2S Albumin Storage Protein Research Articles

Onset of Nut Allergy in a Pediatric Cohort: Clinical and Molecular Patterns in the AFRUSEN Study.

Nut allergy is a growing problem, yet little is known about its onset in children. Objective: To characterize the onset of nut allergy in children in southern Europe. The study population comprised consecutive patients up to 14 years of age who visited allergy departments with an initial allergic reaction to peanut, tree nut, or seed. The allergy work-up included a clinical history, food challenge, skin prick testing, determination of whole-extract sIgE, and ImmunoCAP ISAC-112 assay. Of the 271 children included, 260 were first diagnosed with nut allergy at a mean age of 6.5 years and at a mean (SD) of 11.8 (21.2) months after the index reaction. The most common culprit nuts at onset were walnut (36.5%), peanut (28.5%), cashew (10.4%), hazelnut (8.5%), pistachio (5.4%), and almond (5%). Onset of peanut allergy was more frequent in children ≤6 years and walnut in those aged >6 years (P=.032). In 65% of cases, the allergic reaction occurred the first time the patient consumed the nut, and 35% of reactions were anaphylactic. Overall, polysensitization to nuts was detected by skin prick testing in 64.9% of patients, although this rate was lower among walnut-allergic children (54.7%) and peanut-allergic children (54.1%) (P<.0001). Sensitization to 2S albumins was predominant (75%), especially Jug r 1 (52.8%), whereas sensitization to lipid transfer proteins was less relevant (37%). In the population we assessed, the onset of nut allergy occurred around 6 years of age, slightly later than that reported in English-speaking countries. Walnut was the main trigger, followed by peanut. 2S albumin storage proteins, especially Jug r 1, were the most relevant allergens. This study will help guide management and may contribute to preventive strategies in pediatric nut allergy.

Gene expression and spatiotemporal localization of antifungal chitin-binding proteins during Moringa oleifera seed development and germination.

Chitin-binding proteins behave as storage and antifungal proteins in the seeds of Moringa oleifera. Moringa oleifera is a tropical multipurpose tree. Its seed constituents possess coagulant, bactericidal, fungicidal, and insecticidal properties. Some of these properties are attributed to a group of polypeptides denominated M. oleifera chitin-binding proteins (in short, Mo-CBPs). Within this group, Mo-CBP2, Mo-CBP3, and Mo-CBP4 were previously purified to homogeneity. They showed high amino acid similarity with the 2S albuminstorageproteins. These proteins also presented antimicrobial activity against human pathogenic yeast and phytopathogenic fungi. In the present study, the localization and expression of genes that encode Mo-CBPs and the biosynthesis and degradation of the corresponding proteins during morphogenesis and maturation of M. oleifera seeds at 15, 30, 60, and 90days after anthesis (DAA) and germination, respectively, were assessed. The Mo-CBP transcripts and corresponding proteins were not detected at 15 and 30days after anthesis (DAA). However, they accumulated at the latter stages of seed maturation (60 and 90 DAA), reaching the maximum level at 60 DAA. The degradation kinetics of Mo-CBPs during seed germination by in situ immunolocalization revealed a reduction in the protein content 48h after sowing (HAS). Moreover, Mo-CBPs isolated from seeds at 60 and 90 DAA prevented the spore germination of Fusarium spp. Taken together, these results suggest that Mo-CBPs play a dual role as storage and defense proteins in the seeds of M. oleifera.

Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children

Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children

2S Albumin Storage Proteins: What Makes them Food Allergens?

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response.

2S storage protein gene of Douglas-fir: characterization and activity of promoter in transgenic tobacco seeds

To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir ( Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.

The expression and processing of two recombinant 2S albumins from soybean ( Glycine max) in the yeast Pichia pastoris

Soybean seeds contain two 2S albumin storage proteins (AL1 and AL3) which may contribute to their industrial processing quality and allergenicity. We show that these proteins (AL1 and AL3) are well expressed by the methylotrophic yeast Pichia pastoris and that one of the secreted proteins (AL3) has a similar conformation and stability to that purified from soybean seeds. Further, we show that the subunits are post-translationally processed within the same loop region as the native protein but with some differences in the precise sites. This internal processing provides useful information on the endoproteolytic activity in P. pastoris. We also show that, similar to many plant allergens, the 2S albumins from soybean are stable to heat and chemical treatments.

