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M Meldal

Publications and source records attributed to M Meldal.

At least 55 records · Page 3Linked to original sources
PubMed1997

Synthetic methods of glycopeptide assembly, and biological analysis of glycopeptide products.

The technology of glycopeptide synthesis has recently developed into a fully mature science capable of creating diverse glycopeptides of biological interest, even in combinatorial displays. This has allowed biochemists to investigate substrate specificity in the biosynthetic processing and immunology of various protein glycoforms. The construction of all the mucin core structures and a variety of cancer-related glycopeptides has facilitated detailed analysis of the interaction between MHC-bound glycopeptides and T cell receptors. Novel dendritic neoglycopeptide ligands have been shown to demonstrate high affinity for carbohydrate receptors and these interactions are highly dendrimer specific. Large complex N-linked oligosaccharides have been introduced into glycopeptides using synthetic or chemoenzymatic procedures, both methods affording pure glycopeptides corresponding to a single glycoform in preparative quantities. The improved availability of glycosyl transferases has led to increased use of chemoenzymatic synthesis. Chemical ligation has been introduced as a method of attaching glycans to peptide templates. Combinatorial synthesis and the analysis of resin-bound glycopeptide libraries have been successfully carried out by applying the ladder synthesis principle. Direct quantitative glycosylation of peptide templates on solid phase has paved the way for the synthesis of templated glycopeptide mixtures as libraries of libraries.

Amino Acid Sequence↗
PubMed1996

The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the structure of the phosphomannan backbone.

The phosphomannan core of the exopolysaccharide of Pichia (Hansenula) holstii NRRL Y-2448 was isolated after hydrolytic removal of the oligosaccharide phosphate side-chains. The core polysaccharide and its dephosphorylated derivative were subjected to extensive 1D and 2D NMR spectroscopy which yielded information on the linkage sites and on the sequence of the mannosyl residues in the major oligosaccharide repeating unit. The most probable structure for the repeating unit was -[6-O-PO3H2-alpha-D-Man-(1-->3)-alpha-D-Man-(1-->2)-alpha-D-Man-(1 -->2)]-alpha-D-Man-(1-->6)-[alpha-D-Man-(1-->2)]-alpha-D-Man-(1-->6)-. A semiquantitative conformational analysis was performed by Monte Carlo simulations and the result was confirmed by comparison with the experimentally determined NMR data. The distance distribution for the phosphate groups was determined from the modeling and was found to cover the expected range of distances for phosphorylated high-mannose oligosaccharides.

Carbohydrate Sequence↗
PubMed1996

Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms.

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH > 5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.

Amino Acid Sequence↗
PubMed1996

T cell recognition of Tn-glycosylated peptide antigens.

The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attached at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67-76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.

Amino Acid Sequence↗
PubMed1996

Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase using synthetic glycopeptide substrates.

The factors determining glycosylation of mucin type glycoproteins are not well understood. In the present work, we investigated the role of the peptide moiety and of the presence of O-glycan chains on O-glycosylation by UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyl-transferase (ppGalNAc-T). We used purified ppGalNAc-T from bovine colostrum and a series of synthetic glycopeptide and peptide substrates most of which contained sequences derived from the tandem repeat region of MUC2 mucin. The rate of incorporation of GalNAc into Thr was significantly greater than toward Ser residues. The presence of one or two GalNAc-Thr moieties in the substrate significantly reduced enzyme activity, and this effect was more pronounced when the disaccharide Gal beta 1-3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Gal beta 1-3GalNAc-Thr occurs at a slower rate than primary glycosylation of carbohydrate-free peptide. Analysis of products by HPLC showed that the enzyme was selective in glycosylating peptides or glycopeptides with the PTTTPIST sequence in that the preferred primary glycosylation site was the third Thr from the amino-terminal end; secondary glycosylation depended on the site of the primary glycosylation. Negatively but not positively charged amino acids on the carboxy-terminal side of the putative secondary glycosylation site resulted in high activity suggesting charge-charge interactions of substrates with the enzyme. These studies indicate that O-glycosylation by bovine colostrum ppGalNAc-T is a selective process dependent on both the amino acid sequence and prior glycosylation of peptide substrates.

