Tetrameric Inhibitor Research Articles

Adaptive polymorphism of tetrameric alpha-amylase inhibitors in wild emmer wheat

α-Amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. Wheat tetrameric α-amylase inhibitor (WTAI) is a mixture (60 kDa) of 3 units: WTAI-CM2 plus 2 WTAI-CM3 plus WTAI-CM16, where none of the subunits is active on its own. A total of 334 gene sequences were obtained from 14 populations (131 accessions= genotypes) of wild emmer wheat. The frequencies of SNPs in WTAI-CM2, WTAI-CM3 and WTAI-CM16 were 1 out of 87.6, 101.4, and 108.0 bases, where 5, 5 and 4 SNPs were detected in the coding sequence, respectively. The nucleotide sequence of each unit of tetrameric α-amylase inhibitors were much more conserved than that of dimeric or monomeric inhibitors. The wild emmer wheat populations showed diversity on three WTAI loci, both between and within populations. It was revealed that WTAI were naturally selected for across populations by a ratio of dN/dS as expected. The results of purifying and positive selection hypothesis (p<0.05) also showed that the sequences of WTAI were contributed by natural selection, which ensures the protein function conservation as well as the inhibition diversity with insects amylase enzyme. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. Ecological factors have an important evolutionary role in gene differentiation at these loci, and tetrameric α-amylase inhibitors are obviously adaptively selected under different environments.

The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat cultivar Butte 86

BackgroundWheat grains accumulate a variety of low molecular weight proteins that are inhibitors of alpha-amylases and proteases and play an important protective role in the grain. These proteins have more balanced amino acid compositions than the major wheat gluten proteins and contribute important reserves for both seedling growth and human nutrition. The alpha-amylase/protease inhibitors also are of interest because they cause IgE-mediated occupational and food allergies and thereby impact human health.ResultsThe complement of genes encoding alpha-amylase/protease inhibitors expressed in the US bread wheat Butte 86 was characterized by analysis of expressed sequence tags (ESTs). Coding sequences for 19 distinct proteins were identified. These included two monomeric (WMAI), four dimeric (WDAI), and six tetrameric (WTAI) inhibitors of exogenous alpha-amylases, two inhibitors of endogenous alpha-amylases (WASI), four putative trypsin inhibitors (CMx and WTI), and one putative chymotrypsin inhibitor (WCI). A number of the encoded proteins were identical or very similar to proteins in the NCBI database. Sequences not reported previously included variants of WTAI-CM3, three CMx inhibitors and WTI. Within the WDAI group, two different genes encoded the same mature protein. Based on numbers of ESTs, transcripts for WTAI-CM3 Bu-1, WMAI Bu-1 and WTAI-CM16 Bu-1 were most abundant in Butte 86 developing grain. Coding sequences for 16 of the inhibitors were unequivocally associated with specific proteins identified by tandem mass spectrometry (MS/MS) in a previous proteomic analysis of milled white flour from Butte 86. Proteins corresponding to WDAI Bu-1/Bu-2, WMAI Bu-1 and the WTAI subunits CM2 Bu-1, CM3 Bu-1 and CM16 Bu-1 were accumulated to the highest levels in flour.ConclusionsInformation on the spectrum of alpha-amylase/protease inhibitor genes and proteins expressed in a single wheat cultivar is central to understanding the importance of these proteins in both plant defense mechanisms and human allergies and facilitates both breeding and biotechnology approaches for manipulating the composition of these proteins in plants.

Highly potent and long-acting trimeric and tetrameric inhibitors of influenza virus neuraminidase

A set of trimeric and tetrameric derivatives 6– 11 of the influenza virus neuraminidase inhibitor zanamivir 1 have been synthesized by coupling a common monomeric zanamivir derivative 3 onto various multimeric carboxylic acid core groups. These discrete multimeric compounds are all significantly more antiviral than zanamivir and also show outstanding long-lasting protective activity when tested in mouse influenza infectivity experiments.

Divergence in properties of two closely related α‐amylase inhibitors of barley

The only inhibitor of human salivary α‐amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N‐terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu‐C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α‐amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α‐amylase inhibitor family.

