Kunitz-type Soybean Trypsin Inhibitor Research Articles

Structural and functional analysis of miraculin-like protein from Vitis vinifera

The so-called miraculin-like proteins (MLPs) are homologous to miraculin, a homodimeric protein with taste-modifying activity that converts sourness into sweetness. The identity between MLPs and miraculin generally ranges from 30% to 55%, and both MLPs and miraculin are categorized into the Kunitz-type soybean trypsin inhibitor (STI) family. MLP from grape (Vitis vinifera; vvMLP) exhibits significant homology to miraculin (61% identity), suggesting that vvMLP possesses miraculin-like properties. The results of size-exclusion chromatography and sensory analysis illustrated that vvMLP exists as a monomer in solution with no detectable taste-modifying activity. Crystal structure determination revealed that vvMLP exists as a β-trefoil fold, similarly as other MLPs and Kunitz-type protein inhibitors. The conformation of the loops, including the so-called reactive loop in the STI family, was substantially different between vvMLP and STI. Recombinant vvMLP had inhibitory activity against trypsin (Ki = 13.7 μM), indicating that the protein can act as a moderate trypsin inhibitor.

Peptidase compensation in the digestive system of whiteleg shrimp Penaeus vannamei against dietary Kunitz-type soybean trypsin inhibitor

The aim of this study was to assess the regulatory process of digestive peptidases of crustaceans in the presence of soybean trypsin inhibitor (SBTI). This naturally occurring inhibitor in soybean meal was used to inhibit the activity of digestive serine peptidases of the whiteleg shrimp Litopenaeus vannamei. In vitro, SBTI inhibited the total proteolytic activity and chymotrypsin activity by 65%. Trypsin activity was reduced by SBTI from 40% to 15% from 2 to 4 hr of incubation, which is the average time of residence of feed in the shrimp digestive system. During the bioassays, experimental groups were fed with increasing concentration of supplemental SBTI (1 g kg−1 and 2 g kg−1) and digestive gland and faeces of individual specimens were collected daily. At the end of the bioassay, peptidase activity of digestive gland and faeces was shown, revealing differential inhibition after feeding for 5 days. Several serine peptidases were observed in zymograms, showing a compensation effect on the digestive gland through the activation of peptidases from different catalytic type. These results provide evidence that the shrimp digestive gland can overcome the effect of SBTI by two adaptive mechanisms: synthesis of additional peptidases of the serine class and other unidentified peptidases.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop.

Development and Application of a Label-Free Fluorescence Method for Determining the Composition of Gold Nanoparticle–Protein Conjugates

A method was developed for determining the composition of the conjugates between gold nanoparticles and proteins based on the intrinsic fluorescence of unbound protein molecules. The fluorescence was evaluated after separation of the conjugates from the reaction mixture by centrifugation. Gold nanoparticles obtained using the citrate technique (average diameter 24 nm) were conjugated at pH 5.4 with the following four proteins: human immunoglobulin G (IgG), bovine serum albumin (BSA), recombinant streptococcal protein G (protein G), and Kunitz-type soybean trypsin inhibitor (STI). The compositions of these conjugates were determined using the developed method. The conjugate compositions were dependent on the concentration of the added protein, and in all cases reached saturation. The equilibrium dissociation constants of the gold nanoparticle conjugates with IgG, BSA, protein G, STI in the initial section of the concentration dependence curve were 4, 6, 10, and 15 nM, respectively. Close to saturation, the corresponding values were 25, 76, 175, and 100 nM, respectively. The maximal binding capacities of a single gold nanoparticle for IgG, BSA, Protein G, and STI were 52, 90, 500, and 550, respectively, which agrees well with the hypothesis of monolayer immobilization.

Secretion of miraculin through the function of a signal peptide conserved in the Kunitz-type soybean trypsin inhibitor family

Miraculin, a glycoprotein that modifies sour tastes into sweet ones, belongs to the Kunitz-type soybean trypsin inhibitor (STI) family. To clarify the functional relation of miraculin with Kunitz-type STIs, we investigated its subcellular localization and trypsin inhibitory activity. In transgenic Arabidopsis thaliana, miraculin, fused to yellow fluorescent protein, localized to and outside the plasma membrane depending on the putative secretion signal peptide. When transgenic seedlings were cultured in liquid medium, miraculin was present in the supernatant only after cellulase treatment. No trypsin inhibitory activity was detected in native or recombinant miraculin. In conclusion, miraculin is secreted outside the plasma membrane through the function of a signal peptide, conserved in Kunitz-type STIs, whereas its trypsin inhibitory activity may be lost during its evolution.

