A specific wheat germ agglutinin-immunoreactive protein in human placenta

In this study we examined human placenta for the presence of molecules antigenically related to a plant lectin, wheat germ agglutinin. The initial results of immunolocalization using polyclonal antibodies against wheat germ agglutinin showed that human placenta contains protein(s) recognized specifically. Staining of syncytiotrophoblast brush border and cytotrophoblast, granular in appearance was observed in first trimester human placenta. Specific binding was also seen in trophoblast-derived JAr and BeWo carcinoma cells. Isolation of wheat germ agglutinin-immunoreactive material from human placenta was achieved by ion-exchange- and affinity-chromatography on anti-wheat germ agglutinin-immunoglobulin G-Sepharose. The placental protein having molecular mass of 66 kD was identified as specific. The protein of 66 kD was characterized as a calcium-dependent, asialofetuin-binding molecule.     

Identification of protein fragments as pattern features in MALDI-MS analyses of serum

The use of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) to acquire spectral profiles has become a common approach to detect proteomic biomarkers of disease. MALDI-MS signals may represent both intact proteins as well as proteolysis products. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis can tentatively identify the corresponding proteins Here, we describe the application of a data analysis utility called FragMint, which combines MALDI-MS spectral data with LC-MS/MS based protein identifications to generate candidate protein fragments consistent with both types of data. This approach was used to identify protein fragments corresponding to spectral signals in MALDI-MS analyses of unfractionated human serum. The serum also was analyzed by one-dimensional SDS-PAGE and bands corresponding to the MALDI-MS signal masses were excised and subjected to in-gel digestion and LC-MS/MS analysis. Database searches mapped all of the identified peptides to abundant blood proteins larger than the observed MALDI-MS signals. FragMint identified fragments of these proteins that contained the MS/MS identified sequences and were consistent with the observed MALDI-MS signals. This approach should be generally applicable to identify protein species corresponding to MALDI-MS signals.     

Wheat acetyl-CoA carboxylase

The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of ¹⁴C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop.     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

A proteomic approach to the identification and characterisation of protein composition in wheat germ

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0–7.0 and 6.0–9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.     

Differential inhibition of protein synthesis in reticulocyte lysates and wheat germ extracts by a protein isolated from wheat germ extracts.

A protein with a molecular mass of 35-37 kDa has been isolated and partially purified from the postribosomal supernatant of wheat germ by ammonium sulfate precipitation (60-90%), Sephadex G-75, and DEAE-cellulose chromatography. It inhibited endogenous protein synthesis in rabbit reticulocyte lysates but had no effect on translation in wheat germ extracts. At low concentrations (0.34-1.36 ng/15 microliter assay), inhibition was limited to initiation of peptide synthesis. At higher concentrations (13.6 ng/15 microliter assay), elongation was also suppressed.     

Purification and properties of a high specific activity protein kinase from wheat germ

A protein kinase was extensively purified to near-homogeneity from wheat germ by a procedure involving affinity chromatography on casein-Sepharose 4B, gel filtration, and repeated chromatography on carboxymethyl-Sepharose CL-6B. The protein kinase preparations have the highest specific activities (up to 656 nanomoles phosphate incorporated per minute per milligram of protein) yet reported for plant protein kinases. The major polypeptides in purified preparations were revealed as two barely-resolved bands (molecular weight 31,000) on polyacrylamide gel electrophoresis in subunit-dissociating conditions. The molecular size of the protein kinase as determined from gel filtration is 30,000. The protein kinase catalyzes the phosphorylation of casein, phosvitin, and the wheat germ cyclic AMP-binding protein cABPII but not of bovine serum albumin and histones nor of the wheat germ cytokinin-binding protein CBP. The protein kinase has a pH optimum of 7.9 and a K_m value for ATP of 10 micromolar. The protein kinase differs from wheat germ CBP kinase in molecular weight, differential sensitivity to inhibitors, and in substrate specificity.     

Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin-resistant boar seminal plasma O-glycoprotein related to pB1 protein.

The complete covalent structure of a novel boar DQH sperm surface protein resistant to many classical procedures of enzymatic fragmentation was determined. The relative molecular mass of the major form of this protein determined by ESI-MS and MALDI-MS was 13,065.2+/-1.0 and 13,065.1, respectively. However, additional peaks differing by 162 Da (i.e., minus hexose), 365 Da (i.e., minus hexose and N-acetylhexosamine), 146 Da (i.e., plus deoxyhexose), and 291 Da (i.e., plus sialic acid) indicated the heterogeneity due to differences in glycosylation. The complete covalent structure of the protein was determined using automated Edman degradation, MALDI-MS, and post-source decay (PSD) MALDI-MS, and shown to consist of N-terminal O-glycosylated peptide followed by two fibronectin type II repeats. The carbohydrates are O-glycosidically linked to threonine 10, as confirmed by PSD MALDI-MS of the isolated N-terminal glycopeptide. Eight cysteine residues of the protein form four disulfide bridges, the positions of which were assigned from MALDI-MS and Edman degradation data. We conclude that mass spectral techniques provide an indispensable tool for the detailed analysis of the covalent structure of proteins, especially those that are refractory to standard approaches of protein chemistry.     

