Decrease in rice allergenic proteins of polished rice grains by incubating with a miso solution

The effect of miso on allergenic proteins in rice seeds was investigated. When polished rice grains were incubated at 37 degrees C for 30-120 min with a 10% miso solution, but not with heat-treated miso or 1% NaCl, the amount of soluble proteins extracted from the rice grains with 1 M NaCl markedly decreased. SDS-PAGE, immunoblotting and densitometric analyses of these soluble proteins and insoluble proteins indicate that 26 kDa globulin and 14-16 kDa allergens in the grains were decreased to 15-60% during incubation with the miso solution, especially soybean-koji miso, without any large change in the content of major insoluble proteins.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Decrease in Rice Allergenic Proteins of Polished Rice Grains by Incubating with a Miso Solution

The effect of miso on allergenic proteins in rice seeds was investigated. When polished rice grains were incubated at 37°C for 30-120 min with a 10% miso solution, but not with heat-treated miso or 1% NaCl, the amount of soluble proteins extracted from the rice grains with 1 M NaCl markedly decreased. SDS-PAGE, immunoblotting and densitometric analyses of these soluble proteins and insoluble proteins indicate that 26 kDa globulin and 14-16 kDa allergens in the grains were decreased to 15-60% during incubation with the miso solution, especially soybean-koji miso, without any large change in the content of major insoluble proteins.     

Generation of transgenic rice lines with reduced contents of multiple potential allergens using a null mutant in combination with an RNA silencing method

Rice seed proteins are known to be a causative antigen in some patients with food allergy, especially cereal allergy, with clinical symptoms such as eczema and dermatitis. The α-amylase/trypsin inhibitors (14-16 kDa), α-globulin (26 kDa) and β-glyoxalase I (33 kDa) are regarded as major potential allergens of rice (Oryza sativa L.) seed based on specific recognition by serum IgE from allergy patients. In order to suppress the production of these major allergens in rice grains, a mutant in the 'Koshihikari' background lacking the 26 kDa allergen (GbN-1) was used as a host for RNA silencing. A binary vector harboring two RNA interference (RNAi) gene cassettes for suppression of 14-16 kDa and 33 kDa allergens driven by the 13 kDa and 10 kDa prolamin endosperm-specific promoters, respectively, was introduced into the GbN-1 genome by Agrobacterium-mediated transformation. In the most promising transgenic line, the content of the three potential allergens was remarkably reduced to a very faint level without a change in seed phenotype. IgE binding of 15 patients' sera to the transgenic rice seed mostly deficient in the three major allergens was on average only about 10% that of the control wild-type rice, suggesting that these three accounted for the great majority of rice seed causative allergens recognized by patients' IgE and that the sequential allergen deletion/reduction strategy works in the development of hypo-allergenic rice lines.     

Rice Allergenic Protein and Molecular-genetic Approach for Hypoallergenic Rice

Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley α-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker’s asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.     

ATP-stimulated protease activity in brown fat mitochondria: response to a 24-h fast in mice.

Brown fat mitochondria have [3H]casein-hydrolyzing activity at pH 8.0 associated with both membrane and soluble fractions. An ATP-stimulated proteolytic activity inhibited by vanadate and N-ethylmaleimide was found in the soluble fraction. Membrane-associated proteolytic activity was inhibited by phenylmethylsulfonyl fluoride and trypsin inhibitor, suggesting that it is a serine protease. A 24-h fast in mice caused a significant loss of mitochondrial proteins from the tissue, but had no effect on protease activity of isolated mitochondria with or without ATP. The ATP-stimulated release of amino acids or peptides from isolated mitochondria, as measured with fluorescamine, was not influenced by food deprivation. Thus, brown fat mitochondria possess an ATP-stimulated proteolytic pathway that does not appear to be involved in the bulk removal of mitochondrial proteins from brown fat of fasting mice.     