Structural basis for dual inhibitory role of tamarind Kunitz inhibitor (TKI) against factor Xa and trypsin

A Kunitz type dual inhibitor (TKI) of factor Xa (FXa) and trypsin was found in tamarind. It also shows prolongation of blood coagulation time. The deduced 185 amino acid sequence of TKI by cDNA cloning and sequence analysis revealed that it belongs to the Kunitz type soybean trypsin inhibitor (STI) family; however, it has a distorted Kunitz signature sequence due to insertion of Asn15 in the motif. TKI exhibited a competitive inhibitory activity against both FXa (K(i) =220nm) and porcine pancreatic trypsin (K(i) =3.2nm). The crystal structure of TKI shows a β-trefoil fold similar to Kunitz STI inhibitors; however, a distinct mobile reactive site, an inserted residue and loop β7β8 make it distinct from classical Kunitz inhibitors. The crystal structure of TKI-trypsin and a 3D model of TKI-FXa complex revealed that the distinct reactive site loop probably plays a role in dual inhibition. The reactive site of TKI interacts with an active site and two exosites (36 loop and autolysis loop) of FXa. Apart from Arg66 (P1), Arg64 (P3) is one of the most important residues responsible for the specificity of TKI towards FXa. Along with the reactive site loop (β4β5), loops β1 and β7β8 also interact with FXa and could further confer selectivity for FXa. We also present the role of inserted Asn15 in the stabilization of complexes. To the best of our knowledge, this is the first structure of FXa inhibitor belonging to the Kunitz type inhibitor family and its unique structural and sequence features make TKI a novel potent inhibitor. The complete nucleotide of TKI was deposited in the NCBI gene databank with accession no. HQ385502. The atomic coordinates and structure factor files for the structure of TKI and TKI:PPT complex have been deposited in the Protein Data Bank with accession numbers 4AN6 and 4AN7, respectively TKIandTKIbindbyx-ray crystallography(View interaction) TKIandPPTbindbyx-ray crystallography(View interaction).

Human Mesotrypsin Defies Natural Trypsin Inhibitors: From Passive Resistance to Active Destruction.

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.

Structure and Stability of the Potato Cysteine Protease Inhibitor Group (Cv. Elkana)

The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.

A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis

A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin–agarose or a trypsin–agarose column. In both cases, SDS–PAGE showed two bands of M r 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K is for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6×10 −7 and 1.3×10 −5 M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.

Alum augments the experimental allergenicity of Kunitz-type soybean trypsin inhibitor independent of the antigen-adsorption.

In order to inspect the significance of the adsorbing property in the adjuvant activity to enhance IgE production, we immunized BALB/c mice against Kunitz-type soybean trypsin inhibitor (KSTI), the most potent experimental allergen among soybean proteins, associated with Aluminum hydroxide (alum) or DEAE-Sephadex particles. The production of immunoglobulin isotypes was analyzed at the various amounts, 3-3,000 microg per mouse, of the antigen dosages. In our experiments, although alum did not adsorb KSTI significantly, it augmented the total and the antigen-specific IgE without affecting the optimal range of the antigen dosage. On the other hand, alum did not effectively enhance the production of the other immunoglobulin isotypes. The production of immunoglobulin isotypes other than IgE increased dose-dependently on the antigen. These results ensured our previous finding that another protein, ovalbumin, was used as the antigen. We also demonstrated that the adsorption of KSTI by DEAE-Sephadex in the immunizing vehicle resulted in the requirement of more KSTI for accomplishing the equal immunity in BALB/c mice compared to the control. Moreover, we demonstrated that, regardless of the inability to adsorb KSTI, alum exerted its adjuvant activity only when it was co-injected with the antigen. These results showed that some biochemical effect, other than adsorptive activity, to enhance the production of the antigen-specific IgE resides in alum.

Characterization of Recombinant p20 Trypsin Inhibitor, a New Protein from Glycine max.

A 20-kDa protein (p20) having 40% sequence similarity with Kunitz-type soybean trypsin inhibitor (STI) from Glycine max (soybean) cultured cells was expressed in Escherichia coli. The recombinant p20 (rp20) inhibited the activity of trypsin at the same level as STI (rp20, Ki=50nM; STI, Ki=75nM), and both rp20 and STI displayed non-competitive inhibition of trypsin activity. Although STI inhibited the activity of α-chymotrypsin (Ki=140nM) and elastase (Ki=207nM), rp20 did not inhibit the activity of α-chymotrypsin and elastase. These results show that p20 is a novel type of trypsin inhibitor.