Recombinant pronapin precursor produced in Pichia pastoris displays structural and immunologic equivalent properties to its mature product isolated from rapeseed

2S albumin storage proteins from rapeseed (Brassica napus), called napins, consist of two different polypeptide chains linked by disulphide bridges, which are derived by proteolytic cleavage from a single precursor. The precursor form of the napin BnIb (proBnIb) has been cloned using a PCR strategy and sequenced. The amino-acid sequence deduced from the clone includes 31 residues of the small chain and 75 of the large chain, which are connected by the peptide Ser-Glu-Asn. Expression of the cDNA encoding proBnIb has been carried out in the methylotrophic yeast Pichia pastoris. The induced protein was secreted to the extracellular medium at a yield of 80 mg.L(-1) of culture and was purified by means of size-exclusion chromatography and reverse phase-HPLC. Recombinant proBnIb appeared properly folded as its molecular and spectroscopic properties were equivalent to those of the mature heterodimeric protein. As 2S albumin storage proteins from Brassicaceae have been shown to be type I allergy inducers, the immunological activity of the recombinant proBnIb was analysed as a measure of its structural integrity. The immunological properties of the recombinant precursor and the natural napin were indistinguishable by immunoblotting and ELISA inhibition using polyclonal antisera and sera of patients allergic to mustard and rapeseed. In conclusion, the recombinant expression of napin precursors in P. pastoris has been shown to be a successful method for high yield production of homogeneous and properly folded proteins whose polymorphism and complex maturation process limited hitherto their availability.

Isolation and Characterisation of a Reserve Protein from the Seeds of Cereus jamacaru (Cactaceae)

We describe here the isolation and characterisation of a major reserve protein from the seeds of Cereus jamacaru. (Cactaceae). This protein has a molecular mass of 5319 kDa and was isolated by a combination of gel filtration chromatography and reverse phase HPLC. The amino acid composition of the protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The usefulness of this protein as a molecular marker in the Cactaceae is also discussed.

Alcalase rapeseed inhibitors: purification and partial characterization.

Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 degrees C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 degrees C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.

The structure and properties of Napin-seed storage protein from rape (Brassica napus L.)

Purification to homogeneity of 2S albumin storage protein from rape seeds (napin) was achieved and its secondary structure and conformational stability were characterised by circular dichroism (CD), and high-sensitivity differential scanning calorimetry (HS-DSC). Deconvolution of the far-UV CD spectrum of napin revealed about 25% of α-helix and 38% of β-sheet structure at neutral and slightly acid pH. HS-DSC data show a single peak of protein denaturation with maximum at 101°C and 88°C at pH 6.0 and 3.0, respectively. This discloses very high stability of napin tertiary structure. The thermal denaturation of napin is irreversible duc to secondary processes such as hydrophobic aggregation of the unfolded protein. The deconvolution of the transition profile at pH 3.0 carried out with assumption that napin aggregation does not contribute to the transition parameters allows to distinguish two constituent transitions which may be attributed to two different domains in napin folding.

Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae).

We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae). This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg.

Ric c 1 and Ric c 3, the Allergenic 2S Albumin Storage Proteins of Ricinus communis: Complete Primary Structures and Phylogenetic Relationships

The 2S albumin storage protein of Ricinus communis consists of the two heterodimeric proteins Ric c 1 and Ric c 3 each of which is composed of a small and a large subunit linked together by disulphide bridges. The complete primary structures of both heterodimeric proteins were determined by enzymatic degradation and automated Edman degradation. The sequences of all four chains correspond to the known cDNA sequence of the gene of a presumed precursor molecule and to the previously determined partial sequences for Ric c 1 and Ric c 3. In addition, few differences in amino acid positions were found which seem to be related to different varieties of R. communis. Sequence comparisons with 2S albumin from other plant genera revealed high degrees of homology and support the view of a common genetic origin of this protein family. Ric c 1 and Ric c 3 which have 11,212 and 12,032 daltons, respectively, share a similar molecular size, biological function and allergenicity with the 2S albumins from Brassica juncea (Bra j 1E) and Sinapis alba L (Sin a 1). Ric c 1 and Ric c 3 may be classified as isoallergens if, additionally, the high degree of similarity in the position of polar residues is taken into account.

