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1.
J Dairy Sci ; 100(5): 3360-3372, 2017 May.
Article in English | MEDLINE | ID: mdl-28259408

ABSTRACT

ß-Casofensin is a bioactive milk peptide that modulates the intestinal barrier, particularly through its action on goblet cells. ß-Casofensin corresponds to fragment (f) 94-123 of the bovine ß-casein (ß-CN) A2 variant. Fifteen genetic variants of bovine ß-CN (A1-3, B-G, H1-2, I-L) are known, of which the A2, A1, and B forms are the most common. These variants differ from each other by the substitution of one or more amino acids, some of which are localized in f94 to 123. The aim of our study was to compare the intestinal effects of ß-casofensin A2 and its 3 main variants: A1, A3, and B. For this purpose, a solution (0.1 µM; 10 µL/g of body weight, postnatal d 10-20) containing ß-casofensin A2, one of its variants (A1, A3, or B), or drinking water (control; CT) was administered to rat pups orally. After euthanasia (postnatal d 20), intestinal segments were collected for biochemical and histochemical analysis and also used to determine paracellular permeability to fluorescein isothiocyanate-labeled 4-kDa dextran in an Ussing chamber. We also studied the direct effects of ß-casofensin A2 and its A1 variant on the paracellular permeability of jejunum segments of adult rats. ß-Casofensin A2 and its B variant significantly increased the population of goblet cells compared with the CT, A1, and A3 groups. The mucin 2 mRNA level was significantly higher in the ß-casofensin A2 group than in the CT, A3, and B groups. Our results also revealed that the protein expression of zonula occludens-1 and occludin was reduced in the jejunum of rats in the A1, A3, and B groups compared with the CT group. However, the A1 variant was the only peptide to decrease jejunal permeability compared with the CT group. This variant, tested directly in the apical compartment of an Ussing chamber at a concentration of 0.1 nM, also reduced jejunal permeability. In conclusion, the substitution of a single amino acid alters the effect of ß-CN sequence f94 to 123 on goblet cells and on intestinal permeability. A genetic polymorphism of ß-CN can affect the biological activity of peptides derived from this protein. These data should be taken into account in the production of bioactive foods.


Subject(s)
Caseins/chemistry , Milk/chemistry , Animals , Cattle , Genetic Variation , Intestinal Mucosa/metabolism , Peptides , Rats
2.
Mol Nutr Food Res ; 60(4): 823-33, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26719048

ABSTRACT

SCOPE: ß-casofensin, also known as peptide ß-CN(94-123), is a milk bioactive peptide that modulates the intestinal barrier through its action on goblet cells. Here, we evaluated whether oral administration of ß-casofensin can prevent indomethacin-induced injury of the jejunum in rats. METHODS AND RESULTS: Rats received ß-casofensin (0.01-100 µM) or tap water by daily gavage (4 µL/g) for eight days, then two subcutaneous injections of indomethacin (10 mg/kg, days 9 and 10) and were euthanized on day 12. In vitro, we investigated the effects of ß-casofensin on the restitution of a wounded monolayer. Preventive administration of ß-casofensin (100 µM) reduced intestinal macroscopic and microscopic damage induced by indomethacin. ß-casofensin also prevented the depletion of goblet cells and increased myeloperoxidase activity, as well as tumor necrosis factor-ɑ (TNF-ɑ) expression and immunostaining of active caspase-3 in the jejunum of rats treated with indomethacin. In wound healing experiments, ß-casofensin promoted epithelial restitution with no effect on cell proliferation. This effect was inhibited by pre-incubation with an anti-CC chemokine receptor 6 (CCR6) neutralizing antibody. CONCLUSIONS: ß-casofensin exerts protective effects in indomethacin-induced enteritis through preservation of goblet cells and improvement in wound healing. ß-casofensin could therefore become vital in nutritional programs for the prevention of intestinal diseases.


