Isolation and characterisation of a Lactobacillus helveticus ITG LH1 peptidase-rich sub-proteome.

Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.

A peptide derived from bovine beta-casein modulates functional properties of bone marrow-derived macrophages from germfree and human flora-associated mice.

The purpose of this study was to evaluate the immunomodulatory activity of a peptide derived from bovine beta-casein (beta-CN), the beta-CN (193-209) peptide, on mouse macrophages that were obtained either from germfree (GF) or from human flora-associated (HF) mice. Macrophages were derived from bone marrow (BMDM) in the presence of recombinant macrophage colony-stimulating factor and exposed to the peptide or lipopolysaccharide (LPS). Membrane marker expression [F4/80, Mac-1, major histocompatibility complex (MHC) class II antigens] and phagocytic activity were assessed by flow cytometry. Production of tumor necrosis factor-alpha and interleukin (IL)-6 was measured by bioassays and production of IL-1alpha, IL-1beta and IL-12 by ELISA. The expression of cytokine mRNA was determined using semi-quantitative reverse transcription-polymerase chain reaction. The beta-CN (193-209) peptide up-regulated MHC class II antigen expression and phagocytic activity of BMDM from GF and HF mice. Its enhancing effect on phagocytosis was greater than that after LPS stimulation (P < 0.01). The peptide induced notable levels of cytokine mRNA in BMDM from GF and HF mice, but it was a significantly weaker inducer of cytokine secretion than LPS. Nevertheless, although flora implantation had no stimulatory influence on basal MHC class II and basal cytokine levels, cells from HF mice were more susceptible than those from GF mice to the peptide effects on these variables. These results indicate that the beta-CN (193-209) peptide could enhance antimicrobial activity of macrophages without proinflammatory effects.

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.

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.

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.

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: effect on association behavior and protein conformation.

The effect of glycation with lactose on the association behavior and conformational state of bovine beta-lactoglobulin (beta-LG) was studied, using size exclusion chromatography, polyacrylamide gel electrophoresis, proteolytic susceptibility, and binding of a fluorescent probe. Two modification treatments were used, i.e., aqueous solution glycation and dry-way glycation. The results showed that the latter treatment did not significantly alter the nativelike behavior of the protein while the former treatment led to important structural changes. These changes resulted in a specific denatured beta-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation into a high molecular weight species, via noncovalent interactions. The association behavior of glycated beta-LG is discussed with respect to the known multistep denaturation/aggregation process of nonmodified beta-LG.

Formation of stable covalent dimer explains the high solubility at pH 4.6 of lactose-beta-lactoglobulin conjugates heated near neutral pH.

The solubility of lactose-beta-lactoglobulin conjugates at pH 4.6, after heating near neutral pH in phosphate buffer/0.116 M NaCI, was investigated by size exclusion chromatography and compared with unmodified protein. Heated conjugates in the temperature range 65-90 degrees C showed greater solubility at pH 4.6. The proportion of soluble protein increased with the number of bound lactose molecules. Total solubility was obtained for conjugates with nine lactose residues attached per monomer of beta-lactoglobulin. The protective effect of bound sugar toward precipitation was associated with the formation of soluble disulfide cross-linked dimers, highly accessible to trypsin digestion. These results suggested that bound lactose, through steric hindrance and high surface hydrophilicity, prevents the thiol-disulfide exchange reactions of the polymerization-aggregation process of lactose-beta-lactoglobulin conjugates.

Soybean beta-conglycinin alpha subunit is phosphorylated on two distinct serines by protein kinase CK2 in vitro.

Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean beta-conglycinin alpha subunit. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP. Beta-conglycinin alpha subunit (68 kDa) presents seven consensus sequences, but only 0.5-1 mol P/mol alpha subunit was incorporated by CK2. [32P]Phosphorylated beta-conglycinin alpha subunit was cleaved either by cyanogen bromide or by trypsin. 32P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest. The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS). Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70-89, 116-126, and 116-127 sequences. Over the seven consensus sequences of beta-conglycinin alpha subunit, Ser 75 is the only one which was phosphorylated. Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position. Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites.

