Proteose Peptone Component Research Articles

Comparative study on the biological activity of bovine and ovine PP3 and of their hydrolysates

This study investigated the bioactivity of bovine and ovine proteose-peptone (PPT) fractions, specifically the proteose-peptone component 3 (PP3) and its enzymatic hydrolysate. The antioxidant capacity of the above fractions and their activity against Cronobacter sakazakii and rotavirus were evaluated. Results revealed strong interspecies differences on their bioactivity. Thus, antioxidant activity of bovine PPT decreased with the fractionation but increased for ovine PPT, the latter being more active in all cases. Moreover, the antibacterial activity of hydrolyzed bovine PP3 showed high concentration-dependence reaching a maximum of 6.82 logarithmic cycle reduction at 4 mg mL−1, for 24 h of incubation. Conversely, neither intact nor hydrolyzed ovine PP3 were able to reduce the bacterial growth. Finally, both bovine and ovine PP3 neutralized rotavirus in a dose-response way, the former being more effective. Interestingly, the antiviral activity of bovine PP3 increased after hydrolysis, whereas the ovine counterpart lost almost all of its antirotaviral capacity.

Structural and Mechanismic Studies of Lactophoricin Analog, Novel Antibacterial Peptide.

Naturally derived antibacterial peptides exhibit excellent pharmacological action without the risk of resistance, suggesting a potential role as biologicals. Lactophoricin-I (LPcin-I), found in the proteose peptone component-3 (PP3; lactophorin) of bovine milk, is known to exhibit antibiotic activity against Gram-positive and Gram-negative bacteria. Accordingly, we derived a new antibacterial peptide and investigated its structure–function relationship. This study was initiated by designing antibacterial peptide analogs with better antibacterial activity, less cytotoxicity, and shorter amino acid sequences based on LPcin-I. The structural properties of antibacterial peptide analogs were investigated via spectroscopic analysis, and the antibacterial activity was confirmed by measurement of the minimal inhibitory concentration (MIC). The structure and mechanism of the antibacterial peptide analog in the cell membrane were also studied via solution-state nuclear magnetic resonance (NMR) and solid-state NMR spectroscopy. Through 15N one-dimensional and two-dimensional NMR experiments and 31P NMR experiments, we suggest the 3D morphology and antibacterial mechanism in the phospholipid bilayer of the LPcin analog. This study is expected to establish a system for the development of novel antibacterial peptides and to establish a theoretical basis for research into antibiotic substitutes.

Immuno-Detection of C3a, a C3 Complement Activated Product in Mastitis Milk, a Potential Diagnostic Marker.

The sub-clinical form of mastitis is difficult to detect and causes huge economic loss to the dairy industry. It has become a threat to public health at large, thus there is a need for definite diagnosis of the disease. Therefore, this study was undertaken to identify the novel diagnostic marker for the detection of the sub-clinical form of mastitis. Two-dimensional gel analysis of the whey protein fraction of normal and mastitis milk samples revealed the presence of proteose peptone component 3 precursor, Trypsin precursor, complement component-C3, Ig heavy chain precursors and a C-type lectin domain as differentially expressed protein during the early stage of mastitis. Of these proteins identified, complement component-C3 was tested for its diagnostic potential. Western blot analysis of the milk whey of sub-clinical mastitis cases (M+, M++ & M+++) identified the accumulation of C3a, an activated product of complement component-C3. Further, the hemolytic activity of the above milk whey samples positively correlated with the somatic cell count. As C3a is already reported as an anaphylotoxic agent, it chemo tactically attracts lymphocytes at the site of inflammation, the detection of which in the milk whey can be of diagnostic importance for sub-clinical mastitis.

