Sialylglycopeptide Research Articles

Biomimetic Tweezers for N-Glycans: Selective Recognition of the Core GlcNAc2 Disaccharide of the Sialylglycopeptide SGP.

In recent years, glycomics have shown how pervasive the role of carbohydrates in biological systems is and how chemical tools are essential to investigate glycan function and modulate carbohydrate-mediated processes. Biomimetic receptors for carbohydrates can carry out this task but, although significant affinities and selectivities toward simple saccharides have been achieved, targeting complex glycoconjugates remains a goal yet unattained. In this work we report the unprecedented recognition of a complex biantennary sialylglycopeptide (SGP) by a tweezers-shaped biomimetic receptor, which selectively binds to the core GlcNAc2 disaccharide of the N-glycan with an affinity of 170 μM. Because of the simple structure and the remarkable binding ability, this biomimetic receptor can represent a versatile tool for glycoscience, opening the way to useful applications.

Investigation of protecting group for sialic acid carboxy moiety toward sialylglycopeptide synthesis by the TFA-labile protection strategy

The sialylation using sialyl donor with 2,6-dimethylbenzyl (DMBn) ester at C-1 carboxy group rapidly proceeded with good α-selectivity to yield sialyl-N-acetylgalactosaminylated 9-fluorenylmethoxycarbonyl (Fmoc) serine unit. The unit was used for the solid-phase peptide synthesis (SPPS) of the sialyl glycopeptide. The DMBn group was removed during the final deprotection by the trifluoroacetic acid (TFA) treatment keeping the sialyl bond intact and the desired sialyl glycopeptide was successfully obtained. This protecting group strategy provided easier access to sialyl glycoamino acids for sialyl glycopeptides synthesis.

Dual-Functional Ti(IV)-IMAC Material Enables Simultaneous Enrichment and Separation of Diverse Glycopeptides and Phosphopeptides.

Simultaneous enrichment and fractionation of diverse proteins/peptides possessing different post-translational modifications (PTMs) from the same biological samples is highly desirable to reduce sample consumption, avoid complicated sample processing, and enable studies of potential crosstalks between different PTMs. In this work, we report a new approach to enable simultaneous enrichment and separation of glycopeptides, phosphopeptides, and mannose-6-phosphate (M6P) glycopeptides by using a dual-functional Ti(IV)-IMAC material. Moreover, we also made the separation of neutral and sialyl glycopeptides and mono- and multi-phosphopeptides possible by performing different elution processes according to the differences in their electrostatic or hydrophilic properties. These separations are effective and efficient to eliminate the signal suppression from neutral glycopeptides for sialyl glycopeptide detection, allowing separation of mono-phosphopeptides from multi-phosphopeptides, as well as detection of M6P glycopeptides that are free from the abovementioned modifications. This new strategy significantly improves the coverage and identification numbers of glycopeptides, phosphopeptides, and M6P glycopeptides by 1.9, 2.3, and 4.3-fold compared with the conventional method, respectively. This is the first report on simultaneous enrichment and separation of neutral and sialyl glycopeptides, mono- and multi-phosphopeptides, and M6P glycopeptides via dual-functional Ti(IV)- IMAC, revealing novel insights into potential crosstalk among these important PTMs.

Transglycosylation toward naringenin-7-O-glucoside using an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase

Flavonoids are generally glycosylated, and the glycan moieties of flavonoid glycosides are known to greatly affect their physicochemical and biological properties. Thus, the development of a variety of tools for glycan remodeling of flavonoid glycosides is highly desired. An endo-β-N-acetylglucosaminidase mutant Endo-CC N180H, which is developed as an excellent chemoenzymatic tool for creating sialylglycoproteins, was employed for the glycosylation of flavonoids. Endo-CC N180H transferred the sialyl biantennary glycans from the sialylglyco peptide to pNP-GlcNAc and narigenin-7-O-glucoside. The kinetic parameters of Endo-CC N180H towards SGP and pNP-GlcNAc were determined. Flavonoid glucosides harboring a 1,3-diol structure in the glucose moieties acted as substrates of Endo-CC N180H. We proposed that the sialyl biantennary glycan transfer to the flavonoid by Endo-CC N180H could pave the way for the improvement of the inherent biological functions of the flavonoids and creation of novel flavonoid glycoside derivatives for future human health benefits including foods and drugs.

Separation and Purification of Sialylglycopeptide from Egg Yolk Based on Cotton Hydrophilic Chromatography

Abstract The peptide portion of the sialylglycopeptide (SGP) in egg yolk is composed of six amino acids (KVANKT), among which asparagine (N) is modified by a complex sialylated N-glycan. Sialylglycopeptides are mainly obtained from egg yolk through a cumbersome, expensive, and poorly scalable process. In this study, a simple and low-cost method for the separation and purification of sialylglycopeptide from egg yolk was developed based on hydrophilic interaction chromatography with medical absorbent cotton as stationary phase. Fifty egg yolks were treated with phenol to obtain the crude sialylglycopeptide. The latter was then formulated into a 150 mg/mL solution and loaded on a cotton hydrophilic chromatographic column, followed by treatment with 100%, 95%, 85% and 75% acetonitrile (ACN) aqueous solution to remove impurities. Finally, deionized water was applied for the final elution step. By this way, 300 mg of sialylglycopeptide was yielded and its purity was determined by high-performance liquid chromatography to be 95%. The glycan composition and glycosylation site of the sialylglycopeptide were validated by electrospray ionization mass spectrometry and tandem mass spectrometry.

