The CM-Proteins from Cereal Endosperm: Immunochemical Relationships

The CM-proteins, which are salt-soluble proteins that can beextracted with chloroform: methanol (2: 1, v/v), seem to bepresent in the endosperm of all the cereal species investigated.Antibodies raised against a mixture of the barley CM-proteins(A-H) cross-reacted with wheat and rye proteins in Ouchterlonytests and a detailed study was carried out for purified wheat(CM1, CM2. CM3. CM3') and barley (CMa, CMb, CMc, CMd) CM-proteins. [³⁵Sl-Cysteine-labelled endosperm proteins from wheat and barleywere investigated by immuno-precipitation, electrophoresis andfluorography using the antibodies (A-H) and also those to amixture of wheat CM-proteins and to CMd. There was completeantigenic identity for all the wheat proteins and CMd, someof the other proteins showed partial antigenic identity. Previously proposed genetic and biochemical relationships amongthese proteins were confirmed in the present study. Key words: CM-Protein, Cereal endosperm, Immunochemistry     

The Isolation and Characterization of Low Molecular Weight Hydrophobic Salt-Soluble Proteins from Barley

The four main components of the previously designated A-hordeinfraction have been purified to homogeneity and partially characterized.Their amino acid composition and solubility properties clearlydiffer from those of both prolamins and other purified salt-solubleproteins from barley endosperm. Evidence is presented of theirhomology with the previously described CM-proteins from wheatso their designation as CMa, CMb, CMc and CMd is proposed.     

In vivo and in vitro synthesis of CM-proteins (A-hordeins) from barley (Hordeum vulgare L.)

CM-proteins from barley endosperm (CMa, CMb, CMc, CMd), which are the main components of the A-hordein fraction, are synthesized most actively 10 to 30 d after anthesis (maximum at 15–20 d). They are synthesized by membranebound polysomes as precursors of higher apparent molecular weight (13,000–21,000) than the mature proteins (12,000–16,000). The largest in vitro product (21,000) is the putative precursor of protein CMd (16,000), as it is selected with anti-CMd monospecific IgG's, and is coded by an mRNA of greater sedimentation coefficient (9 S) than those encoding the other three proteins (7.5 S). CM-proteins always appear in the soluble fraction, following different homogenization and subcellular fractionation procedures, indicating that these proteins are transferred to the soluble fraction after processing.     

Differential effects of high-lysine mutations on the accumulation of individual members of a group of proteins encoded by a disperse multigene family in the endosperm of barley (Hordeum vulgare L.)

The CM proteins are a group of major salt-soluble endosperm proteins encoded by a disperse multigene family. The effects of high-lysine mutations on the net accumulation in barley endosperm of three members of this group (CMa, CMb, and CMe) have been investigated. Genes CMa, CMb and CMe are located in chromosomes 1, 4, and 3 respectively. Protein CMe has been found to be identical with a previously described trypsin inhibitor. The three proteins have been quantified in the different genetic stocks by HPLC. The different high-lysine mutations have different effects on the expression patterns of the three genes: CMe is markedly decreased and CMa and CMb are increased in mutant Ris? 1508, whereas all three proteins are decreased in Ris? 527 and increased in Ris? 7 with respect to the wild-type Bomi; CMa and CMb are increased and CMe is unaffected in mutant Ris? 56 with respect to the wild-type Carlsberg II; and protein CMe is markedly decreased in Hiproly barley as compared with its sister line CI4362. The implications of these results in connection with the evolution of CM proteins and with the characterization of high-lysine mutations are discussed.     

A tetrameric inhibitor of insect α-amylase from barley

A tetrameric inhibitor that is active against α-amylase from the larvae of the insect Tenebrio molitor, but inactive against the enzyme from human saliva and against the endogenous one, has been described in barley endosperm. The subunits of the inhibitor have been identified as the previously characterized proteins CMa, CMb and CMd, of which only CMa was inhibitory by itself.     

Characterization of cDNA clones of the family of trypsin/α-amylase inhibitors (CM-proteins) in barley (Hordeum vulgare L.)

Summary Recombinants encoding members of the trypsin/-amylase inhibitors family (also designated CM-proteins) were selected from a cDNA library prepared from developing barley endosperm. Inserts in two of the clones, pUP-13 and pUP-38, were sequenced and found to encode proteins which clearly belong to this family, as judged from the extensive homology of the deduced sequences with that of the barley trypsin inhibitor CMe, the only member of the group for which a complete amino acid sequence has been obtained by direct protein sequencing. These results, together with previously obtained N-terminal sequences of purified CM-proteins, imply that there are at least six different members of this dispersed gene family in barley. The relationship of this protein family to the B-3 hordein and to reserve prolamins from related species is discussed in terms of their genome structure and evolution.     

Molecular cloning of the barley seed protein CMd: A variant member of the α-amylase/trypsin inhibitor family of cereals

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of α-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with α-amylases of bacterial origin.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.     

Antifungal Activity of a Bowman–Birk-type Trypsin Inhibitor from Wheat Kernel

A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC₅₀) values ranging from 111.7 to above 500 μg/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens.     