Cloning of cDNA, expression, and chromosomal location of genes encoding the three types of subunits of the barley tetrameric inhibitor of insect ?-amylase

Three cDNA clones from barley developing endosperm, corresponding to proteins BTAI-CMa, BTAI-CMb and BTAI-CMd, which are the three types of subunits of the tetrameric inhibitor of insect alpha-amylases, have been identified and sequenced. The deduced amino acid sequence of BTAI-CMb corresponds to the CM16/CM17 type of subunit in wheat (92/90% identical residues) and has one putative N-glycosylation site (NLT) and a possible kinase-C phosphorylation site (SCR). The BTAI-CMa sequence differs at four amino acid residues from a previously reported one from cv. Bomi and the sequence deduced for BTAI-CMd completes (11 N-terminal residues) and confirms previously available data. The gene for BTAI-CMa (Iat1) is located in the beta arm of barley chromosome 7H (syn.1), while genes for both BTAI-CMb (Iat2) and BTAI-CMd (Iat3) are in the long arm of chromosome 4H. The three genes are expressed in endosperm and their mRNAs are not detected in the other tissues tested, except Iat1, which seems to be expressed at a low level in coleoptile and roots, where it is switched off by 50 microM methyl jasmonate.

Barley Tetrameric Inhibitor of Insect α-amylases. Characterization of an Allelic Variant of the BTAI-CMb Subunit

Abstract An allelic variant, designated BTA1-CMb2, of the -CMb1 subunit of the tetrameric α-amylase inhibitor of Bomi barley has been isolated from the endosperm of Hordeum vulgare L., cv. Hatif de Grignon. BTA1-CMb1 and -CMb2 showed a single, but significant (E → K), difference in the first 23 amino acid residues of their corresponding N-terminal sequences. As in the case of BTAI-CMb1, a glycosylated form of -CMb2 has also been identified and purified. The tetrameric inhibitors of Bomi and Hatif barleys have been reconstituted in vitro from their isolated subunits, and their activities against Tenebrio molitor α-amylase have been tested. No significant differences have been found for inhibitory activity among tetramers with BTAI-CMb1, -CMb2 or their corresponding glycosylated forms.

Cloning of cDNA and chromosomal location of genes encoding the three types of subunits of the wheat tetrameric inhibitor of insect ?-amylase

We have characterized three cDNA clones corresponding to proteins CM1, CM3 and CM16, which represent the three types of subunits of the wheat tetrameric inhibitor of insect alpha-amylases. The deduced amino acid sequences of the mature polypeptides are homologous to those of the dimeric and monomeric alpha-amylase inhibitors and of the trypsin inhibitors. The mature polypeptides are preceded by typical signal peptides. Southern blot analysis of appropriate aneuploids, using the cloned cDNAs as probes, has revealed the location of genes for subunits of the CM3 and of the CM16 type within a few kb of each other in chromosomes 4A, 4B and 4D, and those for the CM 1 type of subunit in chromosomes 7A, 7B and 7D. Known subunits of the tetrameric inhibitor corresponding to genes from the B and D genomes have been previously characterized. No proteins of this class have been found to be encoded by the A genome in hexaploid wheat (genomes AA, BB, DD) or in diploid wheats (AA) and no anti alpha-amylase activity has been detected in the latter, so that the A-genome genes must be either silent (pseudogenes) or expressed at a much lower level.

Wheat tetrameric inhibitors of insect α-amylases: Alloploid heterosis at the molecular level

Tetrameric inhibitors of heterologous alpha-amylases have been characterized in allohexaploid wheat, Triticum aestivum (genomes AABBDD), as well as in Triticum turgidum (AABB) and Triticum tauschii (DD). Their subunits have been identified as the previously described CM proteins. Single oligomeric species were observed in T. Turgidum (subunits CM2, CM3A, and CM16) and in T. tauschii (CM1, CM3D, and CM17) by a two-dimensional electrophoretic method that does not dissociate the inhibitors in the first dimension. Multiple tetrameric species, resulting from different combinations of the subunits contributed by the two ancestral species, are observed by the same procedure in T. aestivum. The three types of subunits were required for significant activity when the inhibitor of T. turgidum was reconstituted from the purified subunits, whereas, in the case of T. tauschii, binary mixtures involving subunit CM1 also had some activity. Additional combinations of the subunits present in these two species, which occur in the allohexaploid T. aestivum, were also reconstituted, and their inhibitory activities ranged from 144% to 33% the activity of the reconstituted inhibitor from T. tauschii. The activity of these inhibitors toward the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) of the insect Tenebrio molitor is much greater than that against the salivary enzyme. These observations, together with the previously established chromosomal locations of genes encoding CM proteins, fit a model of alloploid heterosis at the molecular level.