Subject(s)
Caseins/chemistry , Caseins/pharmacology , Indomethacin/adverse effects , Intestines/drug effects , Peptide Fragments/pharmacology , Wound Healing/drug effects , Administration, Oral , Animals , Cattle , Enteritis/chemically induced , Enteritis/prevention & control , HT29 Cells/drug effects , Humans , Intestinal Mucosa/drug effects , Intestinal Mucosa/pathology , Intestines/pathology , Jejunal Diseases/chemically induced , Jejunal Diseases/prevention & control , Male , Peptide Fragments/administration & dosage , Peptide Fragments/chemistry , Protective Agents/pharmacology , Rats, Wistar
3.
J Dairy Res ; 82(1): 36-46, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25335546

ABSTRACT

We recently reported the identification of a peptide from yoghurts with promising potential for intestinal health: the sequence (94-123) of bovine ß-casein. This peptide, composed of 30 amino acid residues, maintains intestinal homoeostasis through production of the secreted mucin MUC2 and of the transmembrane-associated mucin MUC4. Our study aimed to search for the minimal sequence responsible for the biological activity of ß-CN(94-123) by using several strategies based on (i) known bioactive peptides encrypted in ß-CN(94-123), (ii) in silico prediction of peptides reactivity and (iii) digestion of ß-CN(94-123) by enzymes of intestinal brush border membranes. The revealed sequences were tested in vitro on human intestinal mucus-producing HT29-MTX cells. We demonstrated that ß-CN(108-113) (an ACE-inhibitory peptide) and ß-CN(114-119) (an opioid peptide named neocasomorphin-6) up-regulated MUC4 expression whereas levels of the secreted mucins MUC2 and MUC5AC remained unchanged. The digestion of ß-CN(94-123) by intestinal enzymes showed that the peptides ß-CN(94-108) and ß-CN(117-123) were present throughout 1·5 to 3 h of digestion, respectively. These two peptides raised MUC5AC expression while ß-CN(117-123) also induced a decrease in the level of MUC2 mRNA and protein. In addition, this inhibitory effect was reproduced in airway epithelial cells. In conclusion, ß-CN(94-123) is a multifunctional molecule but only the sequence of 30 amino acids has a stimulating effect on the production of MUC2, a crucial factor of intestinal protection.


Subject(s)
Caseins/pharmacology , Goblet Cells/metabolism , Intestines/cytology , Mucins/biosynthesis , Mucins/drug effects , Peptide Fragments/pharmacology , Amino Acid Sequence , Animals , Caseins/chemistry , Cattle , Gene Expression/drug effects , Goblet Cells/drug effects , HT29 Cells , Humans , Microvilli/enzymology , Molecular Sequence Data , Mucin 5AC/genetics , Mucin-2/biosynthesis , Mucin-2/genetics , Mucin-4/biosynthesis , Peptide Fragments/chemistry , Peptide Hydrolases/metabolism , RNA, Messenger/analysis , Swine , Yogurt/analysis
4.
PLoS One ; 9(12): e115903, 2014.
Article in English | MEDLINE | ID: mdl-25549363

ABSTRACT

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.


Subject(s)
Caseins/chemistry , Cholesterol/chemistry , Mammals/metabolism , Membrane Microdomains/chemistry , Animals , Biological Transport , Caseins/metabolism , Detergents/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Evolution, Molecular , Female , Lactation , Membrane Microdomains/drug effects , Micelles , Rats, Wistar , Species Specificity
5.
Br J Nutr ; 112(4): 557-64, 2014 Aug 28.
Article in English | MEDLINE | ID: mdl-24968280

ABSTRACT

Digestive kinetics are believed to modulate satiety through the modulation of nutrient delivery. We hypothesised that the duration of satiety could be extended by modulating the kinetics of dietary amino acid delivery in overweight subjects, using snacks containing casein and whey protein. In the present study, eighty-two subjects underwent a first satiety test where they received a control snack containing 60 g maltodextrin. For the next 5 d, the subjects consumed a liquid protein snack containing 30 g carbohydrates and 30 g proteins (casein, whey protein or an equal mix of the two; n 26-28 per group). The subjects then underwent a second satiety test after ingesting the protein snack. The time period elapsing between the snack and request for lunch, food intake at lunch and satiety scores were recorded. A subgroup of twenty-four subjects underwent a digestive and metabolic investigation after ingesting their protein snack. Gastric emptying times were 2·5, 4 and 6 h for whey protein, mix and casein, respectively, displaying different kinetics of appearance of dietary N in plasma but without affecting pancreatic and gastrointestinal hormones. Compared with the control snack, proteins extended the duration of satiety (+17 min, P= 0·02), with no difference between the protein groups. The satiating effect of proteins was greater in subjects who ate their lunch early after the snack (below the median value, i.e. 2 h) at the control test (+32 min, P= 0·001). Energy intake at lunch was not modulated by proteins. The satiating effect of proteins is efficient in overweight subjects, especially when the duration of satiety is short, but independently of their digestive and plasma amino acid kinetics.


