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PubMed1996

Fine tuning of epitopic dominance induced by lung cancer on the IgG response to bovine betalactoglobulin: towards a paraneoplastic immune marker.

BACKGROUND: Investigating the humoral immune response to mucosal antigens in patients with lung cancer, we have documented a preferential immunoglobulin G (IgG) binding to cryptic epitopes unmasked by the proteolysis of bovine beta-lactoglobulin (BLG). In contrast, IgG from healthy controls and patients with chronic bronchitis (COPD) bind preferentially to continuous epitopes presented on both native (n) and denaturated (d) forms of this antigen. The present study further characterized the differences in the epitope profiles recognized on BLG. METHODS: The capacity of individual sera from 65 lung cancer patients, tested before and after cancer removal for the patients with early stage lung carcinoma, 65 healthy controls, and 52 patients with COPD, to prevent the binding of pooled IgG fractions from each population as well as murine monoclonal antibodies (MoAb), specific for BLG, to solid phase bound antigen was evaluated in enzyme-linked immunoadsorbent assay using streptavidin-biotin technology. Some of these experiments were also performed with sera from 42 patients diagnosed with other cancers. RESULTS: Compared with control sera and sera from patients with other solid tumors, lung cancer patient sera showed distinct capacities to prevent the binding of murine MoAb as well as human pooled IgG fractions to n- and d-BLG. The inhibition capacities of lung cancer sera changed as soon as five weeks after cancer removal. CONCLUSIONS: The results indicate that the difference in epitope specificity exhibited by lung cancer sera is not restricted to cryptic epitopes, but also affects continuous and discontinuous epitopes, accessible only on the native antigen. A high level of binding discrimination between antibodies from the study populations is also observed at the level of the epitope. This deviation in the epitope specificity of antibodies changes soon after cancer removal, suggesting a tumor-dependent disturbance. Also documented in the Dermatophagoides pteronyssinus model, it opens the way to a new class of paraneoplastic immune markers for this malignancy, with, at first glance, a high specificity level.

Adenocarcinoma↗
PubMed1997

Milk protein-derived opioid receptor ligands.

Milk is mammalian characteristic and is of particular importance for humans: Mother's milk or its substitutes from cows' milk are absolutely essential nutriments for the neonate and cows' milk also represents a basic foodstuff for adults. However, in addition to their well-known nutritive role, milk constituents apparently are also able to carry specific information from the milk producer's to the milk receiver's organism: Thus, a number of milk protein fragments has been shown to behave like opioid receptor ligands able to address opioidergic systems in the adult's or in the neonate's organism. With respect to the proteins, which they are derived off these peptides have been named alpha-casein exorphins or casoxin D (alpha-casein), beta-casomorphins or beta-casorphin (beta-casein), casoxin or casoxin A, B, or C (k-casein), alpha-lactorphins (alpha-lactalbumin), beta-lactorphin (beta-lactoglobulin) or lactoferroxins (lactoferrin). Only casoxins and lactoferroxins display antagonistic properties; the other peptides behave like opioid receptor agonists. Most of the information available so far has been collected about beta-casomorphins. These peptides obviously can be released from beta-casein in the adult's or in the neonate's organism, where they might elicit opioid effects in the frame of a regulatory role as "food hormones". Several synthetic beta-casomorphin derivatives have been shown to be highly specific and potent mu-type opioid receptor ligands which frequently have been used as standard tools in opioid research.

Amino Acid Sequence↗
PubMed1999

Binding of proteins to copolymers of varying hydrophobicity.

Hydrophobic interactions between proteins and amphiphilic polyelectrolytes were studied by frontal analysis continuous capillary electrophoresis (Gao et al., Analytical Chemistry, 1997, Vol. 69, pp. 2945-2951). Binding isotherms were obtained for beta-lactoglobulin and for bovine serum albumin interacting with a series of alternating copolymers of maleic acid and alkyl-vinyl ethers of varying hydrophobicity. Although binding between proteins and copolymers increases with increasing alkyl chain length, a minimum alkyl chain length of 3-4 methylenes is required for significant hydrophobic interactions to occur. These copolymers, like other polyamphiphiles, can form intrapolymer micelles, and the extent of such micellization decreases with increasing degree of carboxylate ionization. Binding results obtained at different pHs suggest that competition exists between intrapolymer micelle formation and protein-polymer hydrophobic interactions.

