Use of Wisteria floribunda agglutinin affinity chromatography in the structural analysis of the bovine lactoferrin N-linked glycosylation

Background

Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(α1-3)Gal(β1-4)GlcNAc (αGal), GalNAc(β1-4)GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with αGal, LacdiNAc, LacNAc [Gal(β1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes, and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.

Methods

For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-MS and HPLC-FD profiling, including sequential exoglycosidase treatments.

Results

This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) containing LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, αGal and sialic acid.

Conclusions

Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, αGal, and Neu5Gc epitopes, than was shown in previous studies.

General significance

Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics.     

Alterations of serum protein N-glycosylation in two mouse models of chronic liver disease are hepatocyte and not B cell driven

N-glycosylation of immunoglobulin G (IgG) has an important impact on the modification of the total serum N-glycome in chronic liver patients. Our aim was to determine the role and magnitude of the alterations in which hepatocytes and B cells are involved in two mouse models of chronic liver disease. Common bile duct ligation (CBDL) and subcutaneous injections with CCl(4) were induced in B cell-deficient and wild-type (WT) mice. IgG depletion was performed with beads covered with protein A/G and the depletions were evaluated by SDS-PAGE and Western blot analysis. N-glycan analysis was performed by improved DSA-FACE technology. Structural analysis of the mouse serum N-glycans was performed by exoglycosidase digests and MALDI-TOF mass spectrometry of permethylated glycans. The alterations seen in B cell-deficient mice closely resembled the alterations in WT mice, in both the CBDL and the CCl(4) models. N-glycan analysis of the IgG fraction in both mouse models revealed different changes compared with humans. Overall, the impact of IgG glycosylation on total serum glycosylation was marginal. Interestingly, the amount of fibrosis present in CBDL B cell-deficient mice was significantly increased compared with CBDL WT mice, whereas the opposite was true for the CCl(4) model as determined by Sirius red staining. However, this had no major effect on the alteration of N-glycosylation of serum proteins. Alterations of total serum N-glycome in mouse models of chronic liver disease are hepatocyte-driven. Undergalactosylation of IgG is not present in mouse models of chronic liver disease.     

Cloning,Monoclonal Antibody Production,and Bodily Distribution Pattern of a Bovine Lipocalin

A bovine lipocalin, previously identified as a putative odorant-binding protein in bovine colostrum (bcOBP), was cloned and expressed, and its monoclonal antibody was established. bcOBP was constantly secreted into milk on day of parturition until at least 10 d postpartum at a concentration of 181±39 μg/L. Besides milk, bcOBP occurred in the nasal mucus, saliva, amniotic fluid, vaginal discharge, and blood plasma. Despite its low concentration, the distribution pattern and the finding that bcOBP harbored a characteristic sequence motif, CxxxC, which is conserved among insect and mammal pheromone binding proteins, suggest that bcOBP functions as a pheromone carrier. The presence of bcOBP in the plasma at varied concentrations depending on the lactation period does not exclude the possibility that bcOBP is secreted into milk from the blood. Cross-reactivity of the monoclonal antibody indicated presence of proteins homologous to bcOBP in the colostrum of farm animals of Cetartiodactyla.     

Cloning, monoclonal antibody production, and bodily distribution pattern of a bovine lipocalin

A bovine lipocalin, previously identified as a putative odorant-binding protein in bovine colostrum (bcOBP), was cloned and expressed, and its monoclonal antibody was established. bcOBP was constantly secreted into milk on day of parturition until at least 10 d postpartum at a concentration of 181±39 μg/L. Besides milk, bcOBP occurred in the nasal mucus, saliva, amniotic fluid, vaginal discharge, and blood plasma. Despite its low concentration, the distribution pattern and the finding that bcOBP harbored a characteristic sequence motif, CxxxC, which is conserved among insect and mammal pheromone binding proteins, suggest that bcOBP functions as a pheromone carrier. The presence of bcOBP in the plasma at varied concentrations depending on the lactation period does not exclude the possibility that bcOBP is secreted into milk from the blood. Cross-reactivity of the monoclonal antibody indicated presence of proteins homologous to bcOBP in the colostrum of farm animals of Cetartiodactyla.     

Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.     

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.     