Impact of the lysine-188 and aspartic acid-189 inversion on activity of trypsin.

The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity. In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate. The kinetic parameters of the wild-type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates. Compared with trypsin, the mutant K188D/D189K exhibits a 1.5- to 6-fold increase in the Km for the substrates containing arginine and lysine, respectively. This mutant shows a approximately 30-fold decrease of its k(cat) and its second-order rate constant k(cat)/Km decreases approximately 40- and 150-fold for substrates containing arginine and lysine, respectively. Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine.

Engineering of trypsin and its impact on beta-casein processing.

Tryptic processing of beta-casein yields several important nutraceutic and nutritious peptides. However, a final product peptide (1-105) stops the processing, inhibiting the enzyme. In attempt to modulate catalytic properties of this protease, K188 was replaced with aromatic amino acid residues. This aimed amplification of local hydrophobic and electrostatic interactions at the substrate binding site. The catalytic properties of obtained mutants (K188F, K188Y, and K188W) were measured at pH 7, 8, 9, and 10 with synthetic substrates and beta-casein. Kinetic analysis revealed that all the mutants conserve the capacity to split peptide bonds involving arginyl and lysyl residues. However, depending on mutation, the optimum pH of activity changes. As shown only by proteolysis of a natural substrate, produced mutants cleaved near 30 new peptide bonds compared to wild-type trypsin, 8 of them involving asparagine and glutamine amino acids. Some of the new cleavage sites can be related to the nature of the amino acid residue introduced in position 188. Consequently, only the joint use of several methods (synthetic substrate, protein substrate, influence of pH) can help to define better the differences of catalytic properties of wild-type and mutant proteases. Modifications introduced by the mutations are at the origin of the alteration of the specificity of the studied enzymes which are cleaving beta-casein in many places, hydrolysing well the fragment 1-105. Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the new mutants could be used in hydrolysing resistant proteic structures.

Enzymatic phosphorylation of soy globulins by the protein kinase CK2. Determination of the phosphorylation sites of beta-conglycinin alpha subunit by mass spectrometry.

Beta-conglycinin alpha subunit has been phosphorylated using a cAMP-independent protein kinase (CK2) purified from the yeast Yarrowia lipolytica. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Asp/Glu. Only 0.5 to 1 mol P/mol alpha subunit was incorporated although seven consensus sequences are present. Phosphorylated beta-conglycinin alpha subunit (P-alpha) was digested by trypsin. The resulting peptides were analysed by RP-HPLC coupled to electrospray ionisation mass spectrometry (LC-ESMS). Two phosphopeptides were identified corresponding to 70-89 and 116-127 sequences with Ser 75 and Ser 117 phosphorylated respectively. Ser 75 is one of the predicted phosphorylation sites according to the consensus sequence criteria. Ser 117 is inside a very acidic peptide but does not belong to a previously described consensus sequence.

Purification and characterization of the main pepsinogen from the shark, Centroscymnus coelolepis.

The main pepsinogen from the mucosa of the shark, Centroscymnus coelolepis, has been purified and characterized. This zymogen, the most abundant protein in terms of quantity and activity (yield 72%), is a homogeneous monomer of molecular weight 42+/-0.7 kDa, as determined by electrophoresis. The aspartyl proteinase nature of this enzyme was confirmed by the considerable inhibition by pepstatin. Its specificity as to the oxidized B-chain of bovine insulin was determined using electrospray ionization mass spectrometry (ESI-MS) coupled with reversed phase high pressure liquid chromatography (RP-HPLC). The 15-16 Leu-Tyr bond was rapidly cleaved in this substrate, followed by the 24-25 Phe-Phe, 25-26 Phe-Tyr, and 11-12 Leu-Val bonds.

Induction of new physicochemical and functional properties by the glycosylation of whey proteins.

