Evidence for a novel Entamoeba histolytica lectin activity that recognises carbohydrates present on ovalbumin

Entamoeba histolytica, an intestinal amoeba that causes dysentery and liver abscesses, acquires nutrients by engulfing bacteria in the colonic lumen and phagocytoses apoptotic cells during tissue invasion. In preliminary studies to identify ligands that stimulate amoebic phagocytosis, we used ovalbumin immobilized on latex particles as a potential negative control protein. Surprisingly, ovalbumin strongly stimulated E. histolytica particle uptake. Experiments using highly purified ovalbumin confirmed the specificity of this finding. The mechanism of particle uptake was actin-dependent, and the Entamoeba phagosome marker amoebapore A localised to ovalbumin-bead containing vacuoles. The most well described amoebic receptor is a Gal/GalNAc-specific lectin, but d-galactose had no effect on ovalbumin-stimulated phagocytosis. Ovalbumin has a single N-glycosylation site (Asn₂₉₂) and is modified with oligomannose and hybrid-type oligosaccharides. We used both trifluoromethanesulfonic acid and N-glycanase to deglycosylate ovalbumin and tested the effect. Both methods substantially reduced the stimulatory effect of ovalbumin. Biotinylated ovalbumin bound the surface of fixed E. histolytica trophozoites saturably; furthermore, denatured ovalbumin and native ovalbumin both specifically inhibited ovalbumin-biotin binding, but deglycosylated ovalbumin had no effect. Collectively, these data suggest that E. histolytica has a previously unrecognised surface lectin activity that binds to carbohydrates on ovalbumin and stimulates phagocytosis.     

Interdigitated capacitive biosensor based on molecularly imprinted polymer for rapid detection of Hev b1 latex allergen

Allergen protein detection was performed by a surface imprinted layer combined with an interdigitated capacitance (IDC) transducer that allowed label-free measurements. The immobilized imprinted polymers are the probes that bind to rubber allergen proteins extracted from products such as rubber gloves. Copolymers made from methacrylic acid–vinylpyrrolidone–dihydroxyethylene-bisacrylamide (MAA–NVP–DHEBA) are soluble in aqueous solution and eliminate the denaturation of protein. When deposited as a coating onto an IDC microelectrode transduction system, such materials lead to sensors that produce capacitance responses that are clearly dependent on the concentration of the latex protein (10–900 ng ml⁻¹) in pH 7.4 buffer. The biosensor can detect Hev b1 within minutes and with a detection limit of 10 ng ml⁻¹. Different but related hevein allergenic proteins isolated from natural rubber latex from the rubber tree (Hev b1, Hev b2, and Hev b3) were distinguished by the imprinted material, depending on the dimension and conformation of these proteins with a selectivity factor of 4. They recognized Hevea latex proteins better than non-Hev b proteins, such as lysozyme, ovalbumin, and bovine serum albumin, by a factor of 2. Moreover, the sensor exhibited good operational stability of up to 180 days when used continuously at room temperature.     

Effect of Acetylation of Ovalbumin on Its Adsorption Behavior at Solid/Liquid Interface

This paper reports the effect of modification of lysine residues on the adsorption of ovalbumin at alumina/water interface. It has been shown that the pH dependence of the adsorption changes on acetylation of lysine. Thus at pH 7.6 acetylated ovalbumin does not show any affinity for alumina surface although unmodified protein does. It seems that although electrostatic interactions are operative, surface unfolding of proteins and surface hydrophobicity of protein also control the adsorption of ovalbumin onto alumina.     

Analysis of molecular interactions in heat-induced aggregation of a non-inhibitory serpin ovalbumin using a molecular chaperone

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 microM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 microM by BiP, lower than that for RCMLA (Kd = 1.1 microM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd = 21 microM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Screening for plant lectins by latex agglutination tests

The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.     

Protein quantitation of as low as 10-ng/ml concentrations by competitive binding to polystyrene latexes

A protein quantitation method which offers protein detection as low as 10 ng protein/ml and accurate quantitation as low as 30-100 ng protein/ml, depending on the protein, has been designed. The assay, which is relatively quick and simple to perform, utilizes the strong, nonspecific adsorption of proteins onto polystyrene latexes. A competition is created between a marker enzyme and the analyte protein for a limited amount of latex surface area. Due to inactivation of the enzyme upon binding to a hydrophobic latex surface, measurement of enzyme activity allows determination of the bound/free enzyme ratio and thus the competing protein concentration. Considerations of sensitivity and simplicity are suggested to make this assay superior to others presently available.     