Development of a sandwich ELISA assay for measuring bovine soluble type II IL-1 receptor (IL1R2) concentration in serum and milk

The IL1R is composed of two kinds of molecule, type I (IL1R I) and type II (IL1R2). IL1R1 contributes to IL-1 signaling, whereas the IL1R2 has no signaling property and acts as a decoy for IL-1. In this study, we developed a bovine IL1R2-specific sandwich ELISA to examine the sIL1R2 concentration in serum and milk from dairy cows. The concentration of colostral IL-1beta was examined to estimate the correlation to sIL1R2. The results showed that the sIL1R2 concentration in sera and milk changes with the stages of lactation. The serum sIL1R2 concentrations were 5.56+/-0.69 ng/ml (colostrum), 3.14+/-0.72 ng/ml (the early stage of lactation) and 5.76+/-1.25 ng/ml (the late stage of lactation). The milk sIL1R2 concentrations were 1.83+/-0.47 ng/ml (colostrum), 0.73+/-0.22 ng/ml (the early stage of lactation) and 2.92+/-0.56 ng/ml (the late stage of lactation). The concentrations of IL1R2 in sera and milk were significantly higher at the late stage of lactation and colostrum than that of the early stage of lactation. The reduction rates of sIL1R2 levels from the colostrum to the early stage of lactation were 43.6% (serum) and 61% (whey). IL-1beta was detected in all the colostrum (995.9+/-346.6 ng/ml). Significant correlation was observed between the levels of colostral IL-1beta and IL1R2 (r=0.75).     

植物N-糖链检测技术研究进展

N-糖基化作为一种重要的蛋白质翻译后修饰,在胚胎发育、癌症发生发展及免疫防御等诸多复杂的生命活动中发挥着关键作用。近年来,基于质谱的N-糖链的检测及其定量研究在动物方面取得了显著进展,相比之下,植物N-糖基化及N-糖链检测的相关研究要远远滞后,这也是制约植物糖生物学研究发展的关键瓶颈问题之一。对蛋白质N-糖链的释放、定量策略、可视化检测及其在植物中的应用进展进行了归纳总结,以期为指导后续植物N-糖链及N-糖组的定性定量检测提供参考。     

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non–small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.     

Longitudinal study of the dietary selenium intake of exclusively breast-fed infants during early lactation in Korea and Japan.

213 samples of human breast milk were collected from 51 healthy Korean women. Selenium content of the samples was determined by atomic absorption spectrometry with hydride generation. The selenium content of Korean milk decreased with increase of days after birth: The arithmetic mean of selenium content was higher in colostrum (< 4 days) 34 micrograms/kg (SD +/- 11, n = 44) than in transitional milk 21 micrograms/kg (SD +/- 8, n = 78) or in mature milk (> 10 days) 13 micrograms/kg (SD +/- 6, n = 91). The daily dietary selenium intake of 0-1 month aged Korean infants fed on breast milk is estimated to be around 10 micrograms per day (3 micrograms/kg body weight) regardless of days postpartum, resulting from the calculation of our selenium data and daily milk intake during early lactation. The same result on selenium intake for Japanese newborns, as well as Korean infants, is also estimated to be around 10 micrograms per day (3 micrograms/kg body weight) regardless of days postpartum.     

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows has been studied in 10 high-yielding (milk production: 5000 l per lactation) Karan Fries crossbred (Holstein Friesian × Tharparkar) cows. Milk and blood samples were collected from all the experimental animals. Isolation of milk phagocytes (neutrophils and macrophages) and lymphocytes were done by density gradient centrifugation. In vitro phagocytic index of milk neutrophils and macrophages was performed by colorimetric NBT reductive assay. Mitogen-induced milk lymphocyte blastogenic response was estimated by colorimetric MTT (tetrazolium) assay. Total plasma cortisol and prolactin were estimated by enzyme immune assay. Highest value of plasma cortisol and prolactin was observed at calving which decreased significantly (p < 0.01) on 15th day postpartum for both prolactin and cortisol. Immune activity of milk leukocytes was highest on day 0 colostrum and decreased significantly (p < 0.01) on 7th day postpartum. A significant (p < 0.01) rise of plasma prolactin was observed around 135th and 225th days postpartum, whereas a peak level of plasma cortisol was observed at 105th, 180^th, and 270th days postpartum. Phagocytic index of milk neutrophils and macrophages remains almost in a steady state during mid-lactation period (between 100 and 200 days postpartum). A decline in increasing trend of milk phagocytic activity was observed during late lactation. Mitogen-induced milk lymphocyte blastogenic response was highest on day 0 colostrum which decreased significantly (p < 0.01) on 15th day postpartum. Con A-induced milk lymphocyte blastogenic response showed an increasing trend from 120th to 210th days postpartum. Upon correlation study, it showed that the plasma cortisol has a negative effect on milk leukocyte activity, while prolactin has a positive effect, though the effect is lactation stage specific.     

