Inhibition of neutral proteases from polymorphonuclear (PMN) neutrophils by salicylazosulfapyridine (SASP)

Summary Cytochemical methods were used to demonstrate the inhibitory effect of salicylazosulfapyridine (SASP) on the activity of neutral proteases produced by neutrophilic granulocytes (PMN proteases). The SASP metabolites (5-aminosalicylic acid and sulfapyridine), produced by splitting of SASP by bacteria in the colon, did not inhibit the activity of PMN proteases. Paradoxically, sulfapyridine intensified PMN protease activity. A similar effect however could not be demonstrated for 5-aminosalicylic acid.     

Cys-His proteases are among the wired proteins of the cell

Integrated cell protein degradation can be paced by the transfer of reductive energy, as revealed by experimental agents of informative actions. The peptidolytic pair of Cys-His proteases can undergo oxidative reactions to inactive derivatives and inhibitory metal binding. Proton-dependent ionizations can modify ongoing activity. If the reaction rate of a Cys-His protease were found responsive to the ranges of metal/redox/proton factors regulated within the cell, then these factors might serve to link the peptidolytic reaction rate to cell controls. Here, cathepsin B (cat B) was found to be inhibited by Zn2+, Fe3+, and Cu2+ (1-50 microM) under excess GSH or DTT protease activators (6 mM). Under DTT or GSH (6 mM) the initial inhibitory action of Zn2+ is stable indefinitely; however, the inhibitory actions of Fe3+ and Cu2+ are reversed over approximately 1h. The 12-14 min half time of reversal of initial protease inhibition is correlated with the measured reduction of Fe3+ to Fe2+ by DTT or GSH (pH 5.5 or 6.5). Endogenous Fe2+ concentrations (100 microM) inhibit cat B only marginally. However, the inhibitory threshold of several microM Fe3+ is only a few percent oxidation of the endogenous pool. Without metals cat B reaction is reportedly proportional to GSH concentration, and is inhibited by increasing GSSG/GSH redox ratio. Following activation with GSH, cat B can be influenced by Fe3+/Fe2+, Cu2+/Cu+, and GSSG/GSH ratios and concentrations. Results are interpreted in relation to properties of the thiolate-imidazolium pair as illustrated by Dock modeling of their shared Fe3+ binding. It is proposed that the interaction of Cys-His with 1 electron transition between Fe2+ and Fe3+ serves as a sensor, signal integrator and switch wiring cat B reaction rate to the transfer of reductive energy in the presence of excess GSH. Speciated metals might also serve among electron acceptors transferring from reduced protease to oxygen. Results provide a model for pharmacologic redox switching of protease functions with metal-interactive drugs, and other nano-technology engineering.     

Purification and degradation of purified neurofilament proteins by the brain calcium-activated neutral proteases

The effect of the three forms (CANP1, CANP2 & CANP3) of calf brain calcium activated neutral protease (CANP) on the hydrolysis of purified neurofilament triplet proteins was investigated. It was observed that: each of the purified neurofilament proteins, was hydrolyzed slowly by CANP2 whereas the hydrolysis of 150 KDa and 68 kDa proteins by CANP1 & CANP3 was rapid; when assembled neurofilaments were used as a substrate, again differences in the rate and extent of degradation of the triplet proteins by the three proteases were observed. For example, little cleavage of the 68 kDa protein by CANP2 and CANP3 was noted whereas 210 kDa and 150 kDa proteins remained largely intact. CANP1 degraded the 150 kDa and 68 kDa proteins more rapidly than 210 kDa protein, where only a slight effect was noted. These data provide further proof of the existence of three different forms of CANP in the brain, and indications of the resistance of 210 kDa protein to proteolysis which may be compatible with its proposed special role in crossbridge formation.     

Two carbohydrate binding sites in the H(CC)-domain of tetanus neurotoxin are required for toxicity

Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.     

Cell-wall lytic enzymes (autolysins) of Chlamydomonas reinhardtii are (hydroxy)proline-specific proteases

Two stage specific cell-wall lytic enzymes (autolysins) from different strains of the unicellular, biflagellated green alga Chlamydomonas reinhardtii were isolated and purified to homogeneity. Quantitative and specific photometric assays for biological activity were worked out to follow fractionation and to establish lytic specificity and kinetics. The autolysins were studied for enzymatic properties and screened for biological activity towards several wall components obtained by salt extractions of sporangia and zoospores from C. reinhardtii. The autolysins are proteolytic enzymes, fragmenting proline- or hydroxyproline-containing polypeptides in structures like connective tissue. They attack predominantly selected domains within the walls of zoosporangia or gametes. The sporangial autolysins are not only site- and strain-specific but also stage-specific, whereas the gamete autolysins lyse cell walls of gametes as well as those of sporangia and zoospores.     

