Component PP3 Research Articles

Highly sensitive periodic acid/Schiff detection of bovine milk glycoproteins electrotransferred after nondenaturing electrophoresis, urea electrophoresis, and isoelectric focusing

Due to its lack of sensitivity, periodic acid/Schiff (PAS) staining of gels is not considered to be relevant for the detection of glycoproteins other than mucin-type glycoproteins after nondenaturing or urea polyacrylamide gel electrophoresis (PAGE). A simple and highly sensitive method was developed for the detection of bovine milk glycoproteins separated by nondenaturing PAGE, urea-PAGE, and isoelectric focusing. After electrophoresis, proteins were electrotransferred onto a polyvinylidene difluoride (PVDF) membrane and then glycoproteins were specifically stained by the Schiff's reagent after periodic acid oxidation. This method allowed the detection of the glycosylated forms of bovine α-lactalbumin with sufficient sensitivity after nondenaturing PAGE or isoelectric focusing of whey proteins. Combination of isoelectric focusing and PAS detection on a PVDF membrane provided a resolutive tool in the characterization of the different glycoforms of κ- casein and also of component PP3 of proteose peptones. Distinction between the non-glycosylated and glycoforms of κ-casein separated by urea-PAGE or isoelectric focusing was performed by com- paring Coomassie blue staining and PAS staining. Moreover, amino-terminal microsequencing of glycoproteins immobilized on the membrane could be performed after PAS staining. bovine milk protein / glycoprotein / electrophoresis / isoelectric focusing / electroblotting / Schiff's reagent

Evidence for membrane affinity of the C-terminal domain of bovine milk PP3 component

Component PP3 is a phosphoglycoprotein isolated from bovine milk with unknown biological function, which displays in its C-terminal region a basic amphipathic α-helix, a feature often involved in membrane association. According to that, the behaviour of PP3 and of a synthetic peptide from the C-terminal domain (residues 113–135) was investigated in lipid environment. Conductance measurements indicated that the peptide was able to associate and form channels in planar lipid bilayers composed of neutral or charged phospholipids. Electrostatic interactions seemed to promote voltage-dependant channel formation but this was not absolutely required since the pore-forming ability of the 113–135 C-terminal peptide was also detected with the zwitterionic lipid bilayer. Additionally, a spectroscopic study using circular dichroism argues that the peptide adopts an α-helical conformation in interaction with neutral or charged micelles. Thus, the conducting aggregates in bilayers might be composed of a bundle of peptides in helical conformation. Besides, similar conductance measurements performed with the whole PP3 protein did not induce any channel fluctuations. However, with the latter, an early breakdown of the bilayers occurred, a finding that can be tentatively explained by a massive incorporation of PP3. In the light of the present results, it could be inferred that PP3 membrane attachment may be achieved by oligomerization of the C-terminal amphipathic helical region.

The structure of the membrane-binding 38 C-terminal residues from bovine PP3 determined by liquid- and solid-state NMR spectroscopy.

The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.

Specific interaction between anionic phospholipids and milk bovine component PP3 and its 119–135 C-terminal fragment

The behaviour of component PP3, a bovine milk protein with emulsifying properties, was investigated at the air–water interface and in a lipidic environment using the monolayer technique. The amphipathic 119–135 C-terminal fragment of PP3 was also tested since we proposed, on the basis of structural analysis, that this region is probably responsible for the surface-active properties of the protein. This hypothesis was confirmed by the tensiometric measurements at the air–water interface in which the addition of the C-terminal peptide increased the surface pressure with a similar amplitude as the whole protein. Penetration measurements into lipidic monolayers indicated that the insertion of component PP3 and its C-terminal peptide is the highest with anionic phospholipids in a gel state. Moreover, the electrostatic attractions provided by anionic phospholipids are essential for the peptide interaction. We also showed by Fourier transform infrared spectra study, that the peptide displays a β-type conformational state in aqueous solution and in the presence of solvant or anionic phospholipid (DPPG). In contrast, the protein adopts in aqueous solution an α helical conformation which remains the dominant conformational state in the presence of DPPG although the apparition of β-structure is detected.

