Epitope Analysis of Soybean Major Allergen Gly m Bd 30K Recognized by the Mouse Monoclonal Antibody Using Overlapping Peptides

The epitopes of the major soybean allergen, Gly m Bd 30K, recognized by mouse monoclonal antibodies H6 and F5 were investigated by using synthetic peptides bound to pins. The epitopes are shown to be localized in peptide ³¹QGGCGRGWAFSATGAI-EA⁴⁸ for H6, and in ¹¹⁵ DKVTIDGYETLIMSDEST¹³² for F5.     

大豆(Glycine max(L.)Merr.)致敏蛋白Gly m Bd 30K检测方法及低致敏优异种质鉴定

Gly m Bd 30K蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应.因此,发掘低Gly m Bd 30K蛋白含量优异种质对于培育优质大豆品种具有重要意义.为了获得致敏蛋白Gly m Bd 30K低含量的优异种质,根据Gly m Bd 30K蛋白的190-379aa多肽序列制备多克隆抗体...     

Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.     

Macrophage Migration Inhibitory Factor: Identification of the 30-kDa MIF-Related Protein in Bovine Brain

Macrophage migration inhibitory factor (MIF) is a ubiquitous protein playing various immunologic, enzymatic, and hormonal roles. MIF was originally identified for its capacity to inhibit the random movement of macrophages in vitro. MIF is widely expressed in many tissues with particularly high levels in the nervous system. Using the reversed-phase HPLC, N-terminal microsequence analysis, and database searching, we have identified in bovine brain several MIF-like proteins. According to mass spectral analysis, the molecular masses for three of them were determined as 12,369.2, 12,299.7, and 9,496.2 Da. In addition, we have identified another MIF-related protein (29,568.9 Da) by Western blotting using anti-MIF antibody raised to MIF (having an apparent molecular weight of 12 kDa) isolated to homogeneity from bovine brain cytosol. The modified purification procedure was mainly based on exclusion- and ion-exchange chromatography. Using p-hydroxyphenylpyruvic acid as a substrate, we have demonstrated tautomerase activity of the isolated MIF. The N-terminal sequences for all MIF-like proteins were found to be identical. Several other higher molecular weight putative MIF-related proteins were also revealed in the bovine brain cytosol extract. A multifunctional nature of MIF is suggested to be a result of its occurrence in different oligomerization states in a wide variety of tissues and cells.     

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides.Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane.In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.     

Identification of (Na,K)ATPase inhibitor in brine shrimp,Artemia salina,as long-chain fatty acids

Summary An inhibitory activity to (Na,K)ATPase was found in cell extracts of the brine shrimp, Artemia salina, irrespective of its developmental stages. Organic solvent extraction together with gas chromatographic analysis reveals that the inhibitory activity is due to long-chain, non-esterified fatty acids and their derivatives. Unsaturated fatty acids, especially with cis-configuration, are more effective in inhibition than saturated ones.Abbreviations ATPase adenosine triphosphatase - EDTA ethylenediamine-tetraacetate - TLC thin-layer chromatography     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

Identification of a "glycine-loop"-like coiled structure in the 34 AA Pro,Gly,Met repeat domain of the biomineral-associated protein,PM27

Fracture resistance in biomineralized structures has been linked to the presence of proteins, some of which possess sequences that are associated with elastic behavior. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure and fracture-resistant properties of the calcium carbonate mineral phase within embryonic sea urchin spicules and adult sea urchin spines. In this report, we detail the identification of a repetitive keratin-like "glycine-loop"- or coil-like structure within the 34-AA (AA: amino acid) N-terminal domain, (PGMG)(8)PG, of the spicule matrix protein, PM27. The identification of this repetitive structural motif was accomplished using two capped model peptides: a 9-AA sequence, GPGMGPGMG, and a 34-AA peptide representing the entire motif. Using CD, NMR spectrometry, and molecular dynamics simulated annealing/minimization simulations, we have determined that the 9-AA model peptide adopts a loop-like structure at pH 7.4. The structure of the 34-AA polypeptide resembles a coil structure consisting of repeating loop motifs that do not exhibit long-range ordering. Given that loop structures have been associated with protein elastic behavior and protein motion, it is plausible that the 34-AA Pro,Gly,Met repeat sequence motif in PM27 represents a putative elastic or mobile domain.     

