Lycium barbarum glycopeptide(wolfberry extract)slows N-methyl-N-nitrosourea-induced degradation of photoreceptors

Photoreceptor cell degeneration leads to blindness, for which there is currently no effective treatment. Our previous studies have shown that Lycium barbarum(L. barbarum) polysaccharide(LBP) protects degenerated photoreceptors in rd1, a transgenic mouse model of retinitis pigmentosa. L. barbarum glycopeptide(Lb GP) is an immunoreactive glycoprotein extracted from LBP. In this study, we investigated the potential protective effect of Lb GP on a chemically induced photoreceptor-degenerative mouse model. Wild-type mice received the following: oral administration of Lb GP as a protective pre-treatment on days 1–7;intraperitoneal administration of 40 mg/kg N-methylN-nitrosourea to induce photoreceptor injury on day 7;and continuation of orally administered Lb GP on days 8–14. Treatment with Lb GP increased photoreceptor survival and improved the structure of photoreceptors, retinal photoresponse, and visual behaviors of mice with photoreceptor degeneration. Lb GP was also found to partially inhibit the activation of microglia in N-methyl-N-nitrosourea-injured retinas and significantly decreased the expression of two pro-inflammatory cytokines. In conclusion, Lb GP effectively slowed the rate of photoreceptor degeneration in N-methyl-N-nitrosourea-injured mice, possibly through an anti-inflammatory mechanism, and has potential as a candidate drug for the clinical treatment of photoreceptor degeneration.

Reversal of Apoptotic Resistance by Lycium barbarum Glycopeptide 3 in Aged T Cells

Objective To study whether Lycium barbarurn glycopeplide 3 （LBGP3） affects T cell apoptosis in aged mice. Methods LBGP3 was purified with DEAE cellulose and Sephadex columns. Apoptotic ＂sub-G1 peak＂ was detected by flow cytometry and DNA ladder was resolved by agarose gel electrophoresis. Levels of IFN-7 and IL-10 were measured with specific kits and mRNA expression was detected by RT-PCR. Apoptosis-related proteins of FLIP, FasL, and Bcl-2 were determined by Western blotting. Results LBGP3 was purified from Fructus Lycii water extracts and identified as a 41 kD glycopeptide. Treatment with 200 p.g/mL LBGP3 increased the apoptotic rate of T cells from aged mice and showed a similar DNA ladder pattern to that in young T cells. The reversal of apoptotic resistance was involved in down-regulating the expression of Bcl-2 and FLIP, and up-regulating the expression of FasL. Conclusion Lycium barbarum glycopeptide 3 reverses apoptotic resistance of aged T cells by modulating the expression of apoptosis-related molecules.

Experimental Study on the Enhancement of Murine Splenic Lymphocyte Proliferation by Lycium Barbarum Glycopeptide

In order to investigate the immunoactivity of Lycium Barbarum glycopeptide (LBG), the routinely prepared murine splenic lymphocyte suspension was separately added into the samples with different concentrations (500, 100, 10, 1 μg/ml) of LBG as LBG groups. Blank control group in the absence of Lycium Barbarum glycopeptide or ConA and positive control group in the presence of 0.5 ml ConA but in the absence of LBG were created. 0.5 ml LBG samples with different concentrations in combination with 0.5 ml ConA (10 μg/ml) into each well to observe the synergic effects of LBG and ConA as LBG+ConA groups. After incubation for 72 h at 37 ℃, the samples were analyzed by CFSE-labeled cells combined with flow cytometry, and MTT. Flow cytometry revealed that both LBG could enhance the murine splenic lymphocyte proliferative reaction. Combined use of LBG and ConA had synergic effects. MTT demonstrated that sample A could obviously promote the murine splenic lymphocyte proliferative reaction as compared with control group (P<0.01), while sample B could also enhance the lymphocyte proliferation at a high dose. In combination with ConA, sample A had synergic effects at high dose, while sample B showed obviously synergic effects (P<0.05). It was concluded that both samples (A and B) had strong immunocompetence.

