Degree Of Glycosylation Research Articles

Characterization and film-forming mechanism of egg white/pullulan blend film

Edible films based on pullulan (Pu) and egg white (EW) were prepared in this study. As the egg white proportion increased, the mechanical properties of the blend films initially increased and then decreased. The best mechanical properties occurred at the optimal proportion of Pu:EW = 1:1. The blend films with a higher content of egg white showed a lower degradation speed of moisture and polymers. FTIR spectroscopy illustrated peak displacement, X-ray diffraction patterns showed noticeable changes, and the secondary structure of egg white was altered. These results indicated that pullulan and egg white interacted extensively. The total α-helix and β-sheet content increased with the increment of pullulan content. The decrease of free amino content indicated that the degree of glycosylation increased with the addition of pullulan, which is associated with the Maillard reaction. The apparent color and the scanning electron microscopy images were also assessed in this study.

150 ProBNP glycosylation of threonine 71 is increased with obesity in patients with heart failure

Abstract Funding Acknowledgements The National Heart Foundation of New Zealand Background Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Plasma levels of B-type natriuretic peptide (BNP) or its amino terminal congener (NT-proBNP) are used for HF diagnosis and risk stratification. Circulating levels of both BNP and NT-proBNP are reduced by obesity and this phenomenon is one of the key weaknesses of the diagnostic performance of the natriuretic peptides in HF. Formation of BNP from enzymatic cleavage of proBNP1-108 between residues 76 and 77 by corin and/or furin is influenced by the degree of proBNP glycosylation, therefore we investigated the relationship between proBNP glycosylation, plasma NT-proBNP and body mass index (BMI) in HF patients. Methods Three assays were developed to distinguish between total proBNP (glycosylated plus non-glycosylated proBNP), proBNP not glycosylated at threonine 71 (NG-T71) and proBNP not glycosylated in the central region (NG-C). Intra and inter-assay CVs were <15%, limits of detection were <2 pmol/L and samples diluted in parallel. Results Applying these assays and an NT-proBNP assay to plasma samples from 106 healthy volunteers and 238 patients with HF determined that concentrations (median(IQR)) of proBNP, NG-T71 and NT-proBNP were greater in HF patients compared to controls (24.9 (3.6-55), 9.4 (1.5-21) and 212 (104-409) pmol/L vs 3.0 (1.5-19), 3.0 (1.5-14.5) and 4.7 (2-8) pmol/L respectively, all p < 0.012). NG-C was undetectable in most samples. ProBNP levels in HF patients with BMI above and below 30 kg/m2 were not different (21.9 (2.6-70) pmol/L and 25.7 (3.9-53) pmol/L respectively, p = 0.85), whereas HF patients with BMI > 30 had lower NT-proBNP and NG-T71 levels (121 (64-248) and 3 (1.5-16) pmol/L verse 271 (178-486) and 13.5 (1.5-24.2) pmol/L respectively, p < 0.003) and higher proBNP:NT-proBNP and proBNP:NG-T71 ratios (p = 0.001 and p = 0.02 respectively) than those with BMI < 30. Discussion and Conclusion Using three new assays specific for different glycosylated forms of proBNP we have shown that the processing of proBNP is dysregulated in heart failure compared to controls due to increased glycosylation at threonine 71 of proBNP. Obese patients with HF have even greater dysregulation, demonstrated by decreased concentrations of proBNP that is not glycosylated at T71 (NG-T71), and concomitant decreases in NT-proBNP. Thus, we have shown for the first time that increased BMI is associated with increased proBNP glycosylation at T71 in patients with heart failure. Glycosylation-induced impairment of proBNP processing explains, at least in part, the reduction in plasma concentrations of B-type cardiac natriuretic peptides observed in obesity. Using these assays to evaluate the proBNP profile of larger patient cohorts will further develop understanding of the relationships between BNP production, BMI and heart failure pathogenesis, which would be expected to lead to increased diagnostic performance.

