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Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Metal-chelating peptides (MCP) are considered as indirect antioxidants due to their capacity to inhibit radical chain reaction and oxidation. Here, we propose a new proof of concept for the screening of MCPs present in protein hydrolysates for valorizing their antioxidant properties by using the emerging time-resolved molecular dynamics technology, switchSENSE. This method unveils possible interactions between MCPs and immobilized nickel ions using fluorescence and electro-switchable DNA chips. The switchSENSE method was first set up on synthetic peptides known for their metal-chelating properties. Then, it was applied to soy and tilapia viscera protein hydrolysates. Their Cu 2+ -chelation capacity was, in addition, determined by UV-visible spectrophotometry as a reference method. The switchSENSE method has displayed a high sensitivity to evidence the presence of MCPs in both hydrolysates. Hence, we demonstrate for the first time that this newly introduced technology is a convenient methodology to screen protein hydrolysates in order to determine the presence of MCPs before launching time-consuming separations.

The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response., (© 2020 Federation of European Biochemical Societies.)

The Omega class of glutathione transferases (GSTs) forms a distinct class within the cytosolic GST superfamily because most of them possess a catalytic cysteine residue. The human GST Omega 1 isoform was first characterized twenty years ago, but it took years of work to clarify the roles of the human isoforms. Concerning the kingdom of fungi, little is known about the cellular functions of Omega glutathione transferases (GSTOs), although they are widely represented in some of these organisms. In this study, we re-assess the phylogeny and the classification of GSTOs based on 240 genomes of mushroom-forming fungi (Agaricomycetes). We observe that the number of GSTOs is not only extended in the order of Polyporales but also in other orders such as Boletales. Our analysis leads to a new classification in which the fungal GSTOs are divided into two Types A and B. The catalytic residue of Type-A is either cysteine or serine, while that of Type-B is cysteine. The present study focuses on Trametes versicolor GSTO isoforms that possess a catalytic cysteine residue. Transcriptomic data show that Type-A GSTOs are constitutive enzymes while Type-B are inducible ones. The crystallographic analysis reveals substantial structural differences between the two types while they have similar biochemical profiles in the tested conditions. Additionally, these enzymes have the ability to bind antioxidant molecules such as wood polyphenols in two possible binding sites as observed from X-ray structures. The multiplication of GSTOs could allow fungal organisms to adapt more easily to new environments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chlordecone (CLD) is a chlorinated persistent organic pollutant (POP) whose presence despite the 1993 ban in agriculture areas has caused numerous public health concerns. CLD accumulates in the liver, and the CLD metabolite, chlordecol (CLD-OH) is found in bile, an important site of excretion for cholesterol transported to the liver via lipoproteins. Here, we studied the real-time molecular interaction between CLD and CLD-OH with human serum lipoproteins, LDL and HDL. While no interaction was detected between CLD and HDL, or between CLD-OH and LDL, relatively high specific affinities were observed between CLD and CLD-OH for LDL and HDL, respectively., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Sinorhizobium meliloti is a nitrogen-fixing bacterium forming symbiotic nodules with the legume Medicago truncatula. S. meliloti possesses two BolA-like proteins (BolA and YrbA), the function of which is unknown. In organisms where BolA proteins and monothiol glutaredoxins (Grxs) are present, they contribute to the regulation of iron homeostasis by bridging a [2Fe-2S] cluster into heterodimers. A role in the maturation of iron-sulfur (Fe-S) proteins is also attributed to both proteins. In the present study, we have performed a structure-function analysis of SmYrbA showing that it coordinates diverse divalent metal ions (Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) using His32 and His67 residues, that are also used for Fe-S cluster binding in BolA-Grx heterodimers. It also possesses the capacity to form heterodimers with the sole monothiol glutaredoxin (SmGrx2) present in this species. Using cellular approaches analyzing the metal tolerance of S. meliloti mutant strains inactivated in the yrbA and/or bolA genes, we provide evidence for a connection of YrbA with the regulation of iron homeostasis. The mild defects in M. truncatula nodulation reported for the yrbA bolA mutant as compared with the stronger defects in nodule development previously observed for a grx2 mutant suggest functions independent of SmGrx2. These results help in clarifying the physiological role of BolA-type proteins in bacteria., (© 2020 The Author(s).)

