Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Recombinant Production of Bifidobacterial Endoglycosidases for <em>N</em>-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

Çanakkale Onsekiz Mart University

As more is learned about glycoproteins' roles in human health and disease, the biological functionalities of -linked glycans are becoming more relevant. Protein deglycosylation allows for the selective release of -glycans and facilitates glycoproteomic investigation into their roles as prebiotics or anti-pathogenic factors. To increase throughput and enzyme reusability, this work evaluated several immobilization methods for an endo-β--acetylglucosaminidase recently discovered from the commensal . Ribonuclease B was used as a model glycoprotein to compare -glycans released by the free and immobilized enzyme. Amino-based covalent method showed the highest enzyme immobilization. Relative abundance of -glycans and enzyme activity were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Kinetic evaluation demonstrated that upon immobilization, both V and the K decreased. Optimal pH values of 5 and 7 were identified for the free and immobilized enzyme, respectively. Although a higher temperature (65 vs. 45 °C) favored rapid glycan release, the immobilized enzyme retained over 50% of its original activity after seven use cycles at 45 °C. In view of future applications in the dairy industry, we investigated the ability of this enzyme to deglycosylate whey proteins. The immobilized enzyme released a higher abundance of neutral glycans from whey proteins, while the free enzyme released more sialylated glycans, determined by nano-LC Chip Q-ToF MS.

Immobilization of an Endo-β--acetylglucosaminidase for the Release of Bioactive glycans.

Lactoferrin is a multifunctional glycoprotein found in the milk of most mammals. In addition to its well-known role of binding iron, lactoferrin carries many important biological functions, including the promotion of cell proliferation and differentiation, and as an anti-bacterial, anti-viral, and anti-parasitic protein. These functions differ among lactoferrin homologs in mammals. Although considerable attention has been given to the many functions of lactoferrin, its primary nutritional contribution is presumed to be related to its iron-binding characteristics, whereas the role of glycosylation has been neglected. Given the critical role of glycan binding in many biological processes, the glycan moieties in lactoferrin are likely to contribute significantly to the biological roles of lactoferrin. Despite the high amino acid sequence homology in different lactoferrins (up to 99%), each exhibits a unique glycosylation pattern that may be responsible for heterogeneity of the biological properties of lactoferrins. An important task for the production of biotherapeutics and medical foods containing bioactive glycoproteins is the assessment of the contributions of individual glycans to the observed bioactivities. This review examines how the study of lactoferrin glycosylation patterns can increase our understanding of lactoferrin functionality.

Studying Lactoferrin N-Glycosylation.

Lactoferrin is a highly glycosylated antimicrobial protein that contains multiple glycan types. In this research, recombinantly produced three forms of novel endo-β-N-acetylglucosaminidase (free, genetically attached Glutatiohine-S-transferase and polyhistide) were used for selective release of lactoferrin glycans to understand the contribution of specific glycan types to the antimicrobial function of lactoferrin. Three lactoferrin forms with different glycan profile were obtained by treatment with these fusion tagged enzymes; native, fully deglycosylated and sialylated glycan enriched lactoferrin. The released glycan structures were analyzed and confirmed with mass spectrometry. The results showed that native and sialylated glycans enriched lactoferrin have similar minimum inhibitory concentration (MIC) values against E.coli DH5a (1 mg/ml), whereas the MIC value for fully deglycosylated lactoferrin was 6mg/ml. These results suggest that sialylated glycans play important role in the antimicrobial function of lactoferrin.

Selective deglycosylation of lactoferrin to understand glycans' contribution to antimicrobial activity of lactoferrin.

The Thoroughbred is among the most valuable horse breeds, and its husbandry is a major industry. Mare's milk plays a major role in the health of neonatal foals. Although the main components of mare's milk are broadly characterized, free oligosaccharides (OS), which possess various bioactivities in many mammalian milks, have not been fully profiled in Thoroughbreds. The aim of this study was to identify and quantify OS in Thoroughbred mare's milk during the first week of lactation, when foals typically consume mare's milk exclusively. A total of 48 OS structures (including isomers and anomers), corresponding to 20 unique compositions, were identified by nano LC-Chip QToF-MS and confirmed by tandem mass spectrometry. Neutral OS were the most abundant glycans (58.3%), followed by acidic OS containing Neu5Ac (33.3%), a minor presence of fucosylated OS structures (6.25%) and one structure containing NeuGc (2.1%). Comparison with other well-characterized mammalian milks revealed that mare's milk shared 8 OS structures with human, bovine, pig and goat milk (i.e., 2 sialyllactose isomers, 3 hexose, LNH, LNT, and OS with the composition 3 Hex-1 Neu5Ac). Additionally, there were seven unique OS not previously found in other mammal milks. During the first 7 days of lactation, the percentage of neutral and fucosylated OS increased, whereas acidic OS decreased and the total OS concentration ranged from 217.8 mg·L to 79.8 mg·L.

Thoroughbred mare's milk exhibits a unique and diverse free oligosaccharide profile.