Absolute quantitation of high abundant Fc-glycopeptides from human serum IgG-1

Absolute quantitation of IgG-1 Fc-glycosylation, which is crucial for the clinical practice of glyco-biomarkers and quality control of biopharmaceuticals, has been hindered by the lack of glycopeptide standards. In this study, eleven high abundant IgG-1 Fc-glycopeptides with definite peptide sequences and glycoforms were purified from commercial IgG protein by using two-dimensional hydrophilic interaction liquid chromatographic system. Based on the acquired glycopeptide standards, an absolute quantitation strategy was developed to determine the concentrations of 11 target IgG-1 glycopeptides from pooled human sera. A wide range of Fc-glycopeptide concentrations from 0.60 to 17.61 nmol mL−1 was achieved with excellent accuracy and reproducibility from pooled human sera IgG-1. Compared to conventional relative quantitation, this strategy provides more accurate distribution profiles of 11 high abundant Fc-glycopeptides and degree of glycosylation from pooled human sera IgG-1.

Effects of pH and different sugars on the structures and emulsification properties of whey protein isolate-sugar conjugates

Glycosylation is one of the more studied methods used to improve the emulsification properties of proteins, and glycosylation products are often used as transport carriers for nutrients. In this study, we investigated the structure and emulsifying properties of whey protein isolate (WPI)-sugar conjugates formed with different sugars (glucose, lactose, and dextran) under different pH (2–10) conditions. The results show that pH affected the degree of glycosylation (DG), and alkaline environments were conducive to the formation of WPI-sugar conjugates. As the pH increased from 6 to 10, the protein unfolded, DG, apparent viscosity and emulsifying properties increased. At pH 8–10, the free sulfhydryl content of the WPI-sugar conjugates decreased, disulfide bonds formed, aggregation occurred between protein molecules, the DG, surface hydrophobicity and apparent viscosity decreased, thus the emulsion stability decreased. Different sugars had an effect on DG, with glucose increasing the DG and decreasing surface hydrophobicity and free sulfhydryl content. Dextran increases the apparent viscosity and emulsifying properties of the WPI-sugar conjugates. Therefore, it can be concluded that pH affected the structure and emulsifying properties of the WPI-sugar conjugates, pH has a certain effect on the secondary and tertiary structures of the WPI, the DG and emulsifying properties being better for conjugates formed in alkaline environments. Different sugars affected the emulsifying properties and surface structures of the WPI-sugar conjugates, but had little effect on the secondary and tertiary structures.

TJC4, a Differentiated Anti-CD47 Antibody with Novel Epitope and RBC Sparing Properties

Introduction Tumor cells overexpress CD47 which engages signal-regulatory protein (SIRPa) on macrophages to deliver a "do not eat" signal to avoid being phagocytosed. Blocking CD47 using SIRPa-Fc or anti-CD47 antibodies (Ab) has emerged as a promising strategy to neutralize CD47 and promote tumor eradication. However, targeting CD47 led to significant anemia and thrombocytopenia in both pre-clinical studies and phase I trials as CD47 is also expressed on normal red blood cells (RBCs) and platelets. I-Mab has developed a novel CD47 antibody, TJC4 also known as TJ011133, which was endowed with an RBC sparing property and unique binding epitope, may have better safety profile based on the pre-clinical data. Methods A naïve human single chain variable fragment (ScFv) library was subjected for the binders to human CD47-extracellular domain (ECD). All the binders with unique sequences were converted to full antibodies and screened against human RBCs and tumor cells, leading to the discovery of TJC4. A series of head to head experiments have been performed with other CD47 antibodies to compare the in vitro RBC binding and hemagglutination, ability to block the CD47-SIRPa interaction and enhance the macrophage mediated phagocytosis of tumor cells. Different in vivo tumor models were employed to evaluate the anti-tumor efficacy of TJC4 either by mono or combination treatment. In addition, a comprehensive analysis of the hematological parameters was assessed in cynomolgus monkeys receiving a single intravenous infusion or weekly repeated injections. To explore the underlined mechanism of the RBC sparing properties of TJC4, the binding pose and epitope were identified by X-ray crystallography and the influence of CD47 glycosylation in RBCs were further examined. Results TJC4 is a fully human anti-CD47 IgG4 antibody that shares a similar binding affinity to human and cynomolgus monkey CD47. Like other anti-CD47 antibodies, TJC4 blocks the interaction of CD47 and SIRPa, leading to the enhanced macrophage phagocytosis of various CD47+ tumor cell lines and primary AML cells. Mono-treatment of TJC4 completely eradicated tumor cells in a Raji cell xenograft model and significantly extended the overall survival of treated mice in an AML model. When combined with Rituximab, TJC4 showed a superior efficacy in a DLBCL model over the mono-treatment group. TJC4 has unique RBC sparing properties as evident by the negligible binding to healthy human RBCs and platelets respectively. Single dose or repeat dose treatment of TJC4 minimally and transiently impacts RBCs in cynomolgus monkeys and no other safety findings were observed up to the highest dose (100 mg/kg). No impact was observed in platelets. The unique functional properties of TJC4 can be explained in part by its structure when in complex with CD47, which reveals an almost straight head-to-head binding and a novel conformational epitope that is distinct from other CD47 antibodies. Upon the structural analysis of the binding epitope, we identified a potential N-linked glycosylation site located nearby the two critical epitopes on the CD47 protein. Due to the nature of the high glycosylation degree of membrane proteins by RBCs, the N-linked glycan is hypothesized to function as a "shield" to block the exposure of the epitopes and prevent the TJC4 binding to human RBCs. This hypothesis is validated by the restoration of TJC4 binding to the deglycosylated RBCs after the PNGase treatment. Conclusion In summary, TJC4 is a next generation therapeutic anti-CD47 antibody that is devoid of the hematological liabilities while maintaining anti-tumor efficacy. These attributes of TJC4 differentiate it from other CD47 targeting agents currently in clinical evaluation. Disclosures Meng: I-Mab Biopharma: Employment, Equity Ownership. Wang:I-Mab Biopharma: Employment, Equity Ownership. Guo:I-Mab Biopharma: Employment, Equity Ownership. Cao:I-Mab Biopharma: Employment, Equity Ownership. Shen:I-Mab Biopharma: Employment, Equity Ownership.