Signaling molecule receptors play a central role in quorum sensing and in the coordination onset of specialized metabolite biosynthesis in Streptomyces due to their dual function in signal detection and gene expression control through DNA-binding in the promoter region of their target genes. In Streptomyces ambofaciens the alp biosynthetic gene cluster includes the signaling molecule receptor AlpZ that negatively regulates through a complex regulatory cascade the expression of key genes involved in the kinamycin antibiotic production until its cognate ligand, a yet unidentified signaling molecule, prompts its release from target promoters. Here we use an original molecular dynamics method to evaluate the DNA-binding properties of AlpZ to its target DNA sequence and the impact the signaling molecule has on the interaction. It is the first time this approach is used to characterize a regulator from the γ-butyrolactone receptor family. The observed K D in the nanomolar range indicates that AlpZ-DNA constitute a particularly stable complex. The signaling molecule ably disturbs this binding while kinamycin has no effect on the activity of AlpZ. Regulator size was determined and found to be considerably large regarding protein sequence, indicating that AlpZ regulates gene expression by binding the DNA as a homodimer, and structural modeling comparison with closely related γ-butyrolactone receptors supports this conclusion., (Copyright © 2020 Vicente, Girardet, Hôtel and Aigle.)

In plants, tetrapyrrole biosynthesis occurs in chloroplasts, the reactions being catalysed by stromal and membrane-bound enzymes. The tetrapyrrole moiety is a backbone for chlorophylls and cofactors such as sirohaems, haems and phytochromobilins. Owing to this diversity, the potential cytotoxicity of some precursors and the associated synthesis costs, a tight control exists to adjust the demand and the fluxes for each molecule. After synthesis, haems and phytochromobilins are incorporated into proteins found in other subcellular compartments. However, there is only very limited information about the chaperones and membrane transporters involved in the trafficking of these molecules. After summarizing evidence indicating that glutathione transferases (GST) may be part of the transport and/or degradation processes of porphyrin derivatives, we provide experimental data indicating that tau glutathione transferases (GSTU) bind protoporphyrin IX and haem moieties and use structural modelling to identify possible residues responsible for their binding in the active site hydrophobic pocket. Finally, we discuss the possible roles associated with the binding, catalytic transformation (i.e. glutathione conjugation) and/or transport of tetrapyrroles by GSTUs, considering their subcellular localization and capacity to interact with ABC transporters. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Camelid α-lactalbumin is the only known protein that can undergo nonenzymatic deamidation on two Asn residues. This leads to the generation of a mixture of unusual isoAsp and d-Asp residues that may impact health. The effect of deamidation on camel α-lactalbumin instability was investigated. Circular dichroism showed that the altered protein acquired secondary structure resulting in an increase in α-helix content. In good agreement, the 3D structure of camel α-lactalbumin determined by X-ray crystallography, displayed a short additional α-helix probably induced by deamidation, compared to the human and bovine counterparts. This α-helix was located in the C-terminal region and included residues 101-106. Differential scanning calorimetry together with the susceptibility to thermolysin showed that the deamidation process reinforced the structural stability of the α-lactalbumin at high temperature and its resistance toward proteolysis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bile salts act as steroidal detergents in the gut, and could also interact with peptides and improve their bioavailability, although the mechanism is unclear. The occurrence of direct interaction between milk bioactive peptides, Ile-Asn-Tyr-Trp, Leu-Asp-Gln-Trp, and Leu-Gln-Lys-Trp, and different bile salts in the submicellar or micellar state was investigated by intrinsic fluorescence measurement and dynamic light scattering, above the critical micellar concentration, the latter being determined by isothermal titration calorimetry. The peptides form aggregates, spontaneously. In the presence of bile salts, some released peptide monomers were bound to the micellar surface. The lack of hydrogen bonding involving the C12OH group of the steroid skeleton, and the acidic function of some bile salts, might promote the interaction with the peptides, as could the lack of the C12OH group, rather than that of the C7OH group. At submicellar concentrations, sodium taurochenodeoxycholate and taurodeoxycholate readily interacted with the most hydrophobic peptide Ile-Asn-Tyr-Trp., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Glutathionyl-hydroquinone reductases (GHRs) belong to the recently characterized Xi-class of glutathione transferases (GSTXs) according to unique structural properties and are present in all but animal kingdoms. The GHR ScECM4 from the yeast Saccharomyces cerevisiae has been studied since 1997 when it was found to be potentially involved in cell-wall biosynthesis. Up to now and in spite of biological studies made on this enzyme, its physiological role remains challenging. The work here reports its crystallographic study. In addition to exhibiting the general GSTX structural features, ScECM4 shows extensions including a huge loop which contributes to the quaternary assembly. These structural extensions are probably specific to Saccharomycetaceae. Soaking of ScECM4 crystals with GS-menadione results in a structure where glutathione forms a mixed disulfide bond with the cysteine 46. Solution studies confirm that ScECM4 has reductase activity for GS-menadione in presence of glutathione. Moreover, the high resolution structures allowed us to propose new roles of conserved residues of the active site to assist the cysteine 46 during the catalytic act., Competing Interests: The authors have declared that no competing interests exist.