Proteolytic activation of proteose peptone component 3 by release of a C-terminal peptide with antibacterial properties

The milk protein proteose peptone component 3 (PP3, also known as lactophorin) is a small phosphoglycoprotein, which is exclusively expressed in the lactating mammary gland. A 23-residue synthetic peptide (lactophoricin, Lpcin S), corresponding to the C-terminal amphipathic α-helix of PP3, has previously been shown to permeabilize membranes and display antibacterial activity. Lactophorin readily undergoes proteolytic cleavage in milk and during dairy processing, and it has been suggested that PP3-derived peptides are part of milk's endogenous defense system against bacteria. Here, we report that a 26-residue C-terminal peptide (Lpcin P) can be generated by trypsin proteolysis of PP3 and that structural and functional studies of Lpcin P indicate that the peptide has antibacterial properties. The Lpcin P showed α-helical structure in both anionic and organic solvents, and the amount of α-helical structure was increased in the presence of lipid vesicles. Oriented circular dichroism showed that Lpcin P oriented parallel to the membrane surface. However, the peptide permeabilized calcein-containing vesicles efficiently. Lpcin P displayed antibacterial activity against Streptococcus thermophilus, but not against Staphylococcus aureus and Escherichia coli. The PP3 full-length protein did not display the same properties, which could indicate that PP3 functions as a precursor protein that upon proteolysis, releases a bioactive antibacterial peptide.

PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation

The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the α-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-β-d-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule.

Different proteomic profiles of sweet whey and rennet casein obtained after preparation from raw versus heat-treated skimmed milk

Proteomic profiles of sweet whey and rennet casein fractions prepared from raw or pasteurised (72 °C for 15 s), skimmed milk were studied by proteomics coupled with the detection of protein spots by MALDI TOF mass spectrometry. Proteins were analysed by a modification of the traditional, two-dimensional gel electrophoresis (2-DGE) method under non-reducing running conditions, which potentially permits the visualisation of disulphide-linked protein complexes formed in response to pasteurisation. Separated proteins were stained with Coomassie blue. The relative spot volumes obtained after 2-DGE of fractions from raw or pasteurised milk were compared. A number of different spots in the rennet casein and sweet whey fractions were found to vary in response to pasteurisation. Some of these represented higher molecular mass complexes that increased in the chymosin-precipitated casein fraction, and they were identified by mass spectrometry to contain αS1-casein. Certain fragments of αS1-casein, probably generated as a result of chymosin cleavage, increased in the whey after pasteurisation. The whey content of proteose peptone component 3 (PP3) or lactophorin decreased after pasteurisation, which could indicate increased association of PP3 with for example the milk fat globule membrane after pasteurisation. This shows that gel-based proteome analysis can be used in the characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of pasteurisation.

Age gelation in UHT-processed reconstituted concentrated skim milk

Summary UHT reconstituted concentrated skim milks made from high-heat powder had considerably longer gelation times than those made from medium- or low-heat powders. Addition of hexametaphosphate to the concentrated milk before UHT processing markedly delayed the onset of gelation during storage. Sediment formation was greatest in the UHT concentrated skim milk made using high-heat powder followed by samples made using medium- and low-heat powders, respectively. The extent of proteolysis, as measured by 12% TCA-soluble amino groups, increased at a faster rate in the UHT milks stored at 40°C than in those stored at 22°C but decreased with increasing heat treatment of the milk prior to powder manufacture. The electrophoretic patterns of samples stored at 22°C clearly showed the breakdown of β-casein with a corresponding increase in slower moving bands, presumably γ-casein and proteose-peptone components. However, storage of samples at 40°C resulted in diffused ‘blurred’ protein patterns with some protein material not entering the resolving gel. At 22°C there was some evidence of proteolysis but no evidence of high molecular weight polymer formation, while at 40°C both proteolysis and high molecular weight polymer formation increased with storage time. It appeared that both physico-chemical and proteolytic processes play some part in the mechanism of gelation in UHT reconstituted concentrated skim milk.

Immunostimulation effects of proteose-peptone component 3 fragment on human hybridomas and peripheral blood lymphocytes

Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-gamma production of human PBL was also accelerated 6.0-fold by 50 microg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-alpha production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells.

