New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa

Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.     

Atypical Reactive Center Kunitz-Type Inhibitor from the Sea Anemone Heteractis crispa

The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the K(i) of InhVJ to trypsin and α-chymotrypsin (7.38 × 10(-8) M and 9.93 × 10(-7) M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.     

Members of the Kunitz-type protease inhibitor gene family of potato inhibit soluble tuber invertase in vitro

Summary A soluble proteinaceous invertase inhibitor was purified from tubers of cv. Provita. N-terminal amino acid sequencing of the purified protein indicated that the invertase inhibitor was a member of a family of small tuber proteins known as Kunitz-type protease inhibitors. The purified protein was completely inhibitory to soluble tuber invertase at quantities that did not inhibit trypsin. Based on amino acid sequence information of invertase inhibitor protein, seven candidate cDNA clones were identified in libraries of Provita and Saturna tubers. The candidate cDNA sequences differed from each other between 2% and 5%, having several non-conservative amino acid substitutions when compared with sequence related protease inhibitors. The results suggest invertase as an alternative target of tuber “protease” inhibitors.     

Intramolecular Modulation of Serine Protease Inhibitor Activity in a Marine Cyanobacterium with Antifeedant Properties

Extracts of the Floridian marine cyanobacterium Lyngbya cf. confervoides were found to deter feeding by reef fish and sea urchins (Diadema antillarum). This antifeedant activity may be a reflection of the secondary metabolite content, known to be comprised of many serine protease inhibitors. Further chemical and NMR spectroscopic investigation led us to isolate and structurally characterize a new serine protease inhibitor 1 that is formally derived from an intramolecular condensation of largamide D (2). The cyclization resulted in diminished activity, but to different extents against two serine proteases tested. This finding suggests that cyanobacteria can endogenously modulate the activity of their protease inhibitors.     

Trypsin inhibitors and nutritive value in cereals

Chemical assays demonstrated that rye and barley cultivars contained relatively high levels of trypsin inhibitor activity as compared to oat and wheat cultivars, and there was a low degree of stability to prolonged wet heat treatment. In feeding trials with broiler chicks, incorporation of 67% raw barley or 50% raw rye in the rations enhanced feed intake and weight gains, and the marginal increases in pancreas weight were not reversed by feeding autoclaved cereals. Raw rye cultivars fed at the 75% level in mouse diets reduced weight gains, feed efficiency, protein digestibility, protein efficiency ratio and biological value. Autoclaving to inactivate trypsin inhibitors, or ether extraction to remove the resorcinols, failed to improve the nutritive value of rye diets for mice. It appeared that the protease inhibitors in the four cereals were relatively weak inhibitors of trypsin in the digestive system despite stability to dry heat and acid pH.     

高等植物K+吸收机制研究进展

从高亲和性和低亲和性K⁺吸收两个方面较详细介绍了K⁺营养的吸收机制。高亲和性K⁺吸收机制包括H⁺-K⁺交换ATPase、K⁺-ATPase、H⁺-K⁺ symporter和Na⁺-K⁺ symporter;低亲和性K⁺吸收机制也被称为“协助扩散”,研究表明是通过K⁺通道进行的,但K⁺通道的确切结构尚无定论。综述了植物K⁺吸收的调节,并对相关问题进行了展望。     

Marine Toxins Targeting Ion Channels

This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs), as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs), are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na⁺ (Na_V), Ca²⁺ (Ca_V), and K⁺ (K_V) channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR), and the ATP-activated (P2X_nR) receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of Na_V, Ca_V, or K_V channels opens a pore that is specific for Na⁺, Ca²⁺, or K⁺, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2X_nRs allows the passage of cations (e.g., Na⁺, K⁺, and/or Ca²⁺), whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl⁻ and/or HCO₃⁻). In this regard, the activation of Na_V and Ca_V as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of K_V as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers) of ion channel functions to treat or to alleviate a specific ion channel-linked disease (e.g., channelopaties).     