Component PP3 from bovine milk is a substrate for transglutaminase. Sequence location of putative crosslinking sites.

As the demands for food products of high quality increase, it will become more important to be able to modify the properties of food proteins. One possibility is to use enzymic modifications to improve functional properties and nutritional values. A highly efficient class of enzymic crosslinkers are transglutaminases (TG, EC 2.3.2.13), calcium-dependent enzymes that catalyse an acyl transfer reaction between protein-bound glutaminyl residues and primary amines (for review, see Aeschlimann & Paulsson, 1994). When protein-bound lysyl residues act as acyl acceptors, the reaction leads to the formation of intramolecular and/or intermolecular isopeptide bonds. Although a wide variety of amines such as putrescine, cadaverine or lysyl residues may act as amine donors in this reaction, only a limited number of glutamine residues in certain proteins will act as amine acceptors (Gorman & Folk, 1980).Ikura et al. (1981, 1985) reported that TG can be used to introduce methionine into casein and soyabean proteins and lysine into wheat gluten, thereby showing that TG can also be utilized to improve the nutritional value of food proteins. There have been a number of reports concerning the TG-mediated polymerization of food proteins such as α-lactalbumin and β-lactoglobulin (Aboumahmoud & Savello, 1990; Færgemand et al. 1997), soyabean proteins and casein components (Ikura et al. 1980a, b) and pea legumin (Larré et al. 1993). Recently, improved protein foaming capacity and stability have been demonstrated in TG-catalysed polymers of soyabean protein and whey protein isolate (Yildirim et al. 1996).Bovine PP3 (for review, see Girardet & Linden, 1996) is a phosphorylated glycoprotein isolated from the proteose peptone fraction of milk (Sørensen & Petersen, 1993a). The primary structure of PP3 has been determined (Sørensen & Petersen, 1993b) and comprises a polypeptide backbone of 135 amino acid residues containing five phosphorylated serines, two threonine-linked O-glycosylations, and one N-glycosylation. Immunological studies have shown that PP3 is present in the milk fat globule membrane, and that PP3 forms multimeric aggregates in bovine milk (Sørensen et al. 1997). Several functions have been suggested and investigated for bovine PP3 and fractions enriched with PP3, including emulsification (Shimizu et al. 1989), inhibition of lipolysis (Girardet et al. 1993) and mitogenesis (Mati et al. 1993).Previously, we have localized the potential TG-reactive glutamines in the four bovine caseins (Christensen et al. 1996) and in milk osteopontin (Sørensen et al. 1994). In the present study we have shown that component PP3 is a substrate for guinea-pig liver TG containing both reactive glutamine and lysine residues. In addition, we have localized the glutamine residues that act as amine acceptors.

Conformational Studies of a Synthetic Peptide from the Putative Lipid-binding Domain of Bovine Milk Component PP3

In bovine milk, a glycosylated phosphoprotein, component PP3, is known for its remarkable emulsifying properties and its capability to inhibit lipolytic activities. The determination of its primary structure is not sufficient to explain these properties. Secondary structure predictions of component PP3 and of its homologous proteins were achieved using a combination of multiple predictive methods. Based on this study, the f 119–135 region of component PP3 was proposed to be likely to adopt an amphipathic helical conformation, which is a lipid-binding motif. The conformation of the synthetic peptide corresponding to the C-terminal f 119–135 part of bovine component PP3 was analyzed by circular dichroism experiments using various media. The circular dichroism data indicated that the peptide was able to form an amphipathic α-helix structure in trifluoroethanol as well as in the presence of sodium dodecyl sulfate or acidic and neutral lipids, but not in water. Moreover, the conformation of this peptide is solvent dependent because it was found to adopt a β-sheet structure for low concentrations of sodium dodecyl sulfate or a low molar ratio of acidic lipid to peptide. Tensiometric measurements showed that the amphipathic C-terminal region of component PP3 is highly tensioactive and, thus, must be responsible for the particular behavior of the protein in emulsions.

