Søren K. Rasmussen

Phytate: impact on environment and human nutrition. A challenge for molecular breeding

Phytic acid (PA) is the primary storage compound of phosphorus in seeds accounting for up to 80% of the total seed phosphorus and contributing as much as 1.5% to the seed dry weight. The negatively charged phosphate in PA strongly binds to metallic cations of Ca, Fe, K, Mg, Mn and Zn making them insoluble and thus unavailable as nutritional factors. Phytate mainly accumulates in protein storage vacuoles as globoids, predominantly located in the aleurone layer (wheat, barley and rice) or in the embryo (maize). During germination, phytate is hydrolysed by endogenous phytase(s) and other phosphatases to release phosphate, inositol and micronutrients to support the emerging seedling. PA and its derivatives are also implicated in RNA export, DNA repair, signalling, endocytosis and cell vesicular trafficking. Our recent studies on purification of phytate globoids, their mineral composition and dephytinization by wheat phytase will be discussed. Biochemical data for purified and characterized phytases isolated from more than 23 plant species are presented, the dephosphorylation pathways of phytic acid by different classes of phytases are compared, and the application of phytase in food and feed is discussed.

Nucleotide sequence of a cDNA coding for the NADPH-protochlorophyllide oxidoreductase (PCR) of barley (Hordeum vulgare L.) and its expression in Escherichia coli

SummaryThe primary structure of the NADPH-protochlorophyllide oxidoreductase of barley has been deduced from the nucleotide sequence of a cloned full-length cDNA. This cDNA hybridizes to a 1.7 kb RNA whose steady-state level in dark-grown seedlings is drastically reduced upon illumination. The predicted amino acid sequence (388 residues in length) includes a transit peptide of 74 amino acids whose end point has been delimited by sequencing the N-terminus of the mature protein. Expression of the cDNA inEscherichia coli leads to the synthesis of an enzymatically active precursor of the NADPH-protochlorophyllide oxidoreductase. Activity of this protein in bacterial lysates is completely dependent on the presence of NADPH and protochlorophyllide and requires light.

cDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis

Abstract A cDNA library of RNA from barley leaves inoculated with Erysiphe graminis was screened using labelled cDNA enriched for specific sequences by subtractive hybridization against RNA from non-inoculated leaves. This resulted in isolation of several clones representing pathogen induced genes. By cross-hybridization and sequence analysis, one of the cDNAs (pBT6-3) was found to be a partial clone representing a putative peroxidase, for which a full-length cDNA clone (pBH6-301) was subsequently isolated. The predicted amino acid sequence revealed a 21 amino acid signal peptide and a 294 amino acid mature protein (31 kDa) and shows 56 % amino acid identity to a basic peroxidase from turnip, 89 % to a putative peroxidase from wheat, but only 38% to the amino acid sequence derived from the cDNA clone (pcD1311) of a second putative barley peroxidase expressed in leaves. Northern blot analysis showed that the pBT6-3 (pBH6-301) transcript is elevated as early as 4 h after inoculation with E. graminis f. sp. hordei and that two maxima in transcript levels appear, which can be correlated with penetration attempts by the fungus. The amount of the pcD1311 transcript was also found to increase in inoculated leaves but at a later time point.

cDNA, amino acid and carbohydrate sequence of barley seed-specific peroxidase BP 1

The major peroxidase of barley seed BP 1 was characterized. Previous studies showed a low carbohydrate content, low specific activity and tissue-specific expression, and suggested that this basic peroxidase could be particularly useful in the elucidation of the structure-function relationship and in the study of the biological roles of plant peroxidases (S.K. Rasmussen, K.G. Welinder and J. Hejgaard (1991) Plant Mol Biol 16: 317–327). A cDNA library was prepared from mRNA isolated from seeds 15 days after flowering. Full-length clones were obtained and showed 3′ end length variants, a G+C content of 69% in the translated region, a 90% G or C preference in the wobble position of the codons and a typical signal peptide sequence. N-terminal amino acid sequencing and sequence analysis of tryptic peptides verified 98% of the sequence of the mature BP 1 which contains 309 amino acid residues. BP 1 is the first characterized plant peroxidase which is not blocked by pyroglutamate. BP 1 polymorphism was observed. BP 1 is less than 50% identical to other plant peroxidases which, taken together with its developmentally dependent expression in the endosperm 15–20 days after flowering, suggests a unique biological role of this enzyme. The barley peroxidase is processed at the C-terminus and might be targeted to the vacuole. The single site of glycosylation is located near the C-terminus in the N-glycosylation sequon -Asn-Cys-Ser- in which Cys forms part of a disulphide bridge. The major glycan is a typical plant modified-type structure, Manα1-6(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc. The BP 1 gene was RFLP-mapped on barley chromosome 3, and we propose Prx5 as the name for this new peroxidase locus.

Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins CLONING AND CHARACTERIZATION OF FIVE MAJOR MOLECULAR FORMS

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P1-P1′ Gln-Gln, and WSZ2b at P2-P1 Leu-Arg—one of two overlapping reactive centers. WSZ1c with P1-P1′ Leu-Gln was the fastest inhibitor of chymotrypsin (k a = 1.3 × 106 m −1 s−1). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k a∼105 m −1 s−1), which has P1-P1′ Leu-Ser—a reactive center common in animal serpins. WSZ2b inhibited plasmin at P1-P1′ Arg-Gln (k a∼103 m −1 s−1). None of the five serpins inhibited Bacillus subtilisin A,Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Sequence homology between barley endosperm protein Z and protease inhibitors of the α1-antitrypsin family

Six cDNA clones encoding parts of protein Z, a major barley endosperm albumin, have been identified. Nucleotide and amino acid sequencing have established a 180 residues long C‐terminal amino acid sequence of protein Z as well as two minor amino acid sequences (14 and 7 residues). These sequences show that barley protein Z is homologous with human α1‐antitrypsin, human otj‐antichymotrypsin, human antithrombin III, mouse contrapsin and chicken ovalbumin (26–32% of the 180 residues in the C‐terminal sequence in identical positions). The sequence homology and specific cleavage of protein Z at a bond corresponding to the reactive site of the inhibitors indicate a possible inhibitory function. Inhibition of microbial or pancreatic serine proteases could, however, not be associated with protein Z.

Biosynthesis of Δ-aminolevulinate in greening barley leaves VI. Activation of glutamate by ligation to RNA

The components involved in the enzymic conversion of glutamate to δ-aminolevulinate have been separated into three fractions; a Blue Sepharose bound, a chlorophyllin-(or heme) Sepharose bound and an unbound fraction. Combination of these three fractions reconstituted δ-aminolevulinate synthesis from glutamate. Participation of a specific RNA in δ-aminolevulinate synthesis was established by isolating a homogeneous RNA from the chlorophyllin-Sepharose bound fraction and reconstituting δ-aminolevulinate synthesis in the presence of the unbound and Blue Sepharose bound fractions. The RNA involved in δ-aminolevulinate synthesis was purified by high-pressure liquid chromatography and preparative gel electrophoresis. In the presence of the Blue Sepharose bound fraction, ATP and Mg2+, glutamate bound covalently to this RNA. Co(III)-ATP-o-phenanthroline bound to the RNA and strongly inhibited glutamyl-RNA formation, whereas heme and Mg-protoporphyrin at 50 μM were only slightly inhibitory. The chlorophyllin-Sepharose bound fraction also contained two other glutamate acceptor RNAs. RNAase A and snake venom phosphodiesterase strongly reduced δ-aminolevulinate synthesis and glutamyl-RNA formation, whereas addition of DNAase or spleen phosphodiesterase was only slightly inhibitory. The RNA became sensitive to the spleen enzyme after phenol extraction of the chlorophyllin-Sepharose bound fraction. E. coli tRNAGlu orwheat germ tRNA did not reconstitute δ-aminolevulinate synthesis when combined with the Blue Sepharose bound and unbound fractions. The RNA involved in δ-aminolevulinate synthesis hybridised to a 3.9 kb Hind III Pst I restriction endonuclease fragment from the barley chloroplast genome located in the large single copy region 38 kb from the large subunit gene for RuBP carboxylase and 12 kb from the inverted repeats. Glutamate 1-semialdehyde aminotransferase was labelled during35S-incorporation into greening barley leaves but not during incorporation into isolated plastids. It is suggested that an NADPH-dependent dehydrogenase involved in the reduction of glutamyl-RNA to glutamate 1-semialdehyde is present in the Blue Sepharose bound fraction.