Dehydroascorbate reductases (DHARs), enzymes belonging to the GST superfamily, catalyse the GSH-dependent reduction of dehydroascorbate into ascorbate in plants. By maintaining a reduced ascorbate pool, they notably participate to H2O2 detoxification catalysed by ascorbate peroxidases (APXs). Despite this central role, the catalytic mechanism used by DHARs is still not well understood and there is no supportive 3D structure. In this context, we have performed a thorough biochemical and structural analysis of the three poplar DHARs and coupled this to the analysis of their transcript expression patterns and subcellular localizations. The transcripts for these genes are mainly detected in reproductive and green organs and the corresponding proteins are expressed in plastids, in the cytosol and in the nucleus, but not in mitochondria and peroxisomes where ascorbate regeneration is obviously necessary. Comparing the kinetic properties and the sensitivity to GSSG-mediated oxidation of DHAR2 and DHAR3A, exhibiting 1 or 3 cysteinyl residues respectively, we observed that the presence of additional cysteines in DHAR3A modifies the regeneration mechanism of the catalytic cysteine by forming different redox states. Finally, from the 3D structure of DHAR3A solved by NMR, we were able to map the residues important for the binding of both substrates (GSH and DHA), showing that DHAR active site is very selective for DHA recognition and providing further insights into the catalytic mechanism and the roles of the additional cysteines found in some DHARs., (© 2016 Authors; published by Portland Press Limited.)

Nonenzymatic deamidation of asparaginyl residues can occur spontaneously under physiological conditions principally when a glycyl residue is at the carboxyl side of Asn and leads to formation of aspartyl and isoaspartyl residues. This modification can change the biological activity of proteins or peptides and trigger an auto-immune response. The α-lactalbumins of members of the Camelidae family are the only of described α-lactalbumins that carry two AsnGly sequences. In the present study, high-resolution mass spectrometry, which enables accurate mass measurement has shown that Asn(16) and Asn(45) underwent a nonenzymatic deamidation, the sequence Asn(45)-Gly(46) being deamidated spontaneously at near-neutral and basic pH and Asn(16)-Gly(17) rather at basic pH. The 16-17 sequence was probably stabilized at near-neutral pH by hydrogen bonds according to the molecular modelisation performed with the camel protein., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The purpose of this study was to improve two common tests used for antioxidant capacity measurements, i.e. the reducing power and chelating ability measurements, for appropriate comparisons between the molecules tested and chosen references, as the usual methods are often performed in a qualitative way rather than a quantitative way. After revision, it was then possible to determine an AERC indice (Ascorbate Equivalent Reducing Capacity) and a CECC (Carnosine Equivalent Chelating Capacity) or EECC (EDTA Equivalent Chelating Capacity) indice according to the standard chosen, by analogy to the TEAC indice (Trolox Equivalent Antioxidant Capacity) already used in many reported works to determine the free radical scavenging activity. Thus, the determination of these relative indices enables the comparison of antioxidative capacities obtained in various studies. The adaptation of these two tests to micro-scales and the calculation of AERC, EECC and CECC were performed on model peptides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS(+) phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS(-) phenotype) and two mutant strains LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS(-) strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS(-) strains.

