Primary structure of the plant serpin BSZ7 having the capacity of chymotrypsin inhibition.

The primary structure of barley grain serpin BSZ7 was deduced from a cDNA encoding 397 amino-acid residues. More than 70% of the residues were confirmed by sequencing peptide fragments. The N-terminus was identified as an acetylated Ala by using mass spectrometry coupled with amino-acid analysis. None of the four putative N-glycosylation sites were found to be glycosylated. The positional identity of BSZ7 with plant and mammalian serpins is 69-72% and 25-32%, respectively.

Primary structure and specificity of the major serine proteinase inhibitor of amaranth (Amaranthus caudatus L.) seeds.

A novel member of the potato inhibitor I family of serine proteinase inactivating proteins has been isolated from seeds of grain amaranth (Amaranthus caudatus L.) and characterized. The mature form of the amaranth trypsin/subtilisin inhibitor (ATSI) with pI approximately 8.3 and molecular mass 7887 Da contains 69 amino acids in a sequence showing 33-51% identity with members of the inhibitor I family from other plant families. A minor form with pI approximately 7.8 and same inhibitory properties lacked the N-terminal dipeptide Ala-Arg. In accordance with the reactive-site bond Lys45-Asp46, which was identified by specific cleavage on a subtilisin column, ATSI is a potent inhibitor of trypsin (Ki approximately 0.34 nM) and more weakly of plasmin (Ki approximately 38 nM) and Factor XIIa (Ki approximately 440 nM). However, ATSI also inactivates chymotrypsin (Ki approximately 0.41 nM), cathepsin G (Ki approximately 122 nM) and several alkaline microbial proteinases, including subtilisin NOVO (Ki approximately 0.37 nM). Interestingly, ATSI contains a Trp residue instead of the highly conserved Arg in position 53 (P8'), which is assumed to play a central role in stabilization of the active-site loop during complex formation. ATSI was immediately inactivated by pepsin and hardly represents an antinutritional component in foods or feeds.

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.

Inhibitory serpins from rye grain with glutamine as P1 and P2 residues in the reactive center.

Six of seven serpins detected in grains of rye (Secale cereale) were purified and characterized. The amino acid sequence close to the blocked N-terminus, the reactive center loop sequence and the second order association rate constant (k(a)') for irreversible complex formation with chymotrypsin were determined for each serpin. Three of four serpins containing the unusual reactive center P2-P1' QQ/S and one with P2-P1' PQ/M were equally efficient inhibitors of chymotrypsin (k(a)' approximately 10(5) M(-1) s(-1)). One serpin with P2-P1' PY/M was a faster inhibitor (k(a)' approximately 10(6) M(-1) s(-1)). Similar but differently organized glutamine-rich reactive centers were recently found in grain serpins cloned from wheat [Ostergaard et al. (2000) J. Biol. Chem. 275, 33272] but not from barley. The prolamin storage proteins of cereal grains contain similar sequences in their glutamine-rich repeats. A possible adaption of hypervariable serpin reactive centers late in Triticeae cereal evolution as defence against insects feeding on cereal grains is discussed.

Two antifungal thaumatin-like proteins from barley grain.

Antifungal activity has been associated with 2 immunochemically distinct proteins, protein R and S (Mr approximately 23 kDa; pI 9-10), which were isolated in pure form from barley grain. The proteins are homologous with thaumatin- and pathogenesis-related proteins of the PR5 family. The proteins inhibit growth of i.a. Trichoderma viride and Candida albicans in microtiter plate assays and act synergistically with barley grain chitinase C. Like maize zeamatin, protein R and S but not chitinase C retarded fungal growth in synergism with nikkomycin Z, a nucleoside-peptide inhibitor of fungal chitin synthesis. Although no inhibition of alpha-amylases or serine proteases could be associated with protein R or S the results indicate that the homologous maize grain bifunctional inhibitor of insect alpha-amylase and trypsin is very similar to or identical with maize zeamatin, which was proposed to have permeabilizing activity towards fungal membranes. Thus, in addition to the intensely sweet properties of thaumatin, multiple unrelated defense functions against insect and fungal pests can now be associated with the family of thaumatin-homologous proteins.

Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf.

Antifungal activity in vitro has been associated with barley leaf and grain proteins which are homologous with pathogenesis related proteins of type 4 (PR-4) from tobacco and tomato and with C terminal domains of potato win and Hevea hevein precursor proteins. One protein (pI approximately 9.3, M(r) approximately 13.7 kDa) from barley grain and two very similar proteins from leaves infected with Erysiphe graminis were isolated by chitin affinity chromatography, but none of the proteins showed chitinase activity in vitro. The leaf proteins were increased several fold in response to either Erysiphe infection or NiCl2 infiltration and accumulated extracellularly. The three barley proteins were found to inhibit growth of Trichoderma harzianum in microtiter plate assays using approximately 10 micrograms/ml concentrations and in lower concentrations in a synergistic way when mixed either with barley chitinase C (a PR-3 type protein) or with barley protein R (a PR-5 type protein). Structurally similar proteins were detected in wheat, rye and oats grain extracts.

