Structure-based design of an indolicidin peptide analogue with increased protease stability

Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analogue CP-11, ILKKWPWWPWRRK-NH(2), with improved activity against Gram-negative bacteria had increased positive charge and amphipathicity while maintaining the short length of the parent molecule. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was determined using nuclear magnetic resonance spectroscopy. CP-11 was found to be an amphipathic molecule with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH(2), was designed to stabilize the lipid-bound structure and to increase protease resistance. The three-dimensional structure of cycloCP-11 was determined under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 associated with phospholipid membranes in a similar manner as indicated by circular dichroism and fluorescence spectra. The minimal inhibitory concentrations of CP-11 and cycloCP-11 for a range of bacteria differed by no more than 2-fold, and they were nonhemolytic at concentrations up to 256 microg/mL. Cyclization was found to greatly increase protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min since the major degradation product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.     

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.     

Effect of Moringa oleifera flower extract on larval trypsin and acetylcholinesterase activities in Aedes aegypti

Aedes aegypti control is crucial to reducing dengue fever. Aedes aegypti larvae have developed resistance to organophosporous insecticides and the use of natural larvicides may help manage larval resistance by increasing elements in insecticide rotation programs. Here, we report on larvicidal activity of Moringa oleifera flower extract against A. aegypti L(1), L(2), L(3), and L(4) as well as the effect of flower extract on gut trypsin and whole-larval acetylcholinesterase from L(4.) In addition, the heated flower extract was investigated for larvicidal activity against L(4) and effect on larval gut trypsin. Moringa oleifera flower extract contains a proteinaceous trypsin inhibitor (M. oleifera flower trypsin inhibitor, MoFTI), triterpene (β-amyrin), sterol (β-sitosterol) as well as flavonoids (kaempferol and quercetin). Larvicidal activity was detected against L(2), L(3), and L(4) (LC(50) of 1.72%, 1.67%, and 0.92%, respectively). Flower extract inhibited L(4) gut trypsin (MoFTI K(i) = 0.6 nM) and did not affect acetylcholinesterase activity. In vivo assay showed that gut trypsin activity from L(4) treated with M. oleifera flower extract decreased over time (0-1,440 min) and was strongly inhibited (98.6%) after 310 min incubation; acetylcholinesterase activity was not affected. Thermal treatment resulted in a loss of trypsin inhibitor and larvicidal activities, supporting the hypothesis that flower extract contains a proteinaceous trypsin inhibitor that may be responsible for the deleterious effects on larval mortality.     

Molecular cloning and partial characterization of a trypsin-like protein in salivary glands of Lygus hesperus (Hemiptera: Miridae)

Trypsin-like enzymes from the salivary gland complex (SGC) of Lygus hesperus Knight were partially purified by preparative isoelectric focusing (IEF). Enzyme active against Nalpha-benzoyl-L-arginine-p-nitroanilide (BApNA) focused at approximately pH 10 during IEF. This alkaline fraction gave a single activity band when analyzed with casein zymograms. The serine proteinase inhibitors, phenylmethylsulfonyl fluoride (PMSF) and lima bean trypsin inhibitor, completely inhibited or suppressed the caseinolytic activity in the crude salivary gland extract as well as the IEF-purified sample. Chicken egg white trypsin inhibitor also inhibited the IEF-purified sample but was not effective against a major caseinolytic band in the crude salivary gland extract. These data indicated the presence of serine proteinases in the SGC of L. hesperus. Cloning and sequencing of a trypsin-like precursor cDNA provided additional direct evidence for serine proteinases in L. hesperus. The encoded trypsin-like protein included amino acid sequence motifs, which are conserved with five homologous serine proteinases from other insects. Typical features of the putative trypsin-like protein from L. hesperus included residues in the serine proteinase active site (His(89), Asp(139), Ser(229)), conserved cysteine residues for disulfide bridges, residues (Asp(223), Gly(252), Gly(262)) that determine trypsin specificity, and both zymogen signal and activation peptides.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Specific formation of trypsin-resistant micelles on a hydrophobic peptide observed with Triton X-100 but not with octylglucoside

