Kunitz Soybean Trypsin Inhibitor Research Articles

Inhibitory modes of Kunitz and Bowman-Birk soybean trypsin inhibitors to tryptic and chymotryptic proteinases of tiger puffer and yellowtail

SUMMARY: An inhibitory effect of Kunitz and Bowman-Birk soybean trypsin inhibitors (K- and BB-SBTI) was investigated on tryptic and chymotryptic proteinases of a brood of tiger puffer Takifugu rubripes and yellowtail Seriola quinqueradiata. These assist to comprehend the differences in dietary soybean meal (SBM) utility by fish species and to develop low cost SBM diets with low contents of fish meal. The K-SBTI strongly inhibited tryptic proteinase and weakly inhibited chymotryptic proteinase from the hepatopancreas and intestine including pyloric ceca of the fishes. The BB-SBTI strongly inhibited both proteinases of the fishes. The K-SBTI inhibition for tryptic protease and the BB-SBTI inhibition for tryptic and chymotryptic proteases were relatively constant in tiger puffer with growth of 12–199 g bodyweight. In yellowtail with growth of 7–672 g bodyweight, the K-SBTI inhibition for tryptic protease and the BB-SBTI inhibition for chymotryptic protease were also relatively constant. The BB-SBTI inhibition for yellowtail tryptic protease typically fell in the stage from 7 g to 57 g bodyweight, and then decreased slightly or was maintained constantly. These results indicate that there are different inhibitory modes of K- and BB-SBTI and there is inter- and intraspecific diversity of tryptic protease conformation between the two species.

Genetic Characterization of a Novel Soybean Kunitz Trypsin Inhibitor.

Genetic and nucleotide sequence studies were performed on a new variant of soybean Kunitz trypsin inhibitor (SKTI) detected in wild soybean (Glycine soja) and showing a slightly slower electrophoretic mobility than the Tia type. The segregation analysis of SKTI bands in F 2 seeds from crosses of the new variant type with Tia or Tib type showed that the variant type is controlled by an allele codominant to Tia and Tib at an SKTI locus. Nucleotide sequence analysis showed that this variant has 217 amino acids composed of 181 amino acid residues of mature SKTI and extra 25 and 11 amino acids at N- and C-terminal regions, respectively. This sequence was identical to that of Tia (=KTi3), except that a G→A transitional mutation occurred at position 500 of Tia, which results in the translational change from Arg to Lys. The result of isoelectric forcusing-PAGE coincided with this change. In addition, three nucleotides GCT were inserted at the N-terminus, which leads to an Ala addition in the precursor of this SKTI protein. From these results, we propose the genetic symbol Tie for the new variant of SKTI.

Identification of distant homologues of fibroblast growth factors suggests a common ancestor for all β-trefoil proteins

Determination of the structures of fibroblast growth factors and interleukin-1s has previously revealed that they both adopt a β-trefoil fold, similar to those found in Kunitz soybean trypsin inhibitors, ricin-like toxins, plant agglutinins and hisactophilin. These families possess distinct functions and occur in different subcellular localisations, and they appear to lack significant similarities in their sequences, ligands and modes of ligand binding. We have analysed the significance of sequence identities observed after structure alignment and provide statistical evidence that these β-trefoil proteins are all homologues, having arisen from a common ancestor. In addition, we have explored the sequence space of all β-trefoil proteins and have determined that the actin-binding proteins fascins, and other proteins of unknown function, are β-trefoil family homologues. Unlike other β-trefoil proteins, the triplicated repeats in each of the four β-trefoil domains of fascins are significantly similar in sequence. This hints at how the β-trefoil fold arose from the duplication of an ancestral gene encoding a homotrimeric single-repeat protein. The combined analysis of structure and sequence databases for detecting significant similarities is suggested as a highly sensitive approach to determining the common ancestry of extremely divergent homologues.