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.

Structure of the O-glycopeptides isolated from bovine milk component PP3.

The heat-stable acid-soluble phosphoglycoprotein component PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptides from the ConA-bound fraction corresponding to the component PP3 were obtained by Pronase digestion and were separated by gel filtration into high and low-molecular-mass glycopeptides. In a previous work, we have investigated the structure of the N-glycans from the high-molecular-mass glycopeptides [Girardet et al. (1995) Eur J Biochem 234: 939-46]. Here, we describe the structure of the O-glycans from the low-molecular-mass glycopeptides. By combining methylation analysis, mass spectrometry, 400 MHz 1H-NMR spectroscopy and peptide sequence analysis, we show that the low-molecular-mass fraction contains several neutral glycopeptides. A mixture of the following three glycan structures linked to the Thr86 has been identified: GalNac alpha1-O-Thr, Gal(beta1-3)GalNAc alpha1-O-Thr and Gal(beta1-4)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc alpha1-O-Thr.

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.

Structure of glycopeptides isolated from bovine milk component PP3.

The heat-stable acid-soluble phosphoglycoprotein component PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptides were released by pronase digestion of the milk component PP3 and were subsequently separated by high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of the glycan and peptide moieties of eight N-glycopeptides have been established by combining methylation analysis, mass spectrometry, 400-MHz 1H-NMR spectroscopy, and peptide sequence analysis. All the analyzed fractions contained biantennary N-acetyllactosamine-type carbohydrate chains, some of them with a GalNAc(beta 1-4)GlcNAc or a NeuAc(alpha 2-6)GalNAc(beta 1-4)GlcNAc group. This particular sequence did or did not replace the Gal(beta 1-4)GlcNAc group usually found in most N-linked glycans. Moreover, the sialylated Gal and GalNAc residues were only found on the Man(alpha 1-3) antenna.

Proteolysis in Samples of Quarter Milk with Varying Somatic Cell Counts. 2. Component PP3 and β-Casein-lP (f29–105 and f29–107) of the Proteose-Peptone Fraction

The proteose-peptone fraction was studied to measure proteolysis in 86 samples of quarter milk from 31 cows with subclinical mastitis. The relative content of component PP3 decreased significantly in the total of proteose-peptone as plasmin activity increased, but the content of this component in milk was not correctly correlated with the plasmin activity (r = .52). Electrophoresis, amino acid composition, and sequence analysis showed that the component β-CN-1P (f29-105/7) of the proteose-peptone fraction was a terminal product of plasminlike activity and was located correctly electrophoretically. Correlation factors between the plasmin activity and content in milk of component β-CN-lP (f29-105/7) was very high (r = .87). This component could be used as indicator of the endogenous proteolysis in milk from cows with subclinical mastitis.

Phosphorylation, glycosylation and amino acid sequence of component PP3 from the proteose peptone fraction of bovine milk

Component PP3 is a phosphorylated glycoprotein with an apparent molecular mass of 28 kDa isolated from the proteose peptone fraction of bovine milk. The function of the protein is not known. The primary structure has been determined and shown to contain 135 amino acid residues (EMBL accession no. P80195). It was phosphorylated at Ser29, Ser34, Ser38, Ser40 and Ser46. Two O-linked carbohydrate groups were found at Thr16 and Thr86, while one N-linked carbohydrate group was present at Asn77. Thr16 was only approximately 50% glycosylated. The amino sugar detected by the amino acid analyser at Thr86 was mainly galactosamine but a small amount of glucosamine was also present. The amino sugars found in the carbohydrate group linked to Asn77 were both glucosamine and galactosamine. A fragment of PP3 has been isolated from milk and shown to correspond to residues 54-135. This fragment was probably generated by plasmin hydrolysing the Arg53-Ser54 bond.