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.

Somatic angiotensin I-converting enzyme (ACE) possesses two catalytic domains and plays a major role in the regulation of blood pressure, thus representing a therapeutic target for the treatment of hypertension. We present a comprehensive surface plasmon resonance (SPR) study of the interaction of human somatic ACE with the pharmacological inhibitors captopril and lisinopril, the bradykinin potentiating peptide BPP-11b, and the food peptidic inhibitors from bovine αs2-casein, F(174)ALPQYLK(181) and F(174)ALPQY(179). SPR binding curves recorded with the high potency inhibitors captopril, lisinopril, and BPP-11b were evaluated both by regression analysis and by kinetic distribution analysis. The results indicated that captopril and lisinopril bound ACE with two K(D)'s differing by a factor 10-20 and >30, respectively (lowest K(D) = 0.1-0.3 nM for both inhibitors). This shows, for the first time in a direct binding assay with the two-domain enzyme, the existence of two binding modes of the pharmacological inhibitors, presumably with the two ACE domains. The BPP-11b-ACE binding curves were complex but showed a predominant interaction with K(D) in the nanomolar range. The caseinopeptides, known to inhibit ACE with an IC₅₀ of 4.3 μM, bound to ACE with K(D) = 3-4 μM. Mapping of the F(174)ALPQY(179) binding site on ACE by sequential binding studies using captopril or BPP-11b indicated that it bound to (or near) the two active sites of ACE, in agreement with the stoichiometry of 2 determined from data fitting. Our results provide a detailed characterization of ACE-inhibitor binding modes and validate SPR for predicting the inhibitory potential of new compounds.

The trend to confer new functional properties to fermented dairy products by supplementation with bioactive peptides is growing in order to encounter the challenge of health-promoting foods. But these functional ingredients have not to be hydrolysed by proteases of bacteria used in the manufacture of these products. One of the two yoghurt bacteria, Streptococcus thermophilus, has long been considered as weakly proteolytic since its only cell wall-associated subtilisin-like protease, called PrtS, is not always present. Nevertheless, a recent study pointed out a possible peptidase activity in certain strains. In this present study, the stability of milk-derived bioactive peptides, e.g. the anxiolytic peptide, αs1-CN-(f91-97), in the presence of two different S. thermophilus strains with PrtS+ or PrtS− phenotype was studied. Both strains appeared to be capable of hydrolysing the αs1-CN-(f91-97) and other bioactive peptides by recurrent removal of N-terminal residues. The hydrolysis was neither due to intracellular peptidases nor to HtrA protease. Results obtained showed that the observed activity originates from the presence at the surface of both strains of an extracellular aminopeptidase activity. Moreover, a cell wall-associated X-prolyl dipeptidyl peptidase activity was also highlighted when β-casomorphin-7 was used as substrate. All of these findings suggest that, in order to use fermented milks as vector of bioactive peptides, the stability of these bioactive peptides in this kind of products implies to carefully characterize the potential action of the surface proteolytic enzymes of S. thermophilus.