Amino Acid Sequence of a New 2S Albumin fromRicinus communisWhich Is Part of a 29-kDa Precursor Protein

The isolation and sequence determination of a new 2S albumin storage protein fromRicinus communisseeds denoted 2S ASP-Ib are described. The fragment approach using selective enzymatic cleavage, Edman degradation, and mass spectrometry was used to demonstrate that the 11-kDa heterodimer protein linked by disulfide bridges has the following structure: short chain, GEREGSSSQQCRQEVQRKDLS-SCERYLRQSSS; long chain, <QQQESQQLQQCC-NQVKQVRDECQCEAIKYIAEDQIQQGQLHGEES-ERVAQRAGEIVSSCGVRCMR. The molecular weight of the intact protein, 11,140 ± 2, determined by matrix-assisted laser desorption mass spectrometry was consistent with the assigned structure. The S- and L-chains are identical to residues 18–49 and 66–130 of the precursor protein predicted by S.D. Irwin, J.N. Keen, J.B.C. Findlay, and J.M. Lord [(1990)Mol. Gen. Genet.222, 400–408], on the basis of the structure of a cDNA isolated using probes based on the sequence of another 2S albumin, described by F.S. Sharief and S.S.L. Li [(1982)J. Biol. Chem.257, 14753–14759], which we denote 2S ASP-Ia. Three of the four termini could have been produced by posttranslational processing by endopeptidase(s) and carboxypeptidase(s) which utilized basic residues as the cleavage sites. Mass spectrometric evidence suggested that the protein presented microheterogeneity at its termini, i.e., truncated forms presumably due to processing heterogeneity. The present characterization of the 2S ASP-Ib protein, the second 2S albumin fromRicinus communisseeds, demonstrates that the 237-residue precursor protein codes for two different heterodimer proteins containing 97 and 99 residues each. This system should be useful for studying the posttranslational processing of plant storage proteins.

Storage protein gene expression in zygotic and somatic embryos of interior spruce

Storage protein gene expression was compared between zygotic and somatic embryos of interior spruce (Picea glauca/engelmanii complex). Somatic embryos, grown on medium containing 40 μM or 10 μM abscisic acid (ABA), and zygotic embryos accumulated IIS legumin, 7S vicilin and 2S albumin storage proteins. Zygotic embryos displayed a rapid, transient period of storage protein accumulation, while somatic embryos differentiated on 40 μM ABA displayed a more prolonged, gradual accumulation, with some accumulation still evident after 9 weeks of maturation. Somatic embryos on 10 μM ABA accumulated storage proteins initially, but these were rapidly degraded as the embryos germinated precociously. Legumin, albumin and vicilin transcripts were detectable in torpedo stage zygotic and somatic embryos, and increased during embryo development. Transcript levels in zygotic embryos increased during cotyledon development, but following maximum dry weight accumulation and moisture loss, transcripts declined rapidly to low levels. In contrast, somatic embryos on 40 μM ABA had high transcript levels for a prolonged period. These levels were still present after 9 weeks of maturation. A decline in storage protein transcripts similar to zygotic embryos was apparent following a mild drying treatment. These results suggest that a decline in storage protein transcripts is stimulated by embryo drying during the later stages of conifer embryogenesis. Low levels of storage protein transcripts also appeared in somatic embryos on 10 μM ABA, but declined during precocious germination. Osmotic stress induced storage protein and storage protein transcript accumulation. This could be partially inhibited by inclusion of the ABA biosynthetic inhibitor, fluridone. However, endogenous ABA levels did not differ significantly between embryos cultured in the presence or absence of fluridone.

Cotton Mat5-A (C164) Gene and Mat5-D cDNAs Encoding Methionine-Rich 2S Albumin Storage Proteins

