S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions.

Dendritic cells (DCs) are antigen-presenting cells that play an essential role in mucosal tolerance. They regularly encounter beneficial intestinal bacteria, but the nature of these cellular contacts and the immune responses elicited by the bacteria are not entirely elucidated. Here, we examined the interactions of Lactobacillus acidophilus NCFM and its cell surface compounds with DCs. L. acidophilus NCFM attached to DCs and induced a concentration-dependent production of IL-10, and low IL-12p70. We further demonstrated that the bacterium binds to DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a DC- specific receptor. To identify the DC-SIGN ligand present on the bacterium, we took advantage of a generated array of L. acidophilus NCFM mutants. A knockout mutant of L. acidophilus NCFM lacking the surface (S) layer A protein (SlpA) was significantly reduced in binding to DC-SIGN. This mutant incurred a chromosomal inversion leading to dominant expression of a second S layer protein, SlpB. In the SlpB-dominant strain, the nature of the interaction of this bacterium with DCs changed dramatically. Higher concentrations of proinflammatory cytokines such as IL-12p70, TNFalpha, and IL-1beta were produced by DCs interacting with the SlpB-dominant strain compared with the parent NCFM strain. Unlike the SlpA-knockout mutant, T cells primed with L. acidophilus NCFM stimulated DCs produced more IL-4. The SlpA-DC-SIGN interaction was further confirmed as purified SlpA protein ligated directly to the DC-SIGN. In conclusion, the major S layer protein, SlpA, of L. acidophilus NCFM is the first probiotic bacterial DC-SIGN ligand identified that is functionally involved in the modulation of DCs and T cells functions.

Identification of phosphorylation sites of equine beta-casein isoforms.

Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro-column. This method turned out to be an efficient tool to separate the CPPs Arg(1)-Lys(34) and Glu(4)-Lys(34) from non-phosphorylated peptides. Purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) and each CPP was hydrolyzed by endoproteinase Glu-C. Finally, the digests were analyzed by RP-HPLC/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) and identified by nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) to locate the phosphorylated sites of the beta-casein isoforms 4P-7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser(9), Ser(23), Ser(24), and Ser(25). Addition of phosphate groups on Ser(18), Thr(12), and Ser(10) led to the formation of the isoforms 5P-7P, respectively. The results indicated that the in vivo phosphorylation of the equine beta-casein follows a sequential way and is not randomly performed.

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.

Susceptibility of phaseolin (Phaseolus vulgaris) subunits to trypsinolysis and influence of dietary level of raw phaseolin on protein digestion in the small intestine of rats.

The aim of the present work was (a) to investigate trypsinolysis of denatured purified T phaseolin (Phaseolus vulgaris) subunits by MS and (b) to test the effect of raw T phaseolin inclusion level in diets fed chronically to rats on digestion in the small intestine. The diets contained casein as the sole protein source, or casein substituted with 33, 67 and 100 % of purified T phaseolin. Rats were fed for 10 d and then euthanised. Digesta and tissues from the first and second halves of the small intestine were prepared for electrophoresis, immunoblotting and densitometry. alpha-Phaseolin subunit for the T phaseolin was more resistant to trypsinolysis than beta-phaseolin subunit. Nearly intact phaseolin subunits (molecular weight, MW 44-54 kDa) and partially digested phaseolin fragments (MW 17-19 and 20-24 kDa) were identified in small intestinal digesta. The concentration of intact phaseolin and of most undigested phaseolin fragments in digesta increased in the second half of the small intestine with increasing phaseolin intake (P < 0.05-0.01). The concentration of phaseolin fragments of a MW of 21-22.5 and 23-24.5 kDa in the mucosa increased linearly (P = 0.016-0.084) when the level of the T phaseolin was increased in the diet. In conclusion, the present work provides evidence that denatured T phaseolin subunits display different trypsinolysis patterns in vitro. Moreover, a high intake of raw T phaseolin impacts digestion in the small intestine of rats.

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood.

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.

Determination of exposed sulfhydryl groups in heated beta-lactoglobulin A using IAEDANS and mass spectrometry.

This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine beta-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In beta-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.

Study of caprine beta-casein using reversed-phase high-performance liquid chromatography and mass spectroscopy: identification of a new genetic variant.

