Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH₂-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH₂-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X₃-Cys-X₃-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.     

A new distinct group of 2 S albumins from rapeseed. Amino acid sequence of two low molecular weight napins.

Two napins (nIa and nIb), isolated from Brassica napus (rapeseed) seeds, have been sequenced. The two proteins show the common structural pattern of the 2 S albumins, since they are composed of two disulfide-linked chains of different size, yet they exhibit an atypical low molecular weight (12.5 kDa vs. 14.5 kDa of the major napins). High sequence similarity has been found between these 2 proteins, but only 54% similarity can be estimated from their comparison with the 14.5 kDa major napins. Thus, nIa and nIb are considered representatives of a new distinct group of rapeseed napins since all the previously known napins exhibit 95% sequence similarity. Unexpectedly, the similarity increases when compared with the 2 S proteins from other species.     

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.     

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: Sequence similarity to PSI-K

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.     

Characterization of a Kunitz trypsin inhibitor with a single disulfide bridge from seeds of Inga laurina (SW.) Willd

Inga laurina is a tree that belongs to the Mimosoideae sub-family of the Leguminosae. A protein inhibitor of trypsin (ILTI) was isolated from its seeds by ammonium sulphate precipitation, ion-exchange chromatography and rechromatography on an HiTrap Q ion-exchange column. By SDS-PAGE, ILTI yielded a single band with a Mr of 20 kDa with or without reduction. ILTI was found to be a single polypeptide chain containing 180 amino acids, the sequence of which was clearly homologous to the Kunitz family of serine protease plant protein inhibitors, and it also showed significant similarity to the seed storage proteins, sporamin and miraculin. However, ILTI displayed major differences to most other Kunitz inhibitors in that it contained only one disulfide bridge, and did not have two polypeptide chains as for the majority of other Kunitz inhibitors purified from Mimosoideae species. ILTI inhibited bovine trypsin with an equilibrium dissociation constant (K(i)) of 6 x 10(-9)M, but did not inhibit chymotrypsin, papain and alpha-amylase. Its amino acid sequence contained a Lys residue at the putative reactive site (position 64). ILTI was stable over a wide range of temperature and pH and in the presence of DTT.     

Hemocyanins in spiders, VI[1]. Comparison of the polypeptide chains of Eurypelma californicum hemocyanin.

The subunits of the hemocyanin from the tarantula, Eurypelma californicum, were isolated, following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing two chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is seven. The amino acid compositions of the seven chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident, if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in the case of chains b and e,, while no end group called be detected in any of the other chains by different methods. The C-terminal end group was found to be valine in both chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the two largest peptides of each chain are of molecular weight near 2400. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications, but resides in major differences of the amino acid sequences.     

Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol proteases of the papain family

A 34,000-Da protein (P34) is one of the four major soybean oil body proteins observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated organic solvent-extracted oil bodies from mature seeds. P34 is processed during seedling growth to a 32,000-Da polypeptide (P32) by the removal of an amino-terminal decapeptide (Herman, E.M., Melroy, D.L., and Buckhout, T.J. (1990) Plant Physiol, in press). A soybean lambda ZAP II cDNA library constructed from RNA isolated from midmaturation seeds was screened with monoclonal antibodies directed against two different epitopes of P34. The isolated cDNA clone encoding P34 contains 1,350 base pairs terminating in a poly(A)+ tail and an open reading frame 1,137 base pairs in length. The open reading frame includes a deduced amino acid sequence which matches 23 of 25 amino-terminal amino acids determined by automated Edman degradation of P34 and P32. The cDNA predicts a mature protein of 257 amino acids and of 28,641 Da. The open reading frame extends 5' from the known amino terminus of P34 encoding a possible precursor and signal sequence segments with a combined additional 122 amino acids. Prepro-P34 is deduced to be a polypeptide of 42,714 Da, indicating that the cDNA clone apparently encodes a polypeptide of 379 amino acids. A comparison of the nucleotide and deduced amino acid sequences in the GenBank Data Bank with the sequence of P34 has shown considerable sequence similarity to the thiol proteases of the papain family. Southern blot analysis of genomic DNA indicated that the P34 gene has a low copy number.     