Subject(s)
Appetite Regulation , Food, Formulated , Milk Proteins/therapeutic use , Overweight/diet therapy , Satiety Response , Snacks , Adult , Beverages , Body Mass Index , Caseins/metabolism , Caseins/therapeutic use , Dietary Carbohydrates/metabolism , Dietary Carbohydrates/therapeutic use , Digestion , Energy Intake , Female , Gastric Emptying , Humans , Lunch , Male , Milk Proteins/metabolism , Overweight/metabolism , Reproducibility of Results , Single-Blind Method , Whey Proteins , Young Adult
6.
Electrophoresis ; 35(11): 1560-70, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24519758

ABSTRACT

Numerous milk components, such as lactoferrin, are recognized as health-promoting compounds. A growing body of evidence suggests that glycans could mediate lactoferrin's bioactivity. Goat milk lactoferrin is a candidate for infant formula supplementation because of its high homology with its human counterpart. The aim of this study was to characterize the glycosylation pattern of goat milk lactoferrin. After the protein was isolated from milk by affinity chromatography, N-glycans were enzymatically released and a complete characterization of glycan composition was carried out by advanced MS. The glycosylation of goat milk lactoferrin was compared with that of human and bovine milk glycoproteins. Nano-LC-Chip-Q-TOF MS data identified 65 structures, including high mannose, hybrid, and complex N-glycans. Among the N-glycan compositions, 37% were sialylated and 34% were fucosylated. The results demonstrated the existence of similar glycans in human and goat milk but also identified novel glycans in goat milk that were not present in human milk. These data suggest that goat milk could be a source of bioactive compounds, including lactoferrin that could be used as functional ingredients for food products beneficial to human nutrition.


Subject(s)
Glycomics/methods , Lactoferrin/analysis , Milk/chemistry , Polysaccharides/analysis , Amino Acid Sequence , Animals , Carbohydrate Sequence , Cattle , Chromatography, High Pressure Liquid/methods , Goats , Humans , Molecular Sequence Data , Tandem Mass Spectrometry/methods
7.
Am J Clin Nutr ; 97(6): 1314-23, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23576048

ABSTRACT

BACKGROUND: The digestive hydrolysis of dietary proteins leads to the release of peptides in the intestinal tract, where they may exert a variety of functions, but their characterization and quantification are difficult. OBJECTIVES: We aimed to characterize and determine kinetics of the formation of peptides present in the jejunum of humans who ingested casein or whey proteins by using mass spectrometry and to look for and quantify bioactive peptides. DESIGN: Subjects were equipped with a double-lumen nasogastric tube that migrated to the proximal jejunum. A sample collection was performed for 6 h after the ingestion of 30 g (15)N-labeled casein (n = 7) or whey proteins (WPs; n = 6). Nitrogen flow rates were measured, and peptides were identified by using mass spectrometry. RESULTS: After casein ingestion, medium-size peptides (750-1050 kDa) were released during 6 h, whereas larger peptides (1050-1800 kDa) were released from WPs in the first 3 h. A total of 356 and 146 peptides were detected and sequenced in the jejunum after casein and WP ingestion, respectively. ß-casein was the most important precursor of peptides, including bioactive peptides with various activities. The amounts of ß-casomorphins (ß-casein 57-, 58-, 59-, and 60-66) and ß-casein 108-113 released on the postprandial window were sufficient to elicit the biological action of these peptides (ie, opioid and antihypertensive, respectively). CONCLUSIONS: Clear evidence is shown of the presence of bioactive peptides in the jejunum of healthy humans who ingested casein. Our findings raise the question about the physiologic conditions under which these peptides can express their bioactivity in humans. This trial was registered at clinicaltrials.gov as NCT00862329.