Several abundant mRNAs are coordinately expressed specifically during the maturation stage of embryogenesis (6, 7). The cotton Mat mRNAs include those of the major vicilin and legumin storage protein genes (6), and representatives of these genes have been sequenced (2, 3). To help define the temporal and spatial regulation of the maturation program of gene expression, we isolated an additional cotton Mat gene, MatS, which encodes mRNAs represented by cDNA clone C164. The mRNAs from the two Mat5 alloalleles in the allotetraploid genome of Gossypium hirsutum make up 2% of the mRNA in maturation stage embryos (6), and the sequences show that they encode methionine-rich 2S albumin storage proteins. Three genomic fragments were recovered, but restriction site analysis and sequencing showed that they contain the same alloallele, Mat5-A, present in the A genome. One was extensively sequenced, as was all of cDNA C 164 and extensive portions of five other cDNA clones (Table I). Figure 1 shows the sequence of clone GC 1 64-24RC of MatS-A. All six cDNAs are identical with each other in the regions sequenced, but they differ significantly from the sequence of Mat5-A. The gene and cDNA sequences are colinear, with eight nucleotide differences, until MatS-A nucleotide 3661 and C164 nucleotide 514. There is no obvious similarity for the next 62 nucleotides until the site of polyadenylation of the cDNAs (Fig. 1). Because there are only two alloallelic genes in G. hirustum (6), we presume that all the sequenced cDNAs are transcribed from the other alloallele, MatS-D. The two MatS alloalleles encode proteins that differ in only three of 139 amino acid residues. Their sequences are very similar to those of the 2S albumin storage protein family (1, 8, 9), and an alignment of the cotton sequences with other 2S albumin preproproteins is unambiguous in its important features (Fig. 2). There is complete identity at the cysteine and leucine residues that are diagnostic of the family and about 44% of the cotton residues are identical with those of Bertholletia (1) and Arabidopsis (9) 2S albumins. The predicted mature form (9; Fig. 2) of the cotton 2S albumin shares with that of Bertholletia a high methionine content. It is 10% in cotton and 19% in Bertholletia, compared with 2 to 3% in other 2S albumins (1, 8, 9). Four of the 10 methionine residues in the cotton protein are at novel positions that may be sites at which other 2S albumins could be engineered for higher methionine content. Because none of the six cDNAs that were examined are transcribed from MatS-A, it is possible that this gene is not active. However, it is more likely that the apparent absence of MatS-A cDNAs is due to failure to process or polyadenylate its transcripts. In the sequence in Figure 1 and in overlapping phage isolate GC164-27 (Table I), there is a class I-like retrotransposon (10) in reverse orientation to the Mat5-A transcription unit (data not shown). The retrotransposon's putative internal domain/3'-long terminal repeat boundary is at MatS-A nucleotide 3791/92, and the 130 nucleotides between this boundary and the Mat5-A/Mat5-D cDNA divergence point can contain only a highly truncated long terminal repeat, if any part of it. We predict that the element, or element-induced rearrangements of Mat5-A sequence, interferes with the processing of the 3' end of MatS-A transcripts. Rearrangement of MatS-A in this region is suggested by the presence of long direct repeats, part of one of which is present in Mat5-D cDNAs (Fig. 1). A sequence very similar to MatS-A nucleotides 907 to 1765 has been found in reverse orientation in the upstream region of an unrelated cotton Lea4 gene (4). Portions of this element are also duplicated at Mat5-A nucleotides 2289 to 2416 (Fig. 1, repeats 1 and 2). Consequently, the sequences that are responsible for transcription of MatS-A are probably between nucleotide 2615 and the transcription start at nucleotide 3146. In this region the sequence of MatS-A has some similarities with those of other 2S albumin storage proteins, in particular two elements (double underlined in Fig. 1) present in similar locations in many of these genes (8, 9). Other elements suggested to be important in various storage protein genes (5) are not obvious in MatS-A. Finally, MatS-A has been compared with two other cotton Mat genes, vicilin (2) and legumin A (3), with which Mat5-A is coordinately expressed in cotton (6, 7), but no similarities are compelling in any sequence pair. ' Supported by a grant from the National Institutes of Health.

Cotton Lea4 (D19) and LeaA2 (D132) Group 1 Lea Genes Encoding Water Stress-Related Proteins Containing a 20-Amino Acid Motif

