Chemical Synthesis of a Glycopeptide Derived from Skp1 for Probing Protein Specific Glycosylation

Abstract

Skp1 is a cytoplasmic and nuclear protein, best known as an adaptor of the SCF family of E3-ubiquitin ligases that label proteins for their degradation. Skp1 in Dictyostelium is posttranslationally modified on a specific hydroxyproline (Hyp) residue by a pentasaccharide, which consists of a Fucα1,2-Galβ-1,3-GlcNAcα core, decorated with two α-linked Gal residues. A glycopeptide derived form Skp1 was prepared to characterize the α-galactosyltransferase (AgtA) that mediates the addition of the α-Gal moieties, and to develop antibodies suitable for tracking the trisaccharide isoform of Skp1 in cells. A strategy was developed for the synthesis of the core trisaccharide-Hyp based on the use of 2-naphthylmethyl (Nap) ethers as permanent protecting groups to allow late stage installation of the Hyp moiety. Tuning of glycosyl donor and acceptor reactivities was critical for achieving high yields and anomeric selectivities of glycosylations. The trisaccharide-Hyp moiety was employed for the preparation of the glycopeptide using microwave-assisted solid phase peptide synthesis. Enzyme kinetic studies revealed that trisaccharide-Hyp and trisaccharide-peptide are poorly recognized by AgtA, indicating the importance of context provided by the native Skp1 protein for engagement with the active site. The trisaccharide-peptide was a potent immunogen capable of generating a rabbit antiserum that was highly selective toward the trisaccharide isoform of full-length Skp1.

O2 sensing–associated glycosylation exposes the F-box–combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases

“…shifts were also compared with those of authentic chemically synthesized Fuc␣1-2Gal␤1-3GlcNAc␣1-4Hyp (27) and are in agreement. They are also in agreement with those of the CASPER database of all Fuc-Gal regioisomers (28) (supplemental Table S3).…”

“…Previous studies suggested that the pentasaccharide consists of Gal␣1,3Fuc␣1,2Gal␤1,3GlcNAc␣1linked to 2S,4R-hydroxyproline, modified by an ␣Gal at an unknown position. However, these linkages were assigned based on indirect evidence, such as exoglycosidase sensitivity and in vitro characterization of the glycosyltransferase substrate preferences and product characterizations (12,(23)(24)(25)(26)(27). The NMR-based data presented here confirm the Fuc␣1,2Gal-and the Gal␣1,3Fuc linkages in the isolated glycopeptide, based on HMBC analysis and chemical shift comparisons with an authentic standard ( Fig.…”

“…1B). Instability in the presence of TFA, formic acid, or acetic acid was also observed during work-up of a synthetic glycopeptide corresponding approximately to the trisaccharide tryptic peptide (27).…”

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

“…The reaction consisted of 6.25 M Toxoplasma Gn-Skp1, 112 nM Dictyostelium FLAG-PgtA, 25 M UDP-Gal, 50 M GDP-Fuc, 120 mM NaCl in 50 mM Tris-HCl (pH 7.5). The reaction was monitored by dot blotting onto nitrocellulose filters and probing with mAb 1C9, which is specific for Dictyostelium Gn-Skp1 relative to other modified glycoforms (15), and a 1:1000 dilution of polyclonal antibody UOK104, which is specific for Dictyostelium FGGn-Skp1 relative to other glycoforms (26). Alexa680-coupled secondary antibodies were applied and detected in an Odyssey infrared scanner (LI-COR).…”

LC-MS/MS-PRM Quantification of IgG glycoforms using stable isotope labeled IgG1 Fc glycopeptide standard

“…One reason for the limited acceptance of IgG glycoform quantification in clinical samples is the dominant production of less specific glycan fragments (oxonium ions) under CID conditions used typically for the fragmentation of glycopeptides 28–30 . Another reason is the lack of synthetic isotope labeled standards (SIS) of glycopeptides which present a substantial synthetic challenge despite recent advances in the chemical and chemoenzymatic synthetic approaches 31–34 . In the course of our study, Li and coworkers have reported a chemoenzymatic synthesis of an array of fucose isotope-labeled Fc glycopeptides for their absolute quantitation but the method is limited to core-fucosylated glycopeptides 35 .…”