Tim Bielfeldt

Specificity of O-glycosylation by bovine colostrum UDP-GalNAc: polypeptide ?-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 α-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 Galβ1–3GalNAc was present. Thus the sequential attachment of a second GalNAc residue in the vicinity of a pre-existing GalNAc-Thr or Galβ1–3GalNAc-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.

Synthesis of the glycosyl amino acids Nα-Fmoc-Ser[Ac4-β-d-Gal p-(1 → 3)-Ac2-α-d-GalN3 p]-OPfp and Nα-Fmoc-Thr[Ac4-β-d-Gal p-(1 → 3)-Ac2-α-d-GalN3 p]-OPfp and the application in the solid-phase peptide synthesis of multiply glycosylated mucin peptides with Tn and T antigenic structures

Abstract Two new glycosyl amino acids N α - Fmoc-Ser[Ac 4 -β- d -Gal p-(1 → 3)- Ac 2 -α- d -GalN 3 p]- OPfp and N α - Fmoc-Thr[Ac 4 -β- d -Gal p-(1 → 3)- Ac 2 -α- d -GalN 3 p]- OPfp were synthesized. Glycosylation of Nα-Fmoc-Ser-OPfp or Nα-Fmoc-Thr-OPfp with protected β- d -Gal -(1 → 3)- d -GalN 3 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.

Multiple column solid phase glycopeptide synthesis

The preparation of the two new building blocks Nα-Fmoc-Ser(Ac3-α-D-GalpNAc)-OPfp 6 and Nα-Fmoc-Thr(Ac3-α-D-GalpNAc)-OPfp 7 and their application in a simultaneous multiple-column solid-phase synthesis of 40 different O-glycopeptides from human intestinal mucin and porcine submaxillary gland mucin are described. All glycopeptides were obtained in excellent purity and were characterised by 1D- and 2D-1H NMR spectroscopy and amino acid analyses.

Solid phase peptide synthesis of mucin glycopeptides

Abstract The synthesis of the new glycosylamino acid building block N α -Fmoc-Thr(Ac 4 -β-D-Galp)-(1→3)-(Ac 2 -α-D-GalpN 3 )-OPfp 3 and its use in a solid phase synthesis of triple glycosylated peptides from human intestinal mucin is described. The azide reduction was performed with thioacetic acid on the polymere bound glycopeptides.

Chapter 4 Chemical Synthesis of Glycopeptides

Publisher Summary This chapter discusses the chemical synthesis of glycopeptides. Glycoproteins are composed of a polypeptide backbone which is glycosylated with one or more carbohydrate chains. The availability of glycopeptides from natural sources is limited because of the low concentration and the microheterogeneity of biological glycoconjugates. The advances in carbohydrate and peptide chemistry in the last decade have created generally applicable methodologies for the synthesis of glycopeptides as model compounds for biochemical and structural investigations. Thus, glycopeptides can be made available with variations in the peptide and the carbohydrate part in superior purity and quantity. The synthesis of glycopeptides requires a combination of synthetic methods from both carbohydrate and peptide chemistry. The glycosylamino acids can be used for incorporation into a peptide chain by application of standard peptide chemistry methods. In case of a solution phase glycopeptide synthesis, the construction of the peptide chain may start on either the C- or N-terminal side of the glycosylamino acid.