A novel protein programmed by the mRNA conserved in dry wheat embryos. The principal site of cysteine incorporation during early germination

If bulk mRNA from dry wheat embryos (wheat germ) is used to direct cell-free incorporation of [35S]cysteine into proteins, a striking proportion of the total radioactivity is channeled into a single protein. During early postimbibition development, when protein synthesis is directed by the mRNA conserved in dry embryos, incorporation of cysteine is preponderantly (20-25%) directed into synthesis of this one protein: the 'early' cysteine-labeled protein (Ec). When conserved mRNA from the dry embryos has been fully degraded, as when cellular or cell-free protein synthesis is directed by the mRNA in germinated embryos, synthesis of Ec is not detected. Reliable detection of Ec requires prior alkylation of wheat embryo proteins, and it was especially interesting to find that when wheat embryo proteins are alkylated by iodo[14C]acetamide, two proteins co-dominate the distribution of radioalkylated products in dodecylsulphate/polyacrylamide gels: Ec and wheat germ agglutinin. Using co-electrophoresis with the isotopically labeled protein to detect a dye-staining counterpart, Ec has been purified by combined cation-exchange and gel-filtration chromatography of alkylated wheat germ proteins. The purified protein can be recovered in milligram quantity (5-10 mg/100 g wheat germ) and compositional analysis shows that it is unusually rich in cysteine (approx. 15%) and glycine (approx. 17%), as is wheat germ agglutinin.     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

Sucrose-phosphate synthase from wheat. Characterization of peptides by immunoblotting analysis

The structure of sucrose-phosphate synthase (SPS: EC 2.4.1.14) from wheat ( Triticum aestivum L. cv. San Agustin was studied using antibodies prepared against the enzyme purified from wheat germ. The antibodies revealed the presence of 55 and 35 kDa polypeptides in wheat germ, endosperm, embryos and whole seed, while in whole wheat leaf, a 90 kDa was detected. It is not clear whether the 35 and 55 kDa polypeptide are truly subunits of SPS or they are the product of protease action, more active in non-photosynthetic tissues than in leaves. The antibodies from wheat germ clearly recognized polypeptides in leaf protein preparations from other plants (barley, soybean, maize) and, weakly in others (peanut, tobacco). It did not recognize any polypeptide in spinach and mustard leaf extracts. In the case of maize leaf, a peptide of higher molecular mass (116 kDa) than the wheat ones was revealed. The results may indicate the presence of different polypeptide compositions for sucrose-phosphate synthase, and suggest the existence of at least two types of this enzyme.     

Wheat germ systems for cell-free protein expression

Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.     

Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.     

Protein translocation across wheat germ microsomal membranes requires an SRP-like component

Different wheat germ extracts were tested for the presence of membranes capable of translocating and processing nascent secretory proteins. One lysate was found in which nascent prehuman-placental lactogen (phPL) was translocated and processed to mature human placental lactogen (hPL). Processing was found to occur concomitant with translocation across membranes. Translocation across the wheat germ membrane required a component which is similar to the mammalian signal recognition particle (SRP). It bound to DEAE–Sepharose, had a sedimentation coefficient of 11S and contained a 7S RNA. In addition to hPL, the plant protein zein and the bacterial protein β-lactamase were translocated across and processed by wheat germ membranes. Transport was found to occur only co-translationally. Our results show that the wheat germ protein translocation system is similar to the mammalian one. Unlike the mammalian SRP, the particle purified from wheat germ did not arrest elongation of nascent secretory proteins.     