Structural Analysis of an Oligosaccharide and Glycopeptide Mixture from Panax Ginseng Root with Inhibition SHP-1 Function

A mixture of oligosaccharide and glycopeptide was isolated from the aqueous extract of Panax ginseng roots. The mixture inhibits protein tyrosinc phosphatase（SHP-1） function, implying it enhances immune activity. The peak molecular mass of the oligosaccharide portion is 1800 calculated via GPC software after separation by HPLC. And the structure of the oligosaccharide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→4）- and （1→6）-linked glucopyranoside, with non-reducing terminals of arabinopyranoside and glucopyranoside. The peak molecular mass of glycopeptide portion is 1900 calculated via GPC software after separation by HPLC. The structure of glycopeptide portion is the backbone of （1→3）- and （1→4）-linked arabinopyranoside, and （1→3,6）-linked glucopyranoside, with non-reducing terminals of galactopyranose and glucopyranoside. The peptide composition is Glu. Asp, Hyp, Set, Arg, Gly , Thr, Pro, Ala, Val, lie, Leu and Lys. The oligosaccharide-peptide linkage is formed by Ara and Hyp.

Enzymatic recovery of glycopeptides from different industrial grades edible bird’s nest and its by-products:nutrient,probiotic and antioxidant activities,and physicochemical characteristics

This study was conducted to recover edible bird’s nest(EBN)hydrolysates from different grades of EBN,including the industrial by-products,using enzymatic treatment.The nutrient,physicochemical properties and antioxidant activities of the recovered hydrolysates at different hydrolysis times were evaluated.Results showed that the recovery yield of enzymatic hydrolysis was above 89%for all grades of EBN and the degree of hydrolysis increased over time.Nitrite content(0.321-0.433 mg/L)was below the permissible tolerance level for all samples.Interestingly,the antioxidant activities(DPPH and ABTS scavenging activities and ferric reducing antioxidant powder(FRAP)activity)were significantly higher(P≤0.05)in hydrolysates recovered from EBN by-products(EBNhC and EBNhD)as compared to the high grade EBN hydrolysates(EBNhA and EBNhB).The in-vitro probiotic activity of EBN and its hydrolysates were examined using the probiotic bacterium Lactobacillus plantarum.Evidently,EBN by-products hydrolysate(EBNhD)recorded the highest number of L.plantarum(1.1×1011 CFU/mL),indicating that low grade EBN has the potential as prebiotic material that promotes probiotic activity.This study demonstrated the concept of using EBN by-products hydrolysates for various applications,such as functional ingredients with enhanced bioactivities,to improve its economic value.

Online Effective Identification of Glycopeptide Using Liquid Chromatography Combined with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS)

The detailed glycan structural analysis of glycoprotein is amenable to glycopeptide enrichment. Here, we develop a simple, effective and economical approach to enrich glycopeptides from proteolytically digested peptide mixtures by chromatographic column packed with graphite carbon and activated charcoal (G/A-column). Glycopeptide from ovalbumin was efficiently enriched by homemade G/A-column using liquid chromatography and the structure of glycopeptide was obtained by tandem mass spectrometry using Fourier transform ion cyclotron resonance mass spectrometry. The results in this study demonstrate that G/A-column can be used to enrich N-glycolpeptides and be benefit for online identification of glycopeptide using LC-MS.

Biomechanics mechanism of the anti-tumor effects of sulfated polysaccharide and sulfated glycopeptide

There are also a variety of cytokines in the tumor microenvironment,which are involved in the change in the hardness of the tumor,thereby affecting the invasion and metastasis of tumor cells.As traditional Chinese medicines,drugs of softening and dissipating firm knot contains different kinds of sulfated polysaccharide and sulfated glycopeptide.By inhibiting the function of cancer-associated fibroblasts,they reduce interstitial fibrosis,thereby reducing the hardness of the tumor and exerting an anti-tumor effect.