The hepatic removal of triglyceride-rich chylomicrons during the postprandial phase represents an important step towards determining the bioavailability of dietary lipids amongst the peripheral tissues. Indeed, elevated postprandial lipemia is often associated with obesity and increased risk of coronary heart disease. The milk protein, lactoferrin, has been shown to inhibit hepatic chylomicron remnant removal by the liver, resulting in increased postprandial lipemia. Despite numerous studies on potential targets for lactoferrin, the molecular mechanisms underlying the effect of lactoferrin remain unclear. We recently demonstrated that the lipolysis stimulated lipoprotein receptor (LSR) contributes to the removal of triglyceride-rich lipoproteins during the postprandial phase. Here, we report that while lactoferrin does not have any significant effect on LSR protein levels in mouse Hepa1-6 cells, this protein colocalizes with LSR in cells but only in the presence of oleate, which is needed to obtain LSR in its active form as lipoprotein receptor. Ligand blotting using purified LSR revealed that lactoferrin binds directly to the receptor in the presence of oleate and prevents the binding of triglyceride-rich lipoproteins. Both C- and N-lobes of lactoferrin as well as a mixture of peptides derived from its hydrolysis retained the ability to bind LSR in its active form. We propose then that the elevated postprandial lipemia observed upon lactoferrin treatment in vivo is mediated in part by its direct interaction with free fatty acid activated LSR, thus preventing clearance of chylomicrons and their remnants through the LSR pathway., (© 2012 The Authors Journal compilation © 2012 FEBS.)

To better understand the mechanism of metal ion transport through the gastrointestinal tract to their absorption sites, isothermal titration calorimetry (ITC) was used to investigate the binding of dicationic metals to β-CN(1-25)4P, a β-casein tetraphosphorylated peptide. ITC technology was found suitable for studying weak bonds between metal ions and phosphopeptides and provided a direct means of thermodynamic and stoichiometric characterisation of complex formation. Thus, one mole of β-CN(1-25)4P binds two moles of Ca(2+), Mg(2+) or Zn(2+) under experimental conditions close to those of the ileum (pH 8, 37°C), with rather low binding affinity constants (K=4900-11,200M(-1)). These low affinities should facilitate the release of metal ions during intestinal absorption. By contrast, Cu(2+) did not bind to β-CN(1-25)4P at pH 8, despite its reported significant affinity towards β-casein and the 1-25 peptide at near-neutral pH., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Equine beta-casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P-7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion-exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro-column. This method turned out to be an efficient tool to separate the CPPs Arg(1)-Lys(34) and Glu(4)-Lys(34) from non-phosphorylated peptides. Purification was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) and each CPP was hydrolyzed by endoproteinase Glu-C. Finally, the digests were analyzed by RP-HPLC/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS) and identified by nano-electrospray ionization tandem mass spectrometry (nESI-MS/MS) to locate the phosphorylated sites of the beta-casein isoforms 4P-7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser(9), Ser(23), Ser(24), and Ser(25). Addition of phosphate groups on Ser(18), Thr(12), and Ser(10) led to the formation of the isoforms 5P-7P, respectively. The results indicated that the in vivo phosphorylation of the equine beta-casein follows a sequential way and is not randomly performed., (Copyright (c) 2010 John Wiley & Sons, Ltd.)

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.

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.

Highly phosphorylated casein with a low molecular mass was isolated from Haflinger mare's milk by RP-HPLC. It accounts for 4.0% of the casein content. Its mass was determined by LC-ESI-MS before and after treatment by alkaline phosphatase. The molecular mass found for the apo-form (10,591 +/- 2 Da) is in agreement with its primary structure, which was established by ESI-MS/MS from tryptic peptides. It appeared that this short protein (94 amino acid residues) is an internally truncated form of the full-length equine beta-casein (226 residues). This low-Mr variant of equine beta-casein displays a large deletion (residues 50-181), due to a cryptic splice site usage occurring within exon 7 during the course of primary transcripts processing. The phosphorylation pattern of this equine beta-casein variant was investigated by LC-ESI-MS and 2-DE. Seven phosphorylation forms were identified with one to seven phosphate groups with pIs ranging between 4.67 and 4.01. The major isoforms carry five and six phosphate groups.

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.