Characterisation of whey proteins of camel (Camelus dromedarius) milk and colostrum

Variation in the composition of whey proteins from camel (Camelus dromedarius) colostrum and milk was recorded over a 192 h period following parturition. Whey proteins were separated by cation-exchange fast protein liquid chromatography and identified by polyacrylamide gel electrophoresis. The main components of whey proteins in camel milk and colostrum were similar to that in bovine, except for the lack in β-lactoglobulin. Serum albumin was the major whey protein present in camel milk, with an average concentration of 10.8 g/l. Camel colostrum was rich in immunoglobulins G, which consist of IgG1, and the enzyme inhibitory antibodies IgG2 and IgG3. The concentration of these proteins decreased rapidly 48 h post partum. Lactophorin (proteose peptone-component 3) and basic whey protein were detected only within 48 h after parturition, reaching a level of 4.9 and 3.1 g/l at 192 h post partum, respectively. The maximum level of lactoferrin (2.3 g/l) was observed at 48 h after parturition. Camel milk and colostrum were shown to be rich in protective proteins, especially IgG2 and IgG3, which revealed to be a potential source of inhibitory antibodies.     

Leptin in bovine colostrum and milk.

We studied leptin content in bovine colostrum, milk and plasma during the first month of lactation, and investigated relationships between selected milk components and milk leptin in five multiparous dairy cows. Colostrum/milk yield and composition were measured on days 0, 10, 20, and 30 of lactation. Leptin was assayed using a multi-species leptin RIA kit. Leptin concentration was 56 % lower in mature milk (day 10) than colostrum (13.90 vs. 6.14 microg/l; p < 0.001), but remained steady over the twenty days afterwards. Daily secretion of leptin into mature milk was 28 % lower than into colostrum (173.2 microg/d vs. 220.0 microg/d; p = 0.09) notwithstanding an 80 % increase in production. Colostrum and milk leptin levels correlated with fat (0.90; p < 0.001) and choline phospholipid (0.76; p < 0.05). Plasma and milk leptin decreased during the first month, but remained higher in milk, and highest in colostrum. Thus, leptin is present in large quantities in colostrum, less so and more variably in untreated milk, and is likely to be decreased in skimmed milk. These findings have implications for the use of untreated milk and colostrum-based (functional) food products.     

Changes in element concentration and distribution in breast-milk fractions of a healthy lactating mother

Daily changes in components of breast milk with number of days of lactation after delivery were demonstrated by determining concentrations and distributions of several elements simultaneously. Concentrations of calcium, copper, magnesium, phosphorus, sulfur, and zinc were determined simultaneously by inductively coupled argon plasma-atomic-emission spectrometry (ICP) for whole milk and milk fractions (skimmed milk and whey) collected from 2 to 196 d postpartum from a healthy lactating mother. Calcium and phosphorus concentrations increased in transitional milk. With days postpartum, the other elements decreased from the highest concentrations in colostrum milk, the modes of decrease being characteristic for each element. Distributions of copper, iron, phosphorus, sulfur, and zinc in whey were determined on a gel-filtration column by HPLC with ICP detection (HPLC-ICP method). Distributions of the five elements and absorbance peaks at 254 and 280 nm changed dramatically day by day at the beginning (colostrum milk), resulting in constant distributions after 30 d (mature milk). These results suggest the important roles of daily changing constituents in breast milk, especially in colostrum milk, in the nutrition of the newborn. Several element peaks on a gelfiltration column were identified by comparison with standard samples.     

The serum-free growth of cultured cells in bovine colostrum and in milk obtained later in the lactation period

Seven established cell lines, including both epithelial cells and fibroblasts (MDCK, Vero, CV-1, NRK, 3T3, F2408, and NIL8) and four early passage cell strains (bovine articular chondrocytes, bovine smooth muscle cells, human foreskin fibroblasts, and rat embryo cells) were cultured in serum-free medium supplemented with milk obtained 1 day after birth (colostrum) or 80 days after birth (older milk). MDCK, Vero, CV-1, NRK, and 3T3 grew readily in colostrum and attained saturation densities ranging from 22% to 63% of that in serum. There was no growth of F2408, NIL8, or the early passage strains in bovine colostrum. None of the 11 cell cultures grew in older milk. The temporal dependence of growth in milk was examined in detail using MDCK cells. Growth equivalent to that in serum occurred in 3% colostrum and in 15% milk obtained 2 days after birth. Milk obtained 3 days and 10 days after birth was not effective as a growth supplement for MDCK cells at any concentration. Those cells, unable to grow in colostrum or in older milk, could be induced to grow if culture dishes were precoated with fibronectin. In addition to fibronectin, it was necessary in some cultures to supplement colostrum or older milk with insulin and/or transferrin in order to achieve growth. In the presence of fibronectin and appropriate factors, the final saturation density attained in colostrum or older milk ranged from 25% to 100% of that in serum. The fibronectin contents of bovine colostrum and milk were determined. The fibronectin level of colostrum was found to be approximately 5% of bovine serum. There was no detectable fibronectin in the 80-day-old milk.     