The effects of Gekko sulfated glycopeptide and basic fibroblast growth factor on human fibrosarcoma HT1080 cells

Background:Fibrosarcoma is a malignant soft tissue tumor of mesenchymal origin.Gekko sulfated glycopeptide(GSPP),an anticancer drug in traditional Chinese medicine,could inhibited the tumor angiogenesis by targeting basic fibroblast growth factor(bFGF).bFGF promoted the proliferation of fibroblasts.Both fibrosarcoma and fibroblasts derived from fibrous connective tissue.This study investigated whether GSPP has the inhibitory effects on human fibrosarcoma HT1080 cells.Materials and methods:The trypan blue exclusion assay was used to determine cell viability and cell numbers.Cells migration was observed by wound-healing and transwell.Results:From the first day to seventh day,HT1080 cells number of GSPP,bFGF,GSPP combined bFGF groups had not change compared with control.HT1080 cells migration distance and the number of migrating cells of GSPP,bFGF,GSPP combined bFGF groups were not significantly reduced.Conclusions:GSPP did not have inhibitory effects on the proliferation and migration of human fibrosarcoma HT1080 cells.Thus further research should be carried out in order to study the mechanism of GSPP and bFGF acting on the tumor stroma.

Different Regulation Effects of Gekko Sulfated Glycopeptide in Breast and Liver Cancer Cell Lines

Background:Our previous work demonstrated that Gekko sulfated glycopeptide extracted from the Chinese gecko Shou gong(Gekko swinhonis Guenther)could inhibit tumor growth by regulating basic fibroblast growth factor.However,basic fibroblast growth factor has opposing effects on growth in breast and liver cancers.Direct effects and mechanisms of Gekko sulfated glycopeptide on tumor growth remain unclear and are ripe for further exploration.Methods:Differential regulation by Gekko sulfated glycopeptide and bFGF were studied in established human breast cancer MCF-7 cells and hepatocarcinoma HepG2 cells.Cell proliferation was evaluated with a Trypan blue exclusion assay.Cell cycle phases were measured by flow cytometry.Basic fibroblast growth factor,transforming growth factor-β1,and p21WAF1/CIP1 mRNA and protein expression levels were detected by real-time PCR(mRNA)and ELISA(protein).Changes in phosphorylated extracellular signal-regulated kinase levels were analyzed by Western blot.Results:Data indicated that Gekko sulfated glycopeptide inhibited the proliferation of HepG2 cells(P<0.001)and also blocked basic fibroblast growth factor-stimulated proliferation of these cells(P=0.001).Gekko sulfated glycopeptide was shown to increase transforming growth factor-β1 and p21WAF1/CiP1 expression(P<0.01)and partially compensate for reductions therein induced by basic fibroblast growth factor.Conversely,in MCF-7 cells,Gekko sulfated glycopeptide alone had no observable effect on transforming growth factor-β1 and p21WAF1/CiP1 expression.Gekko sulfated glycopeptide did,however,enhance basic fibroblast growth factor-induced inhibition of cell proliferation and transforming growth factor-β1 and p21WAF1/CiP1 expression in the MCF-7 cells(P=0.001,P<0.01,P<0.01,respectively).Gekko sulfated glycopeptide was shown to suppress basic fibroblast growth factor secretion in both HepG2 and MCF-7 cell lines(P<0.05 and P<0.01,respectively)and inhibited extracellular signal-regulated kinase 1/2 phosphorylation facilitated by basic fibroblast growth factor.Gekko sulfated glycopeptide alone decreased phosphorylated extracellular signal-regulated kinase in HepG2 cells but did not visibly affect phosphorylated extracellular signal-regulated kinase levels in MCF-7 cells.Conclusions:Gekko sulfated glycopeptide,a basic fibroblast growth factor inhibitor,differentially regulates growth in different cancer cell lines,and these differences may be determined by the opposing effects of basic fibroblast growth factor on transforming growth factor-β1 and p21WAF1/CiP1 levels in breast and liver cancer cells.

