Chemical de‐O‐glycosylation of glycoproteins for application in LC‐based proteomics

We describe a cyclic on‐column procedure for the sequential degradation of complex O‐glycans on proteins or peptides by periodate oxidation of sugars and cleavage of oxidation products by elimination. Desialylated glycoproteins were immobilized to alkali‐stable, reversed‐phase Poros 20 beads followed by two degradation cycles and the eluted apoproteins were either separated by SDS gel electrophoresis or digested with trypsin prior to LC/ESI‐MS. We demonstrate on the peptide and protein level that even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration or by racemization of the Lys/Arg residues. The protocol is applicable on gel‐immobilized glycoproteins after SDS gel electrophoresis. Conversion of O‐glycoproteins into their corresponding apoproteins should result in facilitated accessibility of tryptic cleavage sites, increase the numbers of peptide fragments, and accordingly enhance protein coverage and identification rates within the subproteome of mucin‐type O‐glycoproteins.     

Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed.The biological potency of the glucosylated analogues was compared with that of the micro-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr(5) or Hyp(6) reduces the potency of both [Lys(7)]-dermorphin and [Hyp(6),Lys(7)]-dermorphin. The effect induced by the Tyr(5) glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp(6) residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp(6),Lys(7)]-, [Hyp(betaGlc)(6),Lys(7)]- and [Tyr(betaGlc)(5),Lys(7)]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8-10 times less active than the parent peptide, but its analgesic effect lasts significantly longer.     

Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics

ABSTRACT: BACKGROUND: In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. Results: In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients' serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/-) treatment confirmed the sialylation changes in the ovarian cancer samples. Conclusion: Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.     

Neo-glycopeptides: the importance of sugar core conformation in oxime-linked glycoprobes for interaction studies

Carbohydrate binding proteins, such as lectins, are crucial in numerous biological recognition processes. While binding may be mediated by a single monosaccharide, several lectins have shown exquisite epimer and linkage recognition indicating that a larger structure is essential for optimal interaction. Several approaches have been described for their detailed study, including lectinosorbent assays, microarrays and surface plasmon resonance (SPR). Most of these approaches ignore that the aglycon-bound monosaccharide is often in a non-natural conformation that affects the occurring binding event. In this paper we demonstrate that oxime-bound glycans, employed in such approaches, occur predominantly in the open form (~70%). Through the use of a secondary amine, the aglycon-bound monosaccharide in the resulting neo-glycopeptide probe is forced into the ring-form. Resulting structures were analyzed by means of nuclear magnetic resonance and differential derivatization experiments. The impact of ring closure was further demonstrated through interaction studies using SPR and various lectins with distinct binding specificities.     

Alpha-galactose based neoglycopeptides. Inhibition of verotoxin binding to globotriosylceramide

Solution and solid phase strategies for the synthesis of alpha-galactose based neoglycopeptide derivatives 2-13 were developed. Neoglycopeptides generated were tested for the inhibition of verotoxin binding to globotriosylceramide (Gb3) using ELISA. Among all of the compounds tested, only the lipid derivatives of neoglycopeptides, 11, 12 and 13 were found to be inhibitors, IC50 = 2.0 mM (11b and 12c) and 0.2 mM (11c and 13c). All of the inhibitors (11b, 11c, 12c and 13c) have a similar branching of the two alpha-galactosyl units at the N-terminal glycine residue of a short peptide and a lipid moiety attached at the C-terminal site. Both of these factors seem to be crucial for the inhibition. It is interesting to note that the inhibitors have only a portion of the natural trisaccharide ligand. The secondary groups either may contribute in sub-site oriented interactions with the protein receptors or may mimic the internal sugar units of the cell-surface ligand, Gb3.     

Structural features of model glycopeptides in solution and in membrane phase: a spectroscopic and molecular mechanics investigation

Model glycopeptides of the general formula Boc-Ala-Thr(G-D)-A(1)-A(2)-Leu-Leu-Lys(N)-Ala-OMe, where D = dansyl (dimethyl aminonaphthalenesulphonyl), G = glucosyl and N = naphthyl, while A(1)-A(2) = Ala-Leu or Aib-Aib, and denoted as D-G-Ala-N and D-G-Aib-N, respectively, were used to investigate glycoprotein-membrane interactions. They carry two fluorophores (D and N), covalently linked to the glucose ring and the lysine side chain, respectively, while the threonine side chain is O-glycosylated. CD spectra in different solvent media suggest that both glycopeptides attain an ordered structure, possibly a helix-like conformation. By combining FRET (fluorescence resonance energy transfer) experiments with molecular mechanics data, the most probable structures of both glycopeptides were built up, starting from both a right-handed (rh) alpha- and 3(10)-helix. They were found to populate an alpha-helical conformation, a result further confirmed by the very good agreement between theoretical and experimental quenching efficiency only observed when the backbone chain was in alpha-helix. The association of D-G-Ala-N with model membranes (liposomes) was studied by CD, fluorescence decay, fluorescence anisotropy, and collisional quenching experiments. The binding does not alter the structural features of the peptide because the CD spectral patterns are unaffected by the association. The peptide orientation inside the phospholipidic bilayer is guided by the polar glucose molecule lying in the water phase. The insertion of the hydrophobic backbone chain into the membrane, seeing the probes only partially accessible from the external solution, is characterized by a significant degree of heterogeneity, an increase in vesicles size, and a relevant stabilizing effect on the membrane itself against rupture by methanol.     

