Biomedical subjects

M Meldal

Publications and source records attributed to M Meldal.

At least 19 recordsLinked to original sources

PubMed2001

Solid-phase glycosylation of peptide templates and on-bead MAS-NMR analysis: perspectives for glycopeptide libraries.

The efficient solid-phase glycosylation of amino acid side chains (serine (Ser), threonine (Thr), and tyrosine (Tyr)) in peptides was demonstrated with a variety of glycosyl trichloroacetimidate donors in high yields and purities. A novel photolabile linker, with no chiral centre, was introduced to facilitate analysis by both matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry and nanoprobe magic angle spinning (MAS) NMR spectroscopy. Product analysis by nanoprobe MAS NMR spectroscopy, LC-MS and MALDI-TOF mass spectrometry of the glycosylation reactions indicated that the reactivity order of the hydroxy side-chain functions of amino acids in peptides on the solid-phase was Tyr>Ser>Thr. The nearly quantitative glycosylation yields and the efficient on-bead product analysis by nanoprobe MAS NMR spectroscopy have made a truly solid-phase approach for the synthesis and analysis of glycopeptide libraries possible.

Amino Acid Sequence↗

PubMed2001

Identification of peptides inhibitory to recombinant cysteine proteinase, CPB, of Leishmania mexicana.

We have identified peptides that are relatively resistant to hydrolysis by a recombinant cysteine proteinase, CPB2.8DeltaCTE, of Leishmania mexicana, and yet exhibit inhibition constant (K(i)) values in the nanomolar range. Common to these peptides is a basic-hydrophobic-hydrophobic motif in the P3-P1 sites, which is also present in the pro-region of the enzyme. A nine-amino acid stretch, FAARYLNGA, which has good homology to the pro-region of mammalian cathepsin L was identified as the part of the pro-region most likely to interact with the active site of the parasite enzyme. This peptide is not hydrolyzed by CPB2.8DeltaCTE and inhibited it with a K(i) of 4 microM. Extension of this sequence at both the N- and C-termini and the introduction of ortho-aminobenzoic acid at the N-terminal site reduced the K(i) value to 30 nM. The best substrate for CPB2.8DeltaCTE was also well hydrolyzed by cathepsin L, however the best inhibitor of the parasite enzyme inhibit poorly cathepsin L, with K(i) value two order of magnitude higher than against the parasite enzyme. These promising data provide insights into the peculiar specificity of the parasite enzyme and will aid the design of antiparasitic drugs directed against the leishmanial enzyme.

Amino Acid Sequence↗

PubMed2001

alpha-Ketocarbonyl peptides: a general approach to reactive resin-bound intermediates in the synthesis of peptide isosteres for protease inhibitor screening on solid support.

alpha-Ketocarbonyl peptides were generated from peptide precursors on solid support via a metal-ion-catalyzed transamination. The reaction proceeded to completion within 2 h with glyoxylate as electrophile and copper(II) ions as catalyst in an aqueous acetate buffer at pH 5.5-6.0. The variety of naturally occurring alpha-amino acid substrates gave rise to a diverse set of differentially functionalized ketones. The highly reactive terminal ketocarbonyls were prone to aldol-type dimerization and could be transferred into stable moieties by oxime formation, reduction to the alcohol, or reductive amination, respectively. The alpha-ketocarbonyl peptides were efficient in nucleophilic addition of C-nucleophiles such as phosphono-ylides and allylsilanes.

Combinatorial Chemistry Techniques↗

PubMed2001

Surfactant Mediated Cationic and Anionic Suspension Polymerization of PEG-Based Resins in Silicon Oil: Beaded SPOCC 1500 and POEPOP 1500.

A novel surfactant has been synthesized for use in cationic and anionic ring-opening suspension polymerization of PEG-based macromonomers in silicon oil. A polymer of acrylate esters containing pentamethyldisiloxane and PEG was prepared by radical polymerization. The surfactant can stabilize an emulsion of PEG-based macromonomers, initiator, and solvent in silicon oil such that polymer beads are obtained by ring-opening polymerization, initiated either by a Lewis acid (cationic ring opening) or potassium tert-butoxide (anionic ring opening). The average bead size could be controlled by varying the stirring rate and the amount of surfactant and solvent. The surfactant does not interfere with the polymerization and can be removed together with residual silicon oil by a simple washing procedure.

Journal Article↗

PubMed2001

Radically altered T cell receptor signaling in glycopeptide-specific T cell hybridoma induced by antigen with minimal differences in the glycan group.

A T cell hybridoma raised against the synthetic glycopeptide T(72)(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulated with glycopeptides closely related to the cognate glycopeptide antigen. T(72)(Tn) has an alpha-D-GalNAc group O-linked to the central threonine in the decapeptide VITAFTEGLK, and the hybridoma is known to be highly specific for this carbohydrate group. T(72)(Tn)-pulsed APC induced tyrosine phosphorylation of the TCR-zeta 21- and 23-kDa proteins and the downstream p42/44 MAP kinase and strong IL-2 secretion. APC pulsed with T(72)(alpha-D-GlcNAc), which differs from T(72)(Tn) solely by the orientation of a hydroxy group in the carbohydrate structure, completely failed to induce detectable tyrosine phosphorylation and IL-2 secretion. APC pulsed with S(72)(Tn), which differs from T(72)(Tn) by not having a methyl group in the serine amino acid side chain to which the glycan is attached, induced partial tyrosine phosphorylation of the TCR-zeta 21-kDa protein, no tyrosine phosphorylation of the MAP kinases and no IL-2 production. Molecular modeling of the MHC/glycopeptide complex revealed that the dramatic difference between the stimulatory power of T(72)(Tn) and T(72)(alpha-D-GlcNAc) is mainly due to very small differences in the TCR exposed carbohydrate structure.

