Variants of ß-casofensin, a bioactive milk peptide, differently modulate the intestinal barrier: In vivo and ex vivo studies in rats.

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.

Protective effects of ß-casofensin, a bioactive peptide from bovine ß-casein, against indomethacin-induced intestinal lesions in rats.

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.

The membrane-associated form of α(s1)-casein interacts with cholesterol-rich detergent-resistant microdomains.

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.

Sequential release of milk protein-derived bioactive peptides in the jejunum in healthy humans.

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.

A novel bioactive peptide from yoghurts modulates expression of the gel-forming MUC2 mucin as well as population of goblet cells and Paneth cells along the small intestine.

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.

AlphaS1-casein, which is essential for efficient ER-to-Golgi casein transport, is also present in a tightly membrane-associated form.

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.

Food processing increases casein resistance to simulated infant digestion.

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.

Comparison of electrospray and matrix-assisted laser desorption ionization on the same hybrid quadrupole time-of-flight tandem mass spectrometer: application to bidimensional liquid chromatography of proteins from bovine milk fraction.

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.

Identification in milk of a serum amyloid A peptide chemoattractant for B lymphoblasts.

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.

Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies: the case of casein micelle.

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.