Amino acid and cDNA sequences of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L.)

The amino acid sequence of a methionine-rich 2S seed protein from sunflower (Helianthus annuus L.) and the sequence of a cDNA clone which codes for the entire primary translation product have been determined. The mature protein consists of a single polypeptide chain of 103 amino acids (molecular mass 12133 Da) which contains 16 residues of methionine and 8 residues of cysteine. The cDNA sequence established that the protein is synthesized as a precursor of 141 residues with a typical hydrophobic signal sequence of 25 residues followed by a further 13-residue hydrophobic pro-sequence which is presumably removed by post-translational cleavage. The sequence of the mature protein and that deduced from the cDNA were identical with no evidence of processing at the C-terminus. Comparison of the sunflower methionine-rich protein sequence with sequences of other seed 2S proteins from dicotyledons and monocotyledons showed limited but distinct sequence similarities; in particular the arrangement of the cysteine residues was conserved. The sunflower protein shows 34% identity with the methionine-rich Brazil nut 2S protein and the prepro regions of the precursors of these two proteins show about 50% identity. This similarity indicates that these methionine-rich 2S proteins have diverged as a subclass of the 2S superfamily of proteins which contain only 2-3% methionine. While the related 2S proteins from other dicotyledons are processed to a small and large subunit, the sunflower protein is not cleaved in this way.     

Solution structure of a methionine-rich 2S albumin from sunflower seeds: relationship to its allergenic and emulsifying properties

The three-dimensional structure in aqueous solution of SFA-8, a 2S albumin 103-residue protein from seeds of sunflower (Helianthus anuus L.), has been determined by NMR methods. An almost complete (1)H resonance assignment was accomplished from analysis of two-dimensional (2D) COSY and 2D TOCSY spectra, and the structure was computed by using restrained molecular dynamics on the basis of 1393 upper limit distance constraints derived from NOE cross-correlation intensities measured in 2D NOESY spectra. In contrast with most other 2S albumins, SFA-8 consists of a single polypeptide chain without any cleavage in the segment of residues 30-46. The computed structures exhibited an rmsd radius of 0.52 A for the backbone structural core (residues 11-30 and 46-101) and 1.01 A for the side chain heavy atoms. The resulting structure consists of five amphipathic helices arranged in a right-handed superhelix, a folding motif first observed in nonspecific lipid transfer (nsLTP) proteins, and common to other 2S albumins. In contrast to nsLTP proteins, neither SFA-8 nor RicC3 (a 2S albumin from castor bean) has an internal cavity that is able to host a lipid molecule, which results from an exchange in the pairing of disulfide bridges in the CXC segment. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamin superfamily, which includes a number of important food allergens. Differences in the extension and solvent exposition of the so-called "hypervariable loop" (which connects helices III and IV) in SFA-8 and RicC3 may be responsible for the different allergenic properties of the two proteins. SFA-8 has been shown to form highly stable emulsions with oil/water mixtures. We propose that these properties may be determined partly by a hydrophobic patch at the surface of the protein which consists of five methionines that partially hide the Trp76 residue. The flexibility of the loop which contains Trp76 and the hydrophobicity of the whole environment may favor a conformational change, by which the Trp76 side chain may become inserted into the oil phase.     

Characterization of MRNP34, a novel methionine-rich nacre protein from the pearl oysters

Nacre of the Pinctada pearl oyster shells is composed of 98% CaCO₃ and 2% organic matrix. The relationship between the organic matrix and the mechanism of nacre formation currently constitutes the main focus regarding the biomineralization process. In this study, we isolated a new nacre matrix protein in P. margaritifera and P. maxima, we called Pmarg- and Pmax-MRNP34 (methionine-rich nacre protein). MRNP34 is a secreted hydrophobic protein, which is remarkably rich in methionine, and which is specifically localised in mineralizing the epithelium cells of the mantle and in the nacre matrix. The structure of this protein is drastically different from those of the other nacre proteins already described. This unusual methionine-rich protein is a new member in the growing list of low complexity domain containing proteins that are associated with biocalcifications. These observations offer new insights to the molecular mechanisms of biomineralization.     

Purification and characterization of homogeneous sunflower seed acid phosphatase

Acid phosphatase (EC 3.1.3.2) from sunflower seed was purified 1800-fold to homogeneity using both conventional and affinity chromatographic methods. The purified enzyme was a mixture of two enzyme forms distinguishable by polyacrylamide gel electrophoresis (PAGE). Gel exclusion chromatography, which did not distinguish between the two forms, gave an apparent M, of 103 000. Preparative PAGE permitted the separation of the two forms, and SDS-PAGE showed that they contained equivalent peptide subunits of apparent M, 56 000 and 52 000. Amino acid analysis indicated that both enzyme forms have similar amino acid compositions. Data on substrate specificity and pH dependence is presented. The kinetic constants for hydrolysis of p-nitrophenyl phosphate as catalysed by sunflower seed acid phosphatase were independent of pH in the range 3-5. The enzyme was competitively inhibited by inorganic phosphate and non-competitively inhibited by phosphomycin.     