Animals↗
PubMed1996

Glycopeptide mimics of mammalian Man9GlcNAc2. Ligand binding to mannan-binding proteins (MBPs).

A novel and simple approach for rational design of oligosaccharide mimics has been developed. Mammalian high-mannose triantennary structure Man9GlcNac2 has been subjected to molecular modelling using the NMR data available on structural fragments of the oligosaccharide. The analysis indicated four different low energy conformations, and the spatial arrangement of terminal disaccharides of the oligosaccharide antennae were stimulated with glycopeptides carrying disaccharides by applying weak constraints between the saccharide parts in MD-simulations on a large array of tri- to octaglycopeptides. The five glycopeptides exhibiting the best fit with the four minimum energy confirmations of the oligosaccharide were synthesized by solid phase glycopeptide assembly using glycosylated fluoren-9-ylmethyloxycarbonyl-amino acid-O-pentafluorophenyl esters as building blocks. The glycan was acyl protected alpha-D-Man-(1-->2)-alpha-D-Man and Ser, Thr and Hyp were the glycosylated amino acids. The deprotected and purified glycopeptides were subjected to NMR analysis for characterization, and in order to investigate the cis-trans isomerism of the carbimide bonds to Hyp. The glycopeptides were tested for their ability to inhibit binding of mannan-binding protein to mannan from Saccharomyces cerevisiae. They were found to be weak inhibitors showing no indication of multivalent interaction with the mannan-binding protein.

Carbohydrate Conformation↗
PubMed1995

Synthesis of the glycosyl amino acids N alpha-Fmoc-Ser[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-OPfp and N alpha-Fmoc-Thr[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-OPfp and the application in the solid-phase peptide synthesis of multiply glycosylated mucin peptides with Tn and T antigenic structures.

Two new glycosyl amino acids N alpha-Fmoc-Ser[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-+ ++OPfp and N alpha-Fmoc-Thr[Ac4-beta-D-Galp-(1-->3)-Ac2-alpha-D-GalN3p]-+ ++OPfp were synthesized. Glycosylation of N alpha-Fmoc-Ser-OPfp or N alpha-Fmoc-Thr-OPfp with protected beta-D-Gal-(1-->3)-D-GalN3 donors afforded the glycosyl amino acids containing an activated C-terminus which could be utilized directly for solid-phase glycopeptide synthesis. The transformation of the 2-azido group into the acetamido derivative was achieved quantitatively at the end of the synthesis by treatment of the polymer-bound glycopeptide with thioacetic acid. The versatility of this strategy was demonstrated by the assembly of eight triply glycosylated mucin peptides which were synthesized simultaneously by multiple column techniques. The glycopeptides were prepared in order to investigate the substrate specificity of a galactosyltransferase.

Amino Acid Sequence↗
PubMed1994

Molecular recognition of a Salmonella trisaccharide epitope by monoclonal antibody Se155-4.

The binding site of monoclonal antibody Se155-4, which has been the object of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1, rather than to the tetrasaccharide repeating unit alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp(1-->4) alpha-L-Rhap(1- of the bacterial antigen. Specificity for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi B O-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic oligosaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives showed that complementary surfaces and three antibody-saccharide hydrogen bonds are essential for full binding activity. Both hydroxyl groups of the 3,6-dideoxy-D-galactose residue were obligatory for binding and consistent with the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-dideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mannose, tyvelose were not bound by the antibody. Titration microcalorimetry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy that accompanies binding of the native trisaccharide determinant. The protein sequences determined for the antibody VL and VH domains reveal somatic mutation of the VL germ line gene, implying that this antibody-binding site results from a mature antibody response.

