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PubMed2002

Mutational analysis of the autoprocessing site of subtilisin YaB-G124A.

The potential residue at the autoprocessing site for improving processing efficiency was evaluated from hydrolysis of 19 cleavage-site-mimicking octapeptides, VTTXQTVP (-4 to +4), by the mature subtilisin YaB and YaB-G124A mutants. Both enzymes cleaved the octapeptides mainly at two sites, X-Q (A-site) and Q-T (B-site), at varied preferences. Based on the results above, Met(-1) of YaB-G124A was mutated and, as expected, extracellular enzyme production increased with Gln or Ala replacement, but decreased with Ile or Asp substitution. Together with previous structural studies, our results suggest that autoprocessing is dependent on not only the primary structure, but also the peptide flexibility around the processing site. Cleavage at the B-site resulted in a novel YaB mutant lacking the N-terminus Gln 1, which led the mutant enzyme to less enzymatic activity by 80% and less thermal stability by 20 degrees C, perhaps due to its ligation to the high-affinity calcium ion.

Amino Acid Substitution↗
PubMed1996

Specific IgE and IgG1 responses to subtilisin Carlsberg (Alcalase) in mice: development of an intratracheal exposure model.

The primary objective of this study was to examine the feasibility of using a mouse model to evaluate the immunogenicity of proteins as a potential method to determine occupational exposure guidelines. Mice were intratracheally administered a benchmark protein allergen, subtilisin Carlsberg (Alcalase) in detergent matrix once a week for 4 to 6 weeks and specific IgE and IgG1 levels were determined. In all experiments, specific IgE levels were determined by using a rat basophilic leukemia cell (RBL) release assay, while specific IgG1 was measured by an ELISA. A good correlation was observed between IgE titers determined by the RBL assay and rat passive cutaneous anaphylaxis assay. Intratracheal administration of protease with detergent matrix was found to result in significant IgE and IgG1 responses that were dose related. Detergent matrix was found to enhance the Alcalase-specific IgE and IgG1 response when administered by the intratracheal route. The IgG1 response was much more robust, easier to measure, and found to follow the IgE response. These results suggest that a mouse intratracheal model is a feasible approach to examining the immunogenic potency of enzymes using specific IgE or IgG1 as the end points. Additional development and validation of the mouse model with other types of proteins will be pursued.

Animals↗
PubMed1996

Specific antibody responses to subtilisin Carlsberg (Alcalase) in mice: development of an intranasal exposure model.

An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 x DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-microliters aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.

Administration, Intranasal↗
PubMed1995

Proteolytic processing mechanisms in the biosynthesis of neuroendocrine peptides: the subtilisin-like proprotein convertases.

The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2 (SPC2) and PC1/PC3 (SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member--furin/PACE (SPC1)--is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation of the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps.

Animals↗
PubMed1995

A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. I. Hydrophobic replacements at the position of Met103.

The thermodynamic effects of replacing the Met residue at amino acid position 103 of Streptomyces subtilisin inhibitor with other non-polar aliphatic residues were studied by means of differential scanning calorimetry. All but the Leu mutant, which is as stable as the wild-type but has different cooperative units in the course of unfolding, showed destabilization in terms of free energy. Similar losses in free energy, however, were caused by different reasons, i.e. by increased entropy for the Ala mutant and by decreased enthalpy for the Ile mutant, with a tendency that increases in entropy are accompanied by increases in enthalpy. The gain in entropy that caused the largest loss in free energy for the Gly mutant was unexpectedly smaller than that for the Ala mutant. The changes in enthalpy and entropy induced by the mutations exhibited some correlations with hydrophobicity, while no clear correlation was found between the changes in free energy and hydrophobicity.

Bacterial Proteins↗
PubMed1995

A thermodynamic study of mutant forms of Streptomyces subtilisin inhibitor. II. Replacements at the interface of dimer formation, Val13.

Amino acid replacements in a homodimeric protein, Streptomyces subtilisin inhibitor, at the position of Val13, which is located at the center of the beta-sheet interface of the domain-domain interaction, changed both the overall stability and the denaturational scheme. All the mutant forms lost stability, losses in free energy at 82.21 degrees C at pH 7.0 obtained by calorimetric measurements ranging from 10.3 kcal (mol dimer)-1 for the Gly mutant, which involved a loss in enthalpy without a compensating loss in entropy, to 0.84 kcal (mol dimer)-1 for the Ile mutant, which involved comparable losses in enthalpy and entropy. A gain in enthalpy for the Ala mutant was overcome by a gain in entropy, resulting in a loss in free energy by 6.78 kcal (mol dimer)-1. It was found that decreases in enthalpy and entropy showed good correlation with increasing side-chain hydrophobicity, while no such correlation was present for free energy. Replacements with Gly, Ala, Met and Phe altered the thermal denaturational scheme from N2 reversible 2D, which was the case for the Leu and Ile mutants as well as for the wild-type, to N2 reversible 2N* reversible 2D, where the first step can be regarded as a dissociation of the native dimer followed by a major unfolding in the second step. The free energy acquired by forming the dimer was estimated to be 2.03 kcal mol-1 for the Ala mutant at 66.95 degrees C at pH 9.5 and lower for the other three mutants.