Detection of Potentially Allergenic Material in 12 Hydrolyzed Milk Formulas

Hypoallergenic milk formulas are used as an alternative diet for infants who have allergies to cow's milk when breast-feeding is not possible. These products are based on proteins, which have been heat-treated and hydrolyzed to a different degree in order to cleave anti-body-binding structures. Even extensively hydrolyzed products have occasionally been observed to elicit allergic reactions in sensitized infants, however. Therefore, the parameters of relevance to allergenic potential require more investigation. The objective of the present study was to investigate 12 different hydrolyzed milk formulas for their contents of potentially allergenic protein material, i.e. material that may induce allergenicity or elicit allergic responses in already sensitized individuals. Analytical methods applied were gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE, immunoblotting, dot-immunobinding, and ELISA. Care was taken to assure that all protein fractions were investigated, including supernatants and precipitates following centrifugation of the milk formulas. By gel filtration, protein material with apparent molecular masses of 7 to >30 kDa was detected. Analysis by SDS-PAGE of formula precipitates showed that proteins with a molecular mass above 20 kDa were present even in some of the extensively hydrolyzed formulas. Residual antigenic β-lactoglobulin was found by ELISA in all products. By immunoblotting and dot-immunobinding with antibodies against total whey, caseins, or Kunitz soybean trypsin inhibitor, we observed antigenic material mainly in partially hydrolyzed products. We concluded that SDS-PAGE of formula supernatants and precipitates gave the most differentiated profile of hydrolyzed formulas and that this method is well suited for screening potential allergenicity.

Interpretation of DSC data on protein denaturation complicated by kinetic and irreversible effects

Thermal denaturation of Kunitz soybean trypsin inhibitor (KTI) and ribulose-1,5-biphosphate carboxylase (RBPC) from tobacco leafs was studied by the method of high-sensitivity differential scanning calorimetry (HS-DSC). The dependence of the denaturation temperature on the heating rate reveals in the case of both proteins a non-equilibrium character of the denaturation transition in applied conditions. Developed kinetic approach allows the determination of an equilibrium transition temperature as well as the rate constants of denaturation and renaturation from the complex data of HS-DSC. This method was applied to the analysis of the pH-induced change of the conformational stability of KTI within pH range from 2.0 to 11.0. It allowed the determination of the pH dependencies: of the excess free energy of denaturation, of the activation enthalpy and entropy of denaturation as well as of the denaturation rate constant. Conclusions have been made suggesting the contribution of side-chain hydrogen bonds in the stabilisation of the native and activated states of KTI.

Long‐term effects of soybean protease inhibitors on digestive enzymes, survival and learning abilities of honeybees

AbstractWe have investigated the effects of long‐term ingestion of two serine proteinase inhibitors (PIs), the Kunitz Soybean trypsin inhibitor (SBTI) and the Bowman‐Birk inhibitor (BBI) on survival, learning abilities involved in the foraging behaviour, and digestive physiology of the honeybee (Apis mellifera L., Hymenoptera). A threshold‐dose was established, above which adverse effects of long‐term ingestion of the PIs tested are to be expected. The experiments reported herein could be extended to other PIs or gene products used to confer insect resistance, and be part of a general procedure used to assess the innocuousness of transgenic melliferous plants to honeybees.

Effects of four protease inhibitors on the survival of worker bumblebees, Bombus terrestris L.

To assess risks posed by transgenic pest-resistant plants, a bumblebee bioassay system was developed. Small and large adults of Bombus terrestriswere held individually or in groups of 5, 10 or 20 in cages and survival and rates of pollen, sugar and water consumption determined. Effects on bee survival of Kunitz soybean trypsin inhibitor (SBTI), bovine pancreatic trypsin inhibitor (BPTI) and two potato protease inhibitors, POT-1 and POT-2, were determined. SBTI (10 mg.g -1 ) and POT-1 (10 and 5 mg.g -1 ) reduced survival significantly. Bees fed POT-2 (10 mg .g -1 ) had poorer survival than those fed 0.1 or 0.01 mg.g -1 POT-2. BPTI had no effect. Untreated bee midguts had elastase-like (283.0 ± 16.9 nmol.min -1.g -1 gut), chymotrypsin (148.5 ± 8.4), trypsin (27.2 ± 2.8) and leucine aminopeptidase (258.6 ± 9.6) activities. Elastase-like and chymotrypsin activities were inhibited by SBTI, POT-1 and POT-2, but not BPTI. Trypsin activity was reduced by each inhibitor. Leucine aminopeptidase activity was unaffected. bumblebee / protease inhibitor / food consumption / small-cage bioassay / pest-resistant transgenic plant