大豆蛋白对急性放射性肠炎大鼠血清氨基酸的影响

为确定大豆蛋白对急性放射性肠炎大鼠血清氨基酸的影响 ,用SD大鼠复制急性放射性肠炎动物模型 ,给予含大豆蛋白饲料 ,测量血清氨基酸含量。结果显示 :使用大豆蛋白后细菌移位率显著降低 ,血LPS含量和肠通透性均未见升高 ,回肠绒毛高度、肠全层厚度和粘膜厚度均高于实验对照组 ,血清谷氨酰胺和支链氨基酸的含量均显著高于对照组。说明大豆蛋白使大鼠血清谷氨酰胺和支链氨基酸的含量明显升高 ,可以提高急性放性射肠炎时的肠屏障功能。     

大豆Kunitz型胰蛋白酶抑制剂新类型Tid的全序列分析

大豆ｋｕｎｉｔｚ型胰蛋白酶抑制剂（ＳＢＴｉＡ２）是一种大量存在于大豆（Ｇｌｙｃｉｎｅｍａｘ）中的种子贮藏蛋白．虽然对它的生化特性及结构已有较多的研究,但它在体内的主要功能仍不很清楚．国际上所发现的３个由显性等位基因Ｔｉａ、Ｔｉｂ、Ｔｉｃ编码的大豆ｋｕｎｉｔｚ胰蛋白酶抑制剂的氨基酸顺序已被明确测定,相互间有一到多个氨基酸残基的不同［１］．Ｔｉｄ是从我国１５０００余份大豆资源中筛选到的唯一一份ｋｕｎｉｔｚ型胰蛋白酶抑制剂位点的新类型,遗传分析证明它是另一个ＳＢＴｉＡ２的显性等位基因［２,３］．严…     

Identification of Immunogenic Epitopes of the 170-kDa Subunit Adhesin of Entamoeba histolytica in Patients with Invasive Amebiasis

ABSTRACT. Entamoeba histolytica causes amebic dysentery (AD) and liver abscess (ALA). Little is known about protective immunity to amebiasis, and studies in this area have been complicated by the paucity of defined ameba antigens. We examined the proliferative responses of peripheral blood mononuclear cells (PBMC) from patients with AD and ALA to a recombinant protein containing a portion of the 170 kDa adhesin of E. histolytica (170CR), and to two synthetic peptides (1 and 2) derived from the 170 kDa sequence that were predicted to contain T cell epitopes. A significant number of patients with AD and ALA had PBMC that proliferated to 170CR molecule, and several individuals with ALA and AD had T cells that recognized one or both peptides. Contrarily, individuals from a non-endemic region for amebiasis did not respond to 170CR protein, or to both peptides. In regard to antibody response, nine of fifteen patients with ALA showed antibodies to 170CR protein. These same patients had antibodies to peptide 2. We identified peptides from 170-kDa adhesin that may contain both T and B cell epitopes recognized by some patients with invasive amebiasis. These peptides may be valuable reagents in studies of the immune response to amebiasis.     

Identification of a Glycosylphosphatidylinositol-anchored Plasma Membrane Protein Interacting with the C-terminus of Auxin-binding Protein 1: A Photoaffinity Crosslinking Study

Synthetic peptides corresponding to the C-terminus of auxin-binding protein 1 (ABP1) have been shown to function as auxin agonists. To define a C-terminal receptor, photoaffinity crosslinking experiments were performed using an azido derivative of a C-terminal peptide and plasma membranes from maize (Zea mays L.). The crosslinking reaction was monitored by immunoblotting using anti-ABP1 antibodies. The crosslinked proteins were isolated by 2D gel electrophoresis and identified by mass spectrometric analysis. Further, the noncrosslinked forms of these proteins were also identified. Two proteins with apparent molecular masses of 73 kDa (termed C-terminal peptide-binding protein 1, CBP1) and 35 kDa (CBP2) were specifically linked with the C-terminal peptide. CBP2 is a cytoplasmic protein that consists of two conserved domains that are characteristic of a ricin-type lectin domain. CBP2 remained in the detergent-insoluble particles and was released from the particles by the addition of monosaccharides such as methyl-β-d-galactopyranoside. CBP1 was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that CBP1 is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein. CBP1 was found to be a copper-binding protein, and is highly homologous to Arabidopsis thaliana SKU5 that contributes to directional root growth processes. Further, it is similar to A. thaliana SKS6 that contributes to cotyledon vascular patterning and to Nicotiana tabacum NTP303 that contributes to pollen tube growth. The present results indicate that ABP1 may contribute to directional cell growth processes via the GPI-anchored plasma membrane protein SKU5 and its family members.     