Recent Advances in Glycopeptide and Glycoprotein Synthesis:A Refined Synthetic Probe towards the Biological World

Comprehensive Summary Protein glycosylation is the most complex and diverse form of post-translational modification in human body.Meanwhile,glycosylation of peptides and proteins emerges as a promising strategy to improve the pharmacokinetic profile of peptide-and protein-based therapeutics.Owing to the importance of protein glycosylation,a rigorous evaluation of the relationship between the precise structure and biological function of glycoproteins has to be performed.Recently,chemical synthesis,chemoenzymatic synthesis and semisynthesis strategies have attracted extensive attentions towards the preparation of structurally defined glycopeptides and glycoproteins;the obtained synthetic glycoforms thus enable the thorough investigation of specific effects of protein glycosylation.

Design and fabrication of highly hydrophilic magnetic material by anchoring L-cysteine onto chitosan for efficient enrichment of glycopeptides

Hydrophilic interaction liquid chromatography(HILIC) has been recognized as an effective strategy for glycopeptide enrichment. Hydrophilic materials pave the way to solve the limit of low enrichment capacity and poor selectivity. The present study is the first attempt to combine chitosan(CS) and L-cysteine(L-Cys) to design a novel hydrophilic material focusing on glycopeptide enrichment. CS containing a large number of hydrophilic amino and hydroxyl groups has unique chemical properties, which makes it a very attractive biomaterial for glycopeptide enrichment. The excellent hydrophilicity of zwitterionic molecule L-Cys inspires the idea of anchoring L-Cys onto CS to design a novel hydrophilic material(named as Fe_(3)O_(4)@CS@Au-L-Cys) for the capture of low abundance glycopeptides. To be specific, Au nanoparticles(Au NPs) was introduced into CS-coated Fe_(3)O_(4)via electrostatic interaction and served as bridges to anchor L-Cys onto the surface of CS through strong Au-S bond interaction. The prepared Fe_(3)O_(4)@CS@AuL-Cys exhibited strong affinity, low detection limit(0.5 fmol/μL HRP), high selectivity(HRP/BSA with a molar ratio of 1:1000) for glycopeptides. Moreover, successful application of glycopeptide enrichment in human serum and saliva by Fe_(3)O_(4)@CS@Au-L-Cys was achieved. A satisfactory data set indicates that Fe_(3)O_(4)@CS@Au-L-Cys has promising potential in the application of glycopeptide enrichment in real complex bio-samples and for related glycoproteome research.

Specific enrichment of urinary exosomes and exosomal glycopeptides by coefficient affinity of integrated L-cysteine and titania

Exosome and inclusive cargoes have manifested significant function in different biological events. In particular, glycopeptides in exosome are closely associated with occurrence and development of various diseases. Developing advanced tools is highly desired to enrich glycopeptides from exosomes, and enrich exosomes from complex biological samples as well. In this work, integration of L-cysteine and titania onto the surface of magnetic nanoparticles is designed to realize the coefficient affinity towards exosomes and inclusive glycopeptides. Benefiting from the synergistic affinity, we separate exosomes from human urine concentrate directly, which was proved by the detection of three typical antigen markers of exosomes. Furthermore, hardly any exosomes remained on materials after ultrasonication, which confirmed the good capture performance of Fe_(3)O_(4)@TiO_(2)@L-Cys and high release effect of direct lysis.Moreover, 146 glycopeptides corresponding to 77 glycoproteins were successfully identified from captured exosomes. These satisfactory results will inspire more efforts to be devoted to this field and will be extremely helpful to in-depth information excavation of biological markers, especially disease-related ones, through exosomes and inclusive glycopeptides.

