Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry

The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.     

Molecular cloning of 11S globulin and 2S albumin, the two major seed storage proteins in sesame

Insoluble 11S globulin and soluble 2S albumin, conventionally termed alpha-globulin and beta-globulin, are the two major storage proteins and constitute 80-90% of total seed proteins in sesame. Two full-length cDNA clones were sequenced and deduced to encode sesame 11S globulin and 2S albumin precursors, respectively. Deduced amino acid composition reveals that 2S albumin, but not 11S globulin, is a sulfur-rich protein. Three abundant polypeptides of 50-60 kDa were resolved on SDS-PAGE when seed-purified 11S globulin was prepared in nonreducing conditions. Immunological analysis suggests that these three polypeptides are encoded by homologous genes. Immunodetection on the overexpressed protein of the 11S globulin clone in Escherichia coli indicates that this clone encodes the precursor protein of one of the three purified 11S globulin polypeptides.     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

Characterization of the soluble allergenic proteins of cashew nut (Anacardium occidentale L.)

The allergens associated with cashew food allergy have not been well-characterized. We sought to identify the major allergens in cashew nut by performing IgE immunoblots to dissociated and reduced or nonreduced cashew protein extracts, followed by sequencing of the peptides of interest. Sera from 15 subjects with life-threatening reactions to cashews and 8 subjects who tolerate cashews but have life-threatening reactions to other tree nuts were compared. An aqueous cashew protein extract containing albumin/globulin was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to IgE immunoblotting using patient sera. Selected IgE reactive bands were subjected to N-terminal amino acid sequencing. Each of the 15 sera from cashew-allergic subjects showed IgE binding to the cashew protein extract. The dominant IgE-binding antigens in the reduced preparations included peptides in the 31-35 kD range, consistent with the large subunits of the major storage 13S globulin (legumin-like protein). Low-molecular-weight polypeptides of the 2S albumin family, with similarity to the major walnut allergen Jug r 1, also bound IgE. The sera from eight patients who tolerate cashew but displayed allergies to other tree nuts showed only minimal or no IgE binding to cashew. Cashew food allergy is associated with the presence of IgE directed against the major seed storage proteins in cashew, including the 13S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Thus, the legumin-group proteins and 2S albumins are again identified as major food allergens, which will help further research into seed protein allergenicity.     

Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.     

Gene families encoding isoforms of two major sesame seed storage proteins, 11S globulin and 2S albumin

Sesame (Sesamum indicum L.) seed has been recognized as a nutritional protein source owing to its richness in methionine. Storage proteins have been implicated in allergenic responses to sesame consumption. Two abundant storage proteins, 11S globulin and 2S albumin, constitute 60-70 and 15-25% of total sesame proteins, respectively. Two gene families separately encoding four 11S globulin and three 2S albumin isoforms were identified in a database search of 3328 expressed sequence tag (EST) sequences from maturing sesame seeds. Full-length cDNA sequences derived from these two gene families were completed by PCR using a maturing sesame cDNA library as the template. The amino acid compositions of these deduced storage proteins revealed that the richness in methionine is attributed mainly to two 2S albumin isoforms and partly to one 11S globulin isoform. The presence of four 11S globulin and three 2S albumin isoforms resolved in SDS-PAGE was confirmed by MALDI-MS analyses. The abundance of these isoforms was in accord with the occurrence frequency of their EST sequences in the database. A comprehensive understanding of these storage proteins at the molecular level may also facilitate the identification of allergens in crude sesame products that have caused severe allergic reactions increasingly reported in the past decade.     