盐胁迫下钾吸收途径的抑制对小麦叶片K~+、Na~+含量的影响

为研究盐胁迫下小麦维持钾、钠平衡的生理机制,以耐盐小麦沧麦6005和盐敏感小麦矮抗58为材料,利用TEA、NEM、Ba(NO_3)_2三种药物分别抑制钾离子通道、钾载体及非选择性阳离子通道,测定正常及盐胁迫下小麦叶片K~+、Na~+含量,比较耐盐性不同的小麦品种在K~+、Na~+吸收中的差异。结果显示,盐胁迫下,沧麦6005和矮抗58叶片K~+含量下降,Na~+含量增加;沧麦6005叶片Na~+含量低于矮抗58,K~+/Na~+比值高于矮抗58。正常条件下,NEM、TEA处理均可降低沧麦6005和矮抗58叶片K~+含量,NEM处理较TEA处理效果更为明显;TEA处理显著降低了盐胁迫下矮抗58叶片K~+含量,而NEM处理则明显降低了盐胁迫下沧麦6005的叶片K~+含量;TEA、NEM、Ba(NO_3)_2处理降低了盐胁迫下矮抗58叶片Na~+含量,仅NEM处理降低了沧麦6005叶片Na~+含量。综上所述,正常条件下,钾载体是小麦K~+吸收的主要方式;盐胁迫下,耐盐品种和盐敏感品种K~+吸收途径不同,耐盐品种的NSCCs和钾离子通道具有更强的"拒钠"能力。     

Kunitz Soybean Trypsin Inhibitor is Modified at its C-terminus by Novel Soybean Thiol Protease (Protease T1)

Abstract

Kunitz soybean trypsin inhibitor (KSTI) is hydrolyzed during seed germination to yield amino acids needed to support initial seedling growth. The type of KSTI from Glycine max (L.) Merrill cv. Toyokomachi is KSTI-Ti ^b. The KSTI-Ti ^b from 4-day-old post-germination cotyledons (KSTI-Ti ^b’) has 3 or 4 amino acid residues cleaved off at the C-terminus. This KSTI modification is important to understand the mechanism of degradation in seed reserve proteins by proteases. Protease K1 also cleaves amino acid residues at the C-terminus of KSTI but it removes 5 amino acid residues. Therefore, we presumed the KSTI-Ti ^b’ was produced by a protease other than protease K1. In this study, the protease T1 responsible for cleavage of KSTI-Ti ^b at the C-terminus was purified. The enzyme was estimated to have a molecular mass of 33 kDa from its mobility on SDS-PAGE gels. The N-terminal amino acid sequence of the purified protease T1 corresponded to amino acids Phe-73 to Phe-92 of both thiol protease isoforms A and B from the soybean leaf, and shared 83% identity with the partial amino acid sequence of the membrane-associated cysteine protease from mung bean seedlings, a protease known to perform post-translational cleavage of C-terminal peptides of target proteins. Finally, this enzyme was shown to convert KSTI-Ti ^b to KSTI-Ti ^b’.     

Insights into the Toxicological Properties of a Low Molecular Weight Fraction from Zoanthus sociatus (Cnidaria)

The phylum Cnidaria is an ancient group of venomous animals, specialized in the production and delivery of toxins. Many species belonging to the class Anthozoa have been studied and their venoms often contain a group of peptides, less than 10 kDa, that act upon ion channels. These peptides and their targets interact with high affinity producing neurotoxic and cardiotoxic effects, and even death, depending on the dose and the administration pathway. Zoanthiniaria is an order of the Subclass Hexacorallia, class Anthozoa, and unlike sea anemone (order Actiniaria), neither its diversity of toxins nor the in vivo effects of the venoms has been exhaustively explored. In this study we assessed some toxicological tests on mice with a low molecular weight fraction obtained by gel filtration in Sephadex G-50 from Zoanthus sociatus crude extract. The gel filtration chromatogram at 280 nm revealed two major peaks, the highest absorbance corresponding to the low molecular weight fraction. The toxicological effects seem to be mostly autonomic and cardiotoxic, causing death in a dose dependent manner with a LD₅₀ of 792 μg/kg. Moreover, at a dose of 600 μg/kg the active fraction accelerated the KCl-induced lethality in mice.     