The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide

Carboxypeptidase I from germinated barley (Hordeum vulgare) grain consists of two peptide chains linked by disulfides; the A- and B-chains contain 266 and 148 amino acid residues, respectively (Sorensen, S. B., Breddam, K., and Svendsen, I. (1986) Carlsberg Res. Commun. 51, 475-485). A cDNA library prepared from mRNA isolated from scutella of 2-day germinated barley has now been screened with a mixed oligonucleotide encoding a peptide fragment of the A-chain. Nucleotide sequence analysis of a 1443-nucleotide pair cDNA clone revealed that both chains of the enzyme are translated from a single mRNA. The coding region of the A-chain is located at the 5'-end of the cDNA and is separated from the B-chain coding region by a 165-nucleotide pair linking region. The B-chain coding region is followed by a stop codon, a 187-nucleotide pair 3'-untranslated sequence, and a short polyadenylic acid tail. The results indicate that the A- and B-chains of barley carboxypeptidase I arise by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain. The putative linker peptide is rich in proline, lysine, and arginine residues, has an apparent pI of 11.9, and appears to be excised by cleavage of peptide bonds on the COOH-terminal side of serine residues.     

Molecular cloning and sequencing of the psaD gene encoding subunit II of photosystem I from the cyanobacterium, Synechocystis sp. PCC 6803

Photosystem I reaction center was isolated from the cyanobacterium, Synechocystis sp. PCC 6803, in a form which contains seven different polypeptide subunits. One of the subunits, with a molecular mass of about 16 kDa, was isolated, and protein sequence information was obtained for the amino terminus and several tryptic peptides. Oligonucleotide probes, corresponding to these sequences, were used to probe a genomic library, and the gene, designated psaD, encoding subunit II was cloned and sequenced. The gene encodes a polypeptide with a mass 15,644 Da, which exhibits a high degree of similarity to subunit II from tomato, as well as amino acid sequences reported from barley photosystem I. In addition to this gene, three large open reading frames were identified. Two remain unidentified, and the third is highly homologous to anthranilate synthase, component 1 from Escherichia coli and Saccharomyces cerevisiae.     

Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins

Summary The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3° and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures.The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous.The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa. These data suggest that the polypeptides were formed by cleavage of glutamic acid residues present at regular intervals in the chains of all three mucin glycoproteins. These large immunoreactive peptides were formed by the removal of smaller peptides from the carboxyl terminal end of the deglycosylated mucin glycoprotein chains. Taken collectively, these findings indicate that the polypeptide chains in these mucin glycoproteins are very similar in subunit structure and that there is a high degree of homology between their polypeptide chains.     

Bovine conglutinin is a collagen-like protein

Conglutinin is a bovine plasma protein which is relatively large and asymmetric with elevated contents of glycine and, to some extent, proline. Although its physiologic function is unknown, conglutinin is known to bind, in the presence of calcium, to yeast cell walls and to the solid-phase-inactivated third component of complement. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, isolated conglutinin appeared to consist of a single polypeptide chain (Mr 48 000). Unreduced conglutinin consisted of a single stained band with an apparent molecular weight of approximately 300 000. Cross-linking experiments with glutaraldehyde and dimethyl suberimidate suggested that this Mr 300 000 molecule consists of six of the disulfide-linked polypeptide chains. Amino acid composition revealed hydroxylysine and hydroxyproline together with elevated glycine and proline contents. Digestion of reduced, alkylated conglutinin with bacterial collagenase resulted in formation of a precipitate which consisted of an Mr 24 000 peptide which was digested to Mr 21 000 with large quantities of collagenase. These peptides contained less glycine, proline, hydroxylysine, and hydroxyproline than did the intact protein. The supernatant from this digestion mixture was, however, enriched in these four amino acids, with glycine making up nearly one-third of the total. Prolonged digestion with pepsin at 37 degrees C resulted in an Mr 20 000 peptide which was enriched in glycine, proline, hydroxyproline, and hydroxylysine. Amino-terminal sequence analysis showed that the glycine-X-Y repeating sequence begins at residue 26.(ABSTRACT TRUNCATED AT 250 WORDS)     