Inhibition of coagulation factors by recombinant barley serpin BSZx.

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.

Serpins from wheat grain.

Wheat serpin genes have been identified by Southern blot hybridization with three distinct barley protein Z probes. Immunoblot analysis with a monoclonal antibody towards barley protein Z confirmed expression of related M(r) approximately 40 kDa proteins in wheat grain. The wheat serpins were extracted under reducing conditions and separated from beta-amylase and other seed proteins by thiophilic adsorption and anion-exchange chromatography. One molecular form possessing chymotrypsin inhibitory activity was isolated in a reactive site cleaved form on a chymotrypsin affinity column. N-terminal amino acid sequences of a CNBr fragment and of the C-terminal peptide from the cleaved inhibitor (M(r) 4574 +/- 4 Da) verified homology with barley protein Z and mammalian serpins. The native inhibitory serpin was demonstrated to form an SDS-stable complex with alpha-chymotrypsin.

Synthesis of salt-soluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes.

The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [(14)C] sucrose at specific times after anthesis. Proteins were extracted from isolated endosperms and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Fluorography revealed an early, middle and late synthesis of specific proteins during grain filling. Synthesis of proteins appearing at the later stages responded to increased nitrogen nutrition. Two major components, ß-amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein.

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4.

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.

cDNA cloning, characterization and expression of an endosperm-specific barley peroxidase.

A barley peroxidase (BP 1) of pI ca. 8.5 and Mr 37,000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.

Heterologous expression of three plant serpins with distinct inhibitory specificities.

For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed. BSZx, BSZ4, and WSZ1 were assayed for inhibitory activity against trypsin, chymotrypsin, and cathepsin G, and cleavage sites in the reactive center loop were identified by sequencing. BSZx proved to be a potent inhibitor with specific, overlapping reactive centers either at P1 Arg for trypsin or at P2 Leu for chymotrypsin. At 22 ;C, the apparent rate constant for chymotrypsin inhibition at P2 (ka = 9.4 x 10(5) M-1 s-1) was only four times lower than for trypsin at P1 (ka = 3.9 x 10(6) M-1 s-1), and the apparent inhibition stoichiometries were close to 1. Furthermore, our data suggest that cathepsin G was inhibited by BSZx (ka = 3.9 x 10(6) M-1 s-1) at both the P1 Arg and P2 Leu. These results indicate a unique adaptability of the reactive center loop of BSZx. WSZ1 inhibited chymotrypsin (ka = 1.1 x 10(5) M-1 s-1) and cathepsin G (ka = 7.6 x 10(3) M-1 s-1) at P1 Gln and not, as for BSZx, at the more favorable P2 Leu. BSZ4 inhibited cathepsin G (ka = 2.7 x 10(4) M-1 s-1) at P1 Met but was hydrolyzed by trypsin and chymotrypsin. The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data.

Localization to chromosomes of structural genes for the major protease inhibitors of barley grains.

Wheat-barley chromosome addition lines were compared by isoelectric focusing of protein extracts to identify chromosomes carrying loci for the major immunochemically distinct protease inhibitors of barley grains. Structural genes for the following inhibitors were localized: an inhibitor of both endogenous α-amylase 2 and subtilisin (ASI) on chromosome 2, two chymotrypsin/subtilisin inhibitors (CI-1 and CI-2) on chromosome 5 (long arm) and the major trypsin inhibitor (TI-1) on chromosome 3.

Primary structure and differential expression of beta-amylase in normal and mutant barleys.

The primary structure of barley endosperm beta-amylase, an enzyme which catalyses the liberation of maltose from 1,4-alpha-D-glucans, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 1754 nucleotides long [excluding the poly(A) tail] and codes for a polypeptide of 535 amino acids with a relative molecular mass of 59,663. The deduced amino acid sequence was compared with the sequences of ten peptides obtained from the purified enzyme and unambiguous identification was obtained. The N-terminal region of the deduced sequence was identical to a 12-residue cyanogen-bromide-peptide sequence, indicating that beta-amylase is synthesized as the mature protein. A graphic matrix homology plot shows four glycine-rich repeats, each of 11 residues, preceding the C-terminus. Southern blotting of genomic DNA demonstrates that beta-amylase is encoded by a small gene family, while cDNA sequence analysis indicates the presence of at least two types of mRNA in the endosperm. Dot and northern blot analysis show that Hiproly barley contains greatly increased levels of beta-amylase mRNA compared to the normal cultivar Sundance, whereas Risø mutant 1508 contains only trace amounts. These results correlate well with the deposition of beta-amylase during endosperm development in these lines. Low but similar amounts of beta-amylase mRNAs sequences were detected in leaves and shoots from normal and mutant barleys, demonstrating that the mutant lys3a (1508) and lysl (Hiproly) genes do not affect the expression of beta-amylase in these tissues.