Influence of chemical composition and structural properties on the surface behavior and foam properties of tofu-whey concentrates in acid medium.

This article focuses on the impact of chemical composition and structural properties of tofu-whey concentrates on their surface behavior at the air/water interface and foaming properties in acid medium. Liquid tofu-whey (pH 5.6 ± 0.1) was concentrated at three combinations of temperature and pressure (50 °C-8.0 kPa, 65 °C-16.0 kPa and 80 °C-24.0 kPa), with further dialysis (4 °C, 48 h) and freeze-drying, giving the samples TWC50, TWC65 and TWC80, respectively. The increase of temperature during the concentration step promoted the enrichment of the concentrates in crude protein and calcium, without appreciable changes in the yield, the carbohydrate content and the polypeptide composition. For TWC80, the increase the degree of glycosylation and the intensity of the hydrophobic effect promoted the decrease of molecular flexibility and the formation of compact aggregates mediated by disulfide bridges as was evidenced by tricine-SDS-PAGE, TGA and FTIR assays. These structural differences have a pronounced impact of the pH-dependence of turbidity and solubility of protein and polysaccharides. At pH 4.0 all concentrates evidenced a ζ-potential close to zero, which enhanced their foam ability (overrun >1500%). Nevertheless, at this pH, TWC80 showed both the highest carbohydrate-to-protein mass ratio in the soluble fraction (>1.8) and foam stability (FS). Thus, the improvement of FS at pH 4.0 would be associated to the effective adsorption of compact rich-in-protein aggregates at the air/water interface and the higher content of soluble polysaccharides in the bulk phase. These findings are relevant for the application of tofu-whey concentrates in acidic dispersed systems, such as foams and aerated food emulsions.

Role of prion protein glycosylation in replication of human prions by protein misfolding cyclic amplification

Prion diseases are transmissible neurological disorders associated with the presence of abnormal, disease-related prion protein (PrPD). The detection of PrPD in the brain is the only definitive diagnostic evidence of prion disease and its identification in body fluids and peripheral tissues are valuable for pre-mortem diagnosis. Protein misfolding cyclic amplification (PMCA) is a technique able to detect minute amount of PrPD and is based on the conversion of normal or cellular PrP (PrPC) to newly formed PrPD, sustained by a self-templating mechanism. Several animal prions have been efficiently amplified by PMCA, but limited results have been obtained with human prions with the exception of variant-Creutzfeldt–Jakob-disease (vCJD). Since the total or partial absence of glycans on PrPC has been shown to affect PMCA efficiency in animal prion studies, we attempted to enhance the amplification of four major sporadic-CJD (sCJD) subtypes (MM1, MM2, VV1, and VV2) and vCJD by single round PMCA using partially or totally unglycosylated PrPC as substrates. The amplification efficiency of all tested sCJD subtypes underwent a strong increase, inversely correlated to the degree of PrPC glycosylation and directly related to the matching of the PrP polymorphic 129 M/V genotype between seed and substrate. This effect was particularly significant in sCJDMM2 and sCJDVV2 allowing the detection of PK-resistant PrPD (resPrPD) in sCJDMM2 and sCJDVV2 brains at dilutions of 6 × 107 and 3 × 106. vCJD, at variance with the tested sCJD subtypes, showed the best amplification with partially deglycosylated PrPC substrate reaching a resPrPD detectability at up to 3 × 1016 brain dilution. The differential effect of substrate PrPC glycosylations suggests subtype-dependent PrPC–PrPD interactions, strongly affected by the PrPC glycans. The enhanced PMCA prion amplification efficiency achieved with unglycosylated PrPC substrates may allow for the developing of a sensitive, non-invasive, diagnostic test for the different CJD subtypes based on body fluids or easily-accessible-peripheral tissues.