β-CN-5P and β-CN-4P components of bovine milk proteose–peptone: Large scale preparation and influence on the growth of cariogenic microorganisms

Indigenous proteolytic activity in milk, mostly due to plasmin, gives rise to many casein-derived peptides that subsequently are found in the proteose–peptone fraction of milk where they comprise 10% or more of the total whey protein. Prominent amongst proteose–peptone components are β-CN-5P (β-casein residues, 1–105/107) and β-CN-4P (β-casein residues 1–28). Many peptides have potentially valuable functional or biological properties that differ from those of the parent proteins, and this paper describes simple, rapid and cost-effective preparation of these two milk peptide components in a high degree of purity, and in gramme quantities, for evaluation of such properties. The purification process was more efficient if β-casein was used as starting material. In this work, we prepared 46 g of β-casein from sodium caseinate in a simple rapid DEAE-cellulose ion-exchange chromatography stage. This was followed by in vitro hydrolysis with plasmin and precipitation and gel filtration steps to yield 4.8 g of highly purified β-CN-5P and 1.2 g of β-CN-4P. Utilising either unfractionated sodium caseinate, or milk itself, as starting material was satisfactory but gave less purified material containing other peptide impurities. Peptides similar to these proteose–peptone components have been implicated in the protective effects of milk and dairy products against dental caries in teeth. The mechanism(s) by which this protection occurs is unclear, but some antibiotics are peptides. However, we have found that, even at peptide concentrations as high as 0.5 mg/ml, neither β-CN-5P nor β-CN-4P had any effect on the in vitro growth of cariogenic Streptococcus mutans bacteria, ruling out a simple antibiotic mechanism.

In vitro digestion of novel milk protein ingredients for use in infant formulas: Research on biological functions

Rather than using lengthy clinical studies, in vitro digestion of milk proteins by human neonatal gastric juice at pH values representing those in early neonatal life was investigated. Human milk proteins resisting digestion were immunoglobulins, secretory IgA, α S1-casein, secretory component, osteopontin and MUC-1. Bovine milk proteins resisting digestion at pH 4.0 were immunoglobulins, BSA, caseins, lactadherin, osteopontin, EPV20 and proteose peptone component 3 (PP3). Some proteins were rapidly digested e.g. CGMP. In vitro digestion provides rapid information regarding the susceptibility of proteins to gastric proteases and is a convenient alternative to clinical studies.

Separation and Characterization of Mares’ Milk αs1-, β-, κ-Caseins, γ-Casein-Like, and Proteose Peptone Component 5-Like Peptides

The equine αs1- and β-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The αs1-, β-, and κ-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. κ-Casein was characterized after electrophoresis by glycoprotein-specific staining. To identify αs1-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified αs1-CN. These sequences, that could be estimated to correspond to 62% of the full protein, presented strong identities with regions of αs1-CN primary structures of other species. In particular, 51, 48, 43, and 40% identities were obtained with corresponding regions of sow, dromedary, cow, and human αs1-CN, respectively. On the other hand, trace amounts of equine γ-CN-like and proteose peptone component 5-like peptides were found in the whole CN. They were identified by microsequencing and corresponded to β-CN peptides generated by plasmin action on the whole CN. The equine αs1, β-, and κ-CN were separated by bidimensional electrophoresis in numerous isoelectric variants with apparent isoelectric point distributed between pH 4.4 to 6.3, 4.4 to 5.9, and 3.5 to 5.5, respectively. The β- and κ-CN displayed a more acidic character in the mare than in the cow.

Alternative splicing of lactophorin mRNA from lactating mammary gland of the camel (Camelus dromedarius).