Cell Line↗

PubMed2001

Substrate specificity of recombinant cysteine proteinase, CPB, of Leishmania mexicana.

The primary S(1) subsite specificity of a recombinant cysteine proteinase, CPB2.8 Delta CTE, of Leishmania mexicana was investigated in a systematic way using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg was substituted by all natural amino acids (where Abz is ortho-amino-benzoyl and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine). The peptides from this series with charged side chain amino acids, Cys, Cys(SBzl), and Thr(OBzl) were well hydrolysed. All other substitutions resulted in peptides that were resistant or hydrolysed very slowly and inhibited the enzyme with K(i) values in the range of 9--400 nM. Looking for natural substrates for CPB2.8, we observed that the recombinant enzyme failed to release kinin from human kininogen, an activity earlier observed with cruzipain from Trypanosoma cruzi (Del Nery et al., J. Biol. Chem. 272 (1997) 25713.). This lack of activity seems to be a result of the resistance to hydrolysis of the sequence at the N-terminal site of bradykinin in the human kininogen. The preferences for the S(3), S(2) and S(1)'-S(3)' for some amino acids were also examined using substrates derived from Abz-KLRFSKQ-EDDnp with variations at Lys, Leu, Phe, Ser and Lys, using the amino acids Ala, Phe, Leu, His or Pro. Peptides with Phe at P(1)' presented the highest affinity to the leishmanial enzyme. For comparison, some of the obtained peptides were also assayed with recombinant human cathepsin L and cruzain. The best substrates for CPB2.8 Delta CTE were also well hydrolysed by cathepsin L, however, the best inhibitors of the parasite enzyme have low affinity to cathepsin L. These promising data provide leads for the design of anti-parasitic drugs directed against the leishmanial enzyme.

Amino Acid Sequence↗

PubMed2001

Phosphinic peptide inhibitors of macrophage metalloelastase (MMP-12). Selectivity and mechanism of binding.

Pseudopeptide inhibitors of MMP-12 with a phosphinic dipeptide G psi[PO(2)H-CH(2)]L covering the P1-P1'- positions originating from a combinatorial solid phase library have been identified and kinetically analysed with respect to binding mechanism and selectivity towards MMP-7, MMP-9, MMP-13 and MMP-14. One compound with a low nanomolar dissociation constant for MMP-12 showed significantly lower affinity towards all other MMPs tested compared to MMP-12. Two compounds showed selectivity against MMP-9, MMP-13 and MMP-14. One additional compound showed selectivity against MMP-7. The selectivity of these compounds could partly be rationalized by analysis of homology models of the enzymes. Truncated versions of one inhibitor spanning P2 to P2', P3 to P2' or P2 to P3' showed that interactions on both the prime and the non-prime side are important for binding. A two-step binding mechanism, with a rate limiting second step, was shown for binding of a tryptophane containing inhibitor to MMP-12 by transient state analysis, using the tryptophane residue of the inhibitor as fluorescent probe.

Animals↗

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

The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library approach.

The substrate specificity of CPB2.8DeltaCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S(3) subsite and for hydrophobic residues, both aliphatic and aromatic, in S(2). The S(1) subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S'(1), Arg, Pro and Gly in S'(2) and Lys, Arg and Ser in S'(4). By contrast, a strict preference for the basic residues Arg and Lys was found for S'(3). Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. In addition, there were strict requirements for the amino acids in subsites S(3)--S(1). Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8DeltaCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin-L-like enzymes, -F-R/K-, in P(2) and P(1) were identified (e.g. Y(NO(2))-EKFR down arrow RGK-K(Abz)G, Abz=2-aminobenzoyl; k(cat)K(m)(-1)=4298 mM(-1)s(-1)). However, novel substrates containing the dipeptide -L/I-Q- in P(2) and P(1) were also well hydrolysed (e.g. Y(NO(2))-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1)=2583 mM(-1)s(-1)). The effect of utilising different fluorescent donor--quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor--quencher pair (EDDnp=N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO(2)) resulted in higher k(cat)K(m)(-1) values for analogous substrates.

Amino Acids↗

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

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.

Animals↗

PubMed2000

Alpha-azido acids for direct use in solid-phase peptide synthesis.

Several new alpha-azido acids have been synthesized and their use in solid-phase peptide synthesis has been demonstrated. The azido group allows for high activation of the carboxyl group as an acid chloride without formation of byproducts and with no detectable racemization. An analog of Leu-enkephalin has been prepared and tested in the mouse vas deferens and guinea pig ileum bioassays: it displays moderate activity at the delta-opiod receptor.

Animals↗

PubMed2000

Epitope affinity for MHC class I determines helper requirement for CTL priming.

We show here that priming and memory generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) does not require help if the immunogen binds major histocompatibility complex (MHC) class I molecules with high affinity. This conclusion was based on the study of three chemically distinct optimal length CTL epitopes with high affinity for the restriction element Kb. In contrast, when two subdominant epitopes with intermediate MHC binding affinity were studied, either a class II MHC-restricted T helper cell epitope or administration of antibody to CD40 was required to obtain significant CTL priming. Depending on the epitope, one source of help was much more efficient than the other.

Animals↗

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↗

PubMed2000

Synthesis of a non-natural T-antigen containing glycosphingolipid.

A non natural glycosphingolipid containing the mucin derived, cancer associated disaccharide moiety Galbeta1-->3GalNAc, the T-antigen, alpha linked to a ceramide, has been synthesised via the protected disaccharide glycosyl trichloroacetimidate. The product has potential as a cancer vaccine.

Antigens, Tumor-Associated, Carbohydrate↗

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↗