Hydrophilic adamantane derivatives engineered β-cyclodextrin-based self-assembly materials for highly efficient enrichment of glycopeptides

β-Cyclodextrin(β-CD) based materials have attracted great attention in the separation of hydrophilic glycopeptides due to the abundant hydroxyl groups in its exterior. However, the current materials based on β-CD generally has complex synthesis process and harsh experimental conditions, on the other hand,the interior cavity of β-CD is hydrophobic and is harmful to capture glycopeptides. Herein, a novel hydrophilic material based on β-CD was engineered via a self-assembly process utilizing L-cysteine(L-Cys)or glutathione(GSH) derived adamantane for highly efficient glycopeptide enrichment. It is the first attempt to make use of the hydrophobic interior cavity of β-CD for hydrophilic glycopeptide capture. Taking advantages of strong hydrophilicity and superparamagnetism, the as-prepared materials possess low detection limit, high selectively, and excellent reusability when employed to glycopeptide enrichment. In addition, the feasibility of the hydrophilic material based on β-CD was verified by enriching glycopeptides from human serum and saliva samples. This study provides a heuristic strategy for the application of β-CD-based self-assembly materials in the enrichment of glycopeptides. Importantly, this strategy certified a possible that the change of glycopeptide enrichment sites through host-guest interaction between β-CD and adamantane derivatives with different functional groups.

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Investigations of glycosylated proteins or peptides and their related biological pathways provide new possibilities for illuminating the physiological and pathological mechanisms of glycosylation modification.However,open-ended and in-depth analysis of glycoproteomics is usually subjected to the low-abundance of glycopeptides,heterogeneous glycans,and a variety of interference molecules.In order to alleviate the influence of these obstacles,effective preconcentration of glycopeptides are indispensable.Here,we employed a hydrophilic interaction liquid chromatography(HILIC)-based method to universally capture glycopeptides.Glutathione modified magnetic nanoparticles(Fe304@AuGSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples.The prepared materials showed excellent ability to trap glycopeptides from standard glycoproteins digests,low detection limit(10 fmol/p L),and good selectivity(HRP:BSA=1:100).These results indicated that glutathione-based magnetic nanoparticles synthesized in this work had great potential for glycopeptides enrichment.

Tandem Mass Spectrometric Characterization of Fetuin Sialylated Glycopeptides Enriched by Ti02 Microcolumn

Sialylation of glycoproteins is vital for the function or physicochemical properties of a protein. It becomes more and more important to develop approaches that can be used to efficiently isolate and identify sialylated glycopep- tides or glycoproteins for monitoring changes in glycoproteome. In the present study, we analyze intact structures of the enriched sialylated glycopeptides of bovine fetuin by matrix-assisted laser desorption/ionization-tandem mass spectrometry （MALDI-MS/MS）, without any chemical derivation. The experimental data show that the optimal loading buffer for TiO2 as matrix is 80% acetonitrile/2% TFA （trifluoroacetic acid）/100 mg/mL DHB （2,5-dihydroxybenzoic acid） which is also compatible with MALDI-mass spectrometric analysis. This study indi- cates that the improved enrichment approach combined with MALDI-MS/MS may be a powerful tool to analyze intact structures and components of the sialylated glycopeptides from complex peptide mixture.

Facile Synthesis of Boronic Acid-Functionalized Magnetic Mesoporous Silica Nanocomposites for Highly Specific Enrichment of Glycopeptides

In the work, aminophenylboronic acid （APB）-functionalized magnetic mesoporous silica, which holds the attractive features of high magnetic responsivity and large surface area, was developed to enrich glycopeptides. At first, magnetic mesoporous silica nanocomposites were prepared. And then, the nanocomposites were functioned with glycidoxypropyltrimethoxysilane （GLYMO） for boronic acid immobilization. Due to that the boronic acid group on the surface of magnetic mesoporous silica nanocomposites can form tight yet reversible covalent bond with glycopeptides containing cis-1,2-diols groups, the magnetic mesoporous silica nanocomposites were successfully applied to selective enrichment of glycopeptides. APB functionalized magnetic mesoporous silica was also demonstrated to have high selectivity for the glycopeptides in the presence of a 10-fold excess bovine serum albumin （BSA） over horseradish peroxidase （HRP） in the tryptic digest. We also find that magnetic rnesoporous silica has better sensitivity in HRP digest compared with that of commercial aminophenylboronic acid-functionalized magnetic nanoparticles beads. The limit of detection for glycopeptides from glycoprotein HRP is about 0.01 ng/μL.