Primary structure of A B1 hordein gene from barley

The 873 base pair coding region of a Hor-2 gene of barley and the adjacent 550 base pair upstream and 413 base pair downstream regions were sequenced. The gene is devoid of introns and encodes a 271 amino acid long B1 hordein polypeptide containing a putative 19 amino acid signal peptide. The remaining part of the coding sequence can be divided into three parts. In the 53 residue amino-terminal region there are 9 glutamine-proline blocks with a preferred core sequence PQQP, separated by one or two other residues giving a glutamine proline content of 78%. The second part encodes 164 amino acids, 41% of which are glutamine+proline organised in scattered blocks. Seven cysteine residues are coded for by this portion of the gene. The last part encodes the carboxyterminal 35 amino acids none of which is glutamine.In the 550 base pair upstream region the sequence TATAAATA is found at- 71 base pairs from the initiator methionine. In the 3′ non-coding region three putative polyadenylation signals, AATAAA, are present.Comparison of the gene with 3 partial cDNA clones indicates that the charge polymorphism in the B1 polypeptide group is due to point mutations in the part of the gene corresponding to the carboxy terminal half of the polypeptide. Comparison with the sequence of a second B hordein gene suggests that insertions or deletions of glutamine-proline blocks in the amino-terminal domain are a major source of size polymorphisms in the B hordein family.

Novel MODY3 Mutations in the Hepatocyte Nuclear Factor-1α Gene: Evidence for a Hyperexcitability of Pancreatic β-cells to Intravenous Secretagogues in a Glucose-Tolerant Carrier of a P447L Mutation

One form of maturity-onset diabetes of the young (MODY3) results from mutations in the hepatocyte nuclear factor (HNF)-1α gene, located on chromosome 12q24.2. The primary objective of the present study was to search for genetic variation in the HNF-1α gene in nine nonrelated Danish Caucasian subjects with MODY. Direct sequencing of the coding region and intron-exon boundaries of the HNF-1α gene revealed 2 novel and 1 previously reported missense mutations and 2 novel frameshift mutations in five of nine MODY subjects. These five mutations were found in neither 84 NIDDM patients nor 84 control subjects. One glucose-tolerant lean male with a P447L missense mutation, which in his relatives caused MODY, underwent an oral glucose tolerance test (OGTT), a tolbutamide modified frequently sampled intravenous glucose tolerance test, and a glucagon test to examine for a possible early β-cell abnormality. He had a low insulin secretion rate during an OGTT, but a twofold increase in pancreatic β-cell response after intravenous glucose and a 2.5- to 4-fold increase in β-cell response after either intravenous tolbutamide or intravenous glucagon loads. In conclusion, 1) mutations in the HNF-1α gene are common in Danish Caucasian MODY patients, and 2) early stages in the pathogenesis of MODY3 caused by the P447L mutation may be characterized by a hyperexcitability of β-cells to intravenous secretagogues.

Nucleotide sequences of cDNA clones for C-hordein polypeptides

Two partial cDNA clones, pc hor1-3 and pc-919, encoding the carboxyterminal portion of two different C-hordein polypeptides have been sequenced. pc hor1-3 can be translated into a frame of 74 amino acids, part of which is identical to the amino acid sequence of a C-hordein chymotryptic peptide. The 3′ non-coding region is 174 nucleotides in length and extends to the polyadenylation site. The region has two putative polyadenylation signals, AATAAA, located 27 and 80 nucleotides upstream from the poly(A)-tail. Three RNA species, 1800, 2100 and 4300 bases in length, can be detected in the endosperm using pc hor1-3 as probe. These could code for C-hordein polypeptides of 48, 67 and 144 kD, respectively. Hybridization of pc hor1-3 to restriction endonuclease fragments of nuclear barley DNA suggests that 6–8 genes code for the C-hordein polypeptides.The nucleotide sequence of the second cDNA clone, pc-919, can be translated into 64 amino acids, of which the 22 carboxyterminal amino acid residues are identical to the carboxyterminal reading frame encoded by pc hor1-3. The adjacent 42 amino acid long reading frame shows homology to that deduced from pc hor1-3, but the nucleotide sequence is different and highly homologous to the 5′ coding sequence found in a B-hordein genomic clone. Hybridization using this part of pc-919 as a probe detects RNAs of 1200 and 1400 bases only. Thus pc-919 represents a gene containing sequences related to both Hor1 and the Hor2 locus, and is the first nucleotide sequence data giving evidence that the two loci are related.