Aeroallergens of plant origin: Molecular basis and aerobiological significance

This review presents an update on the sources and molecular basis of aeroallergens of plants, derived from pollen, seeds, leaf and stem detritus and their protein molecules. These aeroallergens are a natural component of the atmosphere, either because of their natural function or human activity. Pollen is a source of allergens within the 10–200 μm size range, and while most allergenic pollen types account for only 20–30% of total annual pollen catch, during their flowering season, they are usually the dominant type. Tree pollen commences the season in winter, with birch pollen counts in Scandinavia being the highest daily pollen counts yet reported and a major allergen, a 14-kDa protein, which is similar to pathogenesis-related proteins. Grass pollen follows in spring, and is unique as its two immunodominant allergens, a 35-kDa glycoprotein and 28–32-kDa protein, are in different cellular sites: the cytosol and surface of pollen grains; and in intracellular starch granules. The allergens at the pollen surface are not inhalable and can interact only with the eyes, nasal and oral cavities. Starch granules are released to the atmospheric aerosol when grains rupture in rainwater. These are a major source of allergen-containing micronic particles, which are important because they are inhalable. At the same time, allergen molecules are present in the aerosol, and these can bind to soot particles, and so be respired deep into the airways. The major Japanese cedar pollen allergen has been detected both within the pollen and in orbicules; particles less than 1 μm that line the anther cavity and can be released into the air when dehiscence occurs. Ragweed is the major cause of late summer hayfever in eastern North America, where its pollen accounts for up to 41% of the annual pollen catch. It is a major source of aeroallergens in both respirable and non-respirable size ranges. As a result of human activity, dusts derived from seeds and cereal grains during transport, storage and milling provide a source of micronic particles, containing potent allergens that can trigger allergic disease.     

Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.     

Molecular chaperones cooperate with PIM1 protease in the degradation of misfolded proteins in mitochondria.

ATP dependent proteolytic degradation of misfolded proteins in the mitochondrial matrix is mediated by the PIM1 protease and depends on the molecular chaperone proteins mt-hsp70 and Mdj1p. Chaperone function is essential to maintain misfolded proteins in a soluble state, a prerequisite for their degradation by PIM1 protease. In the absence of functional mt-hsp70 or Mdj1p misfolded proteins either remain associated with mt-hsp70 or form aggregates and thereby are no longer substrates for PIM1 protease. Mdj1p is shown to regulate the ATP dependent association of an unfolded polypeptide chain with mt-hsp70 affecting binding to as well as release from mt-hsp70. These findings establish a central role of molecular chaperone proteins in the degradation of misfolded proteins by PIM1 protease and thereby demonstrate a functional interrelation between components of the folding machinery and the proteolytic system within mitochondria.     

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.     

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.     

Yeast ubiquitin-conjugating enzymes involved in selective protein degradation are essential for cell viability.

Ubiquitin-mediated proteolysis is a major pathway for selective protein degradation in eukaryotic cells. This proteolysis pathway involves the processive covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by a specific ATP-dependent protease complex. We have cloned the genes and characterized the function of ubiquitin-conjugating enzymes (UBCs) from the yeast Saccharomyces cerevisiae. UBC1, UBC4 and UBC5 enzymes were found to mediate selective degradation of short-lived and abnormal proteins. These enzymes have overlapping functions and constitute a UBC subfamily essential for growth. UBC1 is specifically required at early stages of growth after germination of spores. UBC4 and UBC5 enzymes generate high molecular weight ubiquitin-protein conjugates and comprise a major ubiquitin-conjugation activity in yeast cells. Moreover, these enzymes are central components of the cellular stress response.     