THE HEAT RESISTANCE AND CONFORMATIONAL PLASTICITY OF KUNITZ SOYBEAN TRYPSIN INHIBITOR

The heat resistance of Kunitz soybean trypsin inhibitor (KSTI) and the conformational plasticity of the purified protein were examined in Tris-HCI buffer (0.05M, pH 8). The rate of KSTI inactivation at moderate temperatures 70–100C was slow. The time for 90% inactivation (D-value) of KSTI at 120–150C ranged between 81s–1047s. The activation enthalpy (ΔS) and entropy (ΔS#) change for ultrahigh temperature (UHT) inactivation of KSTI was 107 kj mot-1 and -26 J mol-1, respectively. Fluorescence monitoring of KSTI structure showed a conformational change at 30–70C with a mid-point temperature (Tm) of 55.6C. The heat induced structural change was fully reversible. There was complete regain of the KSTI native structure after heating at 80C, as judged from intrinsic fluorescence measurements. The results are considered in relation to the Lumry-Eyring 2-stage scheme protein inactivation. The UHT resistance of KSTI may be ascribed to its conformation plasticity which allows efficient regain of its native structure after heat unfolding. Other possible causes of heat resistance in biologically active proteins and peptides (BAPP) are briefly discussed.

Enzyme-stabilizing activity of seed trypsin inhibitors during desiccation

Seeds contain protective proteins which are able to preserve glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity upon co-dehydration of the enzyme with heat-stable seed protein extracts. Adenanthera pavonina L. (saga) seed extracts, in particular, have high levels of enzyme-preserving activity which not only alleviate the detrimental effects of desiccation in the presence of Tris–HCl and NaCl, but also stabilize enzyme activity in solution. Purification of Adenanthera seed extracts by reversed-phase and weak anion-exchange perfusion chromatography, followed by N-terminal protein sequencing have identified Kunitz-type trypsin inhibitors as the major proteins in one set of enzyme-preserving fractions. A quantitative comparison using various concentrations of purified proteins has demonstrated that soybean Kunitz trypsin inhibitor is as effective (on a mass basis) as bovine serum albumin in preserving enzyme activity, and may be an important protein permitting enzyme dehydration in a seed undergoing desiccation.

Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stål) in transgenic rice

Proteinase inhibitors are widely distributed in animals, plants and microorganisms and their roles in plants are associated with defense against pests. The utilization of proteinase inhibitors for crop protection has been actively investigated with a variety of proteinase inhibitors. Soybean Kunitz trypsin inhibitor (SKTI), one of the major seed storage protein, is synthesized for a short period during seed development. To investigate the role of SKTI in a plant's defense system against insect predation, a recombinant plasmid containing the full-length cDNA of SKTI under control of the CaMV 35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer method and a large number of transgenic rice plants were regenerated. The integration, expression, and inheritance of this gene was demonstrated in R1 and R2 generations by Southern, northern, and western analyses. Accumulation levels (0.05–2.5% of soluble proteins) of SKTI protein were detected in R1 and R2 plants. Bioassay with R1 and R2 transgenic plants revealed that transgenic plants are more resistant to destructive insect pest of rice, brown planthopper (Nilaparvata lugens Stal), than the control plants. Thus, introduction of SKTI into rice plants can be used to control insect pests.