Subject(s)
Caseins/analysis , Caseins/metabolism , Milk Proteins/analysis , Milk Proteins/metabolism , Peptides/metabolism , Adolescent , Adult , Chromatography, Liquid , Diet , Dietary Proteins/administration & dosage , Digestion , Endorphins/analysis , Endorphins/metabolism , Female , Humans , Hydrolysis , Intestinal Mucosa/metabolism , Jejunum/metabolism , Kinetics , Male , Meals , Nitrogen/analysis , Nitrogen/metabolism , Single-Blind Method , Tandem Mass Spectrometry , Whey Proteins , Young Adult
8.
J Nutr Biochem ; 24(1): 213-21, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22901691

ABSTRACT

Several studies demonstrated that fermented milks may provide a large number of bioactive peptides into the gastrointestinal tract. We previously showed that beta-casomorphin-7, an opioid-like peptide produced from bovine ß-casein, strongly stimulates intestinal mucin production in ex vivo and in vitro models, suggesting the potential benefit of milk bioactive peptides on intestinal protection. In the present study, we tested the hypothesis that the total peptide pool (TPP) from a fermented milk (yoghurt) may act on human intestinal mucus-producing cells (HT29-MTX) to induce mucin expression. Our aim was then to identify the peptide(s) carrying the biological activity and to study its impact in vivo on factors involved in gut protection after oral administration to rat pups (once a day, 9 consecutive days). TPP stimulated MUC2 and MUC4 gene expression as well as mucin secretion in HT29-MTX cells. Among the four peptide fractions that were separated by preparative reversed-phase high-performance liquid chromatography, only the C2 fraction was able to mimic the in vitro effect of TPP. Interestingly, the sequence [94-123] of ß-casein, present only in C2 fraction, also regulated mucin production in HT29-MTX cells. Oral administration of this peptide to rat pups enhanced the number of goblet cells and Paneth cells along the small intestine. These effects were associated with a higher expression of intestinal mucins (Muc2 and Muc4) and of antibacterial factors (lysozyme, rdefa5). We conclude that the peptide ß-CN(94-123) present in yoghurts may maintain or restore intestinal homeostasis and could play an important role in protection against damaging agents of the intestinal lumen.


Subject(s)
Goblet Cells/drug effects , Intestine, Small/cytology , Intestine, Small/drug effects , Mucin-2/metabolism , Paneth Cells/drug effects , Peptides/pharmacology , Yogurt , Amino Acid Sequence , Animals , Caseins/pharmacology , Cell Line/drug effects , Female , Gene Expression Regulation/drug effects , Humans , Intestinal Mucosa/drug effects , Molecular Sequence Data , Mucin-2/genetics , Mucin-4/genetics , Mucin-4/metabolism , Mucins/metabolism , Peptides/isolation & purification , Rats , Rats, Wistar
9.
J Agric Food Chem ; 60(1): 214-9, 2012 Jan 11.
Article in English | MEDLINE | ID: mdl-22129243

ABSTRACT

The behavior of ß-lactoglobulin (ß-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that ß-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.


Subject(s)
Glucose/chemistry , Lactoglobulins/chemistry , Hot Temperature , Hydrogen-Ion Concentration
10.
J Agric Food Chem ; 59(15): 8375-84, 2011 Aug 10.
Article in English | MEDLINE | ID: mdl-21740045

ABSTRACT

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), ß, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, ß-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.