The manner of interaction of the coat peptide of the Pf3 phage (Pf3 peptide) with lipid bilayers has been extensively studied. Presently, we designed a derivative of the Pf3 peptide, referred to as the DDRK peptide, and subjected it to trypsin digestion to understand its physicochemical properties. In the presence of Triton X-100 used for solubilization of the peptide, digestion of DDRK with trypsin caused specific cleavage at the lysine (Lys) residue in its N-terminal region but not at other Lys residues or at the arginine residue. As the N-terminal region of the DDRK peptide is relatively hydrophilic, but its remaining region is hydrophobic, this hydrophobic region of the peptide would be expected to be coated by Triton micelles. Thus, we propose that the presence of such micelles protected against cleavage there, leading to selective cleavage by trypsin of the DDRK peptide at its hydrophilic Lys residue in the N-terminal part of the molecule. However, such a protective effect on the DDRK peptide against trypsin digestion was not observed with octylglucoside. The observed results are important for better understanding of the manner of interaction between detergents and hydrophobic peptides.     

Chemical modification of human placental glutathione transferase by pyridoxal 5'-phosphate.

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.     

Inhibition mechanism of a peanut trypsin-chymotrypsin inhibitor, B-III: determination of the reactive sites for trypsin and chymotrypsin

Peanut inhibitor B-III was found to form two types of complexes with trypsin, T2I and TI, by gel filtration HPLC. Two cleaved peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in the trypsin modified inhibitor (TM-B-III*R*S) (J. Biochem. 93, 479-485 (1983] were resynthesized by the complex formation with 2 mol of trypsin. These results suggest that the two peptide bonds may be the reactive sites for trypsin. TM-B-III*R*S inhibited bovine trypsin as well as native B-III but had little chymotrypsin inhibitory activity. The two peptide bonds, Arg(10)-Arg(11) and Arg(38)-Ser(39), in B-III were cleaved partly by prolonged incubation with a catalytic amount of chymotrypsin. But gel filtration HPLC of the chymotrypsin-inhibitor complex showed the formation of only CI complex. Incubation of TM-B-III*R*S with an equimolar amount of chymotrypsin resulted in the resynthesis of only the Arg(10)-Arg(11) bond. These findings suggest that Arg(10)-Arg(11) may be a true reactive site for chymotrypsin. An inhibition mechanism of B-III against trypsin and chymotrypsin was proposed from the results obtained by the present studies.     

Structural Insights into the Role of the Cyclic Backbone in a Squash Trypsin Inhibitor

MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin.     

Inhibition of rat and bovine trypsins and chymotrypsins by soybean, bovine basic pancreatic, and bovine colostrum trypsin inhibitors.

1. Bovine (Bos taurus) trypsin and trypsin activity in rat (Rattus norvegicus) pancreatic extract were inhibited by soybean trypsin inhibitor and by bovine basic pancreatic and colostrum inhibitors. 2. Bovine alpha-chymotrypsin was inhibited by soybean and bovine basic pancreatic inhibitors but only weakly by colostrum inhibitor. 3. Chymotrypsin activity in rat pancreatic extract was due to at least three different components against all of which the inhibitors were largely ineffective. 4. It is concluded that bovine colostrum inhibitor has a more limited inhibition spectrum than the phylogenetically related basic pancreatic inhibitor which, in turn, is less active against rat than against bovine enzymes.     

Surface-simulation synthesis of the substrate-binding site of an enzyme. Demonstration with trypsin.

From the X-ray co-ordinates of bovine trypsin and its complexes with substrate analogues (benzamidine) and with soya-bean trypsin inhibitor, a peptide (TP) was designed and synthesized by surface-simulation synthesis, a concept previously introduced by this laboratory, to mimic the binding site of trypsin. Also, a control peptide (CTP) was synthesized that contained all the amino acids present in the TP peptide, except that their order was randomized. The radioiodinated TP peptide bound specifically to adsorbents of benzamidine, whereas the control CTP peptide exhibited no binding activity. Conjugates to succinyl (3-carboxypropionyl)-lysozyme of the TP peptide, control CTP peptide and other unrelated peptides were examined by a radiometric binding assay for the ability to bind soya-bean trypsin inhibitor and human alpha 1-antitrypsin. Conjugates of the TP peptide exhibited considerable binding activity to adsorbents of soya-bean trypsin inhibitor or alpha 1-antitrypsin. None of the other peptide conjugates possessed any binding activity. Action of the active-site-directed reagents phenylmethanesulphonyl fluoride and di-isopropyl phosphorofluoridate on free TP and CTP peptides resulted in the modification of a serine residue in the TP peptide whereas the CTP peptide remained unaltered. The TP peptide, either in the free form or as a conjugate on succinyl-lysozyme, had no enzymic activity on protein substrates or on tosylarginine methyl ester. These findings indicated that the binding activity of an enzyme was well mimicked by the surface-stimulation peptide but that reproduction of the catalytic activity was not obtained.     

Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.     

A hemolytic peptide from the mycophilic fungus Sepedonium chrysospermum (Bull.) Fr

The hemolytic activity of an extract of the mycoparasite Sepedonium chrysospermum (teleomorph Hypomyces chrysospermus) was detected and characterized. Extraction of the fungal biomass by methanol yielded a fraction in which the hemolytic activity against human red blood cells corresponded to a peptide with a molecular mass of 7,653.72 Da and an isoelectric point of approximately 5.8. The peptide was temperature resistant, and the hemolysis was only partially inhibited, even after a 30-min pre-incubation at 100°C. Its hemolytic activity was unaffected by treatment with proteolytic enzymes such as trypsin. Among the divalent cations assayed, Hg²⁺ was the strongest inhibitor of hemolysis. The reducing agent, dithiothreitol, and the membrane lipid, cholesterol, demonstrated concentration-dependent inhibitory activities. Finally, hemolytic activity triggered by the peptide was analyzed by scanning electron microscopy, and a pore-forming activity was detected.     

Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis

Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent‐denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Epitope mapping and features of the epitope for monoclonal antibodies inhibiting enzymatic activity of Helicobacter pylori urease

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit.     

Labeling the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum with maleimidylsalicylic acid

Maleimidylsalicylic acid reacts with the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum with high affinity and inhibits the ATPase activity following a pseudo-first-order kinetic with a rate constant of 8.3 m(-1) s(-1). Calcium binding remains unaffected in the maleimide-inhibited ATPase. However, the presence of ATP, ADP, and, to a lesser extent, AMP protects the enzyme against inhibition. Furthermore, ATPase inhibition is accompanied by a concomitant decrease in ATP binding. The stoichiometry of the nucleotide-dependent maleimidylsalicylic acid binding is 6-10 nmol/mg ATPase, which corresponds to the binding of up to one molecule of maleimide/molecule of ATPase. The stoichiometry of maleimide binding is decreased in the presence of nucleotides and in the ATPase previously labeled with fluorescein-5'-isothiocyanate or N-ethylmaleimide A fluorescent peptide was isolated by high performance liquid chromatography after trypsin digestion of the maleimide-labeled ATPase. Analysis of the sequence and mass spectrometry of the peptide leads us to propose Cys(344) as the target for maleimidylsalicylic acid in the inhibition reaction. The effect of Cys(344) modification on the nucleotide site is discussed.     

Trypsin activity reduced by an autocatalytically produced nonapeptide

Trypsin, a serine protease enzyme plays a pivotal role in digestion and is autocatalytic. The crystal structure of a complex formed between porcine trypsin and an auto catalytically produced peptide is reported here. This complex shows a reduction in enzyme activity as compared to native beta-trypsin. The nonapeptide has a lysine, which is recognized by Asp 189 at the specificity pocket. The auto catalytically produced native nonapeptide is bound at the active site cleft like other trypsin inhibitors but the important interactions with the oxyanion hole are absent. The peptide covers only a part of the active site cleft and hence the enzyme activity is reduced rather than being inhibited.     

Activation of Clostridium botulinum type E toxin purified by two different procedures

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.     

Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

When rat brain membranes were incubated with [3H]flunitrazepam in the presence of UV light, predominantly one protein (P51) was irreversibly labeled in cerebellum and at least two proteins (P51 and P55) were labeled in hippocampus. On digestion of membranes with increasing concentrations of trypsin up to 40% of radioactivity irreversibly bound to proteins was removed from the membranes. In addition, P51 was nearly completely degraded to a peptide with apparent molecular weight 39,000 and this peptide was further degraded to a peptide with apparent molecular weight 25,000. In contrast, protein P55 was only partially degraded by trypsin and yielded two proteolytic peptides with apparent molecular weights 42,000 and 45,000 which seemed to be rather stable against further attack by trypsin. Membranes treated with trypsin still had the capacity to bind [3H]-flunitrazepam reversibly with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin. These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane-associated proteins P51 and P55 are differentially protected against degradation by trypsin.     

Limited proteolysis of smooth muscle myosin light chain kinase

Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.