Simultaneous presence of PrtH and PrtH2 proteinases in Lactobacillus helveticus Strains improves breakdown of the pure alphas1-casein.

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α(s1)- and ß-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure ß-casein was observed for all strains, the hydrolysis kinetics of α(s1)-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α(s1)-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

Inhibitory effect of αS1- and αS2-casein hydrolysates on angiotensin I-converting enzyme in human endothelial cells in vitro, rat aortic tissue ex vivo, and renovascular hypertensive rats in vivo.

A great number of milk-derived peptides have been shown to exhibit angiotensin converting enzyme (ACE) inhibitory properties and thus potential utility in the regulation of blood pressure. The present work aimed to investigate the effects of 2 milk trypsin hydrolysates from alpha(S1)- and alpha(S2)-casein (CH1 and CH2, respectively) on ACE activity evaluated in human umbilical vein endothelial cells (HUVEC) in vitro, rat aortic tissues ex vivo, and renovascular hypertensive rat in vivo. Incubation of HUVEC and rat aortic tissues with CH1 or CH2 induced a concentration-dependent inhibition of hydrolysis of the ACE substrate hippuryl-histidyl-leucine (HHL), the hydrolysates being much less potent than perindopril (an ACE inhibitor). However, in contrast to perindopril, CH1 and CH2 failed to modify angiotensin I-induced aortic ring vasoconstriction. The HPLC profiles of rat plasma after intragastric administration were variable among individuals but none of the observed peaks corresponded to peptides comprising CH1 or CH2 or to fragments of these peptides. During 4 wk of cardiovascular monitoring, in hydrolysate-fed renovascular hypertensive rats, systolic blood pressure weakly decreased compared with the control group. However, the CH1-fed hypertensive rats exhibited a decrease of heart rate during the nocturnal period of activity. To conclude, our results show that CH1 and CH2 inhibited ACE activity in HUVEC and rat aortic tissue but failed to antagonize the aortic-constricting effects of the natural agonist angiotensin I. Moreover, we demonstrated that CH1, to a greater extent than CH2, can slightly affect cardiovascular parameters although the ingested bioactive peptides could not be detected in the blood.

Equine alpha S1-casein: characterization of alternative splicing isoforms and determination of phosphorylation levels.

alpha(S1)-Casein was isolated from Haflinger mare's milk by hydrophobic interaction chromatography and displayed great micro-heterogeneity by 2-dimensional electrophoresis, probably because of a variable degree of phosphorylation and alternative splicing events. The aim of the present work was to investigate the complexity of the mare's alpha(S1)-casein. The different isoforms present in milk were submitted to a double treatment of dephosphorylation, first by using alkaline phosphatase and then acid phosphatase to achieve complete dephosphorylation. The apoforms were then analyzed by electrospray ionization mass spectrometry. The results revealed the existence of a full-length protein and 7 variants resulting from posttranscriptional modifications; that is, exon skipping involving exon 7, exon 14, or both and use of a cryptic splice site encoding a glutamine residue. The determination of the different phosphorylation degrees of the native isoforms of alpha(S1)-casein was finally achieved by electrospray ionization mass spectrometry analysis after fractionation of the isoforms by ion-exchange chromatography. Thus, 36 different variants of equine alpha(S1)-casein were identified with several phosphate groups ranging from 2 to 6 or 8 depending on whether exon 7 was skipped.

Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon.

Because of variable degrees of phosphorylation, alternative splicing, and probable instability resulting from nonenzymatic deamidation, equine beta-casein presents a complex pattern by 2-dimensional electrophoresis that needs clarification. beta-Casein prepared from Haflinger mare's milk by hydrophobic interaction chromatography was fractionated by ion-exchange chromatography according to the degree of phosphorylation. Isoforms were identified by mass spectrometry; they corresponded to the full-length protein having 3 to 7 phosphate groups and to the splicing variant involving exon 5 and containing 4 to 7 phosphate groups. Investigations of nonenzymatic deamidation showed that beta-casein did not deamidate spontaneously in stored milk and during the different steps of chromatography, but deamidation could occur when 2-dimensional electrophoresis was performed, increasing the beta-casein pattern complexity. This phenomenon was strongly minimized when the first dimension step was carried out at 10 degrees C instead of at room temperature. Finally, spot attribution on 2-dimensional pattern of beta-casein was achieved by mixing each phosphorylation isoform in its native state with the whole beta-casein fraction.

GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a pleiotropic enzyme that is overexpressed in apoptosis and in several human chronic pathologies. Here, we report that the protein accumulates in mitochondria during apoptosis, and induces the pro-apoptotic mitochondrial membrane permeabilization, a decisive event of the intrinsic pathway of apoptosis. GAPDH was localized by immunogold labeling and identified by matrix-assisted laser desorption/ionization-time of flight and nano liquid chromatography mass spectroscopy/mass spectroscopy in the mitochondrion of various tissues and origins. In isolated mitochondria, GAPDH can be imported and interact with the voltage-dependent anion channel (VDAC1), but not the adenine nucleotide translocase (ANT). The protein mediates a cyclosporin A-inhibitable permeability transition, characterized by a loss of the inner transmembrane potential, matrix swelling, permeabilization of the inner mitochondrial membrane and the release of two pro-apoptotic proteins, cytochrome c and apoptosis-inducing factor (AIF). This novel function of GAPDH might have implications for the understanding of mitochondrial biology, oncogenesis and apoptosis.

Evidence of the glycation and denaturation of LTP1 during the malting and brewing process.

The influence of malting and brewing processes on the chemical and structural modifications occurring on LTP1 was investigated by mass spectrometry and circular dichroism. Proteins were first purified from malt, and samples were collected at various steps of beer processing performed on two barley cultivars. The levels of LTP1 found in malt were not significantly different from the amounts in barley seed. However, in malt, both LTP1b, a post-translational form of LTP1, and a third isoform named LTP1c were isolated. Moreover, both of these proteins were found to be heterogeneously glycated but still exhibited an alpha-helix structure. Both glycated LTP1 and LTP1b were recovered during mashing. It was also shown that glycated LTP1 was unfolded during heat treatment of wort boiling, which is in agreement with the denatured form previously isolated from beer.

Peptides identified during Emmental cheese ripening: origin and proteolytic systems involved.

To determine the proteolytic changes occurring during Emmental cheese ripening, peptides released in cheese aqueous phase were analyzed by reversed-phase HPLC and identified by tandem mass spectrometry sequencing, for which different strategies were illustrated by some examples. Among the 91 peptides identified, most of them arose from alpha(s1)- (51) and beta-caseins (28), and a few arose from alpha(s2)- (9) and kappa-caseins (1). An attempt was made to correlate the released peptides with the proteolytic systems potentially involved during Emmental cheese manufacture. Besides the well-known action of plasmin on beta- and alpha(s2)-caseins, and in the absence of residual fungal coagulant from Endothia parasitica, two other proteinases seem to be involved in the hydrolysis of alpha(s1)-casein in Emmental cheese: cathepsin D originated from milk and cell-envelope proteinase from thermophilic starters. Moreover, peptidases from starters were also active throughout ripening, presumably like those from nonstarter lactic acid bacteria, in contrast to those from propionic acid bacteria.

Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces.

When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.

Identification of a new form of lipid transfer protein (LTP1) in wheat seeds.

Recently, this laboratory has isolated from barley and beer extract an isoform of lipid transfer protein (LTP1), which was not fully sequenced (Jégou, S.; Douliez, J. P.; Mollé, D.; Boivin, P.; Marion, D. J. Agric. Food Chem. 2000, 48, 5023--5029). It was named LTP1b and exhibited a molecular weight 294 Da higher than that of the known LTP1. This paper reports the finding of an LTP1 isoform in wheat that also exhibits an excess of 294 Da compared to the native protein. Amino acid sequencing, reduction and alkylation, and mass spectrometry showed that this new LTP1b possesses the same N-terminal sequence as the native LTP1, suggesting that the difference resides in the binding of an adduct which has a molecular weight of 294 Da. The aim of the present paper is to highlight various biophysical techniques that afford the identification of such an isoform-like LTP1 and to correlate this finding with other isoforms of LTP1 that were isolated from other plants but not fully sequenced.

Influence of pH on the heat-induced proteolysis of casein molecules.

Proteolysis of sodium caseinate solution (24.5 g/l) induced by heat treatment at 120 degrees C at different pH values was studied by measuring nitrogen content and relative fluorescence intensity in the 4% trichloroacetic acid filtrates. The low molar mass pepticles corresponding to the soluble nitrogen were identified using liquid chromatography/tandem mass spectrometry. Increase in proteolysis, deduced from the increase in soluble nitrogen content, was observed with heating time (10, 20 and 30 min) and pH (6.0, 7.0, 8.0 and 9.0). The fluorescence measurements showed that the release of peptides containing tryptophan was minimal at pH approximately 7.0. In parallel, eighteen low molar mass peptides were characterized, of which four came from kappa-casein, nine from beta-casein and five from alphas1-casein. Peptides were preferentially released under alkaline conditions.

Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2.

The 7-kDa lipid transfer proteins, LTP2s, share some amino-acid sequence similarities with the 9-kDa isoforms, LTP1s. Both proteins display an identical cysteine motif and, in this regard, LTP2s have been classified as lipid transfer proteins. However, in contrast with LTP1s, no data are available on their structure, cysteine pairings, lipid transfer and lipid binding properties. We reported on the isolation of two isoforms of 7-kDa lipid transfer protein, LTP2, from wheat seeds and showed for the first time that they indeed display lipid transfer activity. Trypsin and chymotrypsin digestions of the native LTP2 afforded the sequence of both isoforms and assignment of disulfide bonds. The cysteine pairings, Cys10--Cys24, Cys25--Cys60, Cys2--Cys34, Cys36--Cys67, revealed a mismatch at the Cys34-X-Cys36 motif of LTP2 compared to LTP1. Moreover, the secondary structure as determined by circular dichroism suggested an identical proportion of alpha helices, beta sheets and random coils. By analogy with the structure of the LTP1, we discussed what structural changes are required to accommodate the LTP2 disulfide pattern.

Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.

The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 microM and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem. 264, 562-568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two omega-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.

Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides.

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.

Purification and structural characterization of LTP1 polypeptides from beer.

We report on the purification of lipid transfer proteins (LTP) from barley seeds and beer with the aim of investigating the chemical modifications that occur during the brewing process. In seeds, the well-known LTP of 9 kDa (LTP1) has been found together with a second form named LTPb that displays comparable amino acid composition but was not fully sequenced. These two forms have been recovered in beer with marked chemical modifications including disulfide bond reduction and rearrangement and especially glycation by Maillard reaction. The glycation is heterogeneous with variable amounts of hexose units bound to LTPs. Circular dichroism shows that glycated LTP1 having all their disulfide bridges reduced are totally unfolded. These results provide a first basis for understanding how barley LTPs become foam-promoting agents during the malting and brewing process.

Identification of beta-oxidation and thioesterase activities in Staphylococcus carnosus 833 strain.

Staphylococcus carnosus 833, inoculated into sausage meat, increased the level of methyl ketones, which contributed to the cured aroma. These ketones can arise from incomplete beta-oxidation followed by two enzymatic activities: a thioesterase and a decarboxylase. In this study we identified the beta-oxidative pathway (through the measure of 3-hydroxyacyl-CoA dehydrogenase activity) and the thioesterase activity in extracts of S. carnosus cells grown in the presence of different methyl esters. The beta-oxidative system was induced by methyl esters and highest induction was found with a 12-carbon substrate. It was specific for medium chain length fatty acyl CoA substrates. Its maximal activity was observed at the end of stationary growth phase. HPLC analyses of acyl-CoA after incubation of cell extracts with palmitoyl-CoA showed that the beta-oxidation system released preferentially long chain hydroxyacyl-CoAs, enoyl-CoAs, and acyl-CoAs. The time-course of intermediate formation indicated a precursor product relationship indicative of a model of free intermediates which could be further deacylated by a thioesterase. The thioesterase activity was enhanced when S. carnosus was grown in the presence of methyl esters with at least 12 carbons and this enzyme was specific for short chain acyl-CoAs. The maximal activity was reached at the stationary growth phase.

New genetic variants identified in donkey's milk whey proteins.

Novel genetic variants for donkey milk lysozyme and beta-lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N49 --> D, Y52 --> S, and S61 --> N, from the previously published sequence. Three novel genetic variants for donkey beta-lactoglobulins were identified. One of them is a type beta-lactoglobulin I with three amino acid exchanges at E36 --> S, S97 --> T, and V150 --> I (beta-lactoglobulin I B, Mr 18,510 Da). The two others are type beta-lactoglobulins II with two amino acid exchanges at C110 --> P and M118--> T (beta-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D96 --> E, C110 --> P, and M118 -->T (beta-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).

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.

Metabolism of ricinoleic acid into gamma-decalactone: beta-oxidation and long chain acyl intermediates of ricinoleic acid in the genus Sporidiobolus sp.

In order to study differences in gamma-decalactone production in yeast, four species of Sporidiobolus were cultivated with 5% of methyl ricinoleate as the lactone substrate. In vivo studies showed different time courses of intermediates of ricinoleic acid breakdown between the four species. In vitro studies of the beta-oxidation system were conducted with crude cell extracts of Sporidiobolus spp. and with ricinoleyl-CoA (RCoA) as substrate. The beta-oxidation was detected by measuring acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase activities, and acetyl-CoA production. The time courses of the CoA esters resulting from RCoA breakdown by crude extract of Sporidiobolus spp. permit the proposal of different metabolic models in the yeast. These models explained the differences observed during in vivo studies.

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: immunochemical characterization.

Bovine beta-LG was modified by glycation with lactose in a powdered state or in an aqueous solution. An immunological characterization was performed using monoclonal antibodies with defined epitopes. The results showed that the structural changes were confined to the AB loop region of the molecules when glycation was conducted in a restricted water environment and had little consequences on the association state of glycated beta-LG. The protein conformation was much more extensively modified when glycation was performed in an aqueous solution at 60 degrees C, despite a lower glycation extent. These structural changes were located at the dimer interface (AB loop, GH loop, beta-strand I, and alpha-helix). These results allowed us to establish a relationship between the conformational changes and the modification of the association state of the glycated protein (formation of disulfide bridges between the free thiol groups of two monomers), previously described.