Fluorescamine as a reagent for location of proteins after electrophoresis in starch gel or on paper

Fluorescamine (Fluram, Roche) has been used as a reagent for locating proteins after electrophoresis in starch gel and on paper. Attractive features of the method are speed, sensitity, and no destaining of gel or paper is required. The latex protein hevein, which is particularly difficult to stain by conventional methods, was detected very readily in starch gel and on paper with Fluorescamine.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

Binding and phagocytosis of fibronectin-coated beads by BHK cells: receptor specificity and dynamics

Studies on the receptor specificity and dynamics involved in fibroblast phagocytosis of latex beads revealed the following: 1) Ligands other than fibronectin such as concanavalin A (ConA) and serum spreading factor, when coated on latex beads, were found to promote phagocytosis of the beads. This indicates that fibroblast phagocytosis, like spreading, is a ligand-receptor mediated phenomenon not specifically requiring fibronectin (pFN); 2) Anti-pFN antibodies were found to inhibit the ability of cells to ingest pFN-coated beads that previously were bound on the cell surfaces. Consequently, binding of beads to the cell surfaces per se is not a sufficient signal to promote ingestion of the beads; 3) Finally, divalent cations protected receptor function necessary for phagocytosis of pFN-coated beads from proteolysis by trypsin, as previously was found for receptors involved in cell attachment and spreading on pFN-coated culture dishes. Recovery experiments carried out with cells whose surface receptors had been destroyed indicated that there was an internal (or cryptic cell surface) pool of receptors that amounted to at least 50% of the receptors normally found on the cell surface. After complete destruction of the cell surface and cryptic pools of receptors, reappearance of receptors required for bead binding and phagocytosis required several hours and did not occur in the absence of new protein synthesis.     

Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis

Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis‐prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF‐silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF‐silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production.     

Establishment of an in Vitro Model of the Human Placental Barrier by Placenta Slice Culture and Ussing Chamber

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a K _m of 165 μM and a V _max of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a K _d of 0.52 μM by BiP, lower than that for RCMLA (K _d=1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (K _d=21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Conformation and structure of mildly heat-treated ovalbumin in dilute solutions and gel formation at higher protein concentrations

The conformation of heat-denatured ovalbumin aggregates has been examined at several concentrations and pH values, using measurements of optical rotation dispersion (ORD), circular dichroism (CD) and viscosity. The protein was subjected to heating at relatively low temperatures, ranging from 48.5 to 76 degrees; the particular temperature chosen depended on pH. The heat treatment was sufficient to remove the ability of the molecules to absorb heat on re-heating. The denatured molecules were shown to be rather compact, i.e. not much larger than the native molecule, and to retain a significant amount of secondary structure; this was also the case for molecules present in small aggregates. It is suggested that this type of ovalbumin monomer builds three-dimensional networks in denatured solutions at higher concentrations, and that gelation should be looked upon as arising from surface contacts between hydrated globules. The present results also imply that such globules have gelation properties which depend on whether pH is acidic or basic compared to the isoelectric point of the protein.     

Proteins and Polysaccharides In the Nutrition of Paramecium Multimicronucleatun

SYNOPSIS. Paramecium multimicronucleatum has been cultured for 20 years on a medium of salts, vitamins, amino acids, fatty acids, ribosides, and stigmasterol plus a little nondialyzable fraction (NDF) of baker's yeast. Fractionations of NDF identified 2 essentials: (a) in a fraction < 100,000 daltons which contained much protein and replaceable by ovalbumin and (b) in a fraction of < 300,000 daltons; this fraction contained much polysaccharide, replaceable by glycogen, which is > 300,000 daltons. For 2 years now P. multimicronucleatum has grown well with ovalbumin and glycogen replacing NDF. Besides ovalbumin, concanavalin A satisfies the protein requirement; this lectin attaches to sugar residues in glycogen. Studies with a fluorescent dye, PGA-1A, a stilbene derivative, provides further evidence for the polysaccharide requirement. This dye attaches to polysaccharides; when added to glycogen, and this in turn is added to a culture containing ovalbumin, fluorescent blue vacuoles appear within 2–3 h. When dye + glycogen were added to a culture without ovalbumin, no fluorescent vacuoles were found. A protein appears involved in formation of food vacuoles; this fits the pattern for endocytosis described in recent reviews. Besides glycogen, mannan gave good growth. Dextrin and amylopectin gave only fair growth through 7 serial transfers; glucose, maltose and amylose did not sustain growth. Strain 51 of P. tetratrelia , which grows well in NDF medium, grows well when NDF is replaced with ovalbumin and glycogen.     

Studies on the Foaming Property of the Chicken Egg White

The foaming power of the native ovalbumin has been found to be increased very much by various kinds of denaturation. The characters of its surface monolayer were also changed by denaturation procedure. From these resulte, the relation between the foaming power of the protein and the characters of its surface monolayer was discussed.     

PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED,ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES : I. CHEMICAL PROPERTIES,ACTIVATION-INHIBITION,pH-ACTIVITY,AND TEMPERATURE-ACTIVITY CURVES

1. A study has been made of the properties of a hitherto unreported proteolytic enzyme from the latex of the milkweed, Asclepias speciosa. The new protease has been named asclepain by the authors. 2. The results of chemical, diffusion, and denaturation tests indicate that asclepain is a protein. 3. Like papain, asclepain dots milk and digests most proteins, particularly if they are dissolved in concentrated urea solution. Unlike papain, asclepain did not clot blood. 4. The activation and inhibition phenomena of asclepain resemble those of papain, and seem best explained on the assumption that free sulfhydryl in the enzyme is necessary for proteolytic activity. The sulfhydryl of asclepain appears more labile than that of papain. 5. The measurement of pH-activity curves of asclepain on casein, ovalbumin, hemoglobin, edestin, and ovovitellin showed no definite digestion maxima for most of the undenatured proteins, while in urea solution there were well defined maxima near pH 7.0. Native hemoglobin and ovovitellin were especially undigestible, while native casein was rapidly attacked. 6. Temperature-activity curves were determined for asclepain on hemoglobin, casein, and milk solutions. The optimum temperature was shown to increase with decreasing time of digestion.     

Interactions of ovalbumin and of its putative signal sequence with phospholipid monolayers. Possible importance of differing lateral stabilities in protein translocation.

Surface properties of ovalbumin and of its putative signal sequence, and their interactions with phospholipids at an air-water interface, have been studied. The mature protein can form an interfacial film spontaneously from its bulk solution, whereas the signal sequence cannot. Mature ovalbumin also penetrates phospholipid monolayers from the subphase (independently of the type of phospholipid present), whereas its signal sequence does not. The surface stability of a spread film of the signal sequence is, however, higher than that of a film of mature ovalbumin. Above specific threshold concentrations of signal peptide and of mature ovalbumin in mixed films with phospholipids, two separate phases are formed. In such immiscible films, the signal sequence peptide is also able to support a higher lateral surface pressure than mature ovalbumin, at corresponding areas of peptide and mature protein in the mixed monolayers. It is suggested that the differing lateral stabilities of ovalbumin and of its putative signal sequence may be relevant to the translocation of ovalbumin across the membrane of the endoplasmic reticulum, and a scheme for its translocation is proposed that is based on these properties.     

Natural rubber latex protein reduction with an emphasis on enzyme treatment

Natural rubber latex (NRL), derived from the Hevea brasiliensis tree, is a material used to manufacture products in health care, including medical gloves. Proteins are a naturally occurring component of NRL. These proteins, which can be present on the surface of NRL gloves, have been related to hypersensitivity reactions in some humans who come into contact with them. These same proteins also help to maintain the latex colloidal stability during collection and transport prior to manufacture. Consequently, when measures are taken to remove or degrade these proteins, other problems can be introduced, such as destabilization of the latex and changes in its coagulation properties. Practical methods are available to reduce the extractable antigenic protein content of NRL products. We describe here methods of reducing proteins in commercial-grade NRL and finished products. NRL gloves manufactured with adequate leaching can produce products with lower levels of extractable antigenic proteins. Emphasis is given here to enzyme treatment of NRL, as this process is very effective in reducing antigenic proteins in NRL. While this technology adds marginally to the production cost of standard grades of NRL, it is still quite cost-effective when compared with postwashing NRL products or the use of synthetic latex. Moreover, enzyme-treated NRL maintains the excellent physical properties and performance of NRL.     

The ovalbumin gene family: complete sequence and structure of the Y gene.

The "ovalbumin Y" gene, one of three which constitute the ovalbumin gene family in chicken has been completely sequenced. The exact location of exons can be derived from the comparison with the ovalbumin gene sequence and from the map previously established by electron microscopy analysis. During evolution of the Y gene, selective pressure has operated to retain a sequence coding for an ovalbumin-like protein. The location of splice junctions, the length of protein coding exons and the reading phase are as in the ovalbumin gene. The overall homology between the Y and ovalbumin protein coding sequences is 72.6% (resulting in a 58% homology for the amino acid sequences). A significantly high number of base changes within coding sequences are present in clusters, which appear in several cases to be correlated with the occurrence of direct repeats. The 3' untranslated sequences of the Y and ovalbumin mRNAs have diverged much more, and the Y sequence contains a peculiar U(T) rich region. Corresponding introns of the ovalbumin and Y genes differ extensively both in sequence and in length. They share however characteristic biases in their base distribution.     

Ovalbumin-related Protein X Is a Heparin-binding Ov-Serpin Exhibiting Antimicrobial Activities

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys³⁶⁷-His³⁶⁸ is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.