Multiple Column Synthesis of a Library of T-Cell Stimulating Tn-Antigenic Glycopeptide Analogues for the Molecular Characterization of T-Cell–Glycan Specificity

A series of peptides and glycopeptides derived by amino acid and glycosyl amino acid scans through the self peptide from CBA/J mouse haemoglobin Hb (67–76), VITAFNEGLK, was synthesized by multiple column peptide synthesis (MCPS). Investigation of glycopeptide binding to the mouse major histocompatibility class II molecule E^k showed that glycans in position 72 did not interfere with the binding to E^k. Immunization experiments revealed that glycopeptides with the glycan in position 72 were immunogenic. Therefore a series of N-linked and O-linked glycopeptides with the glycan attached in the position 72 either to serine, threonine or asparagine was synthesized by MCPS. The glycan structure was furthermore varied with respect to monosacc haride component, size of oligosaccharide, anomer configuration and stereoche mistry of essential hydroxyl groups in order to investigate the specificity of the interaction with the T-cell receptor. Easy synthesis of ready to use Ser and Thr building blocks corresponding to mucin core 1, the T_n-antigen and its β-anomer were developed using trichloroacetimidates as glycosyl donors and reduction with in situ acetylation of the azide containing glycosylation products. Synthesis of an α-linked GlcNAc-Thr building block was achieved by glycosylation of Fmoc-Thr-OPfp with 2-azido-2-deoxy-3,4,6-tri-O-acetyl-D - glycopyranosyl trichloroacetimidate as a glycosyl donor. Other building blocks were obtained by previously described procedures.     

Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide α-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 -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 Gal1–3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Gal1–3GalNAc-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 aminoterminal 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.Abbreviations Gal G,d-galactose - GalNac N-acetyl-d-galactosamine - HPLC high performance liquid chromatography - ppGalNAc-T UDP-GalNAc: polypeptide -GalNAc-transferase EC 2.4.1.41 - Ser^GalNAc GalNAc-Ser - Thr^GalNac GalNAc-Thr     

Separation of Glycopeptides by High Performance Liquid Chromatography

A method is presented for separation of tryptic glycopeptides-containing oligosaccharides of the N-asparagine-linked type. High performance liquid Chromatography (HPLC) of glycopeptides on a C18 reverse-phase system eluted with a gradient of 0%–50% acetonitrile in 0.1 M NaPO₄ pH 2.2 resolves the two major glycosylation sites from the envelope glycoprotein (G) of vesicular stomatitis virus. Glycopeptides containing N-linked oligosaccharides of the complex type coelute with those containing N-linked oligosaccharides of the neutral, high mannose type, indicating that separation is based upon peptide rather than carbohydrate composition. The contribution of the carbohydrate component to glycopeptide elution, as determined by cleavage of the high mannose oligosaccharides with endo-β-Nacetylglucosaminidase H, is that of a significant, but minor, decrease in peptide retention time. Comparison of the tryptic glycopeptide profiles of G isolated from both wild type and mutant strains of VSV illustrates the rapid, reproducible, and quantitative nature of the technique. Through HPLC analysis of appropriately treated glycopeptides, it is possible to explore both the nature and extent of glycosylation at individual sites in glycoproteins in a single step.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

Conformational investigations on glycosylated threonine-oligopeptides of increasing chain length

Stepwise solution syntheses are described of the homo-oligomers Z-(Thr)_n-NHCH₃ (n=1–4, I _1–4), Z-{[Gal(Ac)₄β]Thr}_n-NHCH₃(n=1–5, II _1–5) and Z-[(Galβ)Thr]n-NHCH₃ (n=1−5, III _1–5). Members of the III _1–5 series were obtained by de-acetylation of the corresponding oligomers of the II _1–5 series. The conformational preferences of the terminally protected homo-peptides of the three series were investigated by FT-IR absorption spectroscopy both in the solid state and in CDCl₃ solution, at various concentrations. Proton NMR measurements in CDCl₃ and in DMSO-d₆ were also carried out and the effect of temperature variation on the chemical shifts of amide protons was determined in DMSO-d₆ (range 298–335 K) and in CDCl₃ (range 298–320 K). CD spectra were recorded in water and in TFE. Solubility problems prevented measurements in CDCl₃ solution for Z-(Thr)₄-NHCH₃ and for the entire III _1–5 series. The existence of unordered structures in the carbohydrate-free oligomers and of more or less extended, organized structures in the glycosylated derivatives is indicated by the NMR and IR measurements. The sugar moieties apparently show a structure-inducing effect on the peptide chain. ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

Teicoplanin biosynthesis genes in Actinoplanes teichomyceticus

The genetic determinants for the biosynthesis of the glycopeptide antibiotic teicoplanin were identified. In order to isolate the corresponding gene cluster, oligonucleotides derived from highly conserved motifs in peptide synthetases were used. These synthetic probes, and gene fragments derived from the balhimycin gene cluster of Amycolatopsis mediterranei, led to the identification of the likely teicoplanin gene cluster centered on a region of ca. 110 kb from the genome of Actinoplanes teichomyceticus, the teicoplanin producer. Partial nucleotide sequences identified partial ORFs likely to encode two glycosyltransferases, three P-450 monooxygenases and one ABC transporter. The corresponding genes have been found in other glycopeptide gene clusters. Furthermore, upstream to the peptide synthetase region a segment was identified with a remarkable similarity to the vanHAX operon, conferring resistance to glycopeptides in enterococci. Thus, in contrast to the other glycopeptide producers thus far analyzed, in A. teichomyceticusthe genes for teicoplanin biosynthesis are closely linked to homologs of glycopeptide resistance commonly found in vancomycin-resistant enterococci.     

Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry.

A mass spectrometry method has been developed for selective detection of glycopeptides at the low (< or = 25) picomole level during chromatography of glycoprotein digests and for differentiation of O-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in Q2, the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc+). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from O-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase F. These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three O-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.     

Subunit composition of the seed globulins of Lupinus albus

The seed globulins separated from Lupinus albus are all oligomeric proteins. Vicilins, i.e. globulins 4,5,6 and 7, and to a minor extent legumins,     

C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

¹³C nuclear magnetic resonance spectroscopy (¹³C n.m.r.) was used to determine the pH titration parameters for the N-terminal N,N-[¹³C]dimethylamino and N,N-[¹³C]monomethylamino groups of glycophorins A^M and A^N, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin A^M.