The objective of this study was to determine the corrected structure of lactophorin, a major whey protein in camel milk. The protein had 60.4% amino acid sequence identity to a proteose peptone component 3 protein from bovine whey and 30.3% identity to the glycosylation-dependent cell adhesion molecule 1 in mice. The N-terminal heterogeneity of the protein was a result of alternative mRNA splicing. About 75% of the protein was expressed as a long variant A with 137 amino acid residues and a molecular mass of 15.7 kDa; about 25% was as a short variant B with 122 amino acid residues and a molecular mass of 13.8 kDa. Both proteins are probably threefold phosphorylated. In contrast to the related proteins, no glycosylation was found in camel lactophorin. Because of this difference, specific interaction with carbohydrate binding proteins, as reported for the murine protein, can be excluded, and a function of the protein other than cell recognition or rotaviral inhibition is proposed. The concentration of lactophorin in camel milk was found to be about three times higher than the concentration of the bovine homologue in bovine milk. Pronounced similarities existed between the primary and secondary structures of bovine and camel proteins. We speculated that camel lactophorin has a similar function to that of bovine protein in milk, which is supposed to be the prevention of fat globule aggregation and the inhibition of spontaneous lipolysis by lipoprotein lipase.

Protein composition of micellar casein obtained by cross-flow micro-filtration of skimmed milk

In this study of the products of cross-flow micro-filtration of unheated skim milk, it was found that some lactoferrin, serum albumin and immunoglobulins were retained with the casein fraction. An acid-soluble casein, identified as proteose peptone component 5, also remained associated with the micellar fraction during micro-filtration.

The Primary Structure of Caprine PP3: Amino Acid Sequence, Phosphorylation, and Glycosylation of Component PP3 from the Proteose-Peptone Fraction of Caprine Milk

Proteose-peptone component 3 is a phosphorylated glycoprotein that was isolated from the proteosepeptone fraction of caprine milk. By mass spectrometric analysis, amino acid sequencing, and polymerase chain reaction analysis, the primary structure has been determined and has been shown to contain 136 amino acids. Phosphorylations were identified at Ser30 and Ser41. A partial glycosylation was present at Thr16, and a N-linked glycosylation was present at Asn78. Galactosamine was the amino sugar detected at Thr16. Glucosamine and galactosamine were the amino sugars found in the carbohydrate group linked to Asn78. The caprine amino acid sequence exhibits 88% identity with the bovine proteose-peptone component 3 sequence. However, when compared with the bovine sequence, the caprine sequence contains an insertion of a serine residue at position 25.

The Localization and Multimeric Nature of Component PP3 in Bovine Milk: Purification and Characterization of PP3 from Caprine and Ovine Milks

The distribution of proteose-peptone component PP3 in bovine whey, milk fat globule membrane, and casein has been investigated with antibodies raised against highly purified PP3. Using Western blot analysis, we show that PP3 is present in the milk fat globule membrane and in whey but is absent in the casein fraction. The proposed multimeric structure of bovine PP3 was analyzed by mass spectrometry and gel filtration. Calibrated gel filtration of acidic whey showed that PP3 eluted at a volume corresponding to 190 kDa, indicating that PP3 exists as a multimeric aggregate in bovine milk. Western blot analysis with anti-bovine PP3 immunoglobulins was used to analyze caprine, ovine, and human milks, and immunoreactive proteins were detected in caprine and ovine milks. Finally, the immunoreactive proteins from caprine and ovine milks were purified and characterized as PP3 analogues by amino acid analysis and N-terminal sequence analysis.

The bovine PP3 gene is homologous to the murine GlyCAM 1 gene

Proteose peptone component 3 (PP3) is a protein synthesized in the bovine mammary gland. A genomic 4.5-kb clone has been sequenced comprising a 2.9-kb PP3 transcriptional unit plus 1 kb of 5′ and 0.6 kb of 3′ flanking sequence. Bovine retroposons of the short interspersed nuclear element sequence class (SINES) and one microsatellite were localized in the PP3 gene. PP3 is homologous with the murine glycosylation-dependent cell-adhesion molecule 1(GlyCAM 1) and this homology also extends to the exon/intron organization of the genes

Study of Mechanism of Lipolysis Inhibition by Bovine Milk Proteose-Peptone Component 3