Effects of piceatannol on the structure and activities of bovine serum albumin: A multi-spectral and molecular modeling studies.

Piceatannol (PIC) displays a wide spectrum of biological activities, such as antioxidation, antibacterial activity and anti-inflammation, but the biochemical and molecular mechanism is not fully understood. In this study, the interaction of PIC with bovine serum albumin (BSA) was studied by fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy, circular dichroism spectroscopy and molecular simulation. The effects of PIC on BSA non-enzymatic glycosylation, fibrillation, thermal stability, and structure information were also studied. The results showed that the formation of PIC-BSA complex by mainly hydrogen-bonding forces resulted in the conformational changes of protein. PIC inhibited the formation of β-sheets structures of BSA. BSA still maintained the esterase-like good activity in the presence of PIC. In addition, PIC significantly reduced the degree of BSA glycosylation. These results provided a basis for the molecular interaction between PIC and protein, and suggested the potential effect of PIC in preventing the progression of diabetes mellitus.

Ability of casein hydrolysate-carboxymethyl chitosan conjugates to stabilize a nanoemulsion: Improved freeze-thaw and pH stability

An oil-in-water (O/W) nanoemulsion stabilized by casein hydrolysates (CHs) and carboxymethyl chitosan (OCC) (CHs-OCC) conjugates was prepared and its stability characteristics were evaluated under environmental conditions. The conjugated reaction between CHs and OCC was easier with a maximum degree of glycosylation (DG) of 40.7% and a browning index (BI) of 1.47 were obtained on the 20th day. This result was confirmed by the bathochromic-shift (350–358 nm) of the conjugates determined by intrinsic tryptophan fluorescence spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Transmission electron microscopy (TEM) revealed that the particles of the nanoemulsion prepared with CHs-OCC (0.5–3.0%, w/v) had a smaller size (227–833 nm) and a spherical structure. In addition, due to the strong steric effect provided by the conjugated polysaccharide chains, the CHs-OCC nanoemulsion exhibited the best stability characteristics; the droplet particle size increased from 227 nm (fresh, 2.5%, w/v) to 427 nm (pH 2.0–7.4 at room temperature, 12 h), 488 nm (63 °C/30 min), 389 nm (95 °C/10 min), 236 nm (0–200 mM NaCl), and 255 nm (1–15-d storage, 4 °C). In particular, the CHs-OCC considerably enhanced the freeze-thaw stability of the nanoemulsion: the particle size after 3 freeze-thaw cycles showed a narrow distribution and did not exhibit the bimodal (0.1–1 μm) appearance, compared with casein. CHs-OCC may be suitable as a natural emulsifier in O/W nanoemulsions.