LEA2 proteins are a diverse group of probable desiccation protectants that are developmentally induced during the postabscission stage of late embryo development by ovule abscission and are environmentally induced, probably by a different mechanism(s), in cultured embryos by ABA, desiccation, and osmotic stress (7, 8). Of the 18 Lea and LeaA mRNAs cloned from cotton (4), mRNAs of the two related genes (4) Lea4 (cDNA clone Dl 9) and LeaA2 (cDNA clone D1 32) have the highest cross-hybridization with embryo RNAs of other plants (K.S. Jakobsen, D.W. Hughes, G.A. Galau, unpublished observations). A recent compilation of Group 1 Lea sequences confirms a very high conservation at the amino acid level in the cDNAs Em of wheat, Dl 9 of cotton, p8B6 of radish, EMB-l of carrot, Emb564 of maize, and B19 of barley (2). Extensive work with a wheat Em gene has defined putative ABA-responsive elements and a leucine zipper-containing protein with an afflnity for these elements (6), and there is some evidence that these elements may be common in other kinds of Lea and other environmentally responsive genes (6, 10). The cotton Lea4 cDNA Dl 9 and the upstream portion of a cotton Lea4 gene have been sequenced (1). The amino acid sequence of cDNA D19 is peculiar in having a carboxyterminal extension of 18 amino acids not found in its homologs (2), but the upstream region of the Lea4 gene does contain elements similar to those defined in a wheat Em gene (6), the only other group 1 Lea gene sequenced (9). To confirm the peculiar carboxy extension and to help define the developmental and environmental responsive regions in the cotton Lea4 gene, both alloalleles of cotton Lea4 and 10 Lea4 cDNAs were sequenced. The principal results are that there is no carboxy extension in the protein and that transcriptionally important sequences are probably within 268 nucleotides of the transcription start. Although the expression of Lea4 and LeaA2 mRNAs cannot be distinguished during the postabscission stage or during environmental induction in embryos (7, 8), earlier in embryo development the LeaA2 mRNAs, but not Lea4 mRNAs, are induced to relatively low levels at the time of transiently high levels of ABA (7). To define the relationship of the proteins encoded by Lea4 and LeaA2, and to help understand the basis of their differential expression during development, both alloalleles of LeaA2 and two LeaA2 cDNAs were also sequenced. LeaA2-encoded proteins are very similar to the one encoded by Lea4, except that they contain a tandem duplication of a 20-amino acid sequence present only once in the Lea4-encoded protein. The two alloalleles of Lea4 were sequenced along with portions of 10 Lea4 cDNA clones (Table I). Two alleles of both alloalleles were identified in this collection (Table II). Figure 1 presents the sequence of the D genome alloallele, Lea4-D9 clone GD 19-9S, as the reference Lea4 sequence. Lea4-D9 is very similar to, but not identical with, the sequence of cotton Lea4 clone gD19 (1). The transcribed region of the other Lea4 alloallele, Lea4A13 clone GD19-13RS, is very similar to that of Lea4-D9 (Fig. 1). However, there is no similarity before Lea4-D9 nucleotide 1822 and they diverge after Lea4-D9 nucleotide 3279. For clarity, the sequence of Lea4-A 13 in these two regions is presented separately in Figure 2. At least part of the difference in the 5' end is due to a repetitive element in Lea4A13; the sequence between nucleotides 1033 and 1932 is present in reverse orientation upstream of the unrelated cotton MatS-A gene which encodes a 2S albumin storage protein (5). After about Lea4-D9 nucleotide 2630 in the 3'-nontranslated region, the genes contain a complex pattern of tandem imperfect repeats of about 80 to 120 nucleotides in length. Multiple insertion/deletion events and substitutions prevent alignment after Lea4-D9 nucleotide 3279, although there are blocks of similar sequence until Lea4-D9 nucleotide 3422 and Lea4-A 13 nucleotide 3732 (comparisons not shown). Lea4 cDNA Dl9 was completely sequenced along with major portions of nine other Lea4 cDNAs (Table I). Eight of the cDNAs, typified by cDNA D47, are transcribed from Lea4-D9. The ninth cDNA, Dl 9, is transcribed from a Lea4D allele that differs from Lea4-D9 in the mRNA region by a single substitution (Table II). At least three polyadenylation sites are used in Lea4-D9 and a fourth site is used in Lea4D19 (Fig. 1). The 10th cDNA, D108, is transcribed from a Lea4-A allele that in the mRNA region differs from Lea4A 13 by a single insertion/deletion event (Table II). Both Lea4 alloalleles encode identical proteins, although there are four nucleotide substitutions in their amino acidcoding regions (Fig. 1). The protein does not contain the carboxy-terminal 18 amino acids that were reported earlier ' Supported by a grant from the National Institutes of Health. 2 Abbreviations: LEA, late embryogenesis abundant; kb, kilobase.