A strategy to improve peptide mass fingerprinting matches through the optimization of matrix-assisted laser desorption/ionization matrix selection and formulation

Peptide mass fingerprinting (PMF) is a powerful technique in which experimentally measured m/z values of peptides that result from a protein digest form the basis for a characteristic fingerprint of the intact protein. Due to its propensity to generate singly-charged ions, along with its relative insensitivity to salts and buffers, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is the MS method of choice for PMF. The qualitative features of a MALDI-MS mass spectrum can be selectively tuned by varying the matrix and the solvent system used to prepare the matrix. The selective tuning of MALDI-MS mass spectra in order to optimize PMF results is addressed in this paper. Carbonic anhydrase, hemoglobin alpha- and beta-chain, and myoglobin were digested with trypsin, and the digest was analyzed with MALDI-MS. 2,5-Dihydroxybenzoic acid (2,5-DHB) and alpha-cyano-4-hydroxycinnamic acid were prepared, using five different solvent systems: (A) 99% acetone; (B) 50% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA); (C) 75% ACN, 0.1% TFA; (D) formic acid:H(2)O: 2-propanol (1:3:2); and (E) H(2)O:MeOH (2:1). Each protein was found to have a different optimum solvent system for PMF. Generally, better PMF results were obtained with 2,5-DHB. The best PMF results were obtained when all of the mass spectral data for a particular protein digest were convolved.     

Purification and Properties of a Protein Which Binds Cytokinin-active 6-Substituted Purines

A protein which binds 6-substituted purines of the cytokinin type with relatively high affinity has been extensively purified from wheat germ. Conventional chromatographic techniques, as well as an affinity matrix to which a cytokinin was covalently coupled, were used in the purification. The wheat germ cytokinin-binding protein (CBF-1) has four unlike subunits and an apparent molecular weight of 183,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

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.     

The development of a matrix-assisted laser desorption/ionization mass spectrometry-based method for the protein fingerprinting and identification of Aeromonas species using whole cells

This report describes the development of a method to detect the waterborne pathogen Aeromonas using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, MALDI-MS was applied to the characterization of seventeen species of Aeromonas. These seventeen species were represented by thirty-two strains, which included type, reference and clinical isolates. Intact cells from each strain were used to generate a reproducible library of protein mass spectral fingerprints or m/z signatures. Under the test conditions used, peak lists of the mass ions observed in each species revealed that three mass ions were conserved among all the seventeen species tested. These common mass ions having an average m/z of 6301, 12,160 or 12,254, and 13,450, can be potentially used as genus-specific biomarkers to identify Aeromonas in unknown samples. A dendrogram generated using the m/z signatures of all the strains tested indicated that the mass spectral data contained sufficient information to distinguish between genera, species, and strains. There are several advantages of using MALDI-MS based protein mass spectral fingerprinting of whole cells for the identification of microorganisms as well as for their differentiation at the sub-species level: (1) the capability to detect proteins, (2) high throughput, and (3) relatively simple sample preparation techniques. The accuracy and speed with which data can be obtained makes MALDI-MS a powerful tool especially suited for environmental monitoring and detection of biological hazards.     

Small protein biomarkers of culture in Bacillus spores detected using capillary liquid chromatography coupled with matrix assisted laser desorption/ionization mass spectrometry

Capillary liquid chromatography (cLC) coupled with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS) was used to compare small proteins and peptides extracted from Bacillus subtilis spores grown on four different media. A single, efficient protein separation, compatible with MALDI-MS analysis, was employed to reduce competitive ionization between proteins, and thus interrogate more proteins than possible using direct MALDI-MS. The MALDI-MS data files for each fraction are assembled as two-dimensional data sets of retention time and mass information. This method of visualizing small protein data required careful attention to background correction as well as mass and retention time variability. The resulting data sets were used to create comparative displays of differences in protein profiles between different spore preparations. Protein differences were found between two different solid media in both phase bright and phase dark spore phenotype. The protein differences between two different liquid media were also examined. As an extension of this method, we have demonstrated that candidate protein biomarkers can be trypsin digested to provide identifying peptide fragment information following the cLC-MALDI experiment. We have demonstrated this method on two markers and utilized acid breakdown information to identify one additional marker for this organism. The resulting method can be used to identify discriminating proteins as potential biomarkers of growth media, which might ultimately be used for source attribution.     

Partial purification and characterization of the proteins from the 40-S ribosomes of Artemia salina and wheat germ.

The proteins were extracted from purified 40-S ribosomes derived from wheat germ and Artemia salina and separated by carboxymethylcellulose ion-exchange chromatography. Approximately four proteins from Artemia and four proteins from wheat germ were separated in a state of high purity. All proteins were identified by co-electrophoresis using a two-dimensional polyacrylamide gel system. A total of 30 unique proteins were found for Artemia and 32 proteins for wheat. The molecular weights of all proteins were estimated by sodium dodecylsulfate gel electrophoresis. Assuming each protein to be present in one copy per 40-S ribosome, the total protein molecular weight was estimated to be 560,000 associated with Artemia 40-S particles and 550,000 associated with wheat germ 40-S ribosomes.