Expression of the soybean (Kunitz) trypsin inhibitor in transgenic tobacco : Effects on larval development of Spodoptera litura

The coding region of the Tia allelic form of the soybean (Kunitz) trypsin inhibitor gene has been introduced, as a transcriptional fusion with the CAMV 35S promoter, into tobacco. Southern analysis of DNA extracted from progeny (Fl) plants confirmed that an intact copy (or copies) of the gene is integrated into the tobacco genome. Gel filtration column chromatography has been used to partially purify the inhibitor from leaves of transgenic tobacco and inhibition assays revealed that the protein can inhibit both bovine trypsin, and trypsin‐like (BApNA‐hydrolysing) activity extracted from Spodoptera litura digestive tracts. SDS‐PAGE and western blotting determined that the inhibitor accumulates as a protein of ca. 20 kD in transgenic leaf tissue. The protein has been purified to homogeneity using reverse‐phase column chromatography, and subsequent N‐terminal sequencing revealed that the inhibitor is processed in tobacco leaf tissue by the removal of the N‐terminal leader sequence. Insect feeding trials, using neonate larvae of S. litura, have been conducted with leaf tissue excised from transgenic progeny plants that either accumulated the inhibitor or from control (non‐transgenic) plants. These trials established that, when compared with insects fed non‐transformed leaf tissue, larvae fed transgenic leaf tissue demonstrated significantly greater mortality, and the survivors grew more slowly in terms of weight gain over time. These results are interpreted with respect to current opinion on the use of proteinase inhibitors as insect pest resistance factors in transgenic plants.

Midgut carboxypeptidase from Helicoverpa armigera (Lepidoptera: Noctuidae) larvae: enzyme characterisation, cDNA cloning and expression

Using synthetic substrates we have characterised carboxypeptidase activity in gut extracts from Helicoverpa armigera larvae. Carboxypeptidase A activity predominates, with only low levels of carboxypeptidase B activity present. Maximum carboxypeptidase A activity occurs over a broad pH range and is inhibited by phenanthroline and potato carboxypeptidase inhibitor. A cDNA clone encoding carboxypeptidase (the first such sequence from a lepidopteran insect) was isolated from a larval gut library. The sequence predicts a secreted polypeptide of Mr 46.6 k with homology to metallocarboxypeptidases from mammalian and invertebrate species. The presence of a serine residue at the active site suggests carboxypeptidase A activity. To further characterise the gene product, the complete cDNA sequence was expressed in insect cells using the baculovirus system. Culture supernatant from these cells contained carboxypeptidase A activity, with no activity against a carboxypeptidase B substrate; the carboxypeptidase B activity in gut extracts must thus be due to a separate enzyme. In agreement with this conclusion, the expressed carboxypeptidase cDNA is a member of a small multigene family. Chronic ingestion of soybean Kunitz trypsin inhibitor by H. armigera larvae results in increased accumulation of carboxypeptidase mRNA in the midgut cells, and an increase in carboxypeptidase A activity detected in gut extract.

Characterization of digestive proteases in the weevil Aubeonymus mariaefranciscae and effects of proteinase inhibitors on larval development and survival

AbstractThe major digestive proteinase activities of a new sugar beet pest, Aubeonymus mariaefranciscae Roudier (Coleoptera: Curculionidae), were characterized. Both larvae and adults of A. mariaefranciscae were found to use a complex proteolytic system for protein digestion based on at least trypsin‐, chymotrypsin‐, elastase‐, cathepsin D, leucine aminopeptidase‐, carboxypeptidase A‐ and carboxypeptidase B‐like activities. An azocaseinolytic activity at pH 5.0–7.0 was identified, that was not affected by specific inhibitors and activators, making its classification in any of the mechanistic classes established not possible. According to this proteolytic profile, several serine proteinase inhibitors were tested in vitro and in vivo to establish their potential as resistance factors against A. mariaefranciscae. Larvae fed from neonate to pupation on diets containing 0.2% (w/w) soybean Bowman‐Birk trypsin‐chymotrypsin inhibitor, soybean Kunitz trypsin inhibitor, turkey egg white trypsin inhibitor, or lima bean trypsin inhibitor endure lower survival rates and display significant delays in the developmental time to pupation and to adult emergence. Interestingly, the most significant levels of mortality (about 90%) occurred with larvae fed on diets containing a combination of two or three inhibitors, suggesting a synergistic toxicity.