A plasminogen-like protein selectively degrades stearoyl-CoA desaturase in liver microsomes

Stearoyl-CoA desaturase (SCD) is an integral membrane protein of the endoplasmic reticulum that is rapidly and selectively degraded when isolated liver microsomes are incubated at 37 degrees C. We previously reported the purification of a 90-kDa microsomal protein with SCD protease activity and characterized the inhibitor sensitivity of the protease. Here we show that the 90-kDa protein is a microsomal form of plasminogen (Pg) and that the purified SCD protease contains a spectrum of plasmin-like derivatives. The 90-kDa protein was identified as Pg by mass spectrometry of its tryptic peptides. The purified SCD protease reacted with Pg antibody, and immunoblotting demonstrated enrichment of Pg by the purification procedure established for the SCD protease. Analysis of microsomes by zymography demonstrated a single band of proteolytic activity at 70-kDa corresponding to the mobility of Pg in nonreduced polyacrylamide gels. When microsomes were incubated at 37 degrees C prior to zymography, an intense band of proteolytic activity developed at 30-kDa. The purified SCD protease displayed a spectrum of proteolytic bands ranging from 70 to 30 kDa. Degradation of SCD by the purified protease and by microsomes was inhibited by bdellin, a plasmin inhibitor from the medicinal leech Hirudo medicinalis. To explore the role of Pg in the degradation of SCD in vivo, we examined SCD expression and degradation in microsomes isolated from Pg-deficient (Pg-/-) mice. Compared with microsomes from wild-type littermate control mice, liver microsomes from Pg-/- mice had significantly higher levels of SCD. Degradation of SCD in microsomes from Pg-/- mice was markedly diminished, whereas liver microsomes from control mice showed rapid SCD degradation similar to that observed in rat liver microsomes. These findings indicate that SCD is degraded by a protease related to Pg and suggest that plasmin moonlights as an intracellular protease.     

The 26S proteasome: ubiquitin-mediated proteolysis in the tunnel

The 26S proteasome is a self-compartmentalizing protease responsible for the degradation of intracellular proteins. This giant intracellular protease is formed by several subunits arranged into two 19S polar caps-where protein recognition and ATP-dependent unfolding occur-flanking a 20S central barrel-shaped structure with an inner proteolytic chamber. Proteins targeted to the 26S proteasome are conjugated with a polyubiquitin chain by an enzymatic cascade before delivery to the 26S proteasome for degradation into oligopeptides. As a self-compartmentalizing protease, the 26S proteasome circumvents proteins not destined for degradation and can be deployed to the cytoplasmic and nuclear compartments. The 26S proteasome is a representative of emerging group of giant proteases, including tricorn protease, multicorn protease, and TPPII (tripeptidyl peptidase II).     

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b ₆ protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.     

Effects of calcium and magnesium on a 41-kDa serine-dependent protease possessing collagen-cleavage activity

We report here the continued characterization of a 41-kDa protease expressed in the early stage of the sea urchin embryo. This protease was previously shown to possess both a gelatin-cleavage activity and an echinoderm-specific collagen-cleavage activity. In the experiments reported here, we have explored the biochemical nature of this proteolytic activity. Pepstatin A (an acidic protease inhibitor), 1,10-phenanthroline (a metalloprotease inhibitor), and E-64 (a thiol protease inhibitor) were without effect on the gelatin-cleavage activity of the 41-kDa species. Using a gelatin substrate gel zymographic assay, the serine protease inhibitors phenylmethylsulfonyl fluoride and benzamide appeared to partially inhibit gelatin-cleavage activity. This result was confirmed in a quantitative gelatin-cleavage assay using the water soluble, serine protease inhibitor [4-(2-aminoethyl)benzenesulfonylfluoride]. The biochemical character of this protease was further explored by examining the effects of calcium and magnesium, the major divalent cations present in sea water, on the gelatin-cleavage activity. Calcium and magnesium competed for binding to the 41-kDa collagenase/gelatinase, and prebound calcium was displaced by magnesium. Cleavage activity was inhibited by magnesium, and calcium protected the protease against this inhibition. These results identify calcium and magnesium as antagonistic agents that may regulate the proteolytic activity of the 41-kDa species.     

Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis

GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.