Daniel Mollé

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, TNFα, and IL-1β 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.

Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

BackgroundDue to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.ResultsThe protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases.ConclusionThis study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale.

Identification of C-terminal peptides of bovine β-casein that enhance proliferation of rat lymphocytes

A casein polypeptidic fraction, obtained from a pepsin-chymosin digestion of caseins, showed a mitogenic effect on primed lymph node (LN) cells and unprimed spleen cells of rats. A biologically active C-terminal sequence of bovine beta-casein (residues 192-209) was characterized. The corresponding synthetic peptide had a stimulatory effect on primed LN cells but failed to enhance proliferation of spleen cells. We prepared two chymosin digests (PA and PB) of bovine beta-casein which contained, respectively, 80% and 95% of the sequence including residues 193-209. They induced a significant proliferative response in both LN and spleen cells. It is therefore possible that other active peptides in the PA preparation may be involved in mitogenic activity.

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.

Application of chromatography and mass spectrometry to the characterization of food proteins and derived peptides.

The following review describes the development of mass spectrometry off-line and on-line coupled with liquid chromatography to the analysis of food proteins. It includes the significant results recently obtained in the field of milk, egg and cereal proteins. This paper also outlines the research carried out in the area of food protein hydrolysates, which are important components in foodstuffs due to their functional properties. Liquid chromatography and mass spectrometry have been particularly used for the characterization of food peptides and especially in dairy products.

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.

Heterogeneity of the bovine κ-casein caseinomacropeptide, resolved by liquid chromatography on-line with electrospray ionization mass spectrometry

Abstract Microheterogeneity occurs in the population of caseinomacropeptides (residues 106–169 of κ-casein) due to variation in the extent and type of oligosaccharide linked to this phosphoglycopeptide. Although caseinomacropeptide A variant (CMPA) was poorly resolved using reversed-phase high-performance liquid chromatography (RP-HPLC) with spectrophotometric detection, it could be analysed with on-line electrospray-ionization mass spectrometry (ESI-MS). From the already established O-linked glycan chains at least fourteen glycosylated forms of CMPA were identified, besides the non-glycosylated and multiphosphorylated (1, 2 or 3 phosphate groups) peptides, giving a maximum number of eighteen known forms. Major subcomponents in CMPA are disialylated species. A maximum of three out of the five potential glycosylation sites were found to be substituted with carbohydrate chains in the most highly glycosylated forms, which may contain up to six N-acetylneuraminic acid residues per molecule. A minor form, diphosphorylated with one disialylated chain, was also detected. From these results, it was shown that the on-line coupling of HPLC with ESI-MS offers a very promising alternative for the analysis of complex mixtures.

Two-step chromatographic procedure for the purification of hen egg white ovomucin, lysozyme, ovotransferrin and ovalbumin and characterization of purified proteins.

An improved procedure is described involving gel permeation and anion-exchange chromatography for the purification of four major hen egg white proteins. The procedure involves a first-step purification of ovomucin and lysozyme by gel permeation on a Superose 6 Prep Grade column. In the second step, anion-exchange chromatography on Q Sepharose Fast Flow led to the isolation of ovotransferrin and ovalbumin from a gel permeation chromatographic peak. The purities were estimated as ca. 80, 100, 80 and 100% for ovomucin, lysozyme, ovotransferrin and ovalbumin, respectively. The purification yield was over 60% for each protein. Further characterization of purified lysozyme revealed that it was fully active and homogeneous in relation to the electrospray ionization mass spectrum. The electrospray ionization mass spectrum showed different ovotransferrin species. The amino acid composition of purified ovomucin was compared to those published previously.

Lactolation of β-lactoglobulin monitored by electrospray ionisation mass spectrometry

Direct monitoring of the Amadori product formation between lactose and β-lactoglobulin was performed by electrospray ionisation mass spectrometry. As expected, the glycation reaction was faster at low water activity than in aqueous system. Heterogeneous β-lactoglobulin glycoforms were observed, with different number of lactose bound: populations of β-lactoglobulin with 1 to 7 and 2 to 11 linked lactose were detected, respectively, after 10 and 22 h of reaction under 65% relative humidity and 50°C. Trypsinolysis of glycated proteins, followed by reverse-phase HPLC coupled to tandem mass spectrometry in the neutral loss scanning mode, indicated that all potential reactive amino groups, except Lys101, were involved in sugar binding. The resulting order of reactivity, as a function of reaction time, was as follows: Lys47,91>α-amino-group, Lys15,70,100>Lys60,69,75,77,83,135,138>Lys8,141.

Phosphopeptides interacting with colloidal calcium phosphate isolated by tryptic hydrolysis of bovine casein micelles

After extended tryptic hydrolysis of large bovine casein micelles, a mineral-rich peptide fraction was recovered by ultracentrifugation. Its mineral part contained 72% of the colloidal Ca and 49% of the colloidal P1 originally present in the native micelle. Colloidal nitrogenous components were also present, amounting to 27% of the original N content. They contained most of the phosphopeptides and 82% of the micellar phosphoseryl residues. These tryptic peptides were characterized by reversed-phase HPLC on-line electrospray ion source-mass spectrometry analysis. Among the peptides produced 14 phosphopeptides were identified: alpha s2-CN(1-24), alpha s2-CN(1-21), alpha s1-CN(43-79), alpha s1-CN(35-79)7P, alpha s1-CN(35-79)8P, alpha s1-CN(37-79), alpha s1-CN(104-119), alpha s1-CN(104-124), beta-CN(1-25), beta-CN(1-28), beta-CN(1-29), beta-CN(30-97), beta-CN(33-97) and beta-CN(29-97). The proportion of the phosphopeptides interacting with colloidal calcium phosphate was correlated with their relative content of phosphoserine residues, since phosphopeptides containing more than four phosphoserine residues were consistently present within this fraction. It also appeared that other types of peptides, some of them hydrophobic in nature, were also partly or completely present within the colloidal fraction, including alpha s1-CN(91-100), alpha s1-CN(152-193), alpha s1-CN(23-34), alpha s1-CN(125-193), alpha s1-CN(125-199), beta-CN(177-209). beta-CN(184-209), beta-CN(114-169) and beta-CN(108-169). Their possible involvement in the micellar backbone is discussed.