Improved strategy for large scale isolation of sialylglycopeptide (SGP) from egg yolk powder

Chicken egg yolk is an easily available source for the isolation of sialylglycopeptides (SGP) carrying homogenous biantennary N-glycans. This approach has gained much attention in the last decade since these SGPs can easily be used for the semi-synthesis of glycoconjugates circumventing laborious full-synthetic methodologies. Here we report an optimised, significantly shorter (one day instead of five) and environmentally friendly procedure for the mg scale isolation of SGP using commercially available egg yolk powder. A single chromatographic step following chloroform/methanol precipitation of proteins and lipids yielded desired approximately 200 mg SGP from 250 g egg yolk powder within a day.•Environmentally friendly procedure for isolation of sialylglycopeptide from Egg yolk powder.•Reduced the protocol from five days down to one.

Acceptor range of endo-β- N -acetylglucosaminidase mutant endo-CC N180H: from monosaccharide to antibody

The endo-β-N-acetylglucosaminidase mutant endo-CC N180H transfers glycan from sialylglycopeptide (SGP) to various acceptors. The scope and limitations of low-molecular-weight acceptors were investigated. Several homogeneous glycan-containing compounds, especially those with potentially useful labels or functional moieties, and possible reagents in glycoscience were synthesized. The 1,3-diol structure is important in acceptor molecules in glycan transfer reactions mediated by endo-CC N180H as well as by endo-M-N175Q. Glycan remodelling of antibodies was explored using core-fucose-deficient anti-CCR4 antibody with SGP and endo-CC N180H. Homogeneity of the glycan in the antibody was confirmed by mass spectrometry without glycan cleavage.

A novel, simplified strategy of relative quantification N-glycan: Quantitative glycomics using electrospray ionization mass spectrometry through the stable isotopic labeling by transglycosylation reaction of mutant enzyme Endo-M-N175Q

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics. The sialylglycopeptide (SGP) of the complex type was used to demonstrate that QMERSIL facilitates the relative quantitation over a linear dynamic range (up to d0/d8=0.02:20) of 3 orders of magnitude. The area ratios of the N-glycan peaks from the QMERSIL method showed a good linearity (d0/d8, R2=0.9999; d8/d0, R2=0.9978). The reproducibility and accuracy assay precisions were all less than 6.12%, and the mean recoveries (%) of SGP spiked in the human plasma were 97.34%. Moreover, the QMERSIL using LC–MS/MS was evaluated with various molar ratios (1:1, 1:5, 5:1) of d0(d8)- MPDPZ-Boc-Asn-GlcNAc-labeled glycans from ribonuclease B, bovine fetuin, and ovalbumin. The ratios of the relative intensity between the isotopically MPDPZ-Boc-Asn-GlcNAc labeled N-glycans were almost equal a close to the theoretical values (1:1, 1:5, 5:1). Finally, this method was used for the relative quantitative comparison of the N-Linked oligosaccharides in human plasma.

Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder

Sialylglycopeptide (SGP) is a complex bi-antennary N-glycan bearing a short peptide fragment that can be isolated from the yolk of hen eggs. This natural product has gained popularity as a starting material for the semi-synthesis of N-glycans. We have found that current isolation methods provide a glycopeptide contaminated with several related structures, one being a glycopeptide having a hexose directly attached to peptide backbone, most like through the hydroxyl containing side chain of the threonine moiety. Furthermore, current methods employ fresh egg yolks that need to be lyophilized and involve several tedious purification steps. Herein, we report a convenient method for the isolation of gram quantities of homogeneous SGP from commercially available egg yolk powder using solid/liquid extraction and HILIC-HPLC purification.

One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody-drug conjugates.

Chemoenzymatic transglycosylation catalyzed by endo-S mutants is a powerful tool for in vitro glycoengineering of therapeutic antibodies. In this paper, we report a one-pot chemoenzymatic synthesis of glycoengineered Herceptin using an egg-yolk sialylglycopeptide (SGP) substrate. Combining this one-pot strategy with novel non-natural SGP derivatives carrying azido or alkyne tags, glycosite-specific conjugation was enabled for the development of new antibody-drug conjugates (ADCs). The site-specific ADCs and semi-site-specific dual-drug ADCs were successfully achieved and characterized with SDS-PAGE, intact antibody or ADC mass spectrometry analysis, and PNGase-F digestion analysis. Cancer cell cytotoxicity assay revealed that small-molecule drug release of these ADCs relied on the cleavable Val-Cit linker fragment embedded in the structure. These results represent a new approach for glycosite-specific and dual-drug ADC design and rapid synthesis, and also provide the structural requirement for their biologic activities.