Selective changes in gangliosides of human milk during lactation: a molecular indicator for the period of lactation

Gangliosides of human milk from women at various periods of lactation were analyzed. GD3 in colostrum, particularly in the early period of lactation, was the major ganglioside, and the molar ratio of GM3 to GD3 was 0.2-0.3 in the milk at 2-6 days postpartum. In contrast, milk from women at 60-390 days postpartum contained GM3 as the major ganglioside and the molar ratio of GM3 to GD3 was more than 3. Milk at 8-40 days postpartum represented an intermediate stage in terms of the ratio of GM3 to GD3. The selective change in the molar ratio of gangliosides was observed as a phenomenon common to all human milk from different individuals at different periods of lactation, indicating that the periods of lactation can be defined on the basis of the ratio. Since glycolipids in human milk are preferentially localized in the milk fat globule membrane, which is derived from the plasma membrane of epithelial cells in the mammary gland, the changes in the ganglioside composition reported in this communication may reflect a qualitative change of the cells in the mammary gland.     

Site-specific N-glycosylation of chicken serum IgG

Avian serum immunoglobulin (IgG or IgY) is functionally equivalent to mammalian IgG but has one additional constant region domain (CH2) in its heavy (H) chain. In chicken IgG, each H-chain contains two potential N-glycosylation sites located on CH2 and CH3 domains. To clarify characteristics of N-glycosylation on avian IgG, we analyze N-glycans from chicken serum IgG by derivatization with 2-aminopyridine (PA) and identified by HPLC and MALDI-TOF-MS. There were two types of N-glycans: (1) high-mannose-type oligosaccharides (monoglucosylated 26.8%, others 10.5%) and (2) biantennary complex-type oligosaccharides (neutral, 29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis of total N-glycans. To investigate the site-specific localization of different N-glycans, chicken serum IgG was digested with papain and separated into Fab [containing variable regions (VH + VL) + CH1 + CL] and Fc (containing CH3 + CH4) fragments. Con A stained only Fc (CH3 + CH4) and RCA-I stained only Fab fractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 + CH4) fragments, and variable regions of Fab contains complex-type N-glycans. MS analysis of chicken IgG-glycopeptides revealed that chicken CH3 domain (structurally equivalent to mammalian CH2 domain) contained only high-mannose-type oligosaccharides, whereas chicken CH2 domain contained only complex-type N-glycans. The N-glycosylation pattern on avian IgG is more analogous to that in mammalian IgE than IgG, presumably reflecting the structural similarity to mammalian IgE.     

Trace elements in human milk: Correlation with blood levels, inter-element correlations and changes in concentration during the first month of lactation

Using inductively coupled plasma mass spectrometry (ICP-MS) based analytical procedures, the concentration of several trace elements (Mn, As, Pb, Co, Ni, Cu, Zn and Se) was determined in human milk samples collected from a group of healthy lactating Portuguese women (n=44), both on the 2nd day postpartum (i.e., colostrum; n=34) and at 1 month postpartum (i.e., mature milk; n=19). Blood samples (n=44), collected on the 2nd day after parturition, were also analyzed for the same trace elements. No major correlations were observed between the levels of the analyzed trace elements in blood and colostrum samples. All the studied elements, except for Co, Pb and Ni, showed a significant trend for a decrease in concentration in milk during the first month of lactation. This trend was more pronounced for Zn and Se, whose levels decreased to approximately 23% and 44% of their initial mean concentration, respectively. With the exception of Co (r=0.607) and Zn (r=0.487), no significant correlations were observed when comparing the levels of each trace element between samples of colostrum and mature milk. Several inter-element correlations were found within each type of milk sample. The most significant were: (i) Se vs Cu (r=0.828) and Se vs Co (r=0.605) in colostrum samples and (ii) Ni vs Pb (r=0.756), Ni vs Mn (r=0.743) and Se vs Co (r=0.714) in mature milk samples. An inverse correlation between Zn and Se was also found in both types of milk sample; however, it only reached statistical significance for mature milk (r=-0.624).     

Concentration of lactoferrin and transferrin throughout lactation in cow's colostrum and milk

The evolution of the concentration of lactoferrin and transferrin was studied in cow's colostrum and milk throughout lactation. The highest concentrations of both proteins were found in the first milking (0.83 mg/ml for lactoferrin and 1.07 mg/ml for transferrin), decreasing sharply during the first days of lactation (colostral period). Thereafter, the concentrations of these proteins decreased slowly, reaching their definitive values during the third week post-partum (0.09 mg/ml for lactoferrin and 0.02 mg/ml for transferrin). The ratio transferrin/albumin in colostrum (first milking), mature milk, milk from mastitic cows and serum was determined, and found to be four times greater in colostrum than in mature or mastitic milk, suggesting a specific transport of transferrin from blood to milk.