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.

Keywords: antibodies; carbohydrate; glycopeptide; glycosylation; posttranslational modification.

Figure 1

Strategy for the chemical synthesis of a glycopeptide derived from Skp1. L is appropriate leaving group.

Figure 2

Acceptor activities of synthetic AgtA substrates. (A) Comparison of AgtA activity towards F-pNP and FGGn-pNP. Reactions were prepared with 10 µM UDP-[³H]Gal (2500 µCi/µmol) and His₆AgtA, and incubated for 60 min. Incorporation was determined using method 1. (B) Activity towards FGGn-pNP and FGGn-Hyp 23 were determined in the presence of 5 µM UDP-[³H]Gal (10,000 µCi/µmol) and of 0.6× His₆AgtA (relative to panel A) for 60 min. Incorporation was determined using method 2. (C) The effect of 23 on the 60-min reaction of 0.06× His₆AgtA with 0.5 mM F-pNP in the presence of 5 µM UDP-[³H]Gal (5000 µCi/µmol), using method 2. (D) Activity towards FGGn-pNP and FGGn-peptide 1 was analyzed using 10 µM UDP-[³H]Gal (2500 µCi/µmol) and 1× His₆AgtA for 180 min. Incorporation was determined using method 1. (E) Similar to D, except that 120-min reactions containing 10 µM UDP-[³H]Gal (5000 µCi/µmol) and 1.5× His₆AgtA were analyzed using method 3. (F) Comparison of activities towards 1 coupled to KLH and FGGn-Skp1. Reactions were conducted using 5 µM UDP-[³H]Gal (10,000 µCi/µmol), the indicated concentrations of FGGn-peptide (0.66 mg/mL KLH-conjugate) or FGGn-Skp1, and 1× or 0.003× His₆AgtA, respectively. Incorporation was determined using method 4. Error bars indicate ± SD.

Figure 3

Specificity of anti-59/KLH UOK104 antibody. (A) Western blot analysis of a panel of Dictyostelium mutants that accumulate the indicated Skp1 glycoforms. Panels were probed with either pAb UOK104 or UOK77 (pan-specific for all Skp1 isoforms) at 1:1000 dilution. Note: An irregularity during placement of the gel on the blot membrane resulted in distortion of relative mobilities of the Skp1 isoforms. (B) The glycan specificity of UOK104 was tested against BSA conjugates of LNFP-I and LNFP-III, which were adsorbed in solutions containing the indicated pmol of glycan equivalents in 50 µL to wells of a 96-well ELISA plate. Coated wells were probed with UOK104 (1:500) or mAb ab3355 (1:10), with specificity for the blood group H type 1 antigen present in LNFP-I/BSA.

Scheme 1

a) 10% TFA in DCM; b) Ac₂O, Pyridine; c) FeCl₃, DCM; d) Ac₂O, pyridine; e) H₂NNHOAc, DMF; f) CCl₃CN, DBU, DCM; g) 9-fluorenylmethyloxycarbonyl-l-trans-4-hydroxyproline, TfOH, Et₂O, 0 °C, 15 min; h) AcSH, pyridine; i) 10% Pd/C, DMF, H₂

Scheme 2

a) NIS, TfOH, DCM:CH₃CN, −30 °C, 30 min; b) H₂NNH.OAc, EtOH:Toluene; c) 4, NIS, TMSOTf, Et₂O, −10 °C, 20 min; d) 10% TFA in DCM; e) Ac₂O, Pyridine; f) DDQ, DCM:H₂O.

Scheme 3

Assembly of Skp1 derived glycopeptide.