Antimicrobial, dehydroascorbate reductase, and monodehydroascorbate reductase activities of defensin from sweet potato [Ipomoea batatas (L.) Lam. 'Tainong 57'] storage roots

A cDNA encoding a small cysteine-rich protein designated defensin (SPD1) was isolated from sweet potato storage roots. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that SPD1 is a member of the plant defensin family. Recombinant SPD1 protein overproduced in Escherichia coli was purified by Ni (2+)-chelated affinity chromatography. A recombinant protein from the storage root cDNA clone effectively inhibited the trypsin activity in a dose-dependent manner. Both the corresponding mRNA and protein level were found to be highest in the storage roots, followed by sprout. SPD1 reduced dehydroascorbate (DHA) in the presence of glutathione to regenerate l-ascorbic acid (AsA). However, without glutathione, SPD1 has very low DHA reductase activity, and AsA was oxidized by AsA oxidase to generate monodehydroascorbate (MDA) free radical. MDA was also reduced by SPD1 to AsA in the presence of NADH, mimicking the MDA reductase catalyzed reaction. These data suggest that SPD1 has both DHA reductase and MDA reductase activities. SPD1 was also shown to inhibit the growth of both fungi and bacteria. SPD1 is apparently the first reported plant defensin exhibiting DHA and MDA activities in vitro.     

Storage proteins from Lathyrus sativus seeds

The proteins from Lathyrus sativus Linn. (chickling vetch or grass pea) seeds were investigated. Protein constitutes approximately 20% of the seed dry weight, >60% of which is composed by globulins and 30% by albumins. A single, 24 kDa polypeptide comprises more than half of the protein present in the albumin fraction. The globulins may be fractionated into three main components, which were named alpha-lathyrin (the major globulin), beta-lathyrin, and gamma-lathyrin. alpha-Lathyrin, with a sedimentation coefficient of approximately 18S, is composed of three main types of unglycosylated subunits (50-66 kDa), each of which produce, upon reduction, a heavy and a light polypeptide chain, by analogy with 11S. beta-Lathyrin, with a sedimentation coefficient of 13S, is composed by a relatively large number of subunits (8-66 kDa). Two major polypeptides are glycosylated and exhibit structural similarity with beta-conglutin from Lupinus albus. One of these possesses an internal disulfide bond. gamma-Lathyrin, with a sedimentation coefficient of approximately 5S, contains two interacting, unglycosylated polypeptides, with no disulfide bonds: the major 24 kDa albumin and the heavier (20 kDa) polypeptide chain of La. sativus lectin.     

Purification and characterization of a mannan-binding lectin specifically expressed in corms of saffron plant (Crocus sativus L.)

Despite the economical interest of Crocus sativus, its biochemistry has been poorly studied. Herein, we have isolated a lectin present in saffron corm by gel-filtration, anion-exchange, and reversed-phase chromatography. One- and two-dimensional PAGE, MALDI-MS, and N-terminal amino acid sequence analyses indicated that the native protein forms noncovalently linked aggregates of about 80 kDa apparent molecular mass, mainly composed of two charged heterogeneous (pI's, 6.69-6.93) basic subunits of approximately 12 kDa. Their N-terminal sequences shared 25% similarity and were homologous to the N- and C-terminal domains of monocotyledonous mannose-binding lectins, respectively. An additional polypeptide of around 28 kDa apparent molecular mass was also detected, probably corresponding to a precursor processed into two mature subunits. In addition, the N-terminal domain subunit exhibited 56% similarity with curculin, a sweet protein with taste-modifying activity. The native lectin specifically interacts with a yeast mannan and is a major corm protein specifically expressed in this organ.     

Cloning of the phloem-specific small heat-shock protein from leaves of rice plants

The N-terminal amino-acid sequence of a major rice phloem-sap protein, designated as RPP23, was determined. The complete amino-acid sequence of RPP23 was deduced from the corresponding rice EST- clone and contained an extra 46 amino acids at the N-terminus, that was apparently cleaved off to form mature RPP23 in sieve tubes. RPP23 shared a similarity to plant small heat-shock proteins (smHSPs), though the N-terminal region of RPP23 was distinct from that of known smHSPs. Immunocytological analyses using leaf sections showed that RPP23 was located only in the phloem regions of leaves, and was present in non-stressed plants. In mature leaves, stronger immunocytological signals were detected in sieve elements than in companion cells.     

Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya)

A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.     

Rice albumin N-terminal (Asp-His-His-Gln) prevents against copper ion-catalyzed oxidations

Chromatographic separation of soluble proteins from rice (Oryza sativa L.) yielded a major albumin protein (16 kDa), with the DHHQVYSPGEQ sequence in the N terminus, showing antioxidant action. The rice albumin was more potent than other rice proteins in preventing Cu2+-induced low-density lipoprotein (LDL) oxidation. Additionally, it also exhibited a remarkable suppression of HOCl oxidation. In a further study, albumin inhibited Cu2+-induced oxidation of LDL in a stoichiometric manner with an EC50 value of 4.3 microM, close to that of serum albumins. Moreover, after digestion with trypsin or chymotrypsin, it maintained its antioxidant action. In an experiment to see the involvement of the N terminus in antioxidant action, a synthetic tetrapeptide, equivalent to the N terminus DHHQ, was found to inhibit Cu2+-induced LDL oxidation or degradation of apolipoprotein B, similar to that of rice albumin. In mechanistic analyses, the action of rice albumin or tetrapeptide is primarily due to the removal of Cu2+, as suggested from its inhibitory effect on Cu2+/diphenylcarbohydrazide (DPCH) complex formation. However, despite its similar inhibitory effect on Cu2+-induced oxidation of LDL, rice albumin was less effective than serum albumin in inhibiting Cu2+/DPCH complex formation, suggesting that the number of Cu2+-binding sites in rice albumin may be less than that in serum albumins. Taken together, rice albumin exerts a potent preventive action against Cu2+-induced oxidations, which is due to the Cu2+ binding by DHHQ in the N-terminal sequence. Such a role as a Cu2+ chelator would add up to the application of rice albumin protein.     

牛杀菌/通透性增加蛋白氮端片段的原核表达

目的 克隆牛杀菌/通透性增强蛋白(BPI)N端cDNA,构建原核表达载体,在大肠杆菌中表达BPI蛋白,并纯化重组蛋白。方法 参照Genbank报道的序列,应用RT-PCR技术,从牛嗜中性粒细胞mRNA中扩增出杀菌/通透性增加蛋白基因,然后将该基因插入原核表达载体pGEX-4T-1中,重组质粒转化大肠杆菌BL21,进行诱导表达。结果 获得BPI N端长度为714 bp的基因片断,序列分析证实该片断中有1个点突变。大肠杆菌中的表达产物为相对分子质量约为52×103的GST-BPI融合蛋白。结论 成功的表达和纯化了BPI重组蛋白。     

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.     

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler)

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.     

Chemical composition and flavor of ecuadorian cocoa liquor

The contribution of the chemical composition to the flavor of cocoa liquor from an Ecuadorian selfed population of clone EET 95 was investigated. Polyphenols, purine alkaloids, organic acids, and sugars were quantified, and the key sensory characteristics of cocoa were scored by a trained panel. Despite the short bean fermentation (2 days) commonly used for Arriba cocoa, acetic acid content was closely correlated to liquor pH, demonstrating its essential role in cocoa liquor acidification. Polyphenols were positively correlated to astringency, bitterness, and the green note and negatively correlated to the fruity character. Alkaloid and polyphenol levels fluctuated significantly within the selfed progeny and tended to be lower than those of the heterozygous clone EET 95 (inbreeding effect). These results support the idea that polyphenols might be essential to the overall perception of cocoa liquor characteristics and indicate that the composition and the sensory quality of cocoa liquor are the result of both a genotypic contribution and the conditions of fermentation and roasting.     

Characterization of globulins from common vetch (Vicia sativa L.)