Amino Acid Sequence↗
PubMed1994

UDPgalactose:glycoprotein-N-acetyl-D-galactosamine 3-beta-D-galactosyltransferase activity synthesizing O-glycan core 1 is controlled by the amino acid sequence and glycosylation of glycopeptide substrates.

In order to investigate the role of the peptide moiety of glycoproteins in the control of O-glycan biosynthesis, UDPgalactose:glycoprotein-N-acetyl-D-galactosamine 3-beta-D-galactosyltransferase (core 1 beta 3-Gal-T) from rat liver was tested for its specificity towards GalNAc-containing glycopeptide substrates. Series of glycopeptides have been synthesized by solid-phase synthesis, protected with an acetyl group on the amino terminal and an amide group on the carboxy terminal, based on variations of the repeat sequences of human intestinal mucin. Most glycopeptides were excellent substrates for core 1 beta 3-Gal-T compared to benzyl alpha-D-galactosamine as indicated by their relatively high Vmax/Km. The enzyme preferred threonine alpha-D-galactosamine Thr(GalNAc) to serine alpha-D-galactosamine. Pro on the carboxy-terminal side adjacent to Thr(GalNAc) was inhibitory. Negatively charged amino acids on either side showed a low Km; substrates with negatively charged amino acids on the amino-terminal side were highly efficient substrates, suggesting charge-charge interactions between enzyme and substrate. Gal beta 1-3GalNAc alpha residues adjacent to Thr(GalNAc) reduced the activity. Product analysis using glycopeptide substrates with three adjacent GalNAc residues showed incorporation of one, two and a small amount of three Gal residues per molecule with an uneven distribution of the potential di-galactosylated isomers. These studies indicate that, in addition to initial glycosylation, the second step in the glycosylation pathways of O-glycans is also controlled by the structure and glycosylation of the peptide core of substrates.

Amino Acid Sequence↗
PubMed1994

Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity.

A solid-phase assay for the complete subsite mapping of the active site of endoproteases has been developed. A library of resin-bound protease substrates was synthesized both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(N,N-dimethylacrylamide) copolymer type of resin that allows proteases to diffuse into the interior and perform their catalytic activity. Anthranilic acid and 3-nitrotyrosine were used as an efficient donor-acceptor pair for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet mixing of the beads and parallel washing procedures. After treatment with subtilisin Carlsberg, fluorescing beads were collected and subjected to peptide sequencing, affording the preferred sequences, their cleavage bond, and a semiquantitative estimation of the turnover. A statistical distribution of preferred amino acids was obtained for each subsite. The result was compared with data from kinetic studies in solution.

Amino Acid Sequence↗
PubMed1994

Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity.

To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81-96)-Y (here named 1) which is immunogenic in H-2k mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule Ek, was synthesized in five different glycosylated forms. The N-terminal serine of HEL(81-96)-Y was derivatized with D-glucose (2), maltotriose (3), and a branched D-glucose pentasaccharide (4). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D-glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non-glycosylated peptide, labeled with 125I, to bind to the two MHC class II molecules, Ak and Ek, was studied using a gel filtration assay. None of them could bind to Ak. Neither 5 nor 6 were able to bind to Ek. Surprisingly 2, 3 and 4 bound better to Ek than did the non-glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to Ek of glycopeptide 4 was found to be due to an increased association rate. The binding of 1 as well as 4 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non-glycosylated peptide. Circular dichroism studies of 1 and 4 indicated a more unordered structure of 4 and a predominant alpha-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-alpha-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.

Amino Acid Sequence↗
PubMed1994

Silyl protection in the solid-phase synthesis of N-linked glycopeptides. Preparation of glycosylated fluorogenic substrates for subtilisins.