Bacterial Proteins↗
PubMed1995

Solution X-ray scattering analysis of cold- heat-, and urea-denatured states in a protein, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI), a homo-dimeric protein with a subunit of 113 residues with two disulfide bonds, is known to exist at low pH in at least three distinct thermodynamic states namely, the native (N), cold-denatured (D') and heat-denatured (D). Small-angle X-ray scattering was used to analyze and to compare overall chain conformations of SSI in typical, N, D', D and urea-denatured states (Durea). Molecular masses were determined from scattering intensities extrapolated to a scattering angle of zero, which showed that SSI exists as a homo-dimer in the N state, but as dissociated monomers in the D', D and Durea states. From Guinier plots of the scattering intensities, radii of gyration (Rg) were determined to be 20.1(+/- 1.8) A for N, and 20.7(+/- 1.3), 25.8(+/- 1.5) and 32 to 35 A for D', D and Durea, respectively. Kratky plots for both N and D' exhibited a bell-shape indicating that the polypeptide chain has a globular part not only in N but also in D', while Kratky plots for D and Durea showed that the polypeptide chain has no globular part either in Durea or D. Combined with the results from circular dichroism and 1H NMR spectra, a picture emerges for the polypeptide chain conformation of SSI such that in N it is a globular dimer close to that in the crystal, in Durea it is totally disordered and expanded nearly to a fully random chain with restrictions only from the disulfide bridges, in D the entire chain is disordered and expanded but with considerable local intra-chain interactions, and in D' the chain consists of a part with a unique tertiary structure and a part disordered and expanded to a degree comparable to D.

Bacterial Proteins↗
PubMed1995

Folding pathway mediated by an intramolecular chaperone: characterization of the structural changes in pro-subtilisin E coincident with autoprocessing.

Mechanisms by which many N-terminal propeptides facilitate folding of proteins are unknown. The maturation of such proteins from their precursors involve three steps, namely: (1) folding of the precursor, (2) autoprocessing of the propeptide from the N terminus and (3) degradation of the cleaved propeptide. Using subtilisin E we have analyzed the mechanism of propeptide-mediated protein folding. Two active site mutations allow us to trap intermediates at stages of autoprocessing and degradation. An analysis of these intermediates has shown the existence of a molten-globule-like intermediate on the folding pathway. After autoprocessing of the propeptide, this intermediate undergoes a structural reorganization which reduces solvent-accessible hydrophobic surface area and increases the amount of its tertiary structure. Removal of the propeptide from the mature enzyme in this intermediate state occurs only by proteolytic degradation and contributes to the stability of the active enzyme.

Circular Dichroism↗
PubMed1996

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

2,2'-Dipyridyl↗
PubMed1999

Engineered Bacillus lentus subtilisins having altered flexibility.

The three-dimensional structures of engineered variants of Bacillus lentus subtilisin having increased enzymatic activity, K27R/N87S/V104Y/N123S/T274A (RSYSA) and N76D/N87S/S103A/V104I (DSAI), were determined by X-ray crystallography. In addition to identifying changes in atomic position we report a method that identifies protein segments having altered flexibility. The method utilizes a statistical analysis of variance to delineate main-chain temperature factors that represent significant departures from the overall variance between equivalent regions seen throughout the structure. This method reveals changes in main-chain mobility in both variants. Residues 125-127 have increased mobility in the RSYSA variant while residues 100-104 have decreased mobility in the DSAI variant. These segments are located at the substrate-binding site and changes in their mobility are believed to relate to the observed changes in proteolytic activity. The effect of altered crystal lattice contacts on segment flexibility becomes apparent when identical variants, determined in two crystal forms, are compared with the native enzyme.

Bacillus↗
PubMed1998

Subtilisin-related serine proteases in the mammalian constitutive secretory pathway.

Proteolytic cleavage is a key step in the activation of many secreted proteins. Since the identification of the prototype yeast enzyme Kex2, seven subtilisin-related serine proteases, together with a number of isoforms, have been identified in mammalian cells, five of which act within the constitutive secretory pathway. Overlapping expression patterns and substrate specificities complicate analysis but individual roles are beginning to emerge for some members of the group.

Animals↗
PubMed1996

Laundry performance of subtilisin proteases.

Effective laundry protease performance against susceptible stains depends upon both the enzyme itself and the environment in which it must work. In order to technically design superior laundry proteases, a model for protease's mechanism of action in detergents was developed which has been substantiated through-the-wash. While evaluation of this model and/or a given protease's effectiveness could be judged by a variety of methods, the utility of using visual wash performance comparisons, analytical, and stain characterization studies is described. Finally, data comparing the performance of wild type Subtilisin proteases with mutants designed via the projected model are given, demonstrating possible utility of the system.