A simplified procedure for gram-scale production of sialylglycopeptide (SGP) from egg yolks and subsequent semi-synthesis of Man3GlcNAc oxazoline

Heterogeneity of glycan structures in native glycoconjugates always hampers precise studies on carbohydrate-involved biological functions. To construct homogeneous glycoconjugates from natural resource of homogeneous glycans is therefore a practical approach to solve this problem. We report here an optimized procedure for gram-scale production of sialylglycopeptide (SGP) containing a disialyl biantennary complex-type N-glycan from egg yolks. Our new procedure simplified the extraction process by treating the egg yolk powder with 40% acetone, avoiding massive emulsification, high-speed centrifugation, and sophisticated chromatography in reported methods. Subsequent semi-synthesis of the N-glycan core Man3GlcNAc oxazoline from SGP was accomplished for the first-time via glyco-trimming and successive oxazoline formation. This efficient semi-synthesis provides an alternative to the pure chemical approach that involves multi-step total synthesis and facilitates the application of endo-glycosidase-enabled chemoenzymatic synthesis of various homogeneous glycoconjugates.

An Efficient Approach for Large-Scale Production of Sialyglycopeptides from Egg Yolks

Glycopeptides are very useful substrates for the synthesis of various complex glycoproteins and glycopeptides that are indispensable materials for the structural and functional investigation of glycoproteins. This report provides an efficient and time- and cost-saving strategy for the large-scale production of a sialylglycopeptide (SGP) that carries an intact N-linked disialyl-biantennary complex-type sialyloligosaccharide. Consequently, 680 mg of SGP was obtained from 100 egg yolks after phenol treatment, Sephadex G-50 gel filtration, and filtration through graphitized carbon cartridges. The chemical structure and the glycosylation site of the isolated SGP were identified by NMR and ESI-MS/MS, and the purity measured with HPLC was over 95%.

Direct detection method of oligosaccharides by high‐performance liquid chromatography with charged aerosol detection

A simple and rapid detection method of oligosaccharides using high-performance liquid chromatography with a charged aerosol detection (HPLC-CAD) was studied. The direct detection of a sialylglycopeptide (SGP) derived from egg yolk was accomplished by HPLC-CAD using an amido-silica column, and its limit of detection was 0.40 pmol [signal-to-noise ratio (S/N) = 3]. The sensitivity of this method was lower than that of the fluorescence detection; however, the method showed approximately 5 times higher sensitivity than that using the conventional UV absorbance detection. Furthermore, this method was used for the analysis of the acid hydrolysis products of SGP. Monosialo- and asialo-oligosaccharides as well as free sialic acid were detected without using fluorescent derivatization. These results indicate that the present method is a new tool for the analysis of oligosaccharides.

Preventive effect of sialylglycopeptide-nondigestive polysaccharide conjugates on salmonella infection.

We have previously reported that sialylglycopeptide (SGP) and its derivatives isolated from egg yolk had a preventive effect on Salmonella infection in vivo; however, their retention time in the gut was rather short. To improve on this, SGP was conjugated with carboxymethyl cellulose (CMC) or carboxymethyl dextran (CMD). The conjugates inhibited the binding of Salmonella enteritidis and Escherichia coli to Caco-2 cells. Infection experiments with mice revealed that the SGP-CMD conjugate (SGP-CMD) had a strong protective effect against Salmonella infection. A turnover experiment in mice administered with radiolabeled SGP-CMD showed that SGP-CMD was more slowly absorbed into the blood and thus remained longer in the intestinal tract than SGP. SGP-CMD itself did not influence the production of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or nitrite ion (NO(2)(-)) by macrophages, although it suppressed that of TNF-alpha and NO(2)(-) in zymosan-treated macrophages, suggesting no causative effects of inflammation in SGP-CMD. SGP-CMD is potentially useful as a food ingredient with a preventive effect on Salmonella infection.

Occurrence of a sialylglycopeptide and free sialylglycans in hen's egg yolk

Free sialylglycans (FSGs) and a sialylglycopeptide (SGP) as components of hen's egg yolk were found and their chemical structures were determined. SGP and FSGs were isolated from fresh egg yolk by treatment with phenol, gel filtration and successive chromatographies on columns of anion- and cation-exchangers. They were localized in the yolk plasma. The glycan moiety of SGP, which was liberated by PNGase digestion, was studied for the chemical structure by HPLC mapping with p-aminobenzoic ethylester-derivatization, sugar composition analysis, 1H nuclear magnetic resonance and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and the glycomoiety was found to be an N-linked disialyl-biantennary glycan. The amino acid sequence of the peptide moiety of SGP was determined to consist of Lys-Val-Ala-Asn-Lys-Thr, the Asn of which is modified with the disialylglycan moiety. FSGs were determined to be two free disialyl-biantennary glycans whose reducing end was either Man β1-4GlcNAc (FSG-I) or Man β1-4GlcNAc β1-4GlcNAc (FSG-II). Since the molar value of SGP present in one egg yolk (2.8 μmol) is comparable to those of well-known major yolk proteins, low density lipoprotein, lipovitellins and phosvitin, it can be considered that SGP is one of the major components in hen's egg yolk.