Analysis of the level of fructosamine in liver diseases

Objective To investigate the level of fructosamine in different liver diseases. Methods From October 2016 to October 2017, 153 patients with liver diseases hospitalized in Tongling Municipal Hospital were selected as study group, including 58 patients with chronic hepatitis B(CHB), 35 patients with liver cirrhosis(LC), 60 patients with primary hepatocellular carcinoma(HCC). Meanwhile, 40 healthy subjects were selected as control group.The total protein(TP), albumin (ALB), cystatin C(CysC), sialic acid(SA) and fasting blood glucose(FBG) were statistically analyzed. Results The difference of fructosamine between the HCC group and the LC group, the CHB group and the control group was statistically significant[(299.1±96.9)μmmol/L, (259.3±41.5)μmmol/L, (248.7±29.5)μmmol/L, (234.9±20.2)μmmol/L](F=11.321, P 0.05). Correlation analysis showed that there was a positive correlation between fructosamine and FBG in the CHB group(r=0.655, P 0.05). Logistic stepwise regression analysis showed that in the HCC group, fructosamine(OR=1.029, 95% CI: 1.007~1.052), CysC (OR=5 454.909, 95% CI: 78.117~380 916.873) and SA(OR=1.155, 95% CI: 1.047~1.275) were independent risk factors of HCC. Conclusion It has positive correlation between fructosamine and FBG in CHB and LC patients, fructosamine may not be related to glycometabolism in HCC patients, but the data of fructosamine reflect the degree of glycosylation of HCC tumor cells. Key words: Liver diseases; Hepatitis B; Liver cirrhosis; Liver neoplasms; Fructosamine; Glycosylation; Sialic acids

A thorny pathway of macrophage activating factor (GcMAF): from bench to bedside

Vitamin D3 Binding Protein (DBP) is a multifunctional glycoprotein whose main role is to transport vitamin D3 and its metabolites, but it also is the precursor of the macrophage activating factor (GcMAF). DBP is converted to GcMAF as a result of site-specific selective deglycosylation under the action of β-galactosidase and sialidase, localized on activated B and T cells, respectively. GcMAF exerts its biological activity primarily as the capability of activating macrophages by enhancing their phagocytic function and producing ROS. Activation results in elevated expression of the specific macrophageal surface receptors involved in the recognition of tumor-associated antigens, as well as in the implementation of direct anticancer activity by inducing the apoptosis or necrosis of tumor cells. Increased interest in GcMAF is associated with its potential to be used in the clinic as a new antitumor drug. Besides its anti-tumor activity, GcMAF exerts a potential against a number of viral and neurodegenerative diseases associated with increased activity of N-acetylgalactosaminidase (nagalase) in the blood serum of patients. Nagalase is an enzyme that completely (rather than selectively) deglycosylates DBP so it cannot be converted to GcMAF, leading to immunodeficiency. Circulating DBP is composed of unmodified and O-glycosylated molecules with the glycosylation degree being dependent on the allelic variants of the gene encoding DBP. The role of DBP in the resistance of organism against a number of diseases is supported by the increased risk of a variety of severe illnesses (amyotrophic lateral sclerosis, colorectal cancer etc.) in patients deficient for GcMAF due to homozygosity for defective DBP alleles. In this review, we also will examine in detail the current data i) on the structure and functions of DBP, as the main precursor of GcMAF, ii) on the main mechanisms of GcMAF anticancer effect, iii) on the tumor strategy for neutralizing GcMAF activity, iv) on the results of GcMAF clinical trials in various cancers; and will discuss the available controversies regarding the positioning of GcMAF as an effective antitumor drug.

Short communication: Application of proteomics for characterization of caseinomacropeptide isoforms before and after desialidation

Caseinomacropeptide (CMP) is an important polypeptide found in cheese whey that has various biological activities and functional properties. Because sialylations play an important role in the functional properties of CMP, the aim of the present work was to characterize CMP isoform heterogeneity in a commercial glycosylated CMP (gCMP) isolate using liquid chromatography- and gel-based proteomics before and after desialidation. Using 2-dimensional gel electrophoresis (2-DGE), we observed a shift in isoelectric point (pI) after enzymatic desialidation, indicating that sialylated gCMP were located at pI 3.0 to 3.1, but desialylated gCMP had repositioned to pI 3.7 to 3.9. We used liquid chromatography/mass spectroscopy (LC-ESI/MS) for further analysis of the glycan chains of gCMP. In total, we identified 19 CMP isoforms, of which 13 were glycosylated and 6 were nonglycosylated. We also detected 3 genetic variants (A, B, and E), with up to 3 glycosylations attached per gCMP. Further, we identified up to 4 isomers, reflecting different sites of glycosylation in the peptide backbone of these isoforms. The dominating gCMP isoform of genetic variant E appeared to contain 4 N-acetyl-neuraminic acid (NeuAc) residues, whereas the dominating gCMP isoforms of variants A and B appeared to contain 2 NeuAc. The identification revealed conversions of isoforms containing different combinations of genetic variants and degrees of glycosylation, sialylation, and phosphorylation to various desialylated counter-isoforms. The content of sialylated gCMP relative to the total CMP content was 37% (wt/wt), which decreased to 1.5% after sialidase treatment, indicating 96% effectivity of the desialidation. This approach can be valuable for future functionality studies specifically addressing the importance of NeuAc.