Subject(s)
Antibodies, Monoclonal/chemistry , Caseins/chemistry , Surface Plasmon Resonance/methods , Animals , Cattle , Hydrophobic and Hydrophilic Interactions , Kinetics , Micelles , Protein Binding , Protein Structure, Tertiary
11.
BMC Cell Biol ; 11: 65, 2010 Aug 12.
Article in English | MEDLINE | ID: mdl-20704729

ABSTRACT

BACKGROUND: Caseins, the main milk proteins, aggregate in the secretory pathway of mammary epithelial cells into large supramolecular structures, casein micelles. The role of individual caseins in this process and the mesostructure of the casein micelle are poorly known. RESULTS: In this study, we investigate primary steps of casein micelle formation in rough endoplasmic reticulum-derived vesicles prepared from rat or goat mammary tissues. The majority of both alphaS1- and beta-casein which are cysteine-containing casein was dimeric in the endoplasmic reticulum. Saponin permeabilisation of microsomal membranes in physico-chemical conditions believed to conserve casein interactions demonstrated that rat immature beta-casein is weakly aggregated in the endoplasmic reticulum. In striking contrast, a large proportion of immature alphaS1-casein was recovered in permeabilised microsomes when incubated in conservative conditions. Furthermore, a substantial amount of alphaS1-casein remained associated with microsomal or post-ER membranes after saponin permeabilisation in non-conservative conditions or carbonate extraction at pH11, all in the presence of DTT. Finally, we show that protein dimerisation via disulfide bond is involved in the interaction of alphaS1-casein with membranes. CONCLUSIONS: These experiments reveal for the first time the existence of a membrane-associated form of alphaS1-casein in the endoplasmic reticulum and in more distal compartments of the secretory pathway of mammary epithelial cells. Our data suggest that alphaS1-casein, which is required for efficient export of the other caseins from the endoplasmic reticulum, plays a key role in early steps of casein micelle biogenesis and casein transport in the secretory pathway.


Subject(s)
Caseins/metabolism , Endoplasmic Reticulum/metabolism , Epithelial Cells/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Animals , Cells, Cultured , Epithelial Cells/cytology , Female , Goats , Humans , Mammary Glands, Animal/cytology , Protein Multimerization , Protein Transport , Rats
12.
Mol Nutr Food Res ; 54(11): 1677-89, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20521278

ABSTRACT

The objective of this study was to determine whether processing could modify the resistance of casein (CN) to digestion in infants. A range of different dairy matrices was manufactured from raw milk in a pilot plant and subjected to in vitro digestion using an infant gut model. Digestion products were identified using MS and immunochemical techniques. Results obtained showed that CNs were able to resist digestion, particularly κ- and αs(2)-CN. Resistant areas were identified and corresponded to fragments hydrophobic at pH 3.0 (gastric conditions) and/or carrying post-translational modifications (phosphorylation and glycosylation). Milk processing led to differences in peptide patterns and heat treatment of milk tended to increase the number of peptides found in digested samples. This highlights the likely impact of milk processing on the allergenic potential of CNs.


Subject(s)
Caseins/metabolism , Digestion , Food Handling/methods , Protein Processing, Post-Translational , Amino Acid Sequence , Chromatography, High Pressure Liquid , Food Hypersensitivity/metabolism , Humans , Hydrolysis , Infant , Lactobacillus delbrueckii/metabolism , Molecular Sequence Data , Peptides/genetics , Streptococcus thermophilus/metabolism , Yogurt/analysis
13.
J Agric Food Chem ; 58(13): 7955-61, 2010 Jul 14.
Article in English | MEDLINE | ID: mdl-20518500

ABSTRACT

Caseinophosphopeptides (CPP) are food mineral-rich components that may resist intestinal enzyme hydrolysis. We wondered whether phosphorylation and/or mineral binding induces resistance of CPP to intestinal hydrolysis. We used intestinal brush-border membrane vesicles to digest different forms of the beta-casein (1-25) peptide: unphosphorylated and phosphorylated carrier of varied cations. The results showed that the activity of alkaline phosphatase seems not to be specific to either the phosphorylation degree or the phosphorylation sites whereas phosphorylations limited the action of peptidases. Studying the mechanism and the kinetics of hydrolysis of the different peptides allows understanding how some cations prevent more CPP from hydrolysis than others. The action of both exo- and endopeptidases was limited for the beta-CN (1-25) peptide bound to zinc or copper. Actually the peptide bound to copper was almost not hydrolyzed during the digestion, suggesting that coordination bond of copper to CPP inhibits the action of both phosphatase and peptidases.