Change in Activity of Soybean Trypsin Inhibitor by Removal of C-terminal Amino Acid Residues during Seed Germination

Kunitz-type soybean trypsin inhibitor (KSTI) is a major factor lowering the nutritional quality of soybean proteins. KSTI might be degraded during protein processing. In the present work, we determined the effect of removal of C-terminal amino acid residues on the inhibitory activity of KSTI in the cotyledons during germination in Japanese soybean cultivars, Toyokomachi and Wasekogane. The type of KSTI in both cultivars was determined by analysis of the complete nucleotide sequences of PCR products amplified from genomic DNA and partial amino acid sequences of both cultivars. These KSTI were identical to Tib in Toyokomachi and Tia in Wasekogane. KSTI from cotyledons at 4 days after germination in both cultivars was separated clearly into two bands Tl and T2 in Toyokomachi and Wl and W2 in Wasekogane upon native-PAGE. Based on the C-terminal amino acid sequences, KSTIs with higher mobility (Tl, Wl) were found to lack 13 amino acid residues at the C-terminus. The KSTI with lower mobility (T2, W2) lacked 14 or 15 amino acid residues at the C-terminus. Further, KSTI with lower mobility showed high inhibitory activity compared with that of KSTI with higher mobility. Thus, the changes in the form of KSTI from Tl or Wl to T2 or W2 by removal of C-terminal amino acid residues during seed germination may change the structure at the active site and consequently increase the inhibitory activity of KSTI.

A novel lectin from Pseudostellaria heterophylla roots with sequence simularity to Kunitz-type soybean trypsin inhibitor

From the roots of the Chinese medicinal herb Pseudostellaria heterophylla a single-chained lectin with a molecular weight of 36 kDa and high hemagglutinating activity was isolated. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 50 mM NaCl. It was adsorbed on SP-Sepharose in 10mM NH 4OAc (pH 4.5) and eluted by approximately 0.5 M NaCl in the same buffer. The hemagglutinating activity of the lectin could not be inhibited by a large variety of monosaccharides, but was largely abrogated by exposure to 0.05 M HCl, 0.05M NaOH or 80°C. However, about 50% of the activity remained after exposure to 0.025M NaOH or 40°C. Despite possession of an N-terminal sequence exhibiting some similarity to thaumatin-like proteins with antifungal activity, the lectin was devoid of antifungal activity. The lectin exerted some inhibitory effect on the glycohydrolases α-glucosidase, β-glucosidase and β-glucuronidase which are involved in HIV infection but had no suppressive action on human immunodeficiency virus-type 1 reverse transcriptase.

A 20-kDa protein with the GTP-binding and trypsin inhibitory activities from Glycine max.

A 20-kDa protein (p20) with a GTP binding activity was purified from the cultured cells of Glycine max (soybean). The amino acid sequence of p20 showed 65% identity in a 23 amino acid overlap against the Kunitz-type trypsin inhibitor of soybean reported. Furthermore, it was found that a Kunitz-type soybean trypsin inhibitor of commercial origin also binds GTP.

Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator

The Kunitz-type trypsin inhibitor from soybean (STI) consists of 181 amino acid residues with two disulfide bridges. Its crystal structures have been determined in complex with porcine pancreatic trypsin in two crystal forms (an orthorhombic form at 1.75 Å resolution and a tetragonal form at 1.9 Å) and in the free state at 2.3 Å resolution. They have been refined to crystallographic R-values of 18.9%, 21.6% and 19.8%, respectively. The three models of STI reported here represent a significant improvement over the partial inhibitor structure in the complex, which was previously determined at a nominal resolution of 2.6 Å by the multiple isomorphous replacement method. This study provides the first high-resolution picture of the complex between a Kunitz-type proteinase inhibitor with its cognate proteinase. Many of the external loops of STI show high B-factors, both in the free and the complexed states, except the reactive site loop whose B-factors are dramatically reduced upon complexation. The reactive site loop of STI adopts a canonical conformation similar to those in other substrate-like inhibitors. The P1 carbonyl group displays no out-of-plane displacement and thus retains a nominal trigonal planar geometry. Modeling studies on the complex between a homologous Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra and the human tissue-type plasminogen activator reveal a new insight into the specific interactions which could play a crucial role in their binding.

Potato cysteine proteinase inhibitor gene family: molecular cloning, characterisation and immunocytochemical localisation studies.

Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.