Factors affecting the protein quality of pigeonpea (Cajanus cajan L.)

Pigeonpea occupies an important place in human nutrition as a source of dietary proteins in several countries. Some of the important factors that affect the protein quality of pigeonpea have been reviewed and summarised in this paper. Among important food legumes, pigeonpea contained the lowest amount of limiting sulphur amino acids, methionine and cystine implicating the importance of these amino acids in protein quality improvement program. Large variation existed in the levels of protease inhibitors of pigeonpea varieties. The concentration of these inhibitors were significantly higher in some of the wild relatives of pigeonpea. Protein digestibility of cooked pigeonpea meal remained low and this could be due to the presence of certain compounds other than trypsin inhibitors. Pigeonpea polyphenolic compounds adversely affected the activity of digestive enzymes and this would affect the protein quality of pigeonpea. The protein quality of pigeonpea was greatly influenced by storage and processing practices.     

Occurrence and Stabilities of Oat Trypsin and Chymotrypsin Inhibitors

Nine chymotrypsin and four trypsin inhibitors have been extracted and separated from ungerminated oat grains. The inhibitors fell into two groups, based on their heat and pH stabilities. Members of the most abundant group are labile and are inactivated at 80 °C or at pHs of 3·3 or lower. Members of the second group are stable and are resistant to boiling for 30 min. On germination, the labile inhibitors are inactivated after 2 days and the stable chymotrypsin inhibitors after 3 days. Most of the labile inhibitors from ungerminated grain are destroyed when incubated at 20 °C for 20 h but addition of PMSF, a serine protease inhibitor, prevented their inactivation. Labile inhibitors in extracts of ungerminated oats are inactivated on incubation with an extract prepared from germinated oats, but not in the presence of PMSF. Most oat chymotrypsin and trypsin inhibitors are heat labile and pH sensitive. These inhibitors are apparently inactivated by serine proteinase(s) already present in ungerminated grain.     

Anti-hyperglycemic Effect of the Aqueous Extract of Banana Infructescence Stalks in Streptozotocin-induced Diabetic Rats

Water extract of banana (Musa sapientum) infructescence stalks has been used in folk medicine in the treatment of diabetes mellitus. This work aims at verifying the claimed effect and elucidating its possible mode of action. The extract was given in replacement of drinking water to diabetic rats, and its mechanism of action was studied by investigating its involvement in glucose transport in Caco-2 monolayers, and in rat jejuna using an in situ perfusion technique. Its effect on the Na⁺/K⁺ ATPase was studied by measuring the amount of inorganic phosphate liberated. The extract reduced significantly blood glucose levels in diabetic rats and glucose transport across rat jejuna and Caco-2 monolayers, and induced a 50 % decrease in their Na⁺/K⁺ ATPase activity. The extract did not induce any further decrease in jejunal glucose uptake in the simultaneous presence of phloridzin and phloretin, respective inhibitors of SGLT1 and GLUT2 transporters nor did it induce a change in the protein expression of SGLT1 and GLUT2. It was concluded that the extract acts by reducing the Na⁺/K⁺ ATPase activity of enterocytes and consequently the sodium gradient required for sugar transport by SGLT1, which leads to down-regulation of GLUT2 and contributes to the observed anti-hyperglycemic effect.     

大豆胰蛋白酶抑制因子的研究进展

大豆胰蛋白酶抑制因子是大豆中的主要抗营养因子,是一种多肽类或蛋白质。该文对大豆胰蛋白酶抑制因子的种类,Kunitz型胰蛋白酶抑制因子和Bowman-Birk型胰蛋白酶抑制因子的结构和特性,大豆胰蛋白酶抑制因子的功能及提取纯化方法进行了综述,旨在为大豆胰蛋白酶抑制因子的进一步开发和利用提供参考。     

Recovery of Protease Inhibitors from Potato Fruit Water by Expanded Bed Adsorption Chromatography in Pilot Scale