The trimethylsilyl (TMS) group was used for protection of the hydroxy groups of three disaccharide 1-amino-alditols and of the glycosylamines of glucose, maltotriose and maltoheptose. The per-O-trimethylsilylated derivatives were coupled with N alpha-Fmoc-Asp(Cl)-OPfp 7 to give six glycosylated building blocks for the solid-phase synthesis of N-linked glycopeptides. Building block 8 was used in the synthesis of five internally quenched fluorescent substrates which were studied by enzymatic hydrolysis with savinase, a subtilisin-type enzyme.

Amino Acid Sequence↗
PubMed1994

Susceptibility of glycans to beta-elimination in Fmoc-based O-glycopeptide synthesis.

In order to investigate the possible extent of beta-elimination occurring in Fmoc-based continuous-flow solid-phase glycopeptide synthesis, the influence of the pKb of the base used for N alpha-deprotection has been studied. A glycosylated pentapeptide was synthesized using 50% morpholine, 10% piperidine or 2% DBU, respectively, in DMF for deprotection. The dehydropentapeptide N alpha-Ac-Thr-Thr-delta Aba-Val-Thr-NH2, which would be formed in the case of beta-elimination, was prepared independently and used as a control in HPLC analysis; however, this product was not formed under any of the deprotection conditions applied. Furthermore, a 23 amino acid long glycopeptide from human intestinal mucin was prepared using 2% DBU as a base for Fmoc cleavage, and similarly no beta-elimination was observed. The glycopeptide products were subjected to a prolonged treatment with sodium hydroxide in methanol/water without significant formation of byproducts, and the pure glycopeptides were isolated and characterized by 1H-NMR spectroscopy.

Amino Acid Sequence↗
PubMed1993

Mutational replacements of the amino acid residues forming the hydrophobic S4 binding pocket of subtilisin 309 from Bacillus lentus.

The amino acid side chains of Ile107, Leu126, and Leu135 participate in the formation of the important hydrophobic S4 binding pocket of the subtilisin Savinase. Ile107 and Leu126, located on each side of the pocket, point toward each other, and Leu135 is situated at the bottom of the pocket. These amino acid residues have been substituted for other hydrophobic amino acid residues by site-directed mutagenesis, and the resulting enzymes have been characterized with respect to their P4 substrate preferences. The Leu126-->Ala or Phe substitutions reduce kcat/KM for the hydrolysis of all substrates to around 5% without altering the substrate preference. It is concluded that Leu126 is an essential structural part of the pocket which cannot be replaced without seriously affecting catalysis, consistent with the fact that Leu126 is conserved among all subtilisins. In contrast, the Ile107-->Gly, Ala, Val, Leu, or Phe and Leu135-->Ala, Val, or Phe substitutions strongly influence the P4 substrate preference, and some of the mutants exhibit large specificity changes for particular substrates when compared to wild-type Savinase. The results can be rationalized on the basis of Ile107 and Leu135 being responsible for steric repulsion of branched aliphatic and aromatic P4 side chains, respectively. Leu135 exclusively interacts with aromatic P4 side chains, and its replacement with less bulky amino acid residues alleviates steric repulsion such that the activity toward this type of substrates is enhanced. Conversely, the introduction of a more bulky amino acid residue at position 135 produces more steric repulsion and reduces the activity toward substrates with aromatic P4 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Multiple column peptide synthesis, Part 2 (1, 2).

A manually operated apparatus for parallel multiple column soli-phase peptide synthesis is described. It employs Fmoc-amino acid-O-Dhbt or -Pfp esters in the continuous flow version of the polyamide method on small packed columns of kieselguhr supported resin in a reaction block of Teflon. The solvents and deprotecting reagents are dispensed from two washers in a parallel fashion and reagent consumption is low. Activated and protected amino acids are transferred from a dispenser tray as solutions, eight at a time. The use of the method is demonstrated by the synthesis of overlapping peptides from a protein structure and of analogous protease substrates. The products have been characterized by HPLC, FAB mass spectroscopy and amino acid analysis.

Amino Acid Sequence↗