The genotypes and the main phosphorylation levels of beta-casein of goat milk were studied using RP-HPLC/ESI-MS. A new variant of caprine beta-casein named E has been characterized using RP-HPLC/ESI-MS, MALDI-MS and NanoESI MS/MS methods. Its sequence differed from that of variant A in the mono amino acid substitution D47 --> Y47, which resulted in a 48 Da experimental mass difference between them. The calculated molecular mass of the new variant E 6 P was estimated as 23,869 Da. Its phosphorylation pattern was similar to that of variant A, the most abundant types being those with 5 and 6 P in similar quantities.

Proteomic analysis of hen egg white.

Hen egg white is an original biological fluid in which major proteins have been widely studied, unlike the minor components. In this study, two-dimensional electrophoresis associated with mass spectrometry enabled the separation of 69 protein spots and their matching with major proteins, which were already known, and with minor proteins. Sixteen proteins were identified, and among them, two had never been previously detected in hen egg white, i.e., Tenp, a protein with strong homology with a bacterial permeability-increasing protein family (BPI), and VMO-1, an outer layer vitelline membrane protein. Thirteen proteins present a very wide polymorphism (ovotransferrin, ovomucoid, clusterin, etc.), some of them up to nine isoforms (ovoinhibitor). Eleven functional protein families were identified (serpin, transferrin, protease inhibitors Kazal, glycosyl hydrolases, lipocalin, bactericidal permeability-increasing protein, clusterin, UPAR/CD59/Ly6/ snake neurotoxin, cysteine protease inhibitor, VMO-1, and folate receptor families). These various biological functions could be interesting for further valorizations. In addition, three spots remain unidentified, probably because these proteins are not yet indexed in the international protein databanks.

Improved absorption of caseinophosphopeptide-bound iron: role of alkaline phosphatase.

Hydrolysis of proteins could lessen their inhibiting effect on the poor absorption of cow's milk iron (Fe), which is responsible for the high incidence of Fe deficiency worldwide. When bound to Fe, caseinophosphopeptides (CPP) derived from milk proteins resist luminal digestion, enhance Fe solubility and could improve its bioavailability; brush border enzyme alkaline phosphatase activity could influence iron absorption by releasing free Fe; this study assessed its role in the absorption of CPP-bound Fe. Rat duodenal loops were perfused with Fe gluconate or Fe bound to the CPP of beta casein [beta-CN (1-25)], with or without the addition of an inhibitor of alkaline phosphatase, Na2WO4. The uptake of Fe-beta-CN (1-25) was greater than Fe gluconate. Na2WO4 enhanced the uptake of Fe-beta-CN (1-25) and not of Fe gluconate. So the release of free, insoluble Fe, by alkaline phosphatase seems to be prevented by providing Fe in the Fe-beta-CN (1-25) complex form. Its good disappearance rate makes beta-CN (1-25)-bound Fe a candidate for food fortification.

Identification and characterization of ovalbumin gene Y in hen egg white.

Ovalbumin gene Y has been known as a member of the ovalbumin gene family since 1982, when its encoding gene was sequenced. In the present study, ovalbumin gene Y has been demonstrated as a new minor protein of hen egg white. This protein has been isolated by isoelectrofocalization and two-dimensional polyacrylamide gel electrophoresis and has been characterized using peptide mass fingerprinting. The concentration ratio of ovalbumin gene Y:ovalbumin is about 13:100. Unlike ovalbumin, ovalbumin gene Y is not phosphorylated, but like ovalbumin, this protein is glycosylated. Ovalbumin gene Y exists as a mixture of three molecular species, which differ in their isoelectric points. The polymorphism of this protein cannot be explained by various glycosylation levels.

Spectroscopic characterization of heat-induced nonnative beta-lactoglobulin monomers.

Previous studies have shown that two altered monomeric species were formed in the early steps of thermal denaturation of bovine beta-lactoglobulin (beta-lg), the well-known Cys121-exposed intermediate (Mcys121), and a new, stable monomer with exposed nonnative Cys119 (Mcys119). In this study, circular dichroism and fluorescence spectroscopies were used to characterize the structural features of these molecules. The structural characteristics of MCys121 after heating and cooling cycles are similar to those of native beta-lg. In contrast, Mcys119 monomer exhibits some characteristics of the well-known molten-globule state. Combined with other published data, these results indicate that heating induces at least two molten globule-like states of beta-lg, a highly reactive Mcys121 that returns to native state after cooling, and a less-reactive Mcys119 that is trapped and stabilized in a molten globule-like state by nonnative disulfide bond.

Heat-induced covalent complex between casein micelles and beta-lactoglobulin from goat's milk: identification of an involved disulfide bond.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.

Quantitative proteomic analysis of bacterial enzymes released in cheese during ripening.

Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by micro-organisms in food in order to better understand their metabolism in situ. While the main objective is the systematic identification of proteins, the next step will be to bridge the gap between identification and quantification of these proteins. For that purpose, a new mass spectrometry-based approach was applied, using isobaric tagging reagent for quantitative proteomic analysis (iTRAQ), which are amine specific and yield labelled peptides identical in mass. Experimental Swiss-type cheeses were manufactured from microfiltered milk using Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 as lactic acid starters. At three ripening times (7, 20 and 69 days), cheese aqueous phases were extracted and enriched in bacterial proteins by fractionation. Each sample, standardised in protein amount prior to proteomic analyses, was: i) analysed by 2D-electrophoresis for qualitative analysis and ii) submitted to trypsinolysis, and labelled with specific iTRAQ tag, one per ripening time. The three labelled samples were mixed together and analysed by nano-LC coupled on-line with ESI-QTOF mass spectrometer. Thirty proteins, both from bacterial or bovine origin, were identified and efficiently quantified. The free bacterial proteins detected were enzymes from the central carbon metabolism as well as stress proteins. Depending on the protein considered, the quantity of these proteins in the cheese aqueous extract increased from 2.5 to 20 fold in concentration from day 7 to day 69 of ripening.

The (193-209) 17-residues peptide of bovine ß-casein is transported through Caco-2 monolayer.

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.

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.

Comparative resistance of food proteins to adult and infant in vitro digestion models.

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.

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.

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.

Lowering of brachial pulse pressure in 9379 hypertensives with type 2 diabetes and reduction of cardiovascular events.

OBJECTIVES: Pulse pressure (PP) is a major risk factor for cardiovascular (CV) events, mainly in diabetic hypertensives. The objectives of the study were to determine which clinical characteristics could predict the fall in PP and the reduction of CV events under treatment. Design and methods. Type 2 diabetic hypertensives (n = 9379) with PP>60 mmHg (mean age 64 years) were included in a cohort study. During the 9 months follow-up, the physician in charge was asked to reinforce treatment in order to lower PP, using preferentially a fixed low-dose perindopril/indapamide combination. RESULTS: After 9 months, PP had fallen by 9.1+/-0.2 mmHg (p<0.001). Multivariate analysis of the determinants of PP reduction showed a significant positive association with administration of fixed ACEI/diuretic combination (p<0.001) and a negative association with glycated hemoglobin (p<0.01). During the 9 months follow-up, 632 CV events occurred. In multivariate analysis, the administration of fixed perindopril/indapamide combination was associated with a lower incidence of CV events (OR = 0.64 [0.48-0.86], p<0.01), independently of CV risk factors. CONCLUSIONS: The reinforcement of therapeutic measures made possible the reduction of PP in type 2 diabetic hypertensives, under conditions of usual care. Administration of a fixed perindopril/indapamide combination therapy was associated with an independent reduction of CV events.

Phaseolin type and heat treatment influence the biochemistry of protein digestion in the rat intestine.

The study aimed to investigate the in vivo digestion of Phaseolus vulgaris phaseolin types differing in their subunit pattern composition. Diets contained either casein as the sole source of protein or a mixture (1:1) of casein and pure Sanilac (S), Tendergreen (T) or Inca (I) phaseolin either unheated or heated. Rats were fed for 11 d with the experimental diets. Their ileal content and mucosa were collected and prepared for electrophoresis, Western blotting, densitometry and MS. Differences in digestion among native phaseolin types were observed for intact phaseolin at molecular weights (MW) of 47-50.5 kDa and for an undigested fragment at MW of 19-21.5 kDa in ileal digesta. In both cases, the concentration of these protein bands was lower for I phaseolin than for S or T phaseolin (P < 0.05). In the mucosa, the concentration of a protein band at MW of 20.5-21.5 kDa was lower for S phaseolin as compared to T or I phaseolin (P < 0.001). The presence of phaseolin subunits and their fragments was confirmed by Western blotting. MS analysis revealed the presence of undigested alpha and beta subunit fragments from phaseolin and endogenous proteins (anionic trypsin I and pancreatic alpha-amylase) in ileal digesta. Thermal treatment improved digestion (P < 0.01), acting on both dietary and endogenous protein components. In conclusion, this study provides evidence for differences in intestinal digestion among phaseolin types, S phaseolin being more resistant and I phaseolin more susceptible. These differences were affected by the origin of the phaseolin subunit precursor. Heat treatment enhanced phaseolin digestion.