Inactivation Kinetics Study of the Kunitz Soybean Trypsin Inhibitor and the Bowman-Birk Inhibitor.

The inactivation of trypsin inhibitors (TIs) in soy flour exhibits a two-phase inactivation behavior. It is sometimes assumed that this behavior is caused by a difference in the heat stabilities of the Kunitz soybean trypsin inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). Kinetics studies with KSTI and BBI in soy flour showed that this two-phase inactivation behavior of TIs could not be explained by the difference in the heat stabilities of KSTI and BBI. Inactivation of KSTI and BBI in an aqueous solution and in a starch matrix followed a first-order reaction. KSTI and BBI in a starch matrix with added cysteine showed a two-phase inactivation behavior. The existence of thiols in soy flour seems to be responsible for the two-phase inactivation of TIs in soy flour. It is suggested that TIs in soy flour are inactivated by sulfhydryl-disulfide interchange during the first inactivation phase and by heat during the second phase.

Glycation of Proteinous Inhibitors: Loss in Trypsin Inhibitory Activity by the Blocking of Arginine and Lysine Residues at Their Reactive Sites

Lysine-type trypsin inhibitors [Japanese quail ovomucoid (QOM) and Bowman−Birk inhibitor (BBI)], and arginine-type trypsin inhibitors [chicken ovomucoid (COM) and Kunitz soybean trypsin inhibitor (KSTI)] were modified with glucose by amino−carbonyl reaction at 50 °C and 65% relative humidity for 15 days. Free amino groups of inhibitors were blocked rapidly and decreased to <20% within 5 days. On the other hand, their free guanidino groups began to decrease after a time lag for a few days, and 6−40% of the free guanidino groups remained after the 15-day incubation. Both lysine- and arginine-type trypsin inhibitors were inactivated by the reaction. A linear relationship between the loss in inhibitory activities for the lysine-type inhibitors but not for the arginine-type inhibitors and the decrease of their free amino groups was observed. The soybean trypsin inhibitors were almost completely inactivated by the 15-day incubation with glucose without severe structural or chemical damage. Denaturation of COM and KSTI incubated with glucose was scarcely observed with immunoassay, though protein polymerization of COM−, QOM−, and BBI−glucose complexes was detected by SDS−PAGE. Keywords: Chicken ovomucoid; Japanese quail ovomucoid; Kunitz soybean trypsin inhibitor; Bowman−Birk inhibitor; amino−carbonyl reaction; Maillard reaction; protein modification; inactivation; trypsin inhibitory activity

Imidazole—a new ligand for metal affinity precipitation

Kunitz soybean trypsin inhibitor (STI) was specifically coprecipitated during precipitation of Cu(II)-loaded copolymers induced by increase in temperature and ionic strength. The copolymers used consisted of 1-vinylimidazole andN-vinylcaprolactam orN- isopropylacrylamide. The elution of STI was achieved by solubilization of the STI-Cu(II)-polymer complex in the presence of an excess of the competing ligand, imidazole, and a subsequent precipitation of the polymer with STI remaining free in solution in a purified form as judged by Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To the best of our knowledge this is the first reported successful metal affinity precipitation of protein in a heterobifunctional format.

Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors

Ingestion of Kunitz soybean trypsin inhibitor (STI) by larval Helicoverpa zea, Agrotis ipsilon, and Trichoplusia ni extended the retention time of food in the digestive tract and increased the level of activity of proteolytic enzymes that were not susceptible to inhibition by STI. The level of enhancement of activity of STI-resistant (STI-R) enzyme(s) was directly influenced by the dosage and timing of exposure to STI. However, not all proteinase inhibitors (PIs) enhanced the level of proteinase inhibitor resistant (PI-R) enzymes, even if those PIs inhibited a significant proportion of enzyme activity. These findings suggest that a complex system may be responsible for the regulation of proteolytic enzymes in the midgut of larval Lepidoptera, and one hypothesis for this regulation is proposed.

Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families

Ingestion of soybean Kunitz trypsin inhibitor (SKTI) by larvae of the phytophagous insect pest Helicoverpa armigera induced production of inhibitor-insensitive protease activity. The induced activity was not due to proteolytic enzymes of different mechanistic classes, but rather to variants of the existing enzymes. Characterization of cDNAs showed that sequences encoding proteins of the serine protease family were abundant in gut tissue of both control and SKTI-fed insects. The majority of serine protease family cDNAs encode enzymes closely homologous to trypsin and chymotrypsin; comparison of these sequences shows variation in amino acid residues within the region which would be in contact with a protein protease inhibitor. More diverged sequences which may not encode active proteases are also present. All the cDNAs examined were found to derive from multigene families; at least 28 different genes are present in the serine protease family. Chronic ingestion of SKTI results in some serine protease-encoding mRNA species increasing in level, whereas others decrease. Chymotrypsin-encoding mRNAs tend to increase in level as a result of SKTI ingestion, but no clear trend is shown by trypsin-encoding mRNAs. It is suggested that multiple, varying protease-encoding genes are an adaptive mechanism for reducing the deleterious effects of plant protease inhibitors.

Effects of two proteinase inhibitors on the digestive enzymes and survival of honey bees ( Apis mellifera)

Two endopeptidase inhibitors, BPTI (bovine pancreatic trypsin inhibitor) and SBTI (Kunitz soybean trypsin inhibitor), were found to significantly reduce the longevity of adult honey bees ( Apis mellifera L.) fed the inhibitors ad lib in sugar syrup at 1.0%, 0.5% or 0.1%, but not at 0.01% or 0.001% (w:v). Bees were taken from frames at emergence, kept in cages at 33 °C, and provided with a pollen/protein diet, water and syrup. In vivo activity levels of three midgut endopeptidases (trypsin, chymotrypsin and elastase) and the exopeptidase leucine aminopeptidase (LAP) were determined in bees fed either BPTI or SBTI at 1.0%, 0.3% or 0.1% (w:v) at two time points: the 8th day after emergence and when 75% of bees had died. LAP activity levels increased significantly in bees fed with either inhibitor at all concentrations. At day 8, bees fed BPTI at all concentrations had significantly reduced levels of trypsin, chymotrypsin and elastase. At the time of 75% mortality, bees fed BPTI at each concentration had reduced trypsin levels, but only those fed the inhibitor at the highest dose level had reduced chymotrypsin or elastase activity. At both time points, only bees fed SBTI at the highest concentration had lowered trypsin, chymotrypsin and elastase activities.

Partial purification and characterization of a novel soybean protease which is inhibited by Kunitz and Bowman-Birk trypsin inhibitors.

A novel serine protease has been partially purified from dry seeds of the soybean (Glycine max) cultivar Keburi by cryoprecipitation at pH 6.4, fractional precipitation with ammonium sulfate, and a series of column chromatographic procedures on DEAE-Sepharose, SP-Sepharose, and Arginine-Sepharose 4B. Some properties of the purified enzyme were studied. The protease hydrolyzed the native storage globulins of soybean seeds, such as the alpha subunit of beta-conglycinin, at a pair of arginine residues, Arg126-Arg127. The proteolysis of the alpha subunit in the purified alpha 2 beta molecule of beta-conglycinin apparently followed first order kinetics. The enzyme was inhibited by both soybean Kunitz trypsin inhibitor and Bowman-Birk proteinase inhibitor in a competitive manner. Moreover, the enzyme could catalyze the specific proteolysis of the A3 polypeptide of the purified G5 glycinin at the Arg99-Gly100 linkage, or the carboxyl side of the Arg98-Arg99 paired basic residues.