Animals↗
PubMed2000

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Amino Acid Sequence↗
PubMed2001

Assessment of the proportion of transgene-bearing sperm by fluorescence in situ hybridization: a novel approach for the detection of germline mosaicism in transgenic male founders.

Genetic mosaicism is frequent among transgenic animals produced by pronuclear microinjection. A successful method for the screening of founder animals for germline mosaicism prior to mating would greatly reduce the costs associated with the propagation of the transgenic lines, and improve the efficiency of transgenic livestock production. With this aim, we have devised a simple method to detect integrated transgenes in individual spermatozoa using fluorescence in situ hybridization (FISH). The experiments reported here were undertaken to investigate the efficiency of this FISH-based approach to accurately evaluate the proportion of transgene-bearing sperm and to be applied for the detection of potential germline mosaics. Sperm samples from mice homozygous and hemizygous for a beta-lactoglobulin transgene were analyzed in a first set of experiments. A high hybridization efficiency was achieved, and the proportions of transgene-positive sperm cells in both homozygous (94.8-98.2%) and hemizygous (49.8-51.9%) animals were close to the expected frequencies (100 and 50%, respectively). To evaluate the sensitivity of the assay more directly, simulated mosaic samples with 5, 10, 15, 20 and 40% of transgene-bearing spermatozoa were then prepared and analyzed by FISH. Significant differences in the frequency of transgene-positive sperm were observed between all mosaic samples, indicating that even small deviations (5%) from the expected 50% transgene transmission rate in a founder animal could be reliably detected with our assay. Therefore, the method proposed represents a novel approach for the identification of germline mosaic founder males in livestock transgenic projects and a much more economic and faster alternative to breeding.

Animals↗
PubMed2001

Improvement of solubility and of emulsifying properties of milk proteins at acid pHs by esterification.

Three milk proteins (beta-lactoglobulin, alpha-lactalbumin and beta-casein) were esterified to different extents with methanol, ethanol and propanol, then their solubility was studied in the pH-range of 3-10. Their emulsifying properties were also checked in the pH-range of 3-7 by measuring the oil-droplet size using laser light scattering. Solubility of all esterified proteins was improved in the acidic pH range (3-6). The magnitude of improvement was depending on the extent of esterification, the nature of the grafted ester group and the nature of the modified protein. Emulsifying activity and stability of esterified milk proteins in the acidic pH range of 3-5 were generally higher compared to the corresponding native proteins. This improvement was associated with the degree of solubility, the degree of esterification, the type of ester group grafted and the nature of the used protein. The highest emulsifying activity improvement was observed for methyl ester derivatives at pH 5 where the native proteins have poor emulsifying properties.

Caseins↗
PubMed2000

Impact of in utero Th2 immunity on T cell deviation and subsequent immediate-type hypersensitivity in the neonate.

It was the aim of this study to analyze the impact of maternal Th2 immune responses on onset and subsequent development of allergen-specific immunity and immediate-type hypersensitivity in early childhood. In a well characterized mouse model of Th2 immunity, BALB / c mice were sensitized to ovalbumin (OVA) before mating followed by allergen aerosol exposure during pregnancy. At the end of pregnancy mice developed allergen-specific Th2 / Th0 immunity and immediate-type hypersensitivity responses to OVA. T cells from these newborns, when restimulated with PMA / ionomycin, demonstrated a lowered capacity to produce IFN-gamma. To assess whether prenatal allergen exposure favors postnatal onset of a Th2-type immune response, these offspring were immunized to a novel antigen by a single injection of beta-lactoglobulin (BLG). In contrast to offspring from non-sensitized mothers, offspring from OVA-sensitized mice showed both higher anti-BLG immunoglobulin titers and higher frequencies of immediate-type skin test responses. Our data suggest that Th2 / Th0 immunity present during pregnancy has a decisive impact on shaping of the Th1 / Th2 T cell profile in the neonate. Furthermore, this effect favors the development of Th2 immune responses, when mice are exposed to a novel antigen during early childhood.

Animals↗
PubMed2002

Characterization of lactosylated proteins of infant formula powders using two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry.

Infant formula powders were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to assess the whey proteins quality, which may be altered by the heat treatment used during the processing conditions. Lactosylation was found to be the major chemical modification occurring in whey proteins. In parallel, a two-dimensional (2-D) gel electrophoresis was performed on the milk sample and the entire protein patterns were analyzed by nano-ESI-MS after cutting the different gel spots and in-gel trypsin digestion. A highly selective and specific tandem MS technique has been developed to characterize and localize up to ten lactosylation sites in beta-lactoglobulin (beta-Lg) and alpha(S2)-casein. alpha-Lactalbumin (alpha-La), with five lactosylated peptides, was found to be an interesting protein marker in the milk powder sample to detect chemical modification induced by the processing/storage conditions.