Neoglycopeptide Synthesis and Purification in Multi-gram Scale: Preparation of O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-Nα-fluoren-9-yl-methoxycarbonyl-hydroxyproline and Its Use in the Pilot-scale Synthesis of the Potent Analgesic Glycopeptide O1.5-β-D-galactopyranosyl[DMet2, Hyp5]enkephalinamide

The preparation of a β-galactosylated hydroxyproline derivative and its use in the multi-gram solid-phase synthesis of the potent analgesic neoglycopeptide O^1.5-β-D -galactopyranosyl[D -Met², Hyp⁵]enkephalinamide is described in this paper. The most closely related impurities have been identified, isolated and characterized. Significant aspects of the synthesis and purification affecting yields and purity of both the building block and the target neoglycopeptide are discussed. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

A new method for quantitative analysis of cell surface glycoproteome

As the altered glycosylation expressions of cell surface proteins are associated with many diseases, glycoproteomics approach has been widely applied to characterization of surface glycosylation alteration. In general, the abundances of proteolytic glycopeptides derived from corresponding glycoproteins can be measured to determine the abundances of glycoproteins. However, this quantification strategy cannot distinguish whether the changes are results from changes of protein abundance or changes in glycosite occupancy. For the accurate and specific quantification of the cell surface glycosylation profile, we proposed a modified cell surface‐capturing strategy where the glycopeptides were submitted to LC‐MS/MS analysis directly for identification of glycoproteins and the non‐glycopeptides were isotopically labelled for quantification of glycoproteins. This strategy was applied to comparatively analyze cell surface glycoproteins of two human cell lines, i.e. Chang Liver and HepG2 cells. Totally 341 glycoproteins were identified with 82.4% specificity for cell membrane proteins and 33 glycoproteins were quantified with significant expression change between the two cell lines. The differential expressions of two selected proteins (EMMPRIN and BCAM) were validated by Western blotting. This method enables specific and accurate analysis of the cell surface glycoproteins and may have broad application in the field of biomarker and drug target discovery.     

Effects of laminin glycopeptides on metastasis—related behaviors of cancer cells

Our previous reports have shown that lamininglycopeptides (LN-GPs),the total glycopeptides prepared from laminin (LN),can prevent the experimental lung metastasis and liver metastasis of mouse cancer cells.In order to explore the anti-metastatic mechanism of LN-GPs,we studied the effects of LN-GPs on metastasisrelated behaviors of cancer cells in vitro.LN-GPs did not affect cell survival.However,LN-GPs inhibited cell attachment and spreading of S180 cells on LN-and Matrigelsubstrate in dose-dependent and time-dependent manners.Moreover,inhibition of cell attachment and spreading on Matrigel substrates were much greater on Matrigel substrate than on LN substrate.In the gresence of LN-GPs,S180 cells on LN substrate changed from a flattened polygonal shape to a round one,the migration of S180 cells on LN substrate decreased,and the number of a highly invasive human pulmonary giant carcinoma PG cells invading Matrigel filter in a Boyden chamber was reduced.LN-GPs thus have multiple inhibitory effects on cancer metastasisrelated behaviors.     

Synthesis and protective anti-infective action of anomeric lipophilic glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine

Anomeric pairs of α-and β-dodecyl, α-and β-(1-pentylhexyl), and α-and β-cyclododecyl glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) were synthesized. The starting β-D-glucosaminides were obtained by the oxazoline method, and the corresponding α-isomers, by the mercuric iodide-catalyzed glycosylation of alcohols with α-glucosaminyl chloride peracetate in nitromethane at ～90°C. No reliable differences between the stimulation of mouse resistance to the infection with Staphylococcus aureus (doses of 2, 20, and 200 μg/mouse) and Escherichia coli (doses of 0.05, 1, and 20 μg/mouse) with the MDP α-and β-glycosides were found.