The current study was conducted to investigate the recovery of native potato protein from potato fruit water (PFW) by expanded bed adsorption (EBA) chromatography. The eluted proteins were concentrated by ultrafiltration and spray-dried into powder. The SDS-PAGE showed that the recovered proteins were potato protease inhibitors (PPIs). The trypsin and chymotrypsin inhibitor activities of the recovered PPIs were 377.93 ± 8.22 and 12.90 ± 0.03 mg g^?1 protein, respectively. The recovery yield of protease inhibitors was 74.88%. The glycoalkaloid assay showed that the recovered PPIs contained 30.31 ± 0.15 μg g^?1 of α-chaconine and 92.77 ± 0.52 μg g^?1 of α-solanine, and these values were much lower than those in potato protein concentrate (PPC) obtained by traditional thermal coagulation. The most abundant amino acid in the PPIs was serine. The results indicated that the EBA can be used to effectively recover native potato protein from PFW.     

Cyclisation Increases the Stability of the Sea Anemone Peptide APETx2 but Decreases Its Activity at Acid-Sensing Ion Channel 3

APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3) and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.     

Relationships between nutrients and sucrose concentrations in sugarcane juice and use of juice analysis for nutrient diagnosis in Japan

Sugarcane is an important economic crop in southwest Japan, but its production is decreasing. To increase sugar production, both sugarcane yield and quality should be improved. Fertilizer management is one of the factors that influence sugarcane quality. We accordingly focused on nutrients present in sugarcane juice and attempted to identify the key factors affecting sugarcane quality. We collected sugarcane samples from 2013 to 2015 from all of the sugar mills in Japan and examined the relationships between juice nutrients and sucrose concentration. Juice analysis over 3 year showed that potassium (K⁺) and chloride (Cl^?) were the most abundant cation and anion in the juice and that both negatively correlated with the sucrose concentration. K⁺ and Cl^? concentrations significantly varied depending on production areas and those with higher K⁺ and Cl^? concentrations had a low sucrose concentration. This finding suggests that sugarcane in those areas may have been supplied with these two ions in excess. Electrical conductivity (EC) in the juice always positively correlated with K⁺ and Cl^? concentrations. EC may thus be a reliable indicator of K⁺ and Cl^? concentrations and could be used for nutrient diagnosis because of its ease of measurement. For improving sugarcane quality, we recommend that potassium chloride, which supplies both K⁺ and Cl^? and is a commonly used potassium fertilizer for sugarcane production in Japan, should be used in lower quantities in a year following one in which the EC of sugarcane juice at harvest is found to be high.     

Protease inhibitor activity is associated to a basic chitinase from potato but not to an acidic one

Summary Several pathogenesis-related proteins, which are produced in plants submitted to stress, have been identified as chitinases. We have previously described that a potato basic chitinase strongly inhibited the activity of an aspartic protease isolated from the same source. In this work, we have tested the activity of two potato chitinases as protease inhibitors. A basic chitinase (ChiB) inhibited trypsin, chymotrypsin, subtilisin, proteinase K and a serine protease ofFusarium sp. An acidic one (ChiA) did not show inhibitory activity. The kinetics of trypsin inhibition by ChiB revealed different patterns of inhibition with azocasein and BAPNA. Metal ions affected differentially both activities, suggesting that ChiB is a bifunctional protein. These results and those reported by other authors suggest a new physiological role for pathogenesis-related proteins. However, results presented in this paper suggest that the protease inhibitor activity is not a general characteristic of potato chitinases.     

New Cyclic Cystine Bridged Peptides from the Sponge Suberites waedoensis

Two new peptides, chujamides A (1) and B (2), were isolated from the marine sponge Suberites waedoensis, which was collected from Korean waters. Based upon the results of the combined spectroscopic analyses, the structures of these compounds were determined to be proline-riched and cyclic cystine bridged dodeca- and undecapeptides. The absolute configurations of all amino acid residues were determined to be l by advanced Marfey’s analysis. The new compounds exhibited weak cytotoxicities against A549 and K562 cell-lines, and compound 2 also demonstrated moderate inhibitory activity against Na⁺/K⁺-ATPase.