Crystal structure and dimerization equilibria of PcoC, a methionine-rich copper resistance protein from Escherichia coli

PcoC is a soluble periplasmic protein encoded by the plasmid-born pco copper resistance operon of Escherichia coli. Like PcoA, a multicopper oxidase encoded in the same locus and its chromosomal homolog CueO, PcoC contains unusual methionine rich sequences. Although essential for copper resistance, the functions of PcoC, PcoA, and their conserved methionine-rich sequences are not known. Similar methionine motifs observed in eukaryotic copper transporters have been proposed to bind copper, but there are no precedents for such metal binding sites in structurally characterized proteins. The high-resolution structures of apo PcoC, determined for both the native and selenomethionine-containing proteins, reveal a seven-stranded beta barrel with the methionines unexpectedly housed on a solvent-exposed loop. Several potential metal-binding sites can be discerned by comparing the structures to spectroscopic data reported for copper-loaded PcoC. In the native structure, the methionine loop interacts with the same loop on a second molecule in the asymmetric unit. In the selenomethionine structure, the methionine loops are more exposed, forming hydrophobic patches on the protein surface. These two arrangements suggest that the methionine motifs might function in protein-protein interactions between PcoC molecules or with other methionine-rich proteins such as PcoA. Analytical ultracentrifugation data indicate that a weak monomer-dimer equilibrium exists in solution for the apo protein. Dimerization is significantly enhanced upon binding Cu(I) with a measured delta(deltaG degrees )相似文献   

Polymorphism and inheritance of seed storage protein in sunflower

The data on polymorphism and inheritance of the seed storage protein helianthinin are presented. The results of hybrid analysis indicate that in the annual sunflower Helianthus annuus, helianthinin synthesis is controlled by at least three loci: HelA, HelB, HelB, and HelC. Codominant alleles controlling different electrophoretic variants of polypeptides were identified at each of the loci. The HelA locus was inherited independently of HelB and HelC in a series of dihybrid crosses. The frequencies of recombination between loci HelB and HelC estimated in F2 and BC of two crossing combinations were respectively 21.8 and 19.0%. Segregation of the Hel-C-controlled variants in the progenies from the crosses of cultured sunflower with annual wild species and forms corresponded to that theoretically expected for Mendelian inheritance. The maternal type of helianthinin inheritance was observed in the progenies from the crosses of inbred H. annuus lines with perennial diploid and polyploid Helianthus species. Altered expression of the HelC locus was detected in some hybrid combinations. These alterations appeared in early (F1, F2) hybrid generations and were similar in different hybrid combinations. They did not depend on the perennial paternal species being more influenced by the maternal genotype and by the mode of obtaining hybrids (in an embryo culture or in the field). These results are explained by "genomic shock" generated by hybridization of genetically incompatible species.     

Purification, characterization and antifungal properties of a lipid-transfer protein from sunflower (Helianthus annuus) seeds

An antifungal protein from Helianthus annuus L. seeds (Ha-AP10) has been purified to homogeneity and characterized. Ha-AP10 purification was performed by gel filtration, cation exchange chromatography and reverse phase HPLC. Its molecular mass was estimated to be 10 kDa and western blot analyses suggest that it has an extracellular location. The N-terminal sequence of Ha-AP10 showed strong homology to some plant lipid-transfer proteins (LTPs). Antifungal tests have demonstrated that Ha-AP10 exerts a fungistatic effect. It completely inhibits the germination of spores of the fungal pathogen Fusarium solani f. sp. eumartii at a concentration of 40 μg ml⁻¹ and produces a 50% growth inhibition at 6.5 μg ml⁻¹ (0.65 μ M ). These data place Ha-AP10 among the most potent antifungal LTPs described so far.     

种子盐溶球蛋白的结构特征

种子球蛋白一直是人类食物的主要来源,随着对种子球蛋白药理作用认识的不断深入,近年来研究重点已从单纯的序列转向结构的研究,种子球蛋白二级结构（如α－螺旋和β－折叠）存在较大程度的相似性,具有较大的保守性且与蛋白质的特殊功能关系密切,而三级结构之间差异明显,属易变异区,主要与蛋白质的一般功能相关。     

Isolation and sequence of a gene encoding a methionine-rich 10-kDa zein protein from maize

We have isolated the gene encoding a methionine-rich 10-kDa zein protein from a lambda EMBL3 maize genomic 'mini' library of the inbred line BSSS-53 and determined its nucleotide sequence. The sequence matches perfectly with a cDNA clone from the inbred line W22 (which has the same restriction fragment length polymorphism as many inbred lines tested) indicating that we have isolated a functional storage protein gene that is very conserved in maize. This comparison also excludes any splicing of any precursor mRNA and therefore any presence of introns. A number of potential regulatory sequences have been located in the flanking regions. The 10-kDa-zein gene represents the last size class in the zein multigene family to be characterized. Its structure allows us now to re-examine the relationship of all the zein proteins and also to compare the structure of a new class of storage proteins that are rich in methionine, an essential amino acid in livestock fodder.     

Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi

Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi. Evidences have shown that Ca(2+) may play an important regulatory role in trypanosomatid flagellar mobility. In these parasites, the response of the cell to variations of Ca(2+) levels is determined by a variety of calcium-modulated proteins. Starting from T. cruzi cDNA lambdagt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected. rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment. In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography. Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca(2+) binding. The Ca(2+) binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca(2+)-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K(a) of 3.3+/-0.5 (10(6)) and 1.9+/-0.2 (10(4)) M(-1). Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca(2+)-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor.     

Characterization and evolutionary relationship of methionine-rich legumin-like protein from buckwheat.

We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B 14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.     

Purification and characterization of arcelin seed protein from common bean

Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common. Both were glycoproteins having similar subunit M_r, deglycosylated M_r, and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity of arcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin affinity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein.     

Transgenic Trifolium repens with foliage accumulating the high sulphur protein,sunflower seed albumin

With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T. repens by Agrobacterium tumefaciens-mediated transformation. The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER). Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (A _ssu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (L _ssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T. repens cv. Haifa. Transgenic T ₀-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting. The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein. We observed that the promoter had a substantive effect on the level of SSA accumulation with A _ssu>CaMV35S>L _ssu. Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited. However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci. During this work, we attempted to improve the efficiency of A. tumefaciens-mediated transformation of T. repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T. repens. T-DNA transfer was in general not enhanced by sonication compared to traditional A. tumefaciens-mediated transformation. Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A. tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event. We concluded from these results that only one plant from each A. tumefaciens-treated cotyledon should be taken to avoid transgenic clones.     

Differential expression of a gene for a methionine-rich storage protein in maize

Summary A methionine-rich 10 kDa zein storage protein from maize was isolated and the sequence of the N-terminal 30 amino acids was determined. Based on the amino acid sequence, two mixed oligonucleotides were synthesized and used to probe a maize endosperm cDNA library. A fulllength cDNA clone encoding the 10 kDa zein was isolated by this procedure. The nucleotide sequence of the cDNA clone predicts a polypeptide of 129 amino acids, preceded by a signal peptide of 21 amino acids. The predicted polypeptide is unique in its extremely high content of methionine (22.5%). The maize inbred line BSSS-53, which has increased seed methionine due to overproduction of this protein, was compared to W23, a standard inbred line. Northern blot analysis showed that the relative RNA levels for the 10 kDa zein were enhanced in developing seeds of BSSS-53, providing a molecular basis for the overproduction of the protein. Southern blot analysis indicated that there are one or two 10 kDa zein genes in the maize genome.     

Isolation of phenolic constituents and characterization of antioxidant markers from sunflower (Helianthus annuus) seed extract

A new compound, benzyl alcohol β-d-apiofuranosyl-(1→6)-β-d-(4-O-caffeoyl) glucopyranoside (1), was isolated from the seed of sunflower (Helianthus annuus), together with eight known phenolic compounds: caffeic acid (2), methyl caffeoate (3), chlorogenic acid (4), 4-O-caffeoylquinic acid (5), 3-O-caffeoylquinic acid (6), methyl chlorogenate (7), 3,5-di-O-caffeoylquinic acid (8), and eriodictyol 5-O-β-d-glucoside (9). Their structures were elucidated on the basis of spectroscopic methods and chemical evidence. The antioxidative effect of the phenolic constituents from the sunflower seeds was also evaluated based on the oxygen-radical absorbance capacity (ORAC), and the fraction containing caffeic acid derivatives showed a high antioxidant potency.     

Extraction,characterization and spontaneous emulsifying properties of pectin from sugar beet pulp

The effects of organic acid extractants on the yield and characteristics of pectin from sugar beet pulp were investigated with citric acid, malic acid and lactic acid at different pH (1.5 and 2.0) and time (1 h and 2 h). The results demonstrated that the yields of pectins were directly correlated with the decrease of pH and reaction time, and the optimum yield of 17.2% was obtained at pH 1.5 and 2 h. Furthermore, the acid type also affected the physicochemical characteristics of pectin, especially on the esterification degree (42–71), galacturonic acid content (60.2–77.8%), emulsion activity (35.2–40.1%) and emulsion stability (62.1–79.4%), and a relatively single pectin mainly consisted of homogalacturonan could be obtained under a suitable reaction condition, which was an excellent crude material for the production of emulsion activity.