Subunits of tetrameric α-amylase inhibitors of Hordeum chilense are encoded by genes located in chromosomes 4Hch and 7Hch

Three proteins (components 1, 2, and 4) of the non-prolamin, 70% ethanol soluble fraction from the endosperm of Hordeum chilense have been identified as putative subunits of the tetrameric inhibitors active against insect α-amylases. In experiments carried out with the synthetic alloploid Tritordeum (H. chilense x Triticum turgidum conv. durum), previously described proteins from T. turgidum, designated CM2, CM3 and CM 16, have been also identified as subunits of α-amylase inhibitors. Genes for components 1 and 4 of H. chilense have been located in chromosomes 4H(ch) and 7H(ch), based on the analysis of H. chilense-T.turgidum addition lines. Subunits of the inhibitors from wheat and from cultivated barley had been previously assigned to chromosomes of the same homoeology groups.

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.

Purification and properties of an α-amylase tetrameric inhibitor from wheat kernel

A family of tetrameric protein inhibitors active against exogenous α-amylases has been extracted from wheat kernel with 150 mM NaCl and purified by gel filtration in the absence and presence of guanidine hydrochloride and by ion-exchange chromatography. The purified isoinhibitor fraction showed four bands of protein with closely related electrophoretic mobilities and isoelectric points. The M r was 48000 in the absence of dissociating agents and dropped to about 12 000 in the presence of guanidine or sodium dodecyl sulphate. Amino acid composition, thermostability and high number of intrachain disulphide bridges were properties closely related to those of monomeric and dimeric protein inhibitors from the same source, but the immunological properties were distinctly different. The tetrameric inhibitor was strongly active against animal α-amylases, and inactive against bacterial, fungal and plant enzymes; the extent of inhibition of yellow mealworm α-amylase was dependent, as for many other wheat protein inhibitors, upon preincubation time, pH and temperature. It is likely that these tetrameric inhibitors, together with wheat inhibitor families of M r 24 000 and 12 000, have a common genetic origin.

Regulation of the Ca 2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca 2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca 2+ (0.1–1 m m), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B. L. (1984) Arch. Biochem. Biophys. 232, 505–512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca 2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca 2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca 2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca 2+. Under these conditions, the high-molecular-weight complex (> 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca 2+-requiring form.

Chymotrypsin Inhibitor I from Potatoes: LARGE SCALE PREPARATION AND CHARACTERIZATION OF ITS SUBUNIT COMPONENTS

Abstract A method for the large scale preparation of chymotrypsin Inhibitor I from potato tubers is described. The method utilizes the inhibitor's stability to acid at elevated temperatures and its dissociation in 4 m guanidine hydrochloride into subunits (protomers) to produce the pure protein with a 47% recovery and a 40-fold purification. Ion exchange chromatography on sulfoethylcellulose in the presence of 0.1 m formic acid in 8 m urea resolves Inhibitor I protomers into two major and two minor types. After reassociation to the tetramer form by dilution, one of the two major rechromatographed protomers was shown to be a powerful inhibitor of both chymotrypsin and trypsin, whereas the other strongly inhibits chymotrypsin but only weakly inhibits trypsin. All four purified protomers resolved from Inhibitor I can be reassociated either individually or hybridized with each other to form tetrameric isoinhibitors. Tetrameric Inhibitor I species prepared from each of the four protomeric types all have an NH2-terminal glutamic acid. However, they differ quantitatively from each other in amino acid composition, reactivity with chymotrypsin and trypsin, digestibility by pepsin, and electrophoretic mobility. Tetrameric Inhibitor I in potatoes is therefore a heterogeneous mixture of isoinhibitors whose properties reflect those of its individual protomers. The molecular weights of tetrameric Inhibitor I and its complex with chymotrypsin were re-evaluated. The molecular weight of Inhibitor I was found to be 39,000 ± 2,000, and that of its complex saturated with chymotrypsin, 140,000 ± 4,600.