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins.

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA(3). B and C hordein polypeptides and the salt-soluble proteins beta-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the alpha-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, beta-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of alpha-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for alpha-amylase, these hormones have the opposite effect on ASI mRNA levels.

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 P(1)-P(1)' Gln-Gln, and WSZ2b at P(2)-P(1) Leu-Arg-one of two overlapping reactive centers. WSZ1c with P(1)-P(1)' Leu-Gln was the fastest inhibitor of chymotrypsin (k(a) = 1.3 x 10(6) m(-1) s(-1)). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k(a) approximately 10(5) m(-1) s(-1)), which has P(1)-P(1)' Leu-Ser-a reactive center common in animal serpins. WSZ2b inhibited plasmin at P(1)-P(1)' Arg-Gln (k(a) approximately 10(3) 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.

A recombinant wheat serpin with inhibitory activity.

A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27-33% identical with human serpins such as alpha 1-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with alpha-chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.

Multiple molecular forms of ß-amylase in seeds and vegetative tissues of barley.

The molecular forms of ß-amylase present in developing, mature, germinating and malted grains of barley (Hordeum vulgare L.), and in vegetative tissues, have been studied using Western-blot analyses and isoelectric focusing of isoenzymes. Five isoforms with different relative molecular masses (Mrs) could be recognised. The major isoform present in the mature grain, called isoform B, had an Mr of about 60 000. This was converted on malting or germination to two lower-Mr forms called C and D. Previous work (R. Lundgard and B. Svensson, 1986, Carlsberg Res. Commun. 51, 487-491) has shown that these result from partial proteolysis of isoform B. Isoenzyme analyses showed complex patterns of bands, with pIs between about 5.0 and 6.0. Two allelic types were present in the eight lines. A number of new bands with a range of pIs appeared during germination and malting.An isoform with the same Mr as D and a minor low-Mr isoform (E) were present in young developing whole caryopses (8-12 d after anthesis), but not in older developing endosperms (14-21 d after anthesis). Isoenzyme analyses also showed different patterns of bands in these two tissues, while hybrid-dot analyses indicated the presence of separate populations of mRNAs. It is suggested that the early endosperm isoforms (D and E) are "green" ß-amylases present in the pericarp and-or testa of the young caryopses.Roots but not shoots or leaves also contained an isoform with the same Mr as D, although the pattern of isoenzymes differed from that present in the seed tissues.The fifth isoform, A, was a diffuse high-Mr form present in small amounts in all seed and vegetative tissues, and may correspond to a constitutively expressed form.These multiple molecular forms of ß-amylase are discussed in relation to the recent report that ß-amylase is encoded by two structural loci, with a total copy number of two to three per haploid genome (Kreis et al, 1988, Genet. Res. Camb. 51, 13-16).

Development of a monoclonal antibody to urinary degradation products from the C-terminal telopeptide alpha 1 chain of type I collagen. Application in an enzyme immunoassay and comparison to CrossLaps ELISA.

A monoclonal antibody MAbA7 was raised against a synthetic peptide having a sequence (EKAHDGGR) specific for a part of the C-telopeptide alpha 1 chain of type I collagen. MAbA7 was labelled with horseradish peroxide and used in a competitive one-step enzyme-linked immunosorbent assay (ELISA) for measurement of urinary type I collagen degradation products. The assay was technically evaluated and preliminary clinical data are presented. The measuring range was 200-7000 micrograms l-1 with a detection limit of 25 micrograms l-1. Within-run and total CVs were 5.5 and 8.0%, respectively. Analytical recovery averaged 96.6% +/- 5.3 (mean +/- 1SD). Values obtained in the ELISA were highly correlated (r = 0.93) to values obtained by a commercially available assay (CrossLaps ELISA) known to measure urinary degradation products derived from the C-telopeptide of type I collagen reflecting the rate of bone resorption. Investigation of the urinary fragments responsible for the immunological response in the two assays revealed, however, that they are not identical. Values obtained in urine samples from postmenopausal women (n = 108) and patients with Paget's disease (n = 6) increased 43% (p < 0.01) and 28-fold (p < 0.001), respectively, when compared to a premenopausal level (n = 50). A decrease in the urinary concentrations of 67% (p < 0.01) was seen after 6 months in urine samples from postmenopausal women (n = 13) receiving hormone replacement therapy (HRT) compared to a group receiving placebo (n = 9). Likewise, the urinary concentrations decreased 88% (p < 0.001) in early postmenopausal women receiving bisphosphonate therapy (n = 11) for a period of 9 months compared to a group receiving placebo (n = 12). These results suggest that the monoclonal antibody and the new assay may be useful for further investigations of the physiological and clinical importance of type I collagen degradation.