Catalysis↗
PubMed1996

Thermitase - kinetic differentiation to the subtilisins.

In the last 12 years more than 80 peptidyl substrates and substrate analogous inhibitors were synthesized in our group and used to characterize thermitase, a serine proteinase of the subtilisin family produced by Thermoactinomyces vulgaris. The kinetic parameters of hydrolysis and inhibition reactions, respectively, with thermitase were determined and compared with those found with related enzymes. Conclusions with respect to the extent of the active site of thermitase and to the specificity of the different subsites could be drawn.

Binding Sites↗
PubMed1996

Crystal structure analysis of subtilisin BPN' mutants engineered for studying thermal stability.

The high resolution crystal structures of four genetically engineered subtilisin BPN' variants (E.C. 3.4.21.14) which vary dramatically in their stability have been determined. The simplest variant, S3, contains two altered residues, N218S and S221C. The N218S change was incorporated for its stabilizing effects and its influence on crystallization; the S221C change, a modification of the active site serine, was included to reduce autolysis. The second variant, S12, includes the two additional stabilizing mutations M50F and Y217K. S15, the third variant, in addition to the 4 single site mutations, has residues 75-83, the high-affinity calcium-binding site, deleted. The final variant S22 incorporates all of the above changes and two additional site specific mutations, T22C and S87C, which form a stabilizing disulfide bridge. The structural changes and influence on stability of each of these mutations are discussed in the context of supporting biophysical studies.

Crystallography, X-Ray↗
PubMed1996

Crystallographic investigations of subtilisin BPN' mutants engineered for studying thermal stability.

Subtilisin BPN' (BPN) is an industrially important serine protease that has been extensively investigated in many laboratories. In an effort to improve the thermal stability of the enzyme, researchers at Procter & Gamble have used site-directed mutagenesis techniques to produce several variants of BPN in which residues at the surface of the enzyme have been substituted. We initiated crystallographic studies to determine the structural consequences of these amino acid substitutions. In the course of this work we obtained excellent crystals that correspond to the C2 crystal form of native BPN that has been previously reported. Since the structure reported in that work was of only medium resolution, high-resolution X-ray data for this crystal form of native BPN have been collected and the refinement of the structure has been extended using these new data. Isomorphous crystals of two variants, Q19E and Q271E, have also been grown, high-resolution X-ray data have been collected for these crystals, and the experimental results are described. The structures of the native enzyme and the Q271E variant have been refined and are described.

Binding Sites↗
PubMed1996

Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL.

As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.

Amino Acid Sequence↗
PubMed1981

[Some experience with the enzymatic digestion of liver tissue by means of subtilisin Carlsberg (author's transl)].

According to the literature, the use of Subtilisin Carlsberg for the enzymatic digestion of tissue is supposed to yield significantly higher findings of barbiturates, alkaline substances, and benzodiazepins than traditional methods. In the case of 11 liver specimens, however, a comparison with a modified acetone method (extraction by ultrasonics) did not lead to an improvement in the results, nor did it facilitate preparation of the specimens.

Forensic Medicine↗
PubMed1996

Production of recombinant proteins in Chinese hamster ovary cells overexpressing the subtilisin-like proprotein converting enzyme furin.

The proprotein processing enzyme furin is the mammalian prototype of a novel family of subtilisin-like serine endoproteases which possess cleavage specificity for sites involving multiple basic amino acid residues and are involved in the processing of precursor proteins of a variety of regulatory peptides and proteins. One of the limiting steps in the engineering of mammalian cells designed for the overproduction of secreted proteins is the endoproteolytic cleavage of the precursor molecule to its mature biologically active form. The extremely low level of endogenous furin is likely the reason why cells are not able to fully mature overexpressed precursor proteins to their mature form. Here, we report a CHO-derived cell line genetically engineered for the production of high levels of recombinant proteins that need such endoproteolytic maturation. First, the human furin cDNA under the control of the cytomegalovirus early promoter and enhancer was introduced and overexpressed in a DHFR-deficient CHO cell line. A permanent cell line CHO-D3-FUR was established that expressed biologically active furin. Subsequently, to demonstrate the capacity of CHO-D3-FUR cells to produce recombinant proteins in a fully matured form, two derivative cell lines were established that overexpressed the von Willebrand factor (vWF) and transforming growth factor beta 1 (TGF beta 1); CHO-D3-vWF and CHO-D3-TGF beta 1, respectively. Both derivative cell lines were able to produce relatively high levels of recombinant protein in a fully matured and biologically active form. Our results illustrate the potential of the CHO-D3-FUR cell line in the production of recombinant secretory proteins that need endoproteolytic activation at the consensus furin cleavage sequence Arg-X-Lys/Arg-Arg.

Animals↗