An Enhanced Stability Nanoparticle Preparation by Corn Protein Hydrolysate-Carboxymethyl Chitosan Maillard Conjugates Loaded with Rutin.

Rutin-loaded corn protein hydrolysate-carboxymethyl chitosan (CPH-NOCC) Maillard conjugate nanoparticles (NPs) with superior stability under different NaCl concentrations (0-0.25 mol/L) and pH levels (7.0 to 3.0) were investigated. Results showed that the degree of glycosylation of 53.3%, browning index of 0.6, and SDS-PAGE lane of CPH-NOCC conjugates were obtained after dry heating for 48 hr (60 °C, 79% RH). The high encapsulation efficiency (EE, 97.8%-98.8%) and CPH-NOCC-rutin (98.8%) was significantly higher than CPH-rutin (97.8%) and CPH/NOCC-rutin (98.4%) NPs illustrated that hydrolysis was positive for zein encapsulation, and conjugation of NOCC to CPH increased the EE. Hairy carbohydrate protrusions on the surface of CPH-NOCC-rutin NPs produced a stronger steric effect and hampered the formation of salt bridges and the particle aggregation under CPH isoelectric point at pH 4.0. Therefore, the CPH-NOCC conjugate NPs may be suitable carrier for hydrophobic bioactive substances in a board range of business foodstuffs.

PSGL-1 is a negative regulator of effector and memory T cells

Abstract Effective T cell responses depend on adhesion mechanisms that guide T cells to sites of infection and inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to impact T cell migration; however, its role in the differentiation of responding T cells has not been investigated. Since PSGL-1 is highly expressed on naïve, effector, and memory T cells with varying degrees of glycosylation that impact ligand binding, we hypothesized that PSGL-1 may regulate T cell fates. Using mice deficient in PSGL-1 and we found that PSGL-1 limited the magnitude of the ensuing T cell response. PSGL-1-deficient mice had an increased accumulation of anti-viral CD4+ and CD8+ T cells after acute LCMV infection. Virus-specific PSGL-1-deficient T cells had enhanced survival and were not hyper proliferative. Furthermore, PSGL-1-deficient T cells had superior effector function, downregulation of PD-1 and other inhibitory receptors and changes in their transcriptional signature. After viral clearance, more CD4+ and CD8+ PSGL-1-deficient T cells survived to form memory cells. Interestingly, memory T cells from PSGL-1-deficient mice sustained PD-1 downregulation implying that PSGL-1 may regulate inhibitory receptors in memory T cells. Our findings highlight a previously unrecognized role for PSGL-1 in negatively regulating T cell differentiation during acute viral infection.

Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms

Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization.

Calreticulin in phagocytosis and cancer: opposite roles in immune response outcomes.

Calreticulin (CRT) is a pleiotropic and highly conserved molecule that is mainly localized in the endoplasmic reticulum. Recently, CRT has gained special interest for its functions outside the endoplasmic reticulum where it has immunomodulatory properties. CRT translocation to the cell membrane serves as an "eat me" signal and promotes efferocytosis of apoptotic cells and cancer cell removal with completely opposite outcomes. Efferocytosis results in a silenced immune response and homeostasis, while removal of dying cancer cells brought about by anthracycline treatment, ionizing-irradiation or photodynamic therapy results in immunogenic cell death with activation of the innate and adaptive immune responses. In addition, CRT impacts phagocyte activation and cytokine production. The effects of CRT on cytokine production depend on its conformation, species specificity, degree of oligomerization and/or glycosylation, as well as its cellular localization and the molecular partners involved. The controversial roles of CRT in cancer progression and the possible role of the CALR gene mutations in myeloproliferative neoplasms are also addressed. The release of CRT and its influence on the different cells involved during efferocytosis and immunogenic cell death points to additional roles of CRT besides merely acting as an "eat me" signal during apoptosis. Understanding the contribution of CRT in physiological and pathological processes could give us some insight into the potential of CRT as a therapeutic target.