Lipase-activated glycopeptide nano-assemblies as an antibiotic nano-adjuvant to inhibit Pseudomonas aeruginosa biofilm and enhance antibacterial activity

Bacterial infections,especially those caused by biofilms,pose a serious threat to public health.In this work,we propose a glycopeptide-based antibiotic nano-adjuvant(GalAc-2) that can synergistically enhance the antibacterial activity of antibiotics through its biofilm inhibition activity.Our glycopeptide nanoparticles can be specifically activated by bacterial infection overexpressed lipase and sequentially enlarged by glycosyl exposure on the surface.The stability and bonding affinity of the nanoparticles were enhanced through the increase in β-sheet conformation.The bacterial growth inhibition rate of the mixture of levofloxacin(LEV) and GalAc-2 at 0.54 μg/m L was over sevenfold higher than that of LEV alone because of the electrostatic and multivalence Lec A interactions that inhibited biofilm formation on the bacterial surface.Finally,the mixture of the antibiotic with GalAc-2 significantly inhibited the abscess exposed to skin infection and significantly reduced the infiltration of immune cells.We believe that GalAc-2 can be further used to enhance broad-spectrum antibiotic sensitization and prolong the lifetime of antibiotics.

Divergent Synthesis of Core m1,Core m2 and Core m3 O-Mannosyi Glycopeptides via a Chemoenzymatic Approach

O-Mannosylation plays a vital role in the regulation of a variety range of biological processes,for instance,brain and muscle development.However,the precise function remains largely unknown due to its innate heterogeneity.In this regard,it is still welcome to develop efficient methods to access diverse structurally-defined glycopeptides.In this study,a diversity-oriented assembly of O-mannosylα-dystroglycan(α-DG)glycopeptides has been achieved via a chemoenzymatic strategy.This strategy features(i)gram scale divergent synthesis of core m1,core m2 and core m3 mannosylated amino acids from judiciously designed protecting group strategies and chemical glycosidation;(i)efficient glycopeptide assembly via the optimized microwave-assisted solid phase peptide synthesis(SPpS);and(ii)enzymatic elaboration of the core glycan structures to install galactosyl and sialyl-galactosyl moieties.The efficiency and flexibility of this chemoenzymatic approach was demonstrated with the construction of 12 glycopeptides with different core m1,core m2 and core m3 mannosyl glycans,including a core m2 glycopeptide bearing a heptasaccharide for the first time.

Application of magnetic solid phase extraction in separation and enrichment of glycoproteins and glycopeptides

The analysis of endogenous glycoproteins and glycopeptides in human body fluids is of great importance for screening and discovering disease biomarkers with clinical significance.However,the presence of interfering substances makes the direct quantitative detection of low-abundance glycoproteins and glycopeptides in human body fluids one of the great challenges in analytical chemistry.Magnetic solid phase extraction(MSPE)has the advantages of easy preparation,low cost and good magnetic responsiveness.Magnetic adsorbents are the core of MSPE technology,and magnetic adsorbents based on different functional materials are widely used in the quantitative analysis of glycoproteins and glycopeptides in human body fluids,making it possible to analyze glycoproteins and glycopeptides with low abundance as well as multiple types,which provides a technical platform for screening and evaluating glycoproteins and glycopeptides in body fluids as disease biomarkers.In this paper,we focus on the recent advances in the application of MSPE technology and magnetic adsorbents for the separation and enrichment of glycoproteins and glycopeptides in human body fluids,and the future trends and application prospects in this field are also presented.

Inherently hydrophilic mesoporous channel coupled with metal oxide for fishing endogenous salivary glycopeptides and phosphopeptides

Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes.For in-depth profiling of glycosylation and phosphorylation,a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels(denoted as Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG).Based on the mechanism of hydrophilic interaction liquid chromatography(HILIC)and metal oxide affinity chromatography(MOAC),the Fe_(3)O_(4)@TiO_(2)@mSiO_(2)-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides.With human saliva collected in successive four days as practical biological sample,endogenous glycopeptides and phosphopeptides are efficiently enriched.Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes.This strategy is anticipated to promote variation analysis of salivary post-translational modifications.