several analogues were synthesized and found to be competitive microM to nM inhibitors of cruzipain in solution. The inhibitory activity was found to be unspecific to cruzipain when compared with cathepsins B and L and specific when compared with kallikrein. One of the inhibitors was docked into the active site of cathepsin B and was found most probably to bind to the enzyme cavity in an unusual manner, owing to the inserted D-amino acid residue.

Amino Acids↗

PubMed1998

PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays.

Permeable resins cross-linked with long PEG chains were synthesized for use in solid-phase enzyme library assays. High molecular weight bis-amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido-PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6-9). Furthermore, acryloyl-sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13-19). N,N-Dimethylacrylamide was used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene: no swelling was observed in diethyl ether. The PEGA resins (Type I) with an amino acid group capacity between 0.07 and 1.0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries were synthesized on the new polymer using a multiple column library synthesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity.

Acetamides↗

PubMed1998

Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid.

ortho-Aminobenzoic acid (Abz) has been used as a convenient fluorescent donor group in internally quenched fluorescent peptides, which are employed as substrates for several proteolytic enzymes. As Abz is usually bound to the N-amino terminal of these peptides, it is of interest to investigate the Abz group fluorescent properties bound to different amino acids. We report in this article the optical absorption and fluorescent properties, in aqueous media, of Abz bound to the alpha-amino group of Ala, Gly, Leu, Ile, Val, Pro, Phe, Arg, Glu, Met, Asn, Tyr, and Trp, with monomethylamidated alpha-carboxyl group. In order to explore the origin of the drastic reduction of Abz attached to Nalpha amino group of prolyl-peptides, we also examined the fluorescence properties of Abz-NHCH3, Abz-N(CH3)2, and Abz-pyrrolidine. Molecular dynamics simulation and NMR data indicated a lack of periplanarity of the Abz-dimethylamide, which could be the origin of low fluorescence quantum yield of Abz-prolyl-peptides.

Amides↗

PubMed1998

The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the phosphorylated side chains.

The exopolysaccharide produced by Pichia (Hansenula) holstii NRRL Y-2448 is composed of a phosphomannan core to which oligosaccharide diester phosphate side chains are appended. The oligosaccharides of the side chains were released as oligosaccharide phosphates and neutral oligosaccharides by mild hydrolysis with aqueous acetic acid and aqueous hydrogen fluoride, respectively. The liberated oligosaccharide phosphates were studied by NMR spectroscopy and by electrospray and fast atom bombardment mass spectrometry. The structures of the neutral oligosaccharides were determined by 1D and 2D NMR spectroscopic experiments. Further insight into the length of the side chains was obtained from a matrix assisted laser desorption ionisation-time of flight mass spectrometric study of high and low molecular weight fragments obtained from partial acid hydrolysis of the native polysaccharide.

Carbohydrate Conformation↗

PubMed1998

Substrate specificity of barley cysteine endoproteases EP-A and EP-B.

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P2-P1-P1'-P2'-tyrosine(NO2)-aspartic acid, in which cleavage occurs between P1 and P1', showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P2. Arginine was preferred to glutamine at P1, whereas proline at P2, P1, or P1' greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower Km values.

Amino Acid Sequence↗

PubMed1998