Milk component 3 was an inhibitor of lipoprotein lipase activity responsible for spontaneous lipolysis occurring in milk stored at 4°C. Experiments using a pH-stat apparatus and emulsified tributyrin showed that component 3 inhibited porcine pancreatic lipase. The lipolytic activity was fully restored by addition of sodium taurodeoxycholate and colipase to the emulsion containing component 3. Inhibition did not seem to be the result of a direct interaction between component 3 and the enzyme. Component 3 had a strong adsorption power superior to that of pancreatic lipase, as shown by tensiometric measurements at an n-tetradecane-water interface. Lipase inhibition by component 3 could be the consequence of a rapid diffusion and preferential adsorption of component 3 at the oil-water interface provoking an important decrease of interfacial tension and avoiding the adsorption of lipase.

Inhibiting and Activating Effects of Skim Milks and Proteose-Peptone Fractions on Spontaneous Lipolysis and Purified Lipoprotein Lipase Activity in Bovine Milk

Effect of heated skim milks on purified milk lipoprotein lipase activity against intralipid was correlated (.75) with their ability to affect spontaneous lipolysis in whole milks. Purified hydrophobic fraction of proteose-peptone that contained principally component 3 inhibited purified lipoprotein lipase activity. In contrast, fractions poor in proteose-peptone component 3 activated lipoprotein lipase activity. Study of interactions between activating and inhibiting effects suggested that the net effect of the milk proteose-peptone fraction on lipoprotein lipase activity resulted mainly from its inhibitory component, rather than from its activating components.

Effects of Different Skim Milk Fractions on Activity of Cow Milk Purified Lipoprotein Lipase

Hydrolysis of a long-chain triglyceride substrate by purified cow milk lipoprotein lipase, in the absence of blood serum, was changed by adding heated skim milks. Preincubation of the substrate with small amounts of several skim milks increased (+ 50 to 300%) lipoprotein lipase activity. By contrast, greater amounts of skim milk increased further or decreased lipoprotein lipase activity (+ 300 to −90%). The inhibitory effect was due to substrate surface protection.Results obtained after removal of casein (by isoelectric precipitation or by ultracentrifugation) and of whey proteins (by heat treatment) showed that the effect of skim milk was due to the proteose-peptone fraction. Fractionation of the proteose-peptone fraction into component 5-enriched fraction and component 3-enriched fractions suggested that the activating effect was due partially to proteose-peptone component 5, and the inhibitory effect resulted from component 3. These effects were over-come by blood serum.

Nomenclature of Proteins of Cow's Milk: Fifth Revision

This report reviews changes the nomenclature of bovine milk proteins necessitated by recent advances of our knowledge. Identification of a number of milk proteins (αs1-, β-, and κ-caseins; α-lactalbumin and β-lactoglobulin) continues to be based upon their primary structures (amino acid sequences). Since our last report, αs2-casein and serum albumin can be added to the list of major milk proteins for primary structure is known. Changes recommended in the nomenclature of caseins are primarily a result of differences within this family of proteins brought about by posttranslational modification. For example, αs0-casein is identical to αs1-casein, and αs3-, αs4-, and αs6-caseins are identical to αs2-casein except for differences in degree of phosphorylation. Additionally, proteose-peptone components 5, 8-slow and 8-fast, and γ1-, γ2-, and γ3-caseins are N-terminal and C-terminal fragments, respectively, of β-casein formed during proteolysis by plasmin. Nomenclature of immunoglobulins remains consistent with guidelines for human proteins and is based largely upon crossreactivity with reference proteins.The minor whey protein lactollin is β2-microglobulin for which the sequence of amino acids is known. An operational definition for proteins associated with the milk fat globule membrane has been developed. Nomenclature initially suggested for these proteins was based upon their electrophoretic behavior under a given set of conditions.Because of increased interest in milk proteins of species other than bovine, the Committee suggests that these be identified as homologs of those already characterized in European, Bos taurus, and Indian, Bos indicus, cattle. Guidelines are given to aid in determining if homology exists. Provisional nomenclature is suggested for use in the interim until homology can be established.