The amino acid sequence of the 9 kDa polypeptide and partial amino acid sequence of the 20 kDa polypeptide of mitochondrial NADH:ubiquinone oxidoreductase.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated enzyme in the respiratory chain and is composed of at least 26 distinct polypeptides. Two hydrophilic subfractions of bovine heart complex I were systematically resolved into individual polypeptides by chromatography. Three polypeptides (51, 24, and 9 kDa) were isolated from the flavoprotein fraction (FP) of complex I, and the complete amino acid sequence of the 9 kDa polypeptide was determined. The 9 kDa polypeptide is composed of 75 amino acids with a molecular weight of 8,437. This protein exhibits no obvious sequence similarity to other proteins. The iron-sulfur protein fraction (IP) of complex I was separated into eight polypeptides, 75, 49, 30, 20, 18, 15, 13 kDa-A, and 13 kDa-B. The 20 kDa polypeptide was recognized as a novel component of IP for the first time. The N-terminal and several peptide sequences of the 20 kDa polypeptide were determined. Comparison of the sequences revealed significant sequence similarities of the 20 kDa polypeptide to the psbG gene products encoded in the chloroplast genome. The conserved sequence in these proteins was also found in the small subunit of the nickel-containing hydrogenases. These results suggest that complex I is related to other redox enzyme complexes.     

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.     

The profilin multigene family of maize: differential expression of three isoforms

Profilin is a small (12–15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays . Two cDNAs isolated from a maize pollen library ( ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA ( ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (>90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30–40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH₂ and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

Rice Allergenic Protein and Molecular-genetic Approach for Hypoallergenic Rice

Allergenic proteins with a molecular mass of about 14 to 16 kDa were isolated from a rice salt-soluble fraction based on the reactivity with IgE antibodies from patients allergic to rice. cDNA clones encoding these allergenic proteins were isolated from a cDNA library of maturing rice seeds, and the deduced amino acid sequences showed considerable similarity to wheat and barley α-amylase/trypsin inhibitors, which have recently been identified as major allergens associated with baker’s asthma. An antisense RNA strategy was applied to repress the allergen gene expression in maturing rice seeds. Immunoblotting and ELISA analyses of the seeds using a monoclonal antibody to a 16-kDa allergen showed that allergen content of seeds from several transgenic rice plants was markedly lower than that of the seeds from parental wild type rice.     

Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.     

Isolation and characterization of a cDNA clone encoding an 18-kDa hydrophobic photosystem I subunit (PSI-L) from barley (Hordeum vulgare L.)

Photosystem I in barley contains a polypeptide with an apparent molecular mass of 14 kDa. The polypeptide is N-terminally blocked to amino acid sequencing, but partial amino acid sequences have been determined from three fragments obtained by chemical and enzymatic cleavage. Using an oligonucleotide probe specifying this amino acid sequence, a full length cDNA clone was isolated. The deduced amino acid sequence does not correspond to any previously identified photosystem I subunit. We designate the novel photosystem I subunit PSI-L and the corresponding nuclear gene PsaL. The cDNA clone encodes a precursor polypeptide of 209 amino acid residues with a deduced molecular mass of 22,210 Da. The precursor has a transit peptide typical of proteins imported into chloroplasts. Based on a putative maturation site, the deduced molecular mass of the mature protein is 18 kDa. The PSI-L polypeptide is hydrophobic and predicted to have at least two membrane-spanning alpha-helices. Northern blot analysis shows that the expression of the PsaL gene is light-induced similar to other of the barley photosystem I genes. Southern blot analysis indicates that PsaL is a single copy gene. Partial amino acid sequences of an N-terminally blocked 9-kDa polypeptide show high sequence similarity to the PSI-G polypeptide of spinach and Chlamydomonas reinhardtii. The gene product of PsaG in spinach has previously been assigned as subunit V (Steppuhn, J., Hermans, J., Nechushtai, R., Ljungberg, U., Thümmler, F., Lottspeich, F., and Herrmann, R. G. (1988) FEBS Lett. 237, 218-224). The present study suggests that PSI-L is equivalent to subunit V and that PSI-G is a subunit migrating closely to PSI-H (subunit VI) and PSI-C (subunit VII).     

Molecular and spectroscopic characterisation of a low molecular weight seed storage protein from yellow mustard (Sinapis alba L.)

• 1.1. A 1.7S protein has been purified from mustard seeds (Sinapis alba L.). This protein, soluble in water and dilute salt solutions, is considered as an albumin and constitutes about 10% of the total soluble protein in mustard seeds.
• 2.2. Its molecular weight is approximately 15,000 and is composed of two polypeptide chains (M_r = 9500 and 5000), linked by two disulfide bridges.
• 3.3. The amino acid compositions of both subunits as well as of the native protein are reported, showing a strong homology with napins from Brassica napus L.
• 4.4. The ultraviolet absorption, fluorescence emission and circular dichroism spectra of the purified protein have been obtained. The mustard protein exhibits about 50% α-helix with a very low β-structure content. Based on its structural characteristics, a zein-like packing is proposed for this protein from mustard seeds.