The proteins from Vicia sativa L. (common vetch) seeds were investigated. Protein comprises approximately 11.4% of the seed fresh weight, >50.8% of which is composed by globulins and 43.6% by albumins. The globulins may be fractionated into two main components, which were named alpha-vicinin (comprising 73% of the total globulin fraction, and hence >37% of the total seed protein) and beta-vicinin. Two minor globulin components are also present, gamma-vicinin and delta-vicinin. alpha-Vicinin, the legumin-like globulin, with a sedimentation coefficient of 10.6 S, is a nonglycosylated, disulfide-bond-containing globulin, composed of a group of subunits with molecular masses ranging from 50 to 78 kDa. Upon reduction, each of these subunits releases a heavy polypeptide chain (34-66 kDa) and a light polypeptide chain (21-23 kDa). beta-Vicinin, the vicilin-like globulin, with a sedimentation coefficient of 7.7 S, is a nonglycosylated globulin that contains no disulfide bonds and consists of two major polypeptides with molecular masses of 58 and 66 kDa. gamma-Vicinin is a minor, glycosylated, disulfide-bond-containing globulin. In the reduced form, it comprises six polypeptide chains with molecular masses of 12, 19, 21, 22, 23, and 31 kDa. Finally, delta-vicinin is a minor, highly glycosylated globulin that exhibits hemagglutinating activity. It is composed of a major 47 kDa polypeptide and two minor (33 and 38 kDa) polypeptides. N-terminal sequencing of the delta-vicinin 47 kDa polypeptide revealed no homology to any other known storage protein.     

火龙果种子清蛋白的理化性质及其胰蛋白酶抑制活性研究

为阐明火龙果种子清蛋白的理化性质和生物学活性,本研究从红心火龙果中纯化获得种子清蛋白(HPA),分析其分子量、氨基酸组成、同源性及胰蛋白酶抑制活性。结果表明,HPA由两条分子量为12.6、13.2 kDa的多肽链(HPA-1,HPA-2)组成。 HPA富含谷氨酸(6.297 g·100g^-1)、精氨酸(3.992 g·100g^-1) 和天冬氨酸(2.694 g·100g^-1)。 HPA与来自甜菜和菠菜2S种子贮藏蛋白具有高度相似性。HPA-2显示出较高的胰蛋白酶抑制活性,比活力为9.19×10³ TIU· mg^-1,纯化倍数为1.86。经大豆胰蛋白酶抑制剂-琼脂糖凝胶柱纯化的组分HPA-2-1是胰蛋白酶竞争性抑制剂,抑制常数(Ki)为0.62 nmol·L^-1, 具有Kunitz型抑制剂的摩尔比曲线。 HPA-2-1在一定的pH值(2~10)和温度(30~70℃)范围内具有稳定的抑制活性,且抑制活性几乎不受二硫苏糖醇(DTT)的影响。本研究为挖掘火龙果种子资源,丰富胰蛋白酶抑制剂的种类提供了参考。     

Surface physicochemical properties of globulin-P amaranth protein

Globulin-P, the polymerized 11S amaranth globulin, is composed of 280 kDa unitary molecules (UM, 23%) and aggregates larger than 500 kDa (A, 70%). Antibodies against these proteins were prepared to study their surface characteristics and to assess their homology with other storage proteins. Results showed that globulin-P unitary molecules and aggregates had similar reactive surfaces. A polypeptide of 56 kDa was found to be the most reactive to the antibodies assayed, followed by the acidic polypeptides. Such results support previous information, according to which these polypeptides appeared to be the most exposed on the molecule surface. Globulin-P fraction presented cross-reactivity with the remaining amaranth protein fractions: 11S-globulin, glutelins, and albumins. Globulin-P and 11S-globulin showed similar reactive surfaces whereas glutelin and albumins presented a lower cross-reactivity. The reactivity of the glutelin fraction depended on its sequence. Globulin-P fraction presented cross-reactivity with quinoa globulins, and to a lesser extent with globulins of sunflower and rice. Moreover, the anti-Gp serum was unable to detect either conformational or sequence epitopes in globulins of soybean, wheat, buckwheat, rice, and rye.