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.     

Apocrine secretion of amylase and exocytosis of trypsin along the midgut of Tenebrio molitor larvae

Amylase and trypsin were purified from Tenebrio molitor midgut larvae and used to raise antibodies in a rabbit. A Western blot of T. molitor midgut homogenates, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using amylase and trypsin antisera, showed only bands co-migrating with the purified enzymes. The antisera were used to localize the enzymes by immunofluorescence and immunogold labeling. Amylase occurs in a few regularly disposed anterior midgut cells. Non-amylase-secreting anterior midgut cells are proposed to be water-absorbing cells based on morphology and dye movements. Amylase is found inside vesicles originating from Golgi areas that seem to fuse together before their release along with the now disorganized apical cytoplasm (apocrine secretion). Trypsin precursors are observed inside small vesicles near the apical plasma membrane of posterior midgut cells, suggesting an exocytic mechanism of secretion, followed by putative trypsin activation. Apocrine secretion is thought to be an adaptation to enhance the dispersion of secretory vesicle contents released from a water-absorbing epithelium, whereas exocytosis is an efficient secretory mechanism in a water-secreting epithelium.     

Genetics of CM-proteins (A-hordeins) in barley

Summary The CM-proteins, which are the main components of the A-hordeins, include four previously described proteins (CMa-1, CMb-1, CMc-1, CMd-1), plus a new one, CMe-1, which has been tentatively included in this group on the basis of its solubility properties and electrophoretic mobility. The variability of the five proteins has been investigated among 38 Hordeum vulgare cultivars and 17 H. spontaneum accessions. Proteins CMa-1, CMc-1 and CMd-1 were invariant within the cultivated species; CMd was also invariant in the wild one. The inheritance of variants CMb-1/CMb-2 and CMe-1/CMe-2,2 was studied in a cross H. spontaneum x H. vulgare. The first two proteins were inherited as codominantly expressed allelic variations of a single mendelian gene. Components CMe-2,2 were jointly inherited and codominantly expressed with respect to CMe-1. Gene CMb and gene(s) CMe were found to be unlinked. The chromosomal locations of genes encoding CM-proteins were investigated using wheat-barley addition lines. Genes CMa and CMc were associated with chromosome 1, and genes CMb and CMd with chromosome 4. These gene locations further support the proposed homoeology of chromosomes 1 and 4 of barley with chromosomes groups 7 and 4 of wheat, respectively. Gene(s) CMe has been assigned to chromosome 3 of barley. The accumulation of protein CMe-1 is totally blocked in the high lysine mutant Riso 1508 and partially so in the high lysine barley Hiproly.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

The peritrophic membrane of Spodoptera frugiperda: secretion of peritrophins and role in immobilization and recycling digestive enzymes

A peritrophin from the Spodoptera frugiperda peritrophic membrane (PM) and microvillar proteins from S. frugiperda anterior midgut cells were isolated and used to raise antibodies in a rabbit. These antibodies, as well as a Tenebrio molitor amylase antibody that cross-reacts with S. frugiperda amylases, and wheat-germ aglutinin were used in immunolocalization experiments performed with the aid of confocal fluorescence and immunogold techniques. The results showed that the peritrophin was secreted by anterior midgut columnar cells in vesicles pinched-off the microvilli (microapocrine secretion). The resulting double membrane vesicles become single membrane vesicles by membrane fusion, releasing peritrophin and part of the amylase and trypsin. The remaining membranes still containing microvillar proteins and membrane-bound amylase and trypsin are incorporated into a jelly-like material associated with PM. Calcofluor-treated larvae lacking a PM were shown to lose the decreasing gradient of trypsin and chymotrypsin observed along the midgut of control larvae. This gradient is thought to be formed by a countercurrent flux of fluid (in the space between PM and midgut cells) that powers enzyme recycling.     

Evaluation of in vitro and in vivo effects of semipurified proteinase inhibitors from theobroma seeds on midgut protease activity of lepidopteran pest insects

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.     

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation.     

Serine protease inhibitors of North American leeches

Serine protease inhibitors in extracts from three North American leeches, Nephelopsis obscura, Erpobdella punctata and Hemopis marmorata have been separated by anion exchange chromatography and the activity pattern against human granulocyte elastase and porcine chymotrypsin and trypsin determined. All three leech species contained a major peak with anti-trypsin activity, but Hemopis was unique in that the trypsin inhibitor was equally active against chymotrypsin. Nephelopsis was rich in anti-elastase activity of two types, one which was also active against chymotrypsin, and one which was a specific elastase inhibitor. Erpobdella contained inhibitors against elastase and chymotrypsin but with major activity against the latter.     

Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests. We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L.) by biolistic bombardment of cultured immature embryos. Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe. BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 1.1% of total extracted protein. No expression was detected in untransformed wheat seeds. Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay. The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance. However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects.     

Purification,characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> L. Wilczek) seeds

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH₄(SO₄)₂ fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.