Animals↗
PubMed1992

Three-dimensional structure and active site of three hydrophobic molecule-binding proteins with significant amino acid sequence similarity.

We review our work on bovine and human retinol-binding protein (RBP), bovine beta lactoglobulin (BLG), and bovine odorant-binding protein (OBP). These three proteins share a sequence similarity high enough to justify the proposal that their three-dimensional structure ought to be quite similar, and they also share the function of similar or even identical hydrophobic ligand binding, although with a very different degree of specificity. Thus they constitute an ideal system to exhaustively explore the question of three-dimensional structure prediction from sequence similarity and the related question of binding site prediction for similar ligands. We have used x-ray diffraction techniques on single crystals of human and bovine RBP, bovine milk BLG, and bovine nasal mucosa OBP to investigate this problem. The results of these crystallographic studies indicate that to the level of resolution so far attained, the three-dimensional structure of these three proteins is reasonably predicted from the sequence similarity. The fold is the same and structural differences are rather subtle. Finally, we present experimental evidence that the binding sites of RBP, BLG, and OBP are in different regions of the molecules. Thus, it appears that although sequence alignment has correctly predicted the protein fold, it has incorrectly predicted the hydrophobic ligand-binding sites.

Amino Acid Sequence↗
PubMed2003

Size-exclusion reaction chromatography (SERC): a new technique for protein PEGylation.

A new, widely applicable process that combines reaction and separation in a single unit operation is described. The process, size-exclusion reaction chromatography (SERC), simultaneously allows control of the extent of reactions in which molecular size is altered and the separation of products and reactants. In SERC, a moving reaction zone is formed by injection of reactants onto a size-exclusion chromatography column. Reactants and products are partitioned differently within the mobile phase, resulting in different linear flow rates through the column. The products are therefore removed selectively from the reaction zone, minimizing their residence time in the reaction zone and allowing their separation in the downstream section of the column. For reactions such as protein PEGylation, in which successive addition of PEG groups to the protein results in significant molecular size increases, SERC potentially offers a method by which a dominant final PEGylated protein size can be produced at high yield. The SERC PEGylation of two model proteins, alpha-lactalbumin and beta-lactoglobulin, is demonstrated and results show that simultaneous reaction and separation was obtained.

Biotinylation↗
PubMed2004

Quantitative analysis of membrane fouling by protein mixtures using MALDI-MS.

Binary aqueous solutions of bovine serum albumin (BSA) and beta-lactoglobulin (bLG) were subject to flux-stepping and constant flux ultrafiltration to identify the apparent critical flux and to study the mechanisms and factors affecting fouling when the membrane is permeable to one protein component. Membranes from these filtration experiments were analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to locate and quantify levels of fouling below and above the apparent critical flux. Hydrophilic (PLTK) regenerated cellulose and hydrophobic (PBTK) polysulfone asymmetric membranes were used, both of 30 kDa nominal molecular weight cut-off. For the hydrophilic PLTK membrane, protein deposition was shown to depend on electrostatic forces, exhibiting little or no fouling when the proteins had the same charge sign as that of the membrane. This was found to apply for both dilute equal mass-per-unit-volume and equimolar binary mixtures. For the PBTK membrane, hydrophobic protein-membrane attractive forces were sufficiently strong to cause deposition of bLG even in the presence of repulsive electrostatic forces. For the PBTK membrane deposition exceeded monolayer coverage below and above apparent critical flux conditions but for the PLTK membrane this generally occurred when the apparent critical flux was exceeded. MALDI-MS was shown to be a facile direct analytical technique for individually quantifying adsorbed proteins on membrane surfaces at levels as low as 50 fmol/mm(2). The high levels of compound specificity inherent to mass spectrometry make this approach especially suited to the quantification of individual components in mixed deposits. In this study, MALDI-MS was found to be successful in identifying and quantifying the protein species responsible for fouling.

Complex Mixtures↗
PubMed2004

Highly selective membranes in protein ultrafiltration.