Raw skim milk was submitted to high pressure (300 to 600 MPa) and temperature (4 to 70 degrees C) treatments for 2 or 5 min. The combined effects of pressure and temperature on milk proteins induced structural changes and polymer and copolymer formation characterized by anion-exchange and size-exclusion fast protein liquid chromatography and electrophoretic techniques. Approximately half of the beta-lactoglobulin formed polymers, and the other half formed large copolymers, mainly with kappa-casein, alpha-lactalbumin via intermolecular disulfide bond exchange, and alpha(s1)-casein via physicochemical interactions, in proportions of 1.0:0.7:0.3:0.1, respectively. Minor whey proteins (serum albumin, immunoglobulins, and lactoferrin) also participated in the formation of the copolymers but to a lesser extent. Two populations of the copolymers were found with apparent molecular masses ranging from 440 to 2000 kDa for the first and more than 2000 kDa for the second. On the contrary, for heated milks the aggregation kinetics obtained by combination of high pressure and thermal treatment were very fast, as no intermediates such as dimers and small size oligomers were observed after pressurization, whatever the temperature studied. Lactosylation of proteins as well as proteolysis were very limited. A beta-casein amino-terminal peptide of 22 kDa was specifically recovered in milk samples treated under the more drastic conditions (500 MPa/55 degrees C per 5 min and 600 MPa/70 degrees C per 5 min) and might have been generated by neutral proteases such as elastase released from somatic cells present in milk. No casein was released from the micelle whatever the combination of high pressure and temperature studied.

A synthetic peptide of 23 residues corresponding to the carboxyterminal 113 to 135 region of component-3 of proteose peptone (PP3) has been investigated with regard to its antibacterial properties. This cationic amphipathic peptide that we refer to as lactophoricin, displayed a growth-inhibitory activity against both gram-positive and gram-negative bacteria. For most of the strains tested, bacterial growth was observed in the presence of lactophoricin except for Streptococcus thermophilus. In that case, lactophoricin exhibited a minimum inhibitory concentration of 10 microM and a minimum lethal concentration of 20 microM. No hemolysis of human red blood cells was detected for peptide concentrations between 2 to 200 microM, indicating that lactophoricin would be noncytotoxic when used in this concentration range.

Streptomyces ambofaciens has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the beta-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against Bacillus subtilis. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production.

Based on the compositional change of the proteose peptone fraction, proteolysis was studied over time following lipopolysaccharide-induced experimental mastitis. Electrophoresis of the proteose peptone fraction revealed many degradation products. Five peptides were identified by amino-terminal sequencing as internal fragments of beta-, kappa-, alpha(s1)-, and alpha(s2)-casein that were generated by somatic cell proteases. Although kappa-casein is considered particularly resistant to endogenous proteolysis, a kappa-casein peptide was electrophoretically isolated in association with a beta-casein fragment. The in vitro kinetic studies of caseinate hydrolysis by elastase, one of the main polymorphonuclear neutrophil (PMN) proteases, suggested that the beta-casein peptide might be generated by elastase. In addition, elastase activity in milk PMN was higher during the inflammation of the mammary gland than prior to infusion.

The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, presented strong identities with regions of alpha(s1)-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 alpha(s1)-CN, respectively. On the other hand, trace amounts of equine gamma-CN-like and proteose peptone component 5-like peptides were found in the whole CN. They were identified by microsequencing and corresponded to beta-CN peptides generated by plasmin action on the whole CN. The equine alpha(s1), beta-, and kappa-CN were separated by bidimensional electrophoresis in numerous isoelectric variants with apparent isoelectric points distributed between pH 4.4 to 6.3, 4.4 to 5.9, and 3.5 to 5.5, respectively. The beta- and kappa-CN displayed a more acidic character in the mare than in the cow.

A combination of proteolysis and dilational rheology has been used to study the behavior of films of beta-casein (beta-CN) and of peptides spread at the oil-water interface. Identification of the peptides produced by trypsin hydrolysis of beta-CN in emulsion at 37 degrees C provided information on the structure of beta-CN adsorbed at the oil-water interface. Good interface properties were observed for beta-CN or its peptides, probably because of the amphipathic nature of beta-CN or a synergistic effect between hydrophilic and hydrophobic peptides. Remarkable surface activity was found for the amphipathic peptide beta-CN (f114-169). Rheological studies had shown that interface films made with peptide fractions or with beta-CN were elastic rather than viscous. Film made with the purified peptide beta-CN (f114-169) was merely elastic at the triolein-water interface. A decrease of the viscoelastic modulus was observed for aging beta-CN film but not for aging peptide films; The beta-CN decrease was related to the flexibility of its structure. When the interface is increased by the dilation of an aqueous droplet plunged into oil, beta-CN may expose new polypeptide trains to cover the increased interface, unlike peptides with simpler structures.