Purification and characterization of a novel isoform of mast cell tryptase from rat tongue.

Rat mast cell tryptase was purified to homogeneity from rat tongue by a series of standard chromatographic procedures. Since the enzyme gave band corresponding to molecular mass of 32-35 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited a molecular mass of 135 kDa on gel filtration, it was presumed to be a noncovalently associated tetramer. The N-terminal amino acid sequence of 50 residues of the enzyme showed the highest degree of homology with the same region in mouse mast cell protease 7 (92%), and less homology to those of tryptases from man and dog, and peritoneal cells of rats and Mongolian gerbils. The inhibitor specificity of rat tongue tryptase was similar to that of rat peritoneal mast cell tryptase free from trypstatin: it was inhibited by alpha 1-antitrypsin, Kunitz-type soybean trypsin inhibitor and Bowman-Birk soybean trypsin inhibitor, but these inhibitors do not inhibit the tryptases from rat skin, human lung, and dog mast cells. Judging from these results, together with other enzymatic properties, the enzyme may be a novel isoform of tryptase in rat tongue. Analysis by differential staining with peroxidase-labeled lectins of the enzyme suggested that it has tri- and/or tetraantennary complex-type oligosaccharides containing a relatively high amount of sialic acid. The immunohistochemical distribution of this enzyme indicated that the reactive antigen was specific in connective tissue but not in mucosal mast cells.

Crystal structure of the complex of porcine pancreatic trypsin with Kunitz-type soybean trypsin inhibitor

Crystal structure of the complex of porcine pancreatic trypsin with Kunitz-type soybean trypsin inhibitor

Thermodynamic Stability Effects of Single Peptide Bond Hydrolysis in Protein Inhibitors of Serine Proteinases

Kunitz type soybean trypsin inhibitor (STI) and basic pancreatic trypsin inhibitor (BPTI) were used as model proteins to measure the thermodynamic consequences of single peptide bond hydrolysis. For each inhibitor the reactive site cleaved form was prepared and, additionally, each inhibitor was selectively cleaved at a Met residue. Selective cleavage generally led to reducing of the T den value from 7 K up to 75 K. For STI cleaved at Met84 a slight stabilization (increase of T den by 1.0 K) was observed. In terms of Δ G den , the difference between the most extreme cleavage effect was 11.44 kcal/mole, much larger than resulting from the theoretical effects of crosslinks. It was found that hydrolysis of a single peptide bond affects not only entropy, but also enthalpy and heat capacity parameters. Moreover, the sign of change is opposite for two inhibitors: Δ H den and Δ S den increase for both cleaved forms of STI, while they decrease for the two nicked forms of BPTI. To understand the stability effects, a thermodynamic cycle analysis was applied based on comparison of stabilities of intact and cleaved protein with peptide bond hydrolysis equilibria in native and denatured states. The cycle revealed a good agreement of the theoretical effect of crosslink elimination with a free energy difference for hydrolysis of a single peptide bond in a denatured protein. It appears that hydrolysis constants for single peptide bonds in a native protein span over at least 20 orders of magnitude. They are very low for peptide bonds placed in α-helices and very high if cleavage reaction leads to a formation of a new secondary structure element.

Conjugation of Succinylated Gelatin to Soybean Trypsin Inhibitor

Kunitz-type soybean trypsin inhibitor (SBTI) and succinylated gelatin fragment (Suc-gel) were conjugated by using water-soluble carbodiimide, and physicochemical and biopharmaceutical properties of the conjugate (Suc-gel-SBTI) were characterized. The conjugate contains about 2.5 mol of Suc-gel with a mean molecular weight of 32,000 per mole of SBTI, based on the content of hydroxyproline. After iv injection of the radiolabeled conjugate or native SBTI into mice, it was found that native SBTI showed rapid elimination from plasma ( t½α = 2 min), whereas Suc-gel-SBTI was slowly ( t½α = 2 min) eliminated. Organ distribution of the two agents at 30 min after iv injection was different: Suc-gel-SBTI accumulated to a large extent in both the liver and the spleen, about 23.4 and 6.9%, respectively which was more than the native SBTI. Native SBTI was taken up more rapidly by the kidney (~ 45%) and excreted into the urine (~34%) before it accumulated in other organs and tissues. In addition, we evaluated the pharmacological effects of these agents in an animal model. Suc-gel-SBTI was more effective than native SBTI against trypsin-induced lethal shock in mice. This polymer-conjugated SBTI seems to exhibit better pharmacological and therapeutic properties than its native counterpart.