Ser(214) is crucial for substrate binding to serine proteases

Highly conserved amino acids that form crucial structural elements of the catalytic apparatus can be used to account for the evolutionary history of serine proteases and the cascades into which they are organized. One such evolutionary marker in chymotrypsin-like proteases is Ser(214), located adjacent to the active site and forming part of the primary specificity pocket. Here we report the mutation of Ser(214) in thrombin to Ala, Thr, Cys, Asp, Glu, and Lys. None of the mutants seriously compromises active site catalytic function as measured by the kinetic parameter k(cat). However, the least conservative mutations result in large increases in K(m) because of lower rates of substrate diffusion into the active site. Therefore, the role of Ser(214) is to promote the productive formation of the enzyme-substrate complex. The S214C mutant is catalytically inactive, which suggests that during evolution the TCN-->AGY codon transitions for Ser(214) occurred through Thr intermediates.     

Cytochemical investigation of neutral proteases in polymorphonuclear (PMN) neutrophils in acute inflammatory diseases

Summary Neutral proteases can be released from PMN neutrophils in blood smears from healthy subjects by incubation with NaCl-borate buffer. The activity of the PMN proteases can be revealed by the degradation of erythrocytes and plasma within ring-shaped areas centered around each neutrophil (halo effect). During the acute stage of various inflammatory diseases (pneumonia, meningitis, cholecystitis, etc.) the activity of neutral PMN proteases is substantially reduced, as reflected by reduced halo formation. After recovery, halo formation returns to normal. Temporary lowering of neutral PMN proteases is thus one of a series of functional defects of PMN neutrophils which are detectable in the course of acute infectious diseases. These include reduced phagocytosis, altered chemotaxis and reduced bactericidal function. The cytochemical test for neutrophilic granulocyte function used in the present investigation is especially practical by comparison with the other techniques: it saves time and is simple to perform.Dedicated to Prof. W. Graumann on the occasion of his 65th birthday     

Rational redesign of neutral endopeptidase binding to merlin and moesin proteins

Neutral endopeptidase (NEP) is a 90‐ to 110‐kDa cell‐surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti‐tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K¹⁹K²⁰K²¹ basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM‐related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E¹⁵⁹Q¹⁶⁰ in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N¹⁵⁹L¹⁶⁰ residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N¹⁵⁹L¹⁶⁰Y¹⁶¹K¹⁶²M¹⁶³ in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP‐binding protein. These data form the basis for further exploration of the details of NEP‐ERM binding and function.     

Glutathione-S-transferases are major cytosolic thyroid hormone binding proteins

Thyroid hormone binding proteins of rat liver cytosol were characterized. Glutathione-S-transferases were identified among major cytosolic proteins adsorbed by thyroxine affinity matrices. The Ya and Yb subunits of the glutathione-S-transferases were also principal proteins of cytosol covalently labeled with 3,3',5-triiodo-L-thyronine (T3) or 3,3',5,5'-tetraiodo-L-thyronine (T4) by photoaffinity methods. T3 and T4, but not L-thyronine or iodinated tyrosines, were bound with high affinity to purified glutathione-S-transferases and were potent inhibitors of their enzymatic activities. These results suggest that glutathione-S-transferases have the potential to function in the intracellular binding and transport of thyroid hormones. The proteins provide a means for regulating the action and metabolism of thyroid hormones by acting as high capacity binding components.     

Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus

The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.     

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3′, a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca²⁺-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3′ by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.     

Evidence that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11)

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.     

Structures and homologies of carbohydrate: phosphotransferase system (PTS) proteins

The bacterial phosphotransferase system (PTS) is the major transport system for many carbohydrates that are phosphorylated concomitantly with the translocation step through the membrane (group translocation). It consists of two general proteins, enzyme I and histidine protein (HPr), and a series of more than 15 substrate-specific enzymes II (EII). The sequences of several of these derived from Gram-positive and Gram-negative bacteria were compared, which allowed the possible identification of the following functional domains: membrane-bound pore, substrate-binding site, linker domains, transphosphorylation domain and primary phosphorylation site. Several EIIs have been analysed in the meantime, also by topological tests, by sequential deletion of the corresponding structural genes, and by construction of intergenic hybrids between different domains of several EIIs. These data suggest evolutionary relationships between different EIIs; they also enable a general model to be constructed of EIIs as carbohydrate transport systems, phosphotransferases, chemoreceptors in chemotaxis and as part of a global regulatory network.