The camel (camelus dromedarius) milk proteose peptone 3 (PP3) was purified successively by size exclusion fast protein liquid chromatography and reversed phase high performance liquid chromatography and then characterized by amino acid residue composition determination and chemical microsequencing after CNBr or trypsin cleavages. In comparison with the previously reported structure of camel milk whey protein, the camel PP3 contains an insertion in the N-terminal region which has approximately 24 residues, whereas the remaining C-terminal regions of these two homologous proteins are essentially identical. The camel PP3 seems to contain a potential O-glycosylation site localized in this insertion and 2 or 3 phosphorylated serine residues. PP3 belongs to the glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) family and could therefore play an immunological role in the camel or its suckling young.

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 alpha-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 beta-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 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.

Substantial progress has been made in our knowledge of the biological properties of mammal milks. Many nutritional, biochemical, immunological, or other biological properties have been studied in mature or industrially processed bovine milk as well as in human milk and colostrum. This article is a critical review of selected publications covering (1) the use of bovine milk or dairy byproducts (processed acid and enzymatic whey fractions) as a serum substitute for cell cultures, (2) specific factors in bovine milk and industrially processed milk the affect cell proliferation, and (3) the known functional and biological roles of two whey proteins: beta-lactoglobulin and the PP3 component.

alpha s1- and beta-Caseins have a sequence cluster -Ser(P)-Ser(P)-Ser(P)-Glu-Glu- which is not present in kappa-casein and the whey PP3 component. The affinity of these phosphoproteins for the iron(III)-iminodiacetic acid (IDA) complex immobilized on Sepharose was studied as a function of pH, urea concentration, calcium ion concentration, enzymatic dephosphorylation and temperature. The affinity of the three polyphosphorylated proteins (alpha s1- and beta-caseins, PP3) was similar. The sequence cluster was not a specific recognition pattern of the iron(III) ion. These three proteins presented a site of high affinity and a site of weak affinity. kappa-Casein, which had only one Ser(P) residue, presented only the site of weak affinity. Their primary site which was absent after dephosphorylation or calcium ion addition required the presence of at least two Ser(P) residues close in space. Their secondary site was sensitive to the presence of urea. It was sensitive to pH variation for PP3 and kappa-casein. The study of the affinity of a few free amino acids towards iron(III)-IDA showed that the secondary site involved tryptophan and tyrosine residues for alpha s1- and beta-caseins, histidine residues for PP3 and cysteine residues for kappa-casein.

Peptidase-1 is a sex-linked enzyme, which can be purified from the liver of the amphibian urodele Pleurodeles waltl. We estimated its apparent molecular mass as 170 kDa by gel filtration chromatography. The enzyme is composed of two subunits with apparent molecular masses of 90 and 99 kDa. It is strongly inhibited by ethylenediaminotetraacetic acid, ethylene glycol bis(beta-aminoethyl ether)-N,N-tetraacetic acid, and 1,10-phenanthroline, indicating that peptidase-1 is a metallopeptidase. Peptidase-1 has optimal activity at 55 degrees C and pH 8.5. This acidic enzyme displays two apparent isoelectric points, at 4.9 and 5.2, and is essentially located in the cytosolic subcellular fraction.

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.

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 beta-CN-1P (f29-105/7) of the proteose-peptone fraction was a terminal product of plasmin-like activity and was located correctly electrophoretically. Correlation factors between the plasmin activity and content in milk of component beta-CN-1P (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.

Milk component 3 was an inhibitor of lipoprotein lipase activity responsible for spontaneous lipolysis occurring in milk stored at 4 degrees 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.