A new ultrafiltration technique based on a multimembrane stack has been developed to fractionate solutes closer in size than conventionally possible. The technique is illustrated here by obtaining a pure protein product from a binary protein mixture. By employing membranes in series without any gaskets or spacers in-between, ultrafiltration is carried out to separate two proteins relatively close in molecular weight or size. Flat YM30 regenerated cellulose membranes, all of the same molecular weight cut-off (MWCO) 30,000, are stacked together in the desired number, and ultrafiltration takes place. The membrane rejection of a protein is amplified with each additional membrane, ultimately resulting in a completely rejected species. Complete purification of the more permeable protein may be achieved regardless of the physicochemical condition that may be optimal or suboptimal for selective separation by a single membrane. Two systems, myoglobin and beta-lactoglobulin, as well as myoglobin and alpha-lactalbumin were studied, under various operating conditions. The solvent flux reduction encountered when each membrane is added may also be avoided, by operating at increased pressure, while still achieving the desired purification. Cleaning in situ is achievable with reproducible experimental results before and after on-line cleaning. The results clearly demonstrate that multimembrane stacks can be used for fractionation of proteins that are quite close in molecular weight/size.

Biotechnology↗
PubMed2006

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography and for the optimal selection of operational conditions.

Elution curves in ionic exchange chromatography (IEC) for a three-protein mixture (alpha-lactoalbumin, ovalbumin, and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Relatively low protein concentrations were used to avoid protein-protein interactions. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. A cost function was built that included the effect of the different production stages, namely fermentation, purification, and concentration. These considered the effect on the performance of IEC; yield, purity, concentration and the time needed to accomplish the separation. Operational conditions in the IEC such as flow rate, ionic strength gradient and the operational time can be selected using this model in order to find the minimum cost for the protein production process depending on the characteristics of the final product desired such as purity and yield. This cost function was successfully used for the selection of the operational conditions as well as the fraction of the product to be collected (peak cutting) in IEC. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.

Chromatography, Ion Exchange↗
PubMed2006

Spectroscopic Studies of water-soluble porphyrins with protein encapsulated in bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: aggregation versus complexation.

We have investigated the interaction of two water-soluble free-base porphyrins (negatively charged meso-tetrakis(p-sulfonatophenyl)porphyrin sodium salt (TSPP) and positively charged meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMpyP)) with two drug-carrier proteins (human serum albumin (HSA) and beta-lactoglobulin (betaLG)) in bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water reverse micelles (RM) by using steady-state and transient-state fluorescence spectroscopy. TSPP exhibited a complex pattern of aggregation on varying the RM size and pH in the absence of the protein: at low omega0 (the ratio of water concentration to AOT concentration, the emission of H-aggregates prevails under acidic or neutral "pH(ext)" conditions. Upon formation of the water-pool, J-aggregates and monomeric diacid species dominate at low "pH(ext)" but only monomer is detected at neutral "pH(ext)". The aggregation number increases with omega0 and the presence of the protein does not seem to contribute to further growth of the aggregate. The presence of protein leads to H-deaggregation but promotes J-aggregation up to a certain protein/porphyrin ratio above which, complexation with the monomer bound to a hydrophobic site of the protein prevails. The effective complex binding constants are smaller than in free aqueous solution; this indicates a weaker binding in these RM probably due to some conformational changes imposed by encapsulation. Only a weak quenching of TMpyP fluorescence is detected due to the presence of protein in contrast to the negative porphyrin.

Albumins↗
PubMed1990

Preparative free-flow isoelectric focusing: modeling and experiments.

Free-flow isoelectric focusing was adapted to preparative scale separations and chemical engineering methods were used to describe the main mechanisms operating in the apparatus. A mixture of human serum albumin (pI 4.6) and beta-lactoglobulin (pI 5.22) was separated in pH gradients, generated with carrier ampholytes of different origin and covering the pH ranges 4-6.5, 3.5-5, 4-5.5 and 4.5-5.0. Best results were obtained in the pH 4-5.5 range. The experimental results have validated the results obtained with a numerical model.

Ampholyte Mixtures↗
PubMed1992

Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel.

It has been found that the double bond of free, unreacted acrylamide in a gel can react with a free -SH group of proteins, forming a cysteinyl-S-propionamide adduct. When beta-lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu-149 to Ile-162. Residue No. 160 was found to be a cysteinyl-S-propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including -NH2 terminus and free -NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the -SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.

Acrylamide↗
PubMed1998

High-resolution density gradient electrophoresis of subcellular organelles and proteins under nondenaturing conditions.

We have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high-resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis 1993, 14, 1011-1018). Isoforms of bovine beta-lactoglobulin, human apo-transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and proteins.

Animals↗
PubMed1998

Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins.

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.

Adsorption↗