Subject(s)
Alkaline Phosphatase/metabolism , Caseins/metabolism , Down-Regulation , Intestines/enzymology , Minerals/metabolism , Peptide Fragments/metabolism , Peptide Hydrolases/metabolism , Transport Vesicles/enzymology , Animals , Hydrolysis , Intestinal Mucosa/metabolism , Phosphorylation , Swine , Transport Vesicles/genetics
14.
Mol Nutr Food Res ; 54(10): 1428-35, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20397193

ABSTRACT

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. ß-casein (ß-CN, 193-209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the ß-CN (193-209) peptide across Caco-2 cell monolayer. In addition, we evaluated the possible routes of the ß-CN (193-209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the ß-CN (193-209) peptide resisted the action of brush-border membrane peptidases, and that it was transported through the Caco-2 cell monolayer. The main route involved in transepithelial transport of the ß-CN (193-209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. Our results demonstrated the transport of an intact long-chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.


Subject(s)
Immunologic Factors/metabolism , Intestinal Absorption , Peptide Fragments/metabolism , Animals , Caco-2 Cells , Caseins/analysis , Caseins/chemistry , Caseins/isolation & purification , Caseins/metabolism , Cattle , Chymosin/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Immunologic Factors/analysis , Immunologic Factors/chemistry , Immunologic Factors/isolation & purification , Membrane Transport Modulators/pharmacology , Membrane Transport Proteins/metabolism , Microvilli/metabolism , Osmolar Concentration , Peptide Fragments/analysis , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Transporter 1 , Protein Isoforms/metabolism , Symporters/antagonists & inhibitors , Tight Junctions/drug effects , Time Factors , Transcytosis/drug effects
15.
Mol Nutr Food Res ; 54(6): 767-80, 2010 Jun.
Article in English | MEDLINE | ID: mdl-19937605

ABSTRACT

IgE-mediated allergy to milk and egg is widespread in industrialised countries and mainly affects infants and young children. It may be connected to an incomplete digestion of dietary proteins causing an inappropriate immune response in the gut. In order to study this, a biochemical model of infant gastroduodenal digestion has been developed, which has reduced levels of protease (eightfold for pepsin and tenfold for trypsin and chymotrypsin), phosphatidylcholine and bile salts, compared with the adult model. This model has been used to study the behaviour of three characterised food-relevant proteins (bovine beta-lactoglobulin (beta-Lg), beta-casein (beta-CN) and hen's egg ovalbumin), all of which are relevant cows' milk and hens' egg allergens. Digestion products were characterised using electrophoresis, immunochemical techniques and MS. These showed that ovalbumin and beta-CN were digested more slowly using the infant model compared with the adult conditions. Resistant fragments of beta-CN were found in the infant model, which correspond to previously identified IgE epitopes. Surprisingly, beta-Lg was more extensively degraded in the infant model compared with the adult one. This difference was attributed to the tenfold reduction in phosphatidylcholine concentration in the infant model limiting the protective effect of this phospholipid on beta-Lg digestion.


Subject(s)
Caseins/metabolism , Digestion , Lactoglobulins/metabolism , Ovalbumin/metabolism , Adult , Antibodies, Monoclonal/immunology , Caseins/immunology , Chromatography, High Pressure Liquid , Humans , Infant , Lactoglobulins/immunology , Ovalbumin/immunology , Phosphatidylcholines/analysis , Tandem Mass Spectrometry
16.
J Chromatogr A ; 1216(12): 2424-32, 2009 Mar 20.
Article in English | MEDLINE | ID: mdl-19174304

ABSTRACT

Recently, two ionization sources, electrospray (ESI) and matrix-assisted laser desorption (MALDI) have been used in parallel to exploit their complementary nature and to increase proteome coverage. In this study, a method using bidimensional (2D) nanoLC coupled online with ESI quadrupole time-of-flight (Q-TOF) with the simultaneous collection of fractions for analyses by LC-MALDI Q-TOF-MS/MS was developed. A total of 39 bovine proteins were identified to a high degree of confidence. To help in differentiating peptide detection following ESI and MALDI with the same mass spectrometer, we compared physico-chemical characteristics of the peptides (molecular mass, charge and size) by principal component analysis (PCA) and analysis of variance on the results of PCA. More hydrophobic peptides with a wider mass coverage were identified when ESI was used, whereas more basic and smaller peptides were identified when MALDI was used. However, the generally accepted differentiation between ESI and MALDI according to the presence of basic amino acids residues Lys and Arg and the ratio Lys/Arg was not shown as significant in this study. Moreover, we pointed out the importance of the type of mass spectrometer used in complement to both ionization sources for achieving a global increase of proteome coverage.