A Review of Soybean (Glycine max) Seed,Pod, and Flower Mycofloras in North America,with Methods and a Key for Identification of Selected Fungi

A review of the fungi associated with soybean seeds, pods, or flowers was conducted in North America. Species of Deuteromycetes are the most common fungi in each of the soybean flower organs followed by the Ascomycetes and Phycomycetes which comprise about one-fourth of the total mycoflora. Eighty genera and about 135 or more species occur in seeds, pods, or flowers. With regard to numbers of taxa from separate mycofloras, 63 genera and about 108 or more species occur in seeds, 65 genera and about 88 or more species occur in pods, and 36 genera and approximately 47 or more species occur in flowers. Most of the fungi which occur in flowers can be cultured from pods, and the majority of those fungi occur in seeds. Methods for and a key are provided for the identification of the 30 most important selected fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors,clorobiocin

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3′ position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5′-adenylyl-β,γ-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented. © 1997 Wiley-Liss Inc.     

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus

ABSTRACT

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

Identification and Characterization of a Host Reversibly Glycosylated Peptide that Interacts with the Tomato leaf curl virus V1 Protein

Monopartite geminiviruses of the genus Begomovirus have two virion-sense genes, V1 and V2. V2 encodes the viral coat protein, but the function of V1 is largely unknown, although some studies suggest that it may play a role in cell-to-cell movement. Yeast two-hybrid technology was used to identify possible host binding partners of V1 from Tomato leaf curl virus (TLCV) to better understand its function. A protein closely related to a family of plant reversibly glycosylated peptides, designated SlUPTG1, was found to interact with V1 in yeast and in vitro. SlUPTG1 may function endogenously in the synthesis of cell wall polysaccharides, since a bacterially expressed form of the protein acted as an autocatalytic glycosyltransferase in␣vitro, a SlUPTG1:GFP fusion protein localized to the cell wall, and expression of SlUPTG1 appeared to be highest in actively dividing tissues. However, expression of SlUPTG1 in a transient TLCV replication assay increased the accumulation of viral DNA, suggesting that this host factor also plays a role in viral infection. Together, these data provide new insight into the role of V1 in TLCV infection and reveal another host pathway which geminiviruses may manipulate to achieve an efficient infection.     

Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

Protein kinase activity on the cell surface of a macrophage-like cell line,J774.1 cells

Protein kinase activity was demonstrated on the cell surface of a murine macrophage-like cell line, J774.1 cells, and was characterized in detail. When intact cells were incubated with [γ-³²P]ATP, a transfer of [³²P]phosphate into acid-insoluble materials of the cells occurred. This reaction was Mg²⁺-dependent but cAMP-independent, and Mg²⁺ could be substituted for by Mn²⁺. The reaction products were found to be proteins, as revealed by SDS-polyacrylamide gel electrophoresis and autoradiography, with phosphomonoester linkages to serine and threonine residues, but not to tyrosine. The results of experiments with chemical and enzymatic treatments as well as Con A-Sepharose column chromatography ruled out the possibility that an acyl-phosphate linkage or phosphomannosylglycopeptide was present in the reaction products. The protein kinase(s) and the reaction products were located on the cell surface of the cells, as shown by the fact that the products were removed by mild trypsinization of cells carefully controlled so that the cells remained in an intact state. Phosphorylation of exogenous proteins (phosvitin and casein) by intact cells further supported the location of the enzyme. The phosphorylated proteins of the cells were found to be metabolically stable and remained on the cell surface even at 120 min after the phosphorylation reaction. Possible roles of ecto-protein kinase activity in macrophage functions and macrophage-activation are also discussed.