Mucin glycoproteins play an important role in protecting the gut epithelium by keeping gut microbes from direct contact with the gut epithelium while allowing for diffusion of small molecules from the lumen to the epithelium. The mucin glycocalyx can be degraded by gut bacteria such as  and , but other bacteria, such as  subsp, cannot consume mucin glycans. Untargeted mass spectrometry profiles were compared to microbiome profiles to assess how different gut microbiomes affect colonic mucin degradation. Samples obtained from nine infants colonized by  EVC001 and from 10 infants colonized by higher levels of mucolytic taxa (controls), including , were compared. Previously performed untargeted nano-high-performance liquid chromatography-chip/time-of-flight mass spectrometry was used to detect and quantify glycans originating from colonic mucin. Colonic mucin-derived -glycans from control infants composed 37.68% (± 3.14% SD) of the total glycan structure pool, whereas colonic mucin-derived -glycans made up of only 1.78% (± 0.038% SD) of the total in  EVC001 samples. The relative abundance of these colonic mucin-derived -glycans in the total glycan pool was higher among control, 26.98% (± 8.48% SD), relative to colonized infants, 1.68% (± 1.12% SD). Key taxa, such as , were significantly and positively correlated with the abundance of these structures, while  were significantly and negatively associated with these structures. These results suggest that colonization of infants by  may diminish colonic glycan degradation and help maintain barrier function in the gastrointestinal tract of infants.

Reduced colonic mucin degradation in breastfed infants colonized by  subsp EVC001.

EndoBI-1 and EndoBI-2 are two endo-  acetylglucosaminidase isoenzymes that cleave  diacetylchitobiosyl moieties found in various types of native  -glycans. These  -glycans are indigestible by human infants and adults due to the lack of responsible glycosyl hydrolases and they act as selective prebiotics for a probiotic microorganism,  subsp  , in the large intestine. The selectivity and the thermostability of EndoBI-1 and EndoBI-2 suggest that these enzymes may be useful for many scientific and industrial applications. In this study, the growing numbers of homologous sequences in different databases were exploited in a comparative approach to investigate structural properties of EndoBI-1 and EndoBI-2 enzymes. Moreover, the complete and partial homology models of these two enzymes were generated and evaluated. Selected models were used for docking studies of the plus subsite ligand of these enzymes for further understanding on the substrate selectivity of EndoBI enzymes.

Structural insights of two novel N-acetyl-glucosaminidase enzymes through in silico methods.

Human milk is the optimal source of infant nutrition. Among many other health benefits, human milk can stimulate the development of a -rich microbiome through human milk oligosaccharides (HMOs). In recent years, the development of novel formulas has placed particular focus on incorporating some of the beneficial functional properties of human milk. These include adding specific glycans aimed to selectively stimulate the growth of . However, the bifidogenicity of human milk remains unparalleled. Dietary glycans are carbohydrate structures conjugated to a wide variety of glycoproteins. These glycans have a remarkable structural similarity to HMOs and, when released, show a strong bifidogenic effect. This review discusses the biocatalytic potential of the endo-β--acetylglucosaminidase enzyme (EndoBI-1) from  subspecies , in releasing glycans inherently present in infant formula as means to increase the bifidogenicity of infant formula. Finally, the potential implications for protein deglycosylation with EndoBI-1 in the development of value added, next-generation formulas are discussed from a technical perspective.

Potential Applications of Endo-β--Acetylglucosaminidases From  Subspecies  in Designing Value-Added, Next-Generation Infant Formulas.

Colostrum is the first milk produced post-partum by mammals and is compositionally distinct from mature milk. Bovine colostrum has a long history of consumption by humans, and there have been a number of studies investigating its potential for applications in human nutrition and health. Extensive characterization of the constituent fractions has identified a wealth of potentially bioactive molecules, their potential for shaping neonatal development, and the potential for their application beyond the neonatal period. Proteins, fats, glycans, minerals, and vitamins are abundant in colostrum, and advances in dairy processing technologies have enabled the advancement of bovine colostrum from relative limitations of a fresh and unprocessed food to a variety of potential applications. In these forms, clinical studies have examined bovine colostrum as having the substantial potential to improve human health. This review discusses the macro-and micronutrient composition of colostrum as well as describing well-characterized bioactives found in bovine colostrum and their potential for human health. Current gaps in knowledge are also identified and future directions are considered in order to elevate the potential for bovine colostrum as a component of a healthy diet for a variety of relevant human populations.

Bovine Colostrum and Its Potential for Human Health and Nutrition.

Morus nigra L. (black mulberry-BM) is a promising nutraceutical fruit containing biologically active polyphenols like anthocyanins, proanthocyanidins, catechins, and stilbenes, with well-established anti-inflammatory, antidiabetic, anti-obesity, and anticancer biofunctions. However, these health-promoting properties in raw fruit are greatly masked due to the presence of soluble and insoluble carbohydrates in excess amounts restricting daily intake of the required dose to achieve targeted effects. In the current study, different protein sources (defatted whey and chickpea flours) were optimized through different conditions to capture polyphenols from BM juice while diminishing its glucose content. To optimize polyphenol-protein interactions, various pHs (3.7, 4.2, and 4.7), matrix concentrations (20, 50, and 80 g protein/L), and incubation times (5, 20, and 45 min) were tested. In the present work, optimized BM polyphenol enriched whey matrix inhibited pro-inflammatory mediators and promoted Nrf-2 dependent cytoprotective enzyme expressions in lipopolysaccharide (LPS) induced macrophages at low doses. In addition, whey proteins were also subjected to an enzymatic deglycosylation process by using recently identified EndoBI-1 enzyme for the specific cleavage of N-glycan core in all glycan types including high mannoses, hybrids as well as complex glycans found on defatted whey proteins. After this process, the polyphenol sorption capacity of deglycosylated whey proteins was found to be significantly higher (37%) than the capacity of non-treated normal whey protein under optimized conditions. In conclusion, deglycosylation of protein matrices could be a novel strategy for efficient sorption/concentration of polyphenols from fruits and vegetables, however, more detailed studies are needed to understand this effect.

Sercan Karav

Department of Molecular Biology and Genetics

Çanakkale Onsekiz Mart University

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Sercan Karav

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Molecular Biology and Genetics

Çanakkale Onsekiz Mart University

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Department of Molecular Biology and Genetics