Subject(s)
Chromatography, Liquid/methods , Mass Spectrometry/methods , Milk Proteins/isolation & purification , Analysis of Variance , Animals , Cattle , Chemical Phenomena , Equipment Design , Hydrophobic and Hydrophilic Interactions , Milk Proteins/chemistry , Peptides/analysis , Peptides/chemistry , Principal Component Analysis
17.
BMC Immunol ; 10: 4, 2009 Jan 23.
Article in English | MEDLINE | ID: mdl-19166592

ABSTRACT

BACKGROUND: Normal mammary gland contains an extravascular population of B lymphoblasts, precursors of the immunoglobulin plasma cells that play a key role in the passive protection of neonates by secreting immunoglobulins to colostrum and milk. We investigated the presence of chemoattractants in the milk by analysing the chemoattractant activity of various fractions of this secretion. Milk chemoattractants are potentially involved in the recruitment of lymphocytes from the maternal bloodstream in lactating mammary glands. RESULTS: The dilution-related lymphoid cell chemoattraction of whey was associated with a < 10 kDa ultrafiltrate. Active fractions were purified by reverse-phase high performance liquid chromatography. Two peptides of 2.7 kDa (DMREANYKNSDKYFHARGNYDAA) and 1 kDa (RPPGLPDKY) were identified as fragments of the SAA protein family, tentatively identified as SAA2. Only the 2.7 kDa synthetic peptide displayed chemotactic activity, at two different optimal concentrations. At the lower concentration (3.7 nM), it attracted B-cell lymphoblasts, whereas at the higher (3.7 microM), it attracted B lymphocytes. Then, the SAA mRNA expression was analysed and we observed more SAA transcripts during lactation than gestation. CONCLUSION: These data are consistent with the SAA23-45 fragment being involved in preplasma B-cell recruitment to the mammary gland and resultant benefit to the neonate.


Subject(s)
B-Lymphocyte Subsets/metabolism , B-Lymphocytes/metabolism , Milk/metabolism , Peptides/metabolism , Serum Amyloid A Protein/metabolism , Amino Acid Sequence , Animals , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Cell Line , Cell Movement/immunology , Chemical Fractionation , Female , Lactation/immunology , Mammary Glands, Animal/metabolism , Mass Spectrometry , Milk/chemistry , Milk Proteins/immunology , Milk Proteins/metabolism , Molecular Sequence Data , Peptides/chemical synthesis , Peptides/immunology , Serum Amyloid A Protein/chemistry , Serum Amyloid A Protein/immunology , Swine , Swine, Miniature , Whey Proteins
18.
Mol Immunol ; 46(6): 1058-66, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18992943

ABSTRACT

In milk, kappa-, beta-, alphas(1)- and alphas(2)-casein (CN) are associated into a supramolecular assembly, the micelle. In this work, CN micelles contained in fresh skim milk were used to produce over 100 monoclonal antibodies. The specificity of these probes was determined using libraries of synthetic peptides and peptides fractionated from tryptic hydrolysis of purified CNs. Although kappa-CN and alphas(2)-CN are minor proteins in the micelle (ratio 1:1:4:4 for kappa, alphas(2), alphas(1), beta) a proportionally high number of clones were produced towards these two proteins (32 for each), compared to 9 and 29 for alphas(1)-CN and beta-CN, respectively. Most of the beta-CN and kappa-CN epitopes were identified, while about 50% of alphas(1)-CN and alphas(2)-CN antibodies were suspected to react to conformational linear or discontinuous epitopes, since no peptide binding could be identified. Antibody binding to the phosphoserine rich regions of the three calcium sensitive CNs was weak or non-existing, suggesting them to be hidden in the micelle structure together with alphas(1)-CN. The C-terminal glycomacropeptide of kappa-CN and the C-terminal moiety of beta-CN were well exposed generating the majority of the antibodies specific for these two proteins. The two major antigenic sites of alphas(2) were alphas(2)-CN (f96-114) and (f16-35). Cross-reaction between alphas(2)-CN specific antibodies with alphas(1)-CN illustrated the tangled structure between the two proteins. Immuno-dominant epitopes identified in the present study totally differ from those known for the purified caseins suggesting they were specific for the micelle supramolecular structure.