Effect of Substrate Ratios and the Species of Sugar on the Antioxidant Activity of Glycosylated Products of the Rana Debris Collagen Peptide

The active peptide (molecular weight < 3500 Da) of the enzymatic hydrolysis from Rana debris is used as a raw material of glycosylation. The peptide can be affected by environmental factors, such as pH and temperature, which can destroy structural and functional properties, so the peptide is usually modified by glycosylation. To investigate the effects of glycosylation on the antioxidant activity of Rana Debris Collagen Peptide (RDCP), glycosylated compounds were prepared using different ratios of RDCP and xylose or glucose by the Maillard reaction. The results indicated that the peptide-xylose compound (PXC) showed higher antioxidant activity than the peptide-glucose compound (PGC), and RDCP and xylose heated at a ratio of 1:4 showed good antioxidant properties. The correlation between the glycosylation degree and antioxidant activity was strong. These results indicated that glycosylation can enhance the antioxidant activity of RDCP. Such glycosylated products can be used in the field of food research.

Enrichment and Assessment of the Contributions of the Major Polyphenols to the Total Antioxidant Activity of Onion Extracts: A Fractionation by Flash Chromatography Approach.

The present study extensively fractionated crude red onion extract in order to identify the polyphenols which contributed most in the total antioxidant capacity of the onion extract using a flash chromatography system. The flash separations produced 70 fractions which were tested for their total phenol content, total flavonoid content, and antioxidant capacities as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Out of these 70 fractions, four fractions which were representatives of the four major peaks of the flash chromatograms, were further analysed for their constituent polyphenols using liquid chromatography tandem mass spectrometry (LC-MS/MS). The main contributor of onion antioxidant capacity is quercetin glycoside followed by quercetin aglycone although quercetin aglycone had higher antioxidant capacity than its glycosidic counterparts. High abundance of quercetin glycosides such as quercetin-3,4′-diglucoside and quercetin-4′-glucoside had compensated for their relatively low antioxidant capacities. A Higher degree of glycosylation resulted in lower antioxidant capacity. The fractionation approach also contributed in enrichment of the onion antioxidant polyphenols. A >9 folds enrichment was possible by discarding the early fractions (fractions 1–15) which contained the main bulk of the extracts, predominantly sugars.

Influences of different pectins on the emulsifying performance of conjugates formed between pectin and whey protein isolate

The effects of different pectins on the conjugates formed between pectin and whey protein isolate (WPI) under the dry-heating condition (80 °C, 79% relative humidity) were investigated in this study. The different pectin samples were sugar beet pectin (SBP), high methyl-esterified citrus pectin (HMCP) and low methyl-esterified citrus pectin (LMCP). The covalent linkage between pectin and WPI was proved by SDS-PAGE. As the incubation time was prolonged, the degree of glycosylation (DG) of WPI-pectin conjugate gradually increased, but its solubility decreased gradually. The glycosylated speed between WPI and SBP was the fastest, while the DG between WPI and LMCP was the highest. Even so, the 24 h-incubated WPI-HMCP conjugate sample had the best emulsifying properties in all of the prepared conjugates. All the results revealed that the HMCP was the most suitable pectin sample for preparing the WPI-pectin conjugate with a superior emulsifying performance.

Isolation and characterization of an aspartic protease able to hydrolyze and decolorize heme proteins from Aspergillus glaucus.

The xerophilic Aspergillus molds, Aspergillus glaucus and Aspergillus repens, have been used in the ripening and fermentation of dried tuna bonito (katsuobushi). These molds, and especially their extracellular hydrolytic enzymes, may also be of wider industrial value. Aspergillus glaucus strain MA0196 produces different types of hydrolytic enzymes, including amylase, serine protease, aspartic protease, lipase and cellulase, depending on the composition of the medium. We characterized several of these enzymes, focusing on a glycosylated aspartic protease. The results showed that the lower the d-glucose concentration in the medium, the higher the degree of protease glycosylation, with excess glycosylation tending to decrease protease activity. The molecular mass of the glycosylated protease as determined by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis was 243 and 253 kDa, respectively. The chemically deglycosylated protease had a molecular mass of only 46 kDa. The amount of myoglobin-decolorizing activity was similar to that of a previously reported aspartic protease from A. repens strain MK82. However, the strain MA0196 protease more broadly hydrolyzed myoglobin and hemoglobins than did the strain MK82 protease. The results of the present study demonstrate the potential utility of Aspergillus molds as a functionally new microbial resource for industrial applications such as the bleaching of heme proteins. © 2018 Society of Chemical Industry.