Nucleophilic primary amino groups of whey proteins (beta-lactoglobulin and alpha-lactalbumin) were modified with reducing sugars in mild heat conditions. After 49 hr of heating (60 degrees C) at pH 6.5, 20-30% of beta-lactoglobulin amino groups were substituted with aldohexoses (galactose, mannose, glucose) and lactose, whereas up to 70% and 90% of beta-lactoglobulin amino groups were modified with ribose and glyceraldehyde, respectively. Gel electrophoresis and reversed-phase HPLC coupled with electrospray ionization mass spectrometry of glycosylated proteins indicated that the substitution was random. Consequently, highly heterogeneous families of glycosylated proteins were generated. Proteins substituted with hexoses and lactose exhibited higher solubility and improved emulsifying properties as compared with nonglycosylated proteins, in the whole pH range studied. In contrast, proteins glycosylated with ribose and glyceraldehyde showed lower solubility close to their isoelectric points. Beta-lactoglobulin modified with ribose and glyceraldehyde displayed substantial differences in denaturation behavior as compared with native protein. When compared with beta-lactoglobulin, glycosylation of alpha-lactalbumin was quicker. There was no difference in glycosylation yields nor rates of alpha-lactalbumin in presence and absence of calcium.

Selective detection of lactolated peptides in hydrolysates by liquid chromatography/electrospray tandem mass spectrometry.

In food processing as well as in human nutrition and physiology, the increasing importance of the Maillard reaction has brought about the need for analytical means to detect and characterize the protein-bound Amadori products. In this paper, we describe a highly selective and sensitive method for detecting peptides glycated with lactose (lactolated peptides) from a complex hydrolysate using electrospray ionization tandem mass spectrometry. Protonated molecular ions of lactolated peptides gave a product-ion spectrum, with the dominant mode of decomposition including cleavage of the O-glycosidic bond followed by dehydration steps giving a characteristic neutral loss of 216 Da. Optimization of the ion marker [M + H]+ - 216, identified as a furylium ion, was investigated. It remained dominant regardless of the nature of the glycated peptides, the collision energy used, or the charge state of the parent ion. An approach for detecting lactolated peptides from protein digest was proposed during reverse-phase high-performance liquid chromatography (HPLC)/electrospray mass spectrometry and reverse-phase HPLC/tandem mass spectrometry using neutral loss scanning. This technique detected picomole amounts of lactolated peptides.

Ionic interactions in nanofiltration of beta casein peptides

Nanofiltration (NF) membrane technology shows interesting potentials for separating organic components on the basis of solute charge and size in the range of 300-1000 g mol-1. Separation properties of two inorganic NF membranes were studied with a set of 10 small peptides (molecular mass range: 300-900 g mol-1; 3 < pI < 10) contained in a well-characterized tryptic beta casein hydrolysate. Peptides transmission strongly depended on ionic interactions in the system. Physicochemical conditions such as ionic strength and especially pH were crucial to the separation, because the membrane and peptides showed amphoteric properties. Thus, the three categories of peptides (acid, basic, neutral) were separated according to their pI because of presumed concentration gradients of charged peptides at the membrane: positive for basic peptides and negative for acid peptides. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a membrane coion and a complete reversion of selectivity when it was a membrane counterion. Copyright 1998 John Wiley & Sons, Inc.

Characterization by ionization mass spectrometry of lactosyl beta-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site.

The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.

Nonenzymatic lactosylation of bovine beta-lactoglobulin under mild heat treatment leads to structural heterogeneity of the glycoforms.

Lactose reacts nonenzymatically with beta-lactoglobulin (beta-LG), the major whey protein, under mild heat treatment and the formation of the complex may be monitored by mass spectrometry. Using Reverse Phase HPLC coupled with Electrospray Ionization MS (ESI-MS) we have measured the global extent of glycosylation and examined the distribution of lactose among the beta-LG glycoforms. Identification of lactosylated sites by trypsinolysis and Tandem MS indicate that, although the glycosylation reaction was non-specific and potentially involved all the reactive sites (alpha- and epsilon-amino groups), beta-LG appeared to have at least two populations of lysine with the distinct ability to react with lactose. These results underline the structural heterogeneity of beta-LG glycoforms, with respect to the number of lactose linked per molecule and to the binding sites involved, which could affect the biological function of beta-LG.