Subject(s)
Antibodies, Monoclonal/immunology , Caseins/immunology , Epitopes/immunology , Milk/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Cattle , Cross Reactions , Epitope Mapping/methods , Female , Mice , Micelles , Molecular Sequence Data , Peptides/immunology , Protein Multimerization
19.
J Nutr ; 138(12): 2342-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19022955

ABSTRACT

Although the chemical and physical modifications to milk proteins induced by technological treatments have been characterized extensively, their nutritional consequences have rarely been assessed in humans. We measured the effect of 2 technological treatments on the postprandial utilization of milk nitrogen (N), pasteurization (PAST) and ultra high temperature (UHT), compared with microfiltration (MF), using a sensitive method based on the use of milk proteins intrinsically labeled with (15)N. Twenty-five subjects were studied after a 1-wk standardization of their diet. On the day of the investigation, they ingested a single test meal corresponding to 500 mL of either MF, PAST, or UHT defatted milk. Serum amino acid (AA) levels as well as the transfer of (15)N into serum protein and AA, body urea, and urinary urea were determined throughout the 8-h postprandial period. The kinetics of dietary N transfer to serum AA, proteins, and urea did not differ between the MF and PAST groups. The transfer of dietary N to serum AA and protein and to body urea was significantly higher in UHT than in either the PAST or MF group. Postprandial deamination losses from dietary AA represented 25.9 +/- 3.3% of ingested N in the UHT group, 18.5 +/- 3.0% in the MF group, and 18.6 +/- 3.7% in the PAST group (P < 0.0001). The higher anabolic use of dietary N in plasma proteins after UHT ingestion strongly suggests that these differences are due to modifications to digestive kinetics and the further metabolism of dietary proteins subsequent to this particular treatment of milk.


Subject(s)
Disinfection/methods , Food Preservation/methods , Milk Proteins/pharmacokinetics , Adult , Amino Acids/blood , Blood Glucose/metabolism , Blood Proteins/metabolism , Female , Hot Temperature , Humans , Male , Nitrogen Isotopes , Postprandial Period/physiology , Urea/blood , Urea/metabolism , Urea/urine , Young Adult
20.
J Agric Food Chem ; 56(17): 8166-73, 2008 Sep 10.
Article in English | MEDLINE | ID: mdl-18698795

ABSTRACT

Caseinomacropeptide (CMP) is a peptide obtained from kappa-casein hydrolysis by gastric proteinases and which exhibits various biological activities. The aim of this study was to analyze the intestinal processing of CMP at the brush border membrane (BBM) level. Intestinal BBM vesicles (BBMV) were used to digest glycosylated and unglycosylated CMP. Our results demonstrated that whatever was the glycosylated state of CMP, they were digested by BBMV intestinal enzymes, from macropeptides to free amino acids. The digestion of unglycosylated and glycosylated CMP throughout the action of exopeptidases was similar, but the activity of endopeptideases on glycosylated CMP was limited, certainly due to the attached O-glycosylations. Consequently, much more peptides were identified from the unglycosylated than from the glycosylated CMP. In addition, the glycosylation core as well as the number of the attached glycosylated chain modified the kinetic of digestion; the most heavily glycosylated forms being the slowest digested.


Subject(s)
Caseins/metabolism , Endopeptidases/metabolism , Exopeptidases/metabolism , Intestines/enzymology , Peptide Fragments/metabolism , Animals , Caseins/chemistry , Glycosylation , Kinetics , Male